| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of immunology, Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030-1601;
Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06519;
Max-Planck Institut fur Immunobiologie, Freiburg, Germany; and
Division of Laboratory Medicine Geisinger Medical Center, Danville, PA 17822
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
gp96, known also as grp94, is one of the most abundant heat shock proteins (HSPs) in the ER (15). The immunological properties of gp96 and other HSPs have gained increasing attention because of a series of observations that tumor-derived HSPs were able to prime tumor-specific CD8+ T cells in the absence of exogenous adjuvant (16). Such a phenomenon has been attributed to the combination of the abilities of gp96 to activate dendritic cells (DCs) (17, 18, 19) and to chaperone antigenic peptides onto cross-presentation pathway to MHC class I molecules (20). Along this line, it was reported that soluble gp96 was able to serve as an endogenous TLR ligand to engage TLR4 and TLR2 on cell surface to activate DCs (23), although this observation could not be substantiated by subsequent biochemical studies (24, 25). Furthermore, direct binding of gp96 to cell surface TLR has not been reported.
To determine the proinflammatory properties of gp96 in vivo, we generated a transgenic (Tg) mouse that constitutively expressed gp96 on cell surfaces in multiple tissues/organs, using a ubiquitous cytomegalovirus promoter (26). The expression of cell surface gp96, named 96tm, was achieved by removing the C-terminal ER-retention signal KDEL of gp96 and fusing it with the transmembrane domain of the platelet-derived growth factor receptor without the signaling cytoplasmic tail (26, 27). The driving logic behind this experiment was that the constant access of gp96 to DCs might lead to breakdown of immunological tolerance should gp96 be indeed proinflammatory. We found that 96tm-expressing Tg mice (96tm-Tg) spontaneously developed lupus-like autoimmune diseases that were dependent on one of the cytosolic adaptor molecules MyD88 for TLR and IL-1/18 receptor signaling. There are at least two possible mechanisms to explain the result, the resolution of which has important implications in understanding how immune response is modulated by the large family of evolutionally conserved HSPs, which have been collectively and increasingly implicated in inflammation, autoimmunity, and anticancer immunity (16, 28, 29). First, 96tm serves as a TLR chaperone to induce hyperresponsiveness of 96tm-Tg mice to TLR ligands such as LPS, which is the cognitive ligand for TLR4. Second, 96tm directly engages the cell surface TLRs to initiate the proinflammatory process, and result in subsequent breakdown of immunological tolerance.
In this study, by crossing 96tm-Tg mice into TLR4-null background, we have formally demonstrated the essential roles of TLR4 in causing lupus-like autoimmune diseases. To understand the underlying mechanisms, we have performed careful analysis of the impact of 96tm on TLR4 signaling. We found that soluble gp96 or 96tm was unable to activate TLR4-dependent pathway. However, 96tm expression confers LPS hyperresponsiveness both in vitro and in vivo. Moreover, we have established the essential roles of commensal microbes in causing autoimmune diseases in 96tm-Tg mice. To prove that chronic activation of TLR4 is sufficient to induce autoimmunity, we have induced LPS hyperresponsiveness by a complete different mechanism, via tlr4 gene amplification. Strikingly, increased tlr4 gene expression alone without any exogenous insult induces a similar lupus-like autoimmune glomerulonephritis (GN). Our results not only resolved the engima of gp96 in innate immunity, but also demonstrated that TLR4 up-regulation at protein or gene level is sufficient to break immunologic tolreance.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
The 96tm-Tg mice were generated in the C57BL/6 background at the Tg mice core facility at the University of Connecticut and have been described previously (26). TLR4 knockout mice were generated and provided by S. Akira (Osaka University, Osaka, Japan) (30) and have been crossed into C57BL/6 background. TIRAP–/– mice have been described before (5).
