Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a dramatic sex bias, affecting 9-times more women than men. Activation of toll-like receptor 7 (TLR7) by self-RNA is a central pathogenic process leading to aberrant production of type-I interferon (IFN) in SLE, but the specific RNA molecules that serve as TLR7 ligands have not been defined. We therefore sought to identify female-specific endogenous RNAs containing canonical TLR7 stimulatory motifs. By leveraging gene expression data and the known sequence specificity of TLR7, we identified the female-specific X-inactive specific transcript (XIST) long non-coding RNA as a uniquely rich source of TLR7 ligands in SLE. XIST RNA stimulated IFNα production by plasmacytoid DCs in a TLR7-dependent manner, and deletion of XIST diminished the ability of whole cellular RNA to activate TLR7. XIST levels were elevated in blood leukocytes from female SLE patients compared to controls, correlated positively with disease activity and the IFN signature, and were enriched in extracellular vesicles released from dying cells in vitro. Importantly, XIST was not IFN-inducible, suggesting that XIST is a driver, rather than a consequence of IFN in SLE. Our work suggests a novel role for XIST RNA as a female-specific danger signal underlying the sex bias in SLE.
Jonathan D. Crawford, Hong Wang, Daniela Trejo-Zambrano, Raffaello Cimbro, C. Conover Talbot Jr., Mekha A. Thomas, Ashley M. Curran, Alexander A. Girgis, John T. Schroeder, Andrea Fava, Daniel W. Goldman, Michelle Petri, Antony Rosen, Brendan Antiochos, Erika Darrah
Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation, which is one of the vital reasons for the development of autoimmune diseases. However, the underlying mechanism is still unclear. Here we explore the function of RFX1 in macrophage polarization by constructing colitis and lupus-like mouse models. Both in vivo and in vitro experiments confirmed that RFX1 can promote M1 and inhibit M2 macrophage polarization. Besides, we also found that RFX1 promoted DNA demethylation of macrophage polarization-related genes by increasing APOBEC3A/Apobec3 expression. Noteworthily, we identified a potential RFX1 inhibitor, adenosine diphosphate (ADP), providing a potential strategy for treating autoimmune diseases.
Shuang Yang, Pei Du, Haobo Cui, Meiling Zheng, Wei He, Xiaofei Gao, Zhi Hu, Sujie Jia, Qianjin Lu, Ming Zhao
Many autoimmune diseases (AIDs) are characterized by persistence of autoreactive B cell responses which is often directly implicated in disease pathogenesis. How and why these cells are generated or how they are maintained for years is largely unknown. Rheumatoid arthritis is among the most common AIDs and characterized by autoantibodies recognizing proteins with post-translational modifications (PTMs). This PTM-directed, autoreactive B cell compartment is ill defined. Here, we visualized the B cell response against the three main types of PTM antigens implicated in RA by spectral flow cytometry. Our results showed extensive cross-reactivity of PTM-directed B cells against all three PTM antigens (citrulline, homocitrulline and acetyllysine). Unsupervised clustering revealed several distinct memory B cell (mBC) populations. PTM-directed cells clustered with the most recently activated, class-switched mBC phenotype, expressing high CD80, low CD24 and low CD21. Notably, patients also harbored large fractions of PTM-directed plasmablasts (PB). Both PTM-directed mBC and PB showed high expression of CXCR3, a receptor for chemokines abundantly present in arthritic joints. Together, our data provide detailed insight into the biology of B cell autoreactivity and its remarkable, seemingly exhaustless persistence in a prominent human AID.
