Effects of a gut-selective integrin-targeted therapy in male mice exposed to early immune activation, a model for the study of autism spectrum disorder.

To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal antibody directed against the integrin alpha4 beta7 (α4ß7 mAb), blocking the leukocyte homing into the gut mucosa. EIA is a double-hit variant of the maternal immune-activation (MIA) model, including both prenatal (Poly I:C) and postnatal (LPS) immune challenges. In C57BL6/J EIA male adult offspring mice, IL-1ß and IL-17A mRNA colonic tissue content increased when compared with controls. Cytofluorimetric analyses of lymphocytes isolated from mesenteric lymph-nodes (MLN) and spleens of EIA mice show increased percentage of total and CD4+α4ß7+, unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in MLN and CD4+α4ß7+ unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in the spleen. Treatment with anti-α4ß7 mAb in EIA male mice was associated with colonic tissue IL-1ß, and IL-17A mRNA content and percentage of CD4+ IL-17A+ and IFN-γ+ lymphocytes in MLN and spleens comparable to control mice. The anti-α4ß7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1ß and decreased CD68 and TGF-ß mRNAs were also observed in hippocampus and prefrontal cortex of EIA male mice together with a reduction of BDNF mRNA levels in all brain regions examined. Anti-α4ß7 mAb treatment restored the expression of BDNF, TGF-ß and CD68 in hippocampus and prefrontal cortex. Improvement of the gut inflammatory status, obtained by a pharmacological agent acting exclusively at gut level, ameliorates some ASD behavioral features and the neuroinflammatory status. Data provide the first preclinical indication for a therapeutic strategy against gut-immune activation in ASD subjects with peripheral increase of gut-derived (α4ß7+) lymphocytes expressing IL-17A.

CXCL4-RNA Complexes Circulate in Systemic Sclerosis and Amplify Inflammatory/Pro-Fibrotic Responses by Myeloid Dendritic Cells.

CXCL4 is an important biomarker of systemic sclerosis (SSc), an incurable autoimmune disease characterized by vasculopathy and skin/internal organs fibrosis. CXCL4 contributes to the type I interferon (IFN-I) signature, typical of at least half of SSc patients, and its presence is linked to an unfavorable prognosis. The mechanism implicated is CXCL4 binding to self-DNA, with the formation of complexes amplifying TLR9 stimulation in plasmacytoid dendritic cells (pDCs). Here, we demonstrate that, upon binding to self-RNA, CXCL4 protects the RNA from enzymatic degradation. As a consequence, CXCL4-RNA complexes persist in vivo. Indeed, we show for the first time that CXCL4-RNA complexes circulate in SSc plasma and correlate with both IFN-I and TNF-α. By using monocyte-derived DCs (MDDCs) pretreated with IFN-α as a model system (to mimic the SSc milieu of the IFN-I signature), we demonstrate that CXCL4-RNA complexes induce MDDC maturation and increase, in particular, pro-inflammatory TNF-α as well as IL-12, IL-23, IL-8, and pro-collagen, mainly in a TLR7/8-dependent but CXCR3-independent manner. In contrast, MDDCs produced IL-6 and fibronectin independently in their CXCL4 RNA-binding ability. These findings support a role for CXCL4-RNA complexes, besides CXCL4-DNA complexes, in immune amplification via the modulation of myeloid DC effector functions in SSc and also during normal immune responses.

CD147 Targeting by AC-73 Induces Autophagy and Reduces Intestinal Fibrosis Associated with TNBS Chronic Colitis.

BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of inflammatory bowel diseases. Medical treatment of intestinal fibrosis is an unmet therapeutic need. CD147 overexpression can induce myofibroblast differentiation associated with extracellular matrix deposition, favouring the development of fibrosis. To understand whether CD147 may promote intestinal fibrosis, we analysed its expression and blocked its function by using its specific inhibitor AC-73 [3-{2-[([1,1'-biphenyl]-4-ylmethyl) amino]-1-hydroxyethyl} phenol] in the murine TNBS [trinitrobenzenesulfonic acid]-chronic colitis model associated with intestinal fibrosis. METHODS: TNBS chronic colitis was induced by weekly intrarectal administration of escalating doses of TNBS. Ethanol-treated and untreated mice were used as controls. Separated groups of TNBS, ethanol-treated or untreated mice received AC-73 or vehicle administered intraperitoneally from day 21 to day 49. At day 49, mice were killed, and colons collected for histological analysis, protein and RNA extraction. CD147, α-SMA and activated TGF-ß1 protein levels, CD147/ERK/STAT3 signalling pathway and autophagy were assessed by Western blot, collagen and inflammatory/fibrogenic cytokines mRNA tissue content by quantitative PCR. RESULTS: In mice with chronic TNBS colitis, CD147 protein level increased during fibrosis development in colonic tissue, as compared to control mice. CD147 inhibition by AC-73 treatment reduced intestinal fibrosis, collagen and cytokine mRNA tissue content, without significant modulation of activated TGF-ß1 protein tissue content. AC-73 inhibited CD147/ERK1/2 and STAT3 signalling pathway activation and induced autophagy. CONCLUSIONS: CD147 is a potential new target for controlling intestinal fibrosis and its inhibitor, AC-73, might represent a potential new anti-fibrotic therapeutic option in IBD.

Diltiazem induces regulatory T cells in vitro by modulating human dendritic cell maturation.

Diltiazem is a calcium channel antagonist that has been commonly associated with currently used immunosuppressants to prevent acute graft rejection in humans. In this study, we examined the possibility that diltiazem may affect human dendritic cell (DC) functions in response to lipopolysaccharide (LPS) stimulation and may induce the generation of DC with the capacity to generate CD4(+) regulatory T cells (Tregs). Blood monocytes were cultured in the presence of diltiazem at the beginning of their differentiation process into DC. Monocyte-derived DCs were stimulated with LPS, and DCs differentiated in the presence of diltiazem showed a decreased interleukin (IL)-12 production and an enhanced IL-10 production. When cultured with CD4(+) CD45RA(+) they were able to enhance the CD4(+) Foxp3(+) T-cell population and to induce slowly proliferating T cells, which showed a significant increase of transforming growth factor (TGF)-ß production. These T cells suppress proliferation of activated autologous T cells, and we show that this effect is attributable to soluble factors, primarily to TGF-ß. Blockade of TGF-ß by specific monoclonal antibodies reversed this inhibitory effect. Herein, we provide new evidence that diltiazem-conditioned monocyte-derived DC induce T cells which acquire a regulatory phenotype and activity similar to those described for Tregs.