Induction of Systemic Autoimmunity by a Xenobiotic Requires Endosomal

Induction of Systemic Autoimmunity by a Xenobiotic Requires Endosomal TLR Trafficking and Signaling from the Late Endosome and Endolysosome but Not Type I This information is current as IFN of September 23, 2021. K. Michael Pollard, Gabriela M. Escalante, Hua Huang, Katarina M. Haraldsson, Per Hultman, Joseph M. Christy, Rahul D. Pawar, Jessica M. Mayeux, Rosana

Gonzalez-Quintial, Roberto Baccala, Bruce Beutler, Downloaded from Argyrios N. Theofilopoulos and Dwight H. Kono J Immunol published online 20 October 2017 http://www.jimmunol.org/content/early/2017/10/20/jimmun ol.1700332 http://www.jimmunol.org/

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 20, 2017, doi:10.4049/jimmunol.1700332 The Journal of Immunology

Induction of Systemic Autoimmunity by a Xenobiotic Requires Endosomal TLR Trafﬁcking and Signaling from the Late Endosome and Endolysosome but Not Type I IFN

K. Michael Pollard,* Gabriela M. Escalante,* Hua Huang,† Katarina M. Haraldsson,† Per Hultman,‡ Joseph M. Christy,* Rahul D. Pawar,* Jessica M. Mayeux,* Rosana Gonzalez-Quintial,† Roberto Baccala,† Bruce Beutler,x Argyrios N. Theoﬁlopoulos,† and Dwight H. Kono†

Type I IFN and nucleic acid–sensing TLRs are both strongly implicated in the pathogenesis of lupus, with most patients expressing IFN-induced genes in peripheral blood cells and with TLRs promoting type I IFNs and autoreactive B cells. About a third of Downloaded from systemic lupus erythematosus patients, however, lack the IFN signature, suggesting the possibility of type I IFN–independent mechanisms. In this study, we examined the role of type I IFN and TLR trafficking and signaling in xenobiotic systemic mercury- induced autoimmunity (HgIA). Strikingly, autoantibody production in HgIA was not dependent on the type I IFN receptor even in NZB mice that require type I IFN signaling for spontaneous disease, but was dependent on the endosomal TLR transporter UNC93B1 and the endosomal proton transporter, solute carrier family 15, member 4. HgIA also required the adaptor protein-3 complex, which transports TLRs from the early endosome to the late endolysosomal compartments. Examination of TLR http://www.jimmunol.org/ signaling pathways implicated the canonical NF-kB pathway and the proinflammatory cytokine IL-6 in autoantibody production, but not IFN regulatory factor 7. These findings identify HgIA as a novel type I IFN–independent model of systemic autoimmunity and implicate TLR-mediated NF-kB proinflammatory signaling from the late endocytic pathway compartments in autoantibody generation. The Journal of Immunology, 2017, 199: 000–000.

he a and b type I IFNs are pleiotropic cytokines with genes induced by type I IFNs (IFN signature) in peripheral diverse effects on the immune system through their ac- blood cells, which in some studies correlate with disease T tions on T and B lymphocytes, macrophages, and den- activity and severity (8–10). However, although virtually all dritic cells (DCs) (1). They are critical mediators of both innate pediatric SLE patients exhibit an IFN signature (11), ∼30% by guest on September 23, 2021 and adaptive immune responses, and are required for the eradi- of adult SLE patients do not, suggesting there may be other cation of certain viral and bacterial infections (2, 3). It is therefore mechanisms or factors that bypass the requirement for IFN. not surprising that type I IFNs play a significant role in autoim- In contrast, most lupus-prone strains exhibit modest to absent elevations in type I IFN or IFN signature compared with human munity (4–6). Indeed, lupus and other autoimmune diseases may SLE (12). Yet, IFN-a/b receptor 1 (Ifnar1)–deficiency and loss of occur following type I IFN treatment for cancer, hepatitis, and type I IFN signaling markedly reduces the production of autoan- other disorders (7). Moreover, patients with systemic lupus tibodies and other disease manifestations (13–15), whereas sus- erythematosus (SLE) and other autoimmune diseases express tained type I IFN exposure exacerbates autoimmunity (16–18). The exception is the MRL strain, for which Ifnar1-deficiency and *Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA IFN-b treatment have the opposite effects on disease (19, 20). 92037; †Department of Immunology and Microbiology, The Scripps Research Insti- tute, La Jolla, CA 92037; ‡Department of Experimental and Clinical Medicine, Link- IFN-g (type II IFN) is also important for the development of lupus x o¨ping University, Linko¨ping 58183, Sweden; and Center for the Genetics of Host and is required in all spontaneous and induced mouse models Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390 examined so far (21–26). ORCIDs: 0000-0003-3182-1947 (G.M.E.); 0000-0002-9089-6706 (J.M.C.); 0000- Although many cell types can produce type I IFN, plasmacytoid 0002-6094-6063 (R.D.P.). DCs (pDCs) have been identified as the most effective (27) and Received for publication March 6, 2017. Accepted for publication September 25, have been implicated as major contributors to lupus. Accordingly, 2017. deletion of DCs, including pDCs (28), defective pDC development This work was supported by the National Institutes of Health (Grants ES007511, ES021464, and ES022625 to K.M.P. and HL114408 to D.H.K). This is manuscript due to IFN regulatory factor 8 (IRF8) deficiency (29), impaired number 29475 from The Scripps Research Institute. pDC function from reduced expression of the transcription factor Address correspondence and reprint requests to Dr. K. Michael Pollard, Department E2-2 (Tcf4) (30), or early transient depletion of pDCs (31) all of Molecular Medicine, MEM125, The Scripps Research Institute, 10550 North Tor- reduced lupus. Furthermore, the disease is also inhibited in mice rey Pines Road, La Jolla, CA 92037. E-mail address: [email protected] deficient for the proton peptide and histidine transporter, solute The online version of this article contains supplemental material. carrier family 15, member 4 (SLC15A4), in which pDCs are un- Abbreviations used in this article: ANA, anti-nuclear Ab; AP-3, adaptor protein-3; cDC, conventional DC; DC, dendritic cell; ER, endoplasmic reticulum; HgIA, able to produce type I IFNs in response to endosomal TLRs mercury-induced autoimmunity; pDC, plasmacytoid DC; poly(I:C), polyinosinic- (TLR3, 7, 9) and B cells are defective in IgG2c class switching polycytidylic acid; SLC15A4, solute carrier family 15, member 4; SLE, systemic (29, 32–35). lupus erythematosus; TSRI, The Scripps Research Institute. The requirement for endosomal TLR signaling in systemic au- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 toimmunity is well established in models of lupus and is consistent

