The Journal of Immunology current issue

IN THIS ISSUE [IN THIS ISSUE]

Wed, 04 Nov 2009 13:05:37 PST

Comment on "Ym1/2 Promotes Th2 Cytokine Expression by Inhibiting 12/15(S)-Lipoxygenase: Identification of a Novel Pathway for Regulating Allergic Inflammation" [LETTERS TO THE EDITOR]

Mabalirajan, U., Agrawal, A., Ghosh, B. — Wed, 04 Nov 2009 13:05:38 PST

Response to Comment on "Ym1/2 Promotes Th2 Cytokine Expression by Inhibiting 12/15(S)-Lipoxygenaes: Identification of a Novel Pathway for Regulating Allergic Inflammation" [LETTERS TO THE EDITOR]

Webb, D. C. — Wed, 04 Nov 2009 13:05:38 PST

Comment on "Reduced Cytotoxic Function of Effector CD8+ T Cells Is Responsible for Indoleamine 2,3-Dioxygenase-Dependent Immune Suppression" [LETTERS TO THE EDITOR]

Sorensen, R. B., Straten, P. t., Andersen, M. H. — Wed, 04 Nov 2009 13:05:38 PST

Role of Gut Commensal Microflora in the Development of Experimental Autoimmune Encephalomyelitis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Mucosal tolerance has been considered a potentially important pathway for the treatment of autoimmune disease, including human multiple sclerosis and experimental conditions such as experimental autoimmune encephalomyelitis (EAE). There is limited information on the capacity of commensal gut bacteria to induce and maintain peripheral immune tolerance. Inbred SJL and C57BL/6 mice were treated orally with a broad spectrum of antibiotics to reduce gut microflora. Reduction of gut commensal bacteria impaired the development of EAE. Intraperitoneal antibiotic-treated mice showed no significant decline in the gut microflora and developed EAE similar to untreated mice, suggesting that reduction in disease activity was related to alterations in the gut bacterial population. Protection was associated with a reduction of proinflammatory cytokines and increases in IL-10 and IL-13. Adoptive transfer of low numbers of IL-10-producing CD25⁺CD4⁺ T cells (>75% FoxP3⁺) purified from cervical lymph nodes of commensal bacteria reduced mice and in vivo neutralization of CD25⁺ cells suggested the role of regulatory T cells maintaining peripheral immune homeostasis. Our data demonstrate that antibiotic modification of gut commensal bacteria can modulate peripheral immune tolerance that can protect against EAE. This approach may offer a new therapeutic paradigm in the treatment of multiple sclerosis and perhaps other autoimmune conditions.

TNFR2-Deficient Memory CD8 T Cells Provide Superior Protection against Tumor Cell Growth [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kim, E. Y., Teh, S.-J., Yang, J., Chow, M. T., Teh, H.-S. — Wed, 04 Nov 2009 13:05:38 PST

TNF receptor-2 (TNFR2) plays a critical role in promoting the activation and survival of naive T cells during the primary response. Interestingly, anti-CD3 plus IL-2 activated TNFR2^–/– CD8 T cells are highly resistant to activation-induced cell death (AICD), which correlates with high expression levels of prosurvival molecules such as Bcl-2, survivin, and CD127 (IL-7R). We determined whether the resistance of activated TNFR2^–/– CD8 T cells to AICD contributes to more effective protection against tumor cell growth. We found that during a primary tumor challenge, despite initial inferiority in controlling tumor cell growth, TNFR2^–/– mice were able to more effectively control tumor burden over time compared with wild-type (WT) mice. Furthermore, vaccination of TNFR2^–/– mice with recombinant Listeria monocytogenes that express OVA confers better protection against the growth of OVA-expressing E.G7 tumor cells relative to similarly vaccinated WT mice. The enhanced protection against tumor cell growth was not due to more effective activation of OVA-specific memory CD8 T cells in vaccinated TNFR2^–/– mice. In vitro studies indicate that optimally activated OVA-specific TNFR2^–/– CD8 T cells proliferated to the same extent and possess similar cytotoxicity against E.G7 tumor cells as WT CD8 T cells. However, relative to WT cells, activated OVA-specific TNFR2^–/– CD8 T cells were highly resistant to AICD. Thus, the enhanced protection against E.G7 in TNFR2^–/– mice is likely due to the recruitment and activation of OVA-specific memory TNFR2^–/– CD8 T cells and their prolonged survival at the tumor site.

Dendritic Cell Function in Allostimulation Is Modulated by C5aR Signaling [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Regulation of T cell immunity by C5a has been suggested from recent studies. However, the underlying mechanisms, particularly the involved cells and biochemical basis, are not well defined. In this study, the direct modulation of dendritic cell (DC) activation and its function in T cell stimulation by C5a-C5aR interaction and the involved signaling pathways were investigated. We show that DCs from C5aR^–/– mice and normal DCs treated with C5aR antagonist have less-activated phenotype characterized with increased IL-10 and decreased IL-12p70 production in response to LPS stimulation, lowered surface expression of MHC class II, B7.2, and consequently have reduced capacity to stimulate allospecific T cells. Conversely, C5a stimulation up-regulates DC activation and its function in allostimulation. Furthermore, stimulation of C5aR mediates the inhibition of cAMP production and protein kinase A activity and is involved in activation of PI3K/AKT and NF-B signaling in DCs. These results demonstrate that C5a acts directly on C5aR expressed on DCs resulting in the cell activation and subsequently enhances its capacity for allospecific T cell stimulation. It also suggests that NF-B signaling induced by down-regulation of cAMP/ protein kinase A pathway and up-regulation of PI3K/AKT pathway following C5a stimulation may contribute to up-regulation of DC function.

Analysis of the Role of Tripeptidyl Peptidase II in MHC Class I Antigen Presentation In Vivo [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kawahara, M., York, I. A., Hearn, A., Farfan, D., Rock, K. L. — Wed, 04 Nov 2009 13:05:38 PST

Previous experiments using enzyme inhibitors and RNA interference in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I Ag presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared with wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits Ag presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus. The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild-type and TPPII-deficient mice. These data indicate that while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags in vivo.

Immunostimulatory RNA Oligonucleotides Induce an Effective Antitumoral NK Cell Response through the TLR7 [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

RNA oligonucleotides containing immune-activating sequences promote the development of cytotoxic T cell and B cell responses to Ag. In this study, we show for the first time that immunostimulatory RNA oligonucleotides induce a NK cell response that prevents growth of NK-sensitive tumors. Treatment of mice with immunostimulatory RNA oligonucleotides activates NK cells in a sequence-dependent manner, leading to enhanced IFN- production and increased cytotoxicity. Use of gene-deficient mice showed that NK activation is entirely TLR7-dependent. We further demonstrate that NK activation is indirectly induced through IL-12 and type I IFN production by dendritic cells. Reconstitution of TLR7-deficient mice with wild-type dendritic cells restores NK activation upon treatment with immunostimulatory RNA oligonucleotides. Thus, by activating both NK cells and CTLs, RNA oligonucleotides stimulate two major cellular effectors of antitumor immunity. This dual activation may enhance the efficacy of immunotherapeutic strategies against cancer by preventing the development of tumor immune escape variants.

Cupressaceae Pollen Grains Modulate Dendritic Cell Response and Exhibit IgE-Inducing Adjuvant Activity In Vivo [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Pollen is considered a source of not only allergens but also immunomodulatory substances, which could play crucial roles in sensitization and/or the exacerbation of allergies. We investigated how allergenic pollens from different plant species (Japanese cedar and Japanese cypress, which belong to the Cupressaceae family, and birch, ragweed, and grass) modulate murine bone marrow-derived dendritic cell (DC) responses and examined the effect of Cupressaceae pollen in vivo using mice. DCs were stimulated with pollen extracts or grains in the presence or absence of LPS. Cell maturation and cytokine production in DCs were analyzed by flow cytometry, ELISA, and/or quantitative PCR. Pollen extracts suppressed LPS-induced IL-12 production and the effect was greatest for birch and grass. Without LPS, pollen grains induced DC maturation and cytokine production without IL-12 secretion and the response, for which TLR 4 was dispensable, was greatest for the Cupressaceae family. Intranasal administration of Cupressaceae pollen in mice induced an elevation of serum IgE levels and airway eosinophil infiltration. Coadministration of ovalbumin with Cupressaceae pollen grains induced ovalbumin-specific IgE responses associated with eosinophil infiltration. The results suggest that modulation of DC responses by pollen differs among the plant families via (1) the promotion of DC maturation and cytokine production by direct contact and/or (2) the inhibition of IL-12 production by soluble factors. The strong DC stimulatory activity in vitro and IgE-inducing activity in mice support the clinical relevance of Cupressaceae pollen to allergies in humans.

Anergic T Cells Are Metabolically Anergic [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zheng, Y., Delgoffe, G. M., Meyer, C. F., Chan, W., Powell, J. D. — Wed, 04 Nov 2009 13:05:38 PST

Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function. In this report we demonstrate that anergic T cells are metabolically anergic, in that upon full stimulation (signals 1 plus 2) they fail to up-regulate the machinery necessary to support increased metabolism. These findings suggest that one mechanism responsible for the maintenance of T cell anergy is failure to up-regulate the metabolic machinery. Furthermore, we demonstrate that by blocking leucine, glucose, and energy metabolism, T cell activation is mitigated. Additionally, inhibition of these metabolic pathways during T cell activation leads to anergy in Th1-differentiated cells. Overall, our findings extend the role of T cell metabolism in regulating T cell function.

Ligand Binding but Undetected Functional Response of FcR after Their Capture by T Cells via Trogocytosis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Intercellular transfer of cell surface proteins by trogocytosis is common and could affect T cell responses. Yet, the role of trogocytosis in T cell function is still elusive, and it is unknown whether a molecule, once captured by T cells, harbors the same biological properties as in donor APC. In this study, we showed that FcR as well as the associated FcR subunit could be detected at high levels on murine and human T cells after their intercellular transfer from FcR-expressing APC. Capture of FcR occurred during coculture of T cells with FcR-expressing APC upon Ab- or Ag-mediated T cell stimulation. Once captured by T cells, FcR were expressed in a conformation compatible with physiological function and conferred upon T cells the ability to bind immune complexes and to provision B cells with this source of Ag. However, we were unable to detect downstream signal or signaling-dependent function following the stimulation of FcR captured by T cells, and biochemical studies suggested the improper integration of FcR in the recipient T cell membrane. Thus, our study demonstrates that T cells capture FcR that can efficiently exert ligand-binding activity, which, per se, could have functional consequences in T cell-B cell cooperation.

Activation-Induced CD154 Expression Abrogates Tolerance Induced by Apoptotic Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Gurung, P., Kucaba, T. A., Ferguson, T. A., Griffith, T. S. — Wed, 04 Nov 2009 13:05:38 PST

The decision to generate a productive immune response or tolerance often depends on the context in which T cells first see Ag. Using a classical system of tolerance induction, we examined the immunological consequence of Ag encountered in the presence of naive or activated apoptotic cells. Naive apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154, as tolerance resulted after i.v. injection of either naive or activated apoptotic CD154^–/– T cells, while coinjection of an agonistic anti-CD40 mAb with naive apoptotic T cells induced robust immunity. Dendritic cells fed activated apoptotic T cells in vitro produced IL-12p40 in a CD154-dependent manner, and the use of IL-12p40^–/– mice or mAb-mediated neutralization of IL-12 revealed a link between CD154, IL-12, and the ability of activated apoptotic T cells to induce immunity rather than tolerance. Collectively, these results show that CD154 expression on apoptotic T cells can determine the outcome of an immune response to Ag recognized within the context of the apoptotic cells and suggest that the balance between naive and activated apoptotic T cells may dictate whether a productive immune response is encouraged.

Protein Kinase B/Akt Signals Impair Th17 Differentiation and Support Natural Regulatory T Cell Function and Induced Regulatory T Cell Formation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Protein kinase B (PKB)/Akt signals control T cell proliferation and differentiation but their effect on the generation and function of regulatory T cells (Treg) and Th17 cells is not well understood. In this study, we show that elevated PKB signals antagonize the immunosuppressive effect of TGF-β1 on cell size, CD25 and CD98 expression, and proliferation of CD3-stimulated naive CD4⁺ T cells from wild-type and CD28-deficient mice. Conventional CD4⁺ T cells expressing active PKB are less susceptible to suppression by natural regulatory T cells. Although PKB signals do not affect the development of natural regulatory T cells, they enhance their suppressor capacity. Upon TCR triggering and TGF-β1 costimulation, wild-type and CD28-deficient CD4⁺ T cells transgenic for PKB readily express Foxp3, thereby acquiring suppressor capacity. These effects of elevated PKB signals on T cell function involve a marked and sustained activation of STAT5 and Foxp3 and reduction in nuclear NFATc1 levels. In contrast, PKB signals impair TGF-β1/IL-6-mediated differentiation of naive CD4⁺ T cells into the Th17 lineage. This correlates with an increased signaling of ERK, STAT5, and STAT6. Finally, elevated PKB signals reduced the severity of experimental autoimmune encephalomyelitis in wild-type mice but induced experimental autoimmune encephalomyelitis in mice deficient for CD28. Altogether, these data indicate an important role of PKB signals on control of TGF-β1-mediated T cell responses and, thereby, on tolerizing and inflammatory immune processes.

