Overexpression of Membrane-Bound (CD95L) Exacerbates Autoimmune Disease and Renal Pathology in Pristane-Induced Lupus This information is current as of September 29, 2021. Lukas Bossaller, Vijay A. K. Rathinam, Ramon Bonegio, Ping-I Chiang, Patricia Busto, Adam R. Wespiser, Daniel R. Caffrey, Quan-Zhen Li, Chandra Mohan, Katherine A. Fitzgerald, Eicke Latz and Ann Marshak-Rothstein

J Immunol published online 5 August 2013 Downloaded from http://www.jimmunol.org/content/early/2013/08/03/jimmun ol.1300341 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/08/06/jimmunol.130034 Material 1.DC1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 5, 2013, doi:10.4049/jimmunol.1300341 The Journal of Immunology

Overexpression of Membrane-Bound Fas Ligand (CD95L) Exacerbates Autoimmune Disease and Renal Pathology in Pristane-Induced Lupus

Lukas Bossaller,* Vijay A. K. Rathinam,* Ramon Bonegio,† Ping-I Chiang,* Patricia Busto,‡ Adam R. Wespiser,* Daniel R. Caffrey,* Quan-Zhen Li,x Chandra Mohan,x Katherine A. Fitzgerald,* Eicke Latz,*,{,‖,1 and Ann Marshak-Rothstein‡,1

Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hy- drocarbon oil commonly known as pristane, which induces systemic lupus erythematosus–like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL Downloaded from can also trigger the production of proinflammatory . FasL is a transmembrane with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (DCS) mouse line. DCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and DCS BALB/c http://www.jimmunol.org/ mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, DCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and , a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity. The Journal of Immu- nology, 2013, 191: 000–000.

as ligand (FasL, CD95L) was initially identified as a potent mediated cytotoxicity plays a key role in limiting the expansion by guest on September 29, 2021 proapoptotic type II transmembrane protein belonging to and function of Fas receptor (CD95, TNFRSF6)–positive target the TNF family (1), and it is predominantly expressed populations such as activated T cells, B cells, macrophages, and F + + by CD4 , CD8 , NKT, and NK cytotoxic effector cells (2). FasL- dendritic cells (3–5). Additionally, as with other TNF family members, FasL can also trigger the production of IL-1b and other proinflammatory cytokines and , especially in mac- *Division of Infectious Diseases and Immunology, University of Massachusetts Med- rophages, neutrophils, and other cells of the innate immune sys- ical School, Worcester, MA 01605; †Renal Section, Department of Medicine, Boston tem (6, 7). It follows that FasL is a dangerous molecule and strict University Medical Center, Boston, MA 02118; ‡Division of Rheumatology, Depart- ment of Medicine, University of Massachusetts Medical School, Worcester, MA regulation of its activity is a necessity. FasL expression is con- 01605; xDepartment of Immunology and Medicine, University of Texas Southwestern trolled at a number of levels, including transcription, vesicular { Medical Center, Dallas, TX 75390; Institute of Innate Immunity, University of compartmentalization, and cleavage. The latter depends on the Bonn, 53127 Bonn, Germany; and ‖German Center for Neurodegenerative Diseases, 53127 Bonn, Germany activity of matrix metalloproteinases (MMPs) that recognize a 1E.L. and A.M.-R. contributed equally to this work. cleavage site located in the extracellular region of FasL between the transmembrane and the trimerization domains (8–10). FasL Received for publication February 4, 2013. Accepted for publication June 21, 2013. cleavage releases a soluble isoform whose function is somewhat This work was supported by National Institutes of Health Grants CA90691 and AR050256 (to A.M.-R.), DK090558 (to R.B.), AI057159 (to V.A.K.R), HL093262 controversial. Several studies point to a loss of function of the (to E.L.), and AI083713 (to K.A.F. and E.L.), as well as by the Alliance for Lupus cleavage product whereas data from our own studies and those of Research (to A.M.-R.) and German Research Foundation Grant SFB TR 57 (to E.L.). others indicate that soluble FasL can serve as an antagonist of the Address correspondence and reprint requests to Dr. Ann Marshak-Rothstein, Depart- membrane-bound molecule (11–14). ment of Medicine/Rheumatology, University of Massachusetts Medical School, 364 Plantation Street, LRB 309, Worcester, MA 01605. E-mail address: ann.rothstein@ Exactly what circumstances promote apoptosis and/or the re- umassmed.edu lease of proinflammatory cytokines is unclear, but the functional The online version of this article contains supplemental material. outcome of Fas engagement may reflect the relative levels of the Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; ANA, anti- membrane-bound and soluble forms and/or inherent properties of nuclear Ab; B6, C57BL/6; BMDC, –derived ; DCS mice, the Fas+ target populations. To better understand the significance Fas ligand MMP cleavage site–deleted mice; FasL, Fas ligand; ISG, IFN-stimulated gene; MFI, mean fluorescence intensity; MMP, matrix metalloproteinase; PEC, of FasL cleavage in a physiologically relevant system, we made peritoneal exudate cell; SLE, systemic lupus erythematosus; Sm, Smith; TMPD, a gene-targeted mouse line in which the FasL MMP recognition 2,6,10,14-tetramethylpentadecane; viperin, virus inhibitory protein, endoplasmic site has been mutated to render FasL resistant to MMP-mediated reticulum–associated, IFN-inducible; WT, wild-type. cleavage (15). We refer to these mice as deleted cleavage site Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (DCS) FasL mice. Although it might be anticipated that the failure

