Opposing Impact of B Cell–Intrinsic TLR7 and TLR9 Signals on Autoantibody Repertoire and Systemic Inflammation

Shaun W. Jackson, Nicole E. Scharping, Nikita S. Kolhatkar, Socheath Khim, Marc A. Schwartz, Quan-Zhen Li, Kelly L. Hudkins, Charles E. Alpers, Denny Liggitt and David J. Rawlings
J Immunol May 15, 2014, 192 (10) 4525-4532; DOI: https://doi.org/10.4049/jimmunol.1400098

Abstract

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by autoantibodies targeting nucleic acid–associated Ags. The endosomal TLRs TLR7 and TLR9 are critical for generation of Abs targeting RNA- or DNA-associated Ags, respectively. In murine lupus models, deletion of TLR7 limits autoimmune inflammation, whereas deletion of TLR9 exacerbates disease. Whether B cell or myeloid TLR7/TLR9 signaling is responsible for these effects has not been fully addressed. In this study, we use a chimeric strategy to evaluate the effect of B cell–intrinsic deletion of TLR7 versus TLR9 in parallel lupus models. We demonstrate that B cell–intrinsic TLR7 deletion prevents RNA-associated Ab formation, decreases production of class-switched Abs targeting nonnuclear Ags, and limits systemic autoimmunity. In contrast, B cell–intrinsic TLR9 deletion results in decreased DNA-reactive Ab, but increased Abs targeting a broad range of systemic autoantigens. Further, we demonstrate that B cell–intrinsic TLR9 deletion results in increased systemic inflammation and immune complex glomerulonephritis, despite intact TLR signaling within the myeloid compartment. These data stress the critical importance of dysregulated B cell–intrinsic TLR signaling in the pathogenesis of systemic lupus erythematosus.

Introduction

Despite numerous potential autoantigen targets, patients with systemic lupus erythematosus (SLE) frequently develop a restricted autoantibody repertoire targeting nucleic acid–associated Ags. In addition to exogenous pathogens, the TLR family of germline-encoded, pattern-recognition receptors are able to recognize endogenous ligands. Nucleic acid–containing apoptotic particles promote activation of autoreactive B cells via dual BCR/TLR-mediated signals, thereby explaining the prominence of antinuclear Abs (ANAs) in autoimmunity (1–3). The Myd88-dependent, endosomal receptors TLR7 and TLR9 are critical in this context, with TLR7 required for the generation of Abs targeting RNA and RNA-associated proteins, whereas TLR9 activation promotes production of Abs targeting dsDNA and chromatin (4–7).

Importantly, two alternate, but not mutually exclusive, mechanisms may explain the role of TLR7 and TLR9 in autoimmune pathogenesis in vivo. Either dual BCR/TLR activation may promote a B cell–intrinsic break in tolerance, or immune complex (IC)–mediated TLR activation of plasmacytoid dendritic cells may promote autoimmunity via increased type 1 IFN production (4, 8). In addition, tlr7-deficient lupus-prone mice are significantly protected from autoimmunity, whereas disease is exacerbated in tlr9−/− autoimmune strains (1–3). The mechanisms underlying accelerated autoimmunity in the absence of TLR9 remain unclear. However, tlr9−/− MRL.Mplpr/lpr mice develop greater plasmacytoid dendritic cell activation and increased serum IFN-α levels, suggesting that loss of tlr9 in the myeloid compartment exacerbates autoimmunity on the MRL.Mplpr/lpr background (6).

Although several models (9–13) have implicated B cell Myd88 signaling in autoimmune pathogenesis, the B cell–intrinsic impact of TLR7 and TLR9 has been addressed in only a limited number of studies. The role of B cell–intrinsic TLR7 signaling was evaluated in two studies using the TLR7 transgenic (tlr7Tg) model of spontaneous autoimmunity. First, using a chimeric transplant strategy comparing wild-type (WT) with TLR7 transgenic (tlr7Tg) hematopoietic cells, Walsh et al. (14) demonstrated that tlr7Tg B cells are preferentially recruited into germinal centers (GCs) and generate CD138+ plasmablasts. Second, Hwang et al. (15) used a CD19Cre recombinase system to normalize B cell TLR7 expression in a low-copy tlr7Tg strain crossed with the Sle1 lupus susceptibility locus. B cell–intrinsic TLR7 normalization decreased RNA-associated anti–small nuclear ribonucleoprotein (RNP) titers, but did not have an impact on GC and plasma cell formation and only moderately reduced autoimmune glomerulonephritis. To address the B cell–intrinsic impact of TLR9, a recent study used MRL.Faslpr mixed bone marrow (BM) chimeras in which tlr9 deficiency was primarily limited to the B cell compartment and demonstrated a specific reduction in antinucleosome reactivity. Whether B cell–intrinsic tlr9 deletion accelerated systemic autoimmunity, however, was not addressed in that study (16).

