Essential Requirement for IFN Regulatory Factor 7 in Autoantibody Production but Not Development of Nephritis in Murine Lupus

This information is current as Fumi Miyagawa, Yutaka Tagaya, Keiko Ozato and Hideo of September 28, 2021. Asada J Immunol 2016; 197:2167-2176; Prepublished online 15 August 2016; doi: 10.4049/jimmunol.1502445

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Supplementary http://www.jimmunol.org/content/suppl/2016/08/13/jimmunol.150244 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 52 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/197/6/2167.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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Essential Requirement for IFN Regulatory Factor 7 in Autoantibody Production but Not Development of Nephritis in Murine Lupus

Fumi Miyagawa,* Yutaka Tagaya,† Keiko Ozato,‡ and Hideo Asada*

Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease characterized by the production of autoantibodies against nuclear components. Recent genetic studies of SLE patients have revealed that IFN regulatory factor (IRF) 7 poly- morphisms are associated with an increased risk of SLE, but the precise role of IRF7 in SLE development is not fully understood. We investigated the role of IRF7 in the pathogenesis of SLE using a mouse model and saw a curious dissociation of autoantibody pro- duction and development of glomerulonephritis. SLE was chemically induced into IRF7-deficient mice, and glomerulonephritis with deposits of IgG and lipogranulomas were observed after 10 mo. However, these mice failed to produce anti-dsDNA, ssDNA, ribo- Downloaded from nucleoprotein, and Sm autoantibodies. Following the chemical induction, IRF7-deficient mice expressed substantially lower levels of IFN-stimulated than did wild-type mice, but NF-kB target genes were equally upregulated in both strains. Therefore, the type I IFN pathway seems critical for the autoantibody production, but the NF-kB activation is sufficient for the development of glomerulonephritis in this model. Our study thus demonstrates a specific requirement for IRF7 in autoantibody production and uncovers a new layer of complexity in the pathogenesis of SLE. The Journal of Immunology, 2016, 197: 2167–2176. http://www.jimmunol.org/ ystemic lupus erythematosus (SLE) is a prototypic sys- the distinct clinical features of SLE strongly suggested the po- temic autoimmune disease characterized by the loss of tential pathogenic roles of these autoantibodies (7, 8). S tolerance against nuclear autoantigens in which the pro- The autoantibody production in SLE is allegedly a consequence duction of autoantibodies leads to tissue damages through the of increased cellular apoptosis that leads to the augmented release formation and deposition of immune complexes (ICs). The disease of nuclear autoantigens. Dysregulation of apoptosis and altered involves multiple organs, including skin, kidneys, joints, CNS, and clearance of apoptotic cells observed in SLE patients thus could cardiovascular system, and its clinical manifestations are extremely cause the formation of ICs consisting of autoantibodies and RNA- diverse and variable (1, 2). Although the underlying etiology of or DNA-containing autoantigens (9–11). These ICs in turn could SLE remains largely uncharacterized, several lines of evidence induce tissue inflammation by stimulating the production of IFN-a by guest on September 28, 2021 document a complex interaction between genetic and environ- from plasmacytoid dendritic cells (pDCs) (12). mental factors (1, 3, 4). Previous studies have solidified the pathogenic involvement of The hallmark of SLE is the production of autoantibodies to type I IFN (IFN-a/b) production in SLE. Serum type I IFN levels nuclear components, including dsDNA and ssDNA, respectively, are elevated in SLE patients and correlate well with the disease histones, ribonucleoprotein (RNP), and the Sm Ag (1, 5, 6). The activity (8, 13). Genome-wide transcriptome analyses using correlation of dsDNA, Sm, and RNP Abs with disease activity and microarrays have demonstrated an elevated expression of IFN- stimulated genes (ISGs) (referred to as an IFN signature) in the PBMCs of SLE patients compared with normal controls (14, 15). *Department of Dermatology, Nara Medical University School of Medicine, Kashihara, Moreover, increased serum IFN-a activity has been implicated as † Nara 634-8522, Japan; Institute of Human Virology, University of Maryland School of a heritable trait in families with a history of SLE (16). Medicine, Baltimore, MD 21201; and ‡Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Through recent genome-wide association studies (3, 4, 17), a Health, Bethesda, MD 20892 polymorphism of IFN regulatory factor (IRF) 7 has been associ- ORCIDs: 0000-0002-1342-9282 (Y.T.); 0000-0001-7013-2728 (K.O.); 0000-0003- ated with an increased risk of SLE (18). IRF7 is a member of the 1971-9835 (H.A.). IRF family of transcription factors that induce transcription of Received for publication November 20, 2015. Accepted for publication July 13, IFN-a and the expression of ISGs (19, 20). Phenotypes of Irf72/2 2016. mice indicated that IRF7 is essential for the induction of IFN-a/b This work was supported in part by Japan Society for the Promotion of Science genes under viral infections via MyD88-independent and -dependent Grants-in-Aid for Scientific Research 24591662 and 15K09779 (to F.M.), a grant for basic dermatological research of the Japanese Dermatological Association from pathways. IRF7 thus functions as a master regulator of type I IFN– Shiseido Co. Ltd. (to F.M.), and by a grant from the Lydia O’Leary Memorial Pias dependent immune responses (21). Dermatological Foundation (to F.M.). To investigate the role for IRF7 in the pathogenesis of SLE, we Address correspondence and reprint requests to Dr. Fumi Miyagawa, Department of induced SLE by administrating 2,6,10,14-tetramethylpentadecane Dermatology, Nara Medical University School of Medicine, 840 Shijo, Kashihara, 2/2 Nara 634-8522, Japan. E-mail address: [email protected] (TMPD, pristane) in Irf7 mice (22). Intraperitoneal injection of The online version of this article contains supplemental material. TMPD induces many key immunologic and clinical features of Abbreviations used in this article: ANA, antinuclear Ab; DC, dendritic cell; IC, human SLE in mice, including production of autoantibodies immune complex; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; pDC, plas- against dsDNA, Sm, and RNP, a type I IFN signature, and the macytoid dendritic cell; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; development of IC-mediated glomerulonephritis (22, 23). This TMPD, 2,6,10,14-tetramethylpentadecane; WT, wild-type. lupus model is currently the only animal model that recapitulates Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 the IFN signature during the development of SLE symptoms (23). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502445 2168 REQUIREMENT FOR IRF7 IN AUTOANTIBODY PRODUCTION IN SLE