The C57BL/10ScN mice were obtained from the Frederick Cancer Research Center (Frederick, MD) in 2001 and since then have been bred in the Freiburg facility in Germany. The ScN strain is known to be the progenitor strain of Cr mice, and both mice are equally LPS resistant (31). Cr mice do not express TLR4 mRNA (32), which was due to deletion of the tlr4 gene (33). By PCR, we confirmed that the tlr4 gene was absent in our LPS-resistant ScN mice. In accordance, neither Cr nor ScN expresses TLR4/MD-2 protein complex on cells by FACS analysis on peritoneal cells, splenocytes, collagenase-treated hepatocytes, purified B cells, bone marrow-derived macrophages (BMDMs) or BM-derived mast cells (34, 35) (C. Kalis and M. Freudenberg, unpublished data).
Unlike ScN mice, the Cr mice have an additional single-point mutation (substitution of a C for a G at position 2472) in the IL-12Rβ2 gene, leading to a premature stop codon, which is responsible for IL-12 nonresponsiveness (36). The sequences of IL-12Rβ1 cDNA from both strains are identical. The mice of both Cr and ScN strains produce very similar levels of TNF-
, IL-6, and type I IFN when stimulated in vivo with heat-killed bacteria, bacterial lipopeptides (TLR2-ligands), CpG (a TLR9 ligand), or dsRNA (a ligand for TLR3 and other receptors) and express a comparable degree of resistance to Staphylococcus aureus or lymphocytic choriomeningitis virus (M. Freudenberg, unpublished data).
To restore the TLR4 responsiveness in Cr mice, we generated TCr-5 mice (34), which was described previously. In brief, vector-free mouse tlr4 genomic DNA fragment was injected into C57BL10ScCr oocytes to generate TCr-5. A large part of the DNA was sequenced (91,748 bp; GenBank accession no. AF177767) and the presence of the whole murine wild-type (WT) gene confirmed. There were no indications for the presence of another gene in the fragment or the insertion of transgene occurred in vicinity of a gene that controls the development of autoimmunity.
We used the heterozygous TCr-5 mice for backcrossing to ScN. Although the parental ScN mice were negative for TLR4 gene by genomic PCR and RT-PCR and did not express TLR4/MD-2 by FACS, the parent TCr-5 and the Tg-positive F1 mice expressed the TLR4 gene and comparable levels of TLR4/MD-2 protein on blood monocytes (data not shown). The F1 animals were further backcrossed to ScN. The offsprings (N2), homozygous for the WT IL-12Rβ2 gene and heterozygous for the TLR4 transgene, were then intercrossed. Blood cells of TLR4-positive offspring were screened by FACS for TLR4/MD-2 expression. The TLR4-MD2 expression on monocytes of these mice was either comparable to that of heterozygous TCr-5 or F1 mice or approximately two times higher. The latter animals became parents of the new TScN-5 strain, which were IL-12Rβ2 normal and homozygous for TLR4 transgene. We have been screening these mice regularly for TLR4 expression (via PCR and FACS) and for their phenotype (LPS and IL-12 responsiveness). They exhibited normal IL-12 responsiveness but higher TLR4/MD-2 expression and higher susceptibility to LPS than the WT C57BL/10ScN mice (35).
In summary, C57BL10ScN (deletion of TLR4 gene) is the progenitor of C57BL10ScCr (deletion of TLR4 gene and point mutation in IL-12Rβ2 gene); therefore, the two strains are close to each other genetically. In fact, the only known difference between these two strains is the mutation of IL-12Rβ2. Spontaneous autoimmune diseases in these two strains have not been reported in literature or observed by us (data not shown). Animal experiments were approved by the Animal Care Committee of the University of Connecticut.
