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The Journal of Immunology, 2006, 177: 6880-6888.
Copyright © 2006 by The American Association of Immunologists, Inc.

TLR4 Up-Regulation at Protein or Gene Level Is Pathogenic for Lupus-Like Autoimmune Disease1

Bei Liu*, Yi Yang*, Jie Dai*, Ruslan Medzhitov{dagger}, Marina A. Freudenberg{ddagger}, Ping L. Zhang§ and Zihai Li2,*

* Department of immunology, Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030-1601; {dagger} Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06519; {ddagger} Max-Planck Institut fur Immunobiologie, Freiburg, Germany; and § Division of Laboratory Medicine Geisinger Medical Center, Danville, PA 17822


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLR4 is the receptor for the Gram-negative bacterial cell wall component LPS. TLR4 signaling is controlled by both positive and negative regulators to balance optimal immune response and potential sepsis. Unchecked TLR4 activation might result in autoimmune diseases, a hypothesis that has not been formally resolved. In this study, we found that TLR4 signaling to LPS can be positively enforced by expressing gp96 on cell surfaces through the chaperone function of, but not the direct signaling by, gp96; TLR4 as well as the commensal flora are essential for the production of anti-dsDNA Ab and the immune complex-mediated glomerulonephritis in transgenic mice that express surface gp96. Moreover, a similar constellation of autoimmunity was evident in mice that encode multiple copies of tlr4 gene. Our study has revealed that increased TLR4 signaling alone without exogenous insult can break immunological tolerance. It provides a strong experimental evidence for TLR4 dysregulation as an etiology of lupus-like renal disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Toll-like receptor 4 signaling by LPS initiates a broad range of both innate and adaptive immune responses, including the production of proinflammatory cytokines, increased phagocytosis, Th1 polarization, modulation of regulatory T cells, and ultimately amplified adaptive immunity (1). TLR4 transmits signals via at least four cytosolic adaptor molecules (2), including MyD88 (3, 4), Toll-IL-1R domain-containing adapter protein (TIRAP) (5, 6),3 Toll/IL-1R domain-containing adaptor inducing IFN-β (7), and TRIF-related adapter molecule (8). On the other hand, LPS signaling via TLR4 can be negatively regulated by a number of factors (9), including IL-1R-associated kinase-M (10), TRAIL-R (11), A20 (12), and Ig IL-1R-related molecule (13). TLR4 is also regulated posttranscriptionally by a molecular chaperone gp96 in the lumen of the endoplasmic reticulum (ER) (14). In the absence of gp96, TLR4 fails to export to the cell surface due to its inappropriate folding and assembly.

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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

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-{alpha}, 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{kappa}B{alpha}, p-I{kappa}B{alpha}, 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-{alpha} 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-I{kappa}B{alpha}, anti-phospho-p38, and anti-phospho-Erk or anti-I{kappa}B{alpha}, 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cell surface expression of gp96 confers LPS hyperresponsiveness

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 I{kappa}B{alpha} 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.


Figure 1
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  		FIGURE 1. Expression of membrane-bound gp96 rescues LPS responsiveness of gp96-null cells. A, Schematic diagram of a retrovirus expression vector for full-length gp96 and gp96 fused with the transmembrane domain of a PDGF receptor (96tm). N, M, and C depict N-terminal, middle-, and C-terminal domains of gp96, respectively. LTR, Long terminal repeats; IRES, internal ribosome entry site; EGFP, enhanced GFP. B, Restoration of gp96 expression in the gp96-null pre-B mutants E4.126 by three rounds of spin infection with the gp96 MigR1 retroviruses, followed by flow cytometric analysis of total gp96 by intracellular staining or cell surface gp96. Open histogram, gp96 or TLR4; closed histogram, isotype control. C, Both gp96 and 96tm restore the cell surface expression of TLR4 and responsiveness of mutant cells to LPS. Top panel, Flow cytometry analysis of cell surface TLR4. Bottom panel, Western blot for gp96, phosphorylated I{kappa}B{alpha} as well as total I{kappa}B{alpha} after LPS stimulation (10 µg/ml) for 5 and 15 min.

 

Figure 2
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  		FIGURE 2. Cell surface gp96 and TLR4 do not comodulate in response to LPS. gp96- and 96tm-transduced mutant pre-B cells were stimulated with LPS for various time points, followed by flow cytometric analysis of cell surface TLR4 (A) or gp96 (B). Open histogram, gp96 or TLR4; closed histogram, isotype control.