Using this mouse model, we investigated the role of IRF7 in SLE. In contrast to a naive assumption that SLE may not occur in the absence of IRF7, these mice manifested SLE symptoms upon chemical induction. Interestingly, Irf72/2 mice failed to reca- pitulate all features of SLE. Our study thus depicts a dual re- quirement for two distinct signaling pathways in the development of SLE, only one of which is mediated by IRF7 and is respon- sible for the autoantibody production in SLE. Inhibition of the other pathway, namely the NF-kBpathway,inIrf72/2 mice markedly attenuated the development of glomerulonephritis by the TMPD induction. Collectively, we demonstrate that the IRF7/type I IFN axis is critical for autoantibody production whereas the NF-kB pathway sufficiently controls the develop- ment of glomerulonephritis. This study elucidates the differen- tial contribution of two distinct pathways to the development of SLE and suggests a novel therapeutic approach to control this disease in humans.

Materials and Methods Downloaded from Mice C57BL/6 mice (wild-type [WT]) and Irf72/2 mice were purchased from CLEA Japan (Tokyo, Japan) and RIKEN BRC (Tsukuba, Japan), respec- tively. The mice were housed in a specific pathogen-free facility and bred and used in accordance with protocols approved by the Animal Care and

Use Committee of the Nara Medical University. http://www.jimmunol.org/ Intraperitoneal injection of TMPD Male and female mice between 8 and 12 wk old were used. Age- and gender-matched WT mice or Irf72/2 mice received a single i.p. injection of 0.5 ml of TMPD (pristane) (Funakoshi, Tokyo, Japan) or PBS (vehicle). Ten months later, urine, blood, spleens, peritoneal cells, and kidneys were harvested. In some experiments, blood, spleens, and peritoneal cells were harvested 2 wk after injection.

