Mitochondrial DNA oxidation propagates autoimmunity by enabling plasmacytoid dendritic cells induce Tfh differentiation

Mitochondrial DNA oxidation propagates autoimmunity by enabling plasmacytoid dendritic cells induce Tfh differentiation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondrial DNA oxidation propagates autoimmunity by enabling plasmacytoid dendritic cells induce Tfh differentiation Michael Karin, Hongxu Xian, Masafumi Ohira, Kosuke Watari, Jonathan Brito, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5194985/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jun, 2025 Read the published version in Nature Immunology → Version 1 posted You are reading this latest preprint version Abstract NLRP3 inflammasome activation depends on stress-induced production of oxidized mitochondrial DNA (Ox-mtDNA) fragments that enter the cytoplasm to bind NLRP3 and activate caspase-1. Along with pro-IL-1β processing, caspase-1 generates gasdermin D pores that result in circulatory mtDNA release. Elevated amounts of circulating cell-free (ccf)-mtDNA, which is likely to be oxidized, were documented in the elderly and patients with metabolic and autoimmune disorders and its intra-articular injection elicited arthritis in mice. Investigating whether ccf-mtDNA may promote autoimmunity, we found that induction of sustained Ox-mtDNA release triggered by a prototypical NLRP3 inflammasome activator elicited autoantibody production and glomerulonephritis in mice. Similar autoimmune responses, dependent on plasmacytoid dendritic cells (pDC) and T follicular helper cells (Tfh), were elicited by in-vitro generated Ox-mtDNA but not by non-oxidized mtDNA. Although both mtDNA forms were internalized by pDC and induced interferon-a, only Ox-mtDNA stimulated autocrine IL-1β signaling that induced expression of immunoregulatory and co-stimulatory molecules, including IL-21, that enabled mouse and human pDC convert naïve CD4 + T cells into functional Tfh, supportive of autoantibody production. Highlighting pDC-generated IL-1β as an orchestrator of autoantibody production, these findings suggest that Ox-mtDNA could be a key participant in immune-aging and unravel new therapeutic opportunities. Biological sciences/Immunology/Autoimmunity Biological sciences/Immunology/Signal transduction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Main The borderline between chronic inflammation and autoimmunity is obscure. Specifically, it is unclear whether in addition to inflammaging, persistent innate immune activation breaches self-tolerance and initiates long-lasting autoimmune responses, whose incidence increases with old age 1 , 2 . A key danger signal that triggers sterile inflammation through NLRP3 inflammasome mediated caspase-1 (Casp1) activation is Ox-mtDNA, an alarmin that enters the cytoplasm in response to mitochondrial stress and damage 3 , 4 , 5 . In addition to generating 8-oxo-deoxyguanosine (8-Oxo-dG) containing DNA which binds NLRP3 3, 4 , mtDNA oxidation promotes its cleavage into small fragments that pass through mitochondrial pores by the repair nuclease FEN-1 5 . Along with pro-IL-1β processing, activated Casp1 cleaves gasdermin D (GSDMD) to generate plasma membrane pores through which Ox-mtDNA escapes to the extracellular space and reaches the circulatory system 4 , 5 , 6 . Smoldering NLRP3 inflammasome activation has been linked to inflammaging through poorly defined mechanisms 7 , 8 , 9 . Moreover, mitochondrial dysfunction coupled to oxidative stress is considered as a major hallmark of aging 10 , 11 , 12 . However, whether ccf-Ox-mtDNA drives age-related immunopathologies and immune-aging is unknown. Given the elevated amounts of ccf-mtDNA, which is probably oxidized 6 , in older individuals who are at an elevated autoimmunity risk 2 , 13 , 14 , 15 , 16 , and patients with chronic inflammatory diseases and metabolic disorders, including rheumatoid arthritis 17 , systemic lupus erythematosus (SLE) 18 , 19 , cardiovascular disease 20 , type 2 diabetes mellitus (T2D) 21 , multiple sclerosis (MS) 22 and cancer 23 , we investigated whether Ox-mtDNA fosters the transition from persistent inflammation to maladaptive autoimmunity. Here we show that Ox-mtDNA sensing endows mouse and human pDC with the ability to convert naïve CD4 + T cells into Tfh cells that support pathogenic autoantibody production. Alum induces mtDNA-dependent pathological autoantibody production To address whether ccf-Ox-mtDNA produced during sterile inflammation can promote maladaptive autoimmune responses, we challenged naïve mice with antigen-free alum to induce NLRP3 inflammasome-dependent peritonitis 24 , which is accompanied by substantial generation of ccf-Ox-mtDNA 5 . Remarkably, in addition to sustained production of circulating IL-1β, successive intraperitoneal (i.p.) injections of alum into 8-week-old C57BL/6 (B6) mice increased anti-dsDNA IgG titers (Extended Data Fig. 1 a-c). The same protocol induced splenic B cell follicles harboring nucleosome- and DNA-reactive antibody-forming cells (AFC) that became apparent 58 days after initiation of alum injections but were undetectable in age-matched controls (Fig. 1 a, Extended Data Fig. 1 d and Supplementary Fig. 1a). At the same timepoint, flow cytometry (FC) revealed an increase in splenic class-switched IgM – IgG1 + CD19 + B cells, whereas plasma cells (PC; B220 int CD138 + CD3 − ), class-switched IgM – IgG2b + CD19 + B cells, Tfh cells (CXCR5 + PD-1 + CD44 + CD4 + ) and germinal center (GC) B (B220 + GL7 + CD38 − CD3 − CD11b − CD11c − ) cells were elevated by 100 days after initiation of alum treatment (Extended Data Fig. 1 e,f and Supplementary Fig. 1a-d). Notably, typical and systemic autoimmune features, which in addition to anti-dsDNA IgG production, include elevated BCL6-expressing Tfh cells (CXCR5 + ICOS + Foxp3 − CD4 + ), GC and marginal zone (MZ) B cells (CD21 hi CD23 − B220 + ), which class-switch into IgG + cells 25 , and glomerular enlargement, progressively increased till 130 days after initiation of alum treatment (Fig. 1 b, Extended Data Fig. 1 g-i and Supplementary Fig. 1e,f). To test the role of ccf-DNA in the alum-induced immune response, which was observed in both female and male mice (Extended Data Fig. 1 j), we administered alum followed by i.p. DNase I injections (Extended Data Fig. 2 a). Notably, DNase I treatment inhibited splenomegaly (Extended Data Fig. 2 b), decreased serum anti-dsDNA IgG measured by ELISA or Crithidia luciliae immunofluorescence (IF) test (CLIFT) and anti-chromatin IgG (Fig. 1 c and Extended Data Fig. 2 c), bone marrow (BM) and splenic DNA-reactive CD138 + AFCs located within GL7 + GC (Fig. 1 d,e and Extended Data Fig. 2 d), along with GC B cell numbers and frequencies, Tfh, PC, MZ B (Fig. 1 f and Extended Data Fig. 2 e), proliferative Ki67 + splenic B and CD4 + T cells (Extended Data Fig. 2 f and Supplementary Fig. 1g) and serum IL-21, IL-6, IL-1β and IFN-α (Extended Data Fig. 2 g). Glomerular swelling, proteinuria, IgG and complement (C3) deposition and CD45 + leukocyte infiltration were also blunted (Fig. 1 g-j). Supporting a role for Ox-mtDNA in autoimmunity, DNase I injections reduced the amounts of alum-induced ccf-mtDNA harboring oxidative 8-Oxo-dG lesions (Extended Data Fig. 2 h,i), which was much far abundant than circulating nuclear (n) DNA (Extended Data Fig. 2 j). These results suggested that ccf-DNA, most of which was mitochondrially derived and oxidized, is a major contributor to alum-induced autoimmunity and renal pathology. Alum-induced autoimmunity depends on NLRP3, GSDMD and mtDNA oxidation Macrophages challenged with NLRP3 inflammasome activators release ccf-Ox-mtDNA via Casp1 generated GSDMD pores 5 . Congruently, alum-challenged Nlrp3 −/− mice had less ccf-Ox-mtDNA than wildtype (WT) counterparts, with no change in barely detectable ccf-nDNA (Extended Data Fig. 3 a-d). Fittingly, alum-induced systemic autoimmune features (serum anti-dsDNA IgG, GC reactions, elevated Tfh and proliferative Ki67 + B and CD4 + T cells, class-switched splenic IgG1 + and IgG2b + B cells), serum IL-1β, IL-21, IL-6 and IFN-α, and glomerulonephritis were blunted in Nlrp3 −/− and Gsdmd −/− mice (Fig. 2 a-o). mt- Ogg1 Tg mice, which express a mitochondrially-targeted OGG1, a glycosylase that removes 8-Oxo-dG lesions and are therefore non-responsive to NLRP3 inflammasome activators 5 , also showed diminished GC reactions and reduced splenic Tfh cells (Extended Data Fig. 3 e,f). Moreover, mt-OGG1 lowered splenic and peripheral memory CXCR3 + PD1 + CD4 + T cells and IFN-γ + IL-10 + CXCR5 − CD4 + T cells, which support antibody production 26 , circulating Tfh cells and CD138 + B cells and glomerulonephritis (Extended Data Fig. 3 g-l and Supplementary Fig. 2a,b). These results establish the importance of Ox-mtDNA in the alum-induced autoimmune response. Alum induced ccf-Ox-mtDNA sensing by functionally important peritoneal pDC Single-cell (sc) RNA-Seq provided unbiased and in-depth view of alum’s effects on the peritoneal immune microenvironment. Strikingly, within 24 h after injection, alum led to extensive depletion of peritoneal macrophages, previously reported after i.p. LPS or thioglycolate injections 27 , accompanied by peritoneal influx of DC subsets, monocytes, neutrophils, and NK cells (Fig. 3 a and Supplementary Fig. 3a). Macrophage (F4/80 + CD11b + ) depletion was suppressed in Gsdmd −/− mice, which also displayed reduced IL-1β production (Fig. 3 b), suggesting GSDMD-mediated pyroptosis is pivotal to alum-induced macrophage death and Ox-mtDNA release 5 . Amongst the different peritoneal cell types in alum-injected mice, DCs, which bridge innate and adaptive immunity, were particularly enriched for inflammation - and Il1b -related pathways (Fig. 3 c). Peritoneal DC classification showed no changes in transitory (t) DC, an insignificant decline in cDC2 and alum-induced increases in cDC1, pDC-like, and CD40 + MHCII + CD80 + pDC, whose number and frequency were reduced after DNase I injections, or in Nlrp3 −/− , Gsdmd −/− , mt- Ogg1 Tg and Cmpk2 ΔMye [myeloid restricted cytidine/uridine monophosphate kinase 2 (CMPK2) deficiency that blocks TLR4-stimulated mtDNA synthesis] mice 28 (Fig. 3 d-i and Supplementary Fig. 3b-d), all of which produce less Ox-mtDNA 4 , 5 , 28 (Extended Data Fig. 4 a-f). By contrast, PBS-injected mice had very few peritoneal pDC (Fig. 3 d-i). Compared to splenic pDC at steady state, alum-induced peritoneal pDC showed upregulation of IFN-stimulated genes (ISGs), Il1b, Pycard and P2rx7 mRNAs (Fig. 3 j) and Casp1 activation (FLICA hi ) (Fig. 3 k). Alum induced pDC also expressed IFN-α, IRF7, CD40, MHC II, CD80 and IL-21 proteins (Fig. 3 l), although the intensity of IL-21 intracellular staining in pDC was lower than in alum-induced Tfh cells (Fig. 3 m). Since FLICA-detected Casp1 activity was much lower in cDC1 and cDC2 (Fig. 3 k), and amongst the alum-induced DC subsets, peritoneal pDC expressed higher amounts of inflammasome-related Nek7 and P2rx7 mRNAs (Fig. 3 n), we examined pDC’s role in alum-stimulated autoimmunity. Diphtheria toxin (DT)-induced splenic and peritoneal pDC depletion in BDCA2 -DTR (DT receptor) transgenic mice 29 (Extended Data Fig. 4 g,h), diminished alum-induced splenomegaly (Extended Data Fig. 4 i), anti-dsDNA and anti-chromatin IgGs (Extended Data Fig. 4 j), serum IL-21 and IFN-α (Extended Data Fig. 4 k), splenic GC reactions, Tfh cells, class-switched IgG1 + and IgG2b + B cells (Extended Data Fig. 4 l,m), and glomerulonephritis (Extended Data Fig. 4 n). DT-treated DTR − mice, however, still exhibited alum-induced autoimmunity, suggesting that the above effects, were due to pDC depletion. This is consistent with the recently demonstrated amelioration of autoimmunity by Litifilimab, a humanized pDC depleting BDCA2 antibody 30 . pDC intrinsic IL-1β signaling induces functionally important Tfh differentiation We established an ex vivo model in which peritoneal or splenic pDC isolated after alum injections were co-cultured with naïve splenic CD4 + CD25 − CD62L hi CD44 lo T cells (Extended Data Fig. 5 a and Supplementary Fig. 3d, 4a). Either peritoneal or splenic pDC from alum-challenged, but not from PBS-injected, mice induced the generation of CXCR5 + ICOS + Foxp3 − CD44 + CD4 + Tfh cells (Extended Data Fig. 5 a). Of note, peritoneal pDC collected 24 h after two alum injections (72 h apart) were more effective in inducing Tfh differentiation than splenic pDC isolated at the same timepoint, presumably due to early ccf-Ox-mtDNA sensing by peritoneal pDC, which then migrate to the spleen. To test whether Ox-mtDNA directly enables pDC induce Tfh differentiation, we extracted mtDNA and nDNA from fibroblasts and left half of each preparation as is, while the other half was oxidized by UV irradiation (see Methods). Tfh differentiation was triggered by FACS-sorted Flt3L-induced BM-derived CD11c + CD11b − B220 + BST2 + Siglec-H + pDC, but this required incubation with exogenous Ox-mtDNA, while non-oxidized mtDNA was ineffective (Extended Data Fig. 5 b and Supplementary Fig. 4b). Notably, Ox-mtDNA-treated Flt3L-induced cDC1 or cDC2 did not trigger Tfh differentiation (Extended Data Fig. 5 c). Neither oxidized nor non-oxidized nDNA enabled pDC-programmed Tfh differentiation (Extended Data Fig. 5 d), which required direct pDC-T cell contact, because when pDC and T cells were separated via transwell chambers, Tfh were not generated (Extended Data Fig. 5 e). Tfh cells generated in this manner were functional, converting naïve B to GC B cells, as well as proliferative (Ki67 + ) and class-switched IgG-secreting B cells (Extended Data Fig. 5 f-h). Splenic Tfh-containing CD4 + T cells from alum-challenged mice also promoted IgG-secretion and converted naïve B cells into GC B cells but not into CD138 hi B220 lo IgD − CD3 − CD19 + plasmablasts (PB) (Extended Data Fig. 5 i,j). Ox-mtDNA also enabled the conversion of naïve human CD4 + T into BCL6 + Tfh cells by enriched human blood pDC (Extended Data Fig. 5 k,l). To establish the importance of Tfh cells in Ox-mtDNA-driven autoimmunity, Bcl6 f/f / Cd4 - Cre ( Bcl6 ΔCD 4 ) mice with a Tfh cell differentiation defect 31 were used. Naïve CD4 + T cells from Bcl6 ΔCD 4 mice did not differentiate into Tfh cells when co-cultured with pDC plus Ox-mtDNA (Extended Data Fig. 6 a). Compared to Bcl6 f/f mice, Bcl6 ΔCD 4 mice were refractory to alum-induced autoimmunity, manifested by BCL6 upregulation in CD4 + T cells, elevated serum anti-dsDNA IgG, GC reactions, serum IL-21 and glomerulonephritis (Extended Data Fig. 6 b-i), despite exhibiting elevated serum IL-1β and splenomegaly (Extended Data Fig. 6 f,j). Importantly, alum-induced infiltration of peritoneal pDC with activated Casp1 was unaffected by the BCL6 deficiency (Extended Data Fig. 6 k,l), indicating that autoreactive Tfh cells are generated downstream to Ox-mtDNA sensing by pDC and NLRP3 inflammasome activation. Ox-mtDNA sensing by pDC triggers autocrine IL-1β signaling Ox-mtDNA, but not oxidized nDNA (Ox-nDNA) or non-oxidized mtDNA, activated Casp1 in pDC, but not in cDC1, cDC2 or BM derived macrophages (BMDM) (Fig. 4 a,b). pDC incubation with Ox-mtDNA induced IL-1β, IL-21, IL-6, CD40 and ICOSL (Fig. 4 c,d), proteins known to stimulate Tfh cell differentiation 32 , 33 , 34 , which were not induced in Ox-mtDNA treated cDC1 or cDC2 (Fig. 4 e-g). Non-oxidized mtDNA did not induce IL-21 or ICOSL, although it induced IFN-α as effectively as Ox-mtDNA (Fig. 4 c,d). Both mtDNA forms, but not nDNA, triggered TLR9-dependent NF-κB activation (Fig. 4 h), although the response to Ox-mtDNA, the only DNA type that activated Casp1, was stronger and relied on IL1R expression in pDC (Fig. 4 i), implicating autocrine IL-1β signaling. IL-21 and IL-6 inductions were also abrogated in Il1b −/− or Il1r −/− BM-derived pDC (Fig. 4 j), that were no longer capable of inducing Tfh differentiation (Fig. 4 k). Ox-mtDNA had no effect on naïve CD4 + T cells in the absence of pDC (Fig. 4 k). CD4 + T cell intrinsic IL-1R signaling was also needed for Tfh generation (Fig. 4 l), consistent with its role in vaccine-related induction of Tfh and antibody responses 35 , 36 . However, IFNAR-deficient naïve CD4 + T cells converted to Tfh as effectively as WT CD4 + T cells when incubated with pDC and Ox-mtDNA (Fig. 4 m), suggesting that pDC-derived IFN-α does not directly contribute to Tfh differentiation. Despite its relatively low expression level, pDC-generated IL-21 was essential for induction of BCL6 + Tfh differentiation (Fig. 4 n,o). In-vitro oxidized mtDNA is sufficient for in-vivo induction of autoimmunity To test if cell- and protein-free Ox-mtDNA triggers autoimmunity in-vivo , we injected B6 mice with equal amounts of non-oxidized and oxidized mtDNA prepared as above (Fig. 5 a). When examined 24 h post-injection, both mtDNA preparations induced alum-like peritoneal influx of DCs enriched for inflammation - and Il1b -related pathways (Fig. 5 b), that included pDC (Fig. 5 c,d). However, scRNA-seq analysis showed Ox-mtDNA to be more effective than non-oxidized mtDNA in inducing NF-κB- and inflammasome-related mRNAs ( Nfkbia, Irak1, Nfkbiz, P2rx7, Casp1, Gsdmd, Nfkb2, Il1b, Nlrp3, Tlr9 ) in pDC (Fig. 5 e), in which certain inflammatory genes ( Nfkbiz, P2rx7, Nek7, Tnf, Tlr9) were more highly expressed than in other DC subsets (Fig. 5 f). To test whether pDC recruited and activated by Ox-mtDNA can program naïve CD4 + T cells to Tfh ex vivo , enriched peritoneal pDC isolated from mtDNA-injected mice were co-cultured with naïve CD4 + T cells. Ox-mtDNA, but not non-oxidized mtDNA, injections enabled pDC induce Tfh differentiation (Extended Data Fig. 7 a). Splenic DCs from DT-treated BDCA2 -DTR − mice subjected to 4 repetitive Ox-mtDNA injections also induced Tfh differentiation, but DCs from DT-treated BDCA2 -DTR + mice were inactive in this assay (Extended Data Fig. 7 b), suggesting that induction of Tfh differentiation is pDC- and Ox-mtDNA- specific. Importantly, a 20-week regimen of repetitive Ox-mtDNA injections induced dsDNA and chromatin antibodies, elevated serum IL-1β, IL-21 and IFN-α, GC reactions, class-switched B cells and splenic Tfh cells (Fig. 5 g-k) while induction of circulating BCL6 + Tfh cells and glomerulonephritis required a 36-week regimen of Ox-mtDNA injections (Fig. 5 l-o). In situ proximity ligation assays (PLA) conducted on spleens isolated after 20 weeks of Ox-mtDNA injections showed interactions between intracellular DNA and either TLR9 or NLRP3 in BST2 + pDC (Fig. 5 p; 100x magnification), whereas injected mtDNA elicited DNA:TLR9 (Fig. 5 q; 10x magnification) but not DNA:NLRP3 interactions (Fig. 5 r; 10x magnification). These results confirm that NLRP3 does bind DNA within pDC after in-vivo Ox-mtDNA challenges. FcγR1 mediates mtDNA uptake followed by TLR9 binding and endosomal escape NLRP3 inflammasome activation and GSDMD cleavage in Ox-mtDNA incubated pDC were required for induction