Cell lines, Abs, chemicals, and other reagents
Pre-B cell line E4.126 was provided by B. Seed (Harvard University, Boston, MA). HEK293 cells stably transfected with TLR4 was a gift from D. Golenbock (University of Massachusetts, Worcester, MA). Most of the Abs used for flow cytometry came from BD Biosciences. Rabbit anti-gp96 pAb against the N-terminal fragment of gp96 and the rat anti-gp96 mAb were obtained from Antigenics and StressGen Biotechnologies, respectively. Rabbit Ab against I
B, p-IB, Erk, p-Erk, p38, and p-p38 were obtained from Cell Signaling Technology. All other chemicals were purchased from Sigma-Aldrich. Cytokine ELISA kits for IL-6, IL-8, IL-12p40, and TNF- were obtained from BD Biosciences.
Antibiotic treatment
Antibiotics were administered daily through drinking water containing 110 mg/L ciprofloxacin, 100 mg/L polymyxin B, and 500 mg/L metronidazole for 11 wk. Sterilization was confirmed by streaking Luria-Bertani plates with the feces of antibiotic-treated mice, followed by colony counts after an overnight culture at 37°C.
Gp96 retroviral and adenoviral expression vector
Full-length gp96 cDNA or 96tm was amplified by PCR, followed by cloning into MigR1 retrovector using EcoRI site, and the proper orientation was confirmed by sequencing. 96tm contains a myc tag immediately before the transmembrane domain as described before (27). Ecotropic gp96 retroviruses were packaged in the Pheonix-Ecotropic cell line as reported previously (37). To construct adenoviral expression vector for 96tm (Ad-96tm), the insert from pCMV-96tm (27) was released by sequential digestion with KpnI, blunted with Klenow, digested with NotI, and ligated into pShuttle vector (BD Clontech). The expression cassette was subcloned into Adeno X system after a double digestion with CeuI and SceI. The propagation of the virus using HEK293 cells was performed strictly according to the manufacturer’s protocol (BD Clontech). Quantifications of adenovirus were performed by optical absorbance and reported as PFU according to the published protocol without significant modifications (38).
Western blot for phosphoproteins
LPS-stimulated or nonstimulated pre-B cells or BMDMs were washed three times with ice-cold PBS and lysed in kinase lysis buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 mM glycerolphosphate, 10 mM sodium fluoride, 1 mM DTT, 1 mM PMSF, and proteinase inhibitor mix) for 30 min at 4°C. The cell lysates were centrifuged at 14,000 rpm for 10 min, and supernatants were collected. Protein concentration was determined by Bradford assay. Fifty micrograms of total cell lysates was resolved on denaturing and reducing 10% SDS-PAGE, and the proteins were transferred from the gel onto Immobilon-P membranes. The membrane was blocked with 5% nonfat milk in PBS for 2 h at room temperature and then incubated with anti-phospho-IB, anti-phospho-p38, and anti-phospho-Erk or anti-IB, anti-p38, and anti-Erk pAb, followed incubated with HRP-conjugated anti-rabbit secondary Ab. Protein bands were visualized by using enhanced chemiluminescent substrate (Pierce).
Flow cytometry and autoimmune assay
Flow cytometry, detection of anti-nuclear Ab (ANA), and ELISA for anti-dsDNA Ab were performed as described previously (26). Fluorescence intensity of ANA staining was graded in the single-blind fashion according to the manufacturer’s protocol: 0, negative; 1, lowest but discernibly positive; 2, clearly positive; 3, bright; 4, brilliant. Five-micrometer-thick cryosectioned kidney sections were fixed with cold acetone, stained first with biotin-anti-mouse IgG, followed by FITC conjugate of streptavidin to demonstrate Ig deposition in the glomeruli. In some experiments, FITC-conjugated goat anti-mouse C3 (ICN Biomedicals) was used. The percentage of glomeruli involvement was determined manually after examining at least 70 nonoverlapping glomeruli per kidney section. Immune complex (IC) deposition was quantitatively graded based on an intensity scale from 1+ to 3+. The IC deposition index was determined by multiplying the percentage involvement with the average glomerular IC deposition grade. Electron microscopic analysis was also done in the single-blinded fashion (by P. L. Zhang); the grading system of dense electron deposits is as follows: 0, no deposits; 1+, mild mesangial deposits; 2+, moderate mesangial deposits; 3+, moderate mesangial deposits with some subendothelial deposits.