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300341 2 FasL OVEREXPRESSION EXACERBATES MURINE SLE to appropriately cleave FasL would perturb normal lymphocyte a resting phenotype during a period of several days (22). Bone marrow– derived dendritic cells (BMDCs) were generated from BALB/c or homeostasis, unmanipulated DCS mice do not exhibit any ap- lpr/lpr parent immune phenotype (data not shown), and they therefore BALB/c bone marrow for 10 d in the presence of recombinant murine GM-CSF (PeproTech). The rested T cells were then added to the BMDCs resemble a similar gene-targeted line described by others (16). in the presence or absence of OVA peptide for 20 h. However, FasL is also expressed at sites of immune privilege, such as the eye, where it has been reported to block both assays + + (migration of Fas endothelial cells) and the influx of Fas proin- To measure cytokines produced locally in the peritoneal cavity 14 d after flammatory cells, thereby protecting the eye from immune-mediated injection with TMPD, the peritoneal cavity was punctured with a 25G5/8 damage (17, 18). Apparently, FasL expression and function in the needle (Becton Dickinson) through the subxiphoidal fat pat and inflated eye is maintained in a remarkably delicate balance between the with 1 ml air. Subsequently, a 22G1 needle was inserted through the same fat pad and 3 ml sterile PBS containing 0.2% BSAwas injected. The mouse was full-length and cleaved isoforms, as we have recently found that rotated 20 times to mix, and the retrieved lavage fluid was stored in aliquots DCS mice develop markedly exacerbated pathology in both immediately at 280˚C until further processing. The Luminex screen was spontaneous and induced murine models of glaucoma (15). done on a Bio-Plex 200 system (Bio-Rad) using a Bio-Plex 23-plex kit FasL/Fas interactions have a profound impact on self-tolerance and (Bio-Rad) per the manufacturer’s instructions. autoimmune development. Failure to express either Fas or FasL leads ELISA to the production of autoantibodies in numerous mouse strains and is associated with accelerated clinical disease in almost all systemic IL-1b protein levels were determined by ELISA (BD Biosciences). IL-18 lupus erythematosus (SLE)–prone murine lines (19). A reported ELISA was performed using rat anti-mouse IL-18 capture Ab and bio- tinylated rat anti-mouse IL-18 detection Ab with recombinant murine IL- exception appears to be C57BL/6 (B6) mice injected with 2,6,10,14- 18 cytokine standard (all MBL International). An anti-mouse albumin Downloaded from tetramethylpentadecane (TMPD), commonly referred to as pristane ELISA kit was used to measure urine proteinuria (Bethyl Laboratories). (20). TMPD-injected mice develop a chronic inflammatory response analysis that eventually presents as an SLE-like autoimmune disease nor- mally associated with autoantibodies reactive to a panel of RNA- RNAwas isolated from total spleen or total peritoneal lavage cells 14 d after associated autoantigens. Somewhat unexpectedly, TMPD-injected TMPD injection with the Qiagen RNeasy Mini kit. The iScript cDNA synthesis kit (Bio-Rad) was used for reverse transcription. Quantitative RT- B6/lpr and B6/gld mice, which harbor loss-of-function mutations http://www.jimmunol.org/ PCR was performed using SYBR Green PCR Master Mix (Bio-Rad) with in Fas and FasL, respectively, were reported to make significantly the following primer pairs: virus inhibitory protein, endoplasmic reticulum– less autoantibody specific for common RNA-associated autoantigens associated, IFN-inducible (viperin), sense, 59-AACCCCCGTGAGTGT- than did TMPD-injected B6 mice (20). These results implicate CAACTA-39,antisense,59-AACCAGCCTGTTTGAGCAGAA-39; b-actin, Fas/FasL interactions in TMPD-induced autoimmune reactions. sense, 59-TGGCATAGAGGTCTTTACGGA-39, antisense, 59- TTGAACA- TGGCATTGTTACCAA-39. The quantitative PCR was performed on a Moreover, the disease-promoting activity of FasL also emerged CFX96 real-time PCR detection system (Bio-Rad). Viperin expression is from a clinical study in which a single nucleotide polymorphism presented relative to b-actin expression. For purified cell populations, cells in the FasL promoter, associated with increased expression of FasL, weresortedbasedonthegatingstrategyemployedinFig.1AeitheronaBD was identified as a risk factor for SLE in an African American FACSVantage DV-1 cell sorter or a BD FACSAria cell sorter (both BD Bio- cohort (21). To further explore the contribution of FasL in SLE, we sciences). The sorting strategy was designed to increase purity by exclusion of by guest on September 29, 2021 B and T cells. Sorted cells were directly harvested into RLT buffer containing have now evaluated the effect of FasL deficiency, as well as FasL 2-ME for subsequent processing with the RNeasy Mini kit (Qiagen). Each overexpression, on TMPD-injected BALB/c mice. RNA sample was adjusted to contain the same quantity by using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific). RNA was then hybridized and quantified with the NanoString nCounter analysis system (NanoString Materials and Methods Technologies, Seattle, WA) per the manufacturer’s protocol. The gene ex- Mice pression data were first normalized to an internal positive control set, secondly to an internal negative control set, and then to six housekeeping , that is, gld/gld Wild-type BALB/c and C57BL/6 mice as well as B6 and BALB/c GAPDH, b-glucuronidase, hypoxanthine phosphoribosyltransferase 1, tubulin 2/2 Rag2 mice were purchased from The Jackson Laboratory and housed in b, phosphoglycerate kinase 1, and clathrin H chain 1. All values were scaled a specific pathogen-free facility at the University of Massachusetts Med- relative to the minimum value (x 2 min(x)) and a pseudocount of 1 was added DCS/DCS 2/2 ical School. The BALB/c FasL (DCS), BALB/c FasL (15), to each value. This operation sets the smallest value in the dataset to 1. All lpr/lpr 2/2 lpr/lpr BALB/c.DO11 .Rag2 (DO11 lpr), and BALB/c.DO11 .DCS/ values were log-transformed and a heat map was generated using the gplots 2/2 DCS.Rag2 (DO11 lpr DCS) mice were bred and maintained at the package within the open source R software environment. University of Massachusetts Medical School. For long-term experiments (during a period of 6 mo), 8- to 12-wk-old mice received a single i.p. in- Ab and autoantibody titers jection of 0.5 ml TMPD (Sigma-Aldrich). For short-term experiments (up to 14 d) mice were injected at 5–6 wk of age, prior to overt onset of au- Anti-nuclear Abs (ANAs) were detected by immunofluorescence on HEp-2 toimmune disease in BALB/c FasL2/2 or B6gld/gld mice. These studies slides (Antibodies, Inc.) with serum diluted at 1:100 using anti-mouse IgG were approved by the Institutional Animal Care and Use Committee. (H+L) DyLight 488–conjugated F(ab)92 (Jackson ImmunoResearch) in Vectashield antifade mounting medium (Vector Laboratories). Images were Flow cytometry captured on a Nikon E600 at 2003 magnification and processed in Adobe Photoshop. Smith (Sm) Ag was detected by ELISA by using SmB,B9,D The following conjugated anti-mouse Abs were used: anti-CD19 (1D3), Ag–coated wells (ImmunoVision) and a goat anti-mouse total IgG HRP anti–Ly-6G (1A8), anti-CD11b (M1/70), anti-CD3 (17A2), anti-CD95L (Sigma-Aldrich) with tetramethylbenzidine substrate (Dako) as detecting (MFL-3), anti-CD95 (Jo-2) (BD Biosciences); anti-CD19 (1D3), anti-F4/ reagents. The anti-Sm Ag–reactive mAb Y12 was used as a standard. 80 (BM8), anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti-CD45R (RA3-6B2) Total serum IgG1, IgG2a, and IgG2b were measured by ELISA using the (eBiosciences); and anti–TCR-b (H57-597), anti-CD11c (3.9), and anti– following coating Abs: goat anti-mouse anti-IgG2a (SouthernBiotech), goat Ly-6C (ER-MP20) (Serotec). Cells were incubated in CD16/32 (Fc Block; anti-mouse IgG2b (Jackson ImmunoResearch), and goat anti-mouse IgG1 BD Biosciences) prior to staining. Cell death was determined by flow (Jackson ImmunoResearch). Anti-mouse IgG HRP (Sigma-Aldrich) was cytometry using 7-aminoactinomycin D (7-AAD; BD Pharmingen). Cells used as detection Ab and the ELISA was developed with tetrame- were acquired on a LSR II (BD Biosciences) and analyzed with FlowJo thylbenzidine substrate (Dako). IgG1, IgG2b (BD Pharmingen), and IgG2a software (Tree Star). (BioLegend) were used as standards. Cell cultures Autoantigen array DO11 T cells were stimulated with OVA peptide as described previously, Autoantibody reactivity was further examined using an autoantigen pro- expanded in vitro in the presence of IL-2, and then allowed to revert to teonomic array containing 88 autoantigens and 10 control (23). The Journal of Immunology 3

