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Autophagy in Dendritic Cells and B Cells Is Critical for the Inflammatory State of TLR7-Mediated Autoimmunity

This information is current as Chi G. Weindel, Lauren J. Richey, Abhiruchi J. Mehta, of September 29, 2021. Mansi Shah and Brigitte T. Huber J Immunol published online 28 December 2016 http://www.jimmunol.org/content/early/2016/12/28/jimmun ol.1601307 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 28, 2016, doi:10.4049/jimmunol.1601307 The Journal of Immunology

Autophagy in Dendritic Cells and B Cells Is Critical for the Inﬂammatory State of TLR7-Mediated Autoimmunity

Chi G. Weindel,* Lauren J. Richey,† Abhiruchi J. Mehta,‡ Mansi Shah,‡ and Brigitte T. Huber*,‡

Individuals suffering from autoimmune disorders possess a hyperactive cellular phenotype where tolerance to self-antigens is lost. Autophagy has been implicated in both the induction and prevention of autoimmunity, and modulators of this cellular recycling process hold high potential for the treatment of autoimmune diseases. In this study, we determine the effects of a loss of autophagy in dendritic cells (DCs), as well as both B cells and DCs, in a TLR7-mediated model of autoimmunity, similar to systemic lupus erythematosus, where both cell types are critical for disease. Although a loss of DC autophagy slowed disease, the combined loss of autophagy in both cell types resulted in a lethal sepsis-like environment, which included tissue inﬂammation and hyperproduction of inﬂammasome-associated

cytokines. Ablation of B cell signaling reversed this phenotype, indicating that activation of these cells is an essential step in disease Downloaded from induction. Thus, autophagy plays a dichotomous role in this model of disease. The Journal of Immunology, 2017, 198: 000–000.

nflammation is a tightly controlled biological reaction to and release (13–15), and promotes the proliferation and survival of cellular insults. An inflammatory response is required for the adaptive immune cells (16–19). Thus, it is reasonable to think that I efficient clearance of bacterial and viral pathogens, as well as alterations in autophagy contribute to SLE predisposition and/or

removal of cellular debris following tissue damage. Inflammation can disease progression. Based on genetic linkage and cellular func- http://www.jimmunol.org/ also occur in the absence of infection. Detrimental effects of sterile tions, modulators of autophagy have an excellent potential as inflammation are readily observed in autoimmune diseases in which therapeutics for autoimmune diseases where self-tolerance has inflammation is predisposed by genetic susceptibility and com- been lost (reviewed in Refs. 20, 21). However, because autophagy pounded by environmental triggers (1–4). Severe flares of inflam- is critical for multiple aspects of the immune system, its different mation mark the prototypical autoimmune disease, systemic lupus functions may play opposing roles in autoimmunity. Promising erythematosus (SLE). Inflammation in SLE is thought to be driven results have been reported in lupus patients when treated with by antinuclear Abs (ANAs) as a result of a loss of self-tolerance (5). either inhibitors or inducers of autophagic processes (22). Despite the identification of numerous SLE susceptibility genes To better understand the role of autophagy in SLE, we used a via genome-wide association studies, the etiology of this disease mouse model of autoimmunity, mediated by overexpression of the by guest on September 29, 2021 remains poorly understood (6, 7). Notably, several single nucle- RNA-sensing innate immune receptor TLR7 (Tlr7.1 transgenic otide polymorphisms associated with SLE have been mapped to [tg]) (23, 24). We showed recently that B cell autophagy is re- autophagy-related genes (ATGs) (8, 9). Targeted macroautophagy, quired for the induction of autoimmunity (25). In particular, Tlr7.1 the canonical autophagy pathway herein referred to as autophagy, tg mice harboring B cells deficient in autophagy did not make is a key regulator of cellular homeostasis as well as immune ANAs, lacked a type I IFN signature, and did not develop glo- system function; that is, it controls recognition and clearance of merulonephritis (25), all hallmarks of SLE. extracellular pathogens (10–12), mediates cytokine production Although B cells are central in SLE, dendritic cells (DCs) also play an important role in this disease, because they are the main producers of type I IFNs and are essential APCs for T cell acti- *Program in Genetics, Sackler School of Graduate Biomedical Sciences, Tufts Uni- vation, both of which contribute to disease progression in humans versity School of Medicine, Boston, MA 02111; †Division of Laboratory Animal Medicine, Tufts University, Boston, MA 02111; and ‡Department of Integrative and in mouse models (26–30). Autophagy is required for IFN-a Physiology and Pathobiology, Sackler School of Graduate Biomedical Sciences, production by plasmacytoid DCs (pDCs) during viral infection Tufts University School of Medicine, Boston, MA 02111 (11), as well as for Ag presentation by myeloid DCs (mDCs) (12, ORCID: 0000-0001-5090-791X (B.T.H.). 31, 32). Therefore, disrupting autophagy in these cells has the Received for publication August 8, 2016. Accepted for publication November 21, potential to reduce autoimmune symptoms. Thus, we compared 2016. disease in Tlr7.1 tg mice with either a DC-specific ablation of This work was supported in part by National Institutes of Health/Clinical and Trans- autophagy or a combined loss of autophagy in DCs and B cells. lational Science Institute Grant UL1TR001064 (to B.T.H.). As predicted, an autophagy knockout (ko) in DCs slowed Address correspondence and reprint requests to Dr. Brigitte T. Huber, Tufts Univer- sity School of Medicine, Jaharis 523A, 150 Harrison Avenue, Boston, MA 02111. disease progression and reduced IFN-a production; however, E-mail address: [email protected] Tlr7.1 tg mice lacking autophagy in both cell types developed a The online version of this article contains supplemental material. rapid and lethal inflammatory condition, reminiscent of sterile Abbreviations used in this article: ANA, antinuclear Ab; APS, antiphospholipid sepsis, suggesting that autophagy plays a dichotomous role in 2 syndrome; ATG, autophagy-related gene; Bko, Cd19-cre+/ Atg5f/f; CL, cardiolipin; disease progression. The massive inflammatory response in the DBko, Cd11c-cre+/2 Cd19-cre+/2 Atg5f/f; DC, dendritic cell; Dko, Cd11c-cre1/2 Atg5f/f; EMH, extramedullary hematopoiesis; ko, knockout; LAP, LC3-associated latter mice was not driven by ANAs, but instead was driven by phagocytosis; LN, lymph node; mDC, myeloid DC; mtDNA, mitochondrial DNA; autoantibodies against cytoplasmic material, such as cardiolipin MZ, marginal zone; PAS, periodic acid–Schiff; pDC, plasmacytoid DC; SLE, sys- (CL), a diphosphatidylglycerol lipid found in the mitochondrial temic lupus erythematosus; tg, transgenic. membrane (33). These data demonstrate that B cells lacking Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 autophagy maintain the capacity to respond to some self-antigens,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601307 2 AUTOPHAGY CONTROLS INFLAMMASOME ACTIVATION IN LUPUS although unable to produce ANAs. This is further supported by the incubated for 20 min at room temperature. For detection, 50 ml of tetra- finding that inhibition of B cell activation increased survival and methylbenzidine substrate (BD Biosciences, 555214) was added, and reac- reduced cytokine production in these mice, similar to what was tions were stopped with 50 mlof2NH2SO4. For all cytokine ELISAs, plates were read at 450 nm with SoftMax Pro using an EMax (Molecular Devices). observed in Tlr7.1 tg mice lacking B cell autophagy (25). Ig ELISA Materials and Methods Nunc MaxiSorp plates (eBioscience) were coated with either 1 mg/ml Mice unlabeled goat anti-mouse IgG (1030-01) or IgM (1021-01) (South- ernBiotech). Blocking was performed with 1% BSA in borate buffer (pH All mice used in this study were on a C57BL/6J background and were an- 8.4). Sera were serially diluted and incubated at 4˚C overnight. Alkaline alyzed at either 3, 5, or 14 mo of age. C57BL/6J and Cd11c-Cre mice phosphatase–conjugated goat anti-mouse IgG (1030-04), IgM (1021-04), [C57BL/6J-Tg(Itgax-cre,-EGFP)4097Ach/J] (34) mice were purchased from IgG1 (1070-04), IgG2a (1080-04), IgG2b (1090-04), IgG2c (1079-04), or The Jackson Laboratories (Bar Harbor, ME). Tlr7.1 tg mice [C57BL/6-Tg IgG3 (1100-04) at 1 mg/ml were used (SouthernBiotech). Standard curves (Tlr7)1Boll] were produced at the National Institutes of Health and generated were prepared with purified mouse IgG (Santa Cruz Biotechnology, sc- by recombineering the bacterial artificial chromosome clone RP23-139P21 2025), IgM (0101-01), IgG1 (0102-01), IgG2a (0103-01), IgG2b (0104- as previously described (23). Mice were bred at Tufts University School of +/2 f/f +/2 +/2 01), IgG2c (0122-01), or IgG3 (0105-01) (SouthernBiotech). Plates were Medicine; Cd11c-cre Atg5 (Dko) mice and Cd11c-cre Cd19-cre developed with 1 mg/ml phosphatase substrate (Sigma-Aldrich, S0942) in Atg5f/f (DBko) mice were generated by intercrossing Cd11c-Cre and Cd19- +/2 f/f tm1(cre)Cgn tm1Myok development buffer (0.05 M Na2CO3 [pH 9.5]). Plates were read at 405 nm cre Atg5 (Bko) mice [B6.129P2(C) Cd19 Atg5 ](17), with SoftMax Pro using an EMax (Molecular Devices). CL-IgG was de- respectively. Bko mice were obtained from H. Virgin (Washington Univer- tected with Nunc MaxiSorp plates coated with 50 ml per well 0.1 mg/ml sity, St. Louis, MO), at the ninth generation of backcrossing to C57BL/6J, CL (Avanti Polar Lipids) in 100% EtOH and incubated uncovered overnight and were backcrossed at Tufts University for an additional generation. Tlr7.1