We recently developed a murine model of autoimmunity that provides important information regarding how self-reactive B cells are initially activated and can drive generation of pathogenic Abs (12). In this model, B cells, but not other hematopoietic lineages, harbor a mutation that abolishes the expression of Wiskott–Aldrich syndrome (WAS) protein (WASp). In the absence of WASp, peripheral B cells are rendered mildly hyperresponsive to both BCR and TLR ligands. In this setting, WAS-null (was−/−) B cells drive the development of humoral autoimmunity that is characterized by spontaneous GCs, class-switched IgG2c autoantibodies, and IC glomerulonephritis. Disease development is dependent on WT CD4+ T cells and MyD88-dependent B cell–intrinsic TLR signaling. An important advantage of the WASp chimera model is that dysregulated immune responses are limited to the B cell compartment, allowing genetic manipulation in a B cell–intrinsic fashion. In the current study, we detail the relative contributions of B cell TLR signaling in humoral autoimmunity and demonstrate that B cell–intrinsic TLR7 versus TLR9 activation is sufficient to alter the autoantibody repertoire. In addition, we demonstrate that B cell TLR7 and TLR9 signals exert opposing pathogenic and protective effects on systemic inflammation and autoimmune disease.

Materials and Methods

Mice

Ly.5.1+ and Ly5.2+ C57BL/6, μMT, was−/−, tlr7−/−, and tlr9−/− mice were bred and maintained in the specific pathogen–free animal facility of Seattle Children’s Research Institute (Seattle, WA). All animal studies were conducted in accordance with Seattle Children’s Research Institute Institutional Animal Care and Use Committee–approved protocols.

BM transplantation

WT, was−/−, was−/−.tlr7−/−, or was−/−.tlr9−/− donor BM and B cell–deficient (μMT) BM (20:80 ratio, 6 × 106 total BM) were injected into lethally irradiated (450 cGy × 2 doses) μMT recipients. Chimeras were sacrificed at 24–36 wk post transplant. For CD4 depletion assays, mice were treated weekly with i.p. injection of 250 μg anti-CD4 (GK1.5) or isotype control (rat IgG2b) Ab (University of California, San Francisco, Monoclonal Antibody Core) from 5 to 24 wk post transplant, as described (12). Data are representative of four (BWT, BWAS−/−, and BW/TLR9−/−), three (BW/TLR7−/−), or two (BWAS−/− CD4 depletion) independent experimental cohorts.

Flow cytometry and Abs

Flow cytometry was performed as described (12). Abs used were as follows: B220 (RA3-6B2), CD4 (RM4-5), Thy1.2 (53-2.1), CD138 (281-2), CXCR5 (2G8) from BD Biosciences; CD62L (MEL-14), CD11c (N418), Gr-1 (RB6-8C5), Ly5.1 (A20), Ly5.2 (104), CD11b (M1/70), GL7 (GL-7), PD-1 (J43) from eBioscience; goat anti-mouse IgM-, IgG-, IgG2c-HRP conjugated, unlabeled, or isotype from Southern Biotechnology; CD19 (ID3), CD44 (IM7) from BioLegend; PNA (Fl-1071) from Vector Labs; and Fas (Jo2) from BD Pharmingen.

Measurement of autoantibodies

For ANA assays and determination of dsDNA reactivity by kinetoplast staining, diluted serum (1:200 for ANA or 1:50 for kinetoplast) was added to fixed Hep-2 ANA slides (Bio-Rad 30472) or Crithidia luciliae slides (Bio-Rad 31069). FITC-conjugated goat anti-mouse IgG served as the detection Ab, and slides were counterstained with DAPI. Fluorescence images were obtained using a Leica DM6000B microscope, Leica DFL300 FX camera, and Leica Application Suite Advanced Fluorescence software at ×40 with a constant 5 s (ANA) or 2 s (kinetoplast) exposure. ANAs were scored as nuclear homogeneous, nucleolar, cytoplasmic or a combination of these patterns. Kinetoplast reactivity was defined by colocalization of DAPI-positive C. luciliae DNA and IgG FITC staining, with staining intensity scored from 0 to 3. ANA patterns and kinetoplast intensity were scored by two independent observers blinded to genotype.

For specific Ab ELISAs, 96-well Immuno plates (Nunc) were coated with the following: calf thymus dsDNA (100 μg/ml; Sigma-Aldrich D3664-5×2MG); sm/RNP (5 μg/ml; Arotec Diagnostics ATR01-10); phosphoryl choline (PC)–BSA (10 μg/ml; Biosearch Technologies PC-1011-10); or malondialdehyde conjugated with low-density lipoprotein (MDA-LDL) (10 μg/ml; Academy Bio-medical 20P-MD-L110). Plates were blocked for 1 h with 1% BSA/PBS prior to addition of diluted serum for 2 h. Specific Abs were detected using goat anti-mouse IgM-, IgG- or IgG2c-HRP (1:2000 dilution; Southern Biotechnology Associates), and peroxidase reactions were developed using OptEIA TMB substrate (BD Biosciences). Absorbance at 450 nm was read using a Victor 3 plate reader (PerkinElmer), and data were analyzed using GraphPad Prism (GraphPad Software). Autoantigen microarrays were performed at the University of Texas Southwestern Medical Center Microarray Core Facility, Dallas, TX (17).

