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 (α4β7 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-1β 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+α4β7+, unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in MLN and CD4+α4β7+ unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in the spleen. Treatment with anti-α4β7 mAb in EIA male mice was associated with colonic tissue IL-1β, 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-α4β7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1β and decreased CD68 and TGF-β 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-α4β7 mAb treatment restored the expression of BDNF, TGF-β 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 (α4β7+) lymphocytes expressing IL-17A.

Abstract 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-β1 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-β1 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.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by social communication deficits and repetitive/stereotyped behaviours. We evaluated the effects of a chronic treatment with the immunomodulator drug Fingolimod (FTY720 - a non-selective Sphingosine 1-Phosphate Receptor ligand) in an ASD model, the BTBR T(+)tf/J (BTBR) mouse strain. In adult BTBR males, chronic FTY720 treatment (4 weeks) increased social and vocal response during a male-female interaction and hippocampal expression of BDNF and Neuregulin 1, two trophic factors reduced in BTBR when compared to control C57 mice. FTY720 also re-established the expression of IL-1 beta and MnSOD in the hippocampus, whereas it did not modify IL-6 mRNA content. In addition to its central effect, FTY720 modulated the activation state of peripheral macrophages in the BTBR model, both in basal conditions and after stimulation with an immune challenge. Furthermore, IL-6 mRNA colonic content of BTBR mice, reduced when compared with C57 mice, was normalized by chronic treatment with FTY720. Our study, while indicating FTY720 as a tool to attenuate relevant alterations of the BTBR neurobehavioural phenotype, emphasizes the importance of gut mucosal immune evaluation as an additional target that deserve to be investigated in preclinical studies of anti-inflammatory therapeutic approaches in ASD. (C) 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

LL37 exerts a dual pathogenic role in psoriasis. Bound to self-DNA/RNA, LL37 licenses autoreactivity by stimulating plasmacytoid dendritic cells-(pDCs)-Type I interferon (IFN-I) and acts as autoantigen for pathogenic Th17-cells. In systemic lupus erythematosus (SLE), LL37 also triggers IFN-I in pDCs and is target of pathogenic autoantibodies. However, whether LL37 activates T-cells in SLE and how the latter differ from psoriasis LL37-specific T-cells is unknown. Here we found that 45% SLE patients had circulating T-cells strongly responding to LL37, which correlate with anti-LL37 antibodies/disease activity. In contrast to psoriatic Th17-cells, these LL37-specific SLE T-cells displayed a T-follicular helper-(TFH)-like phenotype, with CXCR5/Bcl-6 and IL-21 expression, implicating a role in stimulation of pathogenic autoantibodies. Accordingly, SLE LL37-specific T-cells promoted B-cell secretion of pathogenic anti-LL37 antibodies in vitro. Importantly, we identified abundant citrullinated LL37 (cit-LL37) in SLE tissues (skin and kidney) and observed very pronounced reactivity of LL37-specific SLE T-cells to cit-LL37, compared to native-LL37, which was much more occasional in psoriasis. Thus, in SLE, we identified LL37-specific T-cells with a distinct functional specialization and antigenic specificity. This suggests that autoantigenic specificity is independent from the nature of the autoantigen, but rather relies on the disease-specific milieu driving T-cell subset polarization and autoantigen modifications.

Systemic sclerosis (SSc) is characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an SSc biomarker, predicting unfavorable prognosis and lung fibrosis. CXCL4 binds DNA/RNA and favors interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs), contributing to the type I IFN (IFN-I) signature in SSc patients. However, whether CXCL4 is an autoantigen in SSc is unknown. Here, we show that at least half of SSc patients show consistent antibody reactivity to CXCL4. T-cell proliferation to CXCL4, tested in a limited number of patients, correlates with anti-CXCL4 antibody reactivity. Antibodies to CXCL4 mostly correlate with circulating IFN-α levels and are significantly higher in patients with lung fibrosis in two independent SSc cohorts. Antibodies to CXCL4 implement the CXCL4–DNA complex’s effect on IFN-α production by pDCs; CXCL4–DNA/RNA complexes stimulate purified human B-cells to become antibody-secreting plasma cells in vitro. These data indicate that CXCL4 is indeed an autoantigen in SSc and suggest that CXCL4, and CXCL4-specific autoantibodies, can fuel a harmful loop: CXCL4–DNA/RNA complexes induce IFN-α in pDCs and direct B-cell stimulation, including the secretion of anti-CXCL4 antibodies. Anti-CXCL4 antibodies may further increase pDC stimulation and IFN-α release in vivo, creating a vicious cycle which sustains the SSc IFN-I signature and general inflammation.