at 4˚C. The following day plates were equilibrated at room temperature for Downloaded from tg mice with one copy of Atg5f/+ were crossed with Dko or DBko mice to f/+ 30 min uncovered, allowing for remaining liquid to evaporate. Plates were generate experimental cohorts. Tlr7.1 tg mice with one copy of Atg5 and 3 +/2 blocked with 1 PBS 10% FBS (Atlanta Biologicals, S11550) for 1 h at one copy of Cd19-Cre were bred to DBko mice to generate cohorts room temperature. Standard FC1 was added at 10 mg/ml in 1:3 serial di- containing Tlr7.1 tg DBko Cd19ko mice. All animals were housed, bred, and lutions. Serum was added to the plate at 1:100 in 1:3 serial dilutions at 50 ml studied at Tufts University School of Medicine under approved Institutional per well and incubated for 1.5 h at room temperature. HRP-conjugated goat Animal Care and Use Committee guidelines. anti-mouse IgG (Thermo Fisher, 62-6520) at 1:1000 was used. For detec- Flow cytometry tion, 50 ml of tetramethylbenzidine substrate (BD Biosciences, 555214) was http://www.jimmunol.org/ added, and reactions were stopped with 50 mlof2NH2SO4.Plateswere Single-cell suspensions of spleen and lymph node (LN) were made in 13 read at 450 nm with SoftMax Pro using an EMax (Molecular Devices). PBS (0.137 M NaCl, 2.7 mM KCl, 5.3 mM Na2HPO4, 1.8 mM KH2PO4), 2% FBS (Atlanta Biologicals, S11550) using 70-mm nylon cell strainers Histology (BD Falcon, 352350). Peritoneal cells were isolated by lavage (13 PBS, 2% FBS). Cells were blocked for FCGR2B/CD32 (eBioscience, 14-0161- Tissue were fixed with 10% neutral buffered formalin (Sigma-Aldrich, 85) and stained with the following mouse Abs from eBioscience: CD11c HT501640) and embedded in paraffin; 5-mm sections were cut and stained eFluor (48-0114-82), CD3 eFluor (48-0032-80), CD23 FITC (11-0232-82), with H&E; 2-mm kidney sections were cut and stained with periodic acid– GR1/Ly6G FITC (11-5931-82), PDCA1 PerCP–eFluor 710 (46-3172-80), Schiff (PAS; Rowley Biochemical, SO-429). The pathologist performed a PerCP-Cy5 GR1/Ly6G (45-5931-80), B220 allophycocyanin–eFluor 780 blinded evaluation of kidney sections for membranoproliferative glomeru- (47-0452-82), and allophycocyanin–eFluor CD11b (47-0112-82); and BD lonephritis using the following scoring system: 1, no areas to focal areas of minimal segmental PAS+ mesangial thickening; 2, focal to generalized mild by guest on September 29, 2021 Biosciences: CD69 PerCP-Cy5.5 (551113), CD80 PE (553769), CD11b PE + (01715B), CD138 PE (553714), CD69 PE (553237), CD5 allophycocyanin segmental PAS mesangial thickening; 3, generalized segmental to diffuse moderate to severe PAS+ mesangial thickening with increased glomerular (550035), CD21 allophycocyanin (558658), and allophycocyanin B220 + (553092). Cells were analyzed using an LSR II with FACSDiva software cellularity; 4, generalized diffuse severe PAS mesangial thickening with in- (BD Biosciences). Secondary analysis was performed using FCS Express creased glomerular cellularity, dilation of Bowman’s space, attenuation of 4 research edition (De Novo Software). normally cuboidal parietal epithelium of Bowman’s capsule in male mice, mild thickening of Bowman’s capsule, and small numbers of tubules dilated with Detection of autoantibodies protein casts. Liver inflammation was scored as follows: 0, absent to very slight focal; 1, very slight to slight multifocal or mild focal and suppurative; 2, mild HEp-2 slides (MBL Bion, AN-1012) were used to determine the presence multifocal and suppurative; 3, moderate multifocal or mild multifocal in both of antinuclear IgG. Sera were diluted 1:80, and slides were treated per the parenchymal and portal areas. Liver extramedullary hematopoiesis (EMH) was manufacturer’s recommendations. To identify autoreactive sera, a sec- scored as follows: 0, absent; 1, slight foci (1–2) of minimal megakaryocyte or ondary goat anti-mouse IgG FITC (Sigma-Aldrich, F8264) at 1:250 was myeloid cells; 2, mild multiple foci of both megakaryocytes and myeloid cells; used. Slides were read using a Nikon Eclipse 80i fluorescence microscope 3, numerous moderate foci of both megakaryocytes and myeloid cells in portal and analyzed with NIS-Elements software (Nikon). and parenchymal areas; 4, severe multifocal expansions of both megakaryocytes and myeloid cells in portal and parenchymal areas. Lung megakaryocyte Multiplex immunoassay numbers were given the following scores: 1, megakaryocytes absent; 2, min- Sera were analyzed for 17 cytokines—IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, imal presence of megakaryocytes (1–2); 3, moderate numbers of megakaryo- IL-10, IL-12 (p70), IL-13, IL-17A, IL-18, TNF-a, M-CSF, G-CSF, GM- cytes present; 4, numerous megakaryocytes present throughout the sectioned CSF, IFN-a, and IFN-g—using an eBioscience ProcartaPlex multiplex lung tissue. Lung inflammation was determined as either present or absent. immunoassay, mouse Th1/Th2 cytokine panel (11 plex) (EPX110-20820- 901), IFN-a Simplex (EPX010-26027), IL-1a Simplex (EPX010-20611), Statistical analysis IL-10 Simplex (EPX010-20614), G-CSF Simplex (EPX010-26034), IL- Graphs were generated using Prism (GraphPad Software). Significance for 17A Simplex (EPX010-26001), and M-CSF Simplex (EPX010-26039), in vivo and ex vivo assays of more than two groups were determined by either per the manufacturer’s specifications. All samples were diluted 1:1 and one-way ANOVA followed by a Tukey multiple comparisons test or by a run in duplicate on a Luminex analyzer. Kruskal–Wallis test followed by a Dunn multiple comparisons test, depending Cytokine ELISA on sample distribution. Significance for two groups was determined