Repertoire and Systemic Inflammation and TLR9 Signals on Autoantibody

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

This information is current as Shaun W. Jackson, Nicole E. Scharping, Nikita S. Kolhatkar, of September 28, 2021. Socheath Khim, Marc A. Schwartz, Quan-Zhen Li, Kelly L. Hudkins, Charles E. Alpers, Denny Liggitt and David J. Rawlings J Immunol 2014; 192:4525-4532; Prepublished online 7

April 2014; Downloaded from doi: 10.4049/jimmunol.1400098 http://www.jimmunol.org/content/192/10/4525

Supplementary http://www.jimmunol.org/content/suppl/2014/04/05/jimmunol.140009 http://www.jimmunol.org/ Material 8.DCSupplemental References This article cites 27 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/192/10/4525.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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,x Kelly L. Hudkins,{ Charles E. Alpers,{ Denny Liggitt,‖ and David J. Rawlings*,†,‡

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 Downloaded from 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. The Journal of Immunology, 2014, 192: 4525–4532. http://www.jimmunol.org/

espite numerous potential autoantigen targets, patients activation promotes production of Abs targeting dsDNA and with systemic lupus erythematosus (SLE) frequently chromatin (4–7). D develop a restricted autoantibody repertoire targeting nu- Importantly, two alternate, but not mutually exclusive, mecha- cleic acid–associated Ags. In addition to exogenous pathogens, the nisms may explain the role of TLR7 and TLR9 in autoimmune TLR family of germline-encoded, pattern-recognition receptors pathogenesis in vivo. Either dual BCR/TLR activation may pro- are able to recognize endogenous ligands. Nucleic acid–containing mote a B cell–intrinsic break in tolerance, or immune complex apoptotic particles promote activation of autoreactive B cells via (IC)–mediated TLR activation of plasmacytoid dendritic cells may dual BCR/TLR-mediated signals, thereby explaining the promi- promote autoimmunity via increased type 1 IFN production (4, 8). by guest on September 28, 2021 nence of antinuclear Abs (ANAs) in autoimmunity (1–3). The In addition, tlr7-deficient lupus-prone mice are significantly pro- Myd88-dependent, endosomal receptors TLR7 and TLR9 are tected from autoimmunity, whereas disease is exacerbated in critical in this context, with TLR7 required for the generation of tlr92/2 autoimmune strains (1–3). The mechanisms underlying Abs targeting RNA and RNA-associated proteins, whereas TLR9 accelerated autoimmunity in the absence of TLR9 remain unclear. However, tlr92/2 MRL.Mplpr/lpr mice develop greater plasmacytoid dendritic cell activation and increased serum IFN-a levels, suggesting that loss of tlr9 in the myeloid compartment exacer- *Department of Pediatrics, University of Washington School of Medicine, Seattle, bates autoimmunity on the MRL.Mplpr/lpr background (6). † ‡ WA 98195; Seattle Children’s Research Institute, Seattle, WA 98101; Department Although several models (9–13) have implicated B cell Myd88 of Immunology, University of Washington School of Medicine, Seattle, WA 98195; xDepartment of Immunology, University of Texas Southwestern Medical Center, signaling in autoimmune pathogenesis, the B cell–intrinsic impact { Dallas, TX 75390; Department of Pathology, University of Washington School of of TLR7 and TLR9 has been addressed in only a limited number Medicine, Seattle, WA 98195; and ‖Department of Comparative Medicine, University of Washington School of Medicine, Seattle, WA 98195 of studies. The role of B cell–intrinsic TLR7 signaling was evaluated in two studies using the TLR7 transgenic (tlr7Tg) model of Received for publication January 27, 2014. Accepted for publication March 6, 2014. spontaneous autoimmunity. First, using a chimeric transplant 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.), strategy comparing wild-type (WT) with TLR7 transgenic (tlr7Tg) 5T32AR007108 (to S.W.J.), K12HD043376 (to S.W.J.); by Cancer Research Institute hematopoietic cells, Walsh et al. (14) demonstrated that tlr7Tg Pre-doctoral Training Grants (to N.S.K. and M.A.S.); by the Rheumatology Research B cells are preferentially recruited into germinal centers (GCs) Foundation Scientist Development Award (to S.W.J.); and by the Arnold Lee Smith + Endowed Professorship for Research Faculty Development (to S.W.J.). and generate CD138 plasmablasts. Second, Hwang et al. (15) Cre Address correspondence and reprint requests to Dr. David J. Rawlings, Seattle used a CD19 recombinase system to normalize B cell TLR7 Children’s Research Institute, 1900 Ninth Avenue, Seattle, WA 98101. E-mail expression in a low-copy tlr7Tg strain crossed with the Sle1 lupus address: [email protected] susceptibility locus. B cell–intrinsic TLR7 normalization de- The online version of this article contains supplemental material. creased RNA-associated anti–small nuclear ribonucleoprotein Abbreviations used in this article: ABC, age-associated B cell; ANA, antinuclear Ab; (RNP) titers, but did not have an impact on GC and plasma cell BM, bone