of Tfh differentiation, because the latter was diminished when Nlrp3 −/− or Gsdmd −/− pDC were used (Fig. 6 a). TLR9 in Flt3L-induced pDC was also needed for induction of Tfh differentiation (Fig. 6 a), as well as Casp1 activation and IL-21 and IRF7 induction (Fig. 6 b-d). Consistent with the equally effective induction of IFN-α by non-oxidized and oxidized mtDNAs, both mtDNA types upregulated TLR9-dependent IRF7 (Fig. 6 d), suggesting no difference in their uptake efficacy. In vivo , biotinylated Ox-mtDNA was taken up by peritoneal pDC within 24 h after its i.p. injection, while biotinylated Ox-mtDNA internalization by splenic or BM pDC was not detected at this time point (Extended Data Fig. 8a), suggesting that peritoneal pDC are the first to encounter i.p. injected ccf-Ox-mtDNA. To determine how ccf-Ox-mtDNA reaches NLRP3 in the cytosol, we isolated mature splenic pDC and incubated them with biotin-labeled non-oxidized or in-vitro oxidized mtDNAs (Extended Data Fig. 8b). Both mtDNA types were internalized and colocalized with TLR9 (Extended Data Fig. 8b,c), whose expression was substantially higher in pDC than in cDC1 or cDC2 (Extended Data Fig. 8d), consistent with higher Tlr9 mRNA amounts in pDC (Fig. 3 n, 5 f). These results explained why pDC responded to Ox-mtDNA more vigorously than cDC. Moreover, RNA-seq analysis of FACS-sorted BM-derived pDC after ex-vivo stimulation showed that Ox-mtDNA suppressed the “negative regulation of CpG-DNA responses” 37 and enhanced “hallmarks of NF-κB response to TNF” 38 more robustly than non-oxidized mtDNA, in line with a more enriched autoimmune KEGG SLE pathway (Fig. 6 e). Consistently, Ox-mtDNA, but not Ox-nDNA, induced a more robust NF-κB signaling response than non-oxidized mtDNA in pDC, but not in cDC1 or 2 (Fig. 6 f). Congruently, only Ox-mtDNA engaged NLRP3 to trigger inflammasome assembly evidenced by appearance of DNA:NLRP3 PLA signals (Fig. 6 g) and ASC-containing aggregates in cultured pDC (Fig. 6 h). Serum or IgG removal from the culture medium impaired Ox-mtDNA-stimulated Casp1 activation (Fig. 6 i), suggesting that mtDNA was internalized as a complex with serum IgGs. In line with the known ability of Fc receptors to endocytose antibody-DNA complexes 39 , pDC lacking FcγR1, which presents IgG-DNA complexes to endosomal TLR9 40 , no longer induced Tfh differentiation (Fig. 6 j). Of note, RNA-seq data analysis revealed that both mtDNA forms downregulated endosome organization and vesicle tethering complex pathways (Extended Data Fig. 8e). This was confirmed by presence of both mtDNA forms within ruptured, galectin 8 positive, endosomes 41 , 42 (Extended Data Fig. 8f), which allow mtDNA access to cytosolic sensors, such as NLRP3 4, 5, 6 . We thus conclude that circulating Ox-mtDNA binds (probably non-specifically) preexisting serum IgGs, which enable its internalization via FcγR1 and delivery to endosomal TLR9, whose engagement results in NF-κB mediated priming that licenses NLRP3 inflammasome activation and production of IL-1β, which is secreted via GSDMD pores. In turn, autocrine IL-1β-IL1R signaling further boosts NF-κB signaling (Extended Data Fig. 8g). IL-1R signaling can be targeted to prevent autoimmunity To validate the importance of IL-1R signaling in autoimmunity, irradiated mice were reconstituted with WT or Il1r1 −/− BM cells and challenged with alum (Fig. 7 a). IL-1R ablation blunted generation of dsDNA and chromatin antibodies (Fig. 7 b), IL-21 production (Fig. 7 c), GC formation (Fig. 7 d,e), Tfh expansion, class-switched splenic IgM – IgG1 + CD19 + B cells (Fig. 7 e) and glomerulonephritis (Fig. 7 f-h). Serum IFN-α (Fig. 7 c) and circulating Ox-mtDNA (Fig. 7 i,j), however, remained unaffected, indicating that IL-1β signaling does not contribute to IFN-α or ccf-Ox-mtDNA production. Supporting IL-1β-IL-1R interaction as a potential therapeutic target, treatment of alum challenged mice with IL-1R antagonist (IL-1Ra, anakinra) inhibited splenomegaly, generation of dsDNA antibodies, IL-21 secretion, splenic Tfh, GC generation, B cell isotype switching and glomerulonephritis (Extended Data Fig. 9a-i), with no significant change in circulating Ox-mtDNA amounts (Extended Data Fig. 9j, k). Discussion Our results unravel how oxidation converts IFN-I-inducing mtDNA to a unique and highly immunopathogenic alarmin capable of initiating systemic autoimmune reactions through IFN-I independent mechanisms. As elevated amounts of ccf-mtDNA were detected in elderly individuals 14 , 15 , 16 and patients with metabolic 43 and autoimmune disorders 6 , 18 , 19 , 44 , our results suggest that mtDNA oxidation is a key step in the pathogenesis of inflammaging. Most reports of ccf-mtDNA in the human circulation do not include any information regarding its oxidation state, but our earlier studies show that mtDNA oxidation is obligatory for its cleavage by FEN-1, which enables its cytoplasmic leakage, and binding to NLRP3 and subsequent Casp1 activation 4 , 5 . Although the source and mechanism of ccf-mtDNA production during inflammaging are unknown, we suggest that as long as ccf-mtDNA is generated in response to mitochondrial stress, most of it contains 8-Oxo-dG residues that enable NLRP3 binding, IL-1β induction and GSDMD cleavage, driving progression from sterile inflammation, manifested by splenomegaly and peritonitis, to systemic antibody-mediated autoimmunity. Importantly, non-oxidized mtDNA induces IFN-α in pDC as effectively as Ox-mtDNA but is incapable of inducing autoantibody production when injected into mice. In other words, the difference in the immunogenic properties of oxidized and non-oxidized mtDNAs boils down to their ability and inability to activate NLRP3. The mechanism by which NLRP3-dependent sensing of Ox-mtDNA by pDC circumvents self-tolerance is unprecedented. We show that Ox-mtDNA uptake endows pDC with a new activity: the ability to convert naïve CD4 + T cells into Tfh cells that play a critical role in autoantibody induction in our experimental system (Extended Data Fig. 10). Importantly, Ox-mtDNA also enables human pDC stimulate the generation of Tfh cells, whose elevated abundance has been linked to antibody-dependent autoimmunity 45 , 46 . Originally, pDC were studied for their ability to produce copious amounts of IFN-α 47 , 48 , rather than their T cell priming and differentiation inducing activities. Human pDC activated by CpG oligodeoxynucleotides were reported to be tolerogenic because they induced CD4 + CD25 + Treg cells 49 . In contrast, the ability to secrete IL-6 50, 51 and up-regulate ICOSL 52 , 53 , upon microbial or viral stimulation, suggested that beyond Treg induction, pDC may be capable of promoting Tfh differentiation, at least under certain circumstances 34 . However, whether and how pDC initiate Tfh generation in-vivo was heretofore unknown. Induction of Tfh differentiation depends on pDC-T cell contact, which could be mediated through ICOSL (on pDC):ICOS (on T) and CD40 (on pDC):CD40L (on T) interactions, as well as autocrine and paracrine IL-1β signaling, which induces IL-21 and IL-6 that prime Tfh differentiation 34 , 54 , 55 , 56 . pDC also express MHC molecules (Fig. 3 i,l) but are generally considered as poor antigen presenters 57 , 58 . At this point we don’t know whether antigen presentation has a role in the initiation of Tfh differentiation by Ox-mtDNA stimulated pDC, which warrants further investigation. The key step through which autocrine IL-1β signaling acts is the enhancement of NF-κB activation, beyond the initial amount provided by TLR9 engagement. Both oxidized and non-oxidized mtDNAs are internalized by pDC as complexes with serum IgG molecules via FcγR1, which presents DNA to endosomal TLR9 40 . In addition to IFNα induction, TLR9 signaling accounts for the initial activation of NF-κB, which is required for the so-called “priming stage” that includes NLRP3 and pro-IL-1β induction and precedes NLRP3 inflammasome activation by Ox-mtDNA 4 . Moreover, TLR9 discriminates between non-methylated mtDNA and methylated nDNA 59 , 60 , 61 , which is non-immunogenic in our system, even after its oxidation. Of note, IFNAR deficient CD4 + T cells can still be converted to Tfh, in line with the inability of mtDNA induced IFN-α to drive IL1β-dependent Tfh generation. Consistently, IL-1β was previously found to play a leading role in Tfh generation (through induction of BCL6, ICOS and CXCR5) in response to microbial vaccines, while IFN-I was shown to promote IL-21 production by already differentiated Tfh cells 36 . Studies also show that the autoimmune activities of IFN-α are directed towards other cellular targets, most importantly B cells 44 , 50 . In summary, our work shows that, although much remains to be known about its origin and the time of its production, Ox-mtDNA is an immunogenic alarmin capable of propagating autoimmunity, whose risks increase with old age, paralleling its amounts in the circulation. We suggest that pDC intrinsic IL-1β-IL1R signaling needs to be assessed for its role in systemic autoimmune disorders and if validated as a culprit, should be studied as a target for interception with age-related immunopathologies. Methods Mice Wildtype and Tlr9 −/− mice in the C57BL/6 background were purchased from the Jackson Laboratory. Cmpk2 ΔMye and mt- Ogg1 Tg mice were previously described 5 , 28 , Nlrp3 −/− , Gsdmd −/− , Ilb −/− and Il1r1 −/− mice in the C57BL/6 background were maintained by Hal M. Hoffman (UCSD). BDCA2 -DTR transgenic mice were provided by Elina I. Zuniga (UCSD). Bcl6 f/f / Cd4 - Cre ( Bcl6 ΔCD 4 ) mice were kindly provided by Alexander Dent at Indiana University. All mice were bred and maintained at UCSD and handled in accordance with Institutional Animal Care and Use Committee and NIH guidelines. Gender matched 6–10-week-old mice were used for all experiments. Bones from Il21 −/− , Fcgr1 −/− and Ifn1r1 −/− mice were provided by Drs. Warren Leonard (NIH) and Yi-Guang Chen (Medical College of Wisconsin), Jefferey Ravetch (Rockefeller University) and Kevin King (UCSD), respectively. Bone marrow chimeras Gender-matched 6–8-week-old C57BL/6 mice were lethally γ-irradiated twice with 600 Rad doses, given 4 h apart. Irradiated mice were reconstituted with 5x10 6 BM cells injected intravenously and used 8 weeks later. Alum/mtDNA-induced short-term peritonitis and long-term autoimmunity Mice were allocated randomly and i.p. injected with 1 mg Imject Alum (Thermo Scientific, Cat#77161) in 0.2 mL sterile PBS, or 50 µg mtDNA/Ox-mtDNA/PBS. Peritoneal lavage was performed 4 h post-injection to measure IL-1β by ELISA. Another batch of randomly allocated mice were i.p. injected with alum or PBS and euthanized after 24 h. Their peritoneal cavities were washed with 6 mL cold sterile PBS, followed by 10 min centrifugation at 1200 rpm to separate peritoneal cells and fluid. 10,000 peritoneal cells with viability > 95% were profiled using 10x Genomics’ Chromium Single Cell 3′ V2 chemistry and processed using the 10x Cell Ranger pipeline for scRNA-seq and the rest were FC analyzed. For induction of long-term autoimmunity, randomly allocated gender and age-matched mice were repetitively i.p. injected with alum (1 mg/mouse) or DNA (50 µg/mouse) as indicated. Wherever indicated, 1.25 mg/mouse DNase I (Millipore, Cat#10104159001, Roche), 100 ng/mouse DT (Millipore, Cat#D0564) or 25 mg/kg anakinra were also i.p. administered. Cell Lines and Primary Culture Bone-marrow-derived macrophages (BMDM): Femurs and tibias from C57BL/6 mice > 8 weeks of age of the same gender were used for BMDM generation by culturing BM cells in high glucose DMEM supplemented with 10% FBS, 20% L929-cell conditioned medium, and 100 U/mL penicillin-streptomycin for 7–10 days at 37°C with 5% CO 2 62 . BMDCs were generated by culturing BM cells at 2x10 6 cells/ml for 7 days in 5 ml of RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) containing 100 ng/mL Flt3L and 50 µM β-mercaptoethanol. Cells were either left untreated or activated with DNA as indicated, followed by FC analyses of protein expression/inflammasome activation. RNAs were collected 4 h post-stimulation from FACS sorted pDC for bulk RNA seq analyses. Ig depletion from FBS for BMDC culture: the Pierce™ Protein A/G Magnetic Beads suspension (Thermo Scientific, Cat#88802) was washed three times with PBS, followed by incubation with FBS at 4°C for 24 h with constant mixing, after which IgG-depleted serum FBS was collected and used for BMDC culture. Mouse embryo fibroblasts (MEFs) were cultured in high glucose DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin at 37°C with 5% CO 2 . Human specimens and peripheral blood mononuclear cells (PBMCs) De-identified blood and buffy coats were purchased from San Deigo Blood Bank and StemCell Technologies Human Peripheral Blood Leukopak obtained from healthy adult donors (20–45 years old). PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Human pDC and naïve CD4 + T cells were negatively enriched using EasySep™ Human Plasmacytoid DC Isolation Kit (StemCell Technologies, Cat# 17977) and EasySep™ Human Naïve CD4 + T Cell Isolation Kit (StemCell Technologies, Cat# 19555) respectively. Cell suspension preparation Blood was collected from anesthetized mice via cardiac puncture into a vial containing 1 mM EDTA (Corning), and mononuclear cells were enriched using Lympholyte M (Cedarlane). The serum fraction was used to measure autoantibodies, cytokines and circulating mtDNA. Splenocyte suspensions were obtained by mechanical dissociation in FACS buffer (2 mM EDTA, 2% FBS in PBS) and passed through a 70 µm strainer. For BM, femurs and tibias were flushed using PBS and filtered through a 70 µm strainer. Red blood cells were lysed using RBC Lysis Buffer (ThermoFisher). Cell Purification Peritoneal or splenic pDC were purified using EasySep™ Mouse Plasmacytoid DC Isolation Kit (StemCell Technologies) per manufacturer’s instructions. Unlabeled pDC were enriched by negative selection and pDC (PI − Thy1.2 − CD19 − NK1.1 − CD11c + CD11b − B220 + BST2 + Siglec-H + ) purity was routinely assessed by FC. Splenic DC were enriched using Dendritic Cell Isolation Kit, mouse (Miltenyi Biotec, Cat# 130-100-875). Cultured BMDC were stained and sorted into pDC (PI − CD11c + CD11b − B220 + BST2 + Siglec-H + ), cDC1 (PI − CD11c + B220 − XCR + CD11b − ), cDC2 (PI − CD11c + B220 − XCR1 − CD11b + ), using BD Aria II or Fusion (BD Biosciences). Naïve splenic CD4 + T cells (CD62 + CD44 − CD25 − CD4 + ) were negative-selected using EasySep™ Mouse Naïve CD4 + T Cell Isolation Kit (StemCell Technologies, Cat#19765) or FACS-sorted. Total splenic CD4 + T cells were enriched with a CD4 + T Cell Isolation Kit, mouse (Miltenyi Biotec, Cat# 130-104-454). B cells were purified by negative selection using CD43 (Ly-48) MicroBeads, mouse (Miltenyi Biotec, Cat#130-049-801). T or B cells were FC analyzed 3 days after co-culture in RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) supplemented with 50 µM β-mercaptoethanol. pDC and CD4 + T cell co-culture assay Purified/sorted mouse pDC (3×10 4 cells/well) or human pDC (1×10 4 cells/well) were seeded with naïve mouse splenic CD4 + T cells (1.5×10 5 cells/well) or human naïve CD4 + T cells (5×10 4 cells/well) into 24-well plates. 50 µg mtDNAs or nDNAs were added into RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) supplemented with 50 µM β-mercaptoethanol. Cells were co-cultured for 3 days after which Tfh differentiation was FC analyzed. Flow Cytometry (FC) Antibodies were from Biolegend, BD Biosciences, ThermoFisher and Cell Signaling Technologies (Supplementary Table 1) and were used at 1:200 dilution. Cell suspensions were incubated with Purified Rat Anti-Mouse CD16/CD32 (BD Pharmingen™ Cat#553141) to block nonspecific binding for 15 min at 4°C, followed by incubation with antibody cocktails for cell surface protein staining for 45 min at 4°C. PI or eBioscience™ Fixable Viability Dye eFluor™ 780 (ThermoFisher, Catalog# 65-0865-14) was used to exclude dead cells. Human cells were incubated with human gamma-globulin (ThermoFisher, Cat# Catalog # 31879) to block non-specific binding for 15 min at 4°C, followed by antibodies staining cell surface protein in human FACS buffer (2 mM EDTA, 2% Donor equine serum in PBS) for 30 min at RT. For intracellular staining, cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining kit (Life Technologies Cat# 00-5523-00) according to manufacturer’s instructions. Cells were analyzed on a Beckman Coulter Cyan ADP flow cytometer followed by data analysis with FlowJo software (Treestar, Inc.). To determine anti-DNA/nucleosome antibody-forming cells (AFCs), biotin-tagged mtDNA or recombinant nucleosomes (BPS Bioscience, Catalog #52048) were incubated with single cell suspensions after CD16/CD32 Fc blocking, for 15 min at 4°C, followed by cell surface protein staining for 45 min at 4°C and Streptavidin, Alexa Fluor™ 488 conjugate (ThermoFisher, Cat# S11223) in FACS buffer for additional incubation 30 min at 4°C. Cytokine and IgG Measurements Secreted cytokines from mouse sera or culture supernatants of FACS-purified pDC (3×10 5 cells/well in 96-well plates) were measured using mouse ELISA kits following manufacturer’s instructions. FACS-purified pDC were incubated in RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) supplemented with 50 µM β-mercaptoethanol in the presence or absence of non-oxidized or UV-oxidized mtDNA (50 µg) for 4 h at 37°C. Supernatants were collected and analyzed. Graphs depicting cytokine measurements represent individual wells from several independent experiments. Relative titers of anti-dsDNA (Chondrex Cat#3031), anti-nucleosome (Creative Diagnostics, Cat#DEIA-BJ2361) in sera and total IgG (ThermoFisher, Cat#88-50400-22) amounts in B cell culture supernatants were measured by ELISA. Urinary protein was measured using Mouse Proteinuria ELISA Kit (MyBioSource, Cat#MBS723873). DNA Isolation and in-vitro preparation of Ox-DNA Mitochondrial isolation from MEF was carried out as described 5 , 63 . Briefly, PBS-washed MEF were resuspended in pre-chilled mitochondrial extraction buffer 1 (220 mM mannitol, 70 mM sucrose, 20 mM HEPES-KOH, pH 7.5, 1 mM EDTA and 2 mg/mL BSA) and passed through a 25-G syringe (BD Biosciences) 20x on ice. The homogenized cells were centrifuged at 1000×g for 15 min at 4°C yielding nuclear pellets. The post-nuclear supernatant was further centrifuged at 10,000×g for 10 min at 4°C to pellet mitochondria from supernatant cytosolic fraction. mtDNA was purified from the mitochondrial fraction while nDNA was extracted from the nuclear pellets using AllPrep DNA/RNA Mini Kit (Qiagen, Cat#80204) according to manufacturer’s instructions, followed by 10x UV irradiation at 1,000 mJ/cm 2 to generate Ox-mtDNA or Ox-nDNA 64 , 65 . Biotin-mtDNAs were prepared using Label IT Tracker Intracellular Nucleic Acid Localization Kit, Biotin Kit (Mirus Bio, Cat#MIR 7024), per manufacturer’s instructions. Measurements of circulating mtDNA Seral DNA was isolated using QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat#55114) according to manufacturer’s instructions, eluted in 300 µL dd H 2 O and quantified using spectrophotometric analysis at 260/280 nm with Nanodrop. qPCR was performed using mtDNA ( D-loop , Cox1 , non-NUMT ), and nDNA ( Tert , 18S , B2m ) primers (Supplementary Table 2). Ct values of mtDNA abundance obtained from the alum-challenged group served as normalization controls. For measurement of Ox-mtDNA, 8-Oxo-dG content was quantified using 8-hydroxy 2-deoxyguanosine ELISA Kit (Abcam, Cat#ab201734), per manufacturer’s instructions. To determine mtDNA oxidative damage, Fpg-sensitive qPCR analysis was used as described 5 (Fpg removes oxidized purines from DNA and creates single-strand breaks, which block PCR amplification. Differences in qPCR cycles between Fpg-treated and untreated DNAs are therefore a specific indicator of oxidative base damage). Briefly, 500 ng of purified ccf-DNA were incubated with 8 units of Fpg (New England BioLabs Inc., Cat#M0240S) in 1×NEBuffer 1 and 100 µg/mL BSA in 50 µL at 37°C for 1 h. Fpg was then inactivated at 60°C for 5 min, followed by qPCR to detect Fpg-sensitive cleavage sites. Data are presented as the ratio of Fpg-insensitive DNA, calculated as the quotient of signal intensities in Fpg-treated relative to untreated DNA. Relative mtDNA amounts were determined by qPCR in a CFX96 thermal cycler (Biorad) as described 28 . Data are presented in arbitrary units and calculated by the 2ˆ(−ΔΔCT) method. Primers were provided by Integrated DNA technologies. Histological evaluation and immunohistochemistry Spleen and kidneys were fixed in 10% formalin for 24 h and embedded in paraffin. 