BM transplantation
BM cells (2 x 106) in PBS were injected via tail vein into recipient mice that were lethally irradiated (550 cGy twice with an interval of 4 h) 24 h before transfer.
Statistical analysis
Error bars represent SEM. Student’s t test and ANOVA were used for statistical analysis. Values of p < 0.05 were considered to represent statistically significant differences.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
The autoimmune process in 96tm-Tg mice could be initiated directly by cell surface gp96 or indirectly by a protein substrate chaperoned by 96tm. Understanding the chaperone function of 96tm should help us to differentiate the two possibilities. All nucleated cells express high level of gp96. To separate the roles of 96tm from the endogenous gp96, we took advantage of a gp96 mutant B cell line, E4.126 that was used originally by Randow and Seeds (14) to uncover the function of gp96 as a chaperone for TLR. Because of the insertional mutagenesis of both alleles of gp96, no functional gp96 was expressed in E4.126 cells. TLR4 failed to fold properly and transport to cell surface. We generated retroviral vectors that express either full-length gp96 or 96tm (Fig. 1A). As expected, E4.126 transduced with empty vector did not express gp96 (Fig. 1B). Consequently, no cell surface TLR4 was detectable (Fig. 1C), and these cells were unresponsive to LPS stimulations (Fig. 1C). However, retroviral expression of either full-length gp96 or 96tm in E4.126 resulted in the restoration of cell surface TLR4 and the responsiveness to LPS, as demonstrated by the phosphorylation of IB and its degradation (Fig. 1C). The relative potency of 96tm is comparable to the native gp96 in restoring TLR4 function. No differences in the kinetics of the downstream signaling were observed. It is well known that cell surface TLR4 undergoes conformational changes and decreases rapidly in response to LPS (39), which is responsible partially for LPS tolerance. We took the advantage of this phenomenon to address whether cell surface gp96 formed complexes with TLR4. We demonstrated the same degree/kinetics of down-regulation of cell surface TLR4 from both gp96- and 96tm-transduced cells in response to LPS (Fig. 2A). By comparison, the expression of surface 96tm was unaltered after treatment with LPS (Fig. 2B). We conclude, therefore, that there were no qualitative or quantitative differences between gp96 and 96tm in folding TLR4. TLR4 and 96tm did not comodulate in response to LPS, suggesting that these two molecules do not form complexes on the cell surface, although this possibility could not be ruled out entirely by this experiment.
|
|
in response to LPS in the dose-dependent manner, which correlated well with the increased phosphorylation of p38 and Erk (Fig. 3, A and B). As expected, when mice were challenged with LPS systemically, the serum level of IL-6 in Tg mice was significantly higher than that in WT mice (Fig. 3C). The increased sensitivity of Tg mice to LPS was also demonstrated experimentally by a condition called LPS tolerance, which is characterized by a reduction of proinflammatory cytokines in mice previously exposed to LPS, in response to LPS rechallenge. We found that IL-6 production by Tg mice was considerably reduced upon second exposure to LPS 1 wk after the first injection; the magnitude of the reduction was more pronounced in Tg mice. However, increased IL-6 production by Tg mice was again manifested when the interval between first and second LPS injection was >4 wk (Fig. 3C).