marrow; EM, effector/memory (cell); GBM, glomerular basement membrane; GC, germinal center; IC, immune complex; MDA-LDL, malondialdehyde formation and only moderately reduced autoimmune glomerulo- conjugated with low-density lipoprotein; mMT, B cell–deficient; PC, phosphoryl nephritis. To address the B cell–intrinsic impact of TLR9, a recent lpr choline; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; TFH, Th fol- study used MRL.Fas mixed bone marrow (BM) chimeras in licular cell; WAS, Wiskott–Aldrich syndrome; WASp, WAS protein; WT, wild-type. which tlr9 deficiency was primarily limited to the B cell com- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 partment and demonstrated a specific reduction in antinucleosome www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400098 4526 B CELL–INTRINSIC TLR7 AND TLR9 SIGNALS IN LUPUS reactivity. Whether B cell–intrinsic tlr9 deletion accelerated sys- For specific Ab ELISAs, 96-well Immuno plates (Nunc) were coated with temic autoimmunity, however, was not addressed in that study (16). the following: calf thymus dsDNA (100 mg/ml; Sigma-Aldrich D3664- 3 m We recently developed a murine model of autoimmunity that 5 2MG); sm/RNP (5 g/ml; Arotec Diagnostics ATR01-10); phosphoryl choline (PC)–BSA (10 mg/ml; Biosearch Technologies PC-1011-10); or provides important information regarding how self-reactive B cells malondialdehyde conjugated with low-density lipoprotein (MDA-LDL) are initially activated and can drive generation of pathogenic Abs (10 mg/ml; Academy Bio-medical 20P-MD-L110). Plates were blocked (12). In this model, B cells, but not other hematopoietic lineages, for 1 h with 1% BSA/PBS prior to addition of diluted serum for 2 h. harbor a mutation that abolishes the expression of Wiskott– Specific Abs were detected using goat anti-mouse IgM-, IgG- or IgG2c-HRP (1:2000 dilution; Southern Biotechnology Associates), and Aldrich syndrome (WAS) protein (WASp). In the absence of peroxidase reactions were developed using OptEIA TMB substrate (BD WASp, peripheral B cells are rendered mildly hyperresponsive to Biosciences). Absorbance at 450 nm was read using a Victor 3 plate 2 2 both BCR and TLR ligands. In this setting, WAS-null (was / ) reader (PerkinElmer), and data were analyzed using GraphPad Prism B cells drive the development of humoral autoimmunity that is (GraphPad Software). Autoantigen microarrays were performed at the 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 Downloaded from 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. http://www.jimmunol.org/ Materials and Methods Mice Ly.5.1+ and Ly5.2+ C57BL/6, mMT, was2/2, tlr72/2, and tlr92/2 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 by guest on September 28, 2021 WT, was2/2, was2/2.tlr72/2,orwas2/2.tlr92/2 donor BM and B cell– deﬁcient (mMT) BM (20:80 ratio, 6 3 106 total BM) were injected into lethally irradiated (450 cGy 3 2 doses) mMT recipients. Chimeras were sacriﬁced at 24–36 wk post transplant. For CD4 depletion assays, mice were treated weekly with i.p. injection of 250 mg 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,BWAS2/2, and BW/TLR92/2), three (BW/TLR72/2), or two (BWAS2/2 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 FIGURE 1. B cell–intrinsic TLR7 and TLR9 signals promote ANA For ANA assays and determination of dsDNA reactivity by kinetoplast production. (A) Upper panel, ANA staining patterns were scored as ho- staining, diluted serum (1:200 for ANA or 1:50 for kinetoplast) was added to mogeneous, nucleolar, or cytoplasmic by two independent, blinded ob- fixed Hep-2 ANA slides (Bio-Rad 30472) or Crithidia luciliae slides (Bio- servers. Unfilled portion of circle represents ANA-negative animals. The Rad 31069). FITC-conjugated goat anti-mouse IgG served as the detection number in the circle represents the total number of mice analyzed. Lower Ab, and slides were counterstained with DAPI. Fluorescence images were panel, Representative Hep-2 ANA immunofluorescence staining showing obtained using a Leica DM6000B microscope, Leica DFL300 FX camera, combined homogeneous/nucleolar staining in BWAS2/2, homogeneous 3 and Leica Application Suite Advanced Fluorescence software at 40 with pattern with nucleolar sparing in BW/TLR72/2, and nucleolar and cytoplasmic a constant 5 s (ANA) or 2 s (kinetoplast) exposure. ANAs were scored as 2 2 staining in BW/TLR9 / .Scalebars,50mm. (B) Anti-dsDNA IgG Ab by ELISA nuclear homogeneous, nucleolar, cytoplasmic or a combination of these C patterns. Kinetoplast reactivity was defined by colocalization of DAPI- and kinetoplast staining. ( ) Anti-sm/RNP IgG and IgG2c Ab by ELISA. C. luciliae (B and C) Mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. (A–C) Total positive DNA and IgG FITC staining, with staining intensity 2 2 2 2 scored from 0 to 3. ANA patterns and kinetoplast intensity were scored by mice analyzed: BWT (n = 6), BWA S / (n =18),BW/TLR7 / (n =11),and two independent observers blinded to genotype. BW/TLR92/2 (n = 16), pooled from four independent experimental cohorts. The Journal of Immunology 4527