Spleen and kidney immunofluorescence staining

Mouse spleens and kidneys were embedded in OCT compound and snap frozen over liquid nitrogen. Then 10-μm sections were cut on a cryostat, mounted on Superfrost Plus slides, and fixed in −20°C acetone for 20 min. After rehydration in staining buffer (PBS, 1% goat serum, 1% BSA, 0.1% Tween 20), slides were stained with the following: B220-PE, CD4-FITC, and GL7-allophycocyanin (spleen); or, IgG-FITC, IgG2c-FITC, or C3-FITC (kidney). Images were acquired using a Leica DM6000B microscope, Leica DFL300 FX camera, and Leica Application Suite Advanced Fluorescence software. For glomerular IC quantification, images were obtained using a constant exposure and scored from 0 to 3 by two independent observers blinded to genotype.

Histopathology

Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin; tissue sections were stained with H&E (lung, liver, pancreas) or Jones’ methenamine silver–periodic acid–Schiff (kidney) according to standard practices. Immunohistochemistry staining was performed using a Leica Bond–automated immunostainer and HRP-conjugated secondary Abs. Histology images were acquired with a Nikon OptiPhot-2 microscope and a Canon Eos 5D Mark II camera. Tissue sections were examined by a board-certified veterinary pathologist (D.L.), who was blinded to study design. Kidney sections were analyzed by two observers blinded to genotype (K.L.H. and C.E.A.) and scored from 0 to 2 based on degree of mesangial expansion, glomerular basement membrane (GBM) thickening/reduplication, and glomerular hypercellularity. For quantification of MAC-2 area, glomeruli were manually delineated in more than four independent kidney sections and MAC-2+ area as a percentage of glomerular area determined using Image Pro Plus (Media Cybernetics, Rockville, MD).

Statistical evaluation

The p values were calculated using one-way ANOVA, followed by the Tukey multiple comparison test (GraphPad Software).

Results

B cell–intrinsic TLR7 and TLR9 signals alter the autoantibody repertoire

To test the impact of B cell–intrinsic TLR7 and TLR9 deletion in humoral autoimmunity, we generated mixed BM chimeras by transplanting a mix of 20% WT, WAS-null (was−/−), double-deficient was−/−.tlr7−/−, or was−/−.tlr9−/− BM with 80% B cell–deficient μMT BM into lethally irradiated (450 cGy × 2 doses) μMT recipients. After reconstitution, all B cells were donor derived (WT, was−/−, was−/−.tlr7−/−, or was−/−.tlr9−/−), whereas ∼80% of myeloid cells and >97% CD4+ T cells were WT (not shown). With this strategy, in the TLR-deficient cohorts, all B cells, but only ∼20% of myeloid cells, lacked TLR7 or TLR9. Respective B cell chimeras will henceforth be referred to as BWT, BWAS−/−, BW/TLR7−/−, and BW/TLR9−/−.

We initially screened for autoimmunity using fluorescent ANA assays. The majority of BWAS−/− were ANA positive with mixed homogeneous and nucleolar staining patterns, consistent with both DNA and RNA Ab reactivity. In contrast, BW/TLR7−/− ANA staining was homogeneous with no nucleolar reactivity, whereas BW/TLR9−/− showed nucleolar and cytoplasmic, but not homogeneous, ANA reactivity (Fig. 1A). These altered ANA staining patterns suggested a B cell–intrinsic impact of TLR7 versus TLR9 on RNA- and DNA-associated Abs, which we evaluated using specific autoantigen ELISAs. Prior reports have demonstrated persistent anti-dsDNA Ab in tlr9-deficient murine lupus models, an effect attributed to a lack of DNA specificity of standard anti-dsDNA ELISA assays (6, 18). Although we noted anti-dsDNA Ab by ELISA in BW/TLR9−/−, DNA reactivity by highly specific Crithidia luciliae kinetoplast staining was abrogated in BW/TLR9−/−, but unaffected in BW/TLR7−/− (Fig. 1B, Supplemental Fig. 1A). In contrast, RNA-associated anti-sm/RNP Abs were abolished in BW/TLR7−/−. Further, anti-sm/RNP Ab titers of the pathogenic IgG2c subclass were significantly increased in BW/TLR9−/− versus BWAS−/− chimeras, an observation consistent with elevated anti-RNA titers in tlr9−/− MRL.Mplpr/lpr mice (Fig. 1C). Therefore, the requirement for TLR7 in anti-RNA and TLR9 in anti-DNA Ab production is B cell intrinsic; and lack of TLR9 enhances anti-RNA Ab formation in a B cell–intrinsic manner.