Sanne Reijm, Joanneke C. Kwekkeboom, Nienke J. Blomberg, Jolien Suurmond, Diane Van der Woude, Rene E.M. Toes, Hans Ulrich Scherer
OBJECTIVES. Sjögren's Disease (SjD) is a chronic and systemic autoimmune disease characterized by lymphocytic infiltration and the development of dry eyes and dry mouth resulting from the secretory dysfunction of the exocrine glands. SARS-CoV-2 may trigger the development or progression of autoimmune diseases, as evidenced by increased autoantibodies in patients and the presentation of cardinal symptoms of SjD. The objective of the study was to determine whether SARS-CoV-2 induces the signature clinical symptoms of SjD. METHODS. The ACE2-transgenic mice were infected with SARS-CoV-2; SjD profiling was conducted. COVID-19 patients' sera were examined to detect the presence of autoantibodies. Clinical evaluations of convalescent COVID-19 subjects, including minor salivary gland (MSG) biopsies, were collected. Lastly, monoclonal antibodies generated from single B cells of patients were interrogated for ACE2/spike inhibition and nuclear antigens. RESULTS. Mice infected with the virus showed a decreased saliva flow rate, elevated antinuclear antibodies (ANAs) with anti-SSB/La, and lymphocyte infiltration in the lacrimal and salivary glands. Sera of COVID-19 patients showed an increase in ANA, anti-SSA/Ro52, and anti-SSB/La. The male patients showed elevated levels of anti-SSA/Ro52 compared to female patients, and female patients had more diverse ANA patterns. Minor salivary gland biopsies of convalescent COVID-19 subjects showed focal lymphocytic infiltrates in four of six subjects, and 2 of 6 subjects had focus scores >2. Lastly, we found that monoclonal antibodies produced in recovered patients can block ACE2/spike interaction and recognize nuclear antigens. CONCLUSION. Overall, our study shows a direct association between SARS-CoV-2 and SjD. Hallmark features of SjD salivary glands were histologically indistinguishable from convalescent COVID-19 subjects.The results potentially implicate that SARS-CoV-2 could be an environmental trigger for SjD.
Yiran Shen, Alexandria Voigt, Laura Goranova, Mehdi A. Abed, David E. Kleiner, Jose O. Maldonado, Margaret Beach, Eileen Pelayo, John A. Chiorini, William F. Craft, David A. Ostrov, Vijay Ramiya, Sukesh Sukumaran, Ashley N. Brown, Kaley C. Hanrahan, Apichai Tuanyok, Blake M. Warner, Cuong Q. Nguyen
The endosomal Toll-like receptor 7 (TLR7) is a major driver of murine and human systemic lupus erythematosus (SLE). The role of TLR7 in lupus pathogenesis is enhanced when the regulatory role of TLR9 is absent. TLR7 signaling in plasmacytoid DCs (pDC) is generally thought to be a major driver of the IFN response and disease pathology; however, the cell types in which TLR7 acts to mediate disease have not been distinguished. To address this, we selectively deleted TLR7 in either CD11c+ cells or CD19+ cells; using a TLR7-floxed allele, we created on the lupus-prone MRL/lpr background, along with a BM chimera strategy. Unexpectedly, TLR7 deficiency in CD11c+ cells had no impact on disease, while TLR7 deficiency in CD19+ B cells yielded mild suppression of proteinuria and a trend toward reduced glomerular disease. However, in TLR9-deficient MRL/lpr mice with accelerated SLE, B cell–specific TLR7 deficiency greatly improved disease. These results support revision of the mechanism by which TLR7 drives lupus and highlight a cis regulatory interaction between the protective TLR9 and the pathogenic TLR7 within the B cell compartment. They suggest B cell–directed, dual TLR7 antagonism/TLR9 agonism or dual TLR7/9 antagonism as a potential future therapeutic strategy to treat SLE.
Haylee A. Cosgrove, Sebastien Gingras, Minjung Kim, Sheldon Bastacky, Jeremy S. Tilstra, Mark J. Shlomchik
Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities. Cell composition, signaling, epigenetics, and proteomics were evaluated by mass cytometry; droplet-based single-cell transcriptomics and proteomics; and bead-based multiplex soluble mediator levels in plasma. We observed altered whole blood frequencies and enhanced activity in CD8+ T cells, B cells, monocytes, and DCs in Black patients with more active disease. Epigenetic modifications in CD8+ T cells (H3K27ac) could distinguish disease activity level in Black patients and differentiate Black from White patient samples. TLR3/4/7/8/9-related gene expression was elevated in immune cells from Black patients with SLE, and TLR7/8/9 and IFN-α phospho-signaling and cytokine responses were heightened even in immune cells from healthy Black control patients compared with White individuals. TLR stimulation of healthy immune cells recapitulated the ancestry-associated SLE immunophenotypes. This multiomic resource defines ancestry-associated immune phenotypes that differ between Black and White patients with SLE, which may influence the course and severity of SLE and other diseases.