|
Previous works have suggested that gp96 alone might be able to activate DCs via TLR2/TLR4 (23). Our present finding that 96tm expression confers LPS hyperresponsiveness prompted us to re-examine this question by performing careful functional studies. We purified LPS-free gp96 from mouse livers using our stringent good manufacturing practice (LPS content <0.04 EU/µg gp96 by the Limulus amebocyte lysate assay; data not shown). In addition, we engineered an adenovirus expression vector that express 96tm, Ad-96tm. Infection of human lung cancer cell line A549 cells with Ad-96tm, but not with a control adenovirus vector that expresses β-galactosidase (Ad-LacZ), resulted in high level expression of 96tm on the cell surface (Fig. 4A). We then stimulated HEK293 cells that stably express TLR4 with LPS, gp96, A549 cells that were infected with either Ad-LacZ or Ad-96tm, followed by measurement of IL-8 production in the supernatant as an index of TLR4 activation (40). We found that HEK-293-TLR4 secreted significantly high level of IL-8 in response to LPS, but not to gp96 or 96tm-expressing cells (Fig. 4C). Similar results were obtained regardless the source of gp96 (human or mouse gp96) or cell types that were used to display 96tm (data not shown). Additionally, as shown in Fig. 1C, high-level expression of 96tm on pre-B cells did not result in activation of NF-B pathway, unless LPS was present. We suggest that neither LPS-free soluble gp96 nor 96tm was able to reproducibly activate TLR4-dependent signaling in vitro (Fig. 4). This finding is consistent with a recent report that gp96 per se does not activate TLR4 signaling, although it might amplify TLR4 response by chaperoning TLR4 ligand such as LPS (41).
|
Our finding that 96tm expression confers hyperresponsiveness of 96tm-Tg cells to LPS suggests that the underlying mechanism of autoimmunity in 96tm-Tg mice is due to chronic activation of TLR4 by subclinical level of LPS, but not by extracellular gp96 as was postulated previously (23, 26).
To directly test the above hypothesis in vivo, we performed three lines of investigations. First, we crossed 96tm-Tg mice onto TLR4-null background to address the roles of TLR4 in autoimmunity. At 20 wk of age, both male and female 96tm-Tg mice spontaneously developed the signature of lupus-like autoimmune diseases, including the appearance of ANA, anti-dsDNA Ab, and IC-mediated GN (Fig. 5). However, autoimmunity was significantly reduced in TLR4–/–96tm-Tg mice, manifested by the decreased levels of anti-dsDNA Ab, ANA, and IC deposition in the glomeruli. Reduction of IC deposition was confirmed by both immunofluorescence and electron microscopy (EM) (Fig. 5C). Most of the 96tm-Tg kidneys showed evidence of large IC deposits in mesangial and paramesangial areas as well as in subendothelial areas (electron-dense deposit score = 2.33 ± 0.33). IC deposits were absent in WT kidney and significantly reduced in TLR4–/–96tm kidney (score = 1.4 ± 0.25; p < 0.05).
|
|
|
Given that gp96 is also a chaperone for numerous other client proteins including TLR1 and TLR2 (15, 46), 96tm might also have a similar, if not identical, substrate specificity to native gp96. To determine whether TLR4 signaling alone is necessary and sufficient to cause autoimmune GN, we generated a Tg mouse called TCr-5, whose genome contains multiple copies of the tlr4 gene on a TLR4-deficient C57/BL10ScCr (ScCr) background (34). We further crossed TCr-5 mice with TLR4-null C57/BL10ScN (ScN) (47) mice to rescue a loss-of-function mutation in the IL-12Rβ2 gene in the original C57/BL10ScCr mice (36, 47), to generate a mouse named TScN-5. ScCr mice were derived from ScN mice; the only known genetic difference between the two strains is the mutation of IL-12Rβ2 gene in ScCr mice.