FIGURE 1.
FIGURE 1.

B cell–intrinsic TLR7 and TLR9 signals promote ANA production. (A) Upper panel, ANA staining patterns were scored as homogeneous, nucleolar, or cytoplasmic by two independent, blinded observers. Unfilled portion of circle represents ANA-negative animals. The number in the circle represents the total number of mice analyzed. Lower panel, Representative Hep-2 ANA immunofluorescence staining showing combined homogeneous/nucleolar staining in BWAS−/−, homogeneous pattern with nucleolar sparing in BW/TLR7−/−, and nucleolar and cytoplasmic staining in BW/TLR9−/−. Scale bars, 50 μm. (B) Anti-dsDNA IgG Ab by ELISA and kinetoplast staining. (C) Anti-sm/RNP IgG and IgG2c Ab by ELISA. (B and C) Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (A–C) Total mice analyzed: BWT (n = 6), BWAS−/− (n = 18), BW/TLR7−/− (n = 11), and BW/TLR9−/− (n = 16), pooled from four independent experimental cohorts.

Generation of Abs targeting nonnuclear Ags was recently shown to be Myd88 dependent (11, 19), but the specific TLRs responsible for these Ab specificities have not been determined. For this reason, we measured IgG reactivity to apoptotic cell epitopes MDA-LDL and PC. In contrast to abrogation of anti-RNA responses, antiapoptotic IgG responses were decreased in BW/TLR7−/−, but were not absent. Rather, we noted a specific decrease in pathogenic IgG2c subclass titers. Because we previously observed a lack of anti-dsDNA IgG2c in the absence of cognate T cell help (12), we measured anti-PC and anti–MDA-LDL IgG2c subclass titers in BWAS−/− treated weekly with monoclonal CD4-depleting Ab. In the absence of CD4+ T cells, BWAS−/− chimeras developed markedly reduced IgG2c Ab targeting PC and MDA-LDL, findings that mirrored BW/TLR7−/− (Fig. 2A, 2B). Therefore, in addition to the B cell–intrinsic requirement for TLR7 in generating Abs targeting RNA and RNA-associated Ags (Fig. 1C, Ref. 15), our data demonstrate that B cell–intrinsic TLR7 signals are required for generation of class-switched Abs targeting nonnuclear, apoptotic Ags.

FIGURE 2.
FIGURE 2.

B cell–intrinsic TLR7 and TLR9 signals affect autoantibody reactivity to apoptotic and disease-associated Ags. (A) Left panel, Anti–MDA-LDL IgG. Right panel, Anti–MDA-LDL IgG2c in BWAS−/−, BW/TLR7−/−, BW/TLR9−/−, and CD4-depleted BWAS−/− chimeras (BWAS−/− CD4 DEP). Data normalized to BWT anti–MDA-LDL IgG2c titer. (B) Left panel, Anti-PC IgG. Right panel, Anti-PC IgG2c in BWAS−/−, BW/TLR7−/−, BW/TLR9−/−, and CD4-depleted BWAS−/− chimeras (BWAS−/− CD4 DEP). Data normalized to BWT anti-PC IgG2c titer. (A and B) Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Total mice analyzed: BWT (n = 7), BWAS−/− (n = 13), BW/TLR7−/− (n = 13), BW/TLR9−/− (n = 14), and BWAS−/− CD4 DEP (n = 10). (C) Serum IgG Abs from BWT (n = 4), BWAS−/− (n = 6), BW/TLR7−/− (n = 4), and BW/TLR9−/− (n = 8) chimeras determined using an autoantibody array chip containing 88 specific autoantigens. Data are represented as a heat map of Z-scores ranging from −1 (blue) to 3 (red). Representative of two independent microarray analyses.

Finally, to quantify the impact of B cell tlr7 versus tlr9 deficiency on the diversity of the autoantibody repertoire of BWAS−/− chimeras, sera were evaluated for reactivity to 88 distinct autoantigens by microarray. Although BW/TLR7−/− demonstrated a broad reduction in serum Ab titers, the reactivity of BW/TLR9−/− sera was increased relative to BWAS−/− to a wide range of different autoantigens (Fig. 2C). Consistent with our ELISA data, these Abs were predominantly of the pathogenic IgG2c subclass (Supplemental Fig. 1B). Although absolutely required for the generation of anti-dsDNA Abs, TLR9 thus acts B cell intrinsically to limit Ab responses to RNA-associated autoantigens, apoptotic cell epitopes, and a broad range of disease-associated autoantigens.

