NADPH deficiency exacerbates lupus in murine models and in patients, but the mechanisms remain unknown. One hypothesis is that NADPH oxidase suppresses autoimmunity by facilitating dead cell clearance via LC3-associated phagocytosis (LAP). The absence of LAP reportedly causes an autoinflammatory syndrome in aged, non-autoimmune mice. Prior work implicated cytochrome b-245, beta polypeptide (CYBB), a component of the NADPH oxidase complex, and RUN and cysteine-rich domain containing Beclin 1 interacting protein (RUBICON), as requisite for LAP. To test the hypothesis that NADPH oxidase deficiency exacerbates lupus via a defect in LAP, we deleted Rubicon in the B6.Sle1.Yaa and MRL.Faslpr lupus mouse models. Under this hypothesis, RUBICON deficiency should phenocopy NADPH oxidase deficiency, as both work in the same pathway. However, we observed the opposite—RUBICON deficiency resulted in reduced mortality, renal disease, and autoantibody titers to RNA-associated autoantigens. Given that our data contradicts the published role for LAP in autoimmunity, we assessed whether CYBB and RUBICON are requisite for LAP. We found instead that LAP is not dependent on either of these two pathways. Our data thus reveal RUBICON as a novel regulator of SLE, possibly by a B cell-intrinsic mechanism, but do not support a role for LAP in lupus.
Rachael A. Gordon, Christina C. Giannouli, Chirag Raparia, Sheldon I. Bastacky, Anthony Marinov, William Hawse, Richard T. Cattley, Jeremy S. Tilstra, Allison M. Campbell, Kevin M. Nickerson, Anne Davidson, Mark J. Shlomchik
Mechanisms governing entry and exit of immune cells into, and out of, inflamed joints, remain poorly understood. We sought herein to identify the key molecular pathways regulating such migration. Using murine models of inflammation in conjunction with mice expressing a photoconvertible fluorescent protein we characterized the migration of cells from joints to draining lymph nodes (LN) and performed RNA-seq analysis on isolated cells, identifying genes associated with migration and retention. We further refined the gene list to those specific for joint inflammation. RNA-seq data revealed pathways and genes previously highlighted as characteristic of RA in patient studies, validating the methodology. Focusing on gene regulatory pathways associated with cell migration, adhesion and movement, we identified genes involved in the retention of immune cells in the inflamed joint, namely JAM-A, and identified a role for such molecules in T cell differentiation in vivo.Thus, using a combination of novel cell tracking approaches and murine models of inflammatory arthritis we have identified genes, pathways and anatomically specific tissue signatures regulating cell migration in a variety of inflamed sites. This unique skin and joint specific dataset will be an invaluable resource for the identification of novel therapeutic targets for arthritis and other inflammatory disorders.
Catriona T. Prendergast, Robert A. Benson, Hannah E. Scales, Caio S. Bonilha, John J. Cole, Iain McInnes, James M. Brewer, Paul Garside
BACKGROUND. Pathophysiology of type 1 diabetes (T1D) is illustrated by pancreatic islet infiltration of inflammatory lymphocytes, including CD8+ T-cells; however, the molecular factors mediating their recruitment remain unknown. We hypothesized that single-cell RNA-sequencing (scRNA-Seq) analysis of immune cell populations isolated from islets of non-obese diabetic (NOD) mice captured gene expression dynamics providing critical insight into autoimmune diabetes pathogenesis. METHODS. Pancreatic sections from human donors were investigated, including T1D subjects, auto-antibody-positive (aAb+), and non-diabetic controls. Immunohistochemistry was performed to assess islet hormones, and both novel and canonical immune-cell markers that were identified from state-of-the-art workflows after reanalyzing murine scRNA-seq datasets. RESULTS. Computational workflows identified Cadm1-mediated binding among the most significant intercellular interactions among all cell clusters as well as Cadm1 enrichment in macrophages and dendritic cells from pancreata of NOD mice. Immunostaining of human pancreata revealed an increased number of CADM1+GCG+ cells adjacent to CD8+ T-cells in sections from T1D and aAb+ donors compared to non-diabetic subjects. Numbers of CADM1+CD68+ peri-islet myeloid cells adjacent to CD8+ T-cells were also increased in pancreatic sections from both T1D and aAb+ donors compared to non-diabetic subjects. CONCLUSION. Increased detection of CADM1+ cells adjacent to CD8+ T-cells in pancreatic sections of T1D and aAb+ human subjects validated workflows, and indicates CADM1-mediated intercellular contact may facilitate islet infiltration of cytotoxic T lymphocytes and serve as a potential therapeutic target for preventing T1D pathogenesis. FUNDING. The Johns Hopkins All Children’s Foundation IRG Program, NSFC (82071326) and DFG (431549029–SFB 1451, EXC 2030–390661388, and 411422114-GRK 2550).