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700332 2 AUTOIMMUNITY REQUIRES ENDOSOMAL TLRs BUT NOT TYPE I IFN with the genetics and pathogenesis of human SLE (4). Notably, mice Carlsbad, CA). ANAs were scored for intensity (0 to 4+ scale) and pattern with nonfunctional UNC93B1 (Unc93b13d/3d, 3d), which regulates under blinded conditions. ELISAs were used to quantitate anti-chromatin trafficking of TLRs from the endoplasmic reticulum (ER) to (61, 62), ENA5 (Sm, RNP, SS-A/Ro, SS-B/La, Scl70) (Inova Diagnostics), anti-DNA, serum IgG (Immunology Consultants Laboratory, Newburg, endolysosomes, do not respond to ligands of endolysosomal TLR3, OR) (61, 63). A mouse reference serum (Bethyl Laboratories, Mont- TLR7, and TLR9 (36), and consequently 3d lupus-prone B6-Faslpr gomery, TX) was used to generate the standard curve (800–0.04 ng/ml). and BXSB mice fail to develop severe disease (37). Endosomal TLR signaling leads to type I IFN production via IRF7 and the Immunohistology induction of proinflammatory cytokines IL-6, IL-12, proIL-1b,and Sections of kidney and spleen were stained for direct immunofluorescence TNF-a via NF-kB, MAP kinase, and IRF5 pathways (34, 38, 39). as previously described (26). Briefly, 4 mm sections were fixed with eth- Evidence further suggests that TLR-mediated type I IFN and NF- anol, then incubated with serial dilutions of FITC-conjugated goat anti- mouse IgG Abs (Southern Biotechnology, Birmingham, AL). Glomerular kB signaling occurs in distinct endosomal compartments (40) and deposits were graded by the highest dilution of Ab that could detect a involves both endocytic and phagocytic endosomal pathways (41). specific fluorescence (end-point titer). Positive titers ,1:40 were consid- The contribution of these TLR signaling endosomal constituents, ered background. Vessel wall IgG deposits were graded on a 0 to 4+ scale particularly those related to NF-kB, have yet to be defined for au- (25). Slides were examined under blinded conditions. toimmune disease. TLR agonist and HgCl stimulation of cytokine expression Mercury is a widely distributed environmental and industrial 2 pollutant (42), which in both humans and animals induces a milder Mice received 100 mg of polyinosinic-polycytidylic acid [poly(I:C)], 50 mg form of autoimmunity including autoantibodies and membranous of R848 (InvivoGen, San Diego CA), or 40 mg of HgCl2 in 200 ml PBS s.c. Preliminary experiments determined that maximal cytokine expression nephropathy (43–46). The characteristics of mercury-induced au- Downloaded from occurred 6 h after poly(I:C) or HgCl2, and 3 h after R848 injections. These toimmunity (HgIA) are similar to SLE and include lymphocyte exposure periods were used to measure IFN-a (LumiKine Xpress; Invi- proliferation, increased class II MHC expression, hyper- voGen), TNF-a, and IL-6 (ELISA MAX; BioLegend, San Diego, CA) by gammaglobulinemia, polyclonal Abs to self-antigens, notably ELISA. anti-nuclear Abs (ANAs), and to some extent immune complex Flow cytometry deposits in mice (47), as well as necrotizing vasculitis in rats (48– + Single-cell suspensions were prepared from spleens by passage through a 70 52). Furthermore, HgIA requires CD4 T cells, B and T cell co- mm strainer before and after RBC lysis, and two washes with FACS buffer http://www.jimmunol.org/ stimulatory molecules, IFN-g, and susceptible MHC and back- (PBS 2% FCS, 0.5 mM EDTA). Cells (106) were incubated with anti- ground genes (25, 53–57), which strongly supports the possibility mouse CD16/32 (BD Pharmingen, San Diego, CA) to block Fc receptors of related or identical pathogenic mechanisms as spontaneous and Zombie Red (BioLegend) to identify dead cells, followed by washing lupus. with FACS buffer and staining with the following anti-mouse Abs for specific cell-surface markers purchased from BD Pharmingen or BD Ho- In this paper, we dissected the roles of type I IFN and TLR rizon (San Diego, CA): CD3e (FITC, 145-2C11), CD4 (PerCP-Cy5.5, endosomal trafficking and signaling pathways in autoantibody pro- RM4-5), CD8a (BV510, clone 53-6.7); and from BioLegend, CD55 (PE, duction induced by mercury by examining mice with relevant gene clone RIKO-3), and CD44 (BV650, clone IM7). Data were acquired using defects. Our findings showed that xenobiotic autoimmunity differed BD FACS DIVA software and an LSRII flow cytometer, and analyzed with