B cell–intrinsic TLR7 and TLR9 exert opposing signals on immune activation

We previously demonstrated that BWAS−/− chimeras develop systemic inflammation that is dependent on B cell Myd88 signaling (12). B cell TLR7 and TLR9 signaling exerted opposing impacts on immune activation in this model, as splenic inflammation is exacerbated in BW/TLR9−/−, but abrogated in BW/TLR7−/−. Relative to BWAS−/−, BW/TLR9−/− chimeras developed increased splenomegaly, without marked alterations in total B cell numbers (Fig. 3A, 3B). Notably, splenomegaly in BWAS−/− and BW/TLR9−/− resulted from a significant expansion of CD4+ T cells, CD11b+GR1LO monocyte/macrophages, and CD11b+GR1+ neutrophils. Consistent with a role for TLR9 in limiting autoimmune responses, B cell–intrinsic TLR9 deletion resulted in statistically greater numbers of CD4+ T cells and monocyte/macrophages relative to BWAS−/−, with a trend toward more neutrophils (Fig. 3C, 3D).

FIGURE 3.
FIGURE 3.

B cell–intrinsic TLR7 and TLR9 exert opposing signals on immune activation. Splenic monocyte subsets were analyzed in BWT, BWAS−/−, BW/TLR7−/−, and BW/TLR9−/− chimeras at sacrifice. (A) Spleen weight and total splenocyte numbers. (B–D) Number of splenic (B) CD19+ B cells, (C) CD4+ T cells, and (D) CD11b+GR1LO monocyte/macrophages and CD11b+GR1+ neutrophils. (A–D) Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Total mice analyzed: BWT (n = 5), BWAS−/− (n = 12), BW/TLR7−/− (n = 12), and BW/TLR9−/− (n = 13), pooled from four independent experimental cohorts.

Although total B cell numbers were similar, activated B cell subsets, including PNA+FAS+GL7+ GC B cells and B220LOCD138+ plasma cells/plasmablasts, were markedly expanded in spleens of BWAS−/− and BW/TLR9−/− chimeras (Fig. 4A, 4C). In addition, splenic immunofluorescence staining revealed spontaneous GL7+ GCs in BWAS−/− and BW/TLR9−/− mice, which were absent in BW/TLR7−/− (Fig. 4D). Relative to BWAS−/−, BW/TLR9−/− animals exhibited a higher percentage of GC B cells with greater numbers of splenic CD138+ plasma cells/plasmablasts, suggesting that B cell–intrinsic TLR9 signals serve to limit B cell activation. Consistent with this, total serum IgM, IgG, and IgG2c Ab titers were markedly elevated in BW/TLR9−/− mice relative to BW/TLR7−/− mice, with a trend toward greater Ab titers than BWAS−/− chimeras (Fig 4E).

FIGURE 4.
FIGURE 4.

Divergent impact of B cell–intrinsic TLR7 and TLR9 on B cell activation. (A and B) Representative FACS plots (left), total number (middle), and percentage (right) of (A) PNA+FAS+GL7+GC B cells (gated on CD19+ B cells) and (B) CD11b+ CD11c+ ABC cells (gated on CD19+ B cells). (C) Representative FACS plots (left) and total number (right) of B220LOCD138+ plasmablasts/plasma cells (gated on total splenocytes). (A–C) Number in FACS plot represents percentage within gated population. (D) Representative examples of splenic sections stained with B220 (red), CD4 (green), and GL7 (blue). Stars denote spontaneous GCs in BWAS−/− and BW/TLR9−/− chimeras. Images were taken with ×10 objective. Scale bars, 100 μm. (E) Total IgM, IgG, and IgG2c serum titers in BWT, BWAS−/−, BW/TLR7−/−, and BW/TLR9−/− chimeras. (A–C, E) Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Total mice analyzed: BWT (n = 5), BWAS−/− (n = 12), BW/TLR7−/− (n = 12), and BW/TLR9−/− (n = 13), pooled from four independent experimental cohorts.

BWAS−/− and BW/TLR9−/− also developed significant expansion of autoimmune disease–associated CD11b+CD11c+ “age-associated B cells” (ABCs), with ≤75% of total B cells expressing CD11b and CD11c in BW/TLR9−/−, exceeding the percentage of ABCs previously reported in aged NZB/WF1 and Mer−/− autoimmune strains (20, 21). In keeping with the importance of chronic TLR7 activation in ABC generation, this population was absent in BW/TLR7−/− (Fig. 4B).

In addition to B cell activation, CD4+ effector/memory (EM) subsets (CD44HICD62LLO and CD44HICD62LHI) were increased in BWAS−/− and BW/TLR9−/− (with relatively greater T cell activation in BW/TLR9−/−), whereas the number of CD44LOCD62LHI naive CD4 T cells did not differ between experimental groups (Fig. 5A). Consistent with greater GC B cell numbers, PD1+CXCR5+ Th follicular cells (TFH) were also expanded in BWAS−/− and BW/TLR9−/− (Fig. 5B). B cell–intrinsic TLR7 activation appears critical for T cell activation in this model, as CD4+ EM and TFH cell subset expansion is abrogated in BW/TLR7−/−.