Chandan Sona, Yu-Te Yeh, Andreas Patsalos, Laszlo Halasz, Xin Yan, Natalia L. Kononenko, Laszlo Nagy, Matthew N. Poy
Systemic sclerosis (SSc) is a chronic multisystem orphan disease with a highly variable clinical course, significant mortality and a poorly understood complex pathogenesis. We identify an important role for a subpopulation of monocyte/macrophages characterized by surface expression of the scavenger receptor MARCO (MAcrophage Receptor with COllagenous structure) in chronic inflammation and fibrosis in SSc and in preclinical disease model. We show that MARCO+ monocytes and macrophages accumulate in lesional skin and lung in SSc patients and in the bleomycin-induced mouse model of SSc in topographic proximity to activated myofibroblasts. Short-term treatment of mice with a novel nanoparticle composed of a carboxylated FDA-approved biodegradable polymer, poly(lactic-co-glycolic) acid (PLG), which modulates activation and trafficking of MARCO+ inflammatory monocytes, markedly attenuated bleomycin-induced skin and lung inflammation and fibrosis. Mechanistically, in isolated cells in culture PLG nanoparticles inhibited TGF-β-dependent fibrotic responses in vitro. Thus MARCO+ monocytes are potent effector cells of skin and lung fibrosis, and can be therapeutically targeted in SSc using PLG nanoparticles.
Dan Xu, Swati Bhattacharyya, Wenxia Wang, Igal Ifergan, Ming-Yi Alice Chiang Wong, Daniele Procissi, Anjana Yeldandi, Swarna Bale, Roberta G. Marangoni, Craig Horbinski, Stephen D. Miller, John Varga
Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10–12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.
Sarah K. Popp, Federica Vecchio, Debra J. Brown, Riho Fukuda, Yuri Suzuki, Yuma Takeda, Rikako Wakamatsu, Mahalakshmi A. Sarma, Jessica Garrett, Anna Giovenzana, Emanuele Bosi, Antony R.A. Lafferty, Karen J. Brown, Elizabeth E. Gardiner, Lucy A. Coupland, Helen E. Thomas, Beng H. Chong, Christopher R. Parish, Manuela Battaglia, Alessandra Petrelli, Charmaine J. Simeonovic
BACKGROUND. Vaccine-elicited adaptive immunity is a prerequisite for control of SARS-CoV-2 infection. Multiple sclerosis (MS) disease-modifying therapies (DMTs) differentially target humoral and cellular immunity. A comprehensive comparison of MS DMTs on SARS-CoV-2 vaccine-specific immunity is needed, including quantitative and functional B and T cell responses. METHODS. Spike-specific antibody and T cell responses were measured before and following SARS-CoV-2 vaccination in a cohort of 80 subjects, including healthy controls and MS patients in six DMT groups: untreated, glatiramer acetate (GA), dimethyl fumarate (DMF), natalizumab (NTZ), sphingosine-1-phosphate (S1P) receptor modulators, and anti-CD20 monoclonal antibodies. Anti-spike antibody responses were quantified by Luminex assay, high-resolution spike epitope reactivity was mapped by VirScan, and pseudovirus neutralization was assessed. Spike-specific CD4+ and CD8+ T cell responses were characterized by activation-induced marker (AIM) expression, cytokine production, and tetramer analysis. RESULTS. Anti-spike IgG levels were similar between healthy controls, untreated MS, GA, DMF, and NTZ patients, but were significantly reduced in anti-CD20 and S1P-treated patients. Anti-spike seropositivity in anti-CD20 patients was significantly correlated with CD19+ B cell levels and inversely correlated with cumulative treatment duration. Spike epitope reactivity and pseudovirus neutralization was reduced in anti-CD20 and S1P patients, directly correlating with reduced spike receptor binding domain (RBD) IgG levels. Spike-specific CD4+ and CD8+ T cell reactivity remained robust across all groups except in S1P-treated patients in whom post-vaccine CD4+ T cell responses were attenuated. CONCLUSIONS. These findings from a large MS cohort exposed to a wide spectrum of MS immunotherapies have important implications for treatment-specific COVID-19 clinical guidelines. FUNDING. This work was supported by grants from the NIH 1K08NS107619 (JJS), NMSS TA- 1903-33713 (JJS), K08NS096117 (MRW), Westridge Foundation (MRW), Chan Zuckerberg Biohub (JLD), R01AI159260 (JAH), R01NS092835 (SSZ), R01AI131624 (SSZ), R21NS108159 (SSZ), NMSS RG1701-26628 (SSZ), and the Maisin Foundation (SSZ).