5 µm thick sections were stained with hematoxylin and eosin (H&E) or Periodic Acid-Schiff (PAS). Glomerular size was measured. Spleen sections were incubated with Ki67 antibody and positive cells were quantified with ImageJ. Spleen and kidneys were frozen in OCT and cryosectioned at 5 µm thick, followed by fixation with 4% paraformaldehyde (PFA) and chilled acetone, blocked with 5% donkey serum in PBS supplemented with 3% BSA, and stained with DAPI and specific antibody conjugates (Supplementary Table 1). Images were captured on a Leica SP5 microscope at 20X magnification. Crithidia luciliae immunofluorescence test (CLIFT) of sera was determined according to manufacture instructions (Bio-Rad, Catalog#26109) using a Leica SP5 confocal microscope at 20X magnification. IF and confocal microscopy Naïve splenic pDC from B6 mice stimulated ex vivo with 2 µg mtDNA/Ox-mtDNA for 1 h were fixed with 4% PFA, permeabilized in 0.2% Triton X-100, and blocked with 3% BSA/PBS. Primary antibodies were incubated in blocking buffer at 4°C overnight. Secondary Alexa-labelled antibodies were added for 1 h. Nuclei were counterstained with DAPI. Samples were imaged at 100x magnification on Leica SP5 or AXR Confocal, NSPARC Super Resolution microscopes. For the in situ PLA, the Duolink® In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, Cat#DUO92101) was used according to the manufacturer’s instructions. Frozen spleen sections were first fixed with 4% PFA for 15 min, blocked with 5% donkey serum in PBS including CD16/CD32 Fc blocking at RT for 45 min, followed by FITC anti-mouse BST2 antibody (BioLegend, Cat#127008) staining at 4°C overnight, PBS wash, and Donkey anti-Rat IgG (H + L) Alexa Fluor™ 488 antibody (Invitrogen, Cat#A-21208) at RT for 1 h. The sections were then fixed with chilled acetone at -20°C for 2 h, permeabilized with 0.2% Triton X-100 in 3% BSA blocking buffer at RT for 45 min, and subsequently incubated with rabbit TLR9 (Thermo Fisher Scientific Cat#PA5-20203) or NLRP3 antibody (LS Bio Cat#LS-C334192-20) and mouse monoclonal anti-DNA antibody (Millipore Sigma, Cat#CBL186) at 4°C overnight. Rabbit IgG antibody (Cell Signaling Technology, Cat#2729) was used as a negative control. Secondary proximity probes (from Duolink® In Situ Red Starter Kit) were applied to the sections at 37°C for 90 min. Unbound proximity probes were washed. Sections were then incubated with ligation solution at 37°C for 60 min, washed twice and followed by amplification hybridization at 37°C for 120 min. After the PLA, the cells were washed and stained with DAPI before mounting. Isolated pDCs grown on round microscope coverglass pre-coated with Cell-Tak™ (Corning®, Cat#354240, at RT for 30 min) were fixed, permeabilized and Biotin-DNA was first stained with Streptavidin, Alexa Fluor™ 488 Conjugate (Thermo Fisher Scientific Cat# S11223), following by PLA to determine DNA and NLRP3 proximity as described above. Bulk RNA-Seq library preparation, sequencing, and analysis Total RNA was isolated from FACS-sorted pDC treated with mtDNA as indicated. Cells were homogenized with TRIzol reagent (ThermoFisher, Cat#15596026) and purified using RNeasy Mini Kit (Qiagen, Cat#74104). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection performed by Novogene Corporation Inc. Quantified libraries prepared from three biological replicates per group were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. Raw fastq format data were quality checked and trimmed using fastp 0.23.4 with its default parameters, except that automatic adapter detection for pair-end data was enabled with detect_adapter_for_pe . All downstream analyses were based on high-quality clean data. Reads were mapped using STAR 2.7.11b with its default parameters except that outFilterMultimapNmax was set to 1. GRCm39 was used as a reference genome. Aligned reads in the output Bam files from STAR were counted using featureCounts v2.0.1 with its default parameters. Generated raw read count data were analyzed using R version 4.3.3 . Genes with less than 50 total read counts were removed from further analysis. Read count data were normalized, and differential gene expression analysis was performed using R package DESeq2 . Pathway enrichment analysis for each cell was performed using R package fgsea . Gene sets were obtained from the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and MSigDB using R packages genekitr and geneset . scRNA-Seq library preparation, sequencing, and analysis The number of live freshly-collected peritoneal cells was determined and 10,000 cells per sample were run on the 10x Chromium platform (10x Genomics). Library preparation and sequencing were performed by the UCSD IGM Genomics Center using the Chromium Next GEM Single Cell 3' Kit v3.1 according to manufacturer's instructions. Sequencing was performed on the NovaSeq 6000 system (Illumina). Raw fastq files were aligned to the mouse mm10 reference genome using 10x Genomics Cell Ranger 7.1.0 with 10x Genomics Cloud Analysis. Raw count matrices from the 10x Genomics Cell Ranger were analyzed using Seurat v5.0.3 in R version 4.3.3 . For quality control, cells with less than 200 expressed genes, more than 50000 total counts, and more than 10% mitochondrial genes were removed. The data were then normalized using Seurat's NormalizeData function ( LogNormalize method with scale factor 10000). PCA was performed using the top 2000 variable genes, and the top 30 dimensions were used for UMAP. Cell annotation was performed manually using reported marker genes: Igkc, Ebf1, Cd79a, Ly6d , and Iglc2 for B cells; Cd209a, Ctnnd2, Mgl2, Flt3 , and Trerf1 for DC; Dcn, Col1a2, Col3a1 , and Sparc for fibroblasts; Cma1, Cpa3, Tpsb2, Mcpt4 , and Mrgprb1 for mast cells; Fscn1 and Ccr7 for migratory dendritic cells; Spp1, Ccl2, Ccl7, Chil3 , and Vcan for monocytes; S100a9, S100a8, Cxcr2, Il1r2 , and Il1f9 for neutrophils; Gzma, Klra4, Klra8, Klrb1c , and Ncr1 for NK cells; Prg4, Tgfb2, Ltbp1, Alox15 , and C4b for peritoneal macrophages; Themis, Camk4, Trbc2, Cd3g , and Bcl11b for T cells. Cells expressing two or more sets of gene markers of each cell type were considered as doublets and removed. Pathway enrichment analysis for each cell was performed using R package escape . Gene sets were obtained from the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and MSigDB using R packages. After global annotation, only the DC cluster was extracted and subdivided into finer clusters. Annotation for DC subtypes was done manually using reported marker genes: Aif1, Cadm1, Gpr141b, Xcr1, Tlr3, Ppt1, Btla, Itgae , and Cd24a for cDC1; Ltb, Cyp4f16, Pglyrp1, Clec4a4 , and Sirpb1a for cDC2; Ccr9, Iglc3, Klk1, Cox6a2, Smim5, Ly6c2, Ly6d, Siglech , and Runx2 for pDC; Cd33 and Ngfr for pDC-like; Vim, Ccr2, Fxyd5, Cx3cr1, Ccr1 , and Lgals3 for tDC. As reference data for steady-state pDC we obtained published scRNA-seq data (GSE196720) from GEO. The splenic steady state pDC analyses 66 was used as reference and merged with the scRNA seq data we obtained. Alignment of these two different studies was performed using SCTransform function from Seurat package with batch correction using the vars.to.regress argument. Statistical test for compositional analysis was performed using Student's t-test and for differential gene expression analysis was performed using FindMarkers function from Seurat package with test.use argument set to "wilcox". Quantification and statistical analysis No statistical methods were used to pre-determine sample sizes. Data collection and analysis were not performed blinded. No data points were excluded. For scRNA-seq, pre-established criteria for single-cell exclusion, which is, low number of unique genes, abnormally high read count and high mitochondrial gene content, were used. Statistical details of experiments, including statistical tests used, n value, number of experiments and the type of statistical tests, can be found in figure legends. Statistical analysis was performed with at least three biological replicates using GraphPad Prism 9 or R (v.4.1.2) statistical programming language. All graphs show mean ± s.d. Comparisons between two groups were performed by normality test followed by unpaired two-tailed t-test or non-parametric student’s t-test with Mann-Whitney analysis. Comparisons between multiple groups were performed by one-way ANOVA with Tukey’s multiple comparisons test or unless otherwise mentioned. P values less than or equal to 0.05 were considered significant and significance was assigned according to the following breakdown: *p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Declarations Animal procedures described in this study were approved by the University of California, San Diego Institutional Animal Care and Use Committee (IACUC) and complied with all relevant guidelines for the use of non-human vertebrate animals. Data and code availability All data generated or analyzed during this study are included in this manuscript. Raw mouse sequencing data including RNA-seqand scRNA-seq have been deposited at Gene Expression Omnibus (GEO): GSE264619 and GSE264619, which are listed in Deposited data and are publicly available. This study did not generate any original code. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request. Acknowledgements We thank Drs. Juliana Idoyaga, Li-Fan Lu, Virginia Pascual, Simone Caielli, Trever Greene for help and advice. We acknowledge eBioscience, Cell Signaling Technologies, Santa Cruz Technologies, Thermo Fisher, Biolegend, BD Biosciences, STEMCELL Technologies for gifts of reagents, and the UCSD TTSR supported by an NCI Cancer Center Support Grant (CCSG P30CA23100). We are grateful to Dr. Alexander Dent at Indiana University for providing us Bcl6 ff / Cd4 cre mice, Drs. Jeffrey V. Ravetch and Emily Lam at the Rockefeller University, Warren Leonard at the NIH, Yi-Guang Chen at Medical College of Wisconsin, Kevin King at UCSD for Fcgr1 −/− , Il21 −/− and Ifnr1 −/− mouse bones, respectively. Dr. Jay Chung at the NIH kindly provided MEF. We thank the UCSD Nikon Imaging Center and especially Dr. Peng Guo for assistance with AXR Confocal and NSPARC Super Resolution imaging. Cartoons were prepared with BioRender.com software. This publication includes data generated at the UCSD IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased through NIH SIG grant (#S10 OD026929). H.X. was supported by Arthritis National Research Foundation (#1291101). This work was supported by NIH grants R01 DK100640 and R37 AI043477 to M.K., who is an American Cancer Research Society Professor and holds the Ben and Wanda Hildyard Chair for Mitochondrial and Metabolic Diseases, NIH R01 grant AI145314 to E.I.Z. and NIH grants R01 AI155869, R01 DK113592 and P01 HL152958 to H.M.H. Author contributions Conceptualization: H.X. and M.K. Methodology: H.X. and K.W. Investigation: H.X., M.O., K.W., J.S.B. and M.K. Resources: J.O., H.M.H., E.I.Z. and M.K. Formal Analysis: H.X. and M.O. Supervision: M.K. Funding acquisition: E.I.Z., H.M.H. and M.K. and Writing – original draft: H.X. and M.K. Writing – review and editing: E.I.Z., H.M.H. and M.K. Declarations of interests M.K. is a founder of Elgia Pharmaceuticals and received research support from Gossamer Bio and Jansen Pharmaceuticals. M.K. holds an interest in PF-06835375, a Tfh depleting CXCR5 antibody. H.M.H. is a consultant for SOBI and Akros and received research funds from Takeda and Inapill. Other authors declare no competing interests. Data and materials availability RNA-seq and scRNA-seq data were deposited into the Gene Expression Omnibus (GEO) data repository (GSE264619 for bulk RNA-seq and GSE264620 for scRNA-seq). All data are available in the main text or the supplementary materials. Further requests for materials generated in this study should be directed to the corresponding author, Dr. Michael Karin ( [email protected] ). References Goronzy, J.J. & Weyand, C.M. Immune aging and autoimmunity. Cell Mol Life Sci 69, 1615–1623 (2012). Zheng, Y., Liu, Q., Goronzy, J.J. & Weyand, C.M. Immune aging - A mechanism in autoimmune disease. Semin Immunol 69, 101814 (2023). Shimada, K. et al. Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36, 401–414 (2012). Zhong, Z. et al. New mitochondrial DNA synthesis enables NLRP3 inflammasome activation. Nature 560, 198–203 (2018). Xian, H. et al. 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Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat Med 22, 146–153 (2016). Valente, M. et al. Novel mouse models based on intersectional genetics to identify and characterize plasmacytoid dendritic cells. Nat Immunol 24, 714–728 (2023). Additional Declarations Yes there is potential Competing Interest. M.K. is a founder of Elgia Pharmaceuticals and received research support from Gossamer Bio and Jansen Pharmaceuticals. M.K. holds an interest in PF-06835375, a Tfh depleting CXCR5 antibody. H.M.H. is a consultant for SOBI and Akros and received research funds from Takeda and Inapill. Other authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5194985","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":364232202,"identity":"9b40abc9-c9a9-4b64-a6b9-34ac318aedc5","order_by":0,"name":"Michael Karin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYDACHiBOYLABMQ0YGNiI15JGqhYGhsMkaNHtOfzsw8Md5xP7+w9vYPhQdpiwFrOzbcYzEs/cTpxxI62AccY5YrScZzBmSGy7nbhBgseAmbeNKC3sn4FaziVu4D9jwPyXKC1ne0C2HEjcwJBjwMxIlJYzZ4qBWpKNQX452HMunRgt6ZsZf7bZyQJDbOODH2XWhLWggAMkqh8Fo2AUjIJRgAsAAMOGPXr7T3SeAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2758-6473","institution":"University California San Diego","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Karin","suffix":""},{"id":364232203,"identity":"5c7a1999-d2d8-4b4b-a3bb-6020f04fed1c","order_by":1,"name":"Hongxu Xian","email":"","orcid":"","institution":"UCSD","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongxu","middleName":"","lastName":"Xian","suffix":""},{"id":364232204,"identity":"ca7b625d-121f-4d1c-ac91-c380109acbe7","order_by":2,"name":"Masafumi Ohira","email":"","orcid":"","institution":"UCSD","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masafumi","middleName":"","lastName":"Ohira","suffix":""},{"id":364232205,"identity":"2ae9520b-1fa3-4b84-945d-c3e14023e451","order_by":3,"name":"Kosuke Watari","email":"","orcid":"https://orcid.org/0000-0003-0260-2323","institution":"University California San Diego","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kosuke","middleName":"","lastName":"Watari","suffix":""},{"id":364232206,"identity":"586045cf-54ff-4b4c-bf0d-16ace224c15b","order_by":4,"name":"Jonathan Brito","email":"","orcid":"","institution":"University California San Diego","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Brito","suffix":""},{"id":364232207,"identity":"b1e8e1e0-48de-4db0-8d90-080982ad16c5","order_by":5,"name":"Janset Onyuru","email":"","orcid":"","institution":"University California San Diego","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Janset","middleName":"","lastName":"Onyuru","suffix":""},{"id":364232208,"identity":"5b046567-2f78-4a42-9abb-2799dbf9019d","order_by":6,"name":"Elina Zuniga","email":"","orcid":"https://orcid.org/0000-0001-8699-1623","institution":"University of California, San Diego","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elina","middleName":"","lastName":"Zuniga","suffix":""},{"id":364232209,"identity":"aa6226cf-4d2c-4b38-8dea-527fbff8d19b","order_by":7,"name":"Hal Hoffman","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hal","middleName":"","lastName":"Hoffman","suffix":""}],"badges":[],"createdAt":"2024-10-02 21:45:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5194985/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5194985/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41590-025-02179-7","type":"published","date":"2025-06-17T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66550881,"identity":"460ab277-1b09-41fd-8745-c447598c65b0","added_by":"auto","created_at":"2024-10-14 09:04:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":997546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlum injections induce mtDNA-dependent pathological autoantibodies.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were i.p. injected with alum as in Extended Data Fig. 1a (n=5/group, \u003cstrong\u003ea\u003c/strong\u003e), PBS or alum as in Extended Data Fig. 1g (\u003cstrong\u003eb\u003c/strong\u003e), or PBS or alum -/+ DNase I as in Extended Data Fig. 2a (n=7/PBS, n= 9/alum, n=7/alum+DNase I, \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ej\u003c/strong\u003e). \u003cstrong\u003ea\u003c/strong\u003e, Immunofluorescence (IF) analysis of spleen sections stained for IgG (green) and B220 (red) at the indicated timepoints after alum injection. Scale bar, 150 μm. \u003cstrong\u003eb\u003c/strong\u003e, Images and size quantification (n\u0026gt;95/group) of glomeruli in H\u0026amp;E or Periodic acid Schiff-(PAS)-stained kidney sections. Scale bar, 20 μm. \u0026nbsp;\u003cstrong\u003ec\u003c/strong\u003e, Serum titers of anti-dsDNA and anti-nucleosome IgGs. \u003cstrong\u003ed\u003c/strong\u003e, FC plots and percentages of anti-mtDNA antibody forming cells (AFC) in spleens. \u003cstrong\u003ee\u003c/strong\u003e, Spleen sections stained with Biotin-Ox-mtDNA (red), CD169 (green), CD138 (cyan) and GL7 (blue). Scale bars, 50 μm and 25 μm. \u003cstrong\u003ef\u003c/strong\u003e, Percentages of splenic GC B and Tfh cells. \u003cstrong\u003eg\u003c/strong\u003e, Glomeruli in H\u0026amp;E or PAS-stained kidney sections and their sizes (n≥ 86). Scale bars, 20 μm and 100 μm. \u003cstrong\u003eh\u003c/strong\u003e, Urinary protein ELISA. \u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e, Kidney sections stained for IgG or C3 (green), CD45 (red) and DAPI (blue) (\u003cstrong\u003ei\u003c/strong\u003e) and mean fluorescence intensities (MFI) of IgG and C3 deposits and areas (in %) occupied by CD45\u003csup\u003e+\u003c/sup\u003e cells (\u003cstrong\u003ej\u003c/strong\u003e). Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003eResults in (\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e and \u003cstrong\u003ej\u003c/strong\u003e) are mean ± SD. ∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ∗∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Kruskal-Wallis test.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/d8b4c45f10c79edb80c32d6f.png"},{"id":66550882,"identity":"fa222fa6-98e9-4ae7-a71e-f2c47b8f8e28","added_by":"auto","created_at":"2024-10-14 09:04:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":759445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlum-induced autoimmunity depends on NLRP3 and GSDMD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWT and \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n=8/WT PBS; n=14/WT alum; n=9/\u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e alum) or WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n=6/WT PBS; n=10/WT alum; n=8/\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e alum) female mice were treated as in Extended Data Fig. 3a. \u003cstrong\u003ea\u003c/strong\u003e, ELISA of serum anti-dsDNA IgG of WT \u0026amp; \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and WT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003eb\u003c/strong\u003e, B220 (red), CD169 (green) and GL7 (blue) stained WT \u0026amp; \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e spleen sections. Scale bar, 50 μm. \u003cstrong\u003ec\u003c/strong\u003e, IF analysis of GL7 (blue) and B220 (red) stained WT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003espleen sections. Scale bar, 50 μm. \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e, Percentages of splenic GC B and Tfh cells of WT\u0026amp;\u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (\u003cstrong\u003ed\u003c/strong\u003e) or WT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (\u003cstrong\u003ee\u003c/strong\u003e) mice. \u003cstrong\u003ef\u003c/strong\u003e, Ki67 staining and percentages (in %) of splenic Ki67\u003csup\u003e+\u003c/sup\u003e cells (n\u0026gt;62) of WT \u0026amp; \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. Scale bar, 100 μm. \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e, IF analysis of B220 (\u003cstrong\u003eg\u003c/strong\u003e) or CD4 (\u003cstrong\u003eh\u003c/strong\u003e) (red), Ki67 (green) and DAPI (blue) stained WT and \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e spleen sections. Scale bar, 50 μm. \u003cstrong\u003ei\u003c/strong\u003e, FC percentages of WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e IgG1\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e and IgG2b\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e class-switched B cells. \u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e, WT \u0026amp; \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e serum IL-1β, IL-21, IL-6 and IFN-α (\u003cstrong\u003ej\u003c/strong\u003e) and WT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e serum IL-1β and IL-21 (\u003cstrong\u003ek\u003c/strong\u003e). \u003cstrong\u003el\u003c/strong\u003e, Glomeruli sizes of WT \u0026amp; \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n=79/group) and WT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n\u0026gt;122/group). \u003cstrong\u003em\u003c/strong\u003e,\u003cstrong\u003en\u003c/strong\u003e, MFI of renal IgG deposits (n\u0026gt;150 for WT \u0026amp; \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003eand\u003cem\u003e \u003c/em\u003en\u0026gt;120 for\u003cem\u003e \u003c/em\u003eWT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) and C3 (n\u0026gt;114 for WT \u0026amp; \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003eand n\u0026gt;120 for WT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) and WT \u0026amp; \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e renal area (in %) occupied by CD45\u003csup\u003e+\u003c/sup\u003e cells (n\u0026gt;31). \u0026nbsp;\u003cstrong\u003eo\u003c/strong\u003e, WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e urinary protein ELISA.\u003c/p\u003e\n\u003cp\u003eResults in (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003ed-f\u003c/strong\u003e and \u003cstrong\u003ei-o\u003c/strong\u003e) are mean ± SD. ∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ∗∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Two-way ANOVA with Tukey multiple-comparison test.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/1ef22e86b812f8675d0d3041.png"},{"id":66550888,"identity":"9304223e-55ee-411e-bf06-3155860ecb6b","added_by":"auto","created_at":"2024-10-14 09:04:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":628528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlum induced ccf-Ox-mtDNA is sensed by peritoneal pDC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, UMAP plots of peritoneal immune cells in B6 male mice 24 h after PBS or alum injection (n=3). \u003cstrong\u003eb\u003c/strong\u003e, FC plots and percentages of peritoneal macrophages (F4/80\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e, 4 h and 24 h) and IL-1β (4 h) after PBS or alum injections in WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e female mice (WT PBS: n=6; WT Alum: n=6 (4h)/8 (24h); \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e Alum: n=5 (4h)/7 (24h)). \u003cstrong\u003ec\u003c/strong\u003e, Heat maps showing pathway enrichment across cell clusters. Color scales: mean enrichment scores (ES). \u003cstrong\u003ed\u003c/strong\u003e, Relative proportions of DC subclusters. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e, Numbers and percentages of peritoneal pDC (Lin\u003csup\u003e-\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003eBST2\u003csup\u003e+\u003c/sup\u003eSiglec-H\u003csup\u003e+\u003c/sup\u003e, gated as in \u003cstrong\u003eSupplementary Fig. 3d\u003c/strong\u003e) 24 h after PBS or alum injection. Lineage-negative gating (Lin\u003csup\u003e-\u003c/sup\u003e) includes markers for Thy1.2, CD19, and NK1.1 (n=9/PBS, n=6/alum, n=7/DNase I (\u003cstrong\u003ee\u003c/strong\u003e); n=7/WT PBS, n=5/WT alum, n=7/\u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e alum (\u003cstrong\u003ef\u003c/strong\u003e); n=7/WT PBS, n=4/WT alum, n=9/\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e alum (\u003cstrong\u003eg\u003c/strong\u003e); n=8/WT PBS, n=9/WT alum, n=10/mt-\u003cem\u003eOgg1\u003c/em\u003e\u003csup\u003eTg\u003c/sup\u003e alum (\u003cstrong\u003eh\u003c/strong\u003e); n=8/WT PBS, n=9/\u003cem\u003eCmpk2\u003c/em\u003e\u003csup\u003eff\u003c/sup\u003e alum, n=9/\u003cem\u003eCmpk2\u003c/em\u003e\u003csup\u003eΔMye\u003c/sup\u003e alum (\u003cstrong\u003ei\u003c/strong\u003e)). CD40, MHC-II and CD80 expressing pDC were also examined (\u003cstrong\u003ei\u003c/strong\u003e). \u003cstrong\u003ej\u003c/strong\u003e, Volcano plots of differentially expressed genes (DEGs) in alum-recruited peritoneal pDC vs. steady state splenic pDC. DEGs with Log2 change and adjusted p value \u0026lt;0.5 are colored brown or red. \u003cstrong\u003ek\u003c/strong\u003e, Active Casp1 (FLICA\u003csup\u003ehi\u003c/sup\u003e) in peritoneal pDC, cDC1 and cDC2 pre-gated as in \u003cstrong\u003eSupplementary Fig. 3d\u003c/strong\u003e and pooled from 20 mice/group (because pDC number from one mouse is too low) 24 h after alum injections (representative of 3 independent experiments). FMO, fluorescence minus one. \u003cstrong\u003el\u003c/strong\u003e, IFN-a, IRF7, CD40, MHC II, CD80 and IL-21 expression by alum-recruited peritoneal pDC collected as in (\u003cstrong\u003ek\u003c/strong\u003e). \u003cstrong\u003em\u003c/strong\u003e, Intracellular IL-21 in alum-recruited peritoneal pDC from (\u003cstrong\u003ek)\u003c/strong\u003e and splenic Tfh from B6 mice treated as in \u003cstrong\u003eExtended Data Fig. 3a\u003c/strong\u003e. \u003cstrong\u003en\u003c/strong\u003e, Heat maps depicting scaled average expression of NF-kB, TLR7/9 and inflammasome related genes \u003cem\u003eNek7\u003c/em\u003e and \u003cem\u003eP2rx7\u003c/em\u003e of different DC subsets from (\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eResults in (\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e) are mean ± SD. ∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ∗∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001. Mann–Whitney Test (\u003cstrong\u003ed\u003c/strong\u003e) and Two-way ANOVA with Tukey multiple-comparison test (\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/07a32e0d40577b7fd9f0a21a.png"},{"id":66550890,"identity":"6d7ae373-a614-4fd9-a0a8-ea6b5c358d77","added_by":"auto","created_at":"2024-10-14 09:04:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":988804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epDC intrinsic IL-1β signaling induces Tfh differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e, FC analyses of active Casp1 (FLICA\u003csup\u003ehi\u003c/sup\u003e) in pre-gated Flt3L-induced DC subsets (n=4, \u003cstrong\u003ea\u003c/strong\u003e) or BMDM (n=3, \u003cstrong\u003eb\u003c/strong\u003e) 4 h after incubation with different types of DNA. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e, Secreted IL-1β, IL-21 and IFN-a (n=9/None, n=9/mtDNA, n=14/Ox-mtDNA, \u003cstrong\u003ec\u003c/strong\u003e) and histograms of IL-21, IL-6, CD40 and ICOSL expression (n=3, \u003cstrong\u003ed\u003c/strong\u003e) in FACS-sorted Flt3L-induced/pre-gated pDC 4 h after mtDNA or Ox-mtDNA incubation. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e, Histograms of IL-21 (\u003cstrong\u003ee\u003c/strong\u003e), IL-6 (\u003cstrong\u003ef\u003c/strong\u003e) and ICOSL (\u003cstrong\u003eg\u003c/strong\u003e) in pre-gated Flt3L-induced cDC1 or cDC2 treated as above (n=3). \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e, FC analyses of NF-kB p65 phopho-Ser281 in pre-gated WT\u0026amp;\u003cem\u003eTlr9\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e (n=3) (\u003cstrong\u003eh\u003c/strong\u003e) and WT\u0026amp;\u003cem\u003eIl1r1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e (n=4) (\u003cstrong\u003ei\u003c/strong\u003e) pDC incubated with different types of DNA. \u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eIL-21\u003csup\u003ehi\u003c/sup\u003e and IL-6\u003csup\u003ehi\u003c/sup\u003e cell frequencies in pre-gated WT, \u003cem\u003eIl1b\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003eand\u003cem\u003e Il1r1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e pDC incubated -/+ mtDNA or Ox-mtDNA for 4 h (n=4). \u003cstrong\u003ek\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e, Frequencies of Tfh cells generated by co-culture of FACS-sorted WT or \u003cem\u003eIl1b\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e Flt3L-induced pDC with WT naïve splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells (\u003cstrong\u003ek\u003c/strong\u003e), WT or \u003cem\u003eIl1r1\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003enaïve splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells co-cultured with WT pDC (\u003cstrong\u003el\u003c/strong\u003e), -/+ Ox-mtDNA for 3 days (n=4). \u003cstrong\u003em\u003c/strong\u003e, Tfh frequencies after co-culture of naïve WT or \u003cem\u003eIfnar1\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells with FACS-sorted Flt3L-induced pDCs -/+ Ox-mtDNA for 3 days (n=4). \u003cstrong\u003en\u003c/strong\u003e,\u003cstrong\u003eo\u003c/strong\u003e, Naïve WT CD4\u003csup\u003e+\u003c/sup\u003e T cells were co-cultured with WT (n=5/None, n=8/Ox-mtDNA) or \u003cem\u003eIl21\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e (n=7/treatment) FACS-sorted Flt3L-induced pDC, -/+ Ox-mtDNA for 3 days. Tfh frequencies (\u003cstrong\u003en\u003c/strong\u003e) and intracellular BCL6 staining in Tfh (CXCR5\u003csup\u003e+\u003c/sup\u003e) and non-Tfh (CXCR5\u003csup\u003e-\u003c/sup\u003e) CD44\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e cells (\u003cstrong\u003eo\u003c/strong\u003e) are shown.\u003c/p\u003e\n\u003cp\u003eResults in (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e) are mean ± SD. ∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ∗∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. ns, not significant. Kruskal-Wallis test (\u003cstrong\u003ec\u003c/strong\u003e) and two-way ANOVA with Tukey multiple-comparison test (\u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/a449e49776789fed62cac4d0.png"},{"id":66553509,"identity":"06dc6bb4-7c18-4c3b-a8eb-f441730118c1","added_by":"auto","created_at":"2024-10-14 09:12:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":822212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidized mtDNA is sufficient for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein-vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e induction of autoimmunity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Treatment and analysis scheme for \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e. B6 mice were i.p. injected with 50 μg mtDNA extracted from cultured fibroblasts that was either left non-oxidized or subjected to \u003cem\u003ein-vitro \u003c/em\u003eUV-induced oxidation and analyzed 24 h later. PBS injections served as control. \u003cstrong\u003eb\u003c/strong\u003e, Heat maps showing pathway enrichment across cell clusters. Color scales depict enrichment scores. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e, Relative proportions of DC subclusters identified by scRNA-seq (n=3) (\u003cstrong\u003ec\u003c/strong\u003e) and FC determined peritoneal pDC number and percentages (\u003cstrong\u003ed\u003c/strong\u003e) 24 h after mtDNA injections (n=9/PBS, 6/mtDNA, 7/Ox-mtDNA). \u003cstrong\u003ee\u003c/strong\u003e, Heat maps depicting scaled average expression of NF-kB- and inflammasome-related genes in pDC after mtDNA injections. \u003cstrong\u003ef\u003c/strong\u003e, Heat maps depicting scaled average expression of NF-kB- and inflammasome-related genes in different DC subclusters from Ox-mtDNA-injected mice. \u003cstrong\u003eg\u003c/strong\u003e, Treatment and analysis schemes. \u003cstrong\u003eh-k\u003c/strong\u003e. 8-week-old B6 males were repetitively injected with 50 μg non-oxidized or oxidized mtDNA every 72 h and examined for autoimmune signs after 20 weeks. PBS injections (“None”) served as controls. Serum anti-dsDNA and anti-nucleosome IgG titers (\u003cstrong\u003eh\u003c/strong\u003e), IL-1β, IL-21 and IFN-α amounts (\u003cstrong\u003ei\u003c/strong\u003e), GL7 (blue) and B220 (red) stained spleen sections (scale bar, 50 μm, \u003cstrong\u003ej\u003c/strong\u003e) and frequencies of splenic GC B, IgG1\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e, IgG2b\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B and Tfh cells (\u003cstrong\u003ek\u003c/strong\u003e) (n=4/None, 6/mtDNA, 6/Ox-mtDNA). \u003cstrong\u003el\u003c/strong\u003e-\u003cstrong\u003eo\u003c/strong\u003e, B6 females were repetitively injected with non-oxidized or oxidized mtDNA as in (\u003cstrong\u003eg\u003c/strong\u003e) for 36 weeks and analyzed (n=5/treatment). Blood Tfh cells and intracellular BCL6 in circulating Tfh and CD4\u003csup\u003e+\u003c/sup\u003e cells (\u003cstrong\u003el\u003c/strong\u003e), sizes of glomeruli in kidney sections (n\u0026gt;125) (\u003cstrong\u003em\u003c/strong\u003e), urinary protein (\u003cstrong\u003en\u003c/strong\u003e) and MFI of kidney IgG and C3 deposits (n\u0026gt;117) and CD45\u003csup\u003e+\u003c/sup\u003e cell-occupied areas (in %) (n\u0026gt;66) (\u003cstrong\u003eo\u003c/strong\u003e). \u003cstrong\u003ep\u003c/strong\u003e, BST2 (green) stained spleen sections at 100x magnification from mice injected with Ox-mtDNA as in (\u003cstrong\u003eg\u003c/strong\u003e) were subjected to \u003cem\u003ein-situ\u003c/em\u003e PLA (red) of intracellular DNA with control IgG, TLR9 or NLRP3 antibodies. Arrows: PLA signals. Scale bar, 5 μm. \u0026nbsp;\u003cstrong\u003eq\u003c/strong\u003e,\u003cstrong\u003er\u003c/strong\u003e, Spleen sections at 10x magnification from mice injected with mtDNA or Ox-mtDNA as in \u003cstrong\u003eg\u003c/strong\u003e, stained for BST2 (green) and subjected to PLA (red) of intracellular DNA with either TLR9 (\u003cstrong\u003eq\u003c/strong\u003e) or NLRP3 (\u003cstrong\u003er\u003c/strong\u003e). Arrows: PLA signals. Mock PLA of DNA and IgG served as a negative control. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003eResults in (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e and \u003cstrong\u003ek\u003c/strong\u003e-\u003cstrong\u003eo\u003c/strong\u003e) are mean ± SD. ∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ∗∗∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. ns, not significant. Kruskal–Wallis test.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/af66f52ef87305586af168e4.png"},{"id":66553507,"identity":"19538a14-ddb0-47c2-b9f3-5b1f0c11ca14","added_by":"auto","created_at":"2024-10-14 09:12:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1191677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFcgR1 mediates mtDNA uptake followed by binding to TLR9 and endosomal escape.