by a two- tailed Mann–Whitney U test where appropriate. Survival curves were ana- Cytokine levels of serum IL-18 were determined as follows: plates were lyzed using a Mantel–Cox (log-rank) test. Error bars represent SEM. coated with 50 ml of rat anti-mouse IL-18 mAb (MBL International, D047-3) at 1:1000 in 13 PBS and incubated overnight at 4˚C. Plates were blocked for 1hin13 PBS, 1% BSA. Standards were diluted 1:1 starting at 4 ng/ml Results (MBL International, D048-3) and samples, diluted 1:4 in blocking buffer, Loss of DC autophagy extends lifespan and reduces type 1 were added to the plate and incubated overnight at 4˚C. Detection Ab, 50 ml IFNs in TLR7-mediated SLE of polyclonal rat anti-mouse IL-18 (MBL International, D048-6), was used at a 1:2000 dilution in blocking buffer and incubated 1 h at room temperature. To determine whether a deletion of autophagy in DCs would Streptavidin-HRP (R&D Systems, 4800-30-06) was added at 1:1000 and ameliorate TLR7-mediated autoimmunity, we bred the Tlr7.1 tg The Journal of Immunology 3 line to mice containing a Cd11c-Cre–mediated DC-specific con- 40 wk. However, as these mice aged beyond 40 wk, their health ditional deletion of Atg5 (Dko) (see Materials and Methods for a rapidly declined, leading to a precipitous death. Because DCs are full list of the genotypes and abbreviations used). As shown in major producers of IFN-a, we hypothesized that the increased Fig. 1A, a loss of DC autophagy significantly improved the sur- survival of these mice was caused by lower production of this vival of Tlr7.1 tg mice, increasing the median survival from 28 to cytokine and a reduced IFN signature. To test this, we screened Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Tlr7.1 tg mice lacking DC autophagy have increased lifespan and reduced IFN-a.(A) Survival of Tlr7.1 tg mice (red line) compared with Tlr7.1 tg Dko mice (blue line). Mice were observed during 58 wk. Controls (n = 13), Tlr7.1 tg (n = 14), and Tlr7.1 tg Dko (n = 9) mice are shown. *p , 0.05 by a Mantel–Cox log-rank test. (B–G) Sera from age-matched nonmoribund mice between 16 and 20 wk of age were tested for cytokines using multiplex immunoassays: (B) IFN-a;(C) IL-18 and IL-1b,(D) IL-12p70, IL-6, and IL-10, (E) G-CSF, (F) IL-2 and IFN-g,(G) IL-4 and IL-5. Each point is representative of an individual animal. (H) Representative H&E sections of liver for controls (left panels), Tlr7.1 tg (right center panel), and Tlr7.1 tg Dko mice (far right panel). Original magnification 310. (I) HEp-2 slides used to detect ANAs (IgG-FITC) in 16- to 20-wk-old mice. Original magnification 320. Results are based on at least six mice per genotype. *p , 0.05 **p , 0.01, ***p , 0.005 by a Kruskal–Wallis test with a Dunn posttest. 4 AUTOPHAGY CONTROLS INFLAMMASOME ACTIVATION IN LUPUS sera on an array of SLE-associated inflammatory cytokines. capable of producing ANAs in Tlr7.1 tg Dko mice. Similarly, Whereas the Tlr7.1 tg mice presented with a significant increase in spleens from both Tlr7.1 tg and Tlr7.1 tg Dko mice were enlarged IFN-a, this was not seen in Tlr7.1 tg Dko mice, indicating a defect compared with controls (Supplemental Fig. 1A, 1B). Although in type 1 IFN production (Fig. 1B). Alternatively, IL-18 and, to total B cell numbers were unchanged (Supplemental Fig. 1C), the a lesser extent, IL-1b levels were elevated in the latter mice percentage of marginal zone (MZ) B cells was depleted in both (Fig. 1C). These two cytokines require cleavage by caspase-1 for groups of Tlr7.1 tg mice, indicating B cell activation (Supplemental secretion and, therefore, are dependent on inflammasome activa- Fig. 1D). Consistent with this, the percentages of CD80+ Bcells tion (35, 36). Aside from these clear differences, other SLE- were significantly increased in both Tlr7.1 tg and Tlr7.1 Dko mice associated cytokines were similarly elevated in Tlr7.1 tg and (Supplemental Fig. 1E). Tlr7.1 tg Dko mice, including IL-12p70, IL-6, and IL-10 Thus, we conclude that B cells are the primary inducers of (Fig. 1D); growth factors (Fig. 1E) and Th1 and Th2 cytokines disease in these mice and are capable of driving disease pro- were also unchanged (Fig. 1F, 1G). Considering the increased gression, whereas lower production of IFN-a in Tlr7.1 tg Dko mice survival of the Tlr7.1 tg Dko mice, cytokine levels similar to slows disease. Tlr7.1 tg mice with working autophagy were unexpected. Because IFN-a promotes macrophage activation and infiltra- Loss of DC and B cell autophagy results in severe lethality, tion, we next examined the level of tissue inflammation in these splenomegaly, and lymphadenopathy mice to determine whether lower IFN-a production was resulting Because survival was increased in Tlr7.1 tg mice with a DC loss of in a functional decrease in disease progression. Similar levels of autophagy (Dko), likely due to a reduction in IFN-a levels, inflammation and cellular infiltration (Fig. 1H) were present in the whereas B cell activation remained unchanged, we reasoned that Downloaded from livers of Tlr7.1 tg and Tlr7.1 tg Dko mice, indicating comparable combining a loss of DC and B cell autophagy might ablate disease tissue inflammation in both groups. Because SLE-associated tissue completely in Tlr7.1 tg mice. We therefore bred the Tlr7.1 tg line damage is caused by deposition of autoantibodies against DNA to mice containing a combined DC and B cell conditional deletion and RNA, we looked for ANAs in the Tlr7.1 tg Dko mice. of Atg5 (DBko). Surprisingly, we observed a drastically different Staining of HEp-2 cells revealed a similar autoantibody profile in phenotype than predicted; most significantly, these mice suc-