 
Next, we examined the LPS responsiveness of BMDMs from 96tm-Tg mice. We reasoned that Tg cells were more responsive to LPS stimulation than WT cells due to the expression of 96tm on top of the endogenous gp96 in the former. Indeed, 96tm-Tg BMDMs produced significantly more proinflammatory cytokine IL-6, IL-12p40, and TNF-{alpha} 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).


Figure 3
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  		FIGURE 3. 96tm expression confers LPS hyperresponsiveness. A, BMDMs from WT or 96tm-Tg mice were stimulated with LPS for 15 min, followed by Western blot analysis for total as well as phosphorylated p38 and Erk. As an additional loading control, β-actin was used. B, BMDMs from WT or 96tm-Tg mice were stimulated with LPS at 200 ng/ml at the indicated times (left panels), or with LPS for 12 h for IL-6 and IL-12p40, and 3 h for TNF-{alpha} at the indicated concentrations (right panels), followed by ELISA quantification of cytokines in the supernatant. Three independent experiments were performed with the similar findings. *, p < 0.05; **, p < 0.01; ***, p < 0.005. C, Mice were administered i.p. with LPS (1 µg/g body weight), followed 2 mo later by a second dose of LPS (10 µg/g body weight). Serum IL-6 was measured by ELISA 3 h after the first (primary challenge) or the second LPS injection (secondary challenge). Each symbol represents one individual mouse. *, p < 0.05.

 
Neither soluble gp96 nor 96tm was able to activate TLR4-dependent pathway

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-{kappa}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).


Figure 4
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  		FIGURE 4. Soluble gp96 or 96tm does not directly activate TLR4 pathway. A, Surface expression of gp96 on A549 cells by an adenovirus-based 96tm-expression vector. A549 cells were infected with either Ad-LacZ or Ad-96tm, followed by flow cytometry analysis of surface gp96 by Ab against gp96 or a myc tag. Open histogram, gp96 or myc; closed histogram, isotype control. B, gp96 was purified from mouse liver, followed by SDS-PAGE and staining of the gel with Coomassie blue (CB) or immunoblot with anti-gp96 mAb. C, Coculture of 293-TLR4 cells with soluble gp96 or 96tm-expressing A549 cells for 24 h, followed by ELISA analysis of IL-8 in the supernatant. LPS was used as a positive control in this experiment.

 
Essential roles of TLR4 and commensal flora in autoimmunity in 96tm-Tg mice.

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).


Figure 5
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  		FIGURE 5. Cell surface gp96-mediated autoimmune diseases are dependent on TLR4. A, WT, 96tm-Tg, TLR4–/–96tm, and TLR4–/– mice were sacrificed at the age of 20 wk, followed by semiquantitative detection of anti-dsDNA Ab in the sera (1/100). B, ANA in the sera (1/50 dilution) of various groups of mice was quantified. The relative fluorescence intensity from each mouse was plotted. Each symbol represents one individual mouse. C, Kidneys of indicated mice at 20 wk of age were analyzed by immunofluorescence microscopy (x200) after staining of kidney sections with anti-mouse IgG, or by EM (x3000). Five to 10 mice were analyzed in each group. Shown are representative images of one mouse from each group. Arrows point to ultrastructural evidence of protein deposits.

 
Second, we crossed 96tm-Tg mice into TIRAP–/– background to examine the contributions by nucleic acid sensing TLRs, including TLR7 and TLR9, which is important since TLR7 and TLR9 signaling has been shown clearly to activate autoreactive B cells (42, 43, 44, 45). We found that both the serum anti-dsDNA IgG and the renal pathology were significantly reduced in TIRAP–/–96tm mice (Fig. 6, A and B). The reduced renal lesion was obvious ultrastructurally as the electron-dense deposit score of TIRAP–/–96tm kidney was 1.6 ± 0.25, compared with TIRAP-competent 96tm-Tg mice (electron-dense deposit score = 2.33 ± 0.33; p < 0.05) (Fig. 6). TIRAP–/– mice had the electron-dense deposit score of 1.0 ± 0, which was not significantly different from TIRAP–/–96tm mice. Because TIRAP is critical for MyD88-dependent signaling by TLR1, TLR2, TLR4, and TLR6, but not by other TLRs, such as TLR7 and TLR9 (5, 6), we conclude that the latter group of TLRs is unlikely to contribute significantly to autoimmunity in 96tm-Tg mice. Third, we treated 12-wk-old 96tm-Tg mice with a combination of ciprofloxacin, metronidazole, and polymyxin B for 11 wk to test the roles of microbes and LPS. This regimen resulted in near complete decontamination of the gut flora (Fig. 7A) and hence much reduced exposure of mice to LPS, which was further ensured by the treatment of mice with polymyxin B. We found that serum anti-dsDNA IgG (Fig. 7B), ANA (Fig. 7C), as well as autoimmune GN (Fig. 7D), were significantly ameliorated in antibiotic/polymyxin B-treated 96tm-Tg mice. Taken together, our data suggested strongly that TLR4 hyperresponsiveness to commensal flora is essential for the development of autoimmunity in 96tm-Tg mice.