We confirmed that BMDMs from TScN-5 mice indeed expressed high levels of surface TLR4 (Fig. 8A) and were hyperresponsive to LPS stimulation (Fig. 8B), compared with WT C57BL/10ScSn (Sn) mice (47). As predicted, TScN-5, but not Sn, mice developed significant IC-mediated GN (Fig. 8C), demonstrating strongly that TLR4 gene activation was sufficient to cause autoimmunity. To eliminate the possible impact of high levels of TLR4 on the homeostasis of the gut, kidney, or other nonhematopoietic systems of TScN-5 mice, we reconstituted lethally irradiated WT mice with BM from either TScN-5 (TScN-5
WT) or WT Sn (SnWT) mice. These mice were then closely followed for signs of autoimmunity. If enhanced TLR4 signaling alone is sufficient to break tolerance, we would expect to see autoimmunity in TScN-5WT mice, but not in control SnWT mice. Strikingly, as early as 10 wk after the BM reconstitution, anti-dsDNA Ab was significantly elevated in the sera of TScN-5WT mice (Fig. 8D). Immunofluorescence microscopy confirmed the glomerular deposition of immune complexes in the kidney of TScN-5WT mice, but not control SnWT mice (Fig. 8, E and F). These data demonstrated that TLR4 hyperresponsiveness in the hemopoietic system alone is sufficient to induce autoimmunity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
There are at least three possible roles of gp96 in tuning TLR4 responsiveness. The first possibility lies in the roles of gp96 in chaperoning TLR4, which has been established previously in vitro (14) and confirmed functionally by the current study in vivo (Figs. 1 and 3). 96tm was clearly as efficient as gp96 in restoring TLR4 function in gp96 null cells (Fig. 1). This finding is not surprising, because 96tm retains all the functionally important domains for folding TLRs, i.e., the N-terminal ATP/radicicol-binding domain and the C-terminal dimerization domain of gp96 (15). Interestingly, the total level of gp96 (gp96 plus 96tm) was not significantly increased in the 96tm-Tg mice since the 96tm level did not reach even 5% of the level of endogenous gp96 in 96tm+ cells (data not shown). We suggest, therefore, that subtle increase of the expression of gp96, which could be achieved by stress (48) or inflammatory conditions (49), might be sufficient to contribute to the significant amplification of TLR4 responsiveness and subsequent breakdown of tolerance. The second role of gp96 in the context of TLR could be directly acting as TLR4 ligand, which was suggested by a study previously (23). However, our functional studies have demonstrated that neither LPS-free gp96 nor 96tm-expressing cells were able to activate TLR4 consistently (Fig. 4), which is in line with the contention that soluble gp96 per se does not possess intrinsic proinflammatory properties (24, 25). More studies are necessary to demonstrate whether soluble gp96 or membrane-bound gp96 is able to bind cell surface TLR4 in trans. Our current study has not ruled out the third possibility that gp96 might be able to play a role in chaperoning TLR ligands such as LPS to amplify the downstream signaling pathways (29), an idea that has received some experimental support recently (41).
The roles of TLR4 amplification in IC-induced autoimmunity were also demonstrated at the gene level. By overexpressing tlr4 gene alone in the Tg mice, we have revealed that TLR4 hyperresponsiveness is sufficient to induce lupus-like autoimmune disease (Fig. 8). The autoimmune phenotypes seen in 96tm-Tg mice and TScN-5 mice were indistinguishable, highlighting the commonality of pathogenesis in the two models, i.e., the contribution by TLR4 hyperresponsiveness. However, it remains unclear how chronic activation of TLR4 by commensal flora induced autoimmunity in either 96tm-Tg mice or TScN-5 model. We have shown that autoimmunity in 96tm-Tg mice is dependent on TIRAP (Fig. 6), revealing the roles of TLR1, TLR2, TLR4, and TLR6, but not the contribution by nucleic acid-sensing TLRs. Indeed, the TLR9 responses, as assessed by the production of proinflammatory cytokines IL-6 and TNF- by BMDMs in response to unmethylated CpG oligonucleotide stimulations, were not impaired in the absence of either TIRAP (5) or TLR4 (data not shown). However, our results do not completely exclude the mechanism of autoimmunity that is dependent on the recognition of self DNA, RNA complexes or other endogenous danger molecules, particularly since TLR4 stimulation has been shown recently to synergize with other TLR ligands including TLR7 and TLR9 ligands to activate DCs (50). The roles of TLR9 in our model thus warrant further examination, in light of the fact that both positive (51) and negative (52) roles of TLR9 in lupus have been reported recently. Additionally, since both MyD88 and TIRAP are required for secretion of proinflammatory cytokines but not for up-regulation of costimulatory molecules and MHC molecules in response to LPS (5, 6, 53), we suspect that the pathogenicity of autoimmunity in 96tm-Tg mice was primarily driven by increased cytokines but not by augmented costimulation. We are actively studying the roles of other TLRs in both 96tm-Tg mice and TScN-5 mice, the cellular and molecular mediators of autoimmunity in these models, with the focus on professional APCs, regulatory T cells, and a number of proinflammatory cytokines, including IL-6 and IL-12.
