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TLRs in Cryptosporidium parvum infection
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B signaling pathway after C. parvum infection of cholangiocytes (biliary epithelia), but the mechanism for activation was unclear. Continuing their studies, Chen et al. (p. 7447
) showed constitutive expression of TLRs 1–10 in an SV40-transformed normal human cholangiocyte cell line by RT-PCR and immunoblot analyses. Only TLR2 and TLR4 were seen by dual immunofluorescent Ab labeling to accumulate at the C. parvum infection site. Accumulation did not occur after infection of cells stably transfected with dominant negative (DN) mutants of TLR2, TLR4, or MyD88, but parasite attachment and invasion were not altered. TLR2, TLR4, or MyD88 DN mutants inhibited parasite-induced activation of IL-1R-associated kinase, as detected by phosphorylation of myelin basic protein, and phosphorylation of p38, as detected by immunoblotting. In contrast, decreased IB
protein levels and NF-B p65 nuclear translocation following C. parvum infection were blocked in cells transfected with the DN mutants. The increase of IL-8 mRNA expression and secretion and of human 
-defensin 2 mRNA and protein expression, seen in parasite infected cells, were prevented by two inhibitors of NF-B. A significantly higher number of C. parvum was detected in cells deficient in MyD88. The data demonstrate that TLR2 and TLR4 play a key role in cholangiocyte innate immune responses via MyD88 activation of the NF-B signaling pathway. Caspase-1 activation
Procaspase-1 is cleaved by a K+-release stimulus, such as extracellular ATP activation of the P2X7R. The product, caspase-1, activates IL-1 and IL-18 in inflammatory diseases. Although LPS is known to be required for efficient caspase-1 activation, detailed steps in the process are not known. Kahlenberg et al. (p. 7611
) developed an in vitro processing assay to detect the capsase-1 p10 cleavage product in lysates of established murine macrophages and bone marrow-derived macrophages. The active fragment was detected in cells primed with LPS or other TLR4 or TLR2 ligands for 15 min to 4 h and stimulated for 5 min with ATP or another ionophore that induced K+ release. Both LPS priming and ATP stimulation were required for cleavage to occur. Exposure of cells to cyclohexamide before LPS priming attenuated caspase-1 activation, but levels of procaspase-1 and P2X7R proteins and K+ release were not reduced. Treatment of cells with cyclohexamide after LPS priming, but before ATP stimulation, only slightly reduced activation. No activation of procaspase-1 after ATP stimulation was seen in cells exposed to a proteasome inhibitor before, but not after, LPS priming. Blocking the inhibitor of NF-B kinase during the LPS priming step prevented caspase-1 activation. LPS-induced generation of IL-1 was attenuated by treatment of cells with inhibitors of ERK1/2, JNK, p38 MAPK, and PI3K. The authors conclude that LPS priming of TLR2 or TLR4 is required for K+-release stimuli to regulate caspase-1 activation by P2X7R and involves an intact proteasome and NF-B.
Abs and chlamydial infection
Primary urogenital tract infection with the sexually transmitted intracellular bacterium Chlamydia is resolved by a CD4+ T cell response. However, a clear role for an Ab response has not been established. Morrison and Morrison (p. 7536
) injected naive B cell knockout mice with immune serum from wild-type mice infected with Chlamydia muridarum. Immune serum did not alter the course of primary infection or level of chlamydial shedding in CD4+ T cell-depleted mice, but it did decrease chlamydial shedding by 
10-fold in the presence of CD4+ T cells. In contrast, immune serum or mAbs to either of two chlamydial cell surface proteins decreased chlamydial shedding and shortened the duration of secondary infection in Ab-deficient mice depleted of CD4+ T and CD8+ T cells; nonimmune serum or mAb to a cytoplasmic Ag had no effect. Ab titers in serum or vaginal washes of passively immunized Ab-deficient mice did not correlate with the Ab-mediated protection. The data demonstrate that Abs elicited during a primary chlamydial infection cooperate with CD4+ T cells to protect mice against bacterial genital tract reinfection.