Serum samples, diluted 1:100, were detected with Cy3-labeled anti-mouse Software) unless otherwise indicated in the figure legends. A p value IgG and Cy5-labeled anti-mouse IgM (Jackson ImmunoResearch). Net ,0.05 was considered to be statistically significant. fluorescence intensity was normalized using anti-mouse IgG or IgM. Results were plotted using GraphPad Prism software (GraphPad Software, San Diego, CA). The autoantigen heatmap was generated using Gene- Results Pattern software from the Broad Institute. Fas/FasL interactions promote the TMPD-induced inflammatory response Histology Numerous studies have documented the rapid influx of both neu- Kidneys were fixed in paraformaldehyde, embedded in paraffin, and sections were stained with H&E. H&E-stained kidney sections were scored in trophils and into the peritoneum following TMPD in- a blinded fashion to determine a glomerular and interstitial inflammation jection (25, 26). To carefully examine the potential role of FasL in score as described (24). Briefly, a mean glomerular score was calculated for the early events of TMPD-induced inflammation, wild-type (WT), each mouse by grading injury in 50 glomeruli. Glomeruli were scored as DCS, FasL-deficient, and gld mice were injected with TMPD and follows: 0, normal; 1, mesangial expansion; 2, endocapillary proliferation; 3, the cell subsets that accumulated in the peritoneum 14 d later were capillaritis or necrotic changes; 4, crescents. The interstitial score was de- termined by examining 50 high-power fields and scoring the interstitial in- characterized by flow cytometry. Consistent with previous reports, + flammation on a scale from 0 to 4 as absent or involving ,25, 25–50, or in WT mice TMPD injection led to loss of the resident CD11b F4/ .50% of the interstitium. For immunofluorescence, kidneys were fixed in 80+ macrophages (Fig. 1A, R0) and to a rapid and prolonged ex- 0.7% paraformaldehyde-lysine-periodate solution and frozen in OCT (Tis- travasation of inflammatory monocytes, identified as CD11b+ sueTek) after dehydration in 30% sucrose solution. For the detection of Ly6Chi and Ly6Glo (Fig. 1A, R1), as well as neutrophils, identified glomerular immune complexes, kidney sections were stained with DyLight + int hi 488–labeled goat anti-mouse IgG (minimal x-reactivity; BioLegend). Using as CD11b Ly6C and Ly6G (Fig. 1A, R2), into the peritoneal the NIS-Elements imaging software BR3.10 (Nikon), 20–30 randomly picked cavity (25). Approximately the same number of cells accumulated Downloaded from glomeruli were circled and analyzed for the mean fluorescence intensity in the peritoneum of WT and DCS mice (Fig. 1B). In contrast, mice (MFI) emitted in the green channel over the circled areas. that failed to express FasL (FasL2/2) or expressed a loss-of- Spleen tissue was snap-frozen in OCT and cryostat sections were fixed in + acetone, blocked with 10% rat serum, and stained with biotinylated anti– function point mutation (gld) had significantly fewer CD11b Moma-1 (Abcam) and FITC-labeled anti–Ly-6G. Streptavidin-Alexa Fluor cells, particularly the inflammatory monocytes (R1) and neutrophil 555 (Invitrogen) was used as a secondary reagent. Nuclei were counter- (R2) subsets (Fig. 1B). Furthermore, the peritoneal exudate from the stained with DAPI (Invitrogen). Images were captured on a Nikon E600 FasL-deficient mice included a substantial number of CD11bhi http://www.jimmunol.org/ (kidney immunofluorescence) or on an Olympus IX 70 inverted light mi- lo lo croscope (spleen immunofluorescence). Ly6C Ly6G cells (R3) found only in small number in TMPD- treated WT or DCS mice. The differences in number and type of Statistical analysis exudate cells that accumulated in the FasL-sufficient and FasL-de- Experiments are reported as the means 6 SEM. Statistical comparisons ficient mice demonstrate that FasL plays a major role in promoting were made using a Student t test GraphPad Prism software (GraphPad the early stages of the TMPD-induced inflammatory response. by guest on September 29, 2021

FIGURE 1. Fas/FasL interactions promote TMPD- induced peritoneal inflammation. (A) Phenotype of peritoneal cavity washout cells from untreated mice, as well as day 14 PECs from TMPD-injected mice, ob- tained from the indicated genotypes. Top panel depicts all peritoneal cells gated on live/singlet events stained with a combination of F4/80 and CD11b. The lower panel depicts the CD11b+ cells stained for Ly6C and Ly6G. (B) Total number of cells collected from the peritoneum of untreated BALB/c mice (open bars) or TMPD-treated BALB/c, DCS, FasL2/2, or gld mice (filled bars), as well as cell numbers in the CD11b+, R0, R1, R2, and R3 gates and total number of CD8+ and CD4+ T cells (6SEM, n = 4). *p , 0.05. 4 FasL OVEREXPRESSION EXACERBATES MURINE SLE

