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BackgroundSarcopenia is a condition characterized by the age-related loss of skeletal muscle mass and function. The pathogenesis of the disease is influenced by chronic low-grade inflammation. However, the specific changes in the immune landscape changes of sarcopenic muscle are not yet fully understood.MethodsTo gain insights into the immune cell composition and interactions, we combined single-nucleus RNA sequencing data, bulk RNA sequencing dataset, and comprehensive bioinformatic analyses on the skeletal muscle samples from young, aged, and sarcopenic individuals. Histological staining was then performed on skeletal muscles to validate the distribution of immune cells in clinical samples.ResultsWe analyzed the transcriptomes of 101,862 single nuclei, revealing a total of 10 major cell types and 6 subclusters of immune cell types within the human skeletal muscle tissues. Notable variations were identified in the immune microenvironment between young and aged skeletal muscle. Among the immune cells from skeletal muscle microenvironment, macrophages constituted the largest fraction. A specific marker gene LYVE1 for skeletal muscle resident macrophages was further identified. Cellular subclasses included four distinct groups of resident macrophages, which play different roles in physiological or non-physiological conditions. Utilizing bulk RNA sequencing data, we observed a significant enrichment of macrophage-rich inflammation in sarcopenia.ConclusionsOur findings demonstrate age-related changes in the composition and cross-talk of immune cells in human skeletal muscle microenvironment, which contribute to chronic inflammation in aged or sarcopenia muscle. Furthermore, macrophages emerge as a potential therapeutic target, thus advancing our understanding of the pathogenesis of sarcopenia.

BackgroundPrimary Sjögren’s syndrome (pSS) is a progressive inflammatory autoimmune disease. Immune cell infiltration into glandular lobules and ducts and glandular destruction are the pathophysiological hallmarks of pSS. Macrophages are one of the most important cells involved in the induction and regulation of an inflammatory microenvironment. Although studies have reported that an abnormal tissue microenvironment alters the metabolic reprogramming and polarisation status of macrophages, the mechanisms driving macrophage infiltration and polarisation in pSS remain unclear.MethodsImmune cell subsets were characterised using the single-cell RNA sequencing (scRNA-seq) data of peripheral blood mononuclear cells (PBMCs) from patients with pSS (n = 5) and healthy individuals (n = 5) in a public dataset. To evaluate macrophage infiltration and polarisation in target tissues, labial salivary gland biopsy tissues were subjected to histological staining and bulk RNA-seq (pSS samples, n = 24; non-pSS samples, n = 12). RNA-seq data were analysed for the construction of macrophage co-expression modules, enrichment of biological processes and deconvolution-based screening of immune cell types.ResultsDetailed mapping of PBMCs using scRNA-seq revealed five major immune cell subsets in pSS, namely, T cells, B cells, natural killer (NK) cells, dendritic cells (DCs) and monocyte-macrophages. The monocyte-macrophage subset was large and had strong inflammatory gene signatures. This subset was found to play an important role in the generation of reactive oxygen species and communicate with other innate and adaptive immune cells. Histological staining revealed that the number of tissue-resident macrophages was high in damaged glandular tissues, with the cells persistently surrounding the tissues. Analysis of RNA-seq data using multiple algorithms demonstrated that the high abundance of pro-inflammatory M1 macrophages was accompanied by the high abundance of other infiltrating immune cells, senescence-associated secretory phenotype and evident metabolic reprogramming.ConclusionMacrophages are among the most abundant innate immune cells in PBMCs and glandular tissues in patients with pSS. A bidirectional relationship exists between macrophage polarisation and the inflammatory microenvironment, which may serve as a therapeutic target for pSS.

