Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease that affects millions of people worldwide. Disease pathogenesis involves the synovium, a thin membrane encapsulating the ...joint, mostly composed of cells resembling fibroblasts and macrophages. Th synovium proliferates and forms a pannus of inflamed tissue that persistently recruits immune cells, degrades collagen, and activates bone-resorbing osteoclasts. The infiltrating immune cells interact with the stromal tissue cells, leading to synovial hypertrophy, and ultimately to joint destruction. The cellular composition and molecular functions of stromal fibroblast cells in the synovium are relevant to understanding disease progression. With small quantities of patient tissues, the latest technologies allow us to assay transcriptomics genome-wide at high resolution. Transcriptomics analysis helps us determine the distinct functions of cellular populations in the synovium and reveal gene regulatory factors activated by cytokines elevated in the disease state. Further understanding of basic biology in these cells may reveal fibroblast-specific targets for the treatment of inflammatory diseases mediated by fibroblasts.Here, we use computational analyses of bulk tissue and single cell transcriptomic data, and also genetics data, to advance understanding of mechanisms underlying RA. We integrate genome-wide association study (GWAS) results with gene expression data to understand which cell types express the genes that are associated with risk of developing disease. Next, we analyze transcriptomics data collected from synovial fibroblasts obtained from fresh tissue discarded during joint replacement surgery. Single-cell gene expression profiles reveals the cellular population structure of fibroblasts in the synovium. Within the sublining layer, RA patients have an overabundant fibroblast population negative for CD34 and positive for CD90. The heterogenous synovial cellular populations produce a mixture of signaling molecules that synergistically influence the positive feedback loop between local tissue cells and infiltrating immune cells. To understand the regulation of the synergistic response to tumor necrosis factor (TNF) and interleukin 17 (IL-17A), we use high resolution transcriptomics with time series, dose response, and gene silencing. Our analyses predict and our experiments validate that CUX1 is a key mediator for fibroblast expression of CXCL1, CXCL2, CXCL3. Understanding regulation of transcriptional response to inflammatory factors in synovial fibroblasts has implications for RA as well as other diseases involving chronic inflammation mediated by fibroblasts in connective tissues such as psoriasis. Our approaches for interrogating RA could be applied to these diseases.
Immune checkpoint inhibitor (ICI) therapy has revolutionized oncology, but treatments are limited by immune-related adverse events, including checkpoint inhibitor colitis (irColitis). Little is ...understood about the pathogenic mechanisms driving irColitis, which does not readily occur in model organisms, such as mice. To define molecular drivers of irColitis, we used single-cell multi-omics to profile approximately 300,000 cells from the colon mucosa and blood of 13 patients with cancer who developed irColitis (nine on anti-PD-1 or anti-CTLA-4 monotherapy and four on dual ICI therapy; most patients had skin or lung cancer), eight controls on ICI therapy and eight healthy controls. Patients with irColitis showed expanded mucosal Tregs, ITGAE
CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs and recirculating ITGB2
CD8 T cells. Cytotoxic GNLY
CD4 T cells, recirculating ITGB2
CD8 T cells and endothelial cells expressing hypoxia gene programs were further expanded in colitis associated with anti-PD-1/CTLA-4 therapy compared to anti-PD-1 therapy. Luminal epithelial cells in patients with irColitis expressed PCSK9, PD-L1 and interferon-induced signatures associated with apoptosis, increased cell turnover and malabsorption. Together, these data suggest roles for circulating T cells and epithelial-immune crosstalk critical to PD-1/CTLA-4-dependent tolerance and barrier function and identify potential therapeutic targets for irColitis.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive condition resulting from structural and/or functional defects of the axoneme in motile cilia and sperm flagella. The great majority of ...mutations identified so far involve genes whose defects result in dynein-arm anomalies. By contrast, PCD due to CC/RS defects (those in the central complex CC and radial spokes RSs), which might be difficult to diagnose, remains mostly unexplained. We identified non-ambiguous RSPH3 mutations in 5 of 48 independent families affected by CC/RS defects. RSPH3, whose ortholog in the flagellated alga Chlamydomonas reinhardtii encodes a RS-stalk protein, is mainly expressed in respiratory and testicular cells. Its protein product, which localizes within the cilia of respiratory epithelial cells, was undetectable in airway cells from an individual with RSPH3 mutations and in whom RSPH23 (a RS-neck protein) and RSPH1 and RSPH4A (RS-head proteins) were found to be still present within cilia. In the case of RSPH3 mutations, high-speed-videomicroscopy analyses revealed the coexistence of immotile cilia and motile cilia with movements of reduced amplitude. A striking feature of the ultrastructural phenotype associated with RSPH3 mutations is the near absence of detectable RSs in all cilia in combination with a variable proportion of cilia with CC defects. Overall, this study shows that RSPH3 mutations contribute to disease in more than 10% of PCD-affected individuals with CC/RS defects, thereby allowing an accurate diagnosis to be made in such cases. It also unveils the key role of RSPH3 in the proper building of RSs and the CC in humans.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial ...inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP
). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP
dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP
pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.