Fas and FasL expression by TMPD-induced cells FasL than did the CD4+ cells from any of the other mouse strains hi + To better understand how FasL could be regulating peritoneal (Fig.3A).BecausetheFasL CD4 subset was not found in inflammation in this model, it was important to determine which TMPD-injected BALB/c mice, we conclude that FasL expression subpopulations expressed high levels of Fas and were thereby likely in these cells is regulated by MMP cleavage and might account for the higher numbers of 7-AAD+ cells in the DCS exudate. How- to be targeted by FasL effector populations. To avoid any con- ever, we cannot rule out the potential contribution of the R1 and founding effects of autofluorescence, we compared cell populations R2 subsets, or even other FasL+ cell types, not present in the collected from uninjected BALB/c mice (R0), TMPD-injected 2 2 exudate. BALB/c mice (R1 and R2), and TMPD-injected FasL / mice (R3) to the corresponding populations obtained from Fas-deficient DCS T cells induce BMDCs to make IL-1b lpr mice. The R3-gated cells, found predominantly in the FasL- From previous studies, we know that FasL induces resting peritoneal deficient TMPD-injected mice, resembled the mature R0-gated (R0) cells to rapidly produce IL-1b and other neutrophil-attracting resting peritoneal macrophages found in untreated WT mice; chemokines and then undergo apoptosis (4). FasL can also directly both groups included cells that express F4/80 and relatively high induce neutrophils to produce IL-1b (27). Therefore, cell subsets levels of Fas (Fig. 2A). The inflammatory monocytes (R1) ob- from TMPD-injected mice include populations that are very sen- tained from the TMPD-injected BALB/c and DCS mice expressed sitive to FasL and respond by producing inflammatory mediators. lower levels of Fas, whereas the neutrophils (R2), and especially hi hi To determine whether BALB/c DCS T cells express more FasL the Ly6C Ly6G subset (R2*) found in the TMPD-injected BALB/c than do BALB/c T cells, we compared OVA-specific T cells acti- and DCS mice, expressed high levels of Fas (Fig. 2B). vated in vitro under conditions that favor the development of FasL+ Downloaded from If Fas+ exudate cells are induced to undergo apoptosis by a + T cells. From previous studies, we know that FasL is expressed at FasL effector population, there should be more cell death in the higher levels upon secondary stimulation than after the first stim- TMPD-injected DCS mice than in the TMPD-injected BALB/c ulation, and that the restimulated WT T cells undergo activation- mice. To test this premise, we further analyzed the day 14 exu- induced cell death in vitro whereas Fas-deficient lpr/lpr cells do not dates for percentage of dead cells by determining the number of 7- (28). Therefore, to best study viable T cells, TCR transgenic DO11 + D

AAD cells. In keeping with the expected phenotype of the CS http://www.jimmunol.org/ 2/2 lpr and DO11 lpr DCS cells were stimulated with OVA peptide and and FasL mice, there was a trend toward greater numbers of 7- APCs, expanded in culture for 5 d, and then allowed to revert to AAD+ cells in the DCS mice and fewer numbers of 7-AAD+ cells 2/2 a resting phenotype. The rested cells were then restimulated with in the FasL mice when compared with the WT BALB/c mice plated-bound anti-CD3, and FasL expression was determined by (Fig. 2C). This trend was most significant in the neutrophil pop- FACS. Both populations expressed higher levels of FasL than did ulation, consistent with FACS data showing that neutrophils ex- resting cells, and the DO11 lpr DCS cells expressed more FasL pressed the highest level of Fas. than did the DO11 lpr cells, presumably because FasL was not We also examined the peritoneal exudate cells for FasL ex- cleaved from the surface (Fig. 3B). pression. In contrast to Fas expression, FasL expression is tightly To determine whether the level of FasL expressed by activated regulated and often difficult to detect. Moreover, the point mutation T cells was sufficient to induce inflammatory cytokine production, by guest on September 29, 2021 expressed by gld cells is recognized by most FasL-specific Abs. the rested DO11 cells were also cultured for 20 h with LPS-primed To accurately detect subtle changes in FasL expression, we took BMDCs in the presence of OVA peptide. Culture supernatants were 2/2 advantage of our FasL strain, as it does not express FasL pro- collected and tested for IL-1b by ELISA. The DO11 lpr DCS tein and is therefore an appropriate negative staining control (15). T cells induced more BMDC-derived IL-1b than did the DO11 lpr We were able to detect exceedingly low amounts of FasL on the T cells (Fig. 3C). Furthermore, IL-1b production was dependent R1 and R2 subsets (Supplemental Fig. 1A). In addition to the on the presence of OVA peptide and expression of Fas by the influx of monocytes and neutrophils, the peritoneal cavity of all BMDCs (Fig. 3D). These data show that DCS T cells express TMPD-injected mice contained a small, but substantially elevated, higher levels of functional FasL and that the level of FasL number of CD8+ and CD4+ T cells (Fig. 1B). We could not detect expressed by activated T cells is sufficient to drive myeloid cells to any difference in FasL expression on the CD8+ cells. However, make IL-1b through an Ag- and Fas-dependent mechanism. a small subset of the CD4+ cells, found only in the TMPD-injected If T cells are a relevant source of FasL in TMPD-injected mice, DCS mice, consistently expressed significantly higher levels of then TMPD-injected BALB/c Rag22/2 mice should develop

FIGURE 2. Relative levels of Fas ex- pression by peritoneal cavity washout cells and PECs reflect the extent of cell death in TMPD-injected mice. (A) Fas expression from cell types identified in Fig. 1, obtained from untreated BALB/c and BALB/clpr/lpr mice (R0) and day 14 TMPD-treated mice BALB/clpr/lpr (R1, R2, R3, shaded histo- grams), BALB/c (R1 and R2, line), and FasL2/2 mice (R3, hatched histogram). (B) Fas expression of Ly6G+ neutrophil subsets based on high Ly6C (R2*) and intermediate Ly6C (R2#) expression. (C)Numberofdead cells in total, R1, and R2 PEC populations from untreated (open bars) or day 14 TMPD- treated BALB/c, DCS, or FasL2/2 mice (filled bars). *p , 0.05, **p , 0.01. The Journal of Immunology 5 Downloaded from

