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The pro-inflammatory mediator receptor activator of nuclear factor-kappa B ligand (RANKL) plays a significant role in the development of rheumatoid arthritis; however, its role in inflammatory bowel disease is unknown. Genome-wide association meta-analysis for Crohn's disease (CD) identified a variant near the TNFSF11 gene that encodes RANKL and CD risk allele increased expression of RANKL in specific cell lines. This study aims to elucidate if the RANKL inhibitor denosumab can reduce the severity of experimental colitis and modify the gut microbiota composition using murine dinitrobenzenesulfonic acid (DNBS)-experimental model of colitis mimicking CD. In colitic conditions, denosumab treatment significantly decreased the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α within the colonic mucosa. Moreover, colitis was accompanied by disruption of gut microbiota, and preventative treatment with denosumab modulated this disruption. Denosumab treatment also modified the alpha- and beta diversity of colonic mucosa and fecal microbiota. These results provide a rationale for considering denosumab as a future potential therapy in CD; however, more detailed experimental and clinical studies are warranted., (Copyright © 2020 Khafipour, Eissa, Munyaka, Rabbi, Kapoor, Kermarrec, Khafipour, Bernstein and Ghia.)

Background and Purpose: An alteration in the communication between the innate and adaptive immune cells is a hallmark of ulcerative colitis (UC). Semaphorin-3E (SEMA3E), a secreted guidance protein, regulates various immune responses., Experimental Approach: We investigated the expression of SEMA3E in colonic biopsies of active UC patients and its mechanisms in Sema3e -/- mice using an experimental model of UC., Key Results: SEMA3E level was decreased in active UC patients and negatively correlated with pro-inflammatory mediators. Colonic expression of SEMA3E was reduced in colitic Sema3e +/+ mice, and recombinant (rec-) Plexin-D1 treatment exacerbated disease severity. In vivo rec-SEMA3E treatment restored SEMA3E level in colitic Sema3e +/+ mice. In Sema3e -/- mice, disease severity was increased, and rec-SEMA3E ameliorated these effects. Lack of Sema3e increased the expression of CD11c and CD86 markers. Colitic Sema3e -/- splenocytes and splenic CD11c + cells produced more IL-12/23 and IFN-γ compared to Sema3e +/+ , and rec-SEMA3E reduced their release as much as NF-κB inhibitors, whereas an NF-κB activator increased their production and attenuated the effect of rec-SEMA3E. Colitic Sema3e -/- splenic CD11c + /CD4 + CD25 - T-cell co-cultures produced higher concentrations of IFN-γ and IL-17 when compared to colitic Sema3e +/+ splenic cell co-cultures, and rec-SEMA3E decreased these effects. In vitro, anti-IL-12p19 and -12p35 antibodies and rec-IL-12 and -23 treatment confirmed the crosstalk between CD11c + and CD4 + CD25 - T-cells., Conclusion and Implications: SEMA3E is reduced in colitis and modulates colonic inflammation by regulating the interaction between CD11c + and CD4 + CD25 - T-cells via an NF-κB-dependent mechanism. Thus, SEMA3E could be a potential therapeutic target for UC patients., (© 2019 The British Pharmacological Society.)

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti-TNF, or anti-α4β7 therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled α4β7 integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment.

Background: Neuroimmune interactions are essential to maintain gut homeostasis and prevent intestinal disorders but so far, the impact of enteric glial cells (EGC) on immune cells remains a relatively unexplored area of research. As a dysregulation of critical cytokines such as interleukine-7 (IL-7) was suggested to exacerbate gut chronic inflammation, we investigated whether EGC could be a source of IL-7 in the gastrointestinal tract., Methods: Expression of IL-7 in the rat enteric nervous system was analyzed by immunochemistry and Q-PCR. IL-7 variants were cloned and specific antibodies against rat IL-7 isoforms were raised to characterize their expression in the submucosal plexus. IL-7 isoforms were produced in vitro to analyze their impact on T-cell survival., Key Results: Neurons and glial cells of the rat enteric nervous system expressed IL-7 at both mRNA and protein levels. Novel rat IL-7 isoforms with distinct C-terminal parts were detected. Three of these isoforms were found in EGC or in both enteric neurons and EGC. Exposure of EGC to pro-inflammatory cytokines (IL-1β and/or TNFα) induced an upregulation of all IL-7 isoforms. Interestingly, time-course and intensity of the upregulation varied according to the presence or absence of exon 5a in IL-7 variants. Functional analysis on T lymphocytes revealed that only canonical IL-7 protects T cells from cell death., Conclusions and Inferences: IL-7 and its variants are expressed by neurons and glial cells in the enteric nervous system. Their distinct expression and upregulation in inflammatory conditions suggest a role in gut homeostasis which could be critical in case of chronic inflammatory diseases., (© 2018 John Wiley & Sons Ltd.)