the sera of Tlr7.1 tg and Tlr7.1 tg Dko mice (Fig. 1I). These data cumbed to rapid death with a median survival of 20 wk (Fig. 2A, http://www.jimmunol.org/ indicate that in the absence of DC autophagy B cells are still solid line). A considerable increase in the weight of spleens by guest on September 29, 2021

FIGURE 2. Loss of DC and B cell autophagy results in splenomegaly and lymphadenopathy. (A)SurvivalofTlr7.1 tg mice (red line) compared with Tlr7.1 tg DBko mice (blue line). Mice were observed during 58 wk. Controls (n =13),Tlr7.1 tg (n =13),andTlr7.1 tg DBko (n = 11) mice are shown. *p , 0.05 by Mantel–Cox log-rank test. Spleens and LNs were weighed and normalized as a percentage of body weight at (B) 12–14 wk and at (C) 57 wk for controls. Each point is representative of an individual animal. Results are based on at least three independent experiments. (D and E) H&E sections of spleen (D) white pulp (WP) and red pulp (RF) for controls (left panel), Tlr7.1 tg (center panel), and Tlr7.1 tg DBko mice (right panel). Original magniﬁcation 34. Results are based on at least six mice per genotype. (E) LNs showing nuclei (hematoxylin, blue) and cytoplasm (eosin, pink) for Tlr7.1 (upper panel) and Tlr7.1 tg DBko (lower panel) mice. Original magniﬁcation, 340. (F) Flow cytometry of nucleated cells for spleen (left plot), LNs (middle plot), and peritoneum (right plot). Results are based on at least three independent experiments with mice between 12 and 14 wk of age. *p , 0.05, **p , 0.01, ***p , 0.005 by a Kruskal–Wallis test with a Dunn posttest. The Journal of Immunology 5