University of Texas Southwestern Medical Center Microarray Core Statistical evaluation Facility, Dallas, TX (17). The p values were calculated using one-way ANOVA, followed by the Spleen and kidney immunofluorescence staining Tukey multiple comparison test (GraphPad Software). Mouse spleens and kidneys were embedded in OCT compound and snap Results m frozen over liquid nitrogen. Then 10- m sections were cut on a cryostat, B cell–intrinsic TLR7 and TLR9 signals alter the autoantibody mounted on Superfrost Plus slides, and fixed in 220˚C acetone for 20 min. After rehydration in staining buffer (PBS, 1% goat serum, 1% BSA, 0.1% repertoire Tween 20), slides were stained with the following: B220-PE, CD4-FITC, To test the impact of B cell–intrinsic TLR7 and TLR9 deletion and GL7-allophycocyanin (spleen); or, IgG-FITC, IgG2c-FITC, or C3- in humoral autoimmunity, we generated mixed BM chimeras by FITC (kidney). Images were acquired using a Leica DM6000B micro- transplanting a mix of 20% WT, WAS-null (was2/2), double- scope, Leica DFL300 FX camera, and Leica Application Suite Advanced 2 2 2 2 2 2 2 2 Fluorescence software. For glomerular IC quantification, images were deficient was / .tlr7 / ,orwas / .tlr9 / BM with 80% obtained using a constant exposure and scored from 0 to 3 by two inde- B cell–deficient mMT BM into lethally irradiated (450 cGy 3 2 pendent observers blinded to genotype. doses) mMT recipients. After reconstitution, all B cells were donor 2/2 2/2 2/2 2/2 2/2 Histopathology derived (WT, was ,was .tlr7 ,orwas .tlr9 ), whereas ∼80% of myeloid cells and .97% CD4+ T cells were Tissues were fixed in 10% neutral buffered formalin and embedded in WT (not shown). With this strategy, in the TLR-deficient cohorts, paraffin; tissue sections were stained with H&E (lung, liver, pancreas) or all B cells, but only ∼20% of myeloid cells, lacked TLR7 or Jones’ methenamine silver–periodic acid–Schiff (kidney) according to standard practices. Immunohistochemistry staining was performed using TLR9. Respective B cell chimeras will henceforth be referred to 2 2 2 2 2 2 Downloaded from a Leica Bond–automated immunostainer and HRP-conjugated secondary as BWT,BWAS / ,BW/TLR7 / , and BW/TLR9 / . Abs. Histology images were acquired with a Nikon OptiPhot-2 micro- We initially screened for autoimmunity using fluorescent ANA scope and a Canon Eos 5D Mark II camera. Tissue sections were ex- assays. The majority of BWAS2/2 were ANA positive with mixed amined by a board-certified veterinary pathologist (D.L.), who was blinded to study design. Kidney sections were analyzed by two observers homogeneous and nucleolar staining patterns, consistent with both W/TLR72/2 blinded to genotype (K.L.H. and C.E.A.) and scored from 0 to 2 based on DNAandRNAAbreactivity.Incontrast,B ANA degree of mesangial expansion, glomerular basement membrane (GBM) staining was homogeneous with no nucleolar reactivity, whereas thickening/reduplication, and glomerular hypercellularity. For quantifi- BW/TLR92/2 showed nucleolar and cytoplasmic, but not homoge- http://www.jimmunol.org/ cation of MAC-2 area, glomeruli were manually delineated in more than + neous, ANA reactivity (Fig. 1A). These altered ANA staining four independent kidney sections and MAC-2 area as a percentage of glomerular area determined using Image Pro Plus (Media Cybernetics, patterns suggested a B cell–intrinsic impact of TLR7 versus TLR9 Rockville, MD). on RNA- and DNA-associated Abs, which we evaluated using by guest on September 28, 2021

