The role of the gastrointestinal tract in the pathogenesis of rheumatic diseases

Dysregulation of the intestinal epithelial barrier in genetically susceptible individuals may lead to both intestinal and extraintestinal autoimmune disorders. There is emerging literature on the role of microbiota changes in the pathogenesis of systemic rheumatic diseases such as rheumatoid arthritis, spondyloarthropathies, and connective tissue diseases. Although the role of the gastrointestinal tract in the pathogenesis of spondyloartropathies is well defined and many studies underline the importance of gastrointestinal inflammation in modulating local and systemic inflammation, the data are inconclusive regarding the effect of dysbiosis on rheumatoid arthritis and connective tissue diseases. This review aims to summarize current data on the role of the gastrointestinal involvement and intestinal microbiota in the pathogenesis of systemic rheumatic disease

Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis

Background: Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. Methods: Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. Results: Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. Conclusions: Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour

Role of IL-22 pathway in primary Sjogren’s Syndrome

Objectives In chronic inflammatory disorders, interleukin (IL)-22 may act either as a protective or as a pro-inflammatory cytokine. At mucosal sites, IL-22 is mainly produced by CD4+ T cells and by a subset of mucosal inflammatory natural killer (NK) cells expressing the receptor NKp44 (NKp44+NK cells). The aim of this study was to study the IL-22 pathway in the salivary glands of patients with primary Sjögren's syndrome (pSS) a chronic systemic autoimmune disease characterized by dysregulated innate and adaptive immune responses and by a higher rate of non-Hodgkin lymphomas. Methods Minor salivary gland biopsies were obtained from patients with pSS and fromsubject with non-specific chronic sialoadenitis. Quantitative gene expression analysis by TaqMan real-time PCR, immunohistochemistry and fluorescence-activated cell sorting analysis were performed. Results IL-22 is overexpressed in salivary glands of pSS patients. Aberrant expression of IL-22R1 occurs in the salivary glands and in the PBMC of p-SS. In vitro IL-22 stimulation of isolated IL-22R1-bearing monocytes resulted in the increased expression of STAT3, IL-17 and IL-22 and in Th17 expansion suggesting that an abnormal IL-22 stimulatory pathway may contribute to the perpetuation of local and systemic inflammation in p-SS. Lymphomas of pSS patients also aberrantly expressed IL-22R1 on monocytes and neoplastic B-cells, suggesting a putative role of IL-22 pathway in the pSS associated lymphomagenesis. Rituximab (RTX), which has historically been used for the treatment of B-cell lymphoma, has also been considered to be effective in the therapy of pSS; RTX treatment significantly reduced the number of IL-22+ cells infiltrating the salivary glands of pSS patients and modulates IL-17 expression. Conclusions Our results suggest that, together with IL-17 and IL-23, IL-22 may play a pro-inflammatory role in the pathogenesis of pSS and pSS associated lymphomas.Objectives In chronic inflammatory disorders, interleukin (IL)-22 may act either as a protective or as a pro-inflammatory cytokine. At mucosal sites, IL-22 is mainly produced by CD4+ T cells and by a subset of mucosal inflammatory natural killer (NK) cells expressing the receptor NKp44 (NKp44+NK cells). The aim of this study was to study the IL-22 pathway in the salivary glands of patients with primary Sjögren's syndrome (pSS) a chronic systemic autoimmune disease characterized by dysregulated innate and adaptive immune responses and by a higher rate of non-Hodgkin lymphomas. Methods Minor salivary gland biopsies were obtained from patients with pSS and fromsubject with non-specific chronic sialoadenitis. Quantitative gene expression analysis by TaqMan real-time PCR, immunohistochemistry and fluorescence-activated cell sorting analysis were performed. Results IL-22 is overexpressed in salivary glands of pSS patients. Aberrant expression of IL-22R1 occurs in the salivary glands and in the PBMC of p-SS. In vitro IL-22 stimulation of isolated IL-22R1-bearing monocytes resulted in the increased expression of STAT3, IL-17 and IL-22 and in Th17 expansion suggesting that an abnormal IL-22 stimulatory pathway may contribute to the perpetuation of local and systemic inflammation in p-SS. Lymphomas of pSS patients also aberrantly expressed IL-22R1 on monocytes and neoplastic B-cells, suggesting a putative role of IL-22 pathway in the pSS associated lymphomagenesis. Rituximab (RTX), which has historically been used for the treatment of B-cell lymphoma, has also been considered to be effective in the therapy of pSS; RTX treatment significantly reduced the number of IL-22+ cells infiltrating the salivary glands of pSS patients and modulates IL-17 expression. Conclusions Our results suggest that, together with IL-17 and IL-23, IL-22 may play a pro-inflammatory role in the pathogenesis of pSS and pSS associated lymphomas

