AnnaRita Giardina

Overexpression of interleukin-23, but not interleukin-17, as an immunologic signature of subclinical intestinal inflammation in ankylosing spondylitis

OBJECTIVEnSubclinical gut inflammation is common in spondylarthritis, but the immunologic abnormalities underlying this process are undefined. Perturbation of the interleukin-23 (IL-23)/Th17 axis has emerged as a fundamental trigger of chronic inflammation. This study was undertaken to investigate the expression and tissue distribution of IL-23/Th17-related molecules in Crohns disease (CD) and in subclinical gut inflammation in ankylosing spondylitis (AS).nnnMETHODSnQuantitative gene expression analysis of Th1/Th2 and IL-23/Th17 responses was performed in intestinal biopsy samples obtained from 12 patients with CD, 15 patients with AS, and 13 controls. IL-23 tissue distribution and identification of IL-23-producing cells were evaluated by immunohistochemistry.nnnRESULTSnWe demonstrated a strong and significant up-regulation of IL-23p19 transcripts in the terminal ileum in patients with AS and patients with CD. IL-23 was abundantly produced by infiltrating monocyte-like cells in inflamed mucosa from AS and CD patients. Notably, we also identified Paneth cells as a major source of IL-23 in patients with AS, patients with CD, and normal controls. Unlike CD, in AS patients, IL-23 was not associated with up-regulation of IL-17 and the IL-17-inducing cytokines IL-6 and IL-1beta. Finally, while the Th1-related cytokines interferon-gamma, IL-12p35, and IL-27p28 were overexpressed only in CD patients, IL-4, IL-5, and STAT-6 were also significantly increased in AS patients.nnnCONCLUSIONnOur findings indicate that overexpression of IL-23, but not IL-17, is a pivotal feature of subclinical gut inflammation in AS. Identification of resident Paneth cells as a pivotal source of IL-23 in physiologic and pathologic conditions strongly suggests that IL-23 is a master regulator of gut mucosal immunity, providing a pathophysiologic significance to the reported association between IL-23 receptor polymorphisms and intestinal inflammation.

Long-term anti-TNF therapy and the risk of serious infections in a cohort of patients with rheumatoid arthritis: Comparison of adalimumab, etanercept and infliximab in the GISEA registry

OBJECTIVEnTo evaluate the risk of serious infections (SIs) in RA patients receiving anti-TNF therapy on the basis of the data included in the GISEA register.nnnMETHODSnThe study involved 2769 adult patients with long-standing RA (mean age 53.2±13.4 years; mean disease duration 9.0±8.3 years) enrolled in the GISEA register, who had been treated for at least 6 months with TNF inhibitors or had discontinued therapy due to SI: 837 (30%) treated with infliximab (IFN), 802 (29%) with adalimumab (ADA), and 1130 (41%) with etanercept (ETN).nnnRESULTSn176 patients had experienced at least one of the 226 Sis during the 9 years of treatment with an anti-TNF agent, an overall incidence of 31.8/1000 patient-years (95% CI 25.2-38.3): 23.7/1000 patient-years (95% CI 13.1-34.2) on ADA; 12.8/1000 patient-years (95% CI 6.3-19.4) on ETN and 65.1/1000 patient-years (95% CI 48.4-81.8) on IFN. The risk was higher in the first than in the second year of treatment, but this difference was not statistically significant (p=0.08) (38.9% of the SIs were recorded in the first 12 months of treatment). The risk of SI was significantly different among the three treatment groups (p<0.0001). Multivariate models confirmed that the use of steroids (p<0.046), concomitant DMARD treatment during anti-TNF therapy (p=0.004), advanced age at the start of anti-TNF treatment (p<0.0001), and the use of IFN or ADA rather than ETN (respectively p<0.0001 and p=0.023) were strong and statistically significant predictors of infection.nnnCONCLUSIONSnAnti-TNF therapy is associated with a small but significant risk of SI that is associated with the concomitant use of steroids, advanced age at the start of anti-TNF treatment, and the type of anti-TNF agent.

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 Crohns 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.