Measurement of urine protein by guest on September 28, 2021 Proteinuria was assessed by a urinary test strip (Wako, Osaka, Japan) and graded as 0 (none), 1+ (trace; 10–20 mg/dl), 2+ (30 mg/dl), 3+ (100 mg/dl), 4+ (300 mg/dl), and 5+ (.1000 mg/dl). Direct immunofluorescence Kidneys from WT or Irf72/2 mice treated with TMPD or PBS were har- vested 10 mo after the disease induction, frozen in OCT medium, and stored at 280˚C. Cryosections were prepared at 6 mm thickness and in- FIGURE 1. Development of glomerulonephritis in Irf72/2 mice. (A) cubated with FITC–anti-mouse IgG Ab, FITC–anti-mouse IgM Ab 2/2 (SouthernBiotech, Birmingham, AL), or Alexa Fluor 488–anti-mouse C3 Proteinuria occurred in both WT and Irf7 mice injected with TMPD. Ab (Novus Biologicals, Littleton, CO) and examined by fluorescence Horizontal lines indicate the mean value in each group (n = 10–17 mice per microscopy (Olympus, Tokyo, Japan). group). **p , 0.01. (B and C) Similar increase of glomerular cellularity was For the evaluation of glomerular lesions, images of five glomeruli per observed in WT and Irf72/2 mice injected with TMPD. Glomerular cellu- mouse were captured with a constant exposure time on fluorescence mi- larity was determined as the number of nuclei per glomerular cross-section in croscopy. From captured images, each glomerular lesion was scored based tissue sections stained with H&E. Representative images are shown in (B). , on the involved area as 0 (no staining), 1 ( 25%), 2 (25–50%), 3 (50–75%), The average numbers of nuclei of 20 glomeruli are shown in (C). Error and 4 (.75%). The average severity grade was calculated and defined as bars = SEM. **p , 0.01. Data are representative of two independent ex- the renal score of the mouse. periments with three mice in each. (D and E) Glomerular IgG deposits de- 2 2 Indirect immunofluorescence tected by direct immunofluorescence. WT and Irf7 / mice treated with TMPD were positive for IgG deposits in glomeruli whereas those treated with Hep2 cells (provided by Sei-ichiro Motegi, Department of Dermatology, PBS were negative. Representative images are shown in (D). Glomerular IgG Gunma University, Gunma, Japan) were cultured in eight-well CultureSlides deposits were scored from 0 to 4 and the average values from 9 to 14 mice per (BD Falcon, Tokyo, Japan), fixed with cold acetone, and blocked with 3% E p , F G BSA and 1% FCS in PBS for 1 h. Sera from the mice 10 mo after TMPD or groupareshownin( ). Error bars = SEM. ** 0.01. ( and )Glomerular PBS injection were diluted at 1:100 and slides wereincubatedwithdi- immune complex deposits detected by direct immunofluorescence for com- luted sera overnight. Slides were then incubated with FITC-conjugated plement component C3. WT mice treated with TMPD were positive for C3 2 2 anti-IgG Ab for 30 min, mounted, and examined by fluorescence deposits whereas Irf7 / mice treated with TMPD were negative. Repre- microscopy. sentative images are shown in (F). Glomerular C3 deposits were scored from 0 to 4 and the average values from 5 to 10 mice per group are shown in (G). ELISA Error bars = SEM. Original magnification 3200. **p , 0.01. The sera were obtained 10 mo following TMPD or PBS injection. Serum concentrations of anti-dsDNA IgG, ssDNA IgG (Shibayagi, Gunma, Japan), antinuclear Ab (ANA), RNP, Sm, histone (total Ig [IgG + IgA + IgM]; Alpha by ELISA. In some experiments, the sera were obtained 2 wk after injection Diagnostic International, San Antonio, TX), total IgG, total IgM, IgG1, and the concentrations of TNF-a (Affymetix, Santa Clara, CA) and IL-6 IgG2a, IgG2b, and IgG3 (Life Diagnostics, West Chester, PA) were assayed (R&D Systems, Minneapolis, MN) were determined by ELISA. The Journal of Immunology 2169

Table I. Indirect immunofluorescence staining of Hep2 cells (GL1), allophycocyanin–anti-CD4 (RM4-5), CD8 (53-6.7), and B220 (RA3- 6B2) (BD Pharmingen, Tokyo, Japan) were used for cell surface staining. Strain Tx Positive Staining Staining Patterns Apoptosis was assessed by staining for FITC–annexin V/propidium iodide and FITC–anti-active caspase-3 (clone C92-605) according to the WT PBS 0/10 N/A manufacturer’s protocols (BD Pharmingen). Stained cells were analyzed WT TMPD 14/14 Homogeneous 11/14 on a FACSCalibur flow cytometer (Becton Dickinson, Tokyo, Japan). Speckled 2/14 k Cytoplasmic 1/14 Administration of NF- B inhibitor Irf72/2 PBS 0/11 N/A 2/2 The NF-kB inhibitor (CAS 213546-53-3, SN50 peptide), which inhibits Irf7 TMPD 0/14 N/A translocation of the NF-kB active complex into the nucleus, was purchased 2/2 N/A, not available; Tx, treatment. from Santa Cruz Biotechnology (Santa Cruz, CA). Irf7 mice that were received 0.5 ml of TMPD on day 0 were injected i.p. with 200 ml of PBS, either alone or containing 100 mg of NF-kB inhibitor, on days 0, 1, 4, 7, RNA extraction and real-time PCR and 14. Urine, blood, and kidneys were collected 10 mo later. Single-cell suspensions from spleen cells obtained 2 wk after TMPD or PBS Statistical analysis injection were used. Total RNA was extracted from spleen cells using an RNAeasy Plus kit (Qiagen, Tokyo, Japan) and cDNA was synthesized using a Statistical analyses were carried out using a Kruskal–Wallis test or a , high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). Student t test (Fig. 6E) and p values 0.05 were considered significant. Real-time PCR was performed using TaqMan fast advanced master mix with TaqMan assays (Applied Biosystems). The data were nor- Results malized to the expression levels of GAPDH. 2/2