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 BWAS2/2,BW/TLR72/2,BW/TLR92/2, and CD4-depleted BWAS2/2 chimeras (BWAS2/2 CD4 DEP).DatanormalizedtoBWT anti–MDA-LDL IgG2c titer. (B) Left panel, Anti-PC IgG. Right panel,Anti-PCIgG2cinBWAS2/2,BW/TLR72/2, BW/TLR92/2, and CD4-depleted BWAS2/2 chimeras (BWAS2/2 CD4 DEP). Data normalized to BWT anti-PC IgG2c titer. (A and B) Mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. Total mice analyzed: BWT (n = 7), BWA S2/2 (n =13),BW/TLR72/2 (n =13),BW/TLR92/2 (n = 14), and BWA S2/2 CD4 DEP (n = 10). (C) Serum IgG Abs from BWT (n = 4), BWA S2/2 (n = 6), BW/TLR72/2 (n = 4), and BW/TLR92/2 (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 21 (blue) to 3 (red). Representative of two independent microarray analyses. 4528 B CELL–INTRINSIC TLR7 AND TLR9 SIGNALS IN LUPUS 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/TLR92/2, DNA reactivity by highly specific Cri- thidia luciliae kinetoplast staining was abrogated in BW/TLR92/2, but unaffected in BW/TLR72/2 (Fig. 1B, Supplemental Fig. 1A). In contrast, RNA-associated anti-sm/RNP Abs were abolished in BW/TLR72/2. Further, anti-sm/RNP Ab titers of the pathogenic IgG2c subclass were significantly increased in BW/TLR92/2 versus BWAS2/2 chimeras, an observation consistent with elevated anti- RNA titers in tlr92/2 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. 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 Downloaded from MDA-LDL and PC. In contrast to abrogation of anti-RNA responses, W/TLR72/2 antiapoptotic IgG responses were decreased in B ,butwere FIGURE 3. B cell–intrinsic TLR7 and TLR9 exert opposing signals on not absent. Rather, we noted a specific decrease in pathogenic immune activation. Splenic monocyte subsets were analyzed in BWT,BWAS2/2, IgG2c subclass titers. Because we previously observed a lack of anti- BW/TLR72/2,andBW/TLR92/2 chimeras at sacrifice. (A) Spleen weight and total dsDNA IgG2c in the absence of cognate T cell help (12), we mea- splenocyte numbers. (B–D)Numberofsplenic(B) CD19+ Bcells,(C) WAS2/2 + D + LO sured anti-PC and anti–MDA-LDL IgG2c subclass titers in B CD4 T cells, and ( )CD11bGR1 monocyte/macrophages and http://www.jimmunol.org/ CD11b+GR1+ neutrophils. (A–D)Mean6 SEM. *p , 0.05, **p , 0.01, treated weekly with monoclonal CD4-depleting Ab. In the absence of 2 2 22 + WAS2/2 ***p , 0.001. Total mice analyzed: BWT (n =5),BWAS / (n = 12), BW/TLR7 / CD4 T cells, B chimeras developed markedly reduced IgG2c W/TLR92/2 Ab targeting PC and MDA-LDL, findings that mirrored BW/TLR72/2 (n = 12), and B (n = 13), pooled from four independent experimental cohorts. (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 CD138+ plasma cells/plasmablasts, were markedly expanded in B cell–intrinsic TLR7 signals are required for generation of class- spleens of BWAS2/2 and BW/TLR92/2 chimeras (Fig. 4A, 4C). In switched Abs targeting nonnuclear, apoptotic Ags. addition, splenic immunofluorescence staining revealed sponta- Finally, to quantify the impact of B cell tlr7 versus tlr9 deficiency neous GL7+ GCs in BWAS2/2 and BW/TLR92/2 mice, which were by guest on September 28, 2021 on the diversity of the autoantibody repertoire of BWAS2/2 chi- absent in BW/TLR72/2 (Fig. 4D). Relative to BWAS2/2,BW/TLR92/2 meras, sera were evaluated for reactivity to 88 distinct autoanti- animals exhibited a