Samantha Slight-Webb, Kevin Thomas, Miles Smith, Catriona A. Wagner, Susan Macwana, Aleksandra Bylinska, Michele Donato, Mai Dvorak, Sarah E. Chang, Alex Kuo, Peggie Cheung, Laurynas Kalesinskas, Ananthakrishnan Ganesan, Denis Dermadi, Carla J. Guthridge, Wade DeJager, Christian Wright, Mariko H. Foecke, Joan T. Merrill, Eliza Chakravarty, Cristina Arriens, Holden T. Maecker, Purvesh Khatri, Paul J. Utz, Judith A. James, Joel M. Guthridge
BACKGROUND While B cell depletion is associated with attenuated antibody responses to SARS-CoV-2 mRNA vaccination, responses vary among individuals. Thus, elucidating the factors that affect immune responses after repeated vaccination is an important clinical need.METHODS We evaluated the quality and magnitude of the T cell, B cell, antibody, and cytokine responses to a third dose of BNT162b2 or mRNA-1273 mRNA vaccine in patients with B cell depletion.RESULTS In contrast with control individuals (n = 10), most patients on anti-CD20 therapy (n = 48) did not demonstrate an increase in spike-specific B cells or antibodies after a third dose of vaccine. A third vaccine elicited significantly increased frequencies of spike-specific non-naive T cells. A small subset of B cell–depleted individuals effectively produced spike-specific antibodies, and logistic regression models identified time since last anti-CD20 treatment and lower cumulative exposure to anti-CD20 mAbs as predictors of those having a serologic response. B cell–depleted patients who mounted an antibody response to 3 vaccine doses had persistent humoral immunity 6 months later.CONCLUSION These results demonstrate that serial vaccination strategies can be effective for a subset of B cell–depleted patients.FUNDING The NIH (R25 NS079193, P01 AI073748, U24 AI11867, R01 AI22220, UM 1HG009390, P01 AI039671, P50 CA121974, R01 CA227473, U01CA260507, 75N93019C00065, K24 AG042489), NIH HIPC Consortium (U19 AI089992), the National Multiple Sclerosis Society (CA 1061-A-18, RG-1802-30153), the Nancy Taylor Foundation for Chronic Diseases, Erase MS, and the Claude D. Pepper Older Americans Independence Center at Yale (P30 AG21342).
Hiromitsu Asashima, Dongjoo Kim, Kaicheng Wang, Nikhil Lele, Nicholas C. Buitrago-Pocasangre, Rachel Lutz, Isabella Cruz, Khadir Raddassi, William E. Ruff, Michael K. Racke, JoDell E. Wilson, Tara S. Givens, Alba Grifoni, Daniela Weiskopf, Alessandro Sette, Steven H. Kleinstein, Ruth R. Montgomery, Albert C. Shaw, Fangyong Li, Rong Fan, David A. Hafler, Mary M. Tomayko, Erin E. Longbrake
IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with unclear pathogenesis. We performed single-cell RNA-seq and surface proteome analyses on 61,379 PBMCs from 9 treatment-naïve IgG4-RD patients and 7 age- and sex-matched healthy controls. Integrative analyses were performed for altered gene expression in IgG4-RD, and flow cytometry and immunofluorescence were used for validation. We observed expansion of plasmablasts with enhanced protein processing and activation, which correlated with number of involved organs in IgG4-RD. Increased proportions of CD4+ cytotoxic T lymphocytes (CTLs), CD8+ CTLs-GNLY (granulysin) and γδT cells with enhanced chemotaxis and cytotoxicity but with suppressed inhibitory receptors characterize IgG4-RD. Prominent infiltration of lymphocytes with distinct compositions were found in different organs of IgG4-RD patients. Transcription factors (TFs) including PRDM1/XBP1 and RUNX3 were upregulated in IgG4-RD, promoting the differentiation of plasmablasts and CTLs, respectively. Monocytes in IgG4-RD have stronger expression of genes related to cell adhesion and chemotaxis, which may give rise to profibrotic macrophages in lesions. The gene activation pattern in peripheral immune cells indicated activation of multiple interaction pathways between cell types, in part through chemokines or growth factors and their receptors. Specific upregulation of TFs and expansion of plasmablasts and CTLs may be involved in the pathogenesis of IgG4-RD, and each of these populations are candidate targets for therapeutic interventions in this disease.