Thymic Regulation of Autoimmune Disease by Accelerated Differentiation of Foxp3+ Regulatory T Cells through IL-7 Signaling Pathway [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Chen, X., Fang, L., Song, S., Guo, T. B., Liu, A., Zhang, J. Z. — Wed, 04 Nov 2009 13:05:38 PST

The exact role of adult thymus in autoimmune disease state is poorly understood. We show here that thymus regulated experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, as evidenced by loss of spontaneous recovery in thymectomized EAE mice. There was progressive enrichment for CD4 single-positive Foxp3⁺ regulatory T cells in thymocytes during the course of EAE and they suppressed the disease when adoptively transferred. Thymus was shown to undergo an active process characterized by accelerated differentiation and proliferation of regulatory T (Treg) cells through a mechanism involving increased expression of IL-7 in stromal cells and dynamic expression of IL-7 receptor in thymic Treg cells. This process preceded EAE recovery and selectively affected Treg over non-Treg cells in the thymus, leading to increased output of thymic Treg cells and self-regulation of EAE. The study reveals a novel role of thymus in self-regulation of autoimmune condition.

The Proteasome Inhibitor Bortezomib Enhances the Susceptibility to Viral Infection [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Basler, M., Lauer, C., Beck, U., Groettrup, M. — Wed, 04 Nov 2009 13:05:38 PST

The proteasome, a multicatalytic protease, is responsible for the generation of most MHC class I ligands. Bortezomib, a proteasome inhibitor, is clinically approved for treatment of multiple myeloma and mantle cell myeloma. In the present study, we investigated the effect of bortezomib on viral infection. Infection of bortezomib-treated mice with the lymphocytic choriomeningitis virus (LCMV) led to a decreased cytotoxic T cell response to several LCMV-derived CD8⁺ T cell epitopes. Bortezomib treatment caused a reduced expansion of CD8⁺ T lymphocytes and increased viral titers in LCMV-infected mice. Administration of bortezomib during expansion of CD8⁺ T cells had no influence on the cytotoxic T cell response, suggesting that bortezomib interferes with priming of naive T cells. Indeed, determination of Ag load in spleen 4 days post infection, revealed a reduced presentation of LCMV-derived cytotoxic T cell epitopes on MHC class I molecules. In summary, we show that proteasome inhibition with bortezomib led to an increased susceptibility to viral infection, and demonstrate for the first time, that proteasome inhibitors can alter Ag processing in vivo.

A Major Role for the Minor Capsid Protein of Human Papillomavirus Type 16 in Immune Escape [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Fahey, L. M., Raff, A. B., Da Silva, D. M., Kast, W. M. — Wed, 04 Nov 2009 13:05:39 PST

High-risk human papillomavirus (HPV) infection of the cervical epithelium is causally linked with the generation of cervical cancer. HPV does not activate Langerhans cells (LC), the APC at the site of infection, leading to immune evasion. The HPV protein responsible for inducing this immune escape has not been determined. We demonstrate that LC exposed to the minor capsid protein L2 in HPV16L1L2 virus-like particles do not phenotypically or functionally mature. However, HPV16L1 virus-like particles significantly induce activation of LC. Our data suggest that the L2 protein plays a specific role in the induction of this immune escape of HPV16 through the manipulation of LC. This novel function is the first immune modulating action attributed to the L2 protein and adds significantly to our understanding of the mechanism of HPV immune escape.

Human Follicular Lymphoma CD39+-Infiltrating T Cells Contribute to Adenosine-Mediated T Cell Hyporesponsiveness [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Our previous work has demonstrated that human follicular lymphoma (FL) infiltrating T cells are anergic, in part due to suppression by regulatory T cells. In this study, we identify pericellular adenosine, interacting with T cell-associated G protein-coupled A_2A/B adenosine receptors (AR), as contributing to FL T cell hyporesponsiveness. In a subset of FL patient samples, treatment of lymph node mononuclear cells (LNMC) with specific A_2A/B AR antagonists results in an increase in IFN- or IL-2 secretion upon anti-CD3/CD28 Ab stimulation, as compared with that seen without inhibitors. In contrast, treatment with an A₁ AR antagonist had no effect on cytokine secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ecto-ATPase CD39, we next show that inhibition of CD39 activity using the inhibitor ARL 67156 partially overcomes T cell hyporesponsiveness in a subset of patient samples. Phenotypic characterization of LNMC demonstrates populations of CD39-expressing CD4⁺ and CD8⁺ T cells, which are overrepresented in FL as compared with that seen in normal or reactive nodes, or normal peripheral blood. Thirty percent of the FL CD4⁺CD39⁺ T cells coexpress CD25^high and FOXP3 (consistent with regulatory T cells). Finally, FL or normal LNMC hydrolyze ATP in vitro, in a dose- and time-dependent fashion, with the rate of ATP consumption being associated with the degree of CD39⁺ T cell infiltration. Together, these results support the finding that the ATP-ectonucleotidase-adenosine system mediates T cell anergy in a human tumor. In addition, these studies suggest that the A_2A/B AR as well as CD39 are novel pharmacological targets for augmenting cancer immunotherapy.

Human Late Memory CD8+ T Cells Have a Distinct Cytokine Signature Characterized by CC Chemokine Production without IL-2 Production [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Late memory T cell skewing is observed in the setting of immune recovery after cord blood transplantation, and may be associated with inferior control of viral reactivation and cancers. Therefore, we sought to understand how late memory cells differ functionally from earlier stage memory T cells, and whether surface phenotypes associated with differentiation stages were predictably associated with functional signatures. Higher order cytokine flow cytometry allows characterization of human T cells based on complex phenotypic markers and their differential capacity to simultaneously secrete effector proteins, including cytokines and chemokines. We used 8-color, 10-parameter cytokine flow cytometry to characterize the functional activation of human late memory CD8⁺ T cells defined by CD45RA and CD27 expression (CD27^–CD45RA⁺). We assessed the 15 possible functional signatures of cells defined by production of IL-2, IFN-, TNF-, and MIP-1β alone or in combination, following activation with Ags stimulating bypassing surface proteins (PMA:ionomycin) or through the TCR (e.g., viral Ags). Late memory CD8⁺ T cells produced abundant amounts of CC chemokines (MIP-1β, MIP-1, and RANTES) but not IL-2. IL-2/IFN- coproduction, characteristic of protective immune responses to viral infections, was absent in late memory CD8⁺ T cells. These data demonstrate that functional cytokine signatures are predictably associated with CD8⁺ maturation stages, and that the polarization of late memory CD8⁺ T cells toward CC chemokine production and away from IL-2 production suggests a unique functional role for this subset.

C1q Differentially Modulates Phagocytosis and Cytokine Responses during Ingestion of Apoptotic Cells by Human Monocytes, Macrophages, and Dendritic Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Fraser, D. A., Laust, A. K., Nelson, E. L., Tenner, A. J. — Wed, 04 Nov 2009 13:05:39 PST

C1q, the first component of the classical complement pathway, is also a pattern recognition receptor involved in the recognition and clearance of apoptotic cells. C1q deficiency in humans leads to development of lupus-like autoimmune disease, and it has been speculated that impaired clearance of apoptotic cells may contribute to disease development. Since phagocytes initiate specific and appropriate immune responses as a result of initial ligand-receptor interactions, regulation of gene expression by C1q may also contribute to the sculpting of an immune response to the ingested "self-Ags." In this study, the role of C1q in apoptotic cell clearance and subsequent modulation of cytokine release by phagocytes was assessed including donor matched human monocytes, monocyte-derived macrophages (HMDMs), and dendritic cells (DCs). First, C1q binding is much greater to late compared with early apoptotic cells. Second, C1q binding to apoptotic cells significantly enhanced the levels of ingestion by monocytes but had no effect on HMDM and DC uptake. Third, in the presence of serum, C1q bound to apoptotic cells, activated the complement pathway, leading to C3b deposition, and enhancement of uptake of apoptotic cells by monocytes, HMDMs, and DCs. Finally, although C1q, either immobilized on a plate or bound to apoptotic cells, modulates the LPS-induced cytokine levels released by human monocytes, HMDMs, and DCs toward a more limited immune response, both the degree and direction of modulation differed significantly depending on the differentiation state of the phagocyte, providing further evidence of the integration of these cell- and environment-specific signals in determining appropriate immune responses.

AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-B activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4⁺ T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Mcl-1-Mediated Impairment of the Intrinsic Apoptosis Pathway in Circulating Neutrophils from Critically Ill Patients Can Be Overcome by Fas Stimulation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

Critical Role of TLR2 and TLR4 in Autoantibody Production and Glomerulonephritis in lpr Mutation-Induced Mouse Lupus [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathogenic autoantibodies directed against nuclear Ags and immune complex deposits in damaged organs. Environmental factors have been thought to play a role in the onset of the disease. The recognition of these factors is mediated by TLRs, in particular TLR2 and TLR4 which bind pathogen-associated molecular patterns of Gram⁺ and Gram^– bacteria, respectively. We attempted to determine the role of these TLRs in SLE by creating TLR2- or TLR4-deficient C57BL/6^lpr/lpr mice. These mice developed a less severe disease and fewer immunological alterations. Indeed, in C57BL/6^lpr/lpr-TLR2 or -TLR4-deficient mice, glomerular IgG deposits and mesangial cell proliferation were dramatically decreased and antinuclear, anti-dsDNA, and anti-cardiolipin autoantibody titers were significantly reduced. However, the response against nucleosome remained unaffected, indicating a role of TLR2 and TLR4 in the production of Abs directed against only certain categories of SLE-related autoantigens. Analysis of B cell phenotype showed a significant reduction of marginal zone B cells, particularly in C57BL/6^lpr/lpr-TLR4-deficient mice, suggesting an important role of TLR4 in the sustained activation of these cells likely involved in autoantibody production. Interestingly, the lack of TLR4 also affected the production of cytokines involved in the development of lupus disease.

IL-27 Directly Restrains Lung Tumorigenicity by Suppressing Cyclooxygenase-2-Mediated Activities [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Ho, M.-Y., Leu, S.-J. J., Sun, G.-H., Tao, M.-H., Tang, S.-J., Sun, K.-H. — Wed, 04 Nov 2009 13:05:39 PST

Gene transfer of IL-27 to tumor cells has been proven to inhibit tumor growth in vivo by antiproliferation, antiangiogenesis, and stimulation of immunoprotection. To investigate the nonimmune mechanism of IL-27 that suppresses lung cancer growth, we have established a single-chain IL-27-transduced murine Lewis lung carcinoma (LLC-1) cell line (LLC-1/scIL-27) to evaluate its tumorigenic potential in vivo. Mice inoculated with LLC/scIL-27 displayed retardation of tumor growth. Production of IL-12, IFN-, and cytotoxic T cell activity against LLC-1 was manifest in LLC/scIL-27-injected mice. Of note, LLC-1/scIL-27 exhibited decreased expression of cyclooxygenase-2 (COX-2) and PGE₂. On the cellular level, the LLC/scIL-27 transfectants had reduced malignancy, including down-regulation of vimentin expression and reduction of cellular migration and invasion. The suppression of tumorigenesis by IL-27 on lung cancer cells was further confirmed by the treatment with rIL-27 on the murine LLC-1 and human non-small cell lung carcinoma (NSCLC) cell lines. PGE₂-induced vimentin expression, movement, and invasiveness were also suppressed by the treatment with rIL-27. Our data show that IL-27 not only suppresses expression of COX-2 and PGE₂ but also decreases the levels of vimentin and the abilities of cellular migration and invasion. Furthermore, inoculation of LLC/scIL-27 into immunodeficient NOD/SCID mice also exhibited reduced tumor growth. Our data indicate that IL-27-induced nonimmune responses can contribute to significant antitumor effects. Taken together, the results suggest that IL-27 may serve as an effective agent for lung cancer therapy in the future.