FIGURE 5.
FIGURE 5.

B cell–intrinsic TLR7 and TLR9 exert opposing impacts on T cell activation. (A and B) Representative FACS plots (left) and total number (right) of splenic (A) naive (CD44LOCD62LHI) and EM (CD44HICD62LLO and CD44HICD62LHI) CD4+ T cells and (B) PD1+CXCR5+ TFH cells. Number represents percentage within gated population (gated on splenic CD4+ T cells). Mean ± SEM. ***p < 0.001. Total mice analyzed: BWT (n = 5), BWAS−/− (n = 12), BW/TLR7−/− (n = 12), and BW/TLR9−/− (n = 13), pooled from four independent experimental cohorts.

In conclusion, B cell–intrinsic activation of autoreactive was−/− B cells via TLR7 promotes global immune activation and expansion of activated B and T cell populations; whereas B cell TLR9 signaling serves to limit these processes.

B cell–intrinsic TLR7 signaling promotes systemic autoimmunity

Aged BWAS−/− and BW/TLR9−/− chimeras demonstrated diffuse inflammation and lymphoid infiltrates involving multiple organs—in particular, the lungs and liver—findings that were absent in all BWT and BW/TLR7−/− animals evaluated (Fig. 6A). Immunohistochemistry staining demonstrated that these lymphoid infiltrates were consistent with ectopic lymphoid follicles comprising separate B220+ B cell and CD3+ T cell zones (Fig. 6B). The presence of ectopic lymphoid tissues within target organs has been reported in several human autoimmune diseases, including rheumatoid arthritis and type 1 diabetes, and is correlated with disease severity in autoimmunity (22). We demonstrate that hyperresponsive was−/− B cells are sufficient to recruit WT T cells to ectopic lymphoid follicles in a TLR7-dependent manner, reinforcing the critical role of B cell–intrinsic TLR7 signals in the pathogenesis of systemic autoimmunity.

FIGURE 6.
FIGURE 6.

B cell–intrinsic TLR7 signals promote systemic inflammation and ectopic lymphoid follicles. (A) H&E-stained tissue sections showing prominent lymphoid aggregates surrounding pulmonary vasculature in the lungs (upper panels, ×4) and portal tracts of the liver (lower panels, ×10) in BWAS−/− and BW/TLR9−/− chimeras. Scale bars, 100 μm. (B) Immunohistochemistry of serial lung sections from a representative BW/TLR9−/− mouse showing distinct B220+ B cell follicle and CD3+ T cell zone (*). Image taken with ×10 objective. Scale bars, 100 μm. (A and B) Data are representative of BWT (n = 4), BWAS−/− (n = 10), BW/TLR7−/− (n = 9), and BW/TLR9−/− (n = 11) mice, analyzed from two independent experimental cohorts.

B cell TLR7 deletion eliminates, whereas TLR9 loss exacerbates, IC glomerulonephritis

Given divergent impacts on Ab production and systemic inflammation, we evaluated the role of B cell–intrinsic TLR signaling in autoimmune glomerulonephritis. BWAS−/− chimeras develop inflammatory glomerulonephritis characterized by deposition of class-switched Ab, activated complement, and recruitment of MAC-2+ macrophages (12). In keeping with an overall decrease in Ab titer and systemic inflammation, BW/TLR7−/− chimeras did not develop glomerulonephritis (Fig. 7A), an observation consistent with decreased glomerulonephritis in tlr7−/− MRL/Mplpr/lpr mice (6). Despite absent histologic glomerulonephritis, evaluation of glomerular Ig deposition by immunofluorescence staining showed persistent glomerular IgG in BW/TLR7−/− chimeras (albeit at lower levels than in BWAS−/− and BW/TLR9−/−) (Fig. 7B). However, deposition of the pathogenic IgG2c subclass was absent in BW/TLR7−/− (Fig. 7C). In keeping with the role for IgG2c in complement and Fc receptor activation (23), C3 complement deposition was absent in BW/TLR7−/− and glomerular MAC-2+ macrophages were not increased over BWT controls (Fig. 7D, 7E).

FIGURE 7.
FIGURE 7.