FIGURE 3. DCS T cells express higher levels of FasL than do non-DCS T cells and induce more IL-1b from Fas+ target cells. (A) FasL expression of CD4+ T cells collected from untreated or day 14 TMPD-injected mice of the indicated genotypes (left, representative contour plots; right, percentage of cells in the indicated gate). (B) Histograms for FasL expression on unstimulated (shaded histogram) and anti-CD3–stimulated DO11 lpr T cells (line) or DO11 lpr DCS T cells (broken line). (C) Increasing numbers of DO11 lpr T cells (gray bars) or DO11 lpr DCS T cells (black bars) were cocultured for 20 h in the presence of OVA-peptide with BALB/c WT BMDCs, and IL-1b present in the supernatant was determined by ELISA. White bars depict control http://www.jimmunol.org/ coculture conditions in the absence of OVA peptide and presence of DO11 lpr DCS T cells (2OVA) or in the complete absence of T cells (2T cells). (D) DO11 lpr DCS T cells were cocultured for 20 h in the presence of OVA peptide together with BALB/c WT (open bars) or BALB/clpr/lpr BMDCs (filled bars), and IL-1b present in the supernatant was determined by ELISA. Cell-free membrane-bound FasL-expressing microvesicles (mFasLVes) and poly (deoxyadenylic-thymidylic) acid (dAdT) were used as controls (as described previously in Ref. 32). *p , 0.05. a suboptimal response. In fact, the total number of CD11b+ cells TMPD-treated mice (25). In contrast, both the resident peritoneal was markedly decreased in the Rag22/2 mice, and the Rag22/2 macrophages obtained from untreated mice and the low number mice retained a population of CD11b+Ly6C2 cells (Supplemental of Ly6Clow R3 macrophages isolated from the TMPD-treated Fig. 1B). These data are consistent with the notion that BALB/c mice exhibited a less active phenotype (Fig. 4A, Sup- by guest on September 29, 2021 expression of FasL contributes to the TMPD-induced inflamma- plemental Table 1). We also compared uninduced neutrophils tory response in the peritoneum, although the potential contribu- (isolated from normal bone marrow) to peritoneal neutrophils tion of other T cell cytokines and/or FasL+ cells, for instance isolated from the TMPD mice (R2). The morphology of these cells neutrophils (29), cannot be excluded. is shown in Fig. 4B. Remarkably, the difference between unin- duced neutrophils and TMPD-induced “inflammatory” neutrophils DCS mice display increased cytokine production in response to with regard to ISGs and inflammatory cytokine production was TMPD even greater than the difference between resident peritoneal macro- To explore the effect of the DCS mutation on the production of phages and inflammatory monocytes. cytokines and chemokines in TMPD-injected mice, peritoneal Of note, certain ISGs were differentially regulated in monocytes lavage fluid at 14 d after inoculation was assayed for proin- and neutrophils. For example, IFI204 was more highly induced in flammatory cytokine and content using a Luminex monocytes than in neutrophils, whereas IFIT1 was more highly array. Although most cytokines, including IL-1b and IL-1a, were expressed in neutrophils. This is in agreement with published results detected at comparable levels in DCS mice and BALB/c samples, showing that expression of IFI204 distinguishes the monocytic higher levels of IL-6, IL-9, and MCP-1 were detected in the lineage from the granulocytic lineage (31). In contrast, viperin was peritoneal fluid of the DCS mice compared with those of the uniformly upregulated in both TMPD-induced subsets. BALB/c mice (Supplemental Fig. 1C). The expression profile for the IL-1 family proinflammatory cytokines also revealed clearly distinguishable patterns. IL-18 Monocytes and neutrophils from day 14 TMPD-injected mice appeared to be constitutively expressed in the line- express a strong IFN signature and high levels of IL-1b and age cells but was dramatically upregulated in the TMPD-induced IL-18 neutrophils compared with resting bone marrow–purified neu- To elucidate the immune pathways activated by TMPD challenge, trophils. IL-18, similar to IL-1b, exists as a proform in the cytosol we used a NanoString code set, designed to quantify IFN-stimulated and has to be processed by caspase-mediated cleavage to gain genes (ISGs) and proinflammatory cytokines/chemokines (30). biological activity and be secreted. We have previously shown that We began by comparing the resident macrophage population FasL induces Fas+ BMDCs to transcribe and produce both IL-1b isolated from untreated mice (R0) to the major macrophage- and IL-18 (32). If FasL contributes to IL-18 production in DCS lineage populations identified in the peritoneum of TMPD-injected mice, then IL-18 levels in TMPD-injected mice should correlate BALB/c mice (R1, R3). We found that the inflammatory mono- with the potential capacity to make functional FasL. To address cytes (R1) expressed very high levels of ISGs such as IFN regu- this issue, we screened the peritoneal fluid collected from day 14 latory factor 7, viperin, IFIT1, IFIT2, CXCL10, and CCR2, con- TMPD-treated mice. In fact, the DCS samples contained signifi- sistent with the high levels of type I IFN previously reported for cantly higher levels of IL-18 protein, and the FasL-deficient 6 FasL OVEREXPRESSION EXACERBATES MURINE SLE

FIGURE 4. Both inflammatory monocytes and neutrophils from TMPD-injected mice upregulate ISGs and proinflammatory cytokines. (A) Heatmap depicting the gene expression of total peritoneal cells (PC) or indicated sorted peritoneal cell or sorted bone marrow populations from uninjected (2) or TMPD-treated (+) BALB/c mice. R0 cells (from untreated mice) or R1, R2, and R3 (from TMPD-injected mice) were sorted according to the gating strategy in Fig. 1A after depletion of B cells and T cells. Unactivated neutrophils were isolated from the bone marrow of untreated BALB/c mice. Samples from two different mice for the R1 and R2 groups are shown. (B) H&E stain of cells from the R1, R2, and R3 subsets used for gene expression 3 C profiling (original magnification 400). ( ) IL-18 Downloaded from cytokine concentration of peritoneal wash fluids collected from untreated mice (open bars) or day 14 TMPD-treated mice (filled bars) (6SEM, n = 8). (D) Viperin mRNA expression of total splenocytes from day 14 TMPD-injected mice of the indicated genotypes (6SEM, n = 3). *p , 0.05. http://www.jimmunol.org/

samples contained significantly lower levels of IL-18 when com- Moreover, to our knowledge this dataset characterizes for the first pared with the BALB/c samples (Fig. 4C). These data indicate that time the gene expression profile of all the major cell populations TMPD-induced IL-18 production is at least partially due to likely to be important in the proinflammatory feed-forward loops a process involving Fas/FasL interactions. Moreover, in the total instigated by TMPD. Thus, considering the overall impact of FasL by guest on September 29, 2021 peritoneal sample, IL-1b expression showed nearly a 50-fold in- overexpression on the early stages of TMPD-induced inflammation, crease over the untreated control cells, but IL-1a transcripts were it seemed likely that FasL would also contribute to the long-term not detectably upregulated. Of the three major CD11b+ cell frac- outcomes of TMPD challenge. tions, IL-1b message was most highly upregulated in neutrophils (R2), somewhat upregulated in monocytes (R1), and barely Overexpression of membrane-bound FasL leads to more severe changed in the F4/80+ fraction (R3) compared with their unstim- splenomegaly and neutrophilic infiltrates ulated counterparts. These data demonstrate that both monocytes To determine the effect of FasL overexpression on disease pro- and neutrophils produce significant amounts of proinflammatory gression, we went on to compare WT and DCS mice 6 mo after cytokines following TMPD injection. TMPD injection. As anticipated from chronic immune activation, In contrast to a previous report (33), we did not detect mRNA for there was a significant increase in spleen weight in TMPD-treated type I IFNs in the sorted inflammatory monocytes (or in any of the BALB/c mice compared with age-matched controls. Of note, the other exudate subsets). However, TMPD did not induce the up- spleen weight of the TMPD-treated DCS mice was even further regulation of ISGs in mice lacking a type I IFN receptor (data not increased (Fig. 5), whereas spleens from aged matched, untreated shown), confirming an important role for type I IFNs in this DCS mice were comparable in weight to untreated WT mice (data model. CD11c+ dendritic cells are considered major producers of not shown). Spleen weight was mirrored by a significant increase type I IFNs (34). However, neither the total peritoneal exudate in the number and percentage of splenic CD11b+ cells recovered cells (PECs) nor the total spleen cells (data not shown) expressed from TMPD-injected DCS mice and BALB/c mice. Normally very significant levels of type I IFN mRNA. Thus, the major cellular few neutrophils are detected in the spleens of unmanipulated source of type I IFN after TMPD challenge remains unknown and BALB/c mice. Histopathological examination of spleen sections warrants further investigation. confirmed the increased numbers of neutrophils in DCS mice Having established an IFN signature in the TMPD-injected mice, (Supplemental Fig. 2A). Thus, overexpression of FasL contributes we decided to compare day 14 spleen cells from WT, DCS, and to the long-term effects of TMPD injection. FasL2/2 mice for relative expression of one of the more highly expressed ISGs, viperin. Viperin plays an important role in TLR The autoantibody repertoire induced by TMPD is skewed by signal transduction (35) and the development of SLE-like disease in overexpression of membrane-bound FasL TMPD-injected mice is TLR-dependent. Importantly, at this time The DCS mutation had additional effects on the B cell compart- point, the FasL2/2 mice expressed significantly less viperin than did ment. Although there was considerable variability in the total IgG the FasL-sufficient strains (Fig. 4D). These results demonstrate that isotype titers, the overall ratio of IgG1/IgG2a serum isotype was FasL, either directly or indirectly (perhaps by increasing the number higher in the DCS compared with WT mice, even though total IgG of dead cells and cell debris), promotes the induction of ISGs. titers were comparably elevated in both strains (Fig. 6A). This The Journal of Immunology 7

