Peripheral Blood Of Systemic Sclerosis Patients Research Articles

019 Single cell level protein analysis of autoantigen-reactive B cells in systemic sclerosis and the murine model

Since the antigen affinity of B cells varies from cell to cell, functional analysis in each B cell has been difficult from technical aspects. Especially in autoimmune diseases, promising pathogenic B cells have not been adequately studied to date because of the rarity. Here, we analyzed the single-cell function of autoantigen-reactive B cells in systemic sclerosis (SSc). Topo I-reactive B cells were extracted from peripheral blood of SSc patients. mRNA expression and protein expression analysis, co-culture analysis with T cells in micro-space, and cytokine and antigen binding capacity analysis were performed.

Systemic Sclerosis Perturbs the Architecture of the Immunome.

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive fibrosis of skin and internal organs, and vascular dysfunction. Association of T and B cell subsets has been reported in SSc; however, there is lack of systematic studies of functional relations between immune cell subsets in this disease. This lack of mechanistic knowledge hampers targeted intervention. In the current study we sought to determine differential immune cell composition and their interactions in peripheral blood of SSc patients. Mononuclear cells from blood of SSc patients (n = 20) and healthy controls (n = 10) were analyzed by mass cytometry using a 36-marker (cell surface and intracellular) panel. Transcriptome analysis (m-RNA sequencing) was performed on sorted T and B cell subsets. Unsupervised clustering analysis revealed significant differences in the frequencies of T and B cell subsets in patients. Correlation network analysis highlighted an overall dysregulated immune architecture coupled with domination of inflammatory senescent T cell modules in SSc patients. Transcriptome analysis of sorted immune cells revealed an activated phenotype of CD4 and mucosal associated invariant T (MAIT) cells in patients, accompanied by increased expression of inhibitory molecules, reminiscent of phenotype exhibited by functionally adapted, exhausted T cells in response to chronic stimulation. Overall, this study provides an in-depth analysis of the systemic immunome in SSc, highlighting the potential pathogenic role of inflammation and chronic stimulation-mediated “functional adaptation” of immune cells.

Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and Siglec-3-expressing myeloid dendritic cells in systemic sclerosis patients

ObjectiveTo investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients.MethodsSpontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation.ResultsThe frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function.ConclusionsThese data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis.

A1.13 Increased presence of CD206+macrophage subset in peripheral blood of systemic sclerosis patients

Background and objectives In systemic sclerosis (SSc), the immune-inflammatory infiltrate is primarily made of macrophages and T cells. 1 Recently, alternatively activated macrophages (M2) have been shown to enhance fibrosis through the production of pro-fibrotic molecules (i.e. fibronectin, metalloproteinases and TGFβ. 2 M2 are characterised by an increased expression of specific markers, primarily CD206 (macrophage mannose receptor-1) and macrophage scavenger receptors (CD204 and CD163). The study investigated the presence of M2 in the peripheral blood (PB) of SSc patients. In addition, the capability of SSc serum to induce the expression of M2 markers and the production of pro-fibrotic metalloproteinase (MMP)-9 in cultured PB mononuclear cells (PBMCs) of healthy subjects (HS) were investigated. Materials and methods Eight SSc patients (mean age 65 ± 7 years) who fulfilled the new criteria of EULAR/ACR 3 and five age matched HS were enrolled into the study, after signing an Informed Consent. SSc patients were treated with vasodilators and endothelin-1 blockers. PB from SSc patients and HS was analysed by flow cytometry (FC) to detect the presence of cells positive for CD206, CD204 and CD14 (a marker of monocyte/macrophage lineage). PBMC-derived monocytes isolated from the enrolled HS (2 × 10 6 cells/well) were maintained for 12 h in growth medium at 10% of fetal bovine serum and then stimulated for 48 h with the serum from SSc patients and HS themselves. The expression of CD206, CD204 and CD68 (a macrophage marker) was evaluated by immunocytochemistry (ICC). MMP-9 production was evaluated by zymography. Results In the PB of SSc patients, the CD206 + cell percentage was found significantly higher than that of HS (p + cells co-expressed CD204, belonging to the monocyte/macrophage lineage due to their expression of CD14. SSc serum stimulated PBMC-derived monocytes to express CD206, CD204 and CD68 thus indicating their activation into macrophages. Conversely, serum from HS was unable to stimulate the expression of CD206 and it induced a limited expression of CD204 in these cells. Zymography showed that treatment with SSc serum induced an increased production and secretion of MMP-9 compared to the treatment with HS serum in PBMC-derived macrophages. Conclusion These preliminary results show an increased presence of the CD206 + cell subset in the PB of SSc patients. The ability of SSc serum to induce the expression of this M2 macrophage marker and to increase the production of pro-fibrotic MMP-9 might support a possible involvement of this cell subset in the pathogenesis of SSc. References Wynn TA, Ramalingam TR et al . Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 2012; 18 :1028–40. Fairweather D, Cihakova D et al . Alternatively activated macrophages in infection and autoimmunity. J Autoimmun 2009; 33 :222–30. van den Hoogen F, Khanna D, Fransen J et al . 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthrit Rheum 2013; 65 :2737–47.