B cell–intrinsic TLR7 and TLR9 exert opposing effects on IC glomerulonephritis. (A) Left panels, Representative examples of glomeruli from BWAS−/−, BW/TLR7−/−, and BW/TLR9−/− chimeras stained with Jones’ methenamine silver–periodic acid–Schiff stain. Right panel, Glomerular inflammation scored as follows: (0+) minimal mesangial expansion consistent with radiation injury; (1+) focal glomerular changes with moderate mesangial expansion, GBM thickening/reduplication, and glomerular hypercellularity; or (2+) diffuse glomerular changes with severe mesangial expansion, GBM thickening/reduplication, and glomerular hypercellularity. Pathologic change was scored by two observers blinded to genotype. (B–D) Glomerular IC deposits were determined by immunofluorescence staining for (B) IgG, (C) IgG2c, and (D) complement C3. Representative images are shown (left), together with intensity of glomerular fluorescent staining (right) scored from 0 to 3+ by two independent, blinded observers. (E) Left panels, Representative images of immunohistochemistry staining for glomerular MAC-2+ macrophages. Right panel, MAC-2+ area as a percentage of total glomerular area determined using Image Pro Plus (Media Cybernetics). More than four independent kidney sections were examined per mouse. Scale bars, 50 μm. (A–E) Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Total mice analyzed (A–D, E): BWT (n = 6, 4), BWAS−/− (n = 14, 6), BW/TLR7−/− (n = 11, 9), and BW/TLR9−/− (n = 15, 8), from three (A–D) and two (E) independent experimental cohorts.

In contrast to the protective effect of B cell TLR7 deletion, BWAS−/− and BW/TLR9−/− chimeras developed significant glomerulonephritis characterized by mesangial expansion, GBM thickening, and glomerular hypercellularity (Fig. 7A). In keeping with increased serum IgG2c Ab, BW/TLR9−/− mice demonstrated statistically greater glomerular IgG2c deposition than did BWAS−/−, with a trend toward greater C3 complement deposition and abundant infiltration by MAC-2+ macrophages, likely recruited by glomerular IgG2c ICs binding activating Fc receptors (Fig. 7B–E). Increased IC glomerulonephritis in BW/TLR9−/− parallels accelerated autoimmune disease in tlr9−/− MRL/Mplpr/lpr mice, but demonstrates that the disease-exacerbating effects of TLR9 deletion can occur independently of the lack of TLR9 expression in the majority of the myeloid compartment.

Discussion

The importance of the Myd88-dependent TLRs TLR7 and TLR9 in lupus pathogenesis has been clearly established (1–3). In this context, global deletion of TLR7 in murine lupus models protects against autoimmunity, whereas TLR9 deficiency paradoxically exacerbates disease (4–7). The mechanisms underlying these divergent effects of TLR7 and TLR9 remain unclear, in part because studies involving globally gene-deficient autoimmune strains are unable to address the differential impacts of TLR signaling on B cells versus type 1 IFN–producing plasmacytoid dendritic cells. Our study provides the novel observation that B cell–intrinsic deletion of TLR7 or TLR9 recapitulates several of the key features of global TLR7/TLR9 deficiency in murine lupus models, despite intact TLR signaling within the myeloid compartment.

In this context, our data provide several important new insights into autoimmune pathogenesis. First, this study is, to our knowledge, the first to compare the B cell–intrinsic impact of TLR7 versus TLR9 in parallel models of murine autoimmunity. We demonstrate that, similar to global TLR7/TLR9 deletion in murine lupus models, B cell–intrinsic TLR7 and TLR9 signals are required for in vivo generation of RNA- and DNA-reactive Ab, respectively. In addition, we also establish TLR7 as the major Myd88-dependent receptor responsible for B cell–intrinsic breaks in tolerance to a broad range of nonnuclear, tissue-specific autoantigens.

Second, we identify a critical role for B cell–intrinsic TLR7 signals in sustaining spontaneous autoimmune GC responses. Although the absence of anti-sm/RNP Abs in BW/TLR7−/− may have been predicted from in vitro studies (24), the observation that Abs targeting apoptotic and phospholipid epitopes develop in BW/TLR7−/−, but fail to undergo efficient class switch recombination, suggests an additional role for B cell TLR7 signaling in enhancing GC responses during autoimmune disease—a finding that parallels the requirement for TLR7 in Ab responses to chronic viral infection (25).

Third, we document a striking expansion of effector and TFH CD4+ T cells in BWAS−/− and BW/TLR9−/− animals. Similar expansion of the EM T cell compartment has been documented on the lupus-prone MRL/Mplpr/lpr background (6), although it should be noted that CD4+ T cells in our model are genetically WT and not Fas deficient. Our data, therefore, suggest that BCR/TLR-mediated activation of autoreactive B cells directly promotes T cell proliferation and activation during autoimmunity. These data are consistent with recent reports describing decreased EM T cell expansion in the setting of B cell–intrinsic Myd88 deletion in the MRL.Faslpr and Lyn-deficient models of autoimmunity (11, 13). Our data suggest that B cell TLR7 is likely the major Myd88-dependent receptor driving this CD4+ T cell activation. Surprisingly, dual BCR/TLR7 B cell activation was also sufficient for the establishment of ectopic lymphoid follicles within target organs, as have previously been described in several human autoimmune diseases (22). We predict that, although not formally demonstrated, these expanded EM T cell compartments are enriched for autoreactive T cell clones, supporting the emerging hypothesis that activated autoreactive B cells can initiate breaks in T cell tolerance.