present as a speckled nuclear pattern typical of SmB/D or spliceosome reactivity. In contrast, most TMPD-injected DCS mice exhibited a predominantly cytoplasmic staining pattern, or an atypical nuclear pattern seen infrequently among the TMPD- injected BALB/c mice (Fig. 6B). The absence of Sm-reactive Abs in the TMPD-treated DCS mice was confirmed by an Sm Ag ELISA (Fig. 6C). Untreated DCS mice were followed for up to 12 mo of age and were never seen to develop spontaneous ANAs (data not shown). To further evaluate the autoantibody repertoire, representative serum samples were screened on an autoantigen array (23). The array data recapitulated the overall change in ANA pattern ap- parent from the HEp2 screen. Certain autoantigens were prefer- FIGURE 5. TMPD induces significantly greater splenomegaly and entially targeted in DCS mice, whereas others were predominantly neutrophil accumulation in DCS mice. Spleen weight, total number of targeted in the WT mice. The change in Sm Ag specificity (Fig. splenocytes, and percentage total of the indicated subsets from untreated 6C) was confirmed by loss of small nuclear ribonucleoprotein BALB/c mice or TMPD-injected mice 6 mo after treatment are shown. (U1-snRNP-A, snRNP-BB9, U1-snRNP-C) reactivity as assessed Each dot represents an individual mouse (untreated BALB/c mice, n =7; TMPD BALB/c mice, n = 13; TMPD DCS mice, n = 13). Line indicates by the array (Fig. 6D, Supplemental Fig. 3). Of note, key auto- the mean. *p , 0.05, **p , 0.01, ***p , 0.001. antigens associated with kidney disease, such as C1q (36), were Downloaded from preferentially targeted in DCS mice. TMPD also promoted the production of autoantibodies reactive with neutrophil-associated may reflect elevated levels of Th2-associated cytokines such as IL- autoantigens. Intriguingly, BALB/c anti-neutrophil autoantibodies 9 (Supplemental Fig. 1C). werealmostexclusivelydirectedagainstanAgstoredinthe There was also an effect on the autoantibody specificity, as can azurophilic granules of activated neutrophils, the lipid transfer

be seen from the immunofluorescence staining patterns of HEp2 protein bactericidal permeability/increasing protein (37). In con- http://www.jimmunol.org/ cells. TMPD-injected BALB/c mice frequently make autoanti- trast, DCS autoantibodies did not target bactericidal permeability/ bodies associated with TLR7-driven responses that commonly increasing protein, but reacted preferentially with a neutrophilic by guest on September 29, 2021

FIGURE 6. Ab and autoantibody differ- ences between TMPD-treated DCS and WT mice. (A) Total serum levels of IgG1, IgG2a, and IgG2b in mice 6 mo after TMPD and the calculated IgG2a/IgG1 ratio for BALB/c and DCS mice (BALB TMPD mice, n = 9; TMPD DCS mice, n = 7). Each dot represents an in- dividual mouse. Original magnification 3200. (B) Representative images of HEp2 ANA staining patterns from untreated and TMPD- treated BALB/c and DCS mice. (C) SmB/D autoantibody titers of BALB/c and DCS mice 6 mo after TMPD treatment. (D) MFI (mean 6 SEM; TMPD BALB/c mice, n = 4; TMPD DCS mice, n = 6) for IgG and IgM autoanti- bodies against selected autoantigens as quan- tified by autoantigen array. **p , 0.01. 8 FasL OVEREXPRESSION EXACERBATES MURINE SLE protein stored in the very same granules, myeloperoxidase. The thymoma cell line BW5147 (41), and second, the loss of auto- two strains also differed as far as histone reactivity; WT mice had antibodies reactive with RNA-associated autoantigens in FasL- a stronger reactivity to total histones and the H2a subtype, deficient TMPD-injected mice (20). Moreover, certain SLE whereas histone H3 was predominantly targeted by the DCS mice. patients have lower levels of potentially antagonistic soluble FasL These data demonstrate that the DCS mutation dramatically in their serum than do healthy controls (42). We therefore decided reshapes the autoantibody repertoire of TMPD-treated mice and to further explore the potential contribution of FasL and FasL that Fas/FasL interactions skew the type of ensuing autoimmune cleavage to TMPD-induced inflammation. response. Fas and FasL deficiencies have been repeatedly associated with loss of B cell tolerance, lymphoid hypertrophy, and SLE-like dis- FasL overexpression leads to more severe renal disease ease in numerous autoimmune-prone and nonautoimmune-prone TMPD-injected BALB/c mice develop early signs of renal disease mouse strains, as well as in patients that inherit loss-of-function at ∼6 mo of age and therefore provide a useful model for assessing mutations in either the Fas or FasL genes (1, 7, 43). Presumably the effects of the DCS mutation on SLE-associated glomerulo- autoreactivity in all these instances reflects the absence of FasL- nephritis. Importantly, urine samples collected from the DCS mice induced apoptosis of autoreactive lymphocytes and/or a relevant at 6 mo of age contained significantly higher levels of albumin autoantigen-presenting APC. However, consistent with the corre- than did samples collected from the BALB/c mice (p , 0.01) lation between a gain-of-function FasL variant and increased (Fig. 7A). As a further measure of renal disease, kidneys obtained risk for SLE in an African American cohort (21), we now dem- from BALB/c and DCS mice were subjected to histopathological onstrate that FasL is a two-edged sword in that FasL can also analysis to evaluate glomerular and interstitial pathology. Con- promote both the early inflammatory response as well as the Downloaded from sistent with the proteinuria data, the TMPD-treated DCS mice had subsequent autoimmune disease triggered by TMPD inoculation. greater interstitial and glomerular disease scores than did the Moreover, FasL overexpression can further exacerbate renal pa- TMPD-treated BALB/c mice (Fig. 7B). Frozen sections of kidneys thology in these mice. were also stained with a fluorescent anti-IgG Ab to assess the There are a number of possible mechanisms whereby failure extent of glomerular immune complex deposition. The DCS mice to appropriately cleave FasL, and thereby extend the expression