FlowJo V10 (Tree Star, Ashland, OR). by guest on September 23, 2021 from spontaneous lupus in being type I IFN independent, but was similar in requiring endosomal TLR signaling and endosomal his- Statistics tidine transport. HgIAwas also shown to require the adaptor protein-3 Data are expressed as mean and SE and analyzed by GraphPad Software (AP-3) complex, Nfkb1,andIl6, but not IRF7. Thus, whereas TLR- V5.02 (San Diego, CA). An unpaired two-tailed Mann–Whitney U test was induced NF-kB signaling is thought to occur in early endosomes used for comparison between PBS and HgCl2 exposure within a strain. For (40), our findings indicate that late endosome and endolysosome and multiple comparisons, one-way ANOVA with Tukey posttest was used for not early endosome NF-kB signaling compartments are essential for comparing all possible combinations of HgCl2-exposed or PBS groups. Only p values #0.05 are shown. TLR-mediated HgIA autoantibody production. Results Materials and Methods Systemic autoimmunity induced by HgCl2 is type I IFN Mice independent 2/2 3d/3d NZB/B1Scr, NZB/B1Scr-Ifnar1 , BXSB/Scr, BXSB/Scr-Unc93b1 , 2/2 2 2 To determine if type I IFN is required for HgIA, B6-Ifnar1 mice C57BL/6J-Inept (B6-Inept), B6-Feeble, B10.S/SgMcdJ, and B10.S-Il6 / were as described previously (13, 35, 37, 58). B6, B6Pin.C3-Ap3b1pe/J lacking the common receptor for all type I IFNs were exposed to tm1Bal tm1Agt (pearl), B6.Cg-Nfkb1 /J, and B6.129S2-Ifnar1 /Mmjax mice HgCl2 and autoantibody production was assessed. Strikingly, after 4 were obtained from The Jackson Laboratory (Bar Harbor, ME). All strains wk, ANA and autoantibodies to chromatin, DNA, and ENA5 in were maintained at The Scripps Research Institute (TSRI). HgCl2-exposed Ifnar1-deficient mice were significantly elevated 2/2 Induction of autoimmunity compared with PBS-treated Ifnar mice. The levels of these autoantibodies tended to be lower among Ifnar12/2 mice than in Male and female mice (6–16 wk-old) were injected with 40 mg HgCl2 wild type B6 mice given HgCl . However, this only reached sta- (Mallinckrodt Baker, Phillipsburg, NJ) in 100 ml PBS s.c. 23 per week up 2 tistical significance for anti-ENA5, where levels were low and could to 5 wk; controls received PBS alone (25). The HgCl2 dose is consistent 2/2 with occupational exposure (59, 60). Procedures were approved by the be related to lower total IgG concentrations in Ifnar1 compared TSRI Institutional Animal Care and Use Committee and the TSRI De- with wild type mice (Fig. 1A, Supplemental Fig. 1). Increases in partment of Environmental Health and Safety. Unless indicated, experi- both IgG1 and IgG2a anti-chromatin were observed (Supplemental mental groups were an equal mix of male and female mice. Fig. 2). Thus, significant autoantibody production induced by HgCl2 Serology in B6 mice occurs in the absence of type I IFN signaling. Susceptibility to HgIA is heavily influenced by autoimmune- ANAs were determined by ELISA per the manufacturer’s instructions predisposing genes (64). Therefore, to eliminate the possibility (Inova Diagnostics, San Diego, CA) or indirect immunofluorescence as described (61). Briefly, for the latter, HEp-2 cells on glass slides (Inova) that the less lupus-prone B6 strain might be uniquely type I IFN were incubated with serum diluted 1/100 and then 1/200 dilution of Alexa independent, we examined the effect of type I IFN deficiency on Fluor 488–conjugated goat anti-mouse IgG (H&L) (Molecular Probes, HgIA in the NZB, a strain we previously showed was highly The Journal of Immunology 3 Downloaded from