Irf7 mice develop glomerulonephritis and lipogranuloma Downloaded from Harvesting of peritoneal cells Because of the suspected novel role of IRF7 in the pathogenesis of The peritoneal cavity was lavaged with 2 ml of complete RPMI 1640 plus 10 SLE, we first examined the effect of IRF7 deficiency on the de- U/ml heparin. Cells were collected by centrifugation, depleted of RBCs by velopment of lupus nephritis. SLE was induced in WT and Irf72/2 ACK lysing buffer, and then resuspended in complete RPMI 1640. mice by injection of TMPD, and the consequent kidney dys- Culture of peritoneal cells function and pathological changes were monitored. Proteinuria 2/2 2 2 was detected in both WT and Irf7 mice treated with TMPD but http://www.jimmunol.org/ Freshly isolated peritoneal cells from WT or Irf7 / mice were grown in complete RPMI 1640 in the presence or absence of TMPD. Because of its not in the control mice (Fig. 1A). Consistent with this finding, insolubility in aqueous medium, TMPD was added as the inclusion com- histopathological analyses showed a similar increase of glomer- plexes with b-cyclodextrin (Wako) as described previously (24, 25). ular cellularity in both WT and Irf72/2 mice following TMPD Evaluation of glomerular cellularity induction (Fig. 1B, 1C). Direct immunofluorescence detection revealed glomerular IgG and IgM deposits in both WT and Irf72/2 Glomerular cellularity was evaluated with H&E sections of the kidneys by mice treated with TMPD (Fig. 1D, 1E, Supplemental Fig. 1A, 1B). counting the number of nuclei per glomerular cross-section as previously described (26, 27). However, glomerular C3 deposits were more obvious in TMPD- treated WT mice compared with Irf72/2 mice (Fig. 1F, 1G). These Abs and flow cytometry results suggest that IC was formed only in WT mice after TMPD by guest on September 28, 2021 FITC–anti-CD3 (clone 145-2C11), CD11b (M1/70), CD11c (HL3), Sca-1 treatment; however, IC deposits do not appear to relate to severity (D7), PE–anti-CD19 (1D3), Gr-1 (RB6-8C5), CD80 (16-10A1), CD86 of renal disease.

FIGURE 2. Impaired production of autoantibodies to DNA- and RNA-containing Ags in Irf72/2 mice. (A) Sera from WT or Irf72/2 mice treated either with TMPD or PBS were tested for ANAs by indi- rect immunofluorescence using Hep2 cells (1:100 dilution). Sera from TMPD-treated WT mice were posi- tive for nuclear staining, whereas sera from other groups were negative. Representative images are shown. Original magnification 3200. (B–G) Serum levels of ANA, anti-dsDNA, ssDNA, Sm, RNP, and histone Abs were measured by ELISA for WT or Irf72/2 mice. Sera were collected at 10 mo after the treatment with TMPD or PBS (1:100 dilution). The levels of those Abs were drastically reduced in TMPD-treated Irf72/2 mice but were significantly increased in TMPD- treated WT controls. Horizontal lines indicate the mean values in individual groups. n = 9–16 mice per group. p , 0.05 versus other groups. 2170 REQUIREMENT FOR IRF7 IN AUTOANTIBODY PRODUCTION IN SLE

Lipogranuloma, an ectopic lymphoid tissue that closely re- sembles secondary lymphoid tissues, can be formed in the peri- toneum following i.p. injection of TMPD and is a site of substantial type I IFN production (23, 28). As shown in Supplemental Fig. 1C and 1D, both WT and Irf72/2 mice developed lipogranulomas adherent to the mesothelial surfaces of the peritoneal lining when injected with TMPD but not with PBS. Taken together, TMPD triggered similar pathological changes typical of SLE nephritis in mice in the absence of IRF7. IRF7 is required for the production of antinuclear autoantibodies A bolus i.p. injection of TMPD leads to production of autoanti- bodies against dsDNA and ssDNA, chromatin, Sm, RNP Su, and ribosomal P after 6–10 mo (22, 23), and thus the presence of serum ANAs was analyzed 10 mo after chemical induction in both WT and Irf72/2 mice. Sera from TMPD-treated WT mice produced a homogeneous nuclear staining pattern with Hep2 cells by indirect immunofluorescence, corresponding to DNA and chromatin au- Downloaded from toantibodies, or speckled nuclear staining pattern, characteristic of Sm and RNP autoantibodies (Fig. 2A, Supplemental Fig. 2, Table I). In contrast, sera from TMPD-treated Irf72/2 mice showed no such pattern (Fig. 2A, Table I). Lack of these ANAs in Irf72/2 mice was validated by a specific ELISA (Fig. 2B). Next

we examined lupus-associated autoantibodies (anti-dsDNA, http://www.jimmunol.org/ ssDNA, Sm, RNP, and histone Abs) by ELISA. These autoanti- bodies were virtually undetectable in the sera of TMPD-treated Irf72/2 mice compared with TMPD-treated WT mice (Fig. 2C– G), suggesting a global lack of autoantibody production in Irf72/2 mice in response to the TMPD challenge. IRF7 is dispensable for the increased production of Igs triggered by TMPD

