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Single-cell RNA-seq reveals altered NK cell subsets and reduced levels of cytotoxic molecules in patients with ankylosing spondylitis.

ABSTRACT: Ankylosing spondylitis (AS) is an autoimmune disease with unknown aetiology. To unravel the mechanisms mediating AS pathogenesis, we profiled peripheral blood mononuclear cells (PBMCs) from AS patients and healthy subjects using 10X single-cell RNA sequencing. The frequencies of immune cell subsets were evaluated by flow cytometry. NK cells were purified from PBMCs using isolation kit and were examined for gene expression by RT-qPCR. Plasma levels of cytolytic molecules were examined by enzyme-linked immunosorbent assay. Compared to healthy controls, AS patients showed a significant decrease in total NK cells as well as CD56^dim NK subset, whereas CD56^bright NK cells were increased. Additionally, impaired expression of cytotoxic genes in NK cells of AS patients was observed by bioinformatics algorithm and verified by RT-qPCR and flow cytometry. Consistent with changes in transcriptomics, we found decreased plasma levels of granzymes, but not granulysin, in AS patients. Furthermore, Pearson correlation analysis revealed a negative correlation between plasma GZMB levels and disease activity (r = -0.5275, p = 0.0358). No correlation was observed between plasma cytolytic molecules and biochemical indexes (ESR and CRP). Our findings uncover altered NK cell subsets and cytotoxic profiles in peripheral circulation of AS patients at single-cell resolution.

SUBMITTER: Ren C

PROVIDER: S-EPMC8831943 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Single-cell RNA-seq reveals altered NK cell subsets and reduced levels of cytotoxic molecules in patients with ankylosing spondylitis.

Ren Conglin C Li Mingshuang M Zheng Yang Y Cai Bingbing B Du Weibin W Zhang Helou H Wu Fengqing F Tong Mengsha M Lin Fu F Wang Jinfu J Quan Renfu R

Journal of cellular and molecular medicine 20220106 4

Ankylosing spondylitis (AS) is an autoimmune disease with unknown aetiology. To unravel the mechanisms mediating AS pathogenesis, we profiled peripheral blood mononuclear cells (PBMCs) from AS patients and healthy subjects using 10X single-cell RNA sequencing. The frequencies of immune cell subsets were evaluated by flow cytometry. NK cells were purified from PBMCs using isolation kit and were examined for gene expression by RT-qPCR. Plasma levels of cytolytic molecules were examined by enzyme-l ...[more]

PMID: 34994057

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Project description:Ankylosing spondylitis is a complicated consequence of genetic predisposition and environmental factors. Enthesitis is believed to be the hallmark of ankylosing spondylitis, and the chronic inflammatory state of this disease is perpetuated by the disturbances of both the innate immune system and the acquired immune system. To clarify the alteration of immune system in patients with AS, we conducted a meta-analysis concerning the proportions of major lymphocyte subsets in the peripheral blood of AS patients. We systematically searched PubMed and China National Knowledge Infrastructure (CNKI) for articles related to this subject. A total of 95 articles involving 4,020 AS patients and 3,065 healthy controls were included in the analysis. This meta-analysis is performed on R platform using R package "meta", and Egger's tests were used to determine the presence of publication bias. Results showed that the percentages of T cells, NK cells and NKT cells were not significantly different between AS patients and healthy controls, but B cells were significantly increased. Among the subsets of T cells, the proportions of CD4+ T cells, Th17 cells, Tfh cells as well as Th1/Th2 ratio were significantly increased, while Tregs were significantly decreased. Subgroup analysis showed that the proportions of Th17 among both PBMCs, T cells and CD4+ T cells were significantly elevated, while Tregs were only significantly lower in PBMCs. Subgroup analysis also demonstrated that Tregs defined by "CD4+CD25+FoxP3+", "CD4+CD25+CD127low"or "CD4+CD25+CD127-"were significantly downregulated, indicating that the selection of markers could be critical. Further study is warranted in order to elucidate the complicated interactions between different lymphocyte subsets in AS patients. This study implied that the disequilibrium between Th17 and Tregs, as well as between Th1 and Th2 could contribute to the pathogenesis of ankylosing spondylitis, further cementing the understanding that ankylosing spondylitis is a consequence of disrupted balance of innate immune system and acquired immune system.