A NUP98-HOXD13 Fusion Gene Impairs Differentiation of B and T Lymphocytes and Leads to Expansion of Thymocytes with Partial TCRB Gene Rearrangement [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Choi, C. W., Chung, Y. J., Slape, C., Aplan, P. D. — Wed, 04 Nov 2009 13:05:39 PST

Expression of a NUP98-HOXD13 (NHD13) fusion gene leads to myelodysplastic syndrome in mice. In addition to ineffective hematopoiesis, we observed that NHD13 mice were lymphopenic; the lymphopenia was due to a decrease in both T and B lymphocytes. Although the pro-B cell (B220⁺/CD43⁺) populations from the NHD13 and wild-type mice were similar, the NHD13 mice showed decreased pre-B cells (B220⁺/CD43^–), indicating impaired differentiation at the pro-B to pre-B stage. Thymi from NHD13 mice were smaller and overexpressed Hoxa cluster genes, including Hoxa7, Hoxa9, and Hoxa10. In addition, the NHD13 thymi contained fewer thymocytes, with an increased percentage of CD4^–/CD8^– (double-negative (DN)) cells and a decreased percentage of CD4⁺/CD8⁺ (double-positive) cells; the DN1/DN2 population was increased and the DN3/DN4 population was decreased, suggesting a partial block at the DN2 to DN3 transition. To determine clonality of the thymocytes, we used degenerate RT-PCR to identify clonal Tcrb gene rearrangements. Five of six NHD13 thymi showed an unusual Tcrb gene rearrangement pattern with common, clonal DJ rearrangements, but distinct V-D junctions, suggesting a marked clonal expansion of thymocytes that had undergone a DJ rearrangement, but not completed a VDJ rearrangement. Taken together, these findings demonstrate that expression of the NHD13 transgene inhibits lymphoid as well as myeloid and erythroid differentiation, results in overexpression of Hoxa cluster genes, and leads to a precursor T cell lymphoblastic leukemia/lymphoma.

Regulation of Airway MUC5AC Expression by IL-1{beta} and IL-17A; the NF-{kappa}B Paradigm [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Fujisawa, T., Velichko, S., Thai, P., Hung, L.-Y., Huang, F., Wu, R. — Wed, 04 Nov 2009 13:05:39 PST

Mucin over-production is one of the hallmarks of chronic airway diseases such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis. NF-B activation in airway epithelial cells has been shown to play a positive inflammatory role in chronic airway diseases; however, the role of NF-B in mucin gene expression is unresolved. In this study, we have shown that the proinflammatory cytokines, IL-1β and IL-17A, both of which utilize the NF-B pathway, are potent inducers of mucin (MUC)5AC mRNA and protein synthesis by both well-differentiated primary normal human bronchial epithelial cells and the human bronchial epithelial cell line, HBE1. MUC5AC induction by these cytokines was both time- and dose-dependent and occurred at the level of promoter activation, as measured by a reporter gene assay. These effects were attenuated by the small molecule inhibitor NF-B inhibitor III, as well as p65 small-interfering RNA, suggesting that the regulation of MUC5AC expression by these cytokines is via an NF-B-based transcriptional mechanism. Further investigation of the promoter region identified a putative NF-B binding site at position-3594/-3582 in the promoter of MUC5AC as critical for the regulation of MUC5AC expression by both IL-1β and IL-17A. Chromatin immunoprecipitation analysis confirmed enhanced binding of the NF-B subunit p50 to this region following cytokine stimulation. We conclude that an NF-B-based transcriptional mechanism is involved in MUC5AC regulation by IL-1β and IL-17A in the airway epithelium. This is the first demonstration of the participation of NF-B and its specific binding site in cytokine-mediated airway MUC5AC expression.

HMGB1 Enhances the Proinflammatory Activity of Lipopolysaccharide by Promoting the Phosphorylation of MAPK p38 through Receptor for Advanced Glycation End Products [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Qin, Y.-H., Dai, S.-M., Tang, G.-S., Zhang, J., Ren, D., Wang, Z.-W., Shen, Q. — Wed, 04 Nov 2009 13:05:39 PST

High mobility group box-1 (HMGB1) protein was originally characterized as a nuclear DNA-binding protein, and was described to have an extracellular role when involved in cellular activation and proinflammatory responses. In the present study, we have found that the proinflammatory activity of recombinant HMGB1 proteins is determined by the containing endotoxin level, and HMGB1 that contains few endotoxins fails to stimulate macrophages to secrete proinflammatory cytokines. HMGB1 acts as a ligand of receptor for advanced glycation end products (RAGE) and works in synergy with LPS in activating the macrophages in vitro. In vivo, intra-articular injections of HMGB1 act in synergy with LPS to induce experimental arthritis in mice. HMGB1 promotes the phosphorylation of MAPK p38 and the activation of NF-B through RAGE, and then enhances the expression of proinflammatory cytokines. These results demonstrate that HMGB1 enhances the proinflammatory activity of LPS by promoting the phosphorylation of MAPK p38 and by the activation of NF-B through RAGE.

The Bile Acid Receptor FXR Is a Modulator of Intestinal Innate Immunity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Vavassori, P., Mencarelli, A., Renga, B., Distrutti, E., Fiorucci, S. — Wed, 04 Nov 2009 13:05:39 PST

The farnesoid X receptor (FXR) is a bile acid-regulated nuclear receptor expressed in enterohepatic tissues. In this study we investigated whether FXR is expressed by cells of innate immunity and regulates inflammation in animal models of colitis. Acute (7 days) and chronic (8 wk) colitis were induced in wild-type and FXR^–/– mice by intrarectal administration of trinitrobenzensulfonic acid or by 7-day administration of 5% dextran sulfate in drinking water. The results of this experiment demonstrate that FXR is expressed by and exerts counterregulatory effects on cells of innate immunity. Exposure of LPS-activated macrophages to 6-ethyl chenodeoxycholic acid (6E-CDCA; INT-747) a synthetic FXR ligand, results in a reciprocal regulation of NF-B dependent-genes (TNF-, IL-1β, IL-6, COX-1, COX-2, and iNOS) and induction of SHP, a FXR-regulated gene. FXR activation stabilizes the nuclear corepressor NCoR on the NF-B responsive element on the IL-1β promoter. Colon inflammation in Crohn’s disease patients and in rodent models of colitis is associated with a reduced expression of FXR mRNA. Using two rodent models of colon inflammation, we show that progression of these immune-mediated disorders is exacerbated in FXR^–/– mice (p < 0.01). In vivo treatment with INT-747 attenuates organ injury and immune cell activation. FXR activation increased the colon expression of I-BABP, FXR, and SHP while reducing IL-1β, IL-2, IL-6, TNF-, and IFN- mRNA expression and attenuating disease severity. In aggregate, these findings provide evidence that FXR is an essential component of a network of nuclear receptors that regulate intestinal innate immunity and homeostasis.

Differential Cytokine Production and Bystander Activation of Autoreactive B Cells in Response to CpG-A and CpG-B Oligonucleotides [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Synthetic oligonucleotides containing CpG motifs have been shown to induce proliferation, differentiation, and cytokine production in B cells, macrophages, and dendritic cells through a TLR9-dependent mechanism. A class (CpG-A) and B class (CpG-B) oligonucleotides display distinct physical properties. CpG-A, but not CpG-B, can multimerize to form exceedingly large lattices. CpG-A cannot effectively activate B cells but does induce plasmacytoid dendritic cells to produce high levels of IFN, while CpG-B is a potent B cell mitogen. In this study, we report that CpG-A is internalized by B cells, and CpG-A and CpG-B accumulate in distinct intracellular compartments. When present in the form of an immune complex (CpG-A IC), CpG-A is taken up more efficiently by AM14 IgG2a-specific B cells, and elicits a robust TLR9-dependent B cell proliferative response. B cells proliferating comparably and in a TLR9-dependent fashion in response to CpG-A IC and CpG-B exhibited distinct cytokine profiles. CpG-A IC induced enhanced production of RANTES and markedly reduced levels of IL-6 when compared with CpG-B. We also found that engagement of the AM14 BCR by a protein IC, which cannot by itself induce proliferation, promoted TLR9-dependent but BCR-independent proliferation by bystander CpG-A or fragments of mammalian dsDNA. These data identify direct and indirect mechanisms by which BCR engagement facilitates access of exogenous ligands to TLR9-associated compartments and subsequent B cell activation.

The PPE18 of Mycobacterium tuberculosis Interacts with TLR2 and Activates IL-10 Induction in Macrophage [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

The pathophysiological functions of proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins of Mycobacterium tuberculosis are not well understood. In this study, we demonstrate that one of the PPE proteins, PPE18 can stimulate macrophages to secrete IL-10, known to favor a Th2 type response. The recombinant PPE18 was found to specifically interact with the TLR2 leading to an early and sustained activation of p38 MAPK, which is critical for IL-10 induction. In silico docking analyses and mutation experiments indicate that PPE18 specifically interacts with the leucine rich repeat 11~15 domain of TLR2 and the site of interaction is different from that of a synthetic lipopeptide Pam₃CSK₄ known to activate predominantly ERK 1/2. When PMA-differentiated THP-1 macrophages were infected with a mutant Mycobacterium tuberculosis strain lacking the PPE18, produced poorer levels of IL-10 as compared with those infected with the wild-type strain. In contrast, an M. smegmatis strain overexpressing the PPE18 induced higher levels of IL-10 in infected macrophages. Our data indicate that the PPE18 protein may trigger an anti-inflammatory response by inducing IL-10 production.

CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3{beta} [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Escribano, C., Delgado-Martin, C., Rodriguez-Fernandez, J. L. — Wed, 04 Nov 2009 13:05:39 PST

Chemokine receptor CCR7 regulates chemotaxis and survival in mature dendritic cells (DCs). We studied the role of glycogen synthase kinase-3β (GSK3β) in the regulation of CCR7-dependent survival. We show that GSK3β behaves as a proapoptotic regulator in cultured monocyte-derived human DCs and murine splenic DCs in vitro, and in lymph node DCs in vivo. In keeping with its prosurvival role, stimulation of CCR7 induced phosphorylation/inhibition of GSK3β, which was mediated by the prosurvival regulator Akt1, but it was independent of ERK1/2, a key regulator of chemotaxis. Stimulation of CCR7 also induced translocation of two transcription-factor targets of Akt, prosurvival NF-B and proapoptotic FOXO1, to the nucleus and cytosol, respectively, resulting in DCs with a phenotype more resistant to apoptotic stimuli. We analyzed if GSK3β was able to modulate the mobilizations of these transcription factors. Using pharmacological inhibitors, small interfering RNA, and a construct encoding constitutively active GSK3β, we show that active GSK3β fosters and hampers the translocations to the nucleus of FOXO and NF-B, respectively. Inhibition of GSK3β resulted in the degradation of the NF-B inhibitor IB, indicating a mechanism whereby GSK3 can control the translocation of NF-B to the nucleus. GSK3β and FOXO interacted in vivo, suggesting that this transcription factor could be a substrate of GSK3. The results provide a novel mechanism whereby active GSK3β contributes to regulate apoptosis in DCs. They also suggest that upon stimulation of CCR7, Akt-mediated phosphorylation/inhibition of GSK3β may be required to allow complete translocations of FOXO and NF-B that confer DCs an extended survival.

Localization of Kv1.3 Channels in the Immunological Synapse Modulates the Calcium Response to Antigen Stimulation in T Lymphocytes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

The immunological synapse (IS), a highly organized structure that forms at the point of contact between a T cell and an APC, is essential for the proper development of signaling events, including the Ca²⁺ response. Kv1.3 channels control Ca²⁺ homeostasis in human T cells and move into the IS upon Ag presentation. However, the process involved in channel accumulation in the IS and the functional implications of this localization are not yet known. Here we define the movement of Kv1.3 into the IS and study whether Kv1.3 localization into the IS influences Ca²⁺ signaling in Jurkat T cells. Crosslinking of the channel protein with an extracellular Ab limits Kv1.3 mobility and accumulation at the IS. Moreover, Kv1.3 recruitment to the IS does not involve the transport of newly synthesized channels and it does not occur through recycling of membrane channels. Kv1.3 localization in the IS modulates the Ca²⁺ response. Blockade of Kv1.3 movement into the IS by crosslinking significantly increases the amplitude of the Ca²⁺ response triggered by anti-CD3/anti-CD28-coated beads, which induce the formation of the IS. On the contrary, the Ca²⁺ response induced by TCR stimulation without the formation of the IS with soluble anti-CD3/anti-CD28 Abs is unaltered. The results presented herein indicate that, upon Ag presentation, membrane-incorporated Kv1.3 channels move along the plasma membrane to localize in the IS. This localization is important to control the amplitude of the Ca²⁺ response, and disruption of this process can account for alterations of downstream Ca²⁺-dependent signaling events.

IDO Mediates TLR9-Driven Protection from Experimental Autoimmune Diabetes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Originally predicated on the recognition of an increasing prevalence of allergy, the hygiene hypothesis was later found to accommodate the contrasting epidemiologic trends in developed countries for infectious vs autoimmune diseases. Experimentally, reduced exposure to infections will increase the risk of disease in several models of experimental autoimmunity. Although TLRs were initially considered as stimulatory molecules capable of activating early defense mechanisms against invading pathogens, emerging data suggest that they can also exert a regulatory function. In the present study, we evaluated whether TLR3 and TLR9, recognizing microbial dsDNA and CpG-containing DNA sequences, respectively, play a role in the protection from experimental autoimmune diabetes induced in C57BL/6 mice by streptozotocin. In wild-type animals, the disease was accompanied by up-regulation of IDO in pancreatic lymph nodes and would be greatly exacerbated by in vivo administration of an IDO inhibitor. Conversely, administration of a CpG-containing oligodeoxynucleotide greatly attenuated the disease in an IDO-dependent fashion. TLR9-, but not TLR3-deficient mice developed a more robust disease, an event accompanied by lack of IDO induction in pancreatic lymph nodes. Thus, our data suggest that the TLR9-IDO axis may represent a valuable target in the prevention/therapy of type 1 diabetes.