Background and Aims: A personalized approach to therapy hold great promise to improve disease outcomes. To this end, the identification of different subsets of patients according to the prevalent pathogenic process might guide the choice of therapeutic strategy. We hypothesize that ulcerative colitis [UC] patients might be stratified according to distinctive cytokine profiles and/or to a specific mucosa-associated microbiota. Methods: In a cohort of clinically and endoscopic active UC patients and controls, we used quantitative PCR to analyse the mucosal cytokine mRNA content and 16S rRNA gene sequencing to assess the mucosa-associated microbiota composition. Results: We demonstrate, by means of data-driven approach, the existence of a specific UC patient subgroup characterized by elevated IL-13 mRNA tissue content separate from patients with low IL-13 mRNA tissue content. The two subsets differ in clinical-pathological characteristics. High IL-13 mRNA patients are younger at diagnosis and have a higher prevalence of extensive colitis than low IL-13 mRNA patients.They also show more frequent use of steroid/immunosuppressant/anti-tumour necrosis factor alpha therapy during 1 year of follow-up. The two subgroups show differential enrichment of mucosa-associated microbiota genera with a prevalence of Prevotella in patients with high IL-13 mRNA tissue content and Suiterella and Acidaminococcus in patients with low IL-13 mRNA tissue content. Conclusion: Assessment of mucosal IL-13 mRNA might help in the identification of a patient subgroup that might benefit from a therapeutic approach modulating IL-13.

Background and Aims: In ulcerative colitis (UC), inflammation begins in the rectum and can extend proximally throughout the entire colon. The extension of inflammation is an important determinant of disease course, and may be limited by the action of regulatory T cells (Tregs). In this cross-sectional study, we evaluated the relationship between UC extension and the proportions of CD3+CD4+Foxp3+ and CD3+CD4+LAP+Foxp3-Tregs in the colonic lamina propria (LP) of 79 UC patients and 29 controls. The role of these cells in UC extension was also investigated in the murine oxazolone-induced colitis model. Methods: Patients: Disease extension was classified according to the Montreal classification. Where possible, endoscopic biopsies of involved and uninvolved tissue were obtained from UC patients. Mouse model: Colitis was induced by intrarectal oxazolone administration. Lamina propria mononuclear cells were isolated from patient biopsies and mouse colon tissue using enzymatic method and the percentage of CD3+CD4+Foxp3+ and CD3+CD4+LAP+Foxp3-cells evaluated by immunofluorescence. Confocal microscopy was applied for the visualization and quantification of CD4+LAP+ cells on tissue histological sections. Results: In UC patients with distal colitis the proportion of LP CD3+CD4+Foxp3+ Tregs was significantly higher in inflamed tissue than uninvolved tissue. As opposite, the proportion of LP CD3+CD4+LAP+ Tregs was significantly higher in uninvolved tissue than involved tissue. Both LP CD3+CD4+Foxp3+ and LP CD3+CD4+LAP+ Tregs proportion in involved tissue was significantly higher than in controls irrespective of the extension of inflammation. In mice with oxazolone-induced distal colitis, treatment with LAP-depleting antibody was associated with the development of extensive colitis. Conclusions: Our findings suggest that CD3+CD4+LAP+Foxp3-Tregs limit the extension of inflammatory lesions in UC patients.