SAT0036 Tumor necrosis factor-alpha co-activated T-cells support a profibrotic cytokine milieu on cost of IL-10 expression in patients with systemic sclerosis

BackgroundLow grade inflammation is an increasingly recognized feature in patients suffering from systemic sclerosis (SSc), with substantial infiltration and activation of lymphocytes occurring in SSc affected tissue. However, the transition...

AB0145 Enhanced il-8 production by monocytes in systemic sclerosis patients with pulmonary fibrosis

BackgroundSubstantial evidence supports the implication of immune-activated cells, cytokines and chemokines in the pathogenesis of systemic sclerosis (SSc). In fact, interleukin 6 (IL-6) and IL-8 play a crucial role in...

SAT0033 Allogenic mesenchymal stem cells-mediated immunoregulation involves FAS/FASL-induced T cell apoptosis in systemic sclerosis

BackgroundSystemic infusion of allogenic mesenchymal stem cells (MSC) have shown therapeutic effects on a variety of autoimmune diseases, but the underlying mechanisms of MSC-based immunoregulation are not fully understood.ObjectivesTo investigate...

Interleukin‐13–producing CD8+ T cells mediate dermal fibrosis in patients with systemic sclerosis

Fibrosis is a major contributor to morbidity and mortality in systemic sclerosis (SSc). T cells are the predominant inflammatory infiltrate in affected tissue and are thought to produce cytokines that drive the synthesis of extracellular matrix (ECM) proteins by fibroblasts, resulting in excessive fibrosis. We have previously shown that aberrant interleukin-13 (IL-13) production by peripheral blood effector CD8+ T cells from SSc patients correlates with the extent of skin fibrosis. The present study was undertaken to investigate the role of IL-13 production by CD8+ T cells in dermal fibrosis, an early and specific manifestation of SSc. ECM protein production by normal dermal fibroblasts cocultured with SSc CD8+ T cell supernatants was determined by quantitative polymerase chain reaction and Western blotting. Skin-homing receptor expression and IL-13 production by CD8+ T cells in the peripheral blood of SSc patients were measured by flow cytometry. IL-13+ and CD8+ cells in sclerotic skin were identified by immunohistochemistry. IL-13-producing circulating CD8+ T cells from patients with SSc expressed skin-homing receptors and induced a profibrotic phenotype in normal dermal fibroblasts, which was inhibited by an anti-IL-13 antibody. High numbers of CD8+ T cells and IL-13+ cells were found in the skin lesions of SSc patients, particularly during the early inflammatory phase of the disease. These findings show that IL-13-producing CD8+ T cells are directly involved in modulating dermal fibrosis in SSc. The demonstration that CD8+ T cells homing to the skin early in the course of SSc are associated with accumulation of IL-13 is an important mechanistic contribution to the understanding of the pathogenesis of dermal fibrosis in SSc and may represent a potential target for therapeutic intervention.

Alteration of Th17 and Treg cell subpopulations co-exist in patients affected with systemic sclerosis

Aim of the study has been to understand the relationship between TH17 and Treg cell subsets in patients affected with systemic sclerosis (SSc). Phenotypes and functions of Th17 and Treg cell subsets were analyzed in a series of 36 SSc patients. Th17 cell concentration in the peripheral blood was found to be increased in SSc patients with respect to healthy controls independently from type or stage of disease. After PBMC stimulation with a polyclonal stimulus or Candida albicans antigens the frequency of Th17 T cell clones was significantly higher in SSc patients with respect to controls suggesting the skewing of immune response in SSc patients toward Th17 cell generation/expansion. Concerning the Treg compartment, both CD4+CD25+ and CD8+CD28- Treg subsets showed quantitative and qualitative alteration in the peripheral blood of SSc patients. Collectively, these data highlight the existence of an imbalanced ratio between Th17 and Treg cell subsets in SSc patients.