higher percentage of GC B cells with greater gens by microarray. Although BW/TLR72/2 demonstrated a broad numbers of splenic CD138+ plasma cells/plasmablasts, suggesting reduction in serum Ab titers, the reactivity of BW/TLR92/2 sera was that B cell–intrinsic TLR9 signals serve to limit B cell activation. increased relative to BWAS2/2 to a wide range of different auto- Consistent with this, total serum IgM, IgG, and IgG2c Ab titers antigens (Fig. 2C). Consistent with our ELISA data, these Abs were were markedly elevated in BW/TLR92/2 mice relative to BW/TLR72/2 predominantly of the pathogenic IgG2c subclass (Supplemental mice, with a trend toward greater Ab titers than BWAS2/2 chi- Fig. 1B). Although absolutely required for the generation of anti- meras (Fig 4E). dsDNA Abs, TLR9 thus acts B cell intrinsically to limit Ab BWAS2/2 and BW/TLR92/2 also developed significant expansion responses to RNA-associated autoantigens, apoptotic cell epitopes, of autoimmune disease–associated CD11b+CD11c+ “age-associated and a broad range of disease-associated autoantigens. B cells” (ABCs), with #75% of total B cells expressing CD11b and CD11c in BW/TLR92/2, exceeding the percentage of ABCs previ- B cell–intrinsic TLR7 and TLR9 exert opposing signals on ously reported in aged NZB/WF1 and Mer2/2 autoimmune strains immune activation (20, 21). In keeping with the importance of chronic TLR7 activation We previously demonstrated that BWAS2/2 chimeras develop in ABC generation, this population was absent in BW/TLR72/2 systemic inflammation that is dependent on B cell Myd88 sig- (Fig. 4B). naling (12). B cell TLR7 and TLR9 signaling exerted opposing In addition to B cell activation, CD4+ effector/memory (EM) impacts on immune activation in this model, as splenic inflamma- subsets (CD44HICD62LLO and CD44HICD62LHI) were increased tion is exacerbated in BW/TLR92/2, but abrogated in BW/TLR72/2. in BWAS2/2 and BW/TLR92/2 (with relatively greater T cell acti- Relative to BWAS2/2,BW/TLR92/2 chimeras developed increased vation in BW/TLR92/2), whereas the number of CD44LOCD62LHI splenomegaly, without marked alterations in total B cell numbers naive CD4 T cells did not differ between experimental groups (Fig. 3A, 3B). Notably, splenomegaly in BWAS2/2 and BW/TLR92/2 (Fig. 5A). Consistent with greater GC B cell numbers, PD1+ + + + WAS2/2 resulted from a significant expansion of CD4 T cells, CD11b CXCR5 Th follicular cells (TFH) were also expanded in B GR1LO monocyte/macrophages, and CD11b+GR1+ neutrophils. and BW/TLR92/2 (Fig. 5B). B cell–intrinsic TLR7 activation Consistent with a role for TLR9 in limiting autoimmune responses, appears critical for T cell activation in this model, as CD4+ EM W/TLR72/2 B cell–intrinsic TLR9 deletion resulted in statistically greater and TFH cell subset expansion is abrogated in B . numbers of CD4+ T cells and monocyte/macrophages relative to In conclusion, B cell–intrinsic activation of autoreactive was2/2 BWAS2/2, with a trend toward more neutrophils (Fig. 3C, 3D). B cells via TLR7 promotes global immune activation and ex- Although total B cell numbers were similar, activated B cell pansion of activated B and T cell populations; whereas B cell subsets, including PNA+FAS+GL7+ GC B cells and B220LO TLR9 signaling serves to limit these processes. The Journal of Immunology 4529 Downloaded from http://www.jimmunol.org/