Our present study has established, for the first time, the causal association between TLR4 hyperresponsiveness and lupus-like disease, in the absence of apparent external insult. In the broader context, the epidemiological correlation between autoimmune diseases, such as rheumatic fever (54), vasculitis (55), and immune-mediated GN (56) and infections from bacterial, rickettsial, spirochetal, and other microorganisms has long been recognized. The connection between TLR9 signaling and autoimmunity has been suggested (42, 43, 44, 45). Recently, TLR5 was also implicated in the pathogenesis of lupus since the null mutation of TLR5 seems to confer lupus resistance (57). Mechanistically, a recent study has linked the production of type I IFN in response to TLR ligands and susceptibility of organ-specific autoimmunity (58).
Our study provides a scientific rationale for controlling the commensal flora in the treatment of autoimmune diseases. Overall, our report suggests strongly that one of the underlying mechanisms of lupus is the dysregulated TLR4 signaling, which should stimulate interests and efforts in generating TLR4 antagonists for amelioration of not only sepsis, but also lupus-like autoimmune diseases.
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was supported in part by National Institutes of Health Grants CA90337 and CA100191 (to Z.L.) and SP "Angeborene Immunität" (FR 448/4-3; to M.A.F.). Z.L. is a clinical scholar of the Leukemia and Lymphoma Society. R.M. is an investigator of the Howard Hughes Medical Institute. 
2 Address correspondence and reprint requests to Dr. Zihai Li, Center for Immunotherapy of Cancer and Infectious Diseases, Department of Immunology, University of Connecticut School of Medicine, MC1601, 263 Farmington Avenue, Farmington, CA 06030-1601. E-mail address: zli{at}up.uchc.edu
3 Abbreviations used in this paper: TIRAP, Toll-IL-1R domain-containing adapter protein; ER, endoplasmic reticulum; HSP, heat shock protein; DC, dendritic cell; Tg, transgenic; 96tm-Tg, 96tm-expressing Tg; BMDM, bone marrow-derived macrophage; WT, wild type; ANA, anti-nuclear Ab; IC, immune complex; Ad-LacZ, β-galactosidase; GN, glomerulonephritis; EM, electron microscopy.
Received for publication April 6, 2006. Accepted for publication August 25, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
B pathway. Int. Immunol. 12: 1539-1546. B activation and nitric oxide production. J. Biol. Chem. 278: 31853-31860. This article has been cited by other articles:
|
|
 |
|
  I.-Y. Hwang, C. Park, K. Harrison, and J. H. Kehrl TLR4 signaling augments B lymphocyte migration and overcomes the restriction that limits access to germinal center dark zones J. Exp. Med., November 23, 2009; 206(12): 2641 - 2657. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lartigue, N. Colliou, S. Calbo, A. Francois, S. Jacquot, C. Arnoult, F. Tron, D. Gilbert, and P. Musette Critical Role of TLR2 and TLR4 in Autoantibody Production and Glomerulonephritis in lpr Mutation-Induced Mouse Lupus J. Immunol., November 15, 2009; 183(10): 6207 - 6216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Benakanakere, Q. Li, M. A. Eskan, A. V. Singh, J. Zhao, J. C. Galicia, P. Stathopoulou, T. B. Knudsen, and D. F. Kinane Modulation of TLR2 Protein Expression by miR-105 in Human Oral Keratinocytes J. Biol. Chem., August 21, 2009; 284(34): 23107 - 23115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kliger, O. Levy, A. Oren, H. Ashkenazy, Z. Tiran, A. Novik, A. Rosenberg, A. Amir, A. Wool, A. Toporik, et al. Peptides modulating conformational changes in secreted chaperones: From in silico design to preclinical proof of concept PNAS, August 18, 2009; 106(33): 13797 - 13801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q.