(Fig. 2B, left panel) and LNs (Fig. 2B, right panel) was apparent (Fig. 2E). This latter finding is likely due to the fact that a B cell in these mice. Aged control mice with a double autophagy ko loss of autophagy inhibits development of B1 cells, which are (DBko) also developed significant increases in the weight of mostly found in the peritoneum (17). secondary lymphoid organs over time (Fig. 2C). The phenotype of The early mortality of the Tlr7.1 tg DBko mice, combined with these mice was strongly enhanced by the Tlr7.1 tg, as the spleens the increased size, weight, and cellularity of their secondary of Tlr7.1 tg DBko mice at 12 wk of age accounted for as much as lymphoid organs, suggested excessive levels of tissue inflamma- 12% of total body weight, compared with only 3% in Tlr7.1 tg tion in these mice. Consistent with this, the dramatic cellular mice. Additionally, the spleens of Tlr7.1 tg DBko mice showed a expansion in the spleens and LNs consisted of myeloid cell pop- complete loss of architecture, enhanced myelopoiesis and eryth- ulations; notably, increased numbers of mDCs, neutrophils, ropoiesis, and an expansion of the white pulp as well as the red monocytes, and macrophages were observed (Fig. 3A, 3B; see pulp (Fig. 2D). The LNs of these mice had a similar expansion Supplemental Fig. 2 for myeloid cell gating strategy), whereas of cell populations than did those of their Tlr7.1 tg littermates, Tlr7.1 tg mice only showed a mild increase in splenic mDCs at whereas their peritoneal cavity cell numbers were normal this early time point (Fig. 3A, left plot). Additionally, an increase Downloaded from http://www.jimmunol.org/

FIGURE 3. Loss of B cell and DC autophagy results in a systemic expansion of myeloid lineage by guest on September 29, 2021 cells in Tlr7.1 tg mice. (A and B) Myeloid and granuloid cell populations in the spleen (A)andLNs (B), as well as activated DCs (C and D) and inflammatory monocytes (E and F), were analyzed by flow cytometry. mDCs (CD11c+CD11b+B2202), monocytes (CD11b+GR1low/2CD11c2 [forward scatter/side scatter]), macrophages (CD11b+GR1low/2 CD11c2 [forward scatter/side scatter]) neutrophils (CD11b+GR1+CD11c2), activated DCs (CD11c+B2202CD80+), inflammatory monocytes (CD11b+GR1low/2CD11c2Ly6Chigh). Each point is representative of an individual animal. *p , 0.05, **p , 0.01, ***p , 0.005 by a Kruskal–Wallis test with a Dunn posttest. 6 AUTOPHAGY CONTROLS INFLAMMASOME ACTIVATION IN LUPUS in inflammatory markers was seen on splenic and LN cells of Tlr7. time point. Thus, the lack of IFN-a indicates that the expansion of 1 tg DBko mice, including CD80high DCs (Fig. 3C, 3D) and myeloid cells was not being driven by type 1 IFNs. At this age, Ly6Chigh monocytes (Fig. 3E, 3F). Tlr7.1 tg mice with working autophagy had normal levels of IFN-a, consistent with their early stage in disease progression. Tlr7.1 tg Loss of DC and B cell autophagy promotes inflammasome DBko mice also had increased levels of IL-12p70, IL-6, and IL-10 activation and organ damage (Fig. 4D), as well as multiple T cell cytokines (Fig. 4E, 4F). Taken The inflammatory condition of Tlr7.1 tg mice is well described and together, these data suggest that Tlr7.1 tg DBko mice develop a consists of a myeloid cell expansion, liver and kidney inflammation, severe inflammatory condition, reflective of increased inflamma- as well as elevated cytokines, including IFN-a,TNF-a, and IL-6, some activity, as well as T cell activation. Importantly, their in- seen between 12 and 24 wk (23). To determine whether the lethality flammatory phenotype occurs at an earlier time point and does not and severe secondary lymphoid expansion in Tlr7.1 tg DBko mice include IFN-a, the signature cytokine of SLE; thus, inflammation is was due to an accelerated disease, occurring earlier and beyond that driven by a different mechanism than in Tlr7.1 tg mice with observed in Tlr7.1 tg littermates, we determined the levels of SLE- working autophagy. We conclude that Tlr7.1 tg DBko mice develop associated cytokines in the sera of Tlr7.1 tg and Tlr7.1 tg DBko a lethal inflammatory condition that is distinct from the inflam- mice between 12 and 14 wk of age and assessed organ inflamma- mation present in Tlr7.1 tg mice. tion. As shown in Fig. 4, we detected a dramatically different cy- The distinct cytokine profile of Tlr7.1 tg DBko mice mirrored a tokine profile in the latter mice; namely, IL-18 (Fig. 4A, left panel) condition resembling sterile sepsis, which is typically accompa- and IL-1b (Fig. 4A, right panel) were highly elevated far above the nied by systemic organ damage. Therefore, we surveyed multiple levels seen in Tlr7.1 tg Dko mice (Fig. 1C). These data indicate that organs for signs of inflammation. As expected, the livers of both Downloaded from loss of autophagy in B cells is enhancing the cytokine phenotype Tlr7.1 tg and Tlr7.1 tg DBko mice showed cellular infiltrates and observed in Tlr7.1 tg Dko mice. Increased serum IL-18 was also inflammation (Fig. 5A, Supplemental Table I). Notably, however, observed in Dko and DBko control mice, suggesting increased the latter developed a more widespread condition, with lympho- inflammasome activation in the absence of autophagy. Consistent histiocytic and suppurative inflammation in both the portal and with a highly inflammatory condition, TNF-a was also significantly parenchymal areas (Fig. 5A, far right panel). In addition to in-

elevated in Tlr7.1 tg DBko mice (Fig. 4B). Alternatively, IFN-a was creased inﬂammation, the livers of Tlr7.1 tg DBko mice showed http://www.jimmunol.org/ not signiﬁcantly elevated (Fig. 4C). This was surprising, because increased EMH, consisting of myeloid cells and megakaryocytes Tlr7.1 tg DBko mice showed a severe myeloid expansion at this (Fig. 5B, Supplemental Table I). by guest on September 29, 2021

FIGURE 4. Inﬂammasome-associated cytokines are elevated upon loss of DC and B cell autophagy. Sera from age-matched nonmoribund mice between 12 and 14 wk of age were tested for cytokines using a multiplex immunoassay. (A) IL-18 and IL-1b,(B)TNF-a,(C)IFN-a,(D) IL-12p70, IL-6, and IL-10, (E)IL-2andIFN-g, and (F) IL-4 and IL-5. Each point is representative of an individual animal. *p , 0.05, **p , 0.01, ***p , 0.005 by a Kruskal–Wallis test with a Dunn posttest. The Journal of Immunology 7 Downloaded from