Because the absence of IRF7 partially blocked the TMPD-induced by guest on September 28, 2021 SLE in mice as demonstrated by the lack of ANA production in TMPD-challenged Irf72/2 mice, we next examined whether other SLE-associated responses were impaired in Irf72/2 mice. TMPD is known to cause polyclonal hypergammaglobulinemia, another immunological feature of SLE in mice (23). Thus, we tested whether the ANAs were disproportionally reduced compared with the entire Ig amounts in Irf72/2 mice upon the chemical induc- tion. Serum levels of total IgG and IgM were elevated by TMPD treatment in both WT and Irf72/2 mice (Fig. 3A, 3B), although WT mice showed slightly higher (but not statistically significant) levels than did Irf72/2 mice (Fig. 3A, 3B). Previous studies identified IgG2a and IgG2b as the primary isotypes of autoanti- bodies induced by TMPD (27, 29). Consistent with this, we saw FIGURE 3. Elevated levels of total Igs but selective reduction in IgG2a 2 2 lower levels of total serum IgG2a and IgG2b in Irf72/2 mice than and IgG2b production in TMPD-treated Irf7 / mice. (A and B) Serum 2/2 in WT mice 10 mo after TMPD injection (Fig. 3C–F). Selective levels of total IgG (A) and total IgM (B) from WT and Irf7 mice were reductions in IgG2a and IgG2b production but normal production determined by ELISA 10 mo after the treatment with TMPD or PBS (IgG, of total IgG, total IgM, IgG1, and IgG3 suggest that the global 1:200,000 dilution; IgM, 1:10,000 dilution). Serum IgG and IgM were 2 2 increased by TMPD treatments in both strains. Horizontal lines indicate Ab production and B cell functions were intact in Irf7 / mice the mean values in individual groups (n = 12–20 mice per group). *p , under SLE-like conditions triggered by TMPD, and we conclude 0.05. (C–F) Serum levels of IgG1 (C), IgG2a (D), IgG2b (E), and IgG3 (F) that the ANA production was selectively attenuated in the absence isotypes were determined by ELISA 10 mo after treatment with TMPD or of IRF7. PBS (IgG1, IgG2a, and IgG2b, 1:100,000 dilution; IgG3, 1:50,000 dilu- tion). A selective reduction in the production of IgG2a and IgG2b was Involvement of IRF7 in the alteration in neutrophil and DC observed in TMPD-treated Irf72/2 mice when compared with TMPD- populations treated WT mice. Horizontal lines indicate the mean values in individual To further explore the mechanism on the selective impairment of groups. n = 12–20 mice per group. *p , 0.05. ANA production in Irf72/2 mice, we investigated spleen cell numbers and lymphocyte populations in these mice. Flow cyto- Moreover, no skewed Th polarization was observed in the absence metric analyses revealed nearly comparable numbers of total of IRF7 under these circumstances, as assessed by intracellular spleen cells, CD4 T cells, CD8 T cells, and B cells between WT staining of CD4 T cells (F. Miyagawa, unpublished ob- and Irf72/2 mice 10 mo after the TMPD induction (Fig. 4A, 4B). servation). The numbers of CD11b+ macrophages tended to be The Journal of Immunology 2171 higher in TMPD-treated WT mice than in TMPD-treated Irf72/2 isolated from WT and Irf72/2 mice and challenged ex vivo by mice but were not statistically different (Fig. 4C). The numbers of TMPD in culture. In parallel, peritoneal cells were isolated from Gr-1+ granulocytes, CD11c+B220+ pDCs, and CD11c+CD8a+ in vivo–challenged mice (WT and Irf72/2)2wkaftertreatment CD8a DCs were increased in WT mice but not in Irf72/2 mice with either PBS or TMPD. Apoptosis was assessed by the following TMPD treatment (Fig. 4C, 4D). Flow cytometry of staining of annexin V/propidium iodide and of active caspase-3 peritoneal cells also showed the similar tendency that the number (25). Both ex vivo (Fig. 5A, 5B) and in vivo (Fig. 5C, 5D) of granulocytes, macrophages, pDCs, and CD8a DCs tended to be stimulation by TMPD induced cellular apoptosis similarly in higher in WT mice compared with Irf72/2 mice following TMPD Irf72/2 mice and WT mice, suggesting that the autoantigens treatment, but no statistical difference was reached (Supplemental were abundantly available in challenged Irf72/2 mice, but no Fig. 3). Because no specific roles of IRF7 in T cells, B cells, and autoantibody production ensued. It appears that dysregulation of DCs have been reported (30), these cell types might have been apoptosis was not involved in the pathogenesis of SLE-like recruited by inflammatory responses triggered by the production symptoms in these mice. of type I IFNs. No significant differences were observed in the 2 2 Absence of the expression of ISGs in TMPD-treated Irf7 / expression of two costimulatory molecules, CD80 and CD86, on mice DC populations between WT and Irf72/2 mice after TMPD treatment (Fig. 4E). The production of type I IFN seems integral to the pathogenesis 2/2 of SLE (27, 31), and IRF7 mediates the gene induction by IFNs No impairment of TMPD-induced apoptosis in Irf7 mice (21). We thus studied whether the expression of ISGs (the IFN 2/2 The prevailing paradigm suggests that cellular apoptosis provides signature) is modulated in TMPD-induced Irf7 mice. In this Downloaded from the immune system with autoantigen substrate necessary for a model, upregulation of IFN-a,IFN-b, and ISGs precedes break in tolerance, ultimately leading to the development of (within 2 wk after the peritoneal TMPD challenge) the ap- lupus-like autoimmunity (25). We thus determined whether pearance of lupus autoantibodies and of signs of glomerulo- TMPD-induced apoptosis is diminished in Irf72/2 mice, which nephritis (∼4–6 mo after challenge) following the TMPD are unable to produce ANA. Peritoneal cells were freshly induction (22, 27, 31). Hence, real-time PCR analysis of ISGs http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Changes of neutrophil number and DC subsets in Irf72/2 mice. (A) Total numbers of splenocytes were comparable between WT and Irf72/2 mice treated with TMPD or PBS 10 mo prior (n = 7–13 mice per group). (B–D) Percentages of immune cell subsets identified by the indicated surface markers were determined by flow cytometry. Gr-1+, CD11c+B220+,andCD11c+CD8a+ cells were increased in WT mice but not in Irf72/2 mice following the TMPD treat- ment. n = 4–8 mice per group. *p , 0.05, **p , 0.01 (C and D). (E)Themean fluorescence intensity (MFI) values of the costimulatory molecules on DCs were determined by flow cytometry. n =2–4 mice per group. 2172 REQUIREMENT FOR IRF7 IN AUTOANTIBODY PRODUCTION IN SLE