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), by guest on September 28, 2021 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 BWAS2/2 and BW/TLR92/2 chimeras. Images were taken with 310 objective. Scale bars, 100 mm. (E) Total IgM, IgG, and IgG2c serum titers in BWT,BWAS2/2,BW/TLR72/2, and BW/TLR92/2 chimeras. (A–C, E) Mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. Total mice analyzed: BWT (n = 5), BWAS2/2 (n = 12), BW/TLR72/2 (n = 12), and BW/TLR92/2 (n = 13), pooled from four independent experimental cohorts.

B cell–intrinsic TLR7 signaling promotes systemic inflammatory glomerulonephritis characterized by deposition of autoimmunity class-switched Ab, activated complement, and recruitment of + Aged BWAS2/2 and BW/TLR92/2 chimeras demonstrated diffuse MAC-2 macrophages (12). In keeping with an overall decrease in W/TLR72/2 inflammation and lymphoid infiltrates involving multiple organs— Ab titer and systemic inflammation, B chimeras did not in particular, the lungs and liver—findings that were absent in all develop glomerulonephritis (Fig. 7A), an observation consistent 2/2 lpr/lpr BWT and BW/TLR72/2 animals evaluated (Fig. 6A). Immunohis- with decreased glomerulonephritis in tlr7 MRL/Mp mice tochemistry staining demonstrated that these lymphoid infiltrates (6). Despite absent histologic glomerulonephritis, evaluation of were consistent with ectopic lymphoid follicles comprising sep- glomerular Ig deposition by immunofluorescence staining showed W/TLR72/2 arate B220+ B cell and CD3+ T cell zones (Fig. 6B). The presence persistent glomerular IgG in B chimeras (albeit at lower 2 2 2 2 of ectopic lymphoid tissues within target organs has been reported levels than in BWAS / and BW/TLR9 / ) (Fig. 7B). However, in several human autoimmune diseases, including rheumatoid deposition of the pathogenic IgG2c subclass was absent in 2 2 arthritis and type 1 diabetes, and is correlated with disease severity BW/TLR7 / (Fig. 7C). In keeping with the role for IgG2c in 2 2 in autoimmunity (22). We demonstrate that hyperresponsive was / complement and Fc receptor activation (23), C3 complement de- B cells are sufficient to recruit WT T cells to ectopic lymphoid position was absent in BW/TLR72/2 and glomerular MAC-2+ macro- follicles in a TLR7-dependent manner, reinforcing the critical role phages were not increased over BWT controls (Fig. 7D, 7E). of B cell–intrinsic TLR7 signals in the pathogenesis of systemic In contrast to the protective effect of B cell TLR7 deletion, autoimmunity. BWAS2/2 and BW/TLR92/2 chimeras developed significant glomerulonephritis characterized by mesangial expansion, GBM B cell TLR7 deletion eliminates, whereas TLR9 loss thickening, and glomerular hypercellularity (Fig. 7A). In keeping exacerbates, IC glomerulonephritis with increased serum IgG2c Ab, BW/TLR92/2 mice demonstrated Given divergent impacts on Ab production and systemic inflam- statistically greater glomerular IgG2c deposition than did BWAS2/2, mation, we evaluated the role of B cell–intrinsic TLR signaling with a trend toward greater C3 complement deposition and in autoimmune glomerulonephritis. BWAS2/2 chimeras develop abundant infiltration by MAC-2+ macrophages, likely recruited by 4530 B CELL–INTRINSIC TLR7 AND TLR9 SIGNALS IN LUPUS