On average, the Peruvian population is among the shortest in the
world
1
. Here we show
that Native American ancestry is associated with reduced height in an ethnically
diverse group of Peruvians, ...and identify a novel, population-specific, missense
variant in
FBN1
(E1297G) that is significantly associated with
lower height. Each copy of the minor allele (frequency=4.7%) reduces height by
2.2 cm (4.4 cm in homozygous individuals). This is the largest effect size known
for a common height-associated variant.
FBN1
encodes the
extracellular matrix protein fibrillin-1, a major structural component of
microfibrils. We observed less densely packed fibrillin-1-rich microfibrils with
irregular edges in the skin of individuals homozygous for G1297 compared to
individuals homozygous for E1297. Moreover, we show that E1297G locus is under
positive selection in non-African populations, and the E1297 variant shows
subtle evidence of positive selection within the Peruvian population
specifically. This variant is also significantly more frequent in coastal
Peruvian populations than in populations from the Andes or the Amazon,
suggesting that short stature might be the result of adaptation to factors
associated with the coastal environment in Peru.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Asthma is a chronic disease most commonly associated with allergy and type 2 inflammation. However, the mechanisms that link airway inflammation to the structural changes that define asthma are ...incompletely understood. Using a human model of allergen-induced asthma exacerbation, we compared the lower airway mucosa in allergic asthmatics and allergic non-asthmatic controls using single-cell RNA sequencing. In response to allergen, the asthmatic airway epithelium was highly dynamic and up-regulated genes involved in matrix degradation, mucus metaplasia, and glycolysis while failing to induce injury-repair and antioxidant pathways observed in controls.
-expressing pathogenic T
2 cells were specific to asthmatic airways and were only observed after allergen challenge. Additionally, conventional type 2 dendritic cells (DC2 that express
) and
-expressing monocyte-derived cells (MCs) were uniquely enriched in asthmatics after allergen, with up-regulation of genes that sustain type 2 inflammation and promote pathologic airway remodeling. In contrast, allergic controls were enriched for macrophage-like MCs that up-regulated tissue repair programs after allergen challenge, suggesting that these populations may protect against asthmatic airway remodeling. Cellular interaction analyses revealed a T
2-mononuclear phagocyte-basal cell interactome unique to asthmatics. These pathogenic cellular circuits were characterized by type 2 programming of immune and structural cells and additional pathways that may sustain and amplify type 2 signals, including TNF family signaling, altered cellular metabolism, failure to engage antioxidant responses, and loss of growth factor signaling. Our findings therefore suggest that pathogenic effector circuits and the absence of proresolution programs drive structural airway disease in response to type 2 inflammation.
Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic ...immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.
Abstract Introduction: Immune checkpoint inhibitor (ICI)-related myocarditis (irMyocarditis) is a potentially lethal complication of ICI use that is characterized by the presence of clonally expanded ...T cells in the heart. The antigens recognized by these expanded intracardiac T-cell clones and their relationships to T-cell clones in the blood and tumors of irMyocarditis patients are poorly understood. Methods: Paired single-cell RNA sequencing (scRNA-seq) and T-cell receptor (TCR) sequencing was performed on heart tissue (n = 13) and peripheral blood from patients with irMyocarditis (n = 25) and ICI-treated controls (n = 28) using the 10X Chromium (10X Genomics) system. TCR-β chain sequencing was performed (Adaptive Biotechnologies) on four autopsy cases of patients with available scRNA-seq data and irMyocarditis heart, tumor, and histologically normal tissue. An established high-throughput protocol was used to screen expanded intracardiac TCRs against candidate antigens (Oliveira G, et al. Nature 2021). Briefly, full-length TCRs were cloned, transduced into donor T cells, and screened against autologous or major histocompatibility complex (MHC) Class I-matched antigen presenting cell lines pulsed with peptide pools that covered the full length of the ⍺-myosin, troponin-I, and troponin-T proteins alongside pools of common viral antigens and appropriate controls. Results: scRNA-seq data showed that TCRs shared between heart