Fourth, we demonstrate the importance of autoantibody isotype (in particular, IgG2a/c) in promoting autoimmune nephritis. Despite producing anti-dsDNA Ab, BW/TLR7−/− fail to develop IC glomerulonephritis. These renal protective effects of TLR7 deletion, despite persistent DNA-reactive Ab, are surprising given clinical data implicating anti-dsDNA Abs in lupus nephritis (26). In keeping with this paradox, we observed glomerular IgG deposition by immunofluorescence in BW/TLR7−/−, in the absence of histologic glomerulonephritis. However, deposition of the pathogenic Ig subclass IgG2c was abrogated in the absence of B cell TLR7. In keeping with the role for IgG2c in promoting complement activation and activation of myeloid Fc receptors (23), glomerular C3 deposition and recruitment of inflammatory macrophages were absent in BW/TLR7−/−. These data emphasize the need to consider IgG subclass in addition to Ab specificity in mediating end-organ damage in SLE.

Finally, we show that B cell–intrinsic TLR9 deletion enhances class-switched Abs targeting RNA-associated Ags and broadens the autoantibody repertoire; exacerbates systemic inflammation and expansion of activated CD4+ T cell and myeloid populations; and promotes glomerular deposition of pathogenic IgG2c IC. Although protective roles for TLR9 in autoimmunity have been described in several murine autoimmune models, how TLR9 deletion promotes autoimmunity has not yet been determined. Potential underlying mechanisms include the following: enhanced interaction of TLR7 with the shared endoplasmic reticulum trafficking protein Unc93b1 in the setting of TLR9 deletion (27); facilitation of entry of autoreactive cells into the mature B cell repertoire (16); or decreased generation of TLR9-dependent regulatory B cells (2). Our study makes the important observation that B cell–intrinsic TLR9 deletion can accelerate autoimmunity, despite largely intact myeloid TLR signaling.

Importantly, however, our data do not exclude the possibility that myeloid TLR7 and TLR9 signals may exert additional effects on lupus pathogenesis. Two recent studies, using a cre-recombinase strategy to delete the TLR adaptor Myd88 in CD11c+ dendritic cells, demonstrated that myeloid TLR activation accelerates autoimmunity, likely via enhanced type 1 IFN production by IC-mediated activation of plasmacytoid dendritic cells (11, 13). Given this observation, it will be important to evaluate whether deletion of myeloid TLR7 versus TLR9 exerts similar divergent effects on autoimmune pathogenesis. In this regard, a potential caveat of our study is that reconstitution with an 80:20 ratio of μMT/donor (was−/−, was−/−.tlr7−/−, or was−/−.tlr9−/−) BM, will result in ∼20% myeloid cells also lacking TLR7 or TLR9. However, because the majority of myeloid cells (∼80%) in our model are TLR sufficient, it is highly likely that our data reflect the B cell–intrinsic effects of TLR7 versus TLR9 in autoimmune pathogenesis.

In conclusion, our study provides critical new insights into how dual BCR and TLR signals promote B cell activation resulting in class-switched Ab production, CD4+ T cell activation, and the development of IC glomerulonephritis. B cell TLR7 and TLR9 exert opposing effects in this process, reinforcing the importance of dysregulated B cell TLR signaling in autoimmunity and informing efforts to therapeutically target TLR signals in SLE and other disorders characterized by autoantibody production.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by the National Institutes of Health under award numbers R01HL075453 (to D.J.R.), R01AI084457 (to D.J.R.), R01AI071163 (to D.J.R.), 5T32AR007108 (to S.W.J.), K12HD043376 (to S.W.J.); by Cancer Research Institute Pre-doctoral Training Grants (to N.S.K. and M.A.S.); by the Rheumatology Research Foundation Scientist Development Award (to S.W.J.); and by the Arnold Lee Smith Endowed Professorship for Research Faculty Development (to S.W.J.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ABC
    age-associated B cell
    ANA
    antinuclear Ab
    BM
    bone marrow
    EM
    effector/memory (cell)
    GBM
    glomerular basement membrane
    GC
    germinal center
    IC
    immune complex
    MDA-LDL
    malondialdehyde conjugated with low-density lipoprotein
    μMT
    B cell–deficient
    PC
    phosphoryl choline
    RNP
    ribonucleoprotein
    SLE
    systemic lupus erythematosus
    TFH
    Th follicular cell
    WAS
    Wiskott–Aldrich syndrome
    WASp
    WAS protein
    WT
    wild-type.

  • Received January 27, 2014.
  • Accepted March 6, 2014.

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