BackgroundPrimary Sjogren Syndrome (pSS) is an autoimmune disease characterized by immune cell infiltration. While the presence of follicular T helper (Tfh) cells in the glandular microenvironment has been observed, their biological functions and clinical significance remain poorly understood.MethodsWe enrolled a total of 106 patients with pSS and 46 patients without pSS for this study. Clinical data and labial salivary gland (LSG) biopsies were collected from all participants. Histological staining was performed to assess the distribution of Tfh cells and B cells. Transcriptome analysis using RNA-sequencing (RNA-seq) was conducted on 56 patients with pSS and 26 patients without pSS to uncover the underlying molecular mechanisms of Tfh cells. To categorize patients, we employed the single-sample gene set enrichment analysis (ssGSEA) algorithm, dividing them into low- and high-Tfh groups. We then utilized gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and deconvolution tools to explore functional and immune infiltration differences between the low- and high-Tfh groups.ResultsPatients with pSS had a higher positive rate of the antinuclear antibody (ANA), anti-Ro52, anti-SSA, anti-SSB and hypergammaglobulinaemia and higher levels of serum IgG compared to the non-pSS. Histopathologic analyses revealed the presence of Tfh cells (CD4+CXCR5+ICOS+) in germinal centers (GC) within the labial glands of pSS patients. GSEA, WGCNA, and correlation analysis indicated that the high-Tfh group was associated with an immune response related to virus-mediated IFN response and metabolic processes, primarily characterized by hypoxia, elevated glycolysis, and oxidative phosphorylation levels. In pSS, most immune cell types exhibited significantly higher infiltration levels in the high-Tfh group compared to the low-Tfh group. Additionally, patients in the Tfh-high group demonstrated a higher positive rate of the ANA, rheumatoid factor (RF), and hypergammaglobulinaemia, as well as higher serum IgG levels.ConclusionOur study suggests that Tfh cells may play a crucial role in the pathogenesis of pSS and could serve as potential therapeutic targets in pSS patients.

BackgroundPrimary Sjogren’s syndrome (pSS) is a prototypical systemic autoimmune disease characterised by lymphocyte infiltration and immune-complex deposition in multiple organs. The specific distribution of immune cell populations and their relationship with mitochondria remain unknown.MethodsHistological analysis was performed to assess the specific distribution of innate and adaptive immune cell populations in labial salivary gland (LSG) samples from 30 patients with pSS and 13 patients with non-pSS. The ultrastructural morphometric features of mitochondria within immune cells were observed under the transmission electron microscope (TEM). RNA sequencing was performed on LSG samples from 40 patients with pSS and 7 non-pSS patients. The Single-sample Gene Set Enrichment Analysis (ssGSEA), ESTIMATE, and CIBERSORT algorithms and Pearson correlation coefficients were used to examine the relationship between mitochondria-related genes and immune infiltration. Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify the mitochondria-specific genes and the related pathways based on the immune cell types.ResultsHE staining revealed a massive infiltration of plasma cells with abundant immunoglobulin protein distributed around phenotypically normal-appearing acinar and ductal tissues of patients with pSS. Immunohistochemical analyses revealed that innate immune cells (macrophages, eosinophils and NK cells) were distributed throughout the glandular tissue. Dominant adaptive immune cell infiltration composed of B cells, CD4+T cells and CD8+ T cells or ectopic lymphoid follicle-like structures were observed in the LSGs of patients with pSS. TEM validated the swelling of mitochondria with disorganised cristae in some lymphocytes that had invaded the glandular tissue. Subsequently, bioinformatic analysis revealed that innate and adaptive immune cells were associated with different mitochondrial metabolism pathways. Mitochondrial electron transport and respiratory chain complexes in the glandular microenvironment were positively correlated with innate immune cells, whereas amino acid and nucleic acid metabolism were negatively correlated with adaptive immune cells. In addition, mitochondrial biogenesis and mitochondrial apoptosis in the glandular microenvironment were closely associated with adaptive immune cells.ConclusionInnate and adaptive immune cells have distinct distribution profiles in the salivary gland tissues of patients with pSS and are associated with different mitochondrial metabolic pathways, which may contribute to disease progression.