Mitochondrial Uncoupling Protein 2 Inhibits Mast Cell Activation and Reduces Histamine Content [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Mast cells are immune effector cells that are involved in allergies and inflammation through the release of mediators such as histamine, PGs, and cytokines. Uncoupling protein 2 (UCP2) is a mitochondrial protein that inhibits insulin secretion from β cells, possibly through down-regulation of reactive oxygen species production. We hypothesized that UCP2 could also regulate mast cell activation. In this study, we show that mouse bone marrow mast cells (BMMCs) and human leukemic LAD2 mast cells express UCP2. BMMCs from Ucp2^–^/^– mice exhibited greater histamine release, whereas overexpression of UCP2 in LAD2 cells reduced histamine release after both allergic and nonallergic triggers. Ucp2^–^/^– BMMCs also had elevated histamine content and histidine decarboxylase expression. Histamine content was reduced by overexpression of UCP2 or treatment with the mitochondrial-targeted superoxide dismutase-mimetic (TBAP) tetrakis(4-benzoic acid) porphyrin manganese(III). Furthermore, Ucp2^–^/^– BMMCs also had greater production of both IL-6 and PGD₂ as well as ERK phosphorylation, which is known to regulate PG synthesis. Intradermal administration of substance P, an activator of skin mast cells, and challenge with DNP-human serum albumin after passive sensitization induced significantly greater vascular permeability in the skin of Ucp2^–^/^– mice in vivo. Our results suggest that UCP2 can regulate mast cell activation.

Dec2 Promotes Th2 Cell Differentiation by Enhancing IL-2R Signaling [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Th cell differentiation is precisely regulated by thousands of genes at different stages. In the present study, we demonstrate that Dec2, a transcription factor belonging to the bHLH (basic helix-loop-helix) superfamily, is progressively induced during the course of Th2 differentiation, especially at the late stage. The up-regulated Dec2 can strongly promote Th2 development under Th2-inducing conditions, as evidenced by retrovirus-mediated gene transfer or transgenic manipulation. In addition, an enhancement of Th2 responses is also detectable in Dec2 transgenic mice in vivo. Conversely, RNA interference-mediated suppression of endogenous Dec2 could attenuate Th2 differentiation. Finally, we show that the enhanced Th2 development is at least in part due to substantial up-regulation of CD25 expression elicited by Dec2, thereby resulting in hyperresponsiveness to IL-2 stimulation.

Skin Melanoma Development in ret Transgenic Mice Despite the Depletion of CD25+Foxp3+ Regulatory T Cells in Lymphoid Organs [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) known to mediate self-tolerance were also shown to contribute to tumor progression. In mouse melanoma transplantation models, Treg depletion resulted in the stimulation of antitumor immune responses and tumor eradication. To study Treg in conditions close to the clinical situation, we used a ret transgenic mouse spontaneous melanoma model, which, in contrast to transplantation models, resembles human melanoma regarding clinical development. Significantly higher numbers of Treg were found in skin tumors and metastatic lymph nodes at early stages of melanoma progression compared with more advanced stages accompanied by the elevated CCR4 expression on Treg and higher production of its ligand CCL2 in tumor lesions. Numbers of tumor infiltrating Treg inversely correlated with Treg amounts in the bone marrow, suggesting their possible recruitment to melanoma lesions from this organ. The immunosuppressive function of Treg from transgenic tumor-bearing mice was similar to that from transgenic tumor-free mice or nontransgenic littermates. Although anti-CD25 mAb injections resulted in the efficient Treg depletion from lymphoid organs of transgenic mice, melanoma development was not significantly delayed. Furthermore, the treatment of mice with macroscopical tumors also failed to inhibit tumor progression, which correlated with the inability to deplete intratumoral Treg. We suggest that in the autochthonous melanoma genesis, other immunosuppressive cells could play an important role and replace immunosuppressive, tumor-promoting functions of Treg. Therefore, effective melanoma immunotherapy should include the inhibition of Treg migration into the tumor combined with neutralization of other immunosuppressive cells and factors in the tumor microenvironment.

Potent High-Affinity Antibodies for Treatment and Prophylaxis of Respiratory Syncytial Virus Derived from B Cells of Infected Patients [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (K_d 1–24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.

Regulatory T Cell (Treg) Subsets Return in Patients with Refractory Lupus following Stem Cell Transplantation, and TGF-{beta}-Producing CD8+ Treg Cells Are Associated with Immunological Remission of Lupus [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zhang, L., Bertucci, A. M., Ramsey-Goldman, R., Burt, R. K., Datta, S. K. — Wed, 04 Nov 2009 13:05:40 PST

Compared with conventional drug therapy, autologous hemopoietic stem cell transplantation (HSCT) can induce very-long-term remission in refractory lupus patients. Herein, we show that in posttransplant patients, both CD4⁺CD25^highFoxP3⁺ and an unusual CD8⁺FoxP3⁺ Treg subset return to levels seen in normal subjects; accompanied by almost complete inhibition of pathogenic T cell response to critical peptide autoepitopes from histones in nucleosomes, the major lupus autoantigen from apoptotic cells. In addition to a stably sustained elevation of FoxP3, posttransplant CD8 T cells also maintained markedly higher expression levels of latency-associated peptide (LAP), CD103, PD-1, PD-L1, and CTLA-4, as compared with pretransplant CD8 T cells that were identically treated by a one-time activation and rest in short-term culture. The posttransplant CD8 regulatory T cells (Treg) have autoantigen-specific and nonspecific suppressive activity, which is contact independent and predominantly TGF-β dependent. By contrast, the pretransplant CD8 T cells have helper activity, which is cell contact dependent. Although CD4⁺CD25^high Treg cells return during clinical remission of conventional drug-treated lupus, the posttransplant patient’s CD8 Treg cells are considerably more potent, and they are absent in drug-treated patients in whom CD4 T cell autoreactivity to nucleosomal epitopes persists even during clinical remission. Therefore, unlike conventional drug therapy, hemopoietic stem cell transplantation generates a newly differentiated population of LAP^highCD103^high CD8^TGF-β Treg cells, which repairs the Treg deficiency in human lupus to maintain patients in true immunological remission.

Generation of B Cell Memory to the Bacterial Polysaccharide {alpha}-1,3 Dextran [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Foote, J. B., Kearney, J. F. — Wed, 04 Nov 2009 13:05:40 PST

B1b B cells generate a novel form of memory and provide Ab mediated-protection to persisting bacterial pathogens. To understand how B1b B cells establish memory to polysaccharide Ags, we studied an oligoclonal B cell response to -1,3 dextran (DEX) expressed on Enterobacter cloacae. B cells specific for DEX enrich in the marginal zone (MZ) and B1b B cell populations. After E. cloacae immunization, MZ B cells were responsible for the generation of initial peak DEX-specific Ab titers, whereas, DEX-specific B1b B cells expanded and played an important role in boosted production of DEX-specific Ab titers upon E. cloacae rechallenge. Cell transfer experiments demonstrate that B1b B cells possess the capacity for both robust proliferation and plasma cell differentiation, thus distinguishing themselves from MZ B cells, which uniformly commit to plasma cell differentiation. These results define B1b B cells as the principal reservoir for memory to bacterial-associated polysaccharide Ags.

Mouse Mast Cell Protease 4 Is the Major Chymase in Murine Airways and Has a Protective Role in Allergic Airway Inflammation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

It is widely established that mast cells (MCs) have a harmful role in asthma, for example by secreting various proinflammatory substances stored within their secretory granule. However, in this study, we show that one of the substances stored within MC granule, chymase, in fact has a protective role in allergic airway inflammation, indicating that MCs may possess both harmful and protective activities in connection with this type of disease. Wild-type (WT) mice and mice lacking mouse MC protease 4 (mMCP-4), a chymase that is functionally homologous to human chymase, were sensitized and challenged with OVA, followed by the assessment of airway physiology and inflammatory parameters. Our results show that the airway hyperresponsiveness was significantly higher in mMCP-4^–/– as compared with WT mice. Moreover, the degree of lung tissue inflammation was markedly higher in mice lacking mMCP-4 than in WT controls. Histological analysis revealed that OVA sensitization/challenge resulted in a marked increased in the thickness of the smooth muscle cell (SMC) layer and, notably, that the degree of SMC layer thickening was more pronounced in mMCP-4^–/– animals than in WT controls, thus indicating that chymase may have an effect on airway SMCs. In support of this, mMCP-4-positive MCs were located in the close vicinity of the SMC layer, mainly in the upper airways, and mMCP-4 was shown to be the major chymase expressed in these MCs. Taken together, our results indicate that chymase present in the upper airways protects against allergic airway responses, possibly by regulating SMCs.

FoxP3+ Regulatory T Cells Restrain Splenic Extramedullary Myelopoiesis via Suppression of Hemopoietic Cytokine-Producing T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Lee, J. H., Wang, C., Kim, C. H. — Wed, 04 Nov 2009 13:05:40 PST

Extramedullary myelopoiesis occurs in peripheral organs such as spleen and produces many types of myeloid cells with diverse functions in response to inflammation and infection. It is increased during immune responses and chronic inflammation and is a significant factor in regulating inflammatory diseases and immunity. Increased myeloid cells are found in FoxP3-deficient mice but the mechanism has been unclear. We investigated the mechanism by which FoxP3⁺ regulatory T cells regulate the extramedullary myelopoiesis. We found that Ab or genetic depletion of FoxP3⁺ regulatory T cells greatly increased the number of the myeloid progenitors in spleen during immune responses. Consistently, the splenic myelopoiesis was effectively suppressed by increased numbers of natural or induced FoxP3⁺ regulatory T cells. We demonstrated that myelopoiesis is positively regulated by splenic CD4⁺ T cells that produce myelopoietic cytokines (GM-CSF and IL-3), and these effector CD4⁺ T cells are induced from naive CD4⁺ T cells in response to antigenic stimulation. FoxP3⁺ regulatory T cells were able to effectively suppress the differentiation of naive T cells into myelopoietic cytokine-producing T cells. This suppression was found to be dependent on cell contact but independent of TGFβ. Unlike splenic myelopoiesis, marrow myelopoiesis is not significantly affected by FoxP3⁺ regulatory T cells. We conclude that FoxP3⁺ T cells can negatively regulate splenic extramedullary myelopoiesis by suppressing the naive T cell differentiation into myelopoietic cytokine-producing CD4⁺ T cells. Our results provide new insights into regulation of extramedullary myelopoiesis.

FROUNT Is a Common Regulator of CCR2 and CCR5 Signaling to Control Directional Migration [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Toda, E., Terashima, Y., Sato, T., Hirose, K., Kanegasaki, S., Matsushima, K. — Wed, 04 Nov 2009 13:05:40 PST

FROUNT is a known CCR2-binding protein that facilitates monocyte/macrophage infiltration. Here we report that FROUNT also binds to the C-terminal region of CCR5 and enhances CCR5-mediated cellular chemotaxis. We show that FROUNT overexpression enhances the directionality of chemotaxis, while FROUNT suppression results in impaired responsiveness. Furthermore, we found an increase in consolidated pseudopodium formation in FROUNT-overexpressing cells (FNT cells) on uniform stimulation with CCL4 (MIP1-β), a specific ligand of CCR5. In most FNT cells, one to two pseudopodia directed toward higher chemokine concentration were found, whereas most FNT-suppressed cells had multiple pseudopodia. The data indicate that FROUNT is involved in sensing and amplifying a shallow extracellular chemokine gradient that leads to a limited number of accurate pseudopodia directed toward the chemokine concentration. In addition to its separate roles in CCR2- and CCR5-mediated chemotaxis, FROUNT, as a common regulator of these receptors, possibly plays a crucial role in the recruitment of immune cells expressing these receptors.

Lymph Node Stromal Cells Support Dendritic Cell-Induced Gut-Homing of T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

T cells are imprinted to express tissue-specific homing receptors upon activation in tissue-draining lymph nodes, resulting in their migration to the site of Ag entry. Expression of gut-homing molecules ₄β₇ and CCR9 is induced by retinoic acid, a vitamin A metabolite produced by retinal dehydrogenases, which are specifically expressed in dendritic cells as well as stromal cells in mucosa-draining lymph nodes. In this study, we demonstrate that mesenteric lymph node stromal cell-derived retinoic acid can directly induce the expression of gut-homing molecules on proliferating T cells, a process strongly enhanced by bone marrow-derived dendritic cells in vitro. Therefore, cooperation of sessile lymph node stromal cells with mobile dendritic cells warrants the imprinting of tissue specific homing receptors on activated T cells.