Chromogranin-A (CHGA) is elevated in inflammatory bowel disease (IBD), but little is known about its role in colonic inflammation. IBD is associated with impaired functions of macrophages and increased apoptosis of intestinal epithelial cells. We investigated CHGA expression in human subjects with active ulcerative colitis (UC) and the underlying mechanisms in Chga -/- mice. In UC, CHGA, classically activated macrophage (M1) markers, caspase-3, p53, and its associated genes were increased, while alternatively activated macrophage (M2) markers were decreased without changes in the extrinsic apoptotic pathway. CHGA correlated positively with M1 and the apoptotic pathway and negatively with M2. In the murine dextran sulfate sodium (DSS)-induced colitis, Chga deletion reduced the disease severity and onset, pro-inflammatory mediators, M1, and p53/caspase-3 activation, while it upregulated anti-inflammatory cytokines and M2 markers with no changes in the extrinsic apoptotic markers. Compared to Chga +/+ , M1 and p53/caspase-3 activation in Chga -/- macrophages were decreased in vitro, while M2 markers were increased. CHGA plays a critical role during colitis through the modulation of macrophage functions via the caspase-3/p53 pathway. Strategies targeting CHGA to regulate macrophage activation and apoptosis might be developed to treat UC patients., Key Messages: • Chromogranin-A (CHGA) is pro-hormone and is secreted in the gut. CHGA is elevated in colitis and is associated with the disease severity. The lack of GHGA has beneficial immunomodulatory properties during the development of intestinal inflammation. The lack of CHGA regulates the plasticity of macrophages and p53/caspase activation in colitis. Functional analysis of CHGA may lead to a novel therapy for IBD.

Chromogranin-A (CHGA) is a prohormone secreted by neuroendocrine cells and is a precursor of several bioactive peptides, which are implicated in different and distinctive biological and immune functions. Chromofungin (CHR: CHGA 47-66 ) is a short peptide with antimicrobial effects and encodes from CHGA exon-IV. Inflammatory bowel disease (IBD) is characterized by alterations in the activation of pro-inflammatory pathways, pro-inflammatory macrophages (M1), and nuclear transcription factor kappa B (NF-κB) signaling leading to the perpetuation of the inflammatory process. Here, we investigated the activity of CHR (CHGA Exon-IV) in persons with active ulcerative colitis (UC) and the underlying mechanisms in dextran sulfate sodium (DSS)-colitis in regard to macrophages activation and migration. Tissue mRNA expression of CHR (CHGA Exon-IV) was down regulated in active UC compared to healthy individuals and negatively correlated with pro-inflammatory macrophages (M1) cytokines, toll-like receptors (TLR)-4, and pNF-κB activity. In DSS colitis, CHR (CHGA Exon-IV) expression was reduced, and exogenous CHR treatment decreased the severity of colitis associated with a reduction of M1 macrophages markers and pNF-κB. In vitro, CHR treatment reduced macrophages migration, decreased pro-inflammatory cytokines production and pNF-κB. Targeting CHR may represent a promising new direction in research to define new therapeutic targets and biomarkers associated with IBD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ulcerative colitis (UC) is characterized by a functional dysregulation of alternatively activated macrophage (AAM) and intestinal epithelial cells (IECs) homeostasis. Chromogranin-A (CHGA) secreted by neuroendocrine cells is implicated in intestinal inflammation and immune dysregulation. CHGA undergoes proteolytic processing to generate CHGA-derived peptides. Chromofungin (CHR: CHGA 47-66 ) is a short CHGA-derived peptide encoded by CHGA Exon-IV and is involved in innate immune regulation, but the basis is poorly investigated. We investigated the expression of CHR in colonic tissue of patients with active UC and assessed the effects of the CHR in dextran sulfate sodium (DSS) colitis in mice and on macrophages and human colonic epithelial cells. We found that mRNA expression of CHR correlated positively with mRNA levels of AAM markers and gene expression of tight junction (TJ) proteins and negatively with mRNA levels of interleukin ( IL ) -8, IL-18 , and collagen in patients with active UC. Moreover, AAM markers correlated positively with gene expression of TJ proteins and negatively with IL-8, IL-18 , and collagen gene expression. Experimentally, intracolonic administration of CHR protected against DSS-induced colitis by priming macrophages into AAM, reducing colonic collagen deposition, and maintaining IECs homeostasis. This effect was associated with a significant increase of AAM markers, reduction of colonic IL-18 release and conservation of gene expression of TJ proteins. In vitro , CHR enhanced AAM polarization and increased the production of anti-inflammatory mediators. CHR-treated AAM conditioned medium increased Caco-2 cell migration, viability, proliferation, and mRNA levels of TJ proteins, and decreased oxidative stress-induced apoptosis and proinflammatory cytokines release. Direct CHR treatments had the same effect. In conclusion, CHR treatment reduces the severity of colitis and the inflammatory process via enhancing AAM functions and maintaining IECs homeostasis. CHR is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of colonic inflammation and could lead to new therapeutic strategies for UC.