FIGURE 5. TMPD induced apoptosis in Irf72/2 mice. (A and B) Peritoneal cells were freshly isolated from normal WT 2/2 and Irf7 mice and cultured ex vivo with 200 mM TMPD/ Downloaded from b-cyclodextrin. After 12 h, the cells were harvested, stained with annexin VAb/propidium iodide (A)orwithactivecaspase-3 Ab (B) and analyzed by flow cytometry. The percentages of annexin V+ cells and active caspase-3+ cells were higher in TMPD-treated cells from both strains compared with PBS- treated cells. Data are representative of three independent ex- periments. (C and D) Peritoneal exudate cells were harvested http://www.jimmunol.org/ from WT or Irf72/2 mice 2 wk after TMPD or PBS treatment and stained with annexin V/propidium iodide (C)andactive caspase-3 (D). Data are representative of three independent experiments. by guest on September 28, 2021

was conducted 2 wk after TMPD treatment. Macroscopically, Normal upregulation of NF-kB target genes in TMPD-treated both WT and Irf72/2 mice developed splenomegaly with Irf72/2 mice TMPD treatment (Fig. 6A), as spleens from both induced To explain the dissociation of the development of glomerulone- strains were 1.5-fold heavier than those from PBS-injected phritis and the lack of ANA production and of the augmentation of mice (Fig. 6B). ISG expression in Irf72/2 mice upon chemical induction, we TMPD treatment, but not the PBS injection, caused augmented postulated that there exists a parallel pathway in the pathogenesis expression of a panel of ISGs, including Mx-1, MCP-1, IP-10/ of SLE that is not controlled by IRF7. The NF-kB pathway is a CXCL10, and IRF7, in the splenocytes from WT mice prime candidate (33), as it is known to orchestrate immune and (Fig. 6C). In contrast, the TMPD induction failed to induce ISG expression in Irf72/2 mice despite the development of spleno- inflammatory responses (34). The transcriptional targets of NF-kB megaly. Additionally, IRF5 and IRF8, another set of susceptibility include many proinflammatory (35) and chemokines genes for SLE (32), were not upregulated upon induction in Irf72/2 (36) such as TNF-a, IL-1, IL-6, CXCL1, and CXCL2 (34). We mice (Fig. 6C), suggesting a correlation between the ANA thus determined the expression of CXCL1 and CXCL2 in 2/2 production and augmentation of ISG expression. We also deter- splenocytes from TMPD-treated Irf7 mice and observed a 2/2 mined the surface expression of the ISG Sca-1 on CD19+ Bcells concerted upregulation of these genes in Irf7 mice following from peritoneal cells in TMPD-treated mice. The expression level the TMPD challenge (Fig. 7A). Interestingly, however, this in- 2 2 of Sca-1 was significantly lower in TMPD-treated Irf72/2 mice duction by TMPD was modestly reduced in Irf7 / mice relative compared with TMPD-treated WT mice (Fig. 6D, 6E). to in WT mice, suggesting that NF-kB and IFN pathways need to The Journal of Immunology 2173