Reduced Diabetes in btk-Deficient Nonobese Diabetic Mice and Restoration of Diabetes with Provision of an Anti-Insulin IgH Chain Transgene [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Type 1 diabetes results from T cell-mediated destruction of insulin-producing β cells. Although elimination of B lymphocytes has proven successful at preventing disease, modulation of B cell function as a means to prevent type 1 diabetes has not been investigated. The development, fate, and function of B lymphocytes depend upon BCR signaling, which is mediated in part by Bruton’s tyrosine kinase (BTK). When introduced into NOD mice, btk deficiency only modestly reduces B cell numbers, but dramatically protects against diabetes. In NOD, btk deficiency mirrors changes in B cell subsets seen in other strains, but also improves B cell-related tolerance, as indicated by failure to generate insulin autoantibodies. Introduction of an anti-insulin BCR H chain transgene restores diabetes in btk-deficient NOD mice, indicating that btk-deficient B cells are functionally capable of promoting autoimmune diabetes if they have a critical autoimmune specificity. This suggests that the disease-protective effect of btk deficiency may reflect a lack of autoreactive specificities in the B cell repertoire. Thus, signaling via BTK can be modulated to improve B cell tolerance, and prevent T cell-mediated autoimmune diabetes.

Responsiveness of Stromal Fibroblasts to IFN-{gamma} Blocks Tumor Growth via Angiostasis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Lu, Y., Yang, W., Qin, C., Zhang, L., Deng, J., Liu, S., Qin, Z. — Wed, 04 Nov 2009 13:05:40 PST

The importance of stromal cells for tumor is akin to soil for seed. However, the interaction among these cells is far from understood. In this study, we show that stromal fibroblasts exist not only during tumor progression but also during regression stage, together with immune effector cells. Coinjection of stromal fibroblasts with tumor cells often promotes tumor growth. However, the presence of IFN- significantly impairs the ability of these cells to promote tumor growth due to a reduced angiogenesis. The mechanism relies mainly on the IFN--mediated down-regulation of vascular endothelial growth factor production by fibroblasts. The results reveal a novel link between immune cells and nonbone marrow-derived stromal cells, and define stromal fibroblasts as the main targets of IFN- in tumor immunity.

Histone Acetyltransferase Cofactor Trrap Is Essential for Maintaining the Hematopoietic Stem/Progenitor Cell Pool [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

The pool of hematopoietic stem/progenitor cells, which provide life-long reconstitution of all hematopoietic lineages, is tightly controlled and regulated by self-renewal and apoptosis. Histone modifiers and chromatin states are believed to govern establishment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In this study, we identified a role for the histone acetytransferase cofactor Trrap in the maintenance of hematopietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with induction of p53-independent apoptosis and deregulation of Myc transcription factors. Together, this study has identified a critical role for Trrap in the mechanism that maintains hematopoietic stem cells and hematopoietic system, and underscores the importance of Trrap and histone modifications in tissue homeostasis.

In Vitro Responses to Avian Influenza H5 by Human CD4 T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Cusick, M. F., Wang, S., Eckels, D. D. — Wed, 04 Nov 2009 13:05:40 PST

To address the question of whether human T cells are capable of recognizing novel isolates of influenza virus, in vitro responses to recombinant Ags and synthetic peptides derived from the sequences of H1, H3, and H5 were examined in a cohort of 64 individuals selected from a healthy blood donor population. Humans respond in vitro to H1 and H3 following exposure through natural infection and vaccination. Responses to H5 were well correlated with those to H1 or H3, and thus, a significant repertoire of H5-responsive T cells is present in many individuals; clear nonresponders to H1, H3, and H5, however, do exist. Differences were observed in the cytokine responses to H1, H3, and H5, whereas both IL-2 and IFN- production characteristic of memory responses were observed for H1 and H3, and H5-specific responses elicited primarily IL-2 and little or no IFN-, consistent with a naive T cell phenotype. Responses to all influenza HA were restricted by HLA-DR molecules. To address the structural basis for T cell recognition of H1 and H5, overlapping synthetic peptides were used to identify epitopes and to determine whether recognition of H5 was limited to homologous sequences in H1, the most closely related HA phylogenetically. Although responses were generally correlated, no complete structural overlap was observed. These results suggest that helper T cell cross reactivity between different influenza strains may impart cross-protection to H5N1 strain of influenza.

Chronic CD70-Driven Costimulation Impairs IgG Responses by Instructing T Cells to Inhibit Germinal Center B Cell Formation through FasL-Fas Interactions [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

CD70 provides costimulation that enhances effector T cell differentiation upon binding of its receptor, CD27. During chronic immune activation, CD70 is constitutively expressed on activated immune cells, and this induces T cell-driven disruption of neutralizing Ab responses via an unknown mechanism. We used CD70-transgenic mice to investigate the effect of constitutive expression of CD70 on T cell-dependent B cell responses. CD70 induced up-regulation of the B cell follicle homing chemokine receptor CXCR5 on T cells, enabling not only CD4 but also CD8 T cells to infiltrate the B cell follicles. CD70-transgenic mice failed to develop productive germinal center formation and displayed impaired IgG Ab responses. Defective germinal center B cell differentiation was critically dependent on CD70-mediated CD27 signaling in T cells, and involved Fas-dependent impairment of germinal center B cell differentiation. Thus, CD70-driven costimulation enables T cells to terminate B cell responses, thereby compromising durable Ab production. Our findings imply that the CD70- and CD27-driven costimulatory axis may be involved in shutdown of B cell responses before clearance of Ag. Because CD70 is expressed constitutively in chronic viral infections such as HIV-1 infection, this mechanism may also contribute to defects in humoral immunity associated with this disease.

CD36 and TLR Interactions in Inflammation and Phagocytosis: Implications for Malaria [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

CD36 participates in macrophage internalization of a variety of particles, and has been implicated in inflammatory responses to many of these ligands. To what extent CD36 cooperates with other receptors in mediating these processes remains unclear. Because CD36 has been shown to cooperate with TLR2, we investigated the roles and interactions of CD36 and TLRs in inflammation and phagocytosis. Using Ab-induced endocytosis of CD36 and phagocytosis of erythrocytes displaying Abs to CD36, we show that selective engagement and internalization of this receptor did not lead to proinflammatory cytokine production by primary human and murine macrophages. In addition, CD36-mediated phagocytosis of Plasmodium falciparum malaria-parasitized erythrocytes (PEs), which contain parasite components that activate TLRs, also failed to induce cytokine secretion from primary macrophages. Furthermore, we demonstrate that CD36-mediated internalization did not require TLR2 or the TLR-signaling molecule IRAK4. However, macrophage pretreatment with TLR agonists markedly stimulated particle uptake via CD36. Similarly, PE uptake was unaffected by TLR deficiency, but in wild-type cells was increased by pretreatment with purified P. falciparum glycosylphosphatidylinositols, which activate TLR2. Our findings indicate that CD36 must cooperate with other receptors such as TLRs to participate in cytokine responses. Although purified P. falciparum components activate TLRs, CD36-mediated internalization of intact PEs is not inflammatory. Further, CD36 mediates internalization of particles, including PEs, independently of TLR signaling, but can functionally cooperate with TLRs to enhance internalization.

Outside-In Signal Transmission by Conformational Changes in Integrin Mac-1 [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the _M and β₂ subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals.

IL-33 Amplifies the Polarization of Alternatively Activated Macrophages That Contribute to Airway Inflammation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Alternatively activated macrophages (AAM) play a crucial role in type 2 immunity. Mice deficient in ST2, a receptor for the latest member of the IL-1 family, IL-33, have impaired type 2 immune responses. We therefore reasoned that IL-33/ST2 signaling may be involved in the differentiation and activation of AAM during airway inflammation. We report here that IL-33 changed the quiescent phenotype of alveolar macrophages toward an AAM phenotype that expressed mannose receptor, IL-4R, and produced high levels of CCL24 and CCL17 in an IL-13-dependent manner during IL-33-induced airway inflammation. Neutralization of AAM-derived CCL24 led to an amelioration of IL-33-induced eosinophilia in the lungs. Moreover, depletion of alveolar macrophages reduced IL-33-induced airway inflammation. Additionally, the attenuated OVA-induced airway inflammation in ST2^–/– mice was associated with a decrease in AAM differentiation. In vitro, IL-33 amplified IL-13-induced polarization of alveolar- and bone marrow-derived macrophage toward an AAM phenotype by increasing the expression of arginase I, Ym1, as well as the production of CCL24 and CCL17. IL-13/IL-4R signaling was crucial for IL-33-driven AAM amplification by inducing the expression of ST2L. Finally, we showed that IL-33 was more abundantly expressed in the lung epithelial cells of asthma patients than those from healthy controls, suggesting that IL-33 may be involved in lung macrophage activation in clinical asthma. Taken together, we demonstrate here that IL-33/ST2 plays a significant role in the amplification of AAM polarization and chemokine production which contribute to innate and Ag-induced airway inflammation.

Ca2+ Waves Initiate Antigen-Stimulated Ca2+ Responses in Mast Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Cohen, R., Torres, A., Ma, H.-T., Holowka, D., Baird, B. — Wed, 04 Nov 2009 13:05:41 PST

Ca²⁺ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca²⁺ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca²⁺ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca²⁺ oscillations throughout the cell that usually follow the initiating Ca²⁺ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca²⁺ puffs at the region of cell contact. Initiating Ca²⁺ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca²⁺ release from endoplasmic reticulum stores and to extracellular Ca²⁺, but they do not depend on store-operated Ca²⁺ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca²⁺ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca²⁺ response.

Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inflammatory Activity of Chemerin in a Lung Disease Model [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 04 Nov 2009 13:05:41 PST

Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

Discrete Domains of MARCH1 Mediate Its Localization, Functional Interactions, and Posttranscriptional Control of Expression [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Jabbour, M., Campbell, E. M., Fares, H., Lybarger, L. — Wed, 04 Nov 2009 13:05:41 PST

Within APCs, ubiquitination regulates the trafficking of immune modulators such as MHC class II and CD86 (B7.2) molecules. MARCH1 (membrane-associated RING-CH), a newly identified ubiquitin E3 ligase expressed in APCs, ubiquitinates MHC class II, thereby reducing its surface expression. Following LPS-induced maturation of dendritic cells, MARCH1 mRNA is down-regulated and MHC class II is redistributed to the cell surface from endosomal compartments. Here, we show that MARCH1 expression is also regulated at the posttranscriptional level. In primary dendritic cell and APC cell lines of murine origin, MARCH1 had a half-life of <30 min. MARCH1 degradation appears to occur partly in lysosomes, since inhibiting lysosomal activity stabilized MARCH1. Similar stabilization was observed when MARCH1-expressing cells were treated with cysteine protease inhibitors. Mutational analyses of MARCH1 defined discrete domains required for destabilization, proper localization, and functional interaction with substrates. Taken together, these data suggest that MARCH1 expression is regulated at a posttranscriptional level by trafficking within the endolysosomal pathway where MARCH1 is proteolyzed. The short half-life of MARCH1 permits very rapid changes in the levels of the protein in response to changes in the mRNA, resulting in efficient induction of Ag presentation once APCs receive maturational signals.

CD69 Gene Is Differentially Regulated in T and B Cells by Evolutionarily Conserved Promoter-Distal Elements [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Vazquez, B. N., Laguna, T., Carabana, J., Krangel, M. S., Lauzurica, P. — Wed, 04 Nov 2009 13:05:41 PST

CD69 is a type II C-type lectin involved in lymphocyte migration and cytokine secretion. CD69 expression represents one of the earliest available indicators of leukocyte activation and its rapid induction occurs through transcriptional activation. In this study we examined the molecular mechanism underlying mouse CD69 gene transcription in vivo in T and B cells. Analysis of the 45-kb region upstream of the CD69 gene revealed evolutionary conservation at the promoter and at four noncoding sequences (CNS) that were called CNS1, CNS2, CNS3, and CNS4. These regions were found to be hypersensitive sites in DNase I digestion experiments, and chromatin immunoprecipitation assays showed specific epigenetic modifications. CNS2 and CNS4 displayed constitutive and inducible enhancer activity in transient transfection assays in T cells. Using a transgenic approach to test CNS function, we found that the CD69 promoter conferred developmentally regulated expression during positive selection of thymocytes but could not support regulated expression in mature lymphocytes. Inclusion of CNS1 and CNS2 caused suppression of CD69 expression, whereas further addition of CNS3 and CNS4 supported developmental-stage and lineage-specific regulation in T cells but not in B cells. We concluded CNS1–4 are important cis-regulatory elements that interact both positively and negatively with the CD69 promoter and that differentially contribute to CD69 expression in T and B cells.