had more extensive glomerular IgG deposition (Fig. 7C, Supple- time of the full-length molecule, may promote autoimmunity. One http://www.jimmunol.org/ mental Fig. 2B), perhaps due to autoantibodies reactive with C1q possibility is that greater FasL expression leads to more extensive and/or collagen type IV or other renal components, and such cell death and the release of endogenous danger signals and/or self- deposits most likely promoted renal pathology. Hence, the non- Ags. Increased autoantigen load could also promote the upregu- cleavable mutation in FasL expressed in the DCS mice leads to lation of IFN-inducible genes through TLR-dependent pathways. worsened autoimmune kidney disease. TMPD had previously been shown to induce cell death, especially in peritoneal B cells and dendritic cells, that appeared to be at least Discussion partly attributable to FasL-mediated apoptosis (41). Importantly, TMPD-injected BALB/c mice routinely develop a form or auto- resident peritoneal macrophages in untreated WT mice express immune disease that resembles other genetically programmed high levels of Fas, and i.p. injection of FasL+ tumor lines or FasL- by guest on September 29, 2021 models of SLE. They make similar autoantibodies, develop glo- expressing microvesicles can lead to the death of the resident merular nephritis, and disease progression is regulated by endo- Fas+ macrophages within a matter of hours (4). Therefore, it is not somal TLRs (25, 38). In humans, environmental exposure to surprising that this same resident macrophage population, essen- hydrocarbons is considered a potential risk factor for the devel- tially the R0 population, disappears by day 14 in TMPD-injected opment of autoimmune diseases (39, 40). Moreover, hydrocarbons BALB/c and DCS mice, but persists in the FasL-deficient strains. are widely used to potentiate vaccines, as in the case of the ad- Moreover, 14 d after TMPD injection, the neutrophil R2 cells juvant MF59, where the active ingredient squalene is structurally express high levels of Fas, and the extent of neutrophil death is related to TMPD. Therefore, elucidating mechanisms by which increased in DCS mice and decreased in FasL-deficient mice hydrocarbons promote immune activation, inflammation, and au- compared with controls. These data are consistent with the notion toimmunity is highly relevant. Previous reports have documented, that FasL directly contributes to TMPD-induced cell death. first, the capacity of TMPD to induce the transcription of both Fas However, FasL engagement of resident peritoneal macrophages and FasL in primary peritoneal macrophages and in the murine not only induces apoptosis, but also induces these cells to more

FIGURE 7. Lupus nephritis and proteinuria are exacerbated in TMPD-treated DCS mice. (A) Urine albumin (mg/ml) measured by ELISA and obtained from TMPD-treated BALB/c (n = 17) and DCS mice (n = 11) at 6 mo after TMPD injection. Statistical comparison was made using a two-tailed Mann– Whitney U test. (B) Interstitial and glomerular scores for renal pathology were determined at 6 mo after TMPD injection, as described in Materials and Methods, and reported as mean values (6SEM; untreated BALB/c mice, n = 5; TMPD BALB/c mice, n = 12; TMPD DCS mice, n = 13). Each dot represents an individual mouse. (C) Glomerular IgG deposition was quantified (glomerular MFI of the green channel; representative images are depicted in Supplemental Fig. 2B) from individual glomeruli of TMPD-injected mice 6 mo after treatment. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 9 rapidly transcribe and secrete a number of proinflammatory TMPD-elicited populations to their uninduced counterparts with cytokines/chemokines, including IL-1b, MIP2, MIP1a, MCP-1, regard to a panel of ISGs and proinflammatory cytokines. The and KC, cytokines known to recruit myeloid cells to the perito- TMPD-induced inflammatory monocytes (R1 gate) and especially neal cavity (4). Proinflammatory cytokine production in response the R2 neutrophils (R2 gate) dramatically upregulated IL-1b, to FasL engagement has been demonstrated for many other cell CXCL10, TNF-a, and ISGs. Thus, we propose the designation of types as well (27, 32, 44, 45). Therefore, a second mechanism “inflammatory neutrophils” for the R2 population, as they most whereby FasL promotes inflammation could be by inducing cy- likely represent a major source of the cytokines and chemokines tokine production. responsible for disease progression. Intriguingly, we found that peritoneal fluid collected from the One of the TMPD-induced ISGs, viperin, is rapidly induced in day 14 DCS mice contained higher levels of MCP-1, IL-6, and IL- response to an extensive range of viral infections, and depending on 9 than did fluid collected from the day 14 BALB/c mice. MCP-1 is the circumstances can be downstream of TLR3, TLR4, TLR7, or known to recruit monocytes to sites of inflammation and is likely TLR9 engagement. It is also induced by the retinoic acid–inducible to contribute to the peritoneal inflammation elicited by TMPD. IL- gene I–like receptor and stimulator of IFN genes signaling path- 6isalsoconsideredaproinflammatory cytokine, produced by ways. Importantly, viperin is a critical mediator of TLR7- and macrophages and dendritic cells during an inflammatory response TLR9-dependent production of type I IFNs and thus contributes to and can be directly induced by Fas in fibroblasts (44). It can host defense against viral infections (35). Considering the con- stimulate the production of neutrophils in the bone marrow, sup- nection between TLR7 and TLR9 and the response to nucleic port the growth of B cells, and is known to play a key role in the acid–associated autoantigens, viperin is also likely to play a piv-

TMPD response (46). The role of IL-9 in this context is less clear, otal role in the activation of both dendritic cell and autoreactive Downloaded from but interestingly IL-1 has been shown to promote Th2 production B cell subsets. Viperin also associates with cell membranes and of IL-9 (47). can disrupt lipid rafts. Additionally, it can be found in the endo- Additionally, IL-9 has been shown to contribute to the inflam- plasmic reticulum and in lipid droplet–like vesicles in neutrophils matory response associated with EAE (48), to promote B-1 B cell (60). Therefore, viperin could further modulate cell death as well expansion (49), and to promote the survival of multiple cell types as neutrophil and macrophage function in autoimmune disease.