FIGURE 1. Type I IFN is not required for HgIA in B6 and autoimmune-prone NZB mice. (A) Wild type or Ifnar1-deficient B6 (n = 8 per group) or (B) wild type or Ifnar1-deficient NZB mice (n = 3–12 per group) were given 40 mg HgCl2 (filled circle) or PBS alone (open circle) s.c. twice a week for 4 wk, and sera were obtained 3 d after the final injection. Total IgG, ANA, and anti-chromatin were determined as described in Materials and Methods. Mean and SE (horizontal lines) and p values , 0.05 between groups are indicated. http://www.jimmunol.org/ dependent on type I IFN for spontaneous lupus (13). Remarkably, dicating that trafficking of TLRs from the ER to endolysosomal Ifnar1-deficient NZB mice were similarly susceptible to HgIA as and phagosomal compartments is required for type I IFN–independent wild type NZB mice with increases in serum IgG, ANA, and anti- autoimmunity. 2/2 chromatin (Fig. 1B). All HgCl2-exposed NZB-Ifnar1 and wild TLR7/9-induced responses, critical for the development of auto- type mice were positive for homogeneous ANAs. Finally, com- antibodies, are mediated through the MyD88-TRAF6 complex and parable amounts of glomeruli and vessel wall IgG, IgM, and C3- activation of the IRF7, canonical NF-kB, MAP kinase, and IRF-5 containing immune deposits were detected after exposure to pathways (67). Of these, IRF7 primarily serves to promote the 2/2

HgCl2 in Ifnar1 and wild type NZB mice (Table I). Thus, production of type I IFN, so we hypothesized that it was likely by guest on September 23, 2021 HgIA is type I IFN independent even in a strain that requires type I dispensable for type I–independent autoimmunity. Indeed, IRF7- IFN for spontaneous disease (13). deficient Inept mice, which fail to produce IFN-a upon TLR agonist injection (35), responded to HgCl like B6-Ifnar12/2 mice with Type I IFN–independent autoimmunity is dependent on 2 no significant differences in total serum IgG, ANA, or anti-chromatin endosomal TLR trafficking and signaling, but not on IRF7 autoantibody levels when compared with IRF7-intact wild type mice To dissect the pathogenesis of type I IFN–independent autoim- (Fig. 2B). Similar observations were made for anti-DNA and anti- munity, we next focused on the endosomal TLRs as TLR7 and ENA5 responses (Supplemental Fig. 1) and for IgG1 and IgG2a anti- TLR9 (TLR7/9) are critical for the production of both type I IFNs chromatin subclasses (SupplementalFig.2).Thus,theendosomal and autoantibodies in spontaneous lupus models (65). Accord- TLR-induced pathways critical for HgIA can be inferred to involve ingly, we examined the effect of mercury on lupus-prone BXSB one or more of the proinflammatory pathways. mice expressing the Unc93b13d/3d (3d) mutation that blocks endosomal nucleic acid–sensing TLR trafficking and signaling as Type I IFN–independent autoimmunity requires adaptor well as spontaneous lupus (37, 66). In sharp contrast to type I IFN, protein 3 complex–dependent late endosomes endosomal TLR signaling was required for induction of hyper- To further define the TLR-induced pathways required for type I gammaglobulinemia and autoantibodies by HgCl2 (Fig. 2A), in- IFN–independent autoimmunity, we examined pearl mice deficient

Table I. Immune complex deposition in mercury-exposed Ifnar12/2 and wild type NZB mice