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 LO HI HI LO HI HI + + + (right) of splenic (A) naive (CD44 CD62L ) and EM (CD44 CD62L and CD44 CD62L ) CD4 T cells and (B) PD1 CXCR5 TFH cells. Number represents percentage within gated population (gated on splenic CD4+ T cells). Mean 6 SEM. ***p , 0.001. Total mice analyzed: BWT (n = 5), BWAS2/2 W/TLR72/2 W/TLR92/2 (n = 12), B (n = 12), and B (n = 13), pooled from four independent experimental cohorts. Downloaded from glomerular IgG2c ICs binding activating Fc receptors (Fig. 7B–E). lupus models, B cell–intrinsic TLR7 and TLR9 signals are re- Increased IC glomerulonephritis in BW/TLR92/2 parallels accel- quired for in vivo generation of RNA- and DNA-reactive Ab, erated autoimmune disease in tlr92/2 MRL/Mplpr/lpr mice, but respectively. In addition, we also establish TLR7 as the major demonstrates that the disease-exacerbating effects of TLR9 dele- Myd88-dependent receptor responsible for B cell–intrinsic breaks tion can occur independently of the lack of TLR9 expression in in tolerance to a broad range of nonnuclear, tissue-specific auto- http://www.jimmunol.org/ the majority of the myeloid compartment. antigens. Second, we identify a critical role for B cell–intrinsic TLR7 Discussion signals in sustaining spontaneous autoimmune GC responses. Al- The importance of the Myd88-dependent TLRs TLR7 and TLR9 though the absence of anti-sm/RNP Abs in BW/TLR72/2 may have in lupus pathogenesis has been clearly established (1–3). In this been predicted from in vitro studies (24), the observation that Abs context, global deletion of TLR7 in murine lupus models protects targeting apoptotic and phospholipid epitopes develop in BW/TLR72/2, against autoimmunity, whereas TLR9 deficiency paradoxically but fail to undergo efficient class switch recombination, suggests exacerbates disease (4–7). The mechanisms underlying these di- an additional role for B cell TLR7 signaling in enhancing GC by guest on September 28, 2021 vergent effects of TLR7 and TLR9 remain unclear, in part because responses during autoimmune disease—a finding that parallels the studies involving globally gene-deficient autoimmune strains are requirement for TLR7 in Ab responses to chronic viral infection (25). unable to address the differential impacts of TLR signaling on Third, we document a striking expansion of effector and TFH B cells versus type 1 IFN–producing plasmacytoid dendritic cells. CD4+ T cells in BWAS2/2 and BW/TLR92/2 animals. Similar ex- Our study provides the novel observation that B cell–intrinsic pansion of the EM T cell compartment has been documented on deletion of TLR7 or TLR9 recapitulates several of the key features the lupus-prone MRL/Mplpr/lpr background (6), although it should of global TLR7/TLR9 deficiency in murine lupus models, despite be noted that CD4+ T cells in our model are genetically WT and intact TLR signaling within the myeloid compartment. not Fas deficient. Our data, therefore, suggest that BCR/TLR- In this context, our data provide several important new insights mediated activation of autoreactive B cells directly promotes into autoimmune pathogenesis. First, this study is, to our knowl- T cell proliferation and activation during autoimmunity. These edge, the first to compare the B cell–intrinsic impact of TLR7 data are consistent with recent reports describing decreased EM versus TLR9 in parallel models of murine autoimmunity. We T cell expansion in the setting of B cell–intrinsic Myd88 deletion in demonstrate that, similar to global TLR7/TLR9 deletion in murine the MRL.Faslpr and Lyn-deficient models of autoimmunity (11, 13).