Nucleotide-binding Oligomerization Domain-2 (NOD2) mutations are associated with an increased risk to develop Crohn’s Disease. In previous studies, we have shown that Nod2−/− mice manifest increased proportion of Lamina Propria (LP) CD4+ LAP+ Foxp3− regulatory cells, when compared with Nod2+/+ mice, while CD4+ Foxp3 + regulatory cells were not affected. Here, we investigated the Nod2 gut microbiota, by 16S rRNA pyrosequencing, at steady state and after TNBS-colitis induction in mice reared separately or in cohousing, correlating the microbial profiles with LP regulatory T cells proportion and tissue cytokines content. We found that enrichment of Rikenella and Alistipes (Rikenellaceae) in Nod2−/− mice at 8 weeks of age reared separately was associated with increased proportion of CD4+ LAP+ Foxp3− cells and less severe TNBS-colitis. In co-housed mice the acquisition of Rickenellaceae by Nod2+/+ mice was associated with increased CD4+ LAP+ Foxp3− proportion and less severe colitis. Severe colitis was associated with enrichment of gram-negative pathobionts (Escherichia and Enterococcus), while less severe colitis with protective bacteria (Barnesiella, Odoribacter and Clostridium IV). Environmental factors acting on genetic background with different outcomes according to their impact on microbiota, predispose in different ways to inflammation. These results open a new scenario for therapeutic attempt to re-establish eubiosis in Inflammatory Bowel Disease patients with NOD2 polymorphisms.

Objectives and study Ulcerative colitis (UC) and Crohn's disease (CD) are complex chronic disorders commonly known as inflammatory bowel disease (IBD).It is known that chronic or unresolved inflammation can result in an activated epithelial mesenchymal transition (EMT) that may lead to tissue destruction and fibrosis, both representing a common and severe complication in IBD patients.Specific molecular processes are engaged to initiate EMT, including the activation of several transcription factors.Recently, the zinc finger transcription factor ZNF281 (also known as ZBP-99 or ZNP-99) has been involved in EMT and cancer promotion in colorectal cancer (CRC).The aims of the present study are to investigate: 1) in vitro and ex vivo the expression levels of ZNF281 in intestinal inflammation and 2) in vitro the role of ZNF281 in EMT induced by inflammation.Methods ZNF281 protein expression was analyzed by western blot in mucosal biopsies from inflamed and uninflamed colon of pediatric IBD patients (29 CD, 25 UC) and 16 healthy controls as well as in HT29 cells exposed to cytomix (TNF-alpha + INF-gamma) for 24 and 48 hours.ZNF281 mRNA was also analyzed in HT29 cells exposed to cytomix, by real-time PCR.Furthermore, ZNF281 was silenced and EMT (Snail, Timp1, ColA31, Slug) as well as inflammatory cytokine (IL8, IL17, IL23, IL1beta) genes were evaluated by real-time PCR.Results ZNF281 protein levels were significantly increased in inflamed colon of CD and UC patients as compared to uninflamed tissue of patients and controls (p<0.001).Similarly, ZNF281 protein and mRNA levels were significantly increased in HT29 cells exposed to cytomix compared to unexposed (p<0,05).Moreover, mRNA levels of Snail, Timp1, ColA31, Slug, IL8, IL17, IL23 and IL1beta were strongly decreased in ZNF281-silenced cells exposed to cytomix as compared to controls (cells treated with cytomix and scramble siRNA).Conclusions We show for the first time that zinc finger transcription factor ZNF281, previously known for its role in EMT, is importantly increased in gut inflammation and its deletion causes a fall in expression levels of EMT and inflammatory genes in inflamed cells.We suggest that ZNF281 may actively contribute to fibrosis development during IBD.

Background: A personalized approach to therapy has great promise to improve disease outcomes. Selection of patients as candidates for the early introduction of highly effective therapy can both maximize treatment efficiency and prevent long-term complications. Ulcerative Colitis (UC) has been associated with an atypical Th2 cell response mediated inflammation, but recent findings showed that Th17 response also participates in the inflammatory process. Although some evidences provided a proof-of-concept that IL-13 is an effector cytokine in UC, administration of anti-human IL-13 neutralizing monoclonal antibody, did not significantly improve clinical response vs placebo in UC patients. However, in the same study, the proportion of patients who achieved clinical remission was statistically significantly higher in the anti-IL13 treated group compared with the placebo group, suggesting that UC patients' subgroups might exist. We hypothesize that UC patients might be stratified according to distinctive cytokine profiles Methods: We analyzed tissue cytokine in endoscopic biopsies of 40 UC outpatients (27 males, 13 females, disease duration 117±95.61 (mean±SD) months; median: 108 (range 0–384) months) undergoing colonoscopy for clinical relapse. At the time of endoscopy patients Mayo endoscopic score was 2.07±0.74 and 2 (1–3); mean±SD and median (range), respectively. We quantified by RT-qPCR TNF-α, IFN-γ, IL-17 and IL-13 and analyzed the results by r square of the k means four cluster solution. Results: Only IL-13 and IL-17 mRNA tissue content showed discriminatory ability. A subset of patients (17.5%) showed low IL-13 and IL-17 mRNA tissue content. The remaining patients (82.5%) was distributed in two different clusters characterized by high and low IL-13 mRNA expression in the context of high mRNA IL-17 expression. Patients with low IL-17 and IL-13 mRNA expression showed a shorter disease duration and a lower Mayo endoscopic score when compared to patients with high IL-17 mRNA expression. High IL-13 expression was significantly associated (p<0.05 by Fisher's exact test) with extensive colitis. Conclusions: Data suggest that the majority of UC patients might be classified into two different groups according to the tissue content of IL-13. Disease extension is differently associated with IL-13 mRNA tissue content. Supported by Grant RF-2011–02350689 by Italian Ministry of Health to MB.