Potential involvement of IL-22 and IL-22-producing cells in the inflamed salivary glands of patients with Sjögren'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 natural killer (NK) cells expressing the receptor NKp44 (NKp44+ NK cells). The aim of this study was to investigate the IL-22 expression in the salivary glands of patients with primary Sjögrens syndrome (pSS). Methods Minor salivary gland biopsies were obtained from 19 patients with pSS and 16 with non-specific chronic sialoadenitis. Quantitative gene expression analysis by TaqMan real-time PCR and immunohistochemistry for IL-17, IL-22, IL-23 and STAT3 (signal transducer and activator of transcription) was performed on salivary glands from patients and controls. The cellular sources of IL-22 among infiltrating inflammatory cells were also determined by fluorescence-activated cell sorting analysis and immunohistochemistry. Results IL-22, IL-23 and IL-17 were significantly increased at both protein and mRNA levels in the inflamed salivary glands of patients with pSS. STAT3 mRNA and the tyrosine phosphorylated corresponding protein were also significantly increased in pSS. Th17 and NKp44+ NK cells were the major cellular sources of IL-22 in patients with pSS. 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.

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 α4β7. 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.

Interleukin-22 and interleukin-22-producing NKp44+ natural killer cells in subclinical gut inflammation in ankylosing spondylitis.

OBJECTIVEnThe intestinal inflammation observed in patients with ankylosing spondylitis (AS) is characterized by an overexpression of interleukin-23 (IL-23). IL-23 is known to regulate IL-22 production through lamina propria NKp44+ natural killer (NK) cells, which are thought to be involved in protective mucosal mechanisms. This study was undertaken to evaluate the frequency of NKp44+ NK cells and the expression of IL-22 in the ileum of AS patients.nnnMETHODSnTissue NKp44+ NK cells, NKp46+ NK cells, and IL-22-producing cells were analyzed by flow cytometry. Quantitative gene expression analysis of IL-22, IL-23, IL-17, STAT-3, and mucin 1 (MUC-1) was performed by reverse transcriptase-polymerase chain reaction on ileal samples from 15 patients with AS, 15 patients with Crohns disease (CD), and 15 healthy controls. NKp44, pSTAT-3, and IL-22 expression was analyzed by immunohistochemistry.nnnRESULTSnThe frequency of NKp44+ but not NKp46+ NK cells was increased in the inflamed ileum of AS patients compared to CD patients and controls. The frequency of NKp46+ NK cells was significantly increased only in CD patients. Among CD4+ lymphocytes and NKp44+ NK cell subsets, the latter were the major source of IL-22 on lamina propria mononuclear cells from AS patients. Significant up-regulation of IL-22, IL-23p19, MUC-1, and STAT-3 transcripts in the terminal ileum of patients with AS was observed. Immunohistochemical analysis confirmed the increased IL-22 and pSTAT-3 expression in inflamed mucosa from AS and CD patients.nnnCONCLUSIONnOur findings indicate that overexpression of IL-22, together with an increased number of IL-22-producing NKp44+ NK cells, occurs in the gut of AS patients, where it appears to play a tissue-protective role.

Longterm Retention of Tumor Necrosis Factor-α Inhibitor Therapy in a Large Italian Cohort of Patients with Rheumatoid Arthritis from the GISEA Registry: An Appraisal of Predictors

Objective. To evaluate 4-year retention rates of tumor necrosis factor-α (TNF-α) inhibitors adalimumab, etanercept, and infliximab among patients with longstanding rheumatoid arthritis (RA), as derived from an Italian national registry. Methods. The clinical records of 853 adult patients with RA in the GISEA (Gruppo Italiano Studio Early Arthritis) registry were prospectively analyzed to compare drug survival rates and the baseline factors that may predict adherence to therapy. Results. In 2003 and 2004, 324 patients started treatment with adalimumab, 311 with etanercept, and 218 with infliximab. After 4 years, the global retention rate of anti-TNF-α therapy was 42%. Etanercept survival (51.4%) was significantly better than that of infliximab (37.6%) or adalimumab (36.4%; p < 0.0001). Accordingly, the mean duration of therapy was significantly longer for etanercept (3.1 ± 2 yrs) than for adalimumab (2.6 ± 2 yrs) or infliximab (2.7 ± 2 yrs; p < 0.05). The use of concomitant disease-modifying antirheumatic drugs, mainly methotrexate, and the presence of comorbidities significantly predicted drug continuation (p < 0.01), whereas a high Disease Activity Score did not. Conclusion. The 4-year global drug survival of adalimumab, etanercept, and infliximab was lower than 50%, with etanercept having the best retention rate. The main positive predictor of adherence to anti-TNF-α therapy was the concomitant use of methotrexate. Our study provides further evidence that the real-life treatment of patients with RA may be different from that of randomized clinical trials.

One year study of efficacy and safety of infliximab in the treatment of patients with ocular and neurological Behçet’s disease refractory to standard immunosuppressive drugs

The aim of the study was to assess the long-term efficacy and safety of Infliximab therapy in the treatment of patients with Behçet’s disease refractory to standard immunosuppressive agents. Twenty-one patients that did not respond to corticosteroids and to at least one immunosuppressant (cyclosporin, methotrexate, azathioprine, cyclophosphamide) for the presence of ocular and/or CNS involvement were enrolled. Eighteen patients completed the study up to 54xa0weeks. Stable doses of prednisone (<10xa0mg/day) were permitted, immunosuppressants were discontinued at least 4xa0weeks prior baseline visit. The patients received three infusions of 5xa0mg/kg Infliximab (at weeks 0, 2 and 6) and then infusions of 5xa0mg/kg Infliximab every 8xa0weeks. At each visit data on clinical symptoms, response to therapy and adverse events were collected. The primary outcome of interest was to assess the clinical efficacy (total or partial recovery) of infliximab. Secondary end points were to evaluate quality of life and to monitor the safety of the drug. Eighteen patients achieved a total remission. Two patients achieved a partial remission and relapsed after 3xa0months from discontinuation of therapy. Infliximab was well tolerated throughout the study. A case of non-Hodgkin lymphoma was observed within 6xa0months. Minor side effects were headache, dizziness, tachycardia that regressed spontaneously and did not entail interruption. Anti-nuclear antibodies were not detected during the period of observation.

IL-34 is overexpressed in the inflamed salivary glands of patients with Sjögren’s syndrome and is associated with the local expansion of pro-inflammatory CD14brightCD16+ monocytes

OBJECTIVESnTo investigate the expression of IL-34 in labial salivary glands (LSGs) of patients with primary SS (p-SS) and its role in inducing a pro-inflammatory monocyte phenotype.nnnMETHODSnLSG biopsies were obtained from 20 patients with p-SS and 10 patients with non-Sjögrens sicca syndrome (n-SS). The expression of IL-34, IL-1β, TNF-α, IL-17 and IL-23 was assessed by real-time PCR. IL-34 expression was also investigated in LSGs by immunohistochemistry. The frequencies of subpopulations of CD14(+) monocytes were evaluated by flow cytometry among isolated mononuclear cells from peripheral blood and salivary glands from both patients and controls. The role of recombinant IL-34 on isolated peripheral blood mononuclear cells was also evaluated.nnnRESULTSnIL-34 m-RNA was overexpressed in the inflamed salivary glands of p-SS and associated with increased expression of TNF-α, IL-1β, IL-17 and IL-23p19. The increased expression of IL-34 was confirmed by immunohistochemistry in paraffin-embedded salivary glands from p-SS patients. IL-34 expression was accompanied by the expansion of pro-inflammatory CD14(bright)CD16(+) monocytes in the salivary glands. In vitro stimulation of peripheral blood mononuclear cells with IL-34 induced the expansion of both CD14(+)CD16(-) cells and CD14(bright)CD16(+) cells in p-SS and non-SS subjects.nnnCONCLUSIONnIL-34 seems to be involved in the pathogenesis of salivary gland inflammation in p-SS.

IL-33 is overexpressed in the inflamed arteries of patients with giant cell arteritis

Objective To study the expression of interleukin (IL)-33 and to evaluate its relationship with macrophage polarisation in artery biopsy specimens from patients with giant cell arteritis (GCA). Methods IL-33, ST2, p-STAT-6 and perivascular IL-1 receptor-associated kinase 1 (p-IRAK1) tissue distribution was evaluated by immunohistochemistry. Inducible nitric oxide synthase and CD163 were also used by immunohistochemistry to evaluate the M1 and M2 polarisation, respectively. Quantitative gene expression analysis of IL-33, T-helper (Th)2-related transcription factor STAT6, Th2 cytokines (IL-4, IL-5, IL-25) and interferon (IFN)-γ was performed in artery biopsy samples obtained from 20 patients with GCA and 15 controls. Five additional patients who had received prednisone when the temporal artery biopsy was performed were also enrolled. Results IFN-γ and IL-33 were significantly overexpressed in the inflamed arteries of GCA patients. IL-33 overexpression was not accompanied by a concomitant increase of Th2 cytokines. Neovessels scattered through the inflammatory infiltrates were the main sites of IL-33 expression. The expression of IL-33 receptor ST2 and of p-IRAK1 was also increased in GCA patients. Arteries from glucocorticoid-treated patients had a lower expression of IL-33. IL-33 was accompanied by the expression of p-STAT6 and a clear M2 macrophages polarisation. Conclusions A role for IL-33 in the inflammation of GCA patients is supported by these findings.