While there is growing awareness of a relationship between chromogranin-A (CHGA) and susceptibility to inflammatory conditions, the role of human catestatin [(hCTS); CHGA 352-67 ] in the natural history of established inflammatory bowel disease is not known. Recently, using two different experimental models, we demonstrated that hCTS-treated mice develop less severe acute colitis. We have also shown the implication of the macrophages in this effect. The aims of this study were to determine (1) whether hCTS treatment could attenuate the reactivation of inflammation in adult mice with previously established chronic colitis; (2) whether this effect is mediated through macrophages or the gut microbiota. Quiescent colitis was induced in 7-8-week-old C57BL6 mice using four cycles (2-4%) of dextran sulfate sodium. hCTS (1.5 mg/kg/day) treatment or vehicle started 2 days before the last induction of colitis and continuing for 7 days. At sacrifice, macro- and microscopic scores were determined. Colonic pro-inflammatory cytokines [interleukin (IL)-6, IL-1β, and TNF- α], anti-inflammatory cytokines (IL-10, TGF- β), classically activated (M1) ( iNOS, Mcp1 ), and alternatively activated (M2) ( Ym1, Arg1 ) macrophages markers were studied using ELISA and/or RT-qPCR. In vitro , peritoneal macrophages isolated from naïve mice and treated with hCTS (10 -5  M, 12 h) were exposed to either lipopolysaccharide (100 ng/ml, 12 h) to polarize M1 macrophages or to IL-4/IL-13 (20 ng/ml) to polarize M2 macrophages. M1/M2 macrophage markers along with cytokine gene expression were determined using RT-qPCR. Feces and mucosa-associated microbiota (MAM) samples were collected, and the V4 region of 16 s rRNA was sequenced. Micro- and macroscopic scores, colonic IL-6, IL-1β, TNF- α, and M1 macrophages markers were significantly decreased in the hCTS-treated group. Treatment did not have any effect on colonic IL-10, TGF-β, and M2 markers nor modified the bacterial richness, diversity, or the major phyla in colitic fecal and MAM samples. In vitro , pro-inflammatory cytokines levels, as well as their gene expression, were significantly reduced in hCTS-treated M1 macrophages. hCTS treatment did not affect M2 macrophage markers. These findings suggest that hCTS treatment attenuates the severity of inflammatory relapse through the modulation of the M1 macrophages and the release of pro-inflammatory cytokines.

2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis is an experimental model that mimics Crohn's disease. Appropriateness of reference genes is crucial for RT-qPCR. This is the first study to determine the stability of reference gene expression (RGE) in mice treated with DNBS. DNBS experimental Colitis was induced in male C57BL/6 mice. RNA was extracted from colon tissue and comprehensive analysis of 13 RGE was performed according to predefined criteria. Relative colonic TNF-α and IL-1β mRNA levels were calculated. Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest fluctuation within the inflamed and control groups. Conversely, ribosomal protein large P0 (Rplp0), non-POU domain containing (Nono), TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. TNF-α and IL-1β mRNA levels was dependent on the reference gene used and varied from significant when the most stable genes were used to non-significant when the least stable genes were used. The appropriate choice of RGE is critical to guarantee satisfactory normalization of RT-qPCR data when using DNBS-Model. We recommend using Rplp0, Nono, Tbp, Hprt and Eef2 instead of common reference genes., Competing Interests: The authors declare no competing financial interests.

Enteric glial cells (EGC) have trophic and neuroregulatory functions in the enteric nervous system, but whether they exert a direct effect on immune cells is unknown. Here, we used co-cultures to show that human EGC can inhibit the proliferation of activated T lymphocytes. Interestingly, EGC from Crohn's patients were effective at one EGC for two T cells whereas EGC from control patients required a ratio of 1:1. These data suggest that EGC contribute to local immune homeostasis in the gastrointestinal wall. They also raise the possibility that EGC have particular immunosuppressive properties in inflammatory bowel diseases such as Crohn's disease., (Copyright © 2016. Published by Elsevier B.V.)

The postnatal period is crucial for the development of gastrointestinal (GI) functions. The enteric nervous system is a key regulator of GI functions, and increasing evidences indicate that 1) postnatal maturation of enteric neurons affect the development of GI functions, and 2) microbiota-derived short-chain fatty acids can be involved in this maturation. Although enteric glial cells (EGC) are central regulators of GI functions, the postnatal evolution of their phenotype remains poorly defined. We thus characterized the postnatal evolution of EGC phenotype in the colon of rat pups and studied the effect of short-chain fatty acids on their maturation. We showed an increased expression of the glial markers GFAP and S100β during the first postnatal week. As demonstrated by immunohistochemistry, a structured myenteric glial network was observed at 36 days in the rat