FIGURE 6. No expression of IFN-stimulated genes in Irf72/2 mice. (A) Splenomegaly with WT and Irf72/2 mice 2 wk after i.p. injection of TMPD (reference, PBS-injected mice). Representative images are shown. (B) The spleens were weighed 2 wk after the injection of PBS or TMPD. The av- erage values (6SEM) from seven to nine mice per group are shown. (C) Expression of the ISGs (MX1, MCP-1, IP-10, IRF7) as well as IRF5 and IRF8 were monitored by real-time PCR. Spleens were harvested 2 wk after PBS or TMPD treatment. The data were normalized to GAPDH. Fold changes in reference to PBS-treated WT or Irf72/2 mice are shown. Data are pooled from three independent experiments with triplicates in each experiment. Error bars = SEM. (D and Downloaded from E) Sca-1 surface expression on CD19+ peritoneal cells was determined by flow cytometry 2 wk after TMPD injection. Representative flow cytometric analysis is shown in (D). The mean fluorescence intensity (MFI) of Sca-1 on CD19+ cells was determined by flow cytometry and the average MFI from eight (WT) and nine (Irf72/2) mice is shown in (E). Error , bars = SEM. **p 0.01. http://www.jimmunol.org/

cross-talk for achieving maximum induction of proinflammatory of autoantibodies against DNA- and RNA-containing autoantigens, by guest on September 28, 2021 genes (37). However, ELISA assays showed similar levels of another hallmark of SLE, was profoundly attenuated in Irf72/2 TNF-a and IL-6 production between WT and Irf72/2 mice treated mice. This was surprising because the relevance of anti-dsDNA Ab with TMPD (Fig. 7B, 7C), suggesting that some proinflammatory production in the pathogenesis of human SLE has been reasserted in genes can be fully activated by the NF-kB pathway alone under the literature (1, 38). Other autoantibodies such as anti-Sm Abs have these circumstances. been assigned equally prominent roles (7). These results collectively suggest that the IRF7 deficiency causes Recent findings implicated a relevant role for TLRs in autore- a nearly complete shutdown of the IFN pathway and events caused active B cell stimulation and production of autoantibodies. by the TMPD induction, but the NF-kB pathway has not fully lost Mammalian cells use TLR7, TLR8, and TLR9 to recognize its function in mice, which may explain the partial but not com- pathogen-associated ssRNA and unmethylated CpG DNA, re- plete development of SLE symptoms in the Irf72/2 mice upon spectively, and initiate type I IFN production (39). These TLRs TMPD induction. have received considerable attention due to their ability to rec- ognize endogenous nucleic acids. DNA-containing immune Inhibition of the NF-kB pathway protected TMPD-treated 2 2 complexes within lupus serum have been reported to directly Irf7 / mice from developing glomerulonephritis stimulate TLR9 (40, 41). As well, RNA-containing RNP can di- From the data shown above, we reasoned that the additional rectly act as endogenous self- for TLR7 and TLR8 and 2 2 blockade of the NF-kB pathway in the Irf7 / mice induced by trigger innate immunity (42, 43). Consistent with these findings, TMPD should profoundly block the development of all SLE TLR9-deficient lupus-prone mice fail to generate autoantibodies symptoms, including glomerulonephritis, in these mice. Multiple to DNA-containing autoantigens such as anti-dsDNA and anti- 2 2 injections of NF-kB inhibitor in TMPD-treated Irf7 / mice in- chromatin autoantibodies (44, 45). Nevertheless, there was no deed markedly attenuated the proteinuria and renal pathologic impairment observed in the development of clinical autoimmune 2 2 changes in TMPD-treated Irf7 / mice, confirming the impor- symptoms or of nephritis in these mice (44, 45). In TLR7-deficient tance of the NF-kB pathway in glomerulonephritis (Fig. 7D–F). lupus-prone mice, the generation of autoantibodies to RNA- 2 2 ANA production remained absent in TMPD-treated Irf7 / mice containing Ags such as Sm and RNP is shown to be reduced with NF-kB inhibition (Fig. 7G). along with ameliorated nephritis (45). Therefore, TLR7- and TLR9-deficient mice provide additional evidence in which the Discussion development of lupus nephritis is dissociated from the develop- In this study, we investigated the novel role of IRF7 in the path- ment of autoantibodies against RNA- or DNA-containing auto- ogenesis of SLE using a mouse model induced by TMPD. We were antigens, in addition to our own observation. Conversely, some 2 2 initially disappointed that Irf7 / mice developed SLE-like glo- cytokine-deficient mice demonstrate the lack of lupus nephritis de- merulonephritis, but we were then intrigued because the production spite the production of anti-DNA/RNA nephritogenic autoantibodies. 2174 REQUIREMENT FOR IRF7 IN AUTOANTIBODY PRODUCTION IN SLE Downloaded from http://www.jimmunol.org/