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Naïve WT CD4\u003csup\u003e+\u003c/sup\u003e T cells were co-cultured with WT (n=5),\u003cem\u003e Tlr9\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e, \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e, or \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e (n=4) FACS-sorted Flt3L-induced pDC -/+ Ox-mtDNA for 3 days. Tfh generation was analyzed. \u003cstrong\u003eb-d\u003c/strong\u003e, Frequencies of active Casp1 (FLICA\u003csup\u003ehi\u003c/sup\u003e, \u0026nbsp;n=4/WT None; n=5/\u003cem\u003eTlr9\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e None; n=4/genotype/mtDNA, n=5/genotype/Ox-mtDNA, \u003cstrong\u003eb\u003c/strong\u003e), IL-21\u003csup\u003ehi \u003c/sup\u003e(n=3, \u003cstrong\u003ec\u003c/strong\u003e) and histogram of intracellular IRF7 staining (n=4, \u003cstrong\u003ed\u003c/strong\u003e) in WT and\u003cem\u003e Tlr9\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e Flt3L-induced pDC 4 h after -/+ mtDNA or Ox-mtDNA. \u003cstrong\u003ee\u003c/strong\u003e, GSEA pathway analyses of bulk RNA-Seq data of FACS-sorted BM-Flt3L-induced pDC 4 h after vehicle (None), non-oxidized mtDNA or Ox-mtDNA treatments (n=3). NES, normalized enrichment score. CPG DNA vs Untreated in DC DN, Genes downregulated in CpG DNA (TLR9 agonist) treated versus untreated DC. \u003cstrong\u003ef\u003c/strong\u003e, Percentages of phospho-Ser281 p65 in Flt3L-induced BMDC subsets 4 h after incubation with different types of DNA (n=3). \u003cstrong\u003eg\u003c/strong\u003e, IF of splenic pDC incubated with 2 μg biotin-labeled non-oxidized or oxidized mtDNA for 1 h, followed by staining for biotin (green) and PLA (red) of intracellular DNA with NLRP3. Arrows: PLA signals. Scale bar, 5 μm. \u003cstrong\u003eh\u003c/strong\u003e, IF of splenic pDC treated as in \u003cstrong\u003eg\u003c/strong\u003e and stained for biotin (red) and ASC (green). Arrow: ASC speck. Scale bar, 5 μm. \u003cstrong\u003ei\u003c/strong\u003e, Frequencies of FLICA\u003csup\u003ehi\u003c/sup\u003e cells in Flt3L-induced pDC incubated with Ox-mtDNA for 4 h\u0026nbsp; -/+ fetal bovine serum (FBS) or IgG depleted FBS (n=4). \u003cstrong\u003ej\u003c/strong\u003e, Tfh frequencies in naïve WT CD4\u003csup\u003e+\u003c/sup\u003e T cells co-cultured with WT or\u003cem\u003e Fcgr1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e FACS-sorted Flt3L-induced pDC -/+ Ox-mtDNA for 3 days (n=3).\u003c/p\u003e\n\u003cp\u003eResults in (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e and \u003cstrong\u003ej\u003c/strong\u003e) are mean ± SD. ∗ \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ∗∗∗∗ \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. ns, not significant. Two-way ANOVA with Tukey multiple-comparison test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/101575fb09b20b835c2bc9f1.png"},{"id":66550887,"identity":"c675a0c7-feec-48a9-b2f2-4435de973866","added_by":"auto","created_at":"2024-10-14 09:04:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":901409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1R ablation prevents alum-induced autoimmunity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Generation of female WT and \u003cem\u003eIl1r1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e BM chimeras and treatment scheme for autoimmunity analyses (n=7/WT PBS; n=10/WT Alum; n=8/\u003cem\u003eIl1r1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e Alum). \u003cstrong\u003eb-d\u003c/strong\u003e, Serum titers of anti-dsDNA and anti-nucleosome IgGs (\u003cstrong\u003eb\u003c/strong\u003e), serum IL-21 and IFN-α amounts (\u003cstrong\u003ec\u003c/strong\u003e), IF images of GL7 (blue) and B220 (red) stained spleen sections (Scale bar, 50 μm, \u003cstrong\u003ed\u003c/strong\u003e), percentages of splenic GC B, Tfh and IgG1\u003csup\u003e+\u003c/sup\u003eIgM\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cells (\u003cstrong\u003ee\u003c/strong\u003e), sizes of glomeruli (n\u0026gt;148, \u003cstrong\u003ef\u003c/strong\u003e), MFI quantitation of IgG (n\u0026gt;148) and C3 deposits (n\u0026gt;201) and CD45\u003csup\u003e+\u003c/sup\u003e cell areas % (n\u0026gt;47) in kidney sections (\u003cstrong\u003eg\u003c/strong\u003e), urinary protein amounts (\u003cstrong\u003eh\u003c/strong\u003e), relative amounts of serum ccf-mtDNA (\u003cstrong\u003ei\u003c/strong\u003e) and serum non-Ox-mtDNA (Fpg-resistant) (\u003cstrong\u003ej\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eResults in (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, and \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ej\u003c/strong\u003e) are mean ± SD.\u0026nbsp; ∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ∗∗∗∗ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. ns, not significant. Two-way ANOVA with Tukey multiple-comparison test.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/7449f179cca38be47df4a199.png"},{"id":84861356,"identity":"042f7d78-9d9a-4a25-a2b8-fb0fcf96d3e5","added_by":"auto","created_at":"2025-06-18 07:07:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7739265,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/ddcb9590-8e32-4534-a30c-6f109d91430d.pdf"},{"id":66550884,"identity":"3082bed1-d88a-4203-82fe-df2a8023aa00","added_by":"auto","created_at":"2024-10-14 09:04:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9893260,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/fc686afc4b0b7b35f4be0002.docx"},{"id":66550891,"identity":"10b0c8a2-103a-400e-a771-e1bc2825a906","added_by":"auto","created_at":"2024-10-14 09:04:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5703517,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationFULL.docx","url":"https://assets-eu.researchsquare.com/files/rs-5194985/v1/db63a9f96e5ef5a16228fcad.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nM.K. is a founder of Elgia Pharmaceuticals and received research support from Gossamer Bio and Jansen Pharmaceuticals. M.K. holds an interest in PF-06835375, a Tfh depleting CXCR5 antibody. H.M.H. is a consultant for SOBI and Akros and received research funds from Takeda and Inapill. Other authors declare no competing interests.","formattedTitle":"Mitochondrial DNA oxidation propagates autoimmunity by enabling plasmacytoid dendritic cells induce Tfh differentiation","fulltext":[{"header":"Main","content":"\u003cp\u003eThe borderline between chronic inflammation and autoimmunity is obscure. Specifically, it is unclear whether in addition to inflammaging, persistent innate immune activation breaches self-tolerance and initiates long-lasting autoimmune responses, whose incidence increases with old age\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. A key danger signal that triggers sterile inflammation through NLRP3 inflammasome mediated caspase-1 (Casp1) activation is Ox-mtDNA, an alarmin that enters the cytoplasm in response to mitochondrial stress and damage\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In addition to generating 8-oxo-deoxyguanosine (8-Oxo-dG) containing DNA which binds NLRP3\u003csup\u003e3, 4\u003c/sup\u003e, mtDNA oxidation promotes its cleavage into small fragments that pass through mitochondrial pores by the repair nuclease FEN-1\u003csup\u003e5\u003c/sup\u003e. Along with pro-IL-1β processing, activated Casp1 cleaves gasdermin D (GSDMD) to generate plasma membrane pores through which Ox-mtDNA escapes to the extracellular space and reaches the circulatory system\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Smoldering NLRP3 inflammasome activation has been linked to inflammaging through poorly defined mechanisms\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Moreover, mitochondrial dysfunction coupled to oxidative stress is considered as a major hallmark of aging\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, whether ccf-Ox-mtDNA drives age-related immunopathologies and immune-aging is unknown. Given the elevated amounts of ccf-mtDNA, which is probably oxidized\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, in older individuals who are at an elevated autoimmunity risk\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and patients with chronic inflammatory diseases and metabolic disorders, including rheumatoid arthritis\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, systemic lupus erythematosus (SLE)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, cardiovascular disease\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, type 2 diabetes mellitus (T2D)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, multiple sclerosis (MS)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and cancer\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we investigated whether Ox-mtDNA fosters the transition from persistent inflammation to maladaptive autoimmunity. Here we show that Ox-mtDNA sensing endows mouse and human pDC with the ability to convert na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells into Tfh cells that support pathogenic autoantibody production.\u003c/p\u003e\n\u003ch3\u003eAlum induces mtDNA-dependent pathological autoantibody production\u003c/h3\u003e\n\u003cp\u003eTo address whether ccf-Ox-mtDNA produced during sterile inflammation can promote maladaptive autoimmune responses, we challenged na\u0026iuml;ve mice with antigen-free alum to induce NLRP3 inflammasome-dependent peritonitis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, which is accompanied by substantial generation of ccf-Ox-mtDNA\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Remarkably, in addition to sustained production of circulating IL-1β, successive intraperitoneal (i.p.) injections of alum into 8-week-old C57BL/6 (B6) mice increased anti-dsDNA IgG titers (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c). The same protocol induced splenic B cell follicles harboring nucleosome- and DNA-reactive antibody-forming cells (AFC) that became apparent 58 days after initiation of alum injections but were undetectable in age-matched controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;1a). At the same timepoint, flow cytometry (FC) revealed an increase in splenic class-switched IgM\u003csup\u003e\u0026ndash;\u003c/sup\u003eIgG1\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cells, whereas plasma cells (PC; B220\u003csup\u003eint\u003c/sup\u003eCD138\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003e), class-switched IgM\u003csup\u003e\u0026ndash;\u003c/sup\u003eIgG2b\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cells, Tfh cells (CXCR5\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e) and germinal center (GC) B (B220\u003csup\u003e+\u003c/sup\u003eGL7\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e\u0026minus;\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003e\u0026minus;\u003c/sup\u003e) cells were elevated by 100 days after initiation of alum treatment (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f and Supplementary Fig.\u0026nbsp;1a-d). Notably, typical and systemic autoimmune features, which in addition to anti-dsDNA IgG production, include elevated BCL6-expressing Tfh cells (CXCR5\u003csup\u003e+\u003c/sup\u003eICOS\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e), GC and marginal zone (MZ) B cells (CD21\u003csup\u003ehi\u003c/sup\u003eCD23\u003csup\u003e\u0026minus;\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003e), which class-switch into IgG\u003csup\u003e+\u003c/sup\u003e cells\u003csup\u003e25\u003c/sup\u003e, and glomerular enlargement, progressively increased till 130 days after initiation of alum treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i and Supplementary Fig.\u0026nbsp;1e,f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test the role of ccf-DNA in the alum-induced immune response, which was observed in both female and male mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej), we administered alum followed by i.p. DNase I injections (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Notably, DNase I treatment inhibited splenomegaly (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), decreased serum anti-dsDNA IgG measured by ELISA or \u003cem\u003eCrithidia luciliae\u003c/em\u003e immunofluorescence (IF) test (CLIFT) and anti-chromatin IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), bone marrow (BM) and splenic DNA-reactive CD138\u003csup\u003e+\u003c/sup\u003e AFCs located within GL7\u003csup\u003e+\u003c/sup\u003e GC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), along with GC B cell numbers and frequencies, Tfh, PC, MZ B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), proliferative Ki67\u003csup\u003e+\u003c/sup\u003e splenic B and CD4\u003csup\u003e+\u003c/sup\u003e T cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and Supplementary Fig.\u0026nbsp;1g) and serum IL-21, IL-6, IL-1β and IFN-α (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Glomerular swelling, proteinuria, IgG and complement (C3) deposition and CD45\u003csup\u003e+\u003c/sup\u003e leukocyte infiltration were also blunted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-j). Supporting a role for Ox-mtDNA in autoimmunity, DNase I injections reduced the amounts of alum-induced ccf-mtDNA harboring oxidative 8-Oxo-dG lesions (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh,i), which was much far abundant than circulating nuclear (n) DNA (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). These results suggested that ccf-DNA, most of which was mitochondrially derived and oxidized, is a major contributor to alum-induced autoimmunity and renal pathology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAlum-induced autoimmunity depends on NLRP3, GSDMD and mtDNA oxidation\u003c/h2\u003e \u003cp\u003eMacrophages challenged with NLRP3 inflammasome activators release ccf-Ox-mtDNA via Casp1 generated GSDMD pores\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Congruently, alum-challenged \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice had less ccf-Ox-mtDNA than wildtype (WT) counterparts, with no change in barely detectable ccf-nDNA (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). Fittingly, alum-induced systemic autoimmune features (serum anti-dsDNA IgG, GC reactions, elevated Tfh and proliferative Ki67\u003csup\u003e+\u003c/sup\u003e B and CD4\u003csup\u003e+\u003c/sup\u003e T cells, class-switched splenic IgG1\u003csup\u003e+\u003c/sup\u003e and IgG2b\u003csup\u003e+\u003c/sup\u003e B cells), serum IL-1β, IL-21, IL-6 and IFN-α, and glomerulonephritis were blunted in \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-o). mt-\u003cem\u003eOgg1\u003c/em\u003e\u003csup\u003eTg\u003c/sup\u003e mice, which express a mitochondrially-targeted OGG1, a glycosylase that removes 8-Oxo-dG lesions and are therefore non-responsive to NLRP3 inflammasome activators\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, also showed diminished GC reactions and reduced splenic Tfh cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f). Moreover, mt-OGG1 lowered splenic and peripheral memory CXCR3\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells and IFN-γ\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, which support antibody production\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, circulating Tfh cells and CD138\u003csup\u003e+\u003c/sup\u003e B cells and glomerulonephritis (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-l and Supplementary Fig.\u0026nbsp;2a,b). These results establish the importance of Ox-mtDNA in the alum-induced autoimmune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAlum induced ccf-Ox-mtDNA sensing by functionally important peritoneal pDC\u003c/h3\u003e\n\u003cp\u003eSingle-cell (sc) RNA-Seq provided unbiased and in-depth view of alum\u0026rsquo;s effects on the peritoneal immune microenvironment. Strikingly, within 24 h after injection, alum led to extensive depletion of peritoneal macrophages, previously reported after i.p. LPS or thioglycolate injections\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, accompanied by peritoneal influx of DC subsets, monocytes, neutrophils, and NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;3a). Macrophage (F4/80\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e) depletion was suppressed in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, which also displayed reduced IL-1β production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), suggesting GSDMD-mediated pyroptosis is pivotal to alum-induced macrophage death and Ox-mtDNA release\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Amongst the different peritoneal cell types in alum-injected mice, DCs, which bridge innate and adaptive immunity, were particularly enriched for inflammation\u003cem\u003e-\u003c/em\u003e and \u003cem\u003eIl1b\u003c/em\u003e-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Peritoneal DC classification showed no changes in transitory (t) DC, an insignificant decline in cDC2 and alum-induced increases in cDC1, pDC-like, and CD40\u003csup\u003e+\u003c/sup\u003eMHCII\u003csup\u003e+\u003c/sup\u003eCD80\u003csup\u003e+\u003c/sup\u003e pDC, whose number and frequency were reduced after DNase I injections, or in \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, mt-\u003cem\u003eOgg1\u003c/em\u003e\u003csup\u003eTg\u003c/sup\u003e and \u003cem\u003eCmpk2\u003c/em\u003e\u003csup\u003eΔMye\u003c/sup\u003e [myeloid restricted cytidine/uridine monophosphate kinase 2 (CMPK2) deficiency that blocks TLR4-stimulated mtDNA synthesis] mice\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-i and Supplementary Fig.