FIGURE 5. Multiorgan inflammation is present in Tlr7.1 tg DBko mice. Representative H&E sections of liver (A) showing cellular infiltrates for wild-type (WT; far left), DBko (middle left), Tlr7.1 tg (middle right), and Tlr7.1 tg DBko (far right) mice. WT (n = 6), DBko (n = 6), Tlr7.1 tg (n = 7), and Tlr7.1 tg DBko (n = 10) mice are shown. Original magnification 310. (B) EMH scoring for liver sections. H&E sections of lung (C) showing interstitial thickening and cellular infiltrates for WT (far left), DBko (middle left), Tlr7.1 tg (middle right), and Tlr7.1 tg DBko (far right) mice. Original magnification 310. WT (n = 6), DBko (n = 6), Tlr7.1 tg (n = 7), and Tlr7.1 tg DBko (n = 10) mice are shown. (D) Megakaryocyte infiltrate scoring for lung sections. Each point is representative of an individual animal with mice between 12 and 14 wk of age. ***p , 0.005 by a Kruskal–Wallis test with a Dunn posttest or by a one-way http://www.jimmunol.org/ ANOVA with a Tukey posttest where appropriate.

Unlike the Tlr7.1 tg mice that do not develop severe lung increased inflammasome activity. Interestingly, IL-18 levels in flammation (23), 90% of the Tlr7.1 tg DBko mice showed lung Tlr7.1 tg DBko mice were nearly twice those of Tlr7.1 tg Dko infiltrates of lymphocytes or macrophages, as well as megakar- mice. These data combined with the increased inflammation in yocytes, ranging from mild to severe. Additionally, a thickening of Tlr7.1 tg DBko mice compared with Tlr7.1 tg Dko mice indicated the interstitium was observed, indicative of an inflammatory state that a B cell loss of autophagy enhanced the inflammasome ac- (Fig. 5C, right panel, 5D, summary, Supplemental Table I). In- tivation and septic condition. Because B cells lacking autophagy by guest on September 29, 2021 terestingly, 67% of aged DBko control mice also possessed a were unable to produce ANAa in Tlr7.1 tg mice (25), we sought to slight increase in lung inflammation due to macrophage and confirm this phenotype in Tlr7.1 tg DBko mice. As expected, sera neutrophil infiltrates (Fig. 5C, middle left panel). from DBko and Tlr7.1 tg DBko mice lacked nuclear and nucleolar Tlr7.1 tg mice develop glomerulonephritis, a characteristic of SLE staining on HEp-2 slides (Fig. 6A, middle left and far right panels, (23, 37). Thus, the inflammatory state of Tlr7.1 tg DBko kidneys was Table I), unlike those of their Tlr7.1 tg littermates (Fig. 6A, determined. Early in disease progression the glomeruli of these mice middle right panel, Table I). However, bright staining of either scored more severely on a pathology index than did those of Tlr7.1 tg cytoplasmic components or the nuclear/cellular membrane was mice (Table I). However, their kidneys did not worsen with age; present in the sera of the latter (Fig. 6A, far right panels, Table I). rather, they maintained a lower pathology score, whereas Tlr7.1 tg Because the speckled cytoplasmic pattern resembled mitochon- mice developed more severe kidney pathology as disease progressed drial staining, we tested for Abs against CL, a phospholipid that is (Table I). Overall, our data demonstrate that Tlr7.1 tg DBko mice a major component of the mitochondrial inner membrane, but is have multiple and more widespread organ damage than do Tlr7.1 tg exposed to the cytosol upon mitochondrial damage (38). As shown mice, consistent with a sterile sepsis-like inflammatory condition. in Fig. 6B, anti-CL IgG was increased in Tlr7.1 tg DBko mice. Loss of autophagy promotes B cell recognition of Whereas both Tlr7.1 tg and Tlr7.1 tg DBko mice had simi- mitochondrial Ags in Tlr7.1 tg mice larly elevated total serum IgM levels (Fig. 6C, left graph), the total IgG level of Tlr7.1 tg DBko mice was significantly in- The elevated serum IL-18 and IL-1b present in the Tlr7.1 tg DBko creased (Fig. 6C, right graph), and IgG1 accounted for most IgG mice (Fig. 4A) and Tlr7.1 tg Dko mice (Fig. 1C) indicates in- in these mice (Fig. 6D). Alternatively, IgG2a and IgG2b were the predominant subtypes in Tlr7.1 tg mice (Fig. 6E), as pre- Table I. Kidney pathology and ANA patterns for nonmoribund and viously reported (24, 37). The levels of IgG2c and IgG3 were moribund mice similar for all mice (Fig. 6E). These data indicate that an autophagy ko in B cells prevents production of ANAs, but is 12–14 wk Moribund permissive for production of other autoantibodies. Thus, we Tlr7.1 Tlr7.1 conclude that nuclear and cytoplasmic Ags are processed by Controls Tlr7.1 DBko Tlr7.1 DBko different cellular machinery. Kidney Score 1 1.7 2.3 3.3 2.2 ANA Scores IL-18 hyperproduction and lethality in Tlr7.1 tg DBko mice is Nuclear/nucleolar 0 2030 dependent on BCR function Cytoplasmic 0 0504 Mixed 0 2010 In addition to the presence of anti-CL IgG and high IgG1, B cells Total 0/4 4/5 5/5 4/4 4/4 were increased in the LNs of Tlr7.1 tg DBko mice (Fig. 7A; see 8 AUTOPHAGY CONTROLS INFLAMMASOME ACTIVATION IN LUPUS

FIGURE 6. Autoantibodies against CL and high IgG1 are present in Tlr7.1 tg DBko mice. (A) HEp-2 slides used to detect ANAs (IgG- FITC) in 12- to 14-wk-old mice. Data are representative of four experiments. Controls (n = 4), Tlr7.1 tg (n = 5), and Tlr7.1 tg Atg5ko (n =5) mice are shown. Original magniﬁcation 340. Abs against CL (B), total serum IgM and IgG (C), IgG1 (D), and IgG2a, IgG2b, IgG2c, and IgG3 (E) were determined by ELISA. Each point is representative of an individual animal. Mice were between 12 and 14 wk of age. *p , 0.05, Downloaded from **p , 0.01, ***p , 0.005 by a Kruskal–Wallis test with a Dunn posttest. http://www.jimmunol.org/