-Q. Huang, R. Sobkoviak, A. R. Jockheck-Clark, B. Shi, A. M. Mandelin II, P. P. Tak, G. K. Haines III, C. V. Nicchitta, and R. M. Pope Heat Shock Protein 96 Is Elevated in Rheumatoid Arthritis and Activates Macrophages Primarily via TLR2 Signaling J. Immunol., April 15, 2009; 182(8): 4965 - 4973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shin, Y. Zhang, M. Jagannathan, H. Hasturk, A. Kantarci, H. Liu, T. E. Van Dyke, L. M. Ganley-Leal, and B. S. Nikolajczyk B cells from periodontal disease patients express surface Toll-like receptor 4 J. Leukoc. Biol., April 1, 2009; 85(4): 648 - 655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Lombardi, L. Van Overtvelt, S. Horiot, and P. Moingeon Human Dendritic Cells Stimulated via TLR7 and/or TLR8 Induce the Sequential Production of Il-10, IFN-{gamma}, and IL-17A by Naive CD4+ T Cells J. Immunol., March 15, 2009; 182(6): 3372 - 3379. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Richez, K. Yasuda, A. A. Watkins, S. Akira, R. Lafyatis, J. M. van Seventer, and I. R. Rifkin TLR4 Ligands Induce IFN-{alpha} Production by Mouse Conventional Dendritic Cells and Human Monocytes after IFN-{beta} Priming J. Immunol., January 15, 2009; 182(2): 820 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kiyokawa, S. Akashi-Takamura, T. Shibata, F. Matsumoto, C. Nishitani, Y. Kuroki, Y. Seto, and K. Miyake A single base mutation in the PRAT4A gene reveals differential interaction of PRAT4A with Toll-like receptors Int. Immunol., November 1, 2008; 20(11): 1407 - 1415. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Parks, G. Cooper, M. Dooley, M. Park, E. Treadwell, and G. Gilkeson Childhood agricultural and adult occupational exposures to organic dusts in a population-based case-control study of systemic lupus erythematosus Lupus, August 1, 2008; 17(8): 711 - 719. [Abstract] [PDF]
|
|
|
|
|
|
K. Takahashi, T. Shibata, S. Akashi-Takamura, T. Kiyokawa, Y. Wakabayashi, N. Tanimura, T. Kobayashi, F. Matsumoto, R. Fukui, T. Kouro, et al. A protein associated with Toll-like receptor (TLR) 4 (PRAT4A) is required for TLR-dependent immune responses J. Exp. Med., November 26, 2007; 204(12): 2963 - 2976. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Niessner, M. S. Shin, O. Pryshchep, J. J. Goronzy, E. L. Chaikof, and C. M. Weyand Synergistic Proinflammatory Effects of the Antiviral Cytokine Interferon-{alpha} and Toll-Like Receptor 4 Ligands in the Atherosclerotic Plaque Circulation, October 30, 2007; 116(18): 2043 - 2052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Deocharan, Z. Zhou, K. Antar, L. Siconolfi-Baez, R. H. Angeletti, J. Hardin, and C. Putterman {alpha}-Actinin Immunization Elicits Anti-Chromatin Autoimmunity in Nonautoimmune Mice J. Immunol., July 15, 2007; 179(2): 1313 - 1321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Dai, B. Liu, S. M. Ngoi, S. Sun, A. T. Vella, and Z. Li TLR4 Hyperresponsiveness via Cell Surface Expression of Heat Shock Protein gp96 Potentiates Suppressive Function of Regulatory T Cells J. Immunol., March 1, 2007; 178(5): 3219 - 3225. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