\u0026nbsp;3b-d), all of which produce less Ox-mtDNA\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f). By contrast, PBS-injected mice had very few peritoneal pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-i). Compared to splenic pDC at steady state, alum-induced peritoneal pDC showed upregulation of IFN-stimulated genes (ISGs), \u003cem\u003eIl1b, Pycard\u003c/em\u003e and \u003cem\u003eP2rx7\u003c/em\u003e mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej) and Casp1 activation (FLICA\u003csup\u003ehi\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). Alum induced pDC also expressed IFN-α, IRF7, CD40, MHC II, CD80 and IL-21 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el), although the intensity of IL-21 intracellular staining in pDC was lower than in alum-induced Tfh cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince FLICA-detected Casp1 activity was much lower in cDC1 and cDC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek), and amongst the alum-induced DC subsets, peritoneal pDC expressed higher amounts of inflammasome-related \u003cem\u003eNek7\u003c/em\u003e and \u003cem\u003eP2rx7\u003c/em\u003e mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en), we examined pDC\u0026rsquo;s role in alum-stimulated autoimmunity. Diphtheria toxin (DT)-induced splenic and peritoneal pDC depletion in \u003cem\u003eBDCA2\u003c/em\u003e-DTR (DT receptor) transgenic mice\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg,h), diminished alum-induced splenomegaly (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei), anti-dsDNA and anti-chromatin IgGs (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej), serum IL-21 and IFN-α (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek), splenic GC reactions, Tfh cells, class-switched IgG1\u003csup\u003e+\u003c/sup\u003e and IgG2b\u003csup\u003e+\u003c/sup\u003e B cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el,m), and glomerulonephritis (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en). DT-treated DTR\u003csup\u003e\u0026minus;\u003c/sup\u003e mice, however, still exhibited alum-induced autoimmunity, suggesting that the above effects, were due to pDC depletion. This is consistent with the recently demonstrated amelioration of autoimmunity by Litifilimab, a humanized pDC depleting BDCA2 antibody\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003epDC intrinsic IL-1β signaling induces functionally important Tfh differentiation\u003c/h3\u003e\n\u003cp\u003eWe established an \u003cem\u003eex vivo\u003c/em\u003e model in which peritoneal or splenic pDC isolated after alum injections were co-cultured with na\u0026iuml;ve splenic CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003eCD62L\u003csup\u003ehi\u003c/sup\u003eCD44\u003csup\u003elo\u003c/sup\u003e T cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;3d, 4a). Either peritoneal or splenic pDC from alum-challenged, but not from PBS-injected, mice induced the generation of CXCR5\u003csup\u003e+\u003c/sup\u003eICOS\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e Tfh cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Of note, peritoneal pDC collected 24 h after two alum injections (72 h apart) were more effective in inducing Tfh differentiation than splenic pDC isolated at the same timepoint, presumably due to early ccf-Ox-mtDNA sensing by peritoneal pDC, which then migrate to the spleen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test whether Ox-mtDNA directly enables pDC induce Tfh differentiation, we extracted mtDNA and nDNA from fibroblasts and left half of each preparation as is, while the other half was oxidized by UV irradiation (see Methods). Tfh differentiation was triggered by FACS-sorted Flt3L-induced BM-derived CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003eBST2\u003csup\u003e+\u003c/sup\u003eSiglec-H\u003csup\u003e+\u003c/sup\u003e pDC, but this required incubation with exogenous Ox-mtDNA, while non-oxidized mtDNA was ineffective (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;4b). Notably, Ox-mtDNA-treated Flt3L-induced cDC1 or cDC2 did not trigger Tfh differentiation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Neither oxidized nor non-oxidized nDNA enabled pDC-programmed Tfh differentiation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), which required direct pDC-T cell contact, because when pDC and T cells were separated via transwell chambers, Tfh were not generated (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Tfh cells generated in this manner were functional, converting na\u0026iuml;ve B to GC B cells, as well as proliferative (Ki67\u003csup\u003e+\u003c/sup\u003e) and class-switched IgG-secreting B cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-h). Splenic Tfh-containing CD4\u003csup\u003e+\u003c/sup\u003e T cells from alum-challenged mice also promoted IgG-secretion and converted na\u0026iuml;ve B cells into GC B cells but not into CD138\u003csup\u003ehi\u003c/sup\u003eB220\u003csup\u003elo\u003c/sup\u003eIgD\u003csup\u003e\u0026minus;\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e plasmablasts (PB) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei,j). Ox-mtDNA also enabled the conversion of na\u0026iuml;ve human CD4\u003csup\u003e+\u003c/sup\u003e T into BCL6\u003csup\u003e+\u003c/sup\u003e Tfh cells by enriched human blood pDC (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek,l).\u003c/p\u003e \u003cp\u003eTo establish the importance of Tfh cells in Ox-mtDNA-driven autoimmunity, \u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e/\u003cem\u003eCd4\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e (\u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003eΔCD\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e) mice with a Tfh cell differentiation defect\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e were used. Na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells from \u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003eΔCD\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e mice did not differentiate into Tfh cells when co-cultured with pDC plus Ox-mtDNA (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Compared to \u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice, \u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003eΔCD\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e mice were refractory to alum-induced autoimmunity, manifested by BCL6 upregulation in CD4\u003csup\u003e+\u003c/sup\u003e T cells, elevated serum anti-dsDNA IgG, GC reactions, serum IL-21 and glomerulonephritis (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-i), despite exhibiting elevated serum IL-1β and splenomegaly (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef,j). Importantly, alum-induced infiltration of peritoneal pDC with activated Casp1 was unaffected by the BCL6 deficiency (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek,l), indicating that autoreactive Tfh cells are generated downstream to Ox-mtDNA sensing by pDC and NLRP3 inflammasome activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eOx-mtDNA sensing by pDC triggers autocrine IL-1β signaling\u003c/h3\u003e\n\u003cp\u003eOx-mtDNA, but not oxidized nDNA (Ox-nDNA) or non-oxidized mtDNA, activated Casp1 in pDC, but not in cDC1, cDC2 or BM derived macrophages (BMDM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). pDC incubation with Ox-mtDNA induced IL-1β, IL-21, IL-6, CD40 and ICOSL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d), proteins known to stimulate Tfh cell differentiation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, which were not induced in Ox-mtDNA treated cDC1 or cDC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-g). Non-oxidized mtDNA did not induce IL-21 or ICOSL, although it induced IFN-α as effectively as Ox-mtDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d). Both mtDNA forms, but not nDNA, triggered TLR9-dependent NF-κB activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), although the response to Ox-mtDNA, the only DNA type that activated Casp1, was stronger and relied on IL1R expression in pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei), implicating autocrine IL-1β signaling. IL-21 and IL-6 inductions were also abrogated in \u003cem\u003eIl1b\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or \u003cem\u003eIl1r\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e BM-derived pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej), that were no longer capable of inducing Tfh differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). Ox-mtDNA had no effect on na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells in the absence of pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). CD4\u003csup\u003e+\u003c/sup\u003e T cell intrinsic IL-1R signaling was also needed for Tfh generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el), consistent with its role in vaccine-related induction of Tfh and antibody responses\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, IFNAR-deficient na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells converted to Tfh as effectively as WT CD4\u003csup\u003e+\u003c/sup\u003e T cells when incubated with pDC and Ox-mtDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em), suggesting that pDC-derived IFN-α does not directly contribute to Tfh differentiation. Despite its relatively low expression level, pDC-generated IL-21 was essential for induction of BCL6\u003csup\u003e+\u003c/sup\u003e Tfh differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en,o).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-vitro\u003c/b\u003e \u003cb\u003eoxidized mtDNA is sufficient for\u003c/b\u003e \u003cb\u003ein-vivo\u003c/b\u003e \u003cb\u003einduction of autoimmunity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test if cell- and protein-free Ox-mtDNA triggers autoimmunity \u003cem\u003ein-vivo\u003c/em\u003e, we injected B6 mice with equal amounts of non-oxidized and oxidized mtDNA prepared as above (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). When examined 24 h post-injection, both mtDNA preparations induced alum-like peritoneal influx of DCs enriched for inflammation\u003cem\u003e-\u003c/em\u003e and \u003cem\u003eIl1b\u003c/em\u003e-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), that included pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,d). However, scRNA-seq analysis showed Ox-mtDNA to be more effective than non-oxidized mtDNA in inducing NF-κB- and inflammasome-related mRNAs (\u003cem\u003eNfkbia, Irak1, Nfkbiz, P2rx7, Casp1, Gsdmd, Nfkb2, Il1b, Nlrp3, Tlr9\u003c/em\u003e) in pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), in which certain inflammatory genes (\u003cem\u003eNfkbiz, P2rx7, Nek7, Tnf, Tlr9)\u003c/em\u003e were more highly expressed than in other DC subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). To test whether pDC recruited and activated by Ox-mtDNA can program na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells to Tfh \u003cem\u003eex vivo\u003c/em\u003e, enriched peritoneal pDC isolated from mtDNA-injected mice were co-cultured with na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells. Ox-mtDNA, but not non-oxidized mtDNA, injections enabled pDC induce Tfh differentiation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Splenic DCs from DT-treated \u003cem\u003eBDCA2\u003c/em\u003e-DTR\u003csup\u003e\u0026minus;\u003c/sup\u003e mice subjected to 4 repetitive Ox-mtDNA injections also induced Tfh differentiation, but DCs from DT-treated \u003cem\u003eBDCA2\u003c/em\u003e-DTR\u003csup\u003e+\u003c/sup\u003e mice were inactive in this assay (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), suggesting that induction of Tfh differentiation is pDC- and Ox-mtDNA- specific.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImportantly, a 20-week regimen of repetitive Ox-mtDNA injections induced dsDNA and chromatin antibodies, elevated serum IL-1β, IL-21 and IFN-α, GC reactions, class-switched B cells and splenic Tfh cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-k) while induction of circulating BCL6\u003csup\u003e+\u003c/sup\u003e Tfh cells and glomerulonephritis required a 36-week regimen of Ox-mtDNA injections (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el-o). \u003cem\u003eIn situ\u003c/em\u003e proximity ligation assays (PLA) conducted on spleens isolated after 20 weeks of Ox-mtDNA injections showed interactions between intracellular DNA and either TLR9 or NLRP3 in BST2\u003csup\u003e+\u003c/sup\u003e pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ep; 100x magnification), whereas injected mtDNA elicited DNA:TLR9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eq; 10x magnification) but not DNA:NLRP3 interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003er; 10x magnification). These results confirm that NLRP3 does bind DNA within pDC after \u003cem\u003ein-vivo\u003c/em\u003e Ox-mtDNA challenges.\u003c/p\u003e\n\u003ch3\u003eFcγR1 mediates mtDNA uptake followed by TLR9 binding and endosomal escape\u003c/h3\u003e\n\u003cp\u003eNLRP3 inflammasome activation and GSDMD cleavage in Ox-mtDNA incubated pDC were required for induction of Tfh differentiation, because the latter was diminished when \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e pDC were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). TLR9 in Flt3L-induced pDC was also needed for induction of Tfh differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), as well as Casp1 activation and IL-21 and IRF7 induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-d). Consistent with the equally effective induction of IFN-α by non-oxidized and oxidized mtDNAs, both mtDNA types upregulated TLR9-dependent IRF7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), suggesting no difference in their uptake efficacy. \u003cem\u003eIn vivo\u003c/em\u003e, biotinylated Ox-mtDNA was taken up by peritoneal pDC within 24 h after its i.p. injection, while biotinylated Ox-mtDNA internalization by splenic or BM pDC was not detected at this time point (Extended Data Fig.\u0026nbsp;8a), suggesting that peritoneal pDC are the first to encounter i.p. injected ccf-Ox-mtDNA.\u003c/p\u003e \u003cp\u003eTo determine how ccf-Ox-mtDNA reaches NLRP3 in the cytosol, we isolated mature splenic pDC and incubated them with biotin-labeled non-oxidized or \u003cem\u003ein-vitro\u003c/em\u003e oxidized mtDNAs (Extended Data Fig.\u0026nbsp;8b). Both mtDNA types were internalized and colocalized with TLR9 (Extended Data Fig.\u0026nbsp;8b,c), whose expression was substantially higher in pDC than in cDC1 or cDC2 (Extended Data Fig.\u0026nbsp;8d), consistent with higher \u003cem\u003eTlr9\u003c/em\u003e mRNA amounts in pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). These results explained why pDC responded to Ox-mtDNA more vigorously than cDC. Moreover, RNA-seq analysis of FACS-sorted BM-derived pDC after \u003cem\u003eex-vivo\u003c/em\u003e stimulation showed that Ox-mtDNA suppressed the \u0026ldquo;negative regulation of CpG-DNA responses\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and enhanced \u0026ldquo;hallmarks of NF-κB response to TNF\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e more robustly than non-oxidized mtDNA, in line with a more enriched autoimmune KEGG SLE pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Consistently, Ox-mtDNA, but not Ox-nDNA, induced a more robust NF-κB signaling response than non-oxidized mtDNA in pDC, but not in cDC1 or 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Congruently, only Ox-mtDNA engaged NLRP3 to trigger inflammasome assembly evidenced by appearance of DNA:NLRP3 PLA signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg) and ASC-containing aggregates in cultured pDC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eSerum or IgG removal from the culture medium impaired Ox-mtDNA-stimulated Casp1 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei), suggesting that mtDNA was internalized as a complex with serum IgGs. In line with the known ability of Fc receptors to endocytose antibody-DNA complexes\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, pDC lacking FcγR1, which presents IgG-DNA complexes to endosomal TLR9\u003csup\u003e40\u003c/sup\u003e, no longer induced Tfh differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej). Of note, RNA-seq data analysis revealed that both mtDNA forms downregulated endosome organization and vesicle tethering complex pathways (Extended Data Fig.\u0026nbsp;8e). This was confirmed by presence of both mtDNA forms within ruptured, galectin 8 positive, endosomes\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;8f), which allow mtDNA access to cytosolic sensors, such as NLRP3\u003csup\u003e4, 5, 6\u003c/sup\u003e. We thus conclude that circulating Ox-mtDNA binds (probably non-specifically) preexisting serum IgGs, which enable its internalization via FcγR1 and delivery to endosomal TLR9, whose engagement results in NF-κB mediated priming that licenses NLRP3 inflammasome activation and production of IL-1β, which is secreted via GSDMD pores. In turn, autocrine IL-1β-IL1R signaling further boosts NF-κB signaling (Extended Data Fig.\u0026nbsp;8g).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIL-1R signaling can be targeted to prevent autoimmunity\u003c/h2\u003e \u003cp\u003eTo validate the importance of IL-1R signaling in autoimmunity, irradiated mice were reconstituted with WT or \u003cem\u003eIl1r1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e BM cells and challenged with alum (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). IL-1R ablation blunted generation of dsDNA and chromatin antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), IL-21 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), GC formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed,e), Tfh expansion, class-switched splenic IgM\u003csup\u003e\u0026ndash;\u003c/sup\u003eIgG1\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee) and glomerulonephritis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef-h). Serum IFN-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) and circulating Ox-mtDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei,j), however, remained unaffected, indicating that IL-1β signaling does not contribute to IFN-α or ccf-Ox-mtDNA production. Supporting IL-1β-IL-1R interaction as a potential therapeutic target, treatment of alum challenged mice with IL-1R antagonist (IL-1Ra, anakinra) inhibited splenomegaly, generation of dsDNA antibodies, IL-21 secretion, splenic Tfh, GC generation, B cell isotype switching and glomerulonephritis (Extended Data Fig.\u0026nbsp;9a-i), with no significant change in circulating Ox-mtDNA amounts (Extended Data Fig.\u0026nbsp;9j, k). \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results unravel how oxidation converts IFN-I-inducing mtDNA to a unique and highly immunopathogenic alarmin capable of initiating systemic autoimmune reactions through IFN-I independent mechanisms. As elevated amounts of ccf-mtDNA were detected in elderly individuals\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and patients with metabolic\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and autoimmune disorders\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, our results suggest that mtDNA oxidation is a key step in the pathogenesis of inflammaging. Most reports of ccf-mtDNA in the human circulation do not include any information regarding its oxidation state, but our earlier studies show that mtDNA oxidation is obligatory for its cleavage by FEN-1, which enables its cytoplasmic leakage, and binding to NLRP3 and subsequent Casp1 activation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although the source and mechanism of ccf-mtDNA production during inflammaging are unknown, we suggest that as long as ccf-mtDNA is generated in response to mitochondrial stress, most of it contains 8-Oxo-dG residues that enable NLRP3 binding, IL-1β induction and GSDMD cleavage, driving progression from sterile inflammation, manifested by splenomegaly and peritonitis, to systemic antibody-mediated autoimmunity. Importantly, non-oxidized mtDNA induces IFN-α in pDC as effectively as Ox-mtDNA but is incapable of inducing autoantibody production when injected into mice. In other words, the difference in the immunogenic properties of oxidized and non-oxidized mtDNAs boils down to their ability and inability to activate NLRP3.\u003c/p\u003e \u003cp\u003eThe mechanism by which NLRP3-dependent sensing of Ox-mtDNA by pDC circumvents self-tolerance is unprecedented. We show that Ox-mtDNA uptake endows pDC with a new activity: the ability to convert na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells into Tfh cells that play a critical role in autoantibody induction in our experimental system (Extended Data Fig.