(50). Interestingly, IL-1b promotes the induction of all three Finally, viperin has been found to promote the differentiation of http://www.jimmunol.org/ cytokines, and FasL is known to induce the transcription and Th2 cells (61), and the slight increase in viperin expression may cleavage of IL-1b through a caspase-8–dependent pathway (32). contribute to the shift in the IgG2a/IgG1 serum titers of the We were able to show that in vitro–activated DCS T cells induced TMPD-injected DCS mice. Thus, viperin warrants further inves- myeloid cells to produce more IL-1b than did non-DCS–activated tigation in the context of autoimmune disease. T cells. Among its pleiotropic effects, IL-1 can also drive T cells Owing to the prominent expression of viperin in the TMPD- toward the Th17 lineage (51), and Th17 cells are a prominent activated peritoneal cell populations, revealed by the microarray effector population in SLE (52, 53). Future studies will examine data, we went on to examine viperin expression in the TMPD- whether IL-1b contributes to preferential induction of Th17 cells stimulated spleen cells from the DCS and the FasL-deficient in DCS mice. strains. Viperin expression was modestly increased in the TMPD- by guest on September 29, 2021 FasL can also induce the cleavage of IL-18 (32) and, quite re- treated DCS mice over TMPD-treated BALB/c mice. More re- markably, we found that the IL-18 protein level in peritoneal fluid markably, we found that viperin expression was not detectably collected from day 14 TMPD-injected mice was significantly upregulated in the FasL2/2 mice, at least at this time point, and higher in the DCS compared with the BALB/c group and signif- was significantly lower in the gld mice compared with WT levels. icantly lower in the FasL2/2 group. Elevated levels of IL-18 in the From prior studies, we know that gld mice, expressing a point Fashi R2 neutrophils may well be a source of the IL-18 that mutation in FasL, can make low levels of functional protein accumulates in the peritoneal fluid. IL-18 is a pleiotropic proin- whereas FasL is completely missing in the FasL2/2 mice. Overall, flammatory cytokine initially identified as an IFN-g–inducing these data clearly demonstrate that FasL contributes to the acti- cytokine (54), but depending on the cytokine milieu it can aug- vation of a number of major ISGs, most likely by increasing ment both Th1 and Th2 responses (55). Furthermore, IL-18 is the amount of autoantigen debris and ensuing TLR-dependent increased in patients with SLE (56), and IL-18 injections wors- responses. ened autoimmune glomerulonephritis in the MRL-Faslpr/lpr lupus Despite the pronounced IFN signature, in contrast to a previous mouse model (57). Additionally, FasL itself is induced by IL-18 report (25), our NanoString array did not show an increase in (58), and therefore increased levels of IL-18 may preferentially either IFN-a or IFN-b RNA levels in either the total peritoneal drive a deleterious feed-forward loop in the DCS mice, promoting washout or the sorted populations from the TMPD-activated mice. further FasL-induced apoptosis, inflammation, and tissue damage. Type I IFN is clearly a critical factor in TMPD SLE, as TMPD- Reeves and colleagues (25) have shown that neutrophil re- injected mice that lacked a type I IFN receptor did not upregulate cruitment to the peritoneal cavity is at least partially dependent on ISG expression (L. Bossaller, unpublished observations), failed to IL-1a, whereas the recruitment of inflammatory monocytes de- produce the common SLE autoantibody reactivity, and did not pends on MyD88 and type I IFN, presumably via TLR7. However, develop proteinuria or histological evidence of renal disease (62). they found that TLR7 deficiency does not lead to as complete Further studies are required to identify the major IFN-producing a reduction of inflammatory monocytes as does MyD88 defi- cell types in TMPD-stimulated mice. ciency. Intriguingly, Fas activation has been reported to induce Based on the differences in cell phenotypes and cytokine levels production of the proinflammatory chemokines CXCL1/KC and between the DCS and BALB/c groups detected at day 14, we went CXCL2/MIP2 through a MyD88-dependent, IL-1/caspase–inde- on to examine the long-term effects of FasL overexpression in pendent pathway (59), and this might account for the TLR-inde- TMPD-injected mice. By 6 mo after injection, the spleens of the pendent effect of MyD88 in recruitment. DCS mice were almost 50% larger than the spleens of the BALB/ To better define the properties of the infiltrating monocytes c mice and contained a significantly greater number of neu- and neutrophils in TMPD mice at the day 14 time point, we trophils. SLE patients frequently express a neutrophil signature, used NanoString-based gene expression profiling to compare the and neutrophils can contribute to the disease process through 10 FasL OVEREXPRESSION EXACERBATES MURINE SLE a variety of mechanisms that include proinflammatory cytokine In summary, our studies using both loss-of-function and gain-of- production, release of elastase, myeloperoxidase, and various function FasL mutant mice reveal a critical FasL/Fas signaling axis cathepsins than can cause vascular damage, tissue injury, and in the well-characterized TMPD model of systemic autoimmunity. glomerular destruction (63). Additionally, neutrophil extracellu- These data establish that not only loss of Fas signaling but also lar traps, released by IFN-primed and then immune complex– increased Fas signaling can contribute to SLE, and they point to the activated neutrophils, have been proposed as a key source of potential therapeutic application of Fas/FasL antagonists. autoantigen in the context of SLE (64). Therefore, the discovery of increased numbers of neutrophils in the DCS mice, as well as Acknowledgments the concomitant upregulation of Fas expression on activated We thank Glennice Ryan for assistance with the Luminex analysis, Zhaozhao neutrophils, is likely to be a highly relevant outcome of FasL Jiang and Suzann Carpenter for assistance in the nanostring analysis, and overexpression. Ted Giehl and Marc Barnard for excellent technical help in cell sorting, Based on the previous report from Reeves and colleagues (25), Lisa Waggoner for outstanding technical support, and Lino Teichman, we were particularly interested in examining the effect of FasL Mark Shlomchik, Evelyn Kurt-Jones, and Krishna Moody for advice and overexpression on autoantibody repertoire selection. A careful discussion. comparison of the autoantibodies elicited in the BALB/c and DCS mice revealed intriguing differences in specificity, first apparent in the ANA staining patterns. Interestingly, C1q is known to bind Disclosures apoptotic cells and apoptotic membrane blebs, and it thereby aids The authors have no financial conflicts of interest. in the timely removal of apoptotic debris. Thus, in the presence Downloaded from of anti-C1q Abs, apoptotic cells can no longer be efficiently re- References moved. These apoptotic cells can then progress to secondary ne- 1. Watanabe-Fukunaga, R., C. I. 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