Absolute count of T and B lymphocyte subsets is decreased in systemic sclerosis

BackgroundPrevious reports on lymphocyte subpopulations in systemic sclerosis (SSc) are conflicting. Therefore, we aimed to investigate the lymphocyte subsets in SSc patients who were not on immunosuppressive therapy.MethodsLymphocyte subsets were assessed in the peripheral blood of SSc patients (n = 29) and healthy controls (n = 29) using the four colour flow cytometry method. Correlation studies were also performed in order to assess the relationship between lymphocyte subsets and clinical parameters.ResultsThe absolute count of lymphocytes (P = 0.0042), CD3+ (P = 0.0014), CD4+ (P = 0.0070), CD8+ (P = 0.021), and CD19+ cells (P = 0.024) was significantly decreased in SSc patients when compared to healthy controls. CD4+/CD8+ ratio and the absolute count of CD56+ cells observed in SSc patients did not significantly differ from controls (P = 0.165; P = 0.632, respectively). There was no substantial relationship between the lymphocyte subset levels and clinical features (i.e., SSc subtype, autoantibody profiles, organ involvement), except for a significant inverse correlation of CD19+ cells and the modified Rodnan skin score (r = -0.43, P = 0.020).ConclusionOur data support previous reports indicating that subsets of T lymphocytes as well as B lymphocytes play a role in the pathogenesis of SSc.

Molecular subtypes of systemic sclerosis in association with anti-centromere antibodies and digital ulcers

The objective of this study was to identify molecular profiles that may distinguish clinical subtypes in systemic sclerosis (SSc). Large-scale gene expression profiling was performed on peripheral blood (PB) from 12 SSc patients and 6 healthy individuals. Significance analysis of microarrays, two-way hierarchical cluster analysis and PANTHER (Protein ANalysis THrough Evolutionary Relationships) ontology classification were used to analyze the data. Quantitative PCR was applied for validation in a cohort of 43 SSc patients. The results show that the expression of genes involved in immune defense, cell cycle and signal transduction was significantly elevated in PB of SSc patients (n=12) compared with healthy individuals (n=6). SSc patients could be stratified into subgroups based on differential expression of genes induced by type I interferon (IFN) and genes involved in antimicrobial (AM) activity. Differential expression of type I IFN or AM signature genes was validated and extended in an independent cohort of 31 patients by quantitative PCR. Low expression of IFN response genes was associated with the presence of anti-centromere antibodies, whereas increased expression was associated with the appearance of digital ulcers. In conclusion, patients with SSc can be classified on the basis of differential expression of immune defense genes. Differences in the activity of the type I IFN response program stratify patients into two clinically relevant subgroups.

Peripheral blood T lymphocytes from systemic sclerosis patients show both Th1 and Th2 activation.

Our objective was to investigate the phenotype of helper T cells in the peripheral blood of patients with systemic sclerosis (SSc). PBMC from 15 patients with SSc and 15 sex- and age-matched controls were investigated for lymphocyte subpopulations (CD3, CD4, CD8, CD19, CD16/CD56, CD3-DR); IL-2, IL-4, and IFN-gamma mRNAs; and the relative cytokines in their cytoplasm. The last assay was carried out both in unstimulated and in PMA-activated PBMC. SSc patients presented a higher percentage of activated T cells, CD3+ DR+ (19.7 +/- 9.9 vs 5.1 +/- 2.5%; P < 0.0001); 12 of them presented IFN-gamma mRNA-positive cells; and none IL-2 or IL-4 mRNAs. Under basal conditions, PBMC from six SSc patients contained IL-2, IL-4, and IFN-gamma (i.e., they showed both Th1 and Th2 activation), and 1 IFN-gamma only. PMA-stimulated PBMC of patients differed from those of controls only in the increased percentage of IFN-gamma positive cells (52 +/- 12 vs 37 +/- 11%; P < 0.01). Our study demonstrates that Thl activation occurs in the peripheral blood of SSc patients. This evidence must be faced with from both a pathogenetic and a therapeutical point of view.