Pouchitis is the most common complication following proctocolectomy with ileal pouch-anal anastomosis for ulcerative colitis (UC).To provide a standardized definition of pouchitis clinical, endoscopic and histological markers were grouped and weighted in the pouch disease activity index (PDAI).However, the delay in the assessment of the final score due to the time requested for histological analysis remains the main obstacle to the index implementation in clinical practice so that the use of modified-PDAI (mPDAI) with exclusion of histologic subscore has been proposed.We tested the ability of calprotectin measurement in the pouch endoluminal content to mimic the histologic score as defined in the PDAI, the index that we adopted as gold standard for pouchitis diagnosis.Calprotectin was measured by ELISA in the pouch endoluminal content collected during endoscopy in 40 consecutive patients with J-pouch.In each patient PDAI and mPDAI were calculated and 15% of patients were erroneously classified by mPDAI.ROC analysis of calprotectin values vs. acute histological subscore ≥ 3 identified different calprotectin cut-off values with corresponding sensitivity and specificity allowing the definition and scoring of different range of calprotectin subscores.We incorporated the calprotectin score in the mPDAI obtaining a new score that shows the same specificity as PDAI for diagnosis of pouchitis and higher sensitivity when compared with mPDAI.The use of the proposed new score, once validated in a larger series of patients, might be useful in the early management of patients with symptoms of pouchitis.

Methods:We isolated hASCs from subcutaneous (SAT) and visceral adipose tissue (VAT) (visceral origin: CF in CD subjects and mesenteric in obese subjects) of a well-characterised cohort including 4 groups: inactive CD (in remission), active CD, obese, and obese subjects with associated metabolic morbidities.Each group was composed of least by 3 donors.hASCs were immunophenotyped by flow cytometry using the fluorescein isothiocyanate-conjugated antihuman mouse antibodies (eg, anti-CD34, anti-CD45, anti-CD73, and anti-CD90).Gene expression profile, migration, invasion, and proliferation, as well as phagocytic capacity were analysed in hASCs obtained from different donors.Results: hASCs obtained from CD subjects has shown a significantly greater proliferative, migratory, and more interestingly invasive and phagocytic capacity than those derived from obese subjects, regardless of the presence of other metabolic disturbances (see Figure 1).This distinctive difference in phenotype between CD versus obese hASCs was observed in both active and inactive CD donors.Accordingly, AT inflammation markers (IL-6, TNFα, IL-1β, and MCP-1) indicated that both active and inactive CD subjects present a higher pro-inflammatory environment compared with obese subjects.Remarkably, we demonstrate that CD produces a detrimental effect on not only mesenteric adipose tissue-resident stem cells but also the subcutaneous fat depots.Conclusions: hASCs may have different immuno-modulatory capacities depending on the inflammatory event.Further work will be required to define whether hASCs response is protective or contributes to the associated metabolic derangements of these inflammatory diseases.