Chenyang Lu, Shasha Li, Pingying Qing, Qiuping Zhang, Xing Ji, Zhigang Tang, Chunyan Chen, Tong Wu, Yidan Hu, Yi Zhao, Xiaohui Zhang, Qi He, David A. Fox, Chunyu Tan, Yubin Luo, Yi Liu
Cutaneous lupus erythematosus (CLE) is a disfiguring autoimmune skin disease characterized by an inflammatory infiltrate rich in T cells, which are strongly implicated in tissue damage. How these cells adapt to the skin environment and promote tissue inflammation and damage is not known. In lupus nephritis, we have previously identified an inflammatory gene program in kidney-infiltrating T cells that is dependent on hypoxia-inducible factor-1 (HIF-1), a transcription factor critical for the cellular and developmental response to hypoxia as well as inflammation-associated signals. In our current studies using a mouse model of lupus skin disease, we find that skin-infiltrating CD4+ and CD8+ T cells also express high levels of HIF-1. Skin-infiltrating T cells demonstrate a strong cytotoxic signature at the transcript and protein level, and HIF-1 inhibition abrogates skin and systemic disease in association with decreased T cell cytotoxic activity. We also demonstrate in human CLE tissue that the T cell rich inflammatory infiltrate exhibits increased amounts of HIF-1 and a cytotoxic signature. Granzyme B-expressing T cells are concentrated at sites of skin tissue damage in CLE, suggesting relevance of this pathway to human disease.
Alicia J. Little, Ping-Min Chen, Matthew D. Vesely, Rahanna N. Khan, Jacob Fiedler, James Garritano, Fahrisa M. Islam, Jennifer M. McNiff, Joseph E. Craft
The proportions and phenotypes of immune cell subsets in peripheral blood undergo continual and dramatic remodeling throughout the human lifespan, which complicates efforts to identify disease-associated immune signatures in type 1 diabetes (T1D). We conducted cross-sectional flow cytometric immune profiling on peripheral blood from 826 individuals [stage 3 T1D (n=240), their first-degree relatives (n=310), those with >2 islet autoantibodies (n=24), and autoantibody negative healthy controls (n=252)]. We constructed an immune-age predictive model in healthy participants and observed accelerated immune aging in the T1D cohort (p<0.001). We used generalized additive models for location, shape, and scale (GAMLSS) to obtain age-corrected data for 192 flow cytometry and complete blood count readouts, which can be visualized in our interactive portal (ImmScape; https://ufdiabetes.shinyapps.io/ImmScape/); 46 parameters were significantly associated with age only, 25 with T1D only, and 23 with both age and T1D. Phenotypes associated with accelerated immunological aging in T1D included increased CXCR3+ and PD-1+ frequencies in naïve and memory T cell subsets, despite reduced PD-1 expression levels (MFI) on memory T cells. Phenotypes associated with T1D after age-correction were predictive of T1D status (AUROC=82.3%). Our findings demonstrate advanced immune aging in T1D and highlight disease-associated phenotypes for biomarker monitoring and therapeutic interventions.
Melanie R. Shapiro, Xiaoru Dong, Daniel J. Perry, James M. McNichols, Puchong Thirawatananond, Amanda L. Posgai, Leeana D. Peters, Keshav Motwani, Richard S. Musca, Andrew Muir, Patrick Concannon, Laura M. Jacobsen, Clayton E. Mathews, Clive H. Wasserfall, Michael J. Haller, Desmond A. Schatz, Mark A. Atkinson, Maigan A. Brusko, Rhonda Bacher, Todd M. Brusko
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