\u0026nbsp;10). Importantly, Ox-mtDNA also enables human pDC stimulate the generation of Tfh cells, whose elevated abundance has been linked to antibody-dependent autoimmunity\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Originally, pDC were studied for their ability to produce copious amounts of IFN-α\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, rather than their T cell priming and differentiation inducing activities. Human pDC activated by CpG oligodeoxynucleotides were reported to be tolerogenic because they induced CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Treg cells\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In contrast, the ability to secrete IL-6\u003csup\u003e50, 51\u003c/sup\u003e and up-regulate ICOSL\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, upon microbial or viral stimulation, suggested that beyond Treg induction, pDC may be capable of promoting Tfh differentiation, at least under certain circumstances\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, whether and how pDC initiate Tfh generation \u003cem\u003ein-vivo\u003c/em\u003e was heretofore unknown. Induction of Tfh differentiation depends on pDC-T cell contact, which could be mediated through ICOSL (on pDC):ICOS (on T) and CD40 (on pDC):CD40L (on T) interactions, as well as autocrine and paracrine IL-1β signaling, which induces IL-21 and IL-6 that prime Tfh differentiation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. pDC also express MHC molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei,l) but are generally considered as poor antigen presenters\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. At this point we don\u0026rsquo;t know whether antigen presentation has a role in the initiation of Tfh differentiation by Ox-mtDNA stimulated pDC, which warrants further investigation.\u003c/p\u003e \u003cp\u003eThe key step through which autocrine IL-1β signaling acts is the enhancement of NF-κB activation, beyond the initial amount provided by TLR9 engagement. Both oxidized and non-oxidized mtDNAs are internalized by pDC as complexes with serum IgG molecules via FcγR1, which presents DNA to endosomal TLR9\u003csup\u003e40\u003c/sup\u003e. In addition to IFNα induction, TLR9 signaling accounts for the initial activation of NF-κB, which is required for the so-called \u0026ldquo;priming stage\u0026rdquo; that includes NLRP3 and pro-IL-1β induction and precedes NLRP3 inflammasome activation by Ox-mtDNA\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Moreover, TLR9 discriminates between non-methylated mtDNA and methylated nDNA\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, which is non-immunogenic in our system, even after its oxidation. Of note, IFNAR deficient CD4\u003csup\u003e+\u003c/sup\u003e T cells can still be converted to Tfh, in line with the inability of mtDNA induced IFN-α to drive IL1β-dependent Tfh generation. Consistently, IL-1β was previously found to play a leading role in Tfh generation (through induction of BCL6, ICOS and CXCR5) in response to microbial vaccines, while IFN-I was shown to promote IL-21 production by already differentiated Tfh cells\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Studies also show that the autoimmune activities of IFN-α are directed towards other cellular targets, most importantly B cells\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, our work shows that, although much remains to be known about its origin and the time of its production, Ox-mtDNA is an immunogenic alarmin capable of propagating autoimmunity, whose risks increase with old age, paralleling its amounts in the circulation. We suggest that pDC intrinsic IL-1β-IL1R signaling needs to be assessed for its role in systemic autoimmune disorders and if validated as a culprit, should be studied as a target for interception with age-related immunopathologies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cstrong\u003eMice\u003c/strong\u003e \u003cp\u003eWildtype and \u003cem\u003eTlr9\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice in the C57BL/6 background were purchased from the Jackson Laboratory. \u003cem\u003eCmpk2\u003c/em\u003e\u003csup\u003eΔMye\u003c/sup\u003e and mt-\u003cem\u003eOgg1\u003c/em\u003e\u003csup\u003eTg\u003c/sup\u003e mice were previously described\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, \u003cem\u003eIlb\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and \u003cem\u003eIl1r1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice in the C57BL/6 background were maintained by Hal M. Hoffman (UCSD). \u003cem\u003eBDCA2\u003c/em\u003e-DTR transgenic mice were provided by Elina I. Zuniga (UCSD). \u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e/\u003cem\u003eCd4\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e (\u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003eΔCD\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e) mice were kindly provided by Alexander Dent at Indiana University. All mice were bred and maintained at UCSD and handled in accordance with Institutional Animal Care and Use Committee and NIH guidelines. Gender matched 6\u0026ndash;10-week-old mice were used for all experiments. Bones from \u003cem\u003eIl21\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, \u003cem\u003eFcgr1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and \u003cem\u003eIfn1r1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were provided by Drs. Warren Leonard (NIH) and Yi-Guang Chen (Medical College of Wisconsin), Jefferey Ravetch (Rockefeller University) and Kevin King (UCSD), respectively.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBone marrow chimeras\u003c/strong\u003e \u003cp\u003eGender-matched 6\u0026ndash;8-week-old C57BL/6 mice were lethally γ-irradiated twice with 600 Rad doses, given 4 h apart. Irradiated mice were reconstituted with 5x10\u003csup\u003e6\u003c/sup\u003e BM cells injected intravenously and used 8 weeks later.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAlum/mtDNA-induced short-term peritonitis and long-term autoimmunity\u003c/strong\u003e \u003cp\u003eMice were allocated randomly and i.p. injected with 1 mg Imject Alum (Thermo Scientific, Cat#77161) in 0.2 mL sterile PBS, or 50 \u0026micro;g mtDNA/Ox-mtDNA/PBS. Peritoneal lavage was performed 4 h post-injection to measure IL-1β by ELISA. Another batch of randomly allocated mice were i.p. injected with alum or PBS and euthanized after 24 h. Their peritoneal cavities were washed with 6 mL cold sterile PBS, followed by 10 min centrifugation at 1200 rpm to separate peritoneal cells and fluid. 10,000 peritoneal cells with viability\u0026thinsp;\u0026gt;\u0026thinsp;95% were profiled using 10x Genomics\u0026rsquo; Chromium Single Cell 3\u0026prime; V2 chemistry and processed using the 10x Cell Ranger pipeline for scRNA-seq and the rest were FC analyzed.\u003c/p\u003e \u003cp\u003eFor induction of long-term autoimmunity, randomly allocated gender and age-matched mice were repetitively i.p. injected with alum (1 mg/mouse) or DNA (50 \u0026micro;g/mouse) as indicated. Wherever indicated, 1.25 mg/mouse DNase I (Millipore, Cat#10104159001, Roche), 100 ng/mouse DT (Millipore, Cat#D0564) or 25 mg/kg anakinra were also i.p. administered.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell Lines and Primary Culture\u003c/strong\u003e \u003cp\u003eBone-marrow-derived macrophages (BMDM): Femurs and tibias from C57BL/6 mice\u0026thinsp;\u0026gt;\u0026thinsp;8 weeks of age of the same gender were used for BMDM generation by culturing BM cells in high glucose DMEM supplemented with 10% FBS, 20% L929-cell conditioned medium, and 100 U/mL penicillin-streptomycin for 7\u0026ndash;10 days at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e62\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBMDCs were generated by culturing BM cells at 2x10\u003csup\u003e6\u003c/sup\u003e cells/ml for 7 days in 5 ml of RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) containing 100 ng/mL Flt3L and 50 \u0026micro;M β-mercaptoethanol. Cells were either left untreated or activated with DNA as indicated, followed by FC analyses of protein expression/inflammasome activation. RNAs were collected 4 h post-stimulation from FACS sorted pDC for bulk RNA seq analyses.\u003c/p\u003e \u003cp\u003eIg depletion from FBS for BMDC culture: the Pierce\u0026trade; Protein A/G Magnetic Beads suspension (Thermo Scientific, Cat#88802) was washed three times with PBS, followed by incubation with FBS at 4\u0026deg;C for 24 h with constant mixing, after which IgG-depleted serum FBS was collected and used for BMDC culture.\u003c/p\u003e \u003cp\u003eMouse embryo fibroblasts (MEFs) were cultured in high glucose DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eHuman specimens and peripheral blood mononuclear cells (PBMCs)\u003c/p\u003e \u003cp\u003eDe-identified blood and buffy coats were purchased from San Deigo Blood Bank and StemCell Technologies Human Peripheral Blood Leukopak obtained from healthy adult donors (20\u0026ndash;45 years old). PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Human pDC and na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells were negatively enriched using EasySep\u0026trade; Human Plasmacytoid DC Isolation Kit (StemCell Technologies, Cat# 17977) and EasySep\u0026trade; Human Na\u0026iuml;ve CD4\u0026thinsp;+\u0026thinsp;T Cell Isolation Kit (StemCell Technologies, Cat# 19555) respectively.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell suspension preparation\u003c/strong\u003e \u003cp\u003eBlood was collected from anesthetized mice via cardiac puncture into a vial containing 1 mM EDTA (Corning), and mononuclear cells were enriched using Lympholyte M (Cedarlane). The serum fraction was used to measure autoantibodies, cytokines and circulating mtDNA. Splenocyte suspensions were obtained by mechanical dissociation in FACS buffer (2 mM EDTA, 2% FBS in PBS) and passed through a 70 \u0026micro;m strainer. For BM, femurs and tibias were flushed using PBS and filtered through a 70 \u0026micro;m strainer. Red blood cells were lysed using RBC Lysis Buffer (ThermoFisher).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell Purification\u003c/strong\u003e \u003cp\u003ePeritoneal or splenic pDC were purified using EasySep\u0026trade; Mouse Plasmacytoid DC Isolation Kit (StemCell Technologies) per manufacturer\u0026rsquo;s instructions. Unlabeled pDC were enriched by negative selection and pDC (PI\u003csup\u003e\u0026minus;\u003c/sup\u003eThy1.2\u003csup\u003e\u0026minus;\u003c/sup\u003eCD19\u003csup\u003e\u0026minus;\u003c/sup\u003eNK1.1\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003eBST2\u003csup\u003e+\u003c/sup\u003eSiglec-H\u003csup\u003e+\u003c/sup\u003e) purity was routinely assessed by FC. Splenic DC were enriched using Dendritic Cell Isolation Kit, mouse (Miltenyi Biotec, Cat# 130-100-875). Cultured BMDC were stained and sorted into pDC (PI\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003eBST2\u003csup\u003e+\u003c/sup\u003eSiglec-H\u003csup\u003e+\u003c/sup\u003e), cDC1 (PI\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB220\u003csup\u003e\u0026minus;\u003c/sup\u003eXCR\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003e), cDC2 (PI\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eB220\u003csup\u003e\u0026minus;\u003c/sup\u003eXCR1\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e), using BD Aria II or Fusion (BD Biosciences).\u003c/p\u003e \u003cp\u003eNa\u0026iuml;ve splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD62\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e\u0026minus;\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e) were negative-selected using EasySep\u0026trade; Mouse Na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T Cell Isolation Kit (StemCell Technologies, Cat#19765) or FACS-sorted. Total splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells were enriched with a CD4\u003csup\u003e+\u003c/sup\u003e T Cell Isolation Kit, mouse (Miltenyi Biotec, Cat# 130-104-454). B cells were purified by negative selection using CD43 (Ly-48) MicroBeads, mouse (Miltenyi Biotec, Cat#130-049-801). T or B cells were FC analyzed 3 days after co-culture in RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) supplemented with 50 \u0026micro;M β-mercaptoethanol.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003epDC and CD4\u003csup\u003e+\u003c/sup\u003e T cell co-culture assay\u003c/strong\u003e \u003cp\u003ePurified/sorted mouse pDC (3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) or human pDC (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded with na\u0026iuml;ve mouse splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells (1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) or human na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) into 24-well plates. 50 \u0026micro;g mtDNAs or nDNAs were added into RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) supplemented with 50 \u0026micro;M β-mercaptoethanol. Cells were co-cultured for 3 days after which Tfh differentiation was FC analyzed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFlow Cytometry (FC)\u003c/strong\u003e \u003cp\u003eAntibodies were from Biolegend, BD Biosciences, ThermoFisher and Cell Signaling Technologies (Supplementary Table\u0026nbsp;1) and were used at 1:200 dilution. Cell suspensions were incubated with Purified Rat Anti-Mouse CD16/CD32 (BD Pharmingen\u0026trade; Cat#553141) to block nonspecific binding for 15 min at 4\u0026deg;C, followed by incubation with antibody cocktails for cell surface protein staining for 45 min at 4\u0026deg;C. PI or eBioscience\u0026trade; Fixable Viability Dye eFluor\u0026trade; 780 (ThermoFisher, Catalog# 65-0865-14) was used to exclude dead cells. Human cells were incubated with human gamma-globulin (ThermoFisher, Cat# Catalog # 31879) to block non-specific binding for 15 min at 4\u0026deg;C, followed by antibodies staining cell surface protein in human FACS buffer (2 mM EDTA, 2% Donor equine serum in PBS) for 30 min at RT. For intracellular staining, cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining kit (Life Technologies Cat# 00-5523-00) according to manufacturer\u0026rsquo;s instructions. Cells were analyzed on a Beckman Coulter Cyan ADP flow cytometer followed by data analysis with FlowJo software (Treestar, Inc.).\u003c/p\u003e \u003cp\u003eTo determine anti-DNA/nucleosome antibody-forming cells (AFCs), biotin-tagged mtDNA or recombinant nucleosomes (BPS Bioscience, Catalog #52048) were incubated with single cell suspensions after CD16/CD32 Fc blocking, for 15 min at 4\u0026deg;C, followed by cell surface protein staining for 45 min at 4\u0026deg;C and Streptavidin, Alexa Fluor\u0026trade; 488 conjugate (ThermoFisher, Cat# S11223) in FACS buffer for additional incubation 30 min at 4\u0026deg;C.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCytokine and IgG Measurements\u003c/strong\u003e \u003cp\u003eSecreted cytokines from mouse sera or culture supernatants of FACS-purified pDC (3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well in 96-well plates) were measured using mouse ELISA kits following manufacturer\u0026rsquo;s instructions. FACS-purified pDC were incubated in RPMI-1640 supplemented with 10% (vol/vol) FBS, L-glutamine, penicillin-streptomycin, and HEPES buffer (pH 7.2) supplemented with 50 \u0026micro;M β-mercaptoethanol in the presence or absence of non-oxidized or UV-oxidized mtDNA (50 \u0026micro;g) for 4 h at 37\u0026deg;C. Supernatants were collected and analyzed. Graphs depicting cytokine measurements represent individual wells from several independent experiments. Relative titers of anti-dsDNA (Chondrex Cat#3031), anti-nucleosome (Creative Diagnostics, Cat#DEIA-BJ2361) in sera and total IgG (ThermoFisher, Cat#88-50400-22) amounts in B cell culture supernatants were measured by ELISA. Urinary protein was measured using Mouse Proteinuria ELISA Kit (MyBioSource, Cat#MBS723873).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDNA Isolation and in-vitro preparation of Ox-DNA\u003c/strong\u003e \u003cp\u003eMitochondrial isolation from MEF was carried out as described\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Briefly, PBS-washed MEF were resuspended in pre-chilled mitochondrial extraction buffer 1 (220 mM mannitol, 70 mM sucrose, 20 mM HEPES-KOH, pH 7.5, 1 mM EDTA and 2 mg/mL BSA) and passed through a 25-G syringe (BD Biosciences) 20x on ice. The homogenized cells were centrifuged at 1000\u0026times;g for 15 min at 4\u0026deg;C yielding nuclear pellets. The post-nuclear supernatant was further centrifuged at 10,000\u0026times;g for 10 min at 4\u0026deg;C to pellet mitochondria from supernatant cytosolic fraction. mtDNA was purified from the mitochondrial fraction while nDNA was extracted from the nuclear pellets using AllPrep DNA/RNA Mini Kit (Qiagen, Cat#80204) according to manufacturer\u0026rsquo;s instructions, followed by 10x UV irradiation at 1,000 mJ/cm\u003csup\u003e2\u003c/sup\u003e to generate Ox-mtDNA or Ox-nDNA\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBiotin-mtDNAs were prepared using Label IT Tracker Intracellular Nucleic Acid Localization Kit, Biotin Kit (Mirus Bio, Cat#MIR 7024), per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMeasurements of circulating mtDNA\u003c/strong\u003e \u003cp\u003eSeral DNA was isolated using QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat#55114) according to manufacturer\u0026rsquo;s instructions, eluted in 300 \u0026micro;L dd H\u003csub\u003e2\u003c/sub\u003eO and quantified using spectrophotometric analysis at 260/280 nm with Nanodrop. qPCR was performed using mtDNA (\u003cem\u003eD-loop\u003c/em\u003e, \u003cem\u003eCox1\u003c/em\u003e, \u003cem\u003enon-NUMT\u003c/em\u003e), and nDNA (\u003cem\u003eTert\u003c/em\u003e, \u003cem\u003e18S\u003c/em\u003e, \u003cem\u003eB2m\u003c/em\u003e) primers (Supplementary Table\u0026nbsp;2). Ct values of mtDNA abundance obtained from the alum-challenged group served as normalization controls. For measurement of Ox-mtDNA, 8-Oxo-dG content was quantified using 8-hydroxy 2-deoxyguanosine ELISA Kit (Abcam, Cat#ab201734), per manufacturer\u0026rsquo;s instructions. To determine mtDNA oxidative damage, Fpg-sensitive qPCR analysis was used as described\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e (Fpg removes oxidized purines from DNA and creates single-strand breaks, which block PCR amplification. Differences in qPCR cycles between Fpg-treated and untreated DNAs are therefore a specific indicator of oxidative base damage). Briefly, 500 ng of purified ccf-DNA were incubated with 8 units of Fpg (New England BioLabs Inc., Cat#M0240S) in 1\u0026times;NEBuffer 1 and 100 \u0026micro;g/mL BSA in 50 \u0026micro;L at 37\u0026deg;C for 1 h. Fpg was then inactivated at 60\u0026deg;C for 5 min, followed by qPCR to detect Fpg-sensitive cleavage sites. Data are presented as the ratio of Fpg-insensitive DNA, calculated as the quotient of signal intensities in Fpg-treated relative to untreated DNA. Relative mtDNA amounts were determined by qPCR in a CFX96 thermal cycler (Biorad) as described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Data are presented in arbitrary units and calculated by the 2ˆ(\u0026minus;ΔΔCT) method. Primers were provided by Integrated DNA technologies.