Epitope spreading in SLE
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Generating tolerogenic DCs
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, little IFN-
, and no IL-2. A phosphokinase A inhibitor or an antagonist of the VIP/PACAP receptor VPAC1 reversed the surface phenotype, cytokine profile, and proliferation of stimulated tolerogenic T cells to those of control cells. NF-B nuclear translocation and IB phosphorylation occurred only in control DCs. Inhibition of proliferation of fresh syngeneic T cells by the regulatory T (Treg) cells generated in the presence of VIP/PACAP-DCs required both direct contact and soluble factors. The Treg cells had low expression of Foxp3 and other Treg cell markers. Ag-specific Treg cells were induced in vivo in mice injected with LPS-stimulated, in vitro-generated VIP/DCs loaded with Ag in vitro. Splenic T cells from naive syngeneic mice adoptively transferred with splenic CD4+ T cells from those injected animals had reduced proliferation, IL-2, and IFN- production and increased IL-10 production. Mice transgenic for a specific Ag that were injected with VIP and Ag developed higher numbers of CD11clowCD45RBhigh DCs able to induce Treg cells in vitro than control animals. The authors demonstrate the induction of Treg cells through in vitro and in vivo VIP/PACAP generation of tolerogenic DCs. IL-4 and Be2 cells
The Lund laboratory generated two types of effector B cells (Be1 and Be2) under special in vitro and in vivo conditions. Although both cell types produce "polarized" subsets of cytokines, the factors that control cytokine production are unknown. In a continuation of their previous work, Harris et al. (p. 7103
) cultured naive Ag1-specific BCR transgenic B cells and effector Th1 or Th2 cells, with TCRs specific for a different Ag (Ag2), plus their Ags to generate Be1 or Be2 cells, respectively. IL-4 mRNA was detected in developing Be2, but not in Be1 or naive, cells by quantitative PCR. Expression of IFN-, T-bet, and IL-12R2 mRNAs was suppressed in the Be2 cells compared with Be1 and naive cells. Only the Be1 cells secreted Ab after restimulation. Restimulated Be2 cells generated in cultures containing naive Ag1-specific wild-type B cells and Ag2-specific wild-type Th2 cells plus both Ags produced a high level of IL-4. Be2 cells from cultures substituted with Ag2-specific IL-4–/– Th2 cells produced IFN- but no IL-4. B cells expressing IL-4 and GFP developed in cultures of B cells from mice transgenic for a bicistronic IL-4 reporter plasmid plus Ag2-specific IL-4–/– Th2 cells only after addition of IL-4. Be2 cell development was inhibited by addition of mAbs that block costimulatory signals to cultures containing B cells carrying the IL-4 reporter plasmid and Th2 wild-type effectors plus Ags. Be2 cells that secreted IL-4 after restimulation in vitro developed in IL-4 reporter plasmid transgenic mice infected with a nematode that induced a Th2 immune response; no Be2 cells developed in infected IL-4R–/– or CD4+ T cell-depleted animals. The authors demonstrate the requirement for IL-4 and Th2 cell costimulation in the development of IL-4-producing Be2 cells in vitro and in vivo.
Regulating antimycobacterial immune responses
Live attenuated Mycobacterium bovis bacillus Calmette- Guerin (BCG) is an effective vaccine against widespread infection with Mycobacterium tuberculosis. BCG induces macrophages to release cytokines that regulate signal transduction and control cytokine gene expression via the dsRNA-activated serine/threonine protein kinase (PKR). However, the direct target of PKR is not known. Cheung et al. (p. 7218
) found that exposure of primary human PBMCs to an inhibitor of PKR prevented BCG-induced expression of TNF-, IL-10, and IL-6 mRNAs and secretion of TNF-. BCG-stimulated autophosphorylation of PKR but did not alter PKR mRNA levels. Induction of the cytokines by BCG also was blocked by pretreatment of cells with a specific inhibitor of NF-B activity. Treatment with the PKR inhibitor reduced NF-B binding in EMSAs on nuclear extracts of BCG-stimulated PBMCs. Furthermore, a specific inhibitor of ERK1/2 suppressed BCG induction of TNF- mRNA, and phosphorylation of ERK1/2 was blocked by either the ERK1/2 inhibitor or the PKR inhibitor. PKR induction and phosphorylation of transcription factor eIF2 did not occur in poly(I:C)-treated promonocytes stably transfected with a dominant negative PKR mutant gene, and enhanced expression of TNF- and IL-6 mRNAs in the transfected cells did not occur following BCG treatment. The authors conclude that PKR mediates the induction of cytokines in human PBMCs treated with BCG via activation of transcription factor NF-B and phosphorylation of ERK1/2.
Summaries written by Dorothy L. Buchhagen, Ph.D.
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