A. Rizzo

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.

Growth factor and procollagen type I gene expression in human liver disease

BACKGROUND/AIMSnGrowth factors have been implicated in the pathogenesis of liver fibrosis, a major determinant of the clinical course of chronic liver disease. The aim of this study was to study the relationship of growth factor expression to inflammation and fibrosis in a variety of human liver diseases.nnnMETHODSnWe studied by in situ hybridization the expression of transforming growth factor (TGF) beta 1, platelet-derived growth factor (PDGF) A and PDGF-B, and procollagen type I (pro-I) messenger RNAs (mRNAs) in liver diseases of various etiologies.nnnRESULTSnPro-I mRNA was expressed by mesenchymal cells at sites of inflammation and scarring, where TGF-beta 1 immunoreactivity was often found, and by perisinusoidal cells. TGF-beta 1 and PDGF-A mRNAs were expressed mainly by mononuclear cells and proliferating ductular cells. TGF-beta 1 mRNA was also expressed by perisinusoidal cells. PDGF-A gene expression was more common than that of PDGF-B. Pro-I and TGF-beta 1 expression correlated with both ductular proliferation and tissue inflammation, whereas PDGF-A and PDGF-B only correlated with ductular proliferation.nnnCONCLUSIONSnOur data suggest that TGF-beta 1 and PDGF are involved in human liver inflammation and fibrosis. The expression of growth factor mRNAs in proliferating ductular cells may indicate a role for these cells in liver fibrogenesis and may help explain the pathophysiology of conditions such as biliary atresia progressing to fibrosis despite the absence of marked inflammation.

Bone marrow biopsy in Hodgkin's lymphoma

Abstract:u2002 In the study of patients with Hodgkins lymphoma (HL) the evaluation of bone marrow biopsy (BMB) can be difficult. In this review we analyze the main diagnostic features and the clinical risk factors of BM involvement. Although the role of BMB is criticized by some authors, its value is irreplaceable in the staging of HL and in the diagnosis of primary medullary HL. The Ann Arbor staging committee criteria should be revised and updated in the light of the current immunohistochemical studies that give a fundamental help in the diagnostic process. A single BMB should be adequate for diagnosis in most instances. In cases of suspicious involvement a controlateral BMB could be performed.

Rituximab modulates IL-17 expression in the salivary glands of patients with primary Sjögren’s syndrome

OBJECTIVEnThe aim of this study was to evaluate the role of rituximab (RTX) in modulating the expression of the IL-17/IL-23 pathway in the salivary glands (SGs) of patients with primary SS (pSS).nnnMETHODSnConsecutive SG biopsies were obtained from 15 patients with pSS before and after 1 year of RTX therapy. The SG expression of IL-17, IL-23p19 and p-STAT3 was evaluated by immunohistochemistry at baseline and after RTX therapy. The role of mast cells in pSS patients in modulating the Th17 response and the immunologic effect of RTX on mast cells were also studied in in vitro experiments.nnnRESULTSnIL-17 was overexpressed in the SGs of patients with pSS mainly by infiltrating T cells and mast cells. After RTX therapy, the SG expression of IL-17, but not of IL-23p19 and p-STAT3, was significantly reduced and was accompanied by the depletion of tissue mast cells. In in vitro experiments with heterologous peripheral lymphocytes RTX significantly induced the apoptosis of isolated mast cells. Finally, mast cells isolated from peripheral blood mononuclear cells of pSS patients in vitro significantly increased Th17 lymphocytes.nnnCONCLUSIONnRTX acts on pSS patients by globally reducing the expression of IL-17 and specifically inducing a pronounced apoptotic depletion of mast cells.

Interleukin-36α axis is modulated in patients with primary Sjögren's syndrome.

The aim of this study was to investigate the expression of the interleukin (IL)‐36 axis in patients with primary Sjögrens syndrome (pSS). Blood and minor labial salivary glands (MSG) biopsies were obtained from 35 pSS and 20 non‐Sjögrens syndrome patients (nSS) patients. Serum IL‐36α was assayed by enzyme‐linked immunosorbent assay (ELISA). IL‐36α, IL‐36R, IL‐36RA, IL‐38, IL‐22, IL‐17, IL‐23p19 and expression in MSGs was assessed by reverse transcription–polymerase chain reaction (RT–PCR), and tissue IL‐36α and IL‐38 expression was also investigated by immunohistochemistry (IHC). αβ and γδ T cells and CD68+ cells isolated from MSGs were also studied by flow cytometry and confocal microscopy analysis. IL‐36α was over‐expressed significantly in the serum and in the salivary glands of pSS. Salivary gland IL‐36α expression was correlated with the expression levels of IL‐17, IL‐22 and IL‐23p19. IL‐38, that acts as inhibitor of IL‐36α, was also up‐regulated in pSS. αβ+ CD3+ T cells and CD68+ cells were the major source of IL‐36α in minor salivary glands of pSS. γδ T cells were not significantly expanded in the salivary glands of pSS but produced more IL‐17, as their percentage correlated with the focus score. Higher expression of IL‐36α and IL‐36R was also demonstrated in γδ T cells isolated from pSS compared to controls. In this study we demonstrate that a significant increase in circulating and tissue levels of IL‐36α occurs in pSS patients.

INFLAMMATION IN IRRITABLE BOWEL SYNDROME: MYTH OR NEW TREATMENT TARGET?

Low-grade intestinal inflammation plays a key role in the pathophysiology of irritable bowel syndrome (IBS), and this role is likely to be multifactorial. The aim of this review was to summarize the evidence on the spectrum of mucosal inflammation in IBS, highlighting the relationship of this inflammation to the pathophysiology of IBS and its connection to clinical practice. We carried out a bibliographic search in Medline and the Cochrane Library for the period of January 1966 to December 2014, focusing on publications describing an interaction between inflammation and IBS. Several evidences demonstrate microscopic and molecular abnormalities in IBS patients. Understanding the mechanisms underlying low-grade inflammation in IBS may help to design clinical trials to test the efficacy and safety of drugs that target this pathophysiologic mechanism.

Macrophage phenotype in the subclinical gut inflammation of patients with ankylosing spondylitis

OBJECTIVEnLong-term evolution of subclinical gut inflammation to overt Crohns disease (CD) has been described in AS patients. The aim of this study was to evaluate macrophage polarization occurring in the inflamed gut of patients with AS.nnnMETHODSnTwenty-seven HLA-B27(+) AS patients, 20 CD patients and 17 normal controls were consecutively enrolled. Classic M1 (iNOS(+)IL-10(-)), resolution phase (iNOS(+)IL-10(+)), M2 and CD14(+) macrophages were characterized by immunohistochemistry and flow cytometry. Quantitative gene expression analysis of IFN-γ, IL-4, IL-5, IL-33 and STAT6 was performed by real time PCR.nnnRESULTSnClassic M1 macrophages were expanded in CD and AS, where resolution phase macrophages predominate. A large increase in CD163(+) (M2) macrophages was observed in AS strictly correlated with the expression of IL-33, a Th2 cytokine involved in M2 polarization. Unlike in CD, CD14(+) macrophages were virtually absent in the gut of AS patients and controls.nnnCONCLUSIONnThe absence of CD14(+) macrophages together with the expansion of resolution phase and M2 macrophages is the immunological signature of subclinical ileal inflammation in AS.

Over-expression of paneth cell-derived anti-microbial peptides in the gut of patients with ankylosing spondylitis and subclinical intestinal inflammation

OBJECTIVESnSubclinical gut inflammation has been demonstrated in patients with AS. Altered expression of paneth cell (PC) anti-microbial peptides have been reported in the inflamed ileum of patients with Crohns disease (CD). Here, we investigated the expression of PC-derived peptides in subclinical gut inflammation in AS.nnnMETHODSnMultiple adjacent mucosal biopsies from terminal ileum were obtained from 25 patients with AS, 30 CD and 15 healthy controls (HCs). Expression of human α-defensin 5 (HD-5), phospholipase A2 (PLA2), lysozyme and SOX-9 molecules was assessed by quantitative Taqman RT-PCR on mucosal samples. Immunohistochemistry with anti-human HD-5 antibody and genotyping of relevant NOD2 mutations was also performed.nnnRESULTSnHD-5, PLA2 and lysozyme transcript levels were strongly increased in AS and CD with similar degrees of intestinal inflammation when compared with normal controls. Immunohistochemical evaluation showed a normal number of PCs in both AS patients with chronic gut inflammation and CD patients with less-inflamed ileal samples. Conversely, CD patients with higher degree of gut inflammation had a reduced number of PCs and low expression levels of HD-5.nnnCONCLUSIONnIn this study, we provide evidence that over-expression of PC-derived anti-microbial peptides occurs in the ileum of AS patients with subclinical gut inflammation, likely representing an important early alteration of the mucosal innate immune component and intestinal host defence in AS.

Interleukin (IL)-22 receptor 1 is over-expressed in primary Sjogren's syndrome and Sjögren-associated non-Hodgkin lymphomas and is regulated by IL-18.

The aim of this study was to elucidate more clearly the role of interleukin (IL)‐18 in modulating the IL‐22 pathway in primary Sjögrens syndrome (pSS) patients and in pSS‐associated lymphomas. Minor salivary glands (MSGs) from patients with pSS and non‐specific chronic sialoadenitis (nSCS), parotid glands biopsies from non‐Hodgkin lymphomas (NHL) developed in pSS patients, were evaluated for IL‐18, IL‐22, IL‐22 receptor 1 (IL‐22R1), IL‐22 binding protein (IL‐22BP) and signal transducer and activator of transcription‐3 (STAT‐3) expression. MSGs IL‐22R1‐expressing cells were characterized by confocal microscopy and flow cytometry in pSS, nSCS and healthy controls . The effect of recombinant IL‐18 and IL‐22 on peripheral blood mononuclear cells (PBMCs) from pSS and nSCS was studied by flow cytometry and reverse transcription–polymerase chain reaction (RT‐PCR). MSGs of pSS and NHL were characterized by an imbalance between IL‐22 and IL‐22BP protein expression, with IL‐18 and IL‐22BP being expressed in a mutually exclusive manner and IL‐18 and IL‐22R1 being correlated directly. Aberrant expression of IL‐22R1, induced by IL‐18, was observed only among tissue and circulating myeloid cells of pSS patients and macrophages of NHL tissues of pSS patients, but not nSCS. IL‐22R1 expression on PBMC of pSS was functional, as its stimulation with recombinant IL‐22 significantly up‐regulated the expression of STAT‐3, IL‐17 and IL‐22. An IL‐18‐dependent aberrant expression of IL‐22R1 on cells of haematopoietic origin seems to be a specific immunological signature of patients with pSS and pSS‐associated lymphomas.

Increased expression of interleukin-32 in the inflamed ileum of ankylosing spondylitis patients

OBJECTIVEnTo study the mRNA expression and protein tissue distribution of IL-32 in ileal biopsy specimens from patients with AS.nnnMETHODSnQuantitative gene expression analysis, by real-time PCR, of IL-32, IL-1β, IL-10, TNF-α and IFN-γ was performed on ileal biopsies of 15 AS and 15 Crohns disease (CD) patients and 10 healthy subjects (HSs). IL-32 tissue distribution was evaluated by immunohistochemistry. The effect of IL-32 on the production of IL-10 by intestinal epithelial cell lines was also evaluated.nnnRESULTSnIn the ileal specimens of patients with AS and intestinal chronic inflammation, significant up-regulation of IL-32 at both the mRNA and protein levels was found as compared with non-inflamed AS patients and controls. IL-32 over-expression in AS was accompanied by a significant increase of IL-10 but not of cytokines involved in IL-32 induction. IL-32 stimulates intestinal epithelial cell lines in vitro to produce IL-10.nnnCONCLUSIONnOur findings suggest IL-32 as an important cytokine probably involved in the innate immune response occurring in early phases of intestinal inflammation, where it seems to play a prevalent protective role.