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHistological evaluation and immunohistochemistry\u003c/strong\u003e \u003cp\u003eSpleen and kidneys were fixed in 10% formalin for 24 h and embedded in paraffin. 5 \u0026micro;m thick sections were stained with hematoxylin and eosin (H\u0026amp;E) or Periodic Acid-Schiff (PAS). Glomerular size was measured. Spleen sections were incubated with Ki67 antibody and positive cells were quantified with ImageJ. Spleen and kidneys were frozen in OCT and cryosectioned at 5 \u0026micro;m thick, followed by fixation with 4% paraformaldehyde (PFA) and chilled acetone, blocked with 5% donkey serum in PBS supplemented with 3% BSA, and stained with DAPI and specific antibody conjugates (Supplementary Table\u0026nbsp;1). Images were captured on a Leica SP5 microscope at 20X magnification. \u003cem\u003eCrithidia luciliae\u003c/em\u003e immunofluorescence test (CLIFT) of sera was determined according to manufacture instructions (Bio-Rad, Catalog#26109) using a Leica SP5 confocal microscope at 20X magnification.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIF and confocal microscopy\u003c/strong\u003e \u003cp\u003eNa\u0026iuml;ve splenic pDC from B6 mice stimulated \u003cem\u003eex vivo\u003c/em\u003e with 2 \u0026micro;g mtDNA/Ox-mtDNA for 1 h were fixed with 4% PFA, permeabilized in 0.2% Triton X-100, and blocked with 3% BSA/PBS. Primary antibodies were incubated in blocking buffer at 4\u0026deg;C overnight. Secondary Alexa-labelled antibodies were added for 1 h. Nuclei were counterstained with DAPI. Samples were imaged at 100x magnification on Leica SP5 or AXR Confocal, NSPARC Super Resolution microscopes.\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003ein situ\u003c/em\u003e PLA, the Duolink\u0026reg; In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, Cat#DUO92101) was used according to the manufacturer\u0026rsquo;s instructions. Frozen spleen sections were first fixed with 4% PFA for 15 min, blocked with 5% donkey serum in PBS including CD16/CD32 Fc blocking at RT for 45 min, followed by FITC anti-mouse BST2 antibody (BioLegend, Cat#127008) staining at 4\u0026deg;C overnight, PBS wash, and Donkey anti-Rat IgG (H\u0026thinsp;+\u0026thinsp;L) Alexa Fluor\u0026trade; 488 antibody (Invitrogen, Cat#A-21208) at RT for 1 h. The sections were then fixed with chilled acetone at -20\u0026deg;C for 2 h, permeabilized with 0.2% Triton X-100 in 3% BSA blocking buffer at RT for 45 min, and subsequently incubated with rabbit TLR9 (Thermo Fisher Scientific Cat#PA5-20203) or NLRP3 antibody (LS Bio Cat#LS-C334192-20) and mouse monoclonal anti-DNA antibody (Millipore Sigma, Cat#CBL186) at 4\u0026deg;C overnight. Rabbit IgG antibody (Cell Signaling Technology, Cat#2729) was used as a negative control. Secondary proximity probes (from Duolink\u0026reg; In Situ Red Starter Kit) were applied to the sections at 37\u0026deg;C for 90 min. Unbound proximity probes were washed. Sections were then incubated with ligation solution at 37\u0026deg;C for 60 min, washed twice and followed by amplification hybridization at 37\u0026deg;C for 120 min. After the PLA, the cells were washed and stained with DAPI before mounting. Isolated pDCs grown on round microscope coverglass pre-coated with Cell-Tak\u0026trade; (Corning\u0026reg;, Cat#354240, at RT for 30 min) were fixed, permeabilized and Biotin-DNA was first stained with Streptavidin, Alexa Fluor\u0026trade; 488 Conjugate (Thermo Fisher Scientific Cat# S11223), following by PLA to determine DNA and NLRP3 proximity as described above.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBulk RNA-Seq library preparation, sequencing, and analysis\u003c/strong\u003e \u003cp\u003eTotal RNA was isolated from FACS-sorted pDC treated with mtDNA as indicated. Cells were homogenized with TRIzol reagent (ThermoFisher, Cat#15596026) and purified using RNeasy Mini Kit (Qiagen, Cat#74104). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection performed by Novogene Corporation Inc. Quantified libraries prepared from three biological replicates per group were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.\u003c/p\u003e \u003cp\u003eRaw \u003cem\u003efastq\u003c/em\u003e format data were quality checked and trimmed using \u003cem\u003efastp 0.23.4\u003c/em\u003e with its default parameters, except that automatic adapter detection for pair-end data was enabled with \u003cem\u003edetect_adapter_for_pe\u003c/em\u003e. All downstream analyses were based on high-quality clean data. Reads were mapped using \u003cem\u003eSTAR 2.7.11b\u003c/em\u003e with its default parameters except that \u003cem\u003eoutFilterMultimapNmax\u003c/em\u003e was set to 1. GRCm39 was used as a reference genome. Aligned reads in the output Bam files from STAR were counted using \u003cem\u003efeatureCounts v2.0.1\u003c/em\u003e with its default parameters. Generated raw read count data were analyzed using \u003cem\u003eR version 4.3.3\u003c/em\u003e. Genes with less than 50 total read counts were removed from further analysis. Read count data were normalized, and differential gene expression analysis was performed using \u003cem\u003eR package DESeq2\u003c/em\u003e. Pathway enrichment analysis for each cell was performed using \u003cem\u003eR package fgsea\u003c/em\u003e. Gene sets were obtained from the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and MSigDB using \u003cem\u003eR packages genekitr\u003c/em\u003e and \u003cem\u003egeneset\u003c/em\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003escRNA-Seq library preparation, sequencing, and analysis\u003c/strong\u003e \u003cp\u003eThe number of live freshly-collected peritoneal cells was determined and 10,000 cells per sample were run on the 10x Chromium platform (10x Genomics). Library preparation and sequencing were performed by the UCSD IGM Genomics Center using the Chromium Next GEM Single Cell 3' Kit v3.1 according to manufacturer's instructions. Sequencing was performed on the NovaSeq 6000 system (Illumina). Raw \u003cem\u003efastq\u003c/em\u003e files were aligned to the mouse mm10 reference genome using 10x Genomics Cell Ranger 7.1.0 with 10x Genomics Cloud Analysis. Raw count matrices from the 10x Genomics Cell Ranger were analyzed using \u003cem\u003eSeurat v5.0.3\u003c/em\u003e in \u003cem\u003eR version 4.3.3\u003c/em\u003e. For quality control, cells with less than 200 expressed genes, more than 50000 total counts, and more than 10% mitochondrial genes were removed. The data were then normalized using \u003cem\u003eSeurat's NormalizeData\u003c/em\u003e function (\u003cem\u003eLogNormalize\u003c/em\u003e method with scale factor 10000). PCA was performed using the top 2000 variable genes, and the top 30 dimensions were used for UMAP. Cell annotation was performed manually using reported marker genes: \u003cem\u003eIgkc, Ebf1, Cd79a, Ly6d\u003c/em\u003e, and \u003cem\u003eIglc2\u003c/em\u003e for B cells; \u003cem\u003eCd209a, Ctnnd2, Mgl2, Flt3\u003c/em\u003e, and \u003cem\u003eTrerf1\u003c/em\u003e for DC; \u003cem\u003eDcn, Col1a2, Col3a1\u003c/em\u003e, and \u003cem\u003eSparc\u003c/em\u003e for fibroblasts; \u003cem\u003eCma1, Cpa3, Tpsb2, Mcpt4\u003c/em\u003e, and \u003cem\u003eMrgprb1\u003c/em\u003e for mast cells; \u003cem\u003eFscn1\u003c/em\u003e and \u003cem\u003eCcr7\u003c/em\u003e for migratory dendritic cells; \u003cem\u003eSpp1, Ccl2, Ccl7, Chil3\u003c/em\u003e, and \u003cem\u003eVcan\u003c/em\u003e for monocytes; \u003cem\u003eS100a9, S100a8, Cxcr2, Il1r2\u003c/em\u003e, and \u003cem\u003eIl1f9\u003c/em\u003e for neutrophils; \u003cem\u003eGzma, Klra4, Klra8, Klrb1c\u003c/em\u003e, and \u003cem\u003eNcr1\u003c/em\u003e for NK cells; \u003cem\u003ePrg4, Tgfb2, Ltbp1, Alox15\u003c/em\u003e, and \u003cem\u003eC4b\u003c/em\u003e for peritoneal macrophages; \u003cem\u003eThemis, Camk4, Trbc2, Cd3g\u003c/em\u003e, and \u003cem\u003eBcl11b\u003c/em\u003e for T cells. Cells expressing two or more sets of gene markers of each cell type were considered as doublets and removed. Pathway enrichment analysis for each cell was performed using \u003cem\u003eR package escape\u003c/em\u003e. Gene sets were obtained from the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and MSigDB using R packages. After global annotation, only the DC cluster was extracted and subdivided into finer clusters. Annotation for DC subtypes was done manually using reported marker genes: \u003cem\u003eAif1, Cadm1, Gpr141b, Xcr1, Tlr3, Ppt1, Btla, Itgae\u003c/em\u003e, and \u003cem\u003eCd24a\u003c/em\u003e for cDC1; \u003cem\u003eLtb, Cyp4f16, Pglyrp1, Clec4a4\u003c/em\u003e, and \u003cem\u003eSirpb1a\u003c/em\u003e for cDC2; \u003cem\u003eCcr9, Iglc3, Klk1, Cox6a2, Smim5, Ly6c2, Ly6d, Siglech\u003c/em\u003e, and \u003cem\u003eRunx2\u003c/em\u003e for pDC; \u003cem\u003eCd33\u003c/em\u003e and \u003cem\u003eNgfr\u003c/em\u003e for pDC-like; \u003cem\u003eVim, Ccr2, Fxyd5, Cx3cr1, Ccr1\u003c/em\u003e, and \u003cem\u003eLgals3\u003c/em\u003e for tDC. As reference data for steady-state pDC we obtained published scRNA-seq data (GSE196720) from GEO. The splenic steady state pDC analyses\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e was used as reference and merged with the scRNA seq data we obtained. Alignment of these two different studies was performed using \u003cem\u003eSCTransform\u003c/em\u003e function from Seurat package with batch correction using the \u003cem\u003evars.to.regress\u003c/em\u003e argument. Statistical test for compositional analysis was performed using Student's t-test and for differential gene expression analysis was performed using \u003cem\u003eFindMarkers\u003c/em\u003e function from Seurat package with \u003cem\u003etest.use\u003c/em\u003e argument set to \"wilcox\".\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eQuantification and statistical analysis\u003c/strong\u003e \u003cp\u003eNo statistical methods were used to pre-determine sample sizes. Data collection and analysis were not performed blinded. No data points were excluded. For scRNA-seq, pre-established criteria for single-cell exclusion, which is, low number of unique genes, abnormally high read count and high mitochondrial gene content, were used. Statistical details of experiments, including statistical tests used, n value, number of experiments and the type of statistical tests, can be found in figure legends. Statistical analysis was performed with at least three biological replicates using GraphPad Prism 9 or R (v.4.1.2) statistical programming language. All graphs show mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d. Comparisons between two groups were performed by normality test followed by unpaired two-tailed t-test or non-parametric student\u0026rsquo;s t-test with Mann-Whitney analysis. Comparisons between multiple groups were performed by one-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test or unless otherwise mentioned. P values less than or equal to 0.05 were considered significant and significance was assigned according to the following breakdown: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAnimal procedures described in this study were approved by the University of California, San Diego Institutional Animal Care and Use Committee (IACUC) and complied with all relevant guidelines for the use of non-human vertebrate animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and code availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRaw mouse sequencing data including RNA-seqand scRNA-seq have been deposited at Gene Expression Omnibus (GEO):\u0026nbsp;GSE264619 and GSE264619, which are listed in Deposited data and are publicly available.\u003c/p\u003e\n\u003cp\u003eThis study did not generate any original code.\u003c/p\u003e\n\u003cp\u003eAny additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Juliana Idoyaga, Li-Fan Lu, Virginia Pascual, Simone Caielli, Trever Greene for help and advice. We acknowledge eBioscience, Cell Signaling Technologies, Santa Cruz Technologies, Thermo Fisher, Biolegend, BD Biosciences, STEMCELL Technologies for gifts of reagents, and the UCSD TTSR supported by an NCI Cancer Center Support Grant (CCSG P30CA23100). We are grateful to Dr. Alexander Dent at Indiana University for providing us \u003cem\u003eBcl6\u003c/em\u003e\u003csup\u003eff\u003c/sup\u003e/\u003cem\u003eCd4\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e mice, Drs. Jeffrey V. Ravetch and Emily Lam at the Rockefeller University, Warren Leonard at the NIH, Yi-Guang Chen at Medical College of Wisconsin, Kevin King at UCSD for \u003cem\u003eFcgr1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, \u003cem\u003eIl21\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and \u003cem\u003eIfnr1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse bones, respectively. Dr. Jay Chung at the NIH kindly provided MEF. We thank the UCSD Nikon Imaging Center and especially Dr. Peng Guo for assistance with AXR Confocal and NSPARC Super Resolution imaging. Cartoons were prepared with BioRender.com software. This publication includes data generated at the UCSD IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased through NIH SIG grant (#S10 OD026929). H.X. was supported by Arthritis National Research Foundation (#1291101). This work was supported by NIH grants R01 DK100640 and R37 AI043477 to M.K., who is an American Cancer Research Society Professor and holds the Ben and Wanda Hildyard Chair for Mitochondrial and Metabolic Diseases, NIH R01 grant AI145314 to E.I.Z. and NIH grants R01 AI155869, R01 DK113592 and P01 HL152958 to H.M.H.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: H.X. and M.K. Methodology: H.X. and K.W. Investigation: H.X., M.O., K.W., J.S.B. and M.K. Resources: J.O., H.M.H., E.I.Z. and M.K. Formal Analysis: H.X. and M.O. Supervision: M.K. Funding acquisition: E.I.Z., H.M.H. and M.K. and Writing \u0026ndash; original draft: H.X. and M.K. Writing \u0026ndash; review and editing: E.I.Z., H.M.H. and M.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.K. is a founder of Elgia Pharmaceuticals and received research support from Gossamer Bio and Jansen Pharmaceuticals. M.K. holds an interest in PF-06835375, a Tfh depleting CXCR5 antibody. H.M.H. is a consultant for SOBI and Akros and received research funds from Takeda and Inapill. Other authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq and scRNA-seq data were deposited into the Gene Expression Omnibus (GEO) data repository (GSE264619 for bulk RNA-seq and GSE264620 for scRNA-seq). All data are available in the main text or the supplementary materials. Further requests for materials generated in this study should be directed to the corresponding author, Dr. Michael Karin ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoronzy, J.J. \u0026amp; Weyand, C.M. Immune aging and autoimmunity. Cell Mol Life Sci 69, 1615\u0026ndash;1623 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, Y., Liu, Q., Goronzy, J.J. \u0026amp; Weyand, C.M. Immune aging - A mechanism in autoimmune disease. Semin Immunol 69, 101814 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimada, K. \u003cem\u003eet al.\u003c/em\u003e Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36, 401\u0026ndash;414 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong, Z. \u003cem\u003eet al.\u003c/em\u003e New mitochondrial DNA synthesis enables NLRP3 inflammasome activation. 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Nat Immunol 24, 714\u0026ndash;728 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5194985/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5194985/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNLRP3 inflammasome activation depends on stress-induced production of oxidized mitochondrial DNA (Ox-mtDNA) fragments that enter the cytoplasm to bind NLRP3 and activate caspase-1. Along with pro-IL-1β processing, caspase-1 generates gasdermin D pores that result in circulatory mtDNA release. Elevated amounts of circulating cell-free (ccf)-mtDNA, which is likely to be oxidized, were documented in the elderly and patients with metabolic and autoimmune disorders and its intra-articular injection elicited arthritis in mice. Investigating whether ccf-mtDNA may promote autoimmunity, we found that induction of sustained Ox-mtDNA release triggered by a prototypical NLRP3 inflammasome activator elicited autoantibody production and glomerulonephritis in mice. Similar autoimmune responses, dependent on plasmacytoid dendritic cells (pDC) and T follicular helper cells (Tfh), were elicited by \u003cem\u003ein-vitro\u003c/em\u003e generated Ox-mtDNA but not by non-oxidized mtDNA. Although both mtDNA forms were internalized by pDC and induced interferon-a, only Ox-mtDNA stimulated autocrine IL-1β signaling that induced expression of immunoregulatory and co-stimulatory molecules, including IL-21, that enabled mouse and human pDC convert na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells into functional Tfh, supportive of autoantibody production. Highlighting pDC-generated IL-1β as an orchestrator of autoantibody production, these findings suggest that Ox-mtDNA could be a key participant in immune-aging and unravel new therapeutic opportunities.\u003c/p\u003e","manuscriptTitle":"Mitochondrial DNA oxidation propagates autoimmunity by enabling plasmacytoid dendritic cells induce Tfh differentiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-14 09:04:42","doi":"10.21203/rs.3.rs-5194985/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-immunology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ni","sideBox":"Learn more about [Nature Immunology](http://www.nature.com/ni/)","snPcode":"","submissionUrl":"","title":"Nature Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3d946645-bfa0-463d-8e3a-9f19663988bf","owner":[],"postedDate":"October 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38751087,"name":"Biological sciences/Immunology/Autoimmunity"},{"id":38751088,"name":"Biological sciences/Immunology/Signal transduction"}],"tags":[],"updatedAt":"2025-06-18T07:07:11+00:00","versionOfRecord":{"articleIdentity":"rs-5194985","link":"https://doi.org/10.1038/s41590-025-02179-7","journal":{"identity":"nature-immunology","isVorOnly":false,"title":"Nature Immunology"},"publishedOn":"2025-06-17 04:00:00","publishedOnDateReadable":"June 17th, 2025"},"versionCreatedAt":"2024-10-14 09:04:42","video":"","vorDoi":"10.1038/s41590-025-02179-7","vorDoiUrl":"https://doi.org/10.1038/s41590-025-02179-7","workflowStages":[]},"version":"v1","identity":"rs-5194985","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5194985","identity":"rs-5194985","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}