IC Deposits NZB-Ifnar12/2 NZB Glomerular IgG 4480 6 640a (67 6 13)b 4480 6 640 (267 6 187) Glomerular IgM 2.3 6 0.25 (2.7 6 0.3) 2.5 6 0.29 (2.0 6 0.6) Glomerular C3 2565 6 905 (4267 6 853) 3215 6 635 (2137 6 428) Kidney vessel IgG 3.0 6 0(06 0) 3.0 6 0(06 0) Kidney vessel C3 2.5 6 0.29 (0 6 0) 2.5 6 0.29 (0 6 0) Spleen vessel IgG 2.8 6 0.25 (0 6 0) 3.0 6 0(06 0) Spleen vessel C3 1.8 6 0.25 (0 6 0) 2.5 6 0.29 (0 6 0) See Fig. 1B legend for treatment details. aMean 6 SEM for detecting Ab titer or immunoﬂuorescence score (63). n = 4 per group, p . 0.05 for all unpaired comparisons between mercury-exposed Ifnar12/2 versus wild type NZB mice. bNumbers in parentheses are mean 6 SEM for PBS-treated groups. n = 3 per group. IC, immune complex. 4 AUTOIMMUNITY REQUIRES ENDOSOMAL TLRs BUT NOT TYPE I IFN Downloaded from

FIGURE 2. Type I IFN–independent HgIA requires UNC93B1, but not IRF7. (A) BXSB-3d mice and control BXSB (n = 4–6 per group) or (B) IRF7- deficient B6 (Inept) and control B6 mice (n = 8 per group) were given HgCl2 (filled circle) or PBS (open circle), and total IgG, ANA, and anti-chromatin determined. Methods and analysis are the same as Fig. 1. The same PBS-treated B6 mice are in Fig. 1A as wild type, Inept, and Ifnar2/2 B6 were studied concurrently. http://www.jimmunol.org/ in the AP-3 complex because of a mutation in the AP-3b1 subunit. mercury, autoantibody responses failed to develop and there The AP-3 adaptor complex plays an essential role in transporting was only a modest increase in total IgG compared with wild proteins with an acidic di-leucine motif from the early endosome to type animals (Fig. 4). SLC15A4 deficiency is also associated late endosomes and lysosomes as well as to lysosome-related or- with a B cell intrinsic preferential reduction in IgG2c (equiva- ganelles, such as melanosomes and platelet-dense bodies (68–70). lent to IgG2a) class switching (32, 33). Indeed, this was also In pDCs, IRF7 activation and consequent type I IFN production observed in HgIA for both SLC15A4-deficient (feeble)andAP- requires AP-3–mediated trafficking of TLR7/9 to the late endoly- 3–deficient (pearl) mice as well as type I IFN–deficient by guest on September 23, 2021 sosomal compartments (40), whereas there is controversy about (Ifnar12/2) and IRF7-deficient (Inept) mice (Supplemental Fig. 3). whether AP-3 is needed for proinflammatory cytokine production These observations reveal that type I IFN-independent HgIA re- (35, 40). In contrast, conventional DCs (cDCs) do not require AP-3 quires essentially the same endosomal TLR trafficking and signal- for TLR7/9-mediated induction of proinflammatory cytokines (35). ing components as type I-dependent spontaneous autoimmunity. These observations suggested that HgCl2 autoimmunity would occur in the absence of AP-3 because it is IRF7 and type I IFN independent, therefore presumably not dependent on pDCs, Type I IFN–independent autoimmunity is dependent on NF-kB which promote lupus by endogenous TLR-dependent production and IL-6 of type I IFN (4). Unexpectedly, however, total IgG and autoantibodies were not induced in pearl mice after exposure to To document the requirement for other TLR-induced pathways mercury, clearly demonstrating that trafficking of AP-3 cargos (34) in type I IFN–independent HgIA, we examined the canon- including nucleic acid–sensing TLRs distal to early endosomes ical NF-kB pathway using Nfkb1-deficient B6 mice (71). Indeed, is required to drive autoantibody production (Fig. 3, Sup- Nfkb1 deficiency completely blocked the development of plemental Fig. 1). This dependence upon AP-3 also posed the hypergammaglobulinemia or autoantibodies indicating an abso- question of what role other components of endosomal TLR lute requirement for the canonical NF-kB pathway (Fig. 5A). trafficking and signaling play in type I IFN–independent auto- This supports an essential role for nucleic acid–sensing TLR- immunity. mediated NF-kB signaling in HgIA, although other immuno- logical defects associated with Nfkb1 deficiency could also have Type I IFN–independent autoimmunity is dependent on contributed (71). Slc15a4 To investigate the role of proinflammatory cytokines that could The histidine peptide transporter SLC15A4 contains the acidic mediate type I IFN–independent autoimmunity, we next examined di-leucine motif recognized by AP-3 and is required for proper IL-6, one of several cytokines dependent on NF-kB–mediated acidification of late endosome and lysosome compartments (32, signaling (72). Strikingly, absence of IL-6 in B6 mice exposed to 35). Consequently, it is required for signaling by nucleic acid– mercury resulted in marked suppression of serum IgG, ANA, and sensing TLRs in pDCs, but only partially required in B cells and anti-chromatin autoantibodies similar to mercury-exposed NF-kB not needed for TLR signaling in cDCs (32, 35). Importantly, p50-deficient mice (Fig. 5). Thus, the requirement for endosomal disruption of SLC15A4, either through the feeble mutation (29) TLRs, NF-kB, and IL-6 supports the argument that type I IFN– or gene deletion (32), abrogates development of autoantibodies independent autoimmunity develops via endosomal TLR trafficking and other features of autoimmunity in lupus-prone mice. When and signaling. leading to NF-kB activation and proinflammatory B6 mice homozygous for the feeble mutation were exposed to cytokine expression. The Journal of Immunology 5