and blood are predominantly found in circulating CD8 T cell subsets as compared to circulating CD4 T cell subsets. The gene expression patterns of these shared T-cell clones in circulation appear distinct in fatal and non-fatal irMyocarditis patients, with shared T-cell clones in fatal cases expressing cycling markers (MKI67, STMN1) and the chemokine receptor CXCR3. TCR-β chain sequences most enriched in irMyocarditis tissue relative to control tissues were distinct from those enriched in tumor tissues, and their full-length TCR sequences could be recovered from scRNA-seq data. In total, 52 cardiac-expanded TCRs across eight donors were screened against candidate antigens. None of the screened TCRs recognized the putative cardiac autoantigens. Conclusions: T-cell clones expanded in irMyocarditis are shared in circulation, where the gene expression of these clones may help to distinguish fatal from non-fatal irMyocarditis. TCRs enriched in irMyocarditis appear to be largely distinct from those enriched in tumor and likely recognize currently unknown cardiac autoantigens. Citation Format: Steven Blum, Daniel A. Zlotoff, Neal P. Smith, Isabela J. Kernin, Swetha Ramesh, Giacomo Oliveira, Leyre Zubiri, Joshua Caplin, Nandini Samanta, Sidney Martin, Mike Wang, Alice Tirard, Pritha Sen, Yuhui Song, Katherine Xu, Jaimie L. Barth, Kamil Slowikowski, Mazen Nasrallah, Jessica Tantivit, Kasidet Manakongtreecheep, Benjamin Y. Arnold, John McGuire, Alexander B. Afeyan, Christopher J. Pinto, Daniel McLoughlin, Monica Jackson, PuiYee Chan, Aleigha Lawless, William A. Michaud, Tatyana Sharova, Linda T. Nieman, Justin F. Gainor, Dejan Juric, Mari Mino-Kenudsen, Ryan J. Sullivn, Genevieve M. Boland, James R. Stone, Catherine J. Wu, Molly F. Thomas, Tomas G. Neilan, Kerry L. Reynolds, Alexandra-Chloé Villani. T-cell responses across heart, blood, and tumor in patients with immune checkpoint inhibitor-related myocarditis (irMyocarditis) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7474.
BackgroundAntibodies targeting immune checkpoint inhibitors (ICIs) CTLA-4 and PD-1/PD-L1 have revolutionized the treatment of metastatic solid tumors. However, their use is limited by a high ...incidence of immune-related adverse events. The colon is a frequent target of this immune attack seen in up to 45% of patients on dual PD-1 and CTLA-4 blockade. We leveraged a multi-omics strategy to further our understanding of the cellular and molecular drivers giving rise to ICI-associated colitis and nominate treatment solutions that spare anti-tumor immune response.MethodsWe collected paired endoscopic colon mucosal biopsies and blood specimens from 13 irColitis patients, 8 healthy individuals, and 8 controls on ICIs, and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis.ResultsAnalyses of over 300,000 single epithelial, mesenchymal, and immune single cells revealed that patients with irColitis showed expanded mucosal Tregs, ITGAEHi CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs. We also identified two circulating ITGB2+ CD8 T cell populations associated with irColitis – a CX3CR1Hi population predicted to be intravascular and an EOMESHi KLRG1Hi population. Comparison of dual anti-PD-1/CTLA-4 versus anti-PD1 monotherapy revealed expansion of those two circulating ITGB2Hi CD8 T cell populations, as well as a cytotoxic GNLYHi CD4 T cell subset, and endothelial cells associated with hypoxia gene programs. Cell-cell communication analysis predicted crucial roles for ICAM and CXCR3 ligand-mediated recruitment and retention of these two circulating T cell populations by epithelial, endothelial and myeloid cells during active colitis. In irColitis, we also observed significant epithelial turnover marked by fewer LGR5+ stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Transcriptional programs associated with irColitis were distinct from those in the tumor microenvironment, which may have important therapeutic implications. Finally, by examining many drugs in clinical trials for inflammatory bowel disease, we expand the putative therapeutic options for treating irColitis reported to-date.ConclusionsThis multi-omics approach nominates novel irColitis therapeutic targets and redefines irColitis as a disease not simply marked by the aberrant expansion of CD8 T cells but rather altered global interactions between immune cells and the colon mucosal epithelial or mesenchymal cells.Ethics ApprovalInformed consent was obtained from all patients in accordance with protocols obtained from the Mass General Brigham and/or DANA- Farber/Harvard Cancer Center Institutional Review Boards (DFCI/HCC 11-181 and 13-416, Mass General Brigham 2015P001333).
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