Figure 6
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  		FIGURE 6. TIRAP-dependence of cell surface gp96-mediated lupus GN. A, WT, 96tm-Tg, TIRAP–/–96tm and TIRAP–/– mice were sacrificed at the age of 20 wk, followed by quantification of anti-dsDNA IgG (1/100 dilution) in the sera. B, Ultrastructural analysis of renal pathology by EM (x3000). Five mice were analyzed in each group. Shown are representative images of one mouse from each group. Arrows point to protein deposits. There is a significant difference in the average glomerular protein deposition scores between 96tm-Tg (2.33 ± 0.33) and TIRAP–/–96tm mice (1.6 ± 0.25; p < 0.05).

 

Figure 7
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  		FIGURE 7. Roles of commensal flora in cell surface gp96-mediated lupus GN. Various groups of mice (n = 5 per group) were treated with or without antibiotic mixtures (Abx) continuously for 11 wk at 12 wk old. A, Bacteria colony counts 1 wk after Abx treatment from feces of one treated mouse per group as described in Materials and Methods. B, Serum level of anti-dsDNA IgG (1/100) after 11-wk Abx treatment. C, Semiquantitative detection of ANA in the sera (1/50 dilution). A representative image from each group was shown. D, Examination of kidney pathology and quantification of Ig deposition index in the glomeruli.

 
Tlr4 gene amplification is sufficient to induce autoimmunity

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 (Sn->WT) 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-5->WT mice, but not in control Sn->WT mice. Strikingly, as early as 10 wk after the BM reconstitution, anti-dsDNA Ab was significantly elevated in the sera of TScN-5->WT mice (Fig. 8D). Immunofluorescence microscopy confirmed the glomerular deposition of immune complexes in the kidney of TScN-5->WT mice, but not control Sn->WT mice (Fig. 8, E and F). These data demonstrated that TLR4 hyperresponsiveness in the hemopoietic system alone is sufficient to induce autoimmunity.


Figure 8
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  		FIGURE 8. Tg overexpression of tlr4 leads to autoimmune GN. A, Increased cell surface TLR4 by TScN-5 BMDMs. Close histogram represents staining with isotype control Ab. Open histogram denotes TLR staining. B, BMDMs from TScN-5 and Sn mice were stimulated with LPS for 24 h, followed by quantification of cytokines in the supernatant. C, Representative immunofluorescence images of renal IC deposition from Sn and TsCN mice. All fields are magnified by x200. D, Serum level of anti-dsDNA Ab in various BM chimeric mice (n = 10 for TsCN-5-> WT mice, n = 8 for Sn-> WT mice). *, p < 0.05; **, p < 0.01; ***, p < 0.001. E, Severity of GN based on percentage of glomerular involvement and the amount of IC depositions in the glomerulus. p = 4.148 x 10–7. F, Representative immunofluorescence images of renal IC deposition from one mouse per group. (Magnification, x200.)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have induced TLR4 hyperresponsiveness in vivo via two distinct approaches: enhancing posttranscriptional folding of TLR4 by gp96 (Figs. 1 and 3) and increasing tlr4 gene expression by gene amplification (Fig. 8). Remarkably, both maneuvers resulted in the similar autoimmune GN (Figs. 5 and 8). Importantly, even though 96tm might chaperone other molecules, we demonstrated the essential roles of TLR4 in the pathogenesis of autoimmunity in 96tm-Tg mice (Fig. 5). We conclude, therefore, that increased TLR4 signaling alone is a sufficient and potent trigger to induce autoimmune disease.

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-{alpha} 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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. Back

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 Back

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. Back

Received for publication April 6, 2006. Accepted for publication August 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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