FIGURE 3. Type I IFN–independent HgIA requires an intact AP-3 adaptor complex. Ap3b1-deﬁcient (pearl)(n = 8 per group) and control B6 (n = 8 per group) mice were given HgCl2 (ﬁlled circle) or PBS (open circle), and total IgG, ANA, and anti-chromatin determined. Methods and analysis are the same as Fig. 1. Pearl mice were 60% male and 6–11 wk old.

Soluble TLR agonists are more effective at eliciting pathway and production of the proinflammatory cytokine, IL-6, proinflammatory cytokines than HgCl2 are both required. Taken together, these findings allow new insights into the pathogenesis of HgIA and the endosomal TLR

To understand why HgIA was dependent on a proinflammatory Downloaded from cytokine pathway and not type I IFN, we compared the early in vivo signaling processes needed to induce autoantibodies. IFN-a and proinflammatory cytokine responses of wild type B6 The current paradigm for human idiopathic systemic autoim- mice to s.c. injections of TLR ligands [poly(I:C) and R848] or mune disease argues for a central role for type I IFNs (5), char- acterized by the IFN signature (8, 78), and with special emphasis HgCl2 (Fig. 6). As expected, both TLR3 and TLR7 ligands induced the production of IFN-a and proinflammatory cytokines, on the contribution of innate cells, especially pDCs (1, 5, 79). This IL-6 and TNF-a, consistent with activation of both IRF7 and NF- is supported by animal models of idiopathic (6, 13, 14) and http://www.jimmunol.org/ kB TLR-signaling pathways (67). In contrast, mercury was a pristane-induced SLE (15), which exhibit severe disease pheno- weaker stimulator of systemic cytokine expression than TLR ag- types and dependence on type I IFN, including association of an onists and exhibited a very different cytokine profile. Notably, IFN signature with more rapid onset of disease (80). However, although the IFN gene signature (8, 81) identifies the majority of serum levels of IFN-a and TNF-a were unchanged by HgCl2 exposure, whereas IL-6 levels were significantly increased above patients with severe disease, there is a significant subgroup lacking PBS controls. These findings support the importance of the this signature in which milder disease might be due to type I IFN– proinflammatory signaling pathway and IL-6, particularly in independent mechanisms. In this study, we demonstrate that type I HgIA. IFNs are dispensable for the pathogenesis of HgIA even in the

NZB lupus strain for which development of spontaneous disease is by guest on September 23, 2021 AP-3 is required for T cell activation in HgIA type I IFN dependent. These results provide evidence that the Endosomal TLR trafficking and signaling are not only important accepted paradigm of a pivotal role for type I IFN in systemic for proinflammatory cytokine production, but also contributes to autoimmune disease is not always applicable. adaptive immunity by promoting MHC class II Ag presentation Although our studies do not examine how mercury exposure (73). Indeed, T cell activation is impacted indirectly by deficien- leads to TLR activation and cytokine expression, it is possible that cies in Unc93b1, Slc15a4,orAp3b1 because of reduced TLR- the cell death and proteolysis mediated by mercury releases nucleic mediated activation of APCs and not because of defective Ag acid material that could interact with TLRs (82, 83). Mercury presentation machinery (74–76). As autoantibody responses in exposure of human neutrophil granulocytes also induces the for- HgIA are T cell dependent (55, 63) and associated with increases mation of neutrophil extracellular traps, providing another source in activated/memory (CD44high CD55low) CD4+ T cell subset (77), of TLR agonists (84). These studies are consistent with our ob- we asked if AP-3 deficiency affected T cell activation in mice servation that mercury exposure induces a localized inflammatory exposed to HgCl2. Indeed, whereas HgCl2 exposure in wild type response (85) regulated in part by cathepsin B (61), which is mice increased percentages of activated/memory (CD44high known to be involved in TLR responses (86, 87). Furthermore, the CD55low) CD4+ T cells, in pearl mice, this population was not reported finding that exposure of T cells to mercury induces MHC only reduced prior to HgCl2, but also failed to expand after class II–dependent cytokine production and proliferation in vitro mercury exposure (Fig. 7). This impaired HgCl2-induced T cell only in the presence of APCs (88) is consistent with mercury activation in pearl mice is consistent with defective Ag presen- generating TLR ligands that activate cells to present self-antigens. tation and could contribute to the lack of HgCl2-induced humoral Our findings in UNC93B1-deficient mice showed that mercury autoimmune responses. could not compensate for what appears to be a fundamental requirement for endosomal TLRs in the production of autoantibodies to nucleic acid–containing Ags (66). UNC93B1 is required for Discussion trafficking nucleic acid–sensing TLRs from the ER to the appro- Using a genetics approach, we have made several observations priate endolysosomal and phagolysosomal compartments where about the roles of type I IFN and TLR trafficking and signaling TLRs are processed, engage ligands, and initiate signaling cas- pathways in the production of autoantibodies in xenobiotic cades (89, 90). However, the relative importance of TLR-signaling autoimmunity. Thus, we found that 1) the development of HgIA pathways that induce type I IFN or proinflammatory cytokines, does not require either the type I IFN receptor or IRF-7; 2) and the relevance of specific endocytic and phagocytic compart- autoimmunity is dependent on endosomal trafficking by UNC93B1 ments in autoimmunity have yet to be addressed. We examined and the AP-3 complex; 3) deficiency of the endosomal SLC15A4 these issues by studying the type I IFN–independent HgIA model inhibits HgIA; and 4) signaling through the canonical NF-kB in AP-3– and SLC15A4-deficient mice. 6 AUTOIMMUNITY REQUIRES ENDOSOMAL TLRs BUT NOT TYPE I IFN