Epigenetic Regulation of TLR4 Gene Expression in Intestinal Epithelial Cells for the Maintenance of Intestinal Homeostasis [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Takahashi, K., Sugi, Y., Hosono, A., Kaminogawa, S. — Wed, 04 Nov 2009 13:05:41 PST

Intestinal epithelial cells (IECs) are continuously exposed to large numbers of commensal bacteria but are relatively insensitive to them, thereby averting an excessive inflammatory reaction. In this study, we show that the low responsiveness of human IEC lines to LPS was mainly brought about by a down-regulation of TLR4 gene transcription. Additionally, the presence of an IEC-specific repressor element in the 5' region of the TLR4 gene and binding of a NF to the element was shown. The transcription factor ZNF160, which was expressed more abundantly in a LPS-low responder IEC line than in a LPS-high responder IEC line, repressed TLR4 gene transcription. ZNF160 is known to interact with the scaffold protein KAP1 via its N terminus to recruit histone deacetylase. Histone deacetylation, as well as DNA methylation, at the 5' region of the TLR4 gene was significantly higher in LPS-low responder IEC lines than in a monocyte line or a LPS-high responder IEC line. It was demonstrated that TLR4 gene transcription was repressed by these epigenetic regulations, which were, at least in part, dependent on ZNF160. Down-regulaton of TLR4 gene expression by these mechanisms in IECs possibly contributes to the maintainance of homeostasis in the intestinal commensal system.

Defining the Turkey MHC: Sequence and Genes of the B Locus [IMMUNOGENETICS]

Chaves, L. D., Krueth, S. B., Reed, K. M. — Wed, 04 Nov 2009 13:05:41 PST

The MHC, the most polymorphic and gene dense region in the vertebrate genome, contains many loci essential to immunity. In mammals, this region spans ~4 Mb. Studies of avian species have found the MHC to be greatly reduced in size and gene content with an overall locus organization differing from that of mammals. The chicken MHC has been mapped to two distinct regions (MHC-B and -Y) of a single chromosome. MHC-B haplotypes possess tightly linked genes encoding the classical MHC molecules and few other disease resistance genes. Furthermore, chicken haplotypes possess a dominantly expressed class I and class II B locus that have a significant effect on the progression or regression of pathogenic disease. In this study, we present the MHC-B region of the turkey (Meleagris gallopavo) as a similarly constricted locus, with 34 genes identified within a 0.2-Mb region in near-perfect synteny with that of the chicken MHC-B. Notable differences between the two species are three BG and class II B loci in the turkey compared with one BG and two class II B loci in the chicken MHC-B. The relative size and high level of similarity of the turkey MHC in relation to that of the chicken suggest that similar associations with disease susceptibility and resistance may also be found in turkey.

A Rice-Based Oral Cholera Vaccine Induces Macaque-Specific Systemic Neutralizing Antibodies but Does Not Influence Pre-Existing Intestinal Immunity [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (MucoRice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates.

Long Double-Stranded RNA Induces an Antiviral Response Independent of IFN Regulatory Factor 3, IFN-{beta} Promoter Stimulator 1, and IFN [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

Virus infection elicits a robust innate antiviral response dominated by the production of type 1 IFN. In nonprofessional innate immune cells such as fibroblasts, type 1 IFN is rapidly produced following the recognition of viral dsRNA and the subsequent activation of the constitutively expressed transcription factor IFN regulatory factor 3 (IRF3). Although origin, localization, and length are factors in mediating dsRNA recognition and binding by cellular dsRNA-binding proteins, the biological significance of differential dsRNA binding is unclear, since the subsequent signaling pathways converge on IRF3. In this study, we show a dsRNA length-dependent activation of IRFs, IFNs, and IFN-stimulated genes in mouse fibroblasts. The length dependence was exacerbated in fibroblasts deficient in the mitochondria-associated adaptor IFN-β promoter stimulator 1 and IRF3, suggesting that antiviral gene induction mediated by short and long dsRNA molecules is predominantly IFN-β promoter stimulator 1 and IRF3 dependent and independent, respectively. Furthermore, we provide evidence of an innate antiviral response in fibroblasts in the absence of both IRF3 and type 1 IFN induction. Even with these key modulators missing, a 60–90% inhibition of virus replication was observed following 24-h treatment with short or long dsRNA molecules, respectively. These data provide evidence of a novel antiviral pathway that is dependent on dsRNA length, but independent of the type 1 IFN system.

Resistance to Vaccinia Virus Is Less Dependent on TNF under Conditions of Heterologous Immunity [HOST DEFENSE]

Nie, S., Cornberg, M., Selin, L. K. — Wed, 04 Nov 2009 13:05:41 PST

TNF has been shown to be important for controlling many pathogens. Here, we directly demonstrate using wild-type TNF^–/– and TNFR1^–/– mice that TNF does play a role in protection against vaccinia virus (VV) infection in naive mice. Since VV replication is also partially controlled in lymphocytic choriomeningitis virus (LCMV)-immune C57BL/6J mice through the process of heterologous immunity, we questioned whether TNF was required in mediating this protection. VV-infected LCMV-immune mice that were TNF-deficient as a consequence of genetic deletion or receptor blockade demonstrated normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells and controlled VV infection similar to LCMV-immune mice having TNF function. This indicates that neither TNF nor lymphotoxin, which uses the same receptor, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV made the role of TNF in protection against VV infection much less important, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of patients with anti-TNF compounds is reasonably well tolerated with relatively few infectious complications.

Induction of Cross-Priming of Naive CD8+ T Lymphocytes by Recombinant Bacillus Calmette-Guerin That Secretes Heat Shock Protein 70-Major Membrane Protein-II Fusion Protein [HOST DEFENSE]

Mukai, T., Maeda, Y., Tamura, T., Matsuoka, M., Tsukamoto, Y., Makino, M. — Wed, 04 Nov 2009 13:05:41 PST

Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8⁺ T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8⁺ T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8⁺ T cells, but also CD4⁺ T cells of both types to produce IFN-. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8⁺ T cells by BCG-70M through DC. Thus, the CD8⁺ T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8⁺ T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4⁺ T cells, CD62L^lowCD8⁺ T cells and perforin-producing CD8⁺ T cells were efficiently produced. MMP-II-reactive CD4⁺ and CD8⁺ memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4⁺ T cells, and CD8⁺ T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

A Homolog of Formyl Peptide Receptor-Like 1 (FPRL1) Inhibitor from Staphylococcus aureus (FPRL1 Inhibitory Protein) That Inhibits FPRL1 and FPR [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

The members of the formyl peptide receptor (FPR) family are involved in the sensing of chemoattractant substances, including bacteria-derived N-formylated peptides and host-derived peptides and proteins. We have recently described two chemoattractant receptor inhibitors from Staphylococcus aureus. Chemotaxis inhibitory protein of S. aureus (CHIPS) blocks the formyl peptide receptor (FPR) and the receptor for complement C5a (C5aR), while FPR-like 1 (FPRL1) inhibitory protein (FLIPr) blocks the FPRL1. Here, we describe another staphylococcal chemoattractant-inhibiting protein with 73% overall homology to FLIPr and identical first 25 aa, which we termed FLIPr-like. This protein inhibits neutrophil calcium mobilization and chemotaxis induced by the FPRL1-ligand MMK-1 and FPR-ligand fMLP. While its FPRL1-inhibitory activity lies in the comparable nanomolar range of FLIPr, its antagonism of the FPR is ~100-fold more potent than that of FLIPr and comparable to that of CHIPS. The second N-terminal phenylalanine was required for its inhibition of the FPR, but it was dispensable for the FPRL1. Furthermore, the deletion of the first seven amino acids reduced its antagonism of the FPRL1, and the exchange of the first six amino acids with that of CHIPS-conferred receptor specificity. Finally, studies with cells transfected with several chemoattractant receptors confirmed that FLIPr-like specifically binds to the FPR and FPRL1. In conclusion, the newly described excreted protein from S. aureus, FLIPr-like, is a potent inhibitor of the FPR- and FPRL1-mediated neutrophil responses and may be used to selectively modulate these chemoattractant receptors.

The Natural Cytotoxicity Receptor NKp46 Is Dispensable for IL-22-Mediated Innate Intestinal Immune Defense against Citrobacter rodentium [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

Natural cytotoxicity receptors (including NKp30, NKp44, and NKp46 in humans and NKp46 in mice) are type I transmembrane proteins that signal NK cell activation via ITAM-containing adapter proteins in response to stress- and pathogen-induced ligands. Although murine NKp46 expression (encoded by Ncr1) was thought to be predominantly restricted to NK cells, the identification of distinct intestinal NKp46⁺ cell subsets that express the transcription factor Rorc and produce IL-22 suggests a broader function for NKp46 that could involve intestinal homeostasis and immune defense. Using mice carrying a GFP-modified Ncr1 allele, we found normal numbers of gut CD3^–GFP⁺ cells with a similar cell surface phenotype and subset distribution in the absence of Ncr1. Splenic and intestinal CD3^–NKp46⁺ cell subsets showed distinct patterns of cytokine secretion (IFN-, IL-22) following activation via NK1.1, NKp46, IL-12 plus IL-18, or IL-23. However, IL-22 production was sharply restricted to intestinal CD3^–GFP⁺ cells with the CD127⁺NK1.1^– phenotype and could be induced in an Ncr1-independent fashion. Because NKp46 ligands can trigger immune activation in the context of infectious pathogens, we assessed the response of wild-type and Ncr-1-deficient Rag2^–/– mice to the enteric pathogen Citrobacter rodentium. No differences in the survival or clinical score were observed in C. rodentium-infected Rag2^–/– mice lacking Ncr1, indicating that NKp46 plays a redundant role in the differentiation of intestinal IL-22⁺ cells that mediate innate defense against this pathogen. Our results provide further evidence for functional heterogeneity in intestinal NKp46⁺ cells that contrast with splenic NK cells.

Enterobacter sakazakii Targets DC-SIGN to Induce Immunosuppressive Responses in Dendritic Cells by Modulating MAPKs [HOST DEFENSE]

Mittal, R., Bulgheresi, S., Emami, C., Prasadarao, N. V. — Wed, 04 Nov 2009 13:05:41 PST

Enterobacter sakazakii (ES) is an emerging pathogen that causes meningitis and necrotizing enterocolitis in infants. Dendritic cells (DCs) are professional phagocytic cells that play an essential role in host defense against invading pathogens; however, the interaction of ES with DCs is not known. In this study, we demonstrate that ES targets DC-specific ICAM nonintegrin (DC-SIGN) to survive in myeloid DCs for which outer membrane protein A (OmpA) expression in ES is critical, although it is not required for uptake. In addition, DC-SIGN expression was sufficient to cause a significant invasion by ES in HeLa cells and intestinal epithelial cells, which are normally not invaded by ES. OmpA⁺ ES prevented the maturation of DCs by triggering the production of high levels of IL-10 and TGF-β and by suppressing the activation of MAPKs. Pretreatment of DCs with Abs to IL-10 and TGF-β or of bacteria with anti-OmpA Abs significantly enhanced the maturation markers on DCs. Furthermore, DCs pretreated with various inhibitors of MAPKs prohibited the increased production of proinflammatory cytokines stimulated by LPS or OmpA^– ES. LPS pretreatment followed by OmpA⁺ ES infection of DCs failed to induce maturation of DCs, indicating that OmpA⁺ ES renders the cells in immunosuppressive state to external stimuli. Similarly, OmpA⁺ ES-infected DCs failed to present Ag to T cells as indicated by the inability of T cells to proliferate in MLR. We conclude that ES interacts with DC-SIGN to subvert the host immune responses by disarming MAPK pathway in DCs.

Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces Up-Regulation of Galectin-1 Expression with Potential Anti-Inflammatory Activity [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1β, TNF-, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection.

Expansion of Functionally Skewed CD56-Negative NK Cells in Chronic Hepatitis C Virus Infection: Correlation with Outcome of Pegylated IFN-{alpha} and Ribavirin Treatment [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

NK cells are important innate immune effector cells, normally characterized as CD56⁺CD3^– lymphocytes. In this study, we report that CD56^–CD16⁺ NK cells expand in many patients with chronic hepatitis C virus infection. These CD56^– NK cells were functionally impaired with respect to cytokine production upon target cell recognition, in comparison to CD56^dim and CD56^bright NK cell subsets. In particular, CD56^– NK cells were strikingly defective in their polyfunctional response as measured by the coexpression of MIP-1β, IFN-, TNF-, and CD107a degranulation. The ability of these cells to mediate three or four of these functions was poor; expression of MIP-1β alone dominated their response. CD56^– NK cells retained expression of receptors such as the natural cytotoxicity receptors and NKG2D, whereas the expression of CD57 and perforin was lower when compared with CD56^dim NK cells. Interestingly, pretreatment levels of CD56^– NK cells correlated with the outcome of pegylated IFN- and ribavirin treatment. In patients with CD56^– NK cells in the range of healthy subjects, 80% reached a sustained virological response to treatment, whereas only 25% of patients with levels clearly above those in healthy subjects experienced a sustained virological response. Thus, chronic hepatitis C virus infection is associated with an expansion of CD56^– NK cells functionally skewed toward MIP-1β production only. Furthermore, high levels of these cells reveal a disturbance in innate cellular immunity that is associated with an impaired ability to respond to antiviral treatment with IFN- and ribavirin.