Targeting IL-6 signalling in early rheumatoid arthritis is followed by Th1 and Th17 suppression and Th2 expansion

OBJECTIVES: To investigate the in vitro and ex-vivo effect of IL-6 inhibition on the balance between Th1, Th2, Th17 and Treg cells. METHODS: Ten consecutive adult patients with active early rheumatoid arthritis (ERA) and ten healthy volunteers were included in the study. The percentages of Th1, Th2, Th17 and Treg cells were analysed by flow cytometry in the peripheral blood mononuclear cells obtained from controls and from RA patients at the time of first evaluation and just before the third TCZ infusion. The in vitro effect of TCZ on the different subsets of CD4+ T cells and the expression levels of Th1, Th2, Th17 and Treg-related cytokines was also assessed. RESULTS: Treatment with TCZ, both ex vivo and in vitro, resulted in a significant reduction of the percentage of Th1, Th17 and Treg cells with a concomitant significant increase of Th2 cell subsets. The reduction of the different subsets of T lymphocytes was associated with an intense staining with Annexin V, suggesting an apoptotic-related cell reduction. A significant decrease of Th1, Th17 and Treg cytokines and a concomitant increase of IL-4 was also observed after TCZ treatment in PBMC isolated from RA patients. CONCLUSIONS: TCZ could modify the immune imbalance in RA inducing apoptosis of Th1, Th17 and Treg cells and promoting the appearance of a Th2 response

Evidence that autophagy, but not the unfolded protein response, regulates the expression of IL-23 in the gut of patients with ankylosing spondylitis and subclinical gut inflammation.

OBJECTIVES: Interleukin (IL)-23 has been implicated in the pathogenesis of ankylosing spondylitis (AS). The aim of the study was to clarify the mechanisms underlying the increased IL-23 expression in the gut of AS patients. METHODS: Consecutive gut biopsies from 30 HLA-B27(+) AS patients, 15 Crohn's disease (CD) patients and 10 normal subjects were obtained. Evidence for HLA-B27 misfolding was studied. Unfolded protein response (UPR) and autophagy were assessed by RT-PCR and immunohistochemistry. The contribution of UPR and autophagy in the regulation of IL-23 expression was evaluated in in vitro experiments on isolated lamina propria mononuclear cells (LPMCs). RESULTS: Intracellular colocalisation of SYVN1 and FHCs but not a significant overexpression of UPR genes was observed in the gut of AS patients. Conversely, upregulation of the genes involved in the autophagy pathway was observed in the gut of AS and CD patients. Immunohistochemistry showed an increased expression of LC3II, ATG5 and ATG12 but not of SQSTM1 in the ileum of AS and CD patients. LC3II was expressed among infiltrating mononuclear cells and epithelial cells resembling Paneth cells (PC) and colocalised with ATG5 in AS and CD. Autophagy but not UPR was required to modulate the expression of IL-23 in isolated LPMCs of AS patients with chronic gut inflammation, CD patients and controls. CONCLUSIONS: Our data suggest that HLA-B27 misfolding occurs in the gut of AS patients and is accompanied by activation of autophagy rather than a UPR. Autophagy appears to be associated with intestinal modulation of IL-23 in AS