Project description:Intestinal bacterial dysbiosis has been increasingly linked to ankylosing spondylitis (AS), which is a prototypic and best studied subtype of spondyloarthritis (SpA). Fungi and bacteria coexist in the human gut and interact with each other. Although they have been shown to contribute actively to health or disease, no studies have investigated whether the fungal microbiota in AS patients is perturbed. In this study, fecal samples from 22 AS patients, with clinical and radiographic assessments, and 16 healthy controls (HCs) were collected to systematically characterize the gut microbiota and mycobiota in AS patients by 16S rRNA gene- and ITS2-based DNA sequencing. Our results showed that the microbiota of AS patients was characterized by increased abundance of Proteobacteria and decreased Bacteroidetes, which was contributed by enrichment of Escherichia-Shigella, Veillonella, Lachnospiraceae NK4A136 group, and reduction of Prevotella strain 9, Megamona, and Fusobacterium. The gut mycobiota of AS patients was characterized by higher levels of Ascomycota, especially the class of Dothideomycetes, and decreased abundance of Basidiomycota, which was mainly contributed by the decease of Agaricales. Compared to HCs, decreased ITS2/16S biodiversity ratios and altered bacterial-fungal interkingdom networks were observed in AS patients. Compared with nonsteroidal anti-inflammatory drugs (NSAIDs), treating AS patients with biological agents induced obvious changes in the gut mycobiota, and this result was highly associated with disease activity indexes, including AS disease activity index (ASDAS) C-reactive protein (asCRP), erythrocyte sedimentation rate (ESR), and Bath AS disease activity index (BASDAI). In addition, altered mycobiota in AS patients was also found associated with the degree of radiographic damage. IMPORTANCE The human gut is colonized by diverse fungi (mycobiota), and fungi have long been suspected in the pathogenesis of SpA. Our study unraveled a disease-specific interkingdom network alteration in AS, suggesting that fungi, or the interkingdom interactions between bacteria and fungi, may play an essential role in AS development. However, our study is limited by sample size, and in-depth mechanism studies and additional large-scale investigations characterizing the gut mycobiome in AS patients are needed to form a foundation for research into the relationship between mycobiota dysbiosis and AS development.

Project description:We have compared synovial biopsies from ankylosing spondylitis and undifferentiated spondylitis patients with healthy controls and osteoarthritis patients Objective: In spondylarthropies, whole-genome gene expression profiling studies have been limited to peripheral blood to date. By undertaking a study in knee synovial biopsies from spondylarthropy (SpA) and ankylosing spondylitis (AS) patients we aimed to identified joint-specific candidate genes and pathways. These pathways may mediate systemic inflammation driven joint damaging processes and more specifically, the osteoproliferation that is characteristic of these conditions. Methods: RNA was extracted from six seronegative SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results: 416 differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, osteoblast activity, B-cell associated, matrix catabolic, and metabolic pathways. The inflammatory mediator, MMP3, was strongly upregulated in AS/SpA samples and the Wnt pathway inhibitors DKK3 and Kremen1 were downregulated. Conclusion: Pathways mediating both systemic inflammation as well as local tissue changes were identified. This suggests initial systemic inflammation in spondylarthropies transfers to and persists in the local joint environment, subsequently mediating changes in genes directly involved in the destructive tissue remodelling. Fifteen knee synovial biopsy tissue samples consisting of six seronegative spondyloarthropy (SpA), two ankylosing spondylitis (AS), three osteoarthritis (OA) and four normal control biopsies were obtained from the Synovial Tissue Bank at the Repatriation General Hospital in Adelaide, South Australia with the appropriate ethical approval (Supplementary Table 1). All patients provided informed written consent.

Dataset Information

Single-cell RNA-seq reveals altered NK cell subsets and reduced levels of cytotoxic molecules in patients with ankylosing spondylitis.

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Single-cell RNA-seq reveals altered NK cell subsets and reduced levels of cytotoxic molecules in patients with ankylosing spondylitis.

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