BackgroundPrimary Sjogren’s syndrome (pSS) is a slowly progressive, inflammatory autoimmune disease characterized by lymphocytic infiltration into salivary and lacrimal glands. It becomes more recognized that morphology alterations of epithelial mitochondria are involved in altered cellular bioenergetics in pSS patients. The integrated analysis of the mitochondrial role in the pathogenesis and aberrant immune microenvironment in pSS remains unknown.MethodsThe mitochondria-related genes and gene expression data were downloaded from the MitoMiner, MitoCarta, and NCBI GEO databases. We performed novel transcriptomic analysis and constructed a network between the mitochondrial function and immune microenvironment in pSS-salivary glands by computer-aided algorithms. Subsequently, real-time PCR was performed in clinical samples in order to validate the bioinformatics results. Histological staining and transmission electron microscopy (TEM) were further studied on labial salivary gland samples of non-pSS and pSS patients characterized for mitochondria-related phenotypic observation in the different stages of the disease.ResultsThe bioinformatic analysis revealed that the expression of several mitochondria-related genes was altered in pSS. Quantitative real-time PCR showed that four hub genes, CD38, CMPK2, TBC1D9, and PYCR1, were differentially expressed in the pSS clinical samples. These hub genes were associated with the degree of immune cell infiltration in salivary glands, the mitochondrial respiratory chain complexes, mitochondrial metabolic pathway in gluconeogenesis, TCA cycle, and pyruvate/ketone/lipid/amino acid metabolism in pSS. Clinical data revealed that the gene expression of fission (Fis1, DRP1, and MFF) and fusion (MFN1, MFN2, and OPA1) was downregulated in pSS samples, consistent with the results from the public validation database. As the disease progressed, cytochrome c and Bcl-2 proteins were regionally distributed in salivary glands from pSS patients. TEM revealed cytoplasmic lipid droplets and progressively swollen mitochondria in salivary epithelial cells.ConclusionOur study revealed cross talk between mitochondrial dysfunction and the immune microenvironment in salivary glands of pSS patients, which may provide important insights into SS clinical management based on modulation of mitochondrial function.

BackgroundPrimary Sjögren’s syndrome (pSS) is a chronic systemic autoimmune disease of the exocrine glands characterized by specific pathological features. Previous studies have pointed out that salivary glands from pSS patients express a unique profile of cytokines, adhesion molecules, and chemokines compared to those from healthy controls. However, there is limited evidence supporting the utility of individual markers for different stages of pSS. This study aimed to explore potential biomarkers associated with pSS disease progression and analyze the associations between key genes and immune cells.MethodsWe combined our own RNA sequencing data with pSS datasets from the NCBI Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) via bioinformatics analysis. Salivary gland biopsies were collected from 14 pSS patients, 6 non-pSS patients, and 6 controls. Histochemical staining and transmission electron micrographs (TEM) were performed to macroscopically and microscopically characterize morphological features of labial salivary glands in different disease stages. Then, we performed quantitative PCR to validate hub genes. Finally, we analyzed correlations between selected hub genes and immune cells using the CIBERSORT algorithm.ResultsWe identified twenty-eight DEGs that were upregulated in pSS patients compared to healthy controls. These were mainly involved in immune-related pathways and infection-related pathways. According to the morphological features of minor salivary glands, severe interlobular and periductal lymphocytic infiltrates, acinar atrophy and collagen in the interstitium, nuclear shrinkage, and microscopic organelle swelling were observed with pSS disease progression. Hub genes based on above twenty-eight DEGs, including MS4A1, CD19, TCL1A, CCL19, CXCL9, CD3G, and CD3D, were selected as potential biomarkers and verified by RT-PCR. Expression of these genes was correlated with T follicular helper cells, memory B cells and M1 macrophages.ConclusionUsing transcriptome sequencing and bioinformatics analysis combined with our clinical data, we identified seven key genes that have potential value for evaluating pSS severity.