FIGURE 7. Upregulation of NF-kB target genes in Irf72/2 mice and effectiveness of NF-kB inhibition in preventing nephritis. (A) Expression of NF-kB target genes (CXCL1 and CXCL2) in spleen cells harvested 2 wk after injection was measured by real-time PCR. The data were normalized to GAPDH. Fold changes in reference to PBS-treated WT or Irf72/2 mice are shown. Data are pooled from four independent experiments with triplicates in each by guest on September 28, 2021 experiment. Error bars = SEM. (B and C) Serum levels of TNF-a (B) and IL-6 (C) were determined by ELISA 2 wk after injection of either TMPD or PBS (n = 9–11 mice per group). Horizontal lines indicate the mean value in each group. Error bars = SEM. (D) Proteinuria was decreased in TMPD-treated Irf72/2 mice injected with NF-kB inhibitor after 10 mo. Horizontal lines indicate mean values from individual groups (PBS, n = 5; TMPD without NF-kB inhibitor, n = 10; TMPD plus NF-kB inhibitor, n = 12). (E and F) Glomerular cellularity was determined in TMPD- or PBS-treated Irf72/2 mice with or without NF-kB inhibitor after 10 mo. Glomerular cellularity was determined as the number of nuclei per glomerular cross-section in tissue sections stained with H&E. Representative images are shown in (E) (original magnification 3200). The average numbers of nuclei of 20 glomeruli are shown in (F). Error bars = SEM. Data are representative of two independent experiments with three to four mice in each. The number of nuclei per glomerulus was significantly lower in TMPD-treated Irf72/2 mice with NF-kB inhibition compared with those without. (G) Serum levels of ANA were determined by ELISA. Sera were collected at 10 mo after the treatment with TMPD or PBS (1:100 dilution). ANA levels in TMPD-treated Irf72/2 mice with NF-kB inhibitor were comparable with those in TMPD-treated Irf72/2 mice without NF-kB inhibitor. Horizontal lines indicate the mean values in individual groups. n = 5–8 mice per group. *p , 0.05, **p , 0.01

These examples include IL-6–, IFN-g–, and IL-12p35–deficient independent of Ab secretion, by way of presenting autoantigens mice, all of which are highly resistant to the induction of renal dis- to activate autoreactive T cells and augmenting local proin- ease. Whereas TMPD could induce Abs against both DNA- and flammatory effects (50). RNA-containing Ags in IL-12p352/2 mice (46), only Abs against TMPD induces a lupus-like syndrome in non–autoimmune mice RNA-containing Ags but not against DNA-containing Ags would characterized by the development of autoantibodies against developinIL-62/2 (47) and IFN-g2/2 (48) mice upon TMPD dsDNA and RNPs as well as IC-mediated glomerulonephritis (22). challenge. Collectively, these reports, together with our own obser- Notably, TMPD-induced lupus is reportedly accompanied by ex- vations, suggest that autoantibodies may not be directly responsible cess type I IFN production and upregulation of ISGs (28). Such for inducing renal pathologic changes. IFN signature and the IFN signaling seem pathogenically relevant Our data thus provide reinforcing novel observations to the to the development of lupus because IFN-I a-chain– previous interpretation: although anti-dsDNA autoantibodies and deficient (Ifnar2/2 mice) fail to produce pathogenic antoantibodies anti-Sm autoantibodies may predict nephritis in SLE, they are not and develop no glomerulonephritis (27). Knowing that IRF7 is the necessarily required for renal pathology. However, we admit master regulator of type I IFN–dependent immune responses (21), caution is needed because the association of lupus nephritis and a phenotype of induced SLE in Irf72/2 mice could resemble that these autoantibodies was only dictated in human studies (1, 7, 38) of Ifnar2/2 mice. The diminished IFN production in Irf72/2 mice but was not validated in mice. Investigating the role of antoanti- may most likely account for the decreased number of neutrophils bodies in the MRL/lpr mouse with B cells but no circulating Abs and pDCs upon TMPD treatment. However, significant damages showed that these mice still developed nephritis (49); therefore, to kidney were observed in Irf72/2 mice as shown by severe autoreactive B cells themselves seemed to exert pathogenic effects proteinuria, increased glomerular cellularity, and glomerular IgG The Journal of Immunology 2175 deposits (but not C3 deposits), suggesting that type I IFN signaling 10. Amoura, Z., J. C. Piette, H. Chabre, P. Cacoub, T. Papo, B. Wechsler, J. F. Bach, does not solely account for lupus nephritis in Irf72/2 mice. Cu- and S. Koutouzov. 1997. 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In- Why do Irf7 mice develop glomerulonephritis without in- duction of interferon-a production in plasmacytoid dendritic cells by immune volving autoantibody production when challenged by TMPD? The complexes containing nucleic acid released by necrotic or late apoptotic cells magnitude of apoptosis by the TMPD induction is extensive and and lupus IgG. Arthritis Rheum. 50: 1861–1872. 13. Hooks, J. J., H. M. Moutsopoulos, S. A. Geis, N. I. Stahl, J. L. Decker, and this may provide a partial clue. Such apoptosis may stimulate A. L. Notkins. 1979. Immune interferon in the circulation of patients with au- IRF7-independent inflammatory pathways, perhaps through NF- toimmune disease. N. Engl. J. Med. 301: 5–8. kB. We speculate that renal tissue damage in the TMPD-induced 14. Baechler, E. C., F. M. Batliwalla, G. Karypis, P. M. Gaffney, W. A. Ortmann, K. J. Espe, K. B. Shark, W. J. Grande, K. M. Hughes, V. Kapur, et al. 2003. 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