Adoptive Transfer of T Lymphocytes Sensitized against the Prion Protein Attenuates Prion Invasion in Scrapie-Infected Mice [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

There is to date no effective way of preventing or curing neurodegenerative diseases such as Alzheimer disease or transmissible spongiform encephalopathies. The idea of treating those conditions by immunological approaches has progressively emerged over the last ten years. Encouraging results have been reported in Alzheimer disease and in peripheral forms of mouse prion diseases following passive injection of Abs or active immunization against the peptides or proteins presumably at the origin of those disorders. Still, major difficulties persist due to some characteristics of those conditions such as slow evolution, brain location, uncertainties regarding precise pathogenic pathways, and, above all, the fact that the target Ag is self, meaning that it is poorly immunogenic and potentially harmful if tolerance was transgressed. To analyze some of those difficulties, we are developing adoptive cell transfer approaches. In this study, lymphocytes sensitized against the prion protein in nontolerant Prnp^–/– mice were transferred into histocompatible wild-type recipients which were partly or totally devoid of their own lymphocytes. Under such conditions, we found that the engrafted T lymphocytes resisted peripheral tolerance, remained reactive for several months against epitopes of the prion protein, and significantly attenuated the progression of prions in secondary lymphoid organs with subsequent delay in the evolution of the neurological disease. Interestingly, those protective T lymphocytes secreted lymphokines and migrated more readily into the host CNS but did not appear to be engaged in cooperation with host B cells for Ab production.

Both TRIF- and MyD88-Dependent Signaling Contribute to Host Defense against Pulmonary Klebsiella Infection [HOST DEFENSE]

Cai, S., Batra, S., Shen, L., Wakamatsu, N., Jeyaseelan, S. — Wed, 04 Nov 2009 13:05:42 PST

Klebsiella pneumoniae causes extensive lung damage. TLR signaling involves adaptors TRIF and MyD88. However, the relative contribution of TRIF and MyD88 signaling in host defense against pulmonary K. pneumoniae infection has not been elucidated. Therefore, we investigated the role of TRIF and MyD88 in K. pneumoniae pneumonia. TRIF^–/– mice infected with K. pneumoniae showed impaired survival and reduced bacterial clearance, neutrophil influx, histopathologic evidence of inflammation, and TNF-, IL-6, KC, MIP-2, but not LIX, expression in the lungs. In addition, K. pneumoniae-induced late NF-B activation and phosphorylation of MAPKs was attenuated in the lungs of TRIF^–/– mice. However, MyD88^–/– mice infected with K. pneumoniae showed a much more remarkable phenotype, including impaired survival and reduced bacterial clearance, histopathology, and TNF-, IL-6, KC, MIP-2, and LIX expression with almost no neutrophil influx in the lungs. In MyD88^–/– mice, K. pneumoniae-induced early NF-B and MAPK activation in the lungs was also reduced. Furthermore, the role of MyD88 is dominant over TRIF because TRIF/MyD88 double knockout mice displayed a more pronounced phenotype than TRIF^–/– mice. Moreover, human alveolar macrophages pretreated with MyD88 blocking peptide showed attenuated TNF-, IL-6, and IL-8 expression. Also, C57BL/6 mice pretreated with MyD88 blocking peptide exhibited attenuation in K. pneumoniae-induced neutrophil influx and enhanced bacterial burden in the lungs and dissemination. Overall, this investigation provides new insights into the TRIF and MyD88 signaling triggered by pulmonary K. pneumoniae infection in the lungs and demonstrate the therapeutic potential of MyD88 in reducing excessive neutrophil influx in human disease during Gram-negative bacterial pneumonia.

IL-22 Produced by Human NK Cells Inhibits Growth of Mycobacterium tuberculosis by Enhancing Phagolysosomal Fusion [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:42 PST

We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3^–CD56⁺ NK cells produced IL-22 when exposed to autologous monocytes and -irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.

The Novel Lipopolysaccharide-Binding Protein CRISPLD2 Is a Critical Serum Protein to Regulate Endotoxin Function [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:42 PST

LPS is an immunostimulatory component of Gram-negative bacteria. Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood samples are 607 µg/ml and 290 µg/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2–0.9 µg/ml) and enhance CRISPLD2 secretion (range, 1.5–4.2 µg/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF- and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum concentrations not only are indicators of the degree of a body’s exposure to LPS but also reflect an individual’s LPS sensitivity.

The Bile Acid Sensor Farnesoid X Receptor Is a Modulator of Liver Immunity in a Rodent Model of Acute Hepatitis [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. In this study, we report that activation of the farnesoid X receptor (FXR), a member of the ligand-activated nuclear receptor superfamily and bile sensor highly expressed in the liver, attenuates liver injury in a model of autoimmune hepatitis induced by Con A. We found that FXR gene ablation results in a time-dependent increase of liver expression (up to 20-fold in a 9-mo-old mouse) of osteopontin, a NKT cell-derived extracellular matrix protein and immunoregulatory cytokine. In comparison to wild-type, FXR^–/– mice are more susceptible to Con A-induced hepatitis and react to Con A administration by an unregulated production of osteopontin. Administering wild-type mice with a synthetic FXR agonist attenuated Con A-induced liver damage and liver expression of the osteopontin gene. By in vitro studies, we found that FXR is expressed by primarily isolated NKT cells and its ablation favors ostepontin production in response to Con A. Chromatin immunoprecipitation assay and coimmunoprecipitation experiments demonstrate that the short heterodimer partner (SHP), a nuclear receptor and FXR target, was expressed by NKT cell hybridomas and increased in response to FXR activation. FXR activates SHP that interacts with and inhibits c-Jun binding to the osteopontin promoter. These data indicate that in NKT cells, FXR activation causes a SHP-mediated inhibition of osteopontin production. These data support the notion that the bile acid sensor FXR regulates the activation of liver NKT cells.

Externally Triggered Egress Is the Major Fate of Toxoplasma gondii during Acute Infection [INFLAMMATION]

Tomita, T., Yamada, T., Weiss, L. M., Orlofsky, A. — Wed, 04 Nov 2009 13:05:42 PST

The apicomplexan parasite Toxoplasma gondii expands during acute infection via a cycle of invasion, intracellular replication, and lytic egress. Physiological regulation has not yet been demonstrated for either invasion or egress. We now report that, in contrast to cell culture systems, in which egress occurs only after five or more parasite divisions (2–3 days), intracellular residence is strikingly abbreviated in inflammatory cells in vivo, and early egress (after zero to two divisions) is the dominant parasite fate in acutely infected mice. Adoptive transfer experiments demonstrate rapid, reciprocal, kinetically uniform parasite transfer between donor and recipient compartments, with a t_1/2 of ~3 h. Inflammatory macrophages are major participants in this cycle of lytic egress and reinfection, which drives rapid macrophage turnover. Inflammatory triggering cells, principally macrophages, elicit egress in infected target macrophages, a process we term externally triggered egress (ETE). The mechanism of ETE does not require reactive oxygen or nitrogen species, the mitochondrial permeability transition pore, or a variety of signal transduction mediators, but is dependent on intracellular calcium and is highly sensitive to SB203580, an inhibitor of p38 MAPK as well as a related parasite-encoded kinase. SB203580 both inhibited the initiation of ETE and altered the progression of egress. Parasites recently completing a cycle of egress and reinfection were preferentially restricted in vivo, supporting a model in which ETE may favor host defense by a process of haven disruption. ETE represents a novel example of interaction between a parasite infectious cycle and host microenvironment.

Activation of the Cholinergic Anti-Inflammatory System by Nicotine Attenuates Neuroinflammation via Suppression of Th1 and Th17 Responses [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

The 7 nicotinic acetylcholine receptor (nAChR) was recently described as an anti-inflammatory target in both macrophages and T cells. Its expression by immune cells may explain the epidemiological data claiming a negative link between cigarette smoking and several inflammatory diseases. In this study, we determined the immunological effects of 7 nAChR activation by nicotine. Our results indicate that the 7 nAChR is expressed on the surface of CD4⁺ T cells and that this expression is up-regulated upon immune activation. Nicotine reduced T cell proliferation in response to an encephalitogenic Ag, as well as the production of Th1 (TNF- and IFN-) and Th17 cytokines (IL-17, IL-17F, IL-21, and IL-22). IL-4 production was increased in the same setting. Attenuation of the Th1 and Th17 lineages was accompanied by reduced T-bet (50%) and increased GATA-3 (350%) expression. Overall, nicotine induced a shift to the Th2 lineage. However, 7^–/–-derived T cells were unaffected by nicotine. Furthermore, nicotine reduced NF-B-mediated transcription as measured by IL-2 and IB transcription. In vivo, administration of nicotine (2 mg/kg s.c.) suppressed the severity of CD4⁺ T cell-mediated disease experimental autoimmune encephalomyelitis. 7^–/– mice were refractory to nicotine treatment, although disease severity in those animals was reduced, due to impairment in Ag presentation. Accordingly, CD4⁺ and CD11b⁺ cells infiltration into the CNS, demyelination, and axonal loss were reduced. Our data implicate a role for the 7 nAChR in immune modulation and suggest that 7 nAChR agonists may be effective in the treatment of inflammatory disorders.

Novel Role of Pin1 Induction in Type II Collagen-Mediated Rheumatoid Arthritis [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints and subsequent destruction of cartilage and bone. Inflammatory mediators such as PGs and proinflammatory cytokines contribute to RA progress. Pin1, a peptidyl prolyl isomerase, plays important pathophysiological roles in several diseases, including cancer and neurodegeneration. We found that both Pin1 and cyclooxygenase-2 (COX-2) were highly expressed in ankle tissues of type II collagen-induced RA mice. HTB-94 cells overexpressing Pin1 and primary cultured human chondrocytes showed increased basal expression of proinflammatory proteins (COX-2, inducible NO synthase, TNF-, and IL-1β). Site-directed mutagenesis revealed that Pin1-mediated transcriptional activation of COX-2 was coordinately regulated by NF-B, CREB, and C/EBP. Gel shift, reporter gene, and Western blot analyses confirmed that NF-B, CREB, and C/EBP were consistently activated in chondrocytes overexpressing Pin1. Treatment of RA mice with juglone, a chemical inhibitor of Pin1, significantly reduced RA progress and COX-2 expression in the ankle tissues. Moreover, juglone dose dependently decreased the basal COX-2 expression in primary cultured chondrocytes from RA patients. These results demonstrate that Pin1 induction during RA progress stimulates proinflammatory protein expression by activating NF-B, CREB, and C/EBP, and suggest that Pin1 is a potential therapeutic target of RA.

CXCR2 Is Required for Neutrophilic Airway Inflammation and Hyperresponsiveness in a Mouse Model of Human Rhinovirus Infection [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Human rhinovirus (RV) infection is responsible for the majority of virus-induced asthma exacerbations. Using a mouse model of human RV infection, we sought to determine the requirement of CXCR2, the receptor for ELR-positive CXC chemokines, for RV-induced airway neutrophilia and hyperresponsiveness. Wild-type and CXCR2^–/– mice were inoculated intranasally with RV1B or sham HeLa cell supernatant. Following RV1B infection, CXCR2^–/– mice showed reduced airway and lung neutrophils and cholinergic responsiveness compared with wild-type mice. Similar results were obtained in mice treated with neutralizing Ab to Ly6G, a neutrophil-depleting Ab. Lungs from RV-infected, CXCR2^–/– mice showed significantly reduced production of TNF-, MIP-2/CXCL2, and KC/CXCL1 and lower expression of MUC5B compared with RV-treated wild-type mice. The requirement of TNF- for RV1B-induced airway responses was tested using TNFR1^–/– mice. TNFR1^–/– animals displayed reduced airway responsiveness to RV1B, even when exogenous MIP-2 was added to the airways. We conclude that CXCR2 is required for RV-induced neutrophilic airway inflammation and that neutrophil TNF- release is required for airway hyperresponsiveness.

Recognition of Fungal Protease Activities Induces Cellular Activation and Eosinophil-Derived Neurotoxin Release in Human Eosinophils [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils’ responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils’ innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.

Chemokine-Like Receptor-1 Expression by Central Nervous System-Infiltrating Leukocytes and Involvement in a Model of Autoimmune Demyelinating Disease [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

We examined the involvement of chemokine-like receptor-1 (CMKLR1) in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Upon EAE induction by active immunization with myelin oligodendrocyte glycoprotein amino acids 35–55 (MOG_35–55), microglial cells and CNS-infiltrating myeloid dendritic cells expressed CMKLR1, as determined by flow cytometric analysis. In addition, chemerin, a natural ligand for CMKLR1, was up-regulated in the CNS of mice with EAE. We found that CMKLR1-deficient (CMKLR1 knockout (KO)) mice develop less severe clinical and histologic disease than their wild-type (WT) counterparts. CMKLR1 KO lymphocytes proliferate and produce proinflammatory cytokines in vitro, yet MOG_35–55-reactive CMKLR1 KO lymphocytes are deficient in their ability to induce EAE by adoptive transfer to WT or CMKLR1 KO recipients. Moreover, CMKLR1 KO recipients fail to fully support EAE induction by transferred MOG-reactive WT lymphocytes. The results imply involvement of CMKLR1 in both the induction and effector phases of disease. We conclude that CMKLR1 participates in the inflammatory mechanisms of EAE and represents a potential therapeutic target in multiple sclerosis.