FIGURE 4. Feeble mice are resistant to type I IFN–independent HgIA. Slc15a4-deﬁcient (feeble)(n = 8 per group) and control B6 (n = 6–9 per group) mice were given HgCl2 (ﬁlled circle) or PBS (open circle), and total IgG, ANA, and anti-chromatin determined. Methods and analysis are the same as Fig. 1. Feeble mice were 12-16 wk old.

Recent studies have identified two distinct UNC93B1-mediated naling. From this, it can also be inferred that autoantibody pathways for endosomal TLR engagement depending on the size production is induced in large part by small nucleic acid–con- of the nucleic acid–containing material (41, 91) (Supplemental taining particles or immune complexes, which are the main type Fig. 4). Accordingly, the engulfment of small nucleic acid– of cargoes taken up by endocytosis. Downloaded from containing material alone or in immune complexes by endocy- As noted, a previous study using unfractionated FLT3-derived tosis or FcgR interaction, are directed through an endocytic bone marrow cells reported that TLR-mediated type I IFN sig- pathway dependent on AP-3. The AP-3 complex sorts and naling was generated in AP-3–dependent late endosome and transports protein cargos expressing an acidic di-leucine motif endolysosome compartments (IFN endosome), whereas proin- within the endocytic pathway from the early endosome to the flammatory cytokine signaling occurred in early (NF-kB) endolysosome for degradation, and is required for the development of somes that did not require the AP-3 transport (40). In contrast, http://www.jimmunol.org/ lysosome-related organelles and synaptic vesicles (69). Defi- another study showed that for purified pDCs, both type I IFN and ciency of the AP-3 complex blocks the transport of relevant proinflammatory cytokine production required the AP-3 complex, cargos, including TLRs, from early to late endosomes and sub- whereas purified cDCs did not require AP-3. Our results do not sequent compartments. In contrast, endosomal trafficking and support the finding that most NF-kB and proinflammatory sig- TLR engagement following uptake of large immune particles by naling emanates from early endosomes, and instead support the FcgR in pDCs or macrophages occur through a microtubule- requirement for TLR trafficking to the late endosomes and associated protein 1 L chain 3a-associated phagocytosis path- endolysosomes for both essential proinflammatory signaling and way. This pathway is independent of AP-3, and in addition to L autoimmunity (Supplemental Fig. 4). by guest on September 23, 2021 chain 3a, involves several autophagy genes including VPS34, Endosomal TLRs undergo proteolytic processing by cathepsins Atg5, and Atg7, through a process distinct from canonical and asparagine endopeptidases in acidic conditions, but recently autophagy (92). Our findings show that loss of AP-3 is sufficient furin was shown to cleave TLR7 at neutral pH in both humans and to block autoantibody production, thereby implicating a critical mice (93). Thus, it was postulated that TLR7 could be func- role for the endocytic pathway and AP-3–mediated TLR sig- tional in early endosomes prior to the development of an acidic