Joseph J. Sabatino Jr, Kristen Mittl, William M. Rowles, Kira McPolin, Jayant V. Rajan, Matthew T. Laurie, Colin R. Zamecnik, Ravi Dandekar, Bonny D. Alvarenga, Rita P. Loudermilk, Chloe Gerungan, Collin M. Spencer, Sharon A. Sagan, Danillo G. Augusto, Jessa R. Alexander, Joseph L. DeRisi, Jill A. Hollenbach, Michael R. Wilson, Scott S. Zamvil, Riley Bove
Stromal cells are emerging as key drivers of autoimmunity, in part by producing inflammatory chemokines that orchestrate inflammation. Chemokine expression is regulated transcriptionally but also through post-transcriptional mechanisms, the specific drivers of which are still incompletely defined. CCL2 (MCP1) is a multifunctional chemokine that drives myeloid cell recruitment. During experimental autoimmune encephalomyelitis (EAE), an IL-17-driven model of multiple sclerosis, CCL2 produced by lymph node (LN) stromal cells is essential for immunopathology. Here, we show that Ccl2 mRNA upregulation in human stromal fibroblasts in response to IL-17 requires the RNA binding protein (RBP) insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2, IMP2), which is expressed almost exclusively in non-hematopoietic cells. IMP2 binds directly to CCL2 mRNA, markedly extending its transcript half-life and thus required for efficient CCL2 secretion. Consistent with this, Imp2−/− mice showed reduced CCL2 production in LN during EAE, causing impairments in monocyte recruitment and Th17 cell polarization. Imp2-/- mice were fully protected from CNS inflammation. Moreover, deletion of IMP2 after EAE onset was sufficient to mitigate disease severity. These data show that posttranscriptional control of Ccl2 in stromal cells by IMP2 is required to permit IL-17-driven progression of EAE pathogenesis.
Rami Bechara, Nilesh Amatya, Saikat Majumder, Chunsheng Zhou, Yang Li, Qixing Liu, Mandy J. McGeachy, Sarah L. Gaffen
Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals (“public” chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαβ sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells (“expanded”), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or “private,” TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRβ junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these “innate-like” TCRs differ from previously described immunodominant TCRβ chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.
Peter S. Linsley, Fariba Barahmand-pour-Whitman, Elisa Balmas, Hannah A. DeBerg, Kaitlin J. Flynn, Alex K. Hu, Mario G. Rosasco, Janice Chen, Colin O’Rourke, Elisavet Serti, Vivian H. Gersuk, Keshav Motwani, Howard R. Seay, Todd M. Brusko, William W. Kwok, Cate Speake, Carla J. Greenbaum, Gerald T. Nepom, Karen Cerosaletti
Th17 cells are involved in rheumatoid arthritis (RA) pathogenesis. Our previous studies have revealed that transcription factor Yin Yang 1 (YY1) plays an important role in the pathogenic mechanisms of RA. However, whether YY1 has any role in Th17 cell pathogenicity and what molecular regulatory mechanism is involved remain poorly understood. Here, we found the proportion of pathogenic Th17 (pTh17) cells was significantly higher in RA than in control individuals and showed a potential relationship with YY1 expression. In addition, we also observed YY1 expression was increased in pTh17, and the pTh17 differentiation was hampered by YY1 knockdown. Consistently, knockdown of YY1 decreased the proportion of pTh17 cells and attenuated joint inflammation in collagen-induced arthritis mice. Mechanistically, YY1 could regulate the pathogenicity of Th17 cells through binding to the promoter region of transcription factor T-bet and interacting with T-bet protein. This function of YY1 for promoting pTh17 differentiation was specific to Th17 cells and not to Th1 cells. Moreover, we found miR-124-3p negatively correlated with YY1 in RA patients, and it could bind to 3′-UTR regions of YY1 to inhibit the posttranscriptional translation of YY1. Altogether, these findings indicate YY1 regulation by miR-124-3p could specifically promote Th17 cell pathogenicity in part through interaction with T-bet, and these findings present promising therapeutic targets in RA.
Jinpiao Lin, Jifeng Tang, Junyu Lin, Yujue He, Ziqing Yu, Renquan Jiang, Bin Yang, Qishui Ou
Foxp3+ Tregs are potent immunosuppressive CD4+ T cells that are critical to maintain immune quiescence and prevent autoimmunity. Both the generation and maintenance of Foxp3+ Tregs depend on the cytokine IL-2. Hence, the expression of the IL-2 receptor α-chain (CD25) is not only considered a specific marker, but also a nonredundant requirement for Tregs. Here, we report that Foxp3+ Tregs in the small intestine (SI) epithelium, a critical barrier tissue, are exempt from such an IL-2 requirement, since they had dramatically downregulated CD25 expression, showed minimal STAT5 phosphorylation ex vivo, and were unable to respond to IL-2 in vitro. Nonetheless, SI epithelial Tregs survived and were present at the same frequency as in other lymphoid organs, and they retained potent suppressor function that was associated with high levels of CTLA-4 expression and the production of copious amounts of IL-10. Moreover, adoptive transfer experiments of Foxp3+ Tregs revealed that such IL-2–independent survival and effector functions were imposed by the SI epithelial tissue, suggesting that tissue adaptation is a mechanism that tailors the effector function and survival requirements of Foxp3+ Tregs specific to the tissue environment.
Praveen Prakhar, Jaylene Alvarez-DelValle, Hilary Keller, Assiatu Crossman, Xuguang Tai, Yoo Kyoung Park, Jung-Hyun Park
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