Complement Activation by CpG in a Human Whole Blood Loop System: Mechanisms and Immunomodulatory Effects [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.

Bone Marrow Ly6Chigh Monocytes Are Selectively Recruited to Injured Kidney and Differentiate into Functionally Distinct Populations [INFLAMMATION]

Lin, S. L., Castano, A. P., Nowlin, B. T., Lupher, M. L., Duffield, J. S. — Wed, 04 Nov 2009 13:05:42 PST

Roles for monocyte/macrophages (M) in directing the development of tissue fibrosis are increasingly recognized. Macrophages form a heterogeneous group of inflammatory leukocytes, and the mechanisms by which they acquire heterogeneity and its functional significance are unclear. We used the unilateral ureteral obstruction model of progressive kidney fibrosis to explore macrophage heterogeneity and function further. Unilateral ureteral obstruction kidney Ms form three distinct subpopulations defined by the marker Ly6C, all of which are derived from a single Ly6C^high bone marrow monocyte population selectively recruited to the kidney. Conditional ablation of these Ms in vivo in CD11b-DTR mice is potently antifibrotic. The mRNA transcription profile of these populations is consistent with differential functional roles for each subpopulation, with Ly6C^low macrophages transcribing genes consistent with selective profibrotic or M2-type function. Furthermore, bone marrow chimerism studies indicate that although resident kidney macrophages proliferate markedly to comprise up to 40% of the inflammatory macrophage population, they do not contribute to fibrosis. Our data identify Ly6C as a marker of functionally discrete tissue macrophage subsets and support a model of selective recruitment of Ly6C^high bone marrow monocytes to the kidney that differentiate into three populations of kidney macrophages, including a profibrotic Ly6C^low population.

Nonhemopoietic Cell TLR4 Signaling Is Critical in Causing Early Lipopolysaccharide-Induced Ileus [INFLAMMATION]

Buchholz, B. M., Chanthaphavong, R. S., Bauer, A. J. M. — Wed, 04 Nov 2009 13:05:42 PST

Endotoxin-mediated ileus is poorly understood. Our objective was to mechanistically investigate the role of cell-specific TLR4 expression/signaling in causing gastrointestinal dysmotility. TLR4 chimeras and CSF-1-dependent macrophage-deficient mice were subjected to i.p. ultrapure (UP)-LPS (5 mg/kg). At 6 h, gastric emptying and gastrointestinal transit assessed in vivo motility, and jejunal circular muscle contractility was measured in vitro. Muscularis infiltration of neutrophils and monocytes were counted, and intestinal muscularis inflammatory mediators were quantified by quantitative PCR. Demonstrating TLR4 dependency, UP-LPS-induced gastric stasis and ileus of TLR4^WT mice were absent in mutant TLR4^LPS-d mice. Unexpectedly, engraftment of TLR4-mutant bone marrow into TLR4-competent mice (bmTLR4^LPS-d/TLR4^WT) exhibited a significant transit delay to UP-LPS similar to bmTLR4^WT/TLR4^WT mice. CSF-1^–/– mice were not protected from ileus. Contrary, UP-LPS-treated bmTLR4^WT/TLR4^LPS-d and bmTLR4^LPS-d/TLR4^LPS-d mice had normal transit. No leukocytic infiltration was detected at 6 h. Spontaneous jejunal contractions were markedly suppressed in UP-LPS-treated TLR4-competent mice, but bethanechol-stimulated contractions were not altered by UP-LPS in any group. UP-LPS-induced inflammatory mRNAs in a TLR4-dependent manner, but TLR4 mRNA itself was not significantly altered. In chimera mice, UP-LPS induction of IL-1β and IL-10 were hemopoietic dependent, and GM-CSF was nonhemopoietic dependent, whereas IL-6 and inducible NO synthase were derived from both cell types. Hemopoietic and nonhemopoietic cells contribute to TLR4-sensitive muscularis inflammatory signaling, but nonhemopoietic TLR4 signaling plays an exclusive primary role in causing functional UP-LPS-induced gastric stasis and ileus. Direct LPS suppression of spontaneous contractility participates in mediating early TLR4-transduced dysmotility.

Immunomodulatory Activity of Oenothein B Isolated from Epilobium angustifolium [INFLAMMATION]

Wed, 04 Nov 2009 13:05:43 PST

Epilobium angustifolium has been traditionally used to treat of a number of diseases; however, not much is known regarding its effect on innate immune cells. In this study, we report that extracts of E. angustifolium activated functional responses in neutrophils and monocyte/macrophages. Activity-guided fractionation, followed by mass spectroscopy and NMR analysis, resulted in the identification of oenothein B as the primary component responsible for phagocyte activation. Oenothein B, a dimeric hydrolysable tannin, dose-dependently induced a number of phagocyte functions in vitro, including intracellular Ca²⁺ flux, production of reactive oxygen species, chemotaxis, NF-B activation, and proinflammatory cytokine production. Furthermore, oenothein B was active in vivo, inducing keratinocyte chemoattractant production and neutrophil recruitment to the peritoneum after intraperitoneal administration. Biological activity required the full oenothein B structure, as substructures of oenothein B (pyrocatechol, gallic acid, pyrogallol, 3,4-dihydroxybenzoic acid) were all inactive. The ability of oenothein B to modulate phagocyte functions in vitro and in vivo suggests that this compound is responsible for at least part of the therapeutic properties of E. angustifolium extracts.

Adenosine Blocks IFN-{gamma}-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation [INFLAMMATION]

Barnholt, K. E., Kota, R. S., Aung, H. H., Rutledge, J. C. — Wed, 04 Nov 2009 13:05:43 PST

Macrophages are activated by IFN-, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN- signaling induces phosphorylation of two STAT1 residues: Tyr⁷⁰¹ (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser⁷²⁷ (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN- signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN--stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN--induced STAT1 S727 phosphorylation by >30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A₃ receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25–50%. Further, RAW 264.7 A₃ receptor stimulation with Cl-IB-MECA reduces IFN--induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A₃ receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN--activated mouse and human macrophages. Because STAT1 plays a key role in IFN--induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.

Cysteinyl-Leukotriene Receptor Type 1 Expression and Function Is Down-Regulated during Monocyte-Derived Dendritic Cell Maturation with Zymosan: Involvement of IL-10 and Prostaglandins [INFLAMMATION]

Thivierge, M., Stankova, J., Rola-Pleszczynski, M. — Wed, 04 Nov 2009 13:05:43 PST

TLRs sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). DCs have been shown to produce leukotrienes and, conversely, leukotrienes are known to modulate several DC functions. In this study, we examined the modulation of expression and function of cysteinyl-leukotriene receptor type 1 (CysLT1) on human monocyte-derived DCs during their differentiation and subsequent maturation with zymosan, a TLR2 agonist. Maturation of DCs with zymosan reduced CysLT1 mRNA levels and protein expression in a time-dependent fashion and was associated with a diminution of functional responsiveness to leukotriene D₄ as assessed by intracellular calcium mobilization, CCL2 and CCL3 production, and chemotaxis. The effect of zymosan was mediated by both TLR2 and dectin-1 activation. Zymosan also induced a rapid expression of cyclooxygenase-2 and the production of PGE₂ and IL-10. Addition of an anti-IL-10 neutralizing Ab or inhibitors of cyclooxygenase greatly reduced the ability of zymosan to down-regulate CysLT1 expression. Down-regulation of CysLT1 expression by zymosan could be reproduced by a combination of IL-10 and PGE₂, and was dependent on MAPK activation. Taken together, our findings indicate that zymosan down-regulates CysLT1 expression in DCs with consequently reduced functional responsiveness of the cells to leukotriene D₄ stimulation. This effect is partially dependent on an endogenous production of PGs and IL-10 by DCs.

Selective Prostacyclin Receptor Agonism Augments Glucocorticoid-Induced Gene Expression in Human Bronchial Epithelial Cells [INFLAMMATION]

Wed, 04 Nov 2009 13:05:43 PST

Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57^kip2) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.

Activated CD4+ T Cells Dramatically Enhance Chemotherapeutic Tumor Responses In Vitro and In Vivo [CLINICAL IMMUNOLOGY]

Radfar, S., Wang, Y., Khong, H. T. — Wed, 04 Nov 2009 13:05:43 PST

Chemoimmunotherapy has been widely studied in melanoma, with various degrees of success. One of the most common approaches is the so-called biochemotherapy, which is associated with increased toxicities, but without overall survival benefit. Another conventional strategy is the use of chemotherapy as an immunomodulator to enhance the effect of cancer vaccines or adoptive cell transfer therapy. Based on this approach, recent studies using chemotherapy to prepare the host before the infusion of ex vivo-activated, melanoma Ag-specific tumor-infiltrating lymphocytes and high dose IL-2 resulted in an impressive response rate. However, the development of immunotherapy for the treatment of a broad range of cancer type is still lacking. In this study, we report the development of a simple yet universal approach termed "chemocentric chemoimmunotherapy" that has potential application in the treatment of all cancer types. This technique uses nonspecifically activated CD4⁺ T cells as a chemosensitizer before the administration of chemotherapy. Dramatic enhancement of the cytotoxic effect of chemotherapeutic drugs, either active or nonactive as single agents, was observed both in in vitro and in vivo human tumor xenograft models. Soluble factors secreted from activated CD4⁺ T cells, likely acting on the tumor and its microenvironment, were responsible for the observed effect. Although IFN- played a major role in the therapeutic outcome, it was consistently found to be inferior to the use of activated CD4⁺ T cells in tumor chemosensitization. Our model may provide a plausible mechanism to facilitate further understanding, design and development of improved chemoimmunotherapy in the treatment of cancer.

Targeting a Mimotope Vaccine to Activating Fc{gamma} Receptors Empowers Dendritic Cells to Prime Specific CD8+ T Cell Responses in Tumor-Bearing Mice [CLINICAL IMMUNOLOGY]

Gil, M., Bieniasz, M., Wierzbicki, A., Bambach, B. J., Rokita, H., Kozbor, D. — Wed, 04 Nov 2009 13:05:43 PST

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fc2a) to deliver the antigenic cassette to the activating Fc receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fc2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fc2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

CMV-Specific TCR-Transgenic T Cells for Immunotherapy [CLINICAL IMMUNOLOGY]

Schub, A., Schuster, I. G., Hammerschmidt, W., Moosmann, A. — Wed, 04 Nov 2009 13:05:43 PST

Reactivation of CMV can cause severe disease after allogeneic hemopoietic stem cell transplantation. Adoptive T cell therapy was successfully used for patients who had received transplants from CMV-positive donors. However, patients with transplants from CMV-negative donors are at highest risk, and an adoptive therapy is missing because CMV-specific T cells are not available from such donors. To address this problem, we used retroviral transfer of CMV-specific TCR genes. We generated CMV-specific T cell clones of several HLA restrictions recognizing the endogenously processed Ag pp65. The genes of four TCRs were cloned and transferred to primary T cells from CMV-negative donors. These CMV-TCR-transgenic T cells displayed a broad spectrum of important effector functions (secretion of IFN- and IL-2, cytotoxicity, proliferation) in response to endogenously processed pp65 and could be enriched and expanded by strictly Ag-specific stimulation. Expansion of engineered T cells was accompanied by an increase in specific effector functions, indicating that the transferred specificity is stable and fully functional. Hence, we expect these CMV-TCR-transgenic T cells to be effective in controlling acute CMV disease and establishing an antiviral memory.

Type I Interferons Produced by Resident Renal Cells May Promote End-Organ Disease in Autoantibody-Mediated Glomerulonephritis [CLINICAL IMMUNOLOGY]

Wed, 04 Nov 2009 13:05:43 PST

Increased Type I IFNs or IFN-I have been associated with human systemic lupus erythematosus. Interestingly augmenting or negating IFN-I activity in murine lupus not only modulates systemic autoimmunity, but also impacts lupus nephritis, suggesting that IFN-I may be acting at the level of the end-organ. We find resident renal cells to be a dominant source of IFN-I in an experimental model of autoantibody-induced nephritis. In this model, augmenting IFN-I amplified antibody-triggered nephritis, whereas ablating IFN-I activity ameliorated disease. One mechanism through which increased IFN-I drives immune-mediated nephritis might be operative through increased recruitment of inflammatory monocytes and neutrophils, though this hypothesis needs further validation. Collectively, these studies indicate that an important contribution of IFN-I toward the disease pathology seen in systemic autoimmunity may be exercised at the level of the end-organ.

Apurinic/Apyrimidinic Endonuclease 1 Is a Key Modulator of Keratinocyte Inflammatory Responses [CLINICAL IMMUNOLOGY]

Wed, 04 Nov 2009 13:05:43 PST

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1 and NF-B, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

Beneficial immunomodulation by Streptococcus mutans anti-P1 mAbs is Fc independent and correlates with increased exposure of a relevant target epitope [CORRECTIONS]

Robinette, R. A., Oli, M. W., McArthur, W. P., Brady, L. J. — Wed, 04 Nov 2009 13:05:43 PST