FIGURE 5. HgIA is dependent on the canonical NF-kB pathway and the proinﬂammatory cytokine, IL-6. (A) B6-Nfkb12/2 (n = 8 per group) or (B) B10. 2/2 S-Il6 (n = 8 per group) mice and corresponding wild type controls were given HgCl2 (ﬁlled circle) or PBS (open circle), and total IgG, ANA, and anti- chromatin determined. Methods and analysis are the same as Fig. 1. B6-Nfkb12/2 mice were all female. The Journal of Immunology 7

AP-3 are more broadly applicable to spontaneous and type I IFN-dependent autoimmunity. To further dissect the relationship between the endosomal compartments and type I IFN-independent autoimmunity, we also examined feeble mice that are deficient in SLC15A4, an endosomal histidine peptide transporter that contains an AP-3 di- leucine binding motif (35). SLC15A4 is required for type I IFN and proinflammatory cytokine induction by nucleic acid–sensing TLRs in pDCs, but not cDCs, and is not required for TLR9- induced B cell proliferation (35). Importantly, lupus-prone B6- lpr feeble mice exhibit reduced disease, which was ascribed to defective TLR-induced cytokine production, especially type I IFN, in pDCs (29), and later studies also showed that SLC15A4 had a subtle intrinsic defect in B cells that inhibited isotype switching to IgG2c and autoantibody production (32, 33). Fur- thermore, evidence indicated that this was caused by an alteration in histidine homeostasis that impaired endosomal acidification and activation of TLR7, which then prevented the triggering of a

TLR7-mediated mTOR-IRF7-type I IFN circuit required for au- Downloaded from toantibody production by B cells (32). Our ﬁndings in SLC15A4- deﬁcient mice exposed to mercury were similar in that there was a selective reduction of the IgG2a (equivalent to IgG2c) isotype and reduced total IgG autoantibodies, which in HgIA is primarily of the IgG1 isotype (25). Thus, our data implicate IRF7-independent

TLR signaling in pDCs and B cells as being critical for HgIA http://www.jimmunol.org/ pathogenesis, but do not support the conclusion that SLC15A4 primarily promotes autoantibody production by an IRF7–type I IFN circuit. B6 mice deficient in Nfkb1, the p50 subunit of NF-kB, have significant defects in B cell function including basal and specific Ab responses (71). The dramatic effects of such a gene deficiency on HgIA induction were clearly demonstrated by the total lack of autoantibody response following HgCl2 exposure. This suggested that the requirement for endosomal TLR in type I IFN–independent by guest on September 23, 2021 autoimmunity is linked to signaling through the canonical NF-kB pathway. In support of this possibility, we document the contribution of IL-6, consistent with previous studies showing susceptibility to HgIA, is associated with increased expression of this cytokine (58). NF-kB contributes to the regulation of IL-6 expression,whichisknowntoplayamajorroleinbothidio- pathic (94–96) and induced systemic autoimmunity (58, 97). Taken together, these studies suggest that type I IFN–independent

FIGURE 6. IFN-a and proinﬂammatory cytokine production induced by

TLR agonists and HgCl2. Serum IFN-a (top panel), IL-6 (middle panel), and TNF-a (bottom panel) in B6 (n = 8 per group) mice injected s.c. with

100 mg of poly(I:C) for 6 h; 50 mg of R848 for 3 h; or 40 mg HgCl2 for 6 h in 100 ml PBS. The optimal timepoints for each were determined in preliminary experiments. Agonist or HgCl2 (filled circles) and PBS controls (open circles) are indicated. The p values , 0.05 for comparisons between agonist or HgCl2 and PBS groups are shown. environment. Our finding, however, that type I IFN–independent autoimmunity is dependent on AP-3 does not support a major role for early endosomal signaling from furin-processed + TLR7 in autoimmunity possibly because the optimal binding of FIGURE 7. Activated or memory CD4 T cells are reduced in HgCl2- exposed pearl mice. Percentages of activated or memory (CD44high nucleic acids to TLR occurs in acidic conditions (90). Another CD55low) CD4+ splenic T cells in control B6 and Ap3b1-deficient (pearl) important consideration is how relevant these findings for AP-3 mice (n = 7–10 per group) exposed to 40 mg HgCl2 (filled circle) or PBS in HgIA are to spontaneous autoimmune disease. In this regard, (open circle) s.c. twice a week for 5 wk. Activated or memory CD4+ in preliminary studies, we have confirmed the dependence of T cells were determined by flow cytometry as described in Materials and AP-3 for spontaneous lupus in B6-lpr mice (data not shown), Methods. Mice were 60–70% female. The p values . 0.05 are shown for which suggests that the conclusions in this study related to comparisons between PBS- versus HgCl2-treated Ap3b1-deficient (pearl). 8 AUTOIMMUNITY REQUIRES ENDOSOMAL TLRs BUT NOT TYPE I IFN autoimmunity involves endosomal TLR trafficking and signaling 24. Jacob, C. O., P. H. van der Meide, and H. O. McDevitt. 1987. 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