MUTYH DNA glycosylase removes mismatched adenine opposite 7, 8-dihydro-8-oxoguanine (8-oxoG), which is the major mutagenic lesion induced by oxidative stress. Biallelic mutations in MUTYH are associated with MUTYH-Associated polyposis (MAP) and increased risk in colorectal cancer (CRC). We investigated cancer susceptibility associated with MUTYH inactivation in a mouse model of inflammation-dependent carcinogenesis induced by azoxymethane (AOM) and dextran sulphate (DSS). Mutyh(-/-) mice were more sensitive than wild-type (WT) animals to AOM/DSS toxicity and accumulated DNA 8-oxoG in their gastrointestinal tract. AOM/DSS-induced colonic adenomas were significantly more numerous in Mutyh(-/-) than in WT animals, and frequently showed a tubulo-villous feature along with high-grade dysplasia and larger size lesions. This condition resulted in a greater propensity to develop adenocarcinomas. The colon of untreated Mutyh(-/-) mice expressed higher basal levels of pro-inflammatory cytokines GM-CSF and IFN gamma, and treatment with AOM/DSS induced an early decrease in circulating CD4+ and CD8+ T lymphocytes and an increase in myeloid-derived suppressor cells (MDSCs). Adenomas from Mutyh(-/-) mice had a greater infiltrate of Foxp3+ T regulatory cells, granulocytes, macrophages, MDSCs and strong expression of TGF-beta-latency-associated peptide and IL6. Our findings indicate that MUTYH loss is associated with an increase in CRC risk, which involves immunosuppression and altered inflammatory response. We propose that the AOM/DSS initiation/promotion protocol in Mutyh(-/-) mice provides a good model for MAP.

Background: A CD4+CD25- regulatory T cell population expressing the surface TGF-beta in its latent form LAP+ [latency associated peptide] cells was proved to be protective in experimental colitis and to be suppressive of human peripheral blood [PB] T proliferation. We investigated the frequency and function of lamina propria [LP] CD4+LAP+ T cells in inflammatory bowel disease [IBD] patients.Methods: Specimens from patients undergoing colonoscopy or bowel resection for IBD and colonic cancer were used as source of lamina propria mononuclear cells [LPMC]. The ulcerative colitis [UC] group was divided according to endoscopic activity evaluated with modified Baron Score. IL-17, IFN-gamma, IL-10, LAP, and Foxp3 expression in CD3+CD8- [CD4] or CD3+/CD4+ gated cell population was assessed by immunofluorescence. The ability of FACS-sorted LP CD3+CD8-[CD4] LAP+CD25- to inhibit stimulated autologous PB CD3+CD8-[CD4] LAP- CD25- cells proliferation was assessed.Results: LP CD4LAP+ cells were significantly increased, when compared with controls, in active UC patients and not in Crohn's disease patients. The majority of LP CD4+LAP+ cells were Foxp3-. The percentage of IL-17+ cells in LP CD3+CD8-[CD4] LAP+ cells was significantly higher in active UC patients when compared with controls. LP CD3+CD8-[CD4]LAP+CD25- isolated from UC patients showed reduced or no ability to inhibit autologous PB CD3+CD8-[CD4]LAP-CD25- cell proliferation when compared with controls. Removal of IL-17+ cells from LP CD3+CD8-[CD4] LAP+ cells increases their suppressive ability.Conclusions: The percentage of LP CD4LAP+ cells is increased in active UC, showing reduced suppressor activity due to their increased proportion of intracellular IL-17 expression.

SCOPE:Among food allergies, peanut allergy is frequently associated with severe anaphylactic reactions. In the need for safe and effective therapeutic strategies, probiotics may be considered on the basis of their immunomodulatory properties. The aim of the present study was to investigate the immunological mediators involved in the effects of probiotic VSL#3 oral supplementation on Th2 inflammation and anaphylaxis in a mouse model of peanut allergy.METHODS AND RESULTS:VSL#3 supplementation to peanut-sensitized mice was effective in ameliorating anaphylaxis and Th2-mediated inflammation, by promoting regulatory responses in the jejunum mucosa and in the mesenteric lymph node, as evaluated by ELISA, real-time PCR, histologic, and immunohistochemical analysis. Probiotic-induced TGF-β mediates its protective effects through the induction of regulatory T cells expressing FOXP3 and/or latency-associated peptide, as proven by in vivo blockade of TGF-β in VSL#3-treated mice with a neutralizing monoclonal antibody one day before challenge.CONCLUSION:TGF-β, induced in the gut by VSL#3 supplementation, is capable of reducing the Th2 inflammation associated with food anaphylaxis in a mouse model of peanut sensitization. TGF-β acts through the induction/maintenance of regulatory T cells expressing FOXP3 and/or latency-associated peptide. Probiotics supplementation may represent an effective and safe strategy for treating food allergies in adult population.