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, 34) and portal tracts of the liver (lower panels, 310) in BWAS2/2 and BW/TLR92/2 chimeras. Scale bars, 100 mm. (B) Immunohistochemistry of serial lung sections from a representative BW/TLR92/2 mouse showing distinct B220+ B cell follicle and CD3+ T cell zone (*). Image taken with 310 objective. Scale bars, 100 mm. (A and B) Data are representative of BWT (n = 4), BWAS2/2 (n = 10), BW/TLR72/2 (n = 9), and BW/TLR92/2 (n = 11) mice, analyzed from two independent experimental cohorts. The Journal of Immunology 4531

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. De- spite producing anti-dsDNA Ab, BW/TLR72/2 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/TLR72/2, 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/TLR72/2. These data emphasize the need to con- sider IgG subclass in addition to Ab specificity in mediating end-

organ damage in SLE. Downloaded from 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 http://www.jimmunol.org/ described in several murine autoimmune models, how TLR9 deletion promotes autoimmunity has not yet been determined. Po- tential underlying mechanisms include the following: enhanced interaction of TLR7 with the shared endoplasmic reticulum traf- ficking 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, by guest on September 28, 2021 despite largely intact myeloid TLR signaling. FIGURE 7. B cell–intrinsic TLR7 and TLR9 exert opposing effects on IC glomerulonephritis. (A) Left panels, Representative examples of glo- Importantly, however, our data do not exclude the possibility that meruli from BWAS2/2,BW/TLR72/2, and BW/TLR92/2 chimeras stained with myeloid TLR7 and TLR9 signals may exert additional effects on Jones’ methenamine silver–periodic acid–Schiff stain. Right panel, Glo- lupus pathogenesis. Two recent studies, using a cre-recombinase merular inflammation scored as follows: (0+) minimal mesangial expan- strategy to delete the TLR adaptor Myd88 in CD11c+ dendritic sion consistent with radiation injury; (1+) focal glomerular changes with cells, demonstrated that myeloid TLR activation accelerates au- moderate mesangial expansion, GBM thickening/reduplication, and glo- toimmunity, likely via enhanced type 1 IFN production by IC- merular hypercellularity; or (2+) diffuse glomerular changes with severe mediated activation of plasmacytoid dendritic cells (11, 13). mesangial expansion, GBM thickening/reduplication, and glomerular hy- Given this observation, it will be important to evaluate whether percellularity. Pathologic change was scored by two observers blinded to deletion of myeloid TLR7 versus TLR9 exerts similar divergent genotype. (B–D) Glomerular IC deposits were determined by immuno- effects on autoimmune pathogenesis. In this regard, a potential fluorescence staining for (B) IgG, (C) IgG2c, and (D) complement C3. Representative images are shown (left), together with intensity of glo- caveat of our study is that reconstitution with an 80:20 ratio of m 2/2 2/2 2/2 2/2 2/2 merular fluorescent staining (right) scored from 0 to 3+ by two indepen- MT/donor (was , was .tlr7 ,orwas .tlr9 ) BM, dent, blinded observers. (E) Left panels, Representative images of will result in ∼20% myeloid cells also lacking TLR7 or TLR9. immunohistochemistry staining for glomerular MAC-2+ macrophages. Right However, because the majority of myeloid cells (∼80%) in our panel,MAC-2+ area as a percentage of total glomerular area determined model are TLR sufficient, it is highly likely that our data reflect using Image Pro Plus (Media Cybernetics). More than four independent the B cell–intrinsic effects of TLR7 versus TLR9 in autoimmune kidney sections were examined per mouse. Scale bars, 50 mm. (A–E) Results pathogenesis. 6 , , , are mean SEM. *p 0.05, **p 0.01, ***p 0.001. Total mice ana- In conclusion, our study provides critical new insights into how lyzed (A–D, E): BWT (n = 6, 4), BWA S2/2 (n = 14, 6), BW/TLR72/2 (n = 11, 9), 2 2 dual BCR and TLR signals promote B cell activation resulting in and BW/TLR9 / (n = 15, 8), from three (A–D)andtwo(E) independent class-switched Ab production, CD4+ T cell activation, and the experimental cohorts. development of IC glomerulonephritis. B cell TLR7 and TLR9 exert opposing effects in this process, reinforcing the importance Our data suggest that B cell TLR7 is likely the major Myd88- of dysregulated B cell TLR signaling in autoimmunity and dependent receptor driving this CD4+ T cell activation. Surpris- informing efforts to therapeutically target TLR signals in SLE and ingly, dual BCR/TLR7 B cell activation was also sufficient for the other disorders characterized by autoantibody production. 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 demon- Disclosures strated, these expanded EM T cell compartments are enriched for The authors have no financial conflicts of interest. 4532 B CELL–INTRINSIC TLR7 AND TLR9 SIGNALS IN LUPUS

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