Supplemental Fig. 3 for lymphocyte gating strategy), and these in DCs slowed disease progression but resulted in secondary effects. cells had higher levels of the activation marker CD80 (Fig. 7B, However, when autophagy was absent in both B cells and DCs, a Supplemental Fig. 2A). B cell numbers and activation were sim- severe and lethal inflammatory state was induced. These results are by guest on September 29, 2021 ilarly increased in the spleens (Fig. 7C, 7D). B cell subsets were especially interesting, given that autophagy-associated genes have also altered in these mice, with reduced numbers of follicular and confounding roles in SLE development in humans, where polymor- MZ B cells (Fig. 7E, Supplemental Fig. 2B). Splenic plasmablasts phisms have positive disease association only in specific populations were increased, but plasma cell numbers remained unchanged or are dependent on other functional factors, such as IL-10 (9, 40, 41). (Fig. 7F, Supplemental Fig. 2C). Although total T cell numbers Overall, our observations suggest that autophagy may have opposing were unchanged in Tlr7.1 tg DBko mice, increased T cell acti- roles in the development versus progression of disease. vation (CD69) was seen (Fig. 7G, Supplemental Fig. 2D). The The importance of DC autophagy in disease progression is increase in B cell activation was surprising, given the defects evident by the increased mean survival and reduced IFN-a present reported to be associated with a loss of B cell autophagy (19, 39). in the sera of Tlr7.1 tg Dko mice, slowing down TLR7-mediated To test whether the inflammatory condition of Tlr7.1 tg DBko autoimmune development. However, over time disease was in- mice was due to the hyperactivation of their B cells, we created duced in these mice, and ANA production, liver inflammation, triple ko mice, combining a Cd19 ko with the B cell and DC loss and glomerulonephritis were present at similar levels in aged of autophagy on the Tlr7.1 tg background, and analyzed the sur- mice from both groups. Because elevated type 1 IFN expression vival of these mice. CD19 is critical for BCR function, because its is restricted to pDCs, even in the presence of TLR7 over- loss decreases the B cell response to Ag. As shown in Fig. 7H, loss expression (30), the phenotype of Tlr7.1 tg Dko mice suggests of CD19 rescued Tlr7.1 tg DBko mice from premature death. that a loss of DC autophagy is inhibiting the function of this cell Interestingly, the survival curve of these mice was similar to that type. In support of this, the improved survival of Tlr7.1 tg Dko of Tlr7.1 tg mice with a B cell loss of autophagy, where disease is mice is similar to the increased survival seen in Tlr7 tg mice that prevented (25). Furthermore, aged Tlr7.1 tg DBko Cd19ko mice are unable to develop pDCs due to Tcf4 haplodeficiency (42). showed a reduction in serum IL-18, similar to the levels in DBko These latter mice showed reduced glomerulonephritis, although controls (Fig. 7I). These data suggest that the lethal inflammatory ANA production persisted. It has been shown that type 1 IFN disease in Tlr7.1 tg DBko mice is initiated by B cells responding upregulates TLR7 activity through a positive feedback loop in to intra- or extracellular autoantigenic stimuli and is augmented by B cells (43). The presence of ANA and B cell activation in Tlr7.1 the Tlr7.1 tg and a loss of DC autophagy. tg Dko mice, as well as Tlr7 tg Tcf4 haplodeficient mice, indicates that B cells in Tlr7 tg mice do not require IFN-a produced Discussion by pDCs for autoantibody production. Using a TLR7-mediated model of autoimmunity (Tlr7.1 tg) (23), The clearance of apoptotic and necrotic cells, as well as we have previously shown that loss of B cell autophagy prevents microbes, by macrophages is dependent on LC3-associated disease, indicating that autophagy is required for disease devel- phagocytosis (LAP), a pathway that relies on several compo- opment (25). In this study, we demonstrate that autophagy deficiency nents of the canonical autophagy machinery, including ATG5, The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. The sepsis-like state of Tlr7.1 tg DBko mice is dependent on BCR activation. (A–D) Total LN B cell numbers (A) and activation markers (B220+ CD32 CD80+)(B) and total splenic B cells (C) and activation markers (D) are shown (total B cells, B220+CD32; activated B cells, B220+CD32 CD80+). (E–G) Splenic lymphocyte subsets: (E) follicular (FO) B cells and MZ B cells, (F) plasmablasts and plasma cells, and (G) T cell activation are shown (FO B cells, B220+CD32CD21+CD23+; MZ B cells, B220+CD32CD21low/2CD23+; plasmablasts, B220lowCD138mid;plasmacells,B2202CD138high; and activated T cells, B2202CD3+CD69+). Each point is representative of an individual animal. Results are based on at least three independent experiments with mice between 12 and 14 wk of age. *p , 0.05, **p , 0.01, ***p , 0.005 by a Kruskal–Wallis test with a Dunn posttest. (H) Survival of Tlr7.1 tg DBko mice (blue line) compared with Tlr7.1 tg DBko Cd19ko mice (dotted blue line). Curves of mice were measured during 58 wk. Controls (n = 14), Tlr7.1 tg (n = 9), Tlr7.1 tg DBko (n = 8), and Tlr7.1 tg DBko CD19ko (n = 5) mice are shown. *p , 0.05 by a Mantel–Cox log-rank test. (I) ELISA for serum IL-18 in mice aged 58 wk. *p , 0.05 by a Kruskal–Wallis test with a Dunn posttest.