Difference in the expression of IL-9 and IL-17 correlates with different histological pattern of vascular wall injury in giant cell arteritis

OBJECTIVE: GCA is a large- and medium-vessel arteritis characterized by a range of histological patterns of vascular wall injury. The aim of this study was to immunologically characterize the various histological patterns of GCA. METHODS: Thirty-five consecutive patients with biopsy-proven GCA and 15 normal controls were studied. IL-8, IL-9, IL-9R, IL-17, IL-4, TGF-β and thymic stromal lymphopoietin expression was evaluated by RT-PCR and immunohistochemistry on artery biopsy specimens. Confocal microscopy was used to characterize the phenotypes of IL-9-producing and IL-9R-expressing cells. Five additional patients who had received prednisone when the temporal artery biopsy was performed were also enrolled to evaluate the effect of glucocorticoids on IL-9 and IL-17 expression. RESULTS: IL-17 overexpression was observed mainly in arteries with transmural inflammation and vasa vasorum vasculitis. IL-9 overexpression and Th9 polarization predominated in arteries with transmural inflammation and small-vessel vasculitis. The tissue expression of both IL-9 and IL-17 was correlated with the intensity of the systemic inflammatory response. IL-4, TGF-β and thymic stromal lymphopoietin, which are involved in the differentiation of Th9 cells, were overexpressed in arteries with transmural inflammation and small-vessel vasculitis. IL-9R was also overexpressed in GCA arteries with transmural inflammation and was accompanied by increased expression of IL-8. CONCLUSION: Herein we provide the first evidence that distinct populations of potentially autoreactive T cells, expressing different cytokines (Th17 vs Th9), characterize patients with particular histological subsets of GCA and may thus contribute to the heterogeneity of tissue lesions observed in these patients

The CD68+/H-ferritin+ cells colonize the lymph nodes of the patients with adult onset Still's disease and are associated with increased extracellular level of H-ferritin in the same tissue: Correlation with disease severity and implication for pathogenesis

In this work, we aimed to evaluate the levels of ferritin enriched in H subunits (H-ferritin) and ferritin enriched in L subunits (L-ferritin) and the cells expressing these two molecules in the lymph node (LN) biopsies obtained from adult-onset Still's disease (AOSD) patients, and the possible correlation among these data and the severity of the disease. Ten patients with AOSD underwent LN biopsy. All the samples were stained by immunofluorescence. A statistical analysis was performed to estimate the possible correlation among both H-ferritin and L-ferritin tissue expression and the clinical picture of the disease. Furthermore, the same analysis was performed to evaluate the possible correlation among the number of CD68+/H-ferritin+ or CD68+/L-ferritin+ cells and the clinical picture. Immunofluorescence analysis demonstrated an increased tissue H-ferritin expression in the LNs of AOSD patients. This increased expression correlated with the severity of the disease. An increased number of CD68 macrophages expressing H-ferritin was observed in the LN samples of our patients. Furthermore, we observed that the number of CD68+/H-ferritin+ cells correlated significantly with the severity of the clinical picture. Our data showed an imbalance between the levels of H- and L-ferritin in LNs of AOSD patients and the evidence of an increased number of CD68+/H-ferritin+ cells in the same organs. Furthermore, a correlation among both the tissue H-ferritin levels and the CD68+/H-ferritin+ cells and the clinical picture was observed

Type 3 innate lymphoid cells producing IL-17 and IL-22 are expanded in the gut, in the peripheral blood, synovial fluid and bone marrow of patients with ankylosing spondylitis

Background The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS).Methods ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed.Results ILC3 characterised as Lyn(-)RORc(-)Tbet(+) NKp44(+) cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed alpha 4 beta 7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R(+) cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1.Conclusions Gut-derived IL-17(+) and IL-22(+) ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints