Plasma cells in the mucosa of patients with inflammatory bowel disease produce granzyme B and possess cytotoxic activities.

In both Crohn's disease (CD) and ulcerative colitis (UC), the gut is massively infiltrated with B cells and plasma cells, but the role of these cell types in the pathogenesis of gut tissue damage remains largely unknown. Human B cells express granzyme B (GrB) when cultured with IL-21, a cytokine overproduced in CD and UC mucosa. We therefore examined whether mucosal B cells express GrB and have cytotoxic activity in inflammatory bowel disease (IBD). GrB-expressing CD19(+) and IgA(+) cells were seen in the normal intestinal mucosa, but they were significantly more frequent in both CD and UC. In contrast, only a minority of CD19(+) and IgA(+) cells expressed perforin with no difference between IBD and controls. GrB-producing CD19(+) cells expressed CD27 and were CD38(high) and CD20 negative. CD19(+) B cells from IBD patients induced HCT-116 cell death. IL-21 enhanced GrB expression in control CD19(+) B cells and increased their cytotoxic activity. These data indicate that IBD-related inflammation is marked by mucosal accumulation of cytotoxic, GrB-expressing CD19(+) and IgA(+) cells, suggesting a role for these cells in IBD-associated epithelial damage.

Interleukin-34 sustains inflammatory pathways in the gut.

IBD (inflammatory bowel disease)-related tissue damage occurs in areas which are massively infiltrated with monocytes/macrophages. These cells respond to inflammatory stimuli with enhanced production of cytokines/chemokines. In the present study, we analysed the expression and role of IL (interleukin)-34, a regulator of monocyte/macrophage differentiation, survival and function, in IBD. A significant increase in IL-34 mRNA and protein expression was seen in inflamed mucosa of patients with CD (Crohn's disease) and patients with UC (ulcerative colitis) compared with the uninvolved areas of the same patients and normal controls. IL-34 was up-regulated in LPMCs (lamina propria mononuclear cells) isolated from normal colon by TNF-α (tumour necrosis factor α) and TLR (Toll-like receptor) ligands and was down-regulated in intestinal biopsies and LPMCs of IBD patients upon treatment with infliximab. Treatment of normal LPMCs with IL-34 increased TNF-α expression in an ERK1/2 (extracellular-signal-regulated kinase 1/2)-dependent fashion and neutralization of IL-34 in IBD mucosal explants reduced TNF-α and IL-6 synthesis. In conclusion, our results indicate that IL-34 is up-regulated in IBD and suggest a role for this cytokine in sustaining the inflammatory responses in this disease.

IL-25 prevents and cures fulminant hepatitis in mice through a myeloid-derived suppressor cell-dependent mechanism.

UNLABELLED: Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)-25 in FH. IL-25 expression was evaluated in patients with FH and in livers of mice with FH induced by D-galactosamine (D-Gal) and lipopolysaccharide (LPS). Mice were treated with IL-25 before D-Gal/LPS-induced FH and before or after concanavalin A (ConA)-induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL-25 and analyzed for GR1(+) CD11b(+) cells. CFSE-labeled T cells were cocultured with GR1(+) CD11b(+) cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti-GR1 antibody before IL-25 and D-Gal/LPS administration. IL-25 was constitutively expressed in mouse and human liver and down-regulated during FH. IL-25 prevented D-Gal/LPS-induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1(+) CD11b(+) cells isolated from mice given IL-25 inhibited T-cell proliferation. Consistently, in vivo depletion of GR1(+) cells abrogated the protective effect of IL-25 in experimental D-Gal/LPS-induced FH. IL-25 was both preventive and therapeutic in ConA-induced FH. CONCLUSIONS: IL-25 expression is markedly reduced during human and experimental FH. IL-25 promotes liver accumulation of GR1(+) CD11b(+) cells with immunoregulatory properties.

IL-21 promotes skin recruitment of CD4(+) cells and drives IFN-γ-dependent epidermal hyperplasia.

Psoriasis is a chronic inflammatory disorder of the skin characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. T cell-derived cytokines, such as IFN-γ and IL-17A, play a major role in the psoriasis-associated epidermal hyperplasia, even though factors/mechanisms that regulate the production of these cytokines are not fully understood. We have recently shown that IL-21 is synthesized in excess in psoriatic skin lesions and causes epidermal hyperplasia when injected intradermally in mice. Moreover, in the human psoriasis SCID mouse model, neutralization of IL-21 reduces both skin thickening and expression of inflammatory molecules, thus supporting the pathogenic role of IL-21 in psoriasis. However, the basic mechanism by which IL-21 promotes skin pathology remains unknown. In this study, we show that CD4(+) cells accumulate early in the dermis of IL-21-treated mice and mediate the development of epidermal hyperplasia. Indeed, IL-21 fails to induce skin damage in RAG1-deficient mice and CD4(+) cell-depleted wild-type mice. The majority of CD4(+) cells infiltrating the dermis of IL-21-treated mice express IFN-γ and, to a lesser extent, IL-17A. Studies in cytokine knockout mice show that IFN-γ, but not IL-17A, is necessary for IL-21-induced epidermal hyperplasia. Finally, we demonstrate that IFN-γ-producing CD4(+) cells infiltrating the human psoriatic plaque express IL-21R, and abrogation of IL-21 signals reduces IFN-γ expression in cultures of psoriatic CD4(+) cells. Data indicate that IL-21 induces an IFN-γ-dependent pathogenic response in vivo, thus contributing to elucidate a mechanism by which IL-21 sustains skin-damaging inflammation.

Celiac Disease-Related Inflammation Is Marked by Reduction of Nkp44/Nkp46-Double Positive Natural Killer Cells.

INTRODUCTION AND AIM: Natural killer (NK) cells are a first line of defence against viruses and down-regulation of NK cell cytotoxic receptors represents one of the strategies by which viruses escape the host's immune system. Since onset of celiac disease (CD), a gluten-driven enteropathy, has been associated with viral infections, we examined whether CD-associated inflammation is characterized by abnormal distribution of NK cell receptors involved in recognition of viral-infected cells. MATERIALS AND METHODS: Intraepithelial mononuclear cells, isolated from duodenal biopsies of active and inactive CD patients and healthy controls (CTR) and jejunal specimens of obese subjects undergoing gastro-intestinal bypass, were analysed for NK cell markers by flow-cytometry. Expression of granzyme B, interleukin (IL)-22 and tumor necrosis factor (TNF)-α was as assessed in freshly isolated and toll-like receptor (TLR) ligand-stimulated cells. RESULTS: The percentages of total NK cells and NKT cells did not significantly differ between CD patients and CTR. In active CD, the fractions of NKp30+ NK cells, NKG2D+ NK cells and NKG2D+ NKT cells were significantly increased as compared to inactive CD patients and CTR. In contrast, CD-associated inflammation was marked by diminished presence of NKG2A+ NK cells and NKG2A+ NKT cells. The fractions of NK cells and NKT cells expressing either NKp44 or NKp46 did not differ between CD and controls, but in CD less NK cells and NKT cells co-expressed these receptors. NKp44/NKp46-double positive cells produced granzyme B and IL-22 but not TNF-α and responded to TLR ligands with enhanced expression of granzyme B. CONCLUSIONS: These data indicate that active phase of CD associates with reduced presence of NKp44/NKp46-double positive NK cells and NKT cells in the epithelial compartment.

TNF-α Producing Innate Lymphoid Cells (ILCs) Are Increased in Active Celiac Disease and Contribute to Promote Intestinal Atrophy in Mice.

Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of this study was to characterize the tissue distribution of ILCs in celiac disease (CD), a gluten-driven enteropathy, and analyze their role in gut tissue damage. ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs) isolated from duodenal biopsies of CD patients and healthy controls (CTR) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with peptidoglycan, poly I:C, or CpG, the agonists of TLR2, TLR3, or TLR9 respectively, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Although the percentage of total ILCs did not differ between CD patients and CTR, ILCs producing TNF-α and IFN-γ were more abundant in CD mucosa compared to controls. ILCs expressed TLR2, TLR3 and TLR9 but neither TLR7 nor TLR4. Stimulation of LPMC with poly I:C but not PGN or CpG increased TNF-α and IFN-γ in ILCs. RAG1-deficient mice given poly I:C exhibited increased frequency of TNF-α but not IFN-γ/IL17A-producing ILCs in the gut and depletion of ILCs prevented the poly I:C-driven intestinal damage. Our data indicate that CD-related inflammation is marked by accumulation of ILCs producing TNF-α and IFN-γ in the mucosa. Moreover, ILCs express TLR3 and are functionally able to respond to poly I:C with increased synthesis of TNF-α thus contributing to small intestinal atrophy.

Defective expression of scavenger receptors in celiac disease mucosa.

Celiac disease (CD) is a gluten sensitive enteropathy characterized by a marked infiltration of the mucosa with immune cells, over-production of inflammatory cytokines and epithelial cell damage. The factors/mechanisms that sustain and amplify the ongoing mucosal inflammation in CD are not however fully understood. Here, we have examined whether in CD there is a defective clearance of apoptotic cells/bodies, a phenomenon that helps promote tolerogenic signals thus liming pathogenic responses. Accumulation of apoptotic cells and bodies was more pronounced in the epithelial and lamina propria compartments of active CD patients as compared to inactive CD patients and normal controls. Expression of scavenger receptors, which are involved in the clearance of apoptotic cells/bodies, namely thrombospondin (TSP)-1, CD36 and CD61, was significantly reduced in active CD as compared to inactive CD and normal mucosal samples. Consistently, lamina propria mononuclear cells (LPMC) of active CD patients had diminished ability to phagocyte apoptotic cells. Interleukin (IL)-15, IL-21 and interferon-γ, cytokines over-produced in active CD, inhibited the expression of TSP-1, CD36, and CD61 in normal intestinal LPMC. These results indicate that CD-related inflammation is marked by diminished clearance of apoptotic cells/bodies, thus suggesting a role for such a defect in the ongoing mucosal inflammation in this disorder.

High expression of the "A Disintegrin And Metalloprotease" 19 (ADAM19), a sheddase for TNF-α in the mucosa of patients with inflammatory bowel diseases.

BACKGROUND: Tumor necrosis factor α (TNF-α) plays a major role in the tissue-damaging immune response in inflammatory bowel diseases (IBDs). The tissue concentration of TNF-α is related to the activity of "A Disintegrin And Metalloprotease" (ADAMs), enzymes that process membrane-bound TNF-α and liberate the TNF-α trimer into the extracellular environment. Although IBD-related inflammation is associated with high ADAM17 levels, the contribution of other members of the ADAMs family is not known. In this study, we characterized the expression of other TNF-α convertases (i.e., ADAM9, ADAM10, and ADAM19) in IBD. METHODS: Normal and IBD biopsies were examined for the content of ADAMs by real-time polymerase chain reaction, Western blotting and immunohistochemistry. ADAM19 was also analyzed in intestinal epithelial cells and normal colonic explants stimulated with inflammatory cytokines and in ex vivo biopsies taken from IBD patients before and after a successful infliximab treatment. RESULTS: ADAM19 RNA transcripts and protein were upregulated in patients with ulcerative colitis and, to a lesser extent, in patients with Crohn's disease compared with normal controls. In contrast, ADAM9 and ADAM10 expression did not differ between patients with IBD and controls. Immunohistochemical analysis showed that epithelial cells were the major source of ADAM19 in IBD. ADAM19 expression was increased in colonic epithelial cell lines and normal colonic explants by TNF-α, interleukin 21 and interleukin 6, and was downregulated in IBD tissue by infliximab. CONCLUSIONS: These findings suggest the existence of a positive feedback mechanism involving cytokines and ADAM19 that can amplify cytokine production in IBD.

Lesional accumulation of CD163-expressing cells in the gut of patients with inflammatory bowel disease.

Monocytes/macrophages displaying different markers of activation/differentiation infiltrate the inflamed gut of patients with inflammatory bowel diseases (IBD), but the role that each monocyte/macrophage subpopulation plays in the pathogenesis of IBD is not fully understood. The hemoglobin scavenger receptor CD163, a specific marker of monocytes/macrophages, has been associated with either anti-inflammatory or inflammatory functions of macrophages in several pathologies. In this study we examined the tissue distribution and function of CD163-expressing monocytes/macrophages in IBD. CD163 RNA and protein expression was more pronounced in IBD in comparison to normal controls, with no significant difference between Crohn's disease and Ulcerative colitis. In IBD, over-expression of CD163 was restricted to areas with active inflammation and not influenced by current therapy. Immunohistochemical analysis confirmed the accumulation of CD163-expressing cells in IBD, mostly around and inside blood vessels, thus suggesting that these cells are partly recruited from the systemic circulation. Indeed, FACS analysis of circulating mononuclear cells showed that the fractions of CD163-positive monocytes were increased in IBD patients as compared to controls. Functionally, interleukin-6 up-regulated CD163 expression in lamina propria mononuclear cells and mucosal explants of normal subjects. In IBD blood and mucosal cell cultures, cross-linking of CD163 with a specific monoclonal anti-CD163 antibody enhanced tumor necrosis factor-α synthesis. These findings indicate that IBD mucosa is abundantly infiltrated with CD163-positive cells, which could contribute to amplify the inflammatory cytokine response.

Interleukin-21 in immune and allergic diseases.

Interleukin-21 (IL-21), a cytokine produced by various subsets of activated CD4+ T cells, plays a major role in the control of innate and adaptive immune responses. IL-21 biological activity is mediated by binding of the cytokine to a heterodimeric receptor, composed of a specific subunit, termed IL-21 receptor (IL-21R), and the common γ-chain, that is shared with IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. IL-21 stimulates the proliferation of CD4+ and CD8+ T lymphocytes and regulates the profile of cytokines secreted by these cells, drives the differentiation of B cells into memory cells and Ig-secreting plasma cells, and enhances the activity of natural killer cells. IL-21 controls also the activity of non-immune cells, such as epithelial cells and stromal cells. The demonstration that IL-21 is involved in the immune responses occurring in chronic inflammatory and allergic diseases suggests that either disrupting or enhancing IL-21 signalling may be useful in specific clinical settings.

Targeting IL-23 and Th17-cytokines in inflammatory bowel diseases.

Over the last 15 years, the use of various biological therapies has largely improved the way we manage patients with Inflammatory Bowel Diseases (IBDs). Blockade of cytokine synthesis and/or activity is at the forefront of this new era with the success of inhibitors of tumor necrosis factor (TNF)-α. These therapies are however not effective in all IBD patients and efficacy may wane. Moreover, patients treated with anti-TNF-α antibodies can develop severe side-effects and new immune-mediated diseases. Therefore, a new challenge is to elucidate new inflammatory networks in the IBD tissue and develop novel anti-cytokine compounds, which may act in patients who are resistant to or cannot receive anti-TNF-α therapies. In this article we review the available data supporting the pathogenic role of IL-23 and Th17-related cytokines in IBD, and discuss whether and how compounds that control the activity of these cytokines may enter into the therapeutic armamentarium of IBD.