ATG7, and Beclin (44). It has been shown that defects in this developed autoantibodies (C.G. Weindel, A.J. Mehta, and B.T. Huber, pathway result in increased production of proinﬂammatory cy- unpublished data). These ﬁndings indicate that mDCs, pDCs, and tokines (44, 45). Additionally, mice with a LysM-cre–mediated B cells are dispensable for the clearance of apoptotic debris deletion of LAP components in macrophages, monocytes, neu- through LAP. In addition to the clearance of apoptotic debris, LAP trophils, and mDCs develop an SLE-like disease over time, is also critical for TLR9-dependent IFN-a production following which includes autoantibody production and glomerulonephritis. the uptake of DNA immune complexes in pDCs (15). LAP- Disease progression in these mice was caused by the defective dependent clearance of RNA-containing complexes has not degradation of phagocytized dead cells (45). In contrast, all mice been described; nevertheless, the IFN-a defect in Tlr7.1 tg Dko in our study had functional ATG5 in macrophages, monocytes, mice indicates that TLR7-dependent IFN-a production by and neutrophils. Furthermore, control Dko and DBko mice never pDCsmayalsorelyonLAP. 10 AUTOPHAGY CONTROLS INFLAMMASOME ACTIVATION IN LUPUS

Given the importance of autophagy for B cell activation and response, and increased cell death following activation (19). plasmablast development, it was surprising that the Tlr7.1 tg Endoplasmic reticulum stress can also lead to reactive oxygen DBko mice developed a severe and lethal inflammatory condi- species and mitochondrial damage (54); mitochondrial damage tion. Their pervasive inflammation in the lung, liver, and kidney, and release of mtDNA, in turn, are known inflammasome acti- as well as the increase in inflammatory cytokines and expansion vators (55, 56). Although this would suggest that autophagy- of inflammatory myeloid cells, was reminiscent of a sterile deficient B cells respond to their own damaged mitochondria, systemic inflammatory response, sepsis in the absence of acute thereby augmenting the inflammatory response seen in Tlr7.1 tg infection (reviewed in Ref. 46). Consistent with this, the two Dko mice, our findings that Tlr7.1 tg Bko mice had normal levels cytokines associated with activation of the inflammasome, IL- of serum IgG, IL-18, and IL-1b is not consistent with this hy- 1b andIL-18,werepresentintheseraofthesemice.Interest- pothesis (25). Additionally, the slight but significant increase of ingly, IL-18 levels were more prominently elevated than IL-1b, IL-18 in Dko and DBko controls suggests that the IL-18 pro- which could be due to differences in turnover or assay sensi- duction present is a direct result of a DC-specific loss of ATG5. tivity. It is also possible that IL-1b is produced secondarily to Therefore, Tlr7.1 tg DBko B cells could be responding to ex- IL-18. Notably, IL-1b was undetectable in aged Dko and DBko tracellular Ag, such as the cytoplasmic components of dying DCs. controls (C.G. Weindel and B.T. Huber, unpublished data), It has been shown that B cells do not require autophagy for ac- whereas levels of IL-18 were significantly elevated. The fact that tivation through the BCR in C57BL/6 lpr/lpr mice (57). The IL-18 and IL-1b levels were not increased in Tlr7.1 tg Bko mice notable reduction of disease in Tlr7.1 tg DBko Cd19ko mice, (25) indicates that autophagy requirements for maintaining ho- deficient in BCR function, supports this latter hypothesis. Alter- meostasis and survival are cell type specific. natively, high levels of IL-18 have been shown to result in in- Downloaded from In addition to myeloid cell activation in Tlr7.1 tg DBko mice, we creased Ab production in B cells, which is mitigated by a loss of also observed increased B and T cell activation and elevated IgG. CD19 (58). The decrease in IL-18 levels in Tlr7.1 tg DBko The high serum levels of IgG1 in Tlr7.1 tg DBko mice, compared Cd19ko mice, similar to that of DBko and Dko controls, further with IgG2a and IgG2b in Tlr7.1 tg mice, suggest that autophagy- illustrates the critical role of both B cell function and IL-18 in deficient B cells respond differently to Ag than do B cells with augmenting disease.

working autophagy. Furthermore, the increase in B cell activation Abs against phospholipids such as CL are associated with http://www.jimmunol.org/ and total B cell numbers in Tlr7.1 tg DBko mice indicates that multiple immunological disorders, including antiphospholipid these cells were surviving and undergoing class switch recombi- syndrome (APS), rheumatoid arthritis, and scleroderma. Patients nation in the absence of functioning autophagy (18). Whereas suffering from APS share characteristics of SLE patients, that is, Tlr7.1 tg DBko mice lacked ANAs, they produced autoantibodies hematological malignancies and pregnancy complications (59). against cytoplasmic or membranous determinants, demonstrating Anti-CL Abs have also been found in SLE patients in conjunc- that Abs were being made against organelles. This was substan- tion with ANAs (60, 61). How these autoantibodies develop and tiated by the finding that these mice made anti-CL Abs, indicative contribute to disease pathogenesis remains an unanswered of damaged mitochondria. question. B cell depletion therapy has been shown to significantly It has been shown in some cell types that mitophagy occurs reduce the number of anti-CL Abs in SLE patients (62) and by guest on September 29, 2021 through ATG5/ATG7-independent autophagy (47, 48), and the could, therefore, provide a therapeutic alternative for patients vesicular maintenance of mitochondria is complementary to the with APS. Our study sheds light on potential cellular mecha- autophagy machinery (49). Therefore, the loss of Atg5 may allow nisms contributing to the production of lipid autoantibodies in clearance of damaged mitochondria under basal conditions. these diseases. Consistent with this, control mice lacking DC and B cell autophagy did not make elevated anti-CL Abs, suggesting that the Acknowledgments steady-state maintenance of mitochondria was not perturbed. The We are grateful to Herbert W. “Skip” Virgin for supplying the CD19-cre/ fact that TLR7 overexpression was required for the severe in- ATG5f/f mice, and we thank John F. Kearney and Parisa Kalantari for flammatory state and anti-CL Ab production indicates that ex- interesting discussions. cessive cellular activation and accumulated damage might exhaust the noncanonical autophagy machinery. Alternatively, cellular Disclosures stressors of mitochondria could be initiated prior to apoptosis. In The authors have no financial conflicts of interest. support of this, CL is depleted from damaged mitochondria in neurons prior to mitochondrial expulsion from cells (50); furthermore, in these cells, CL binds directly to LC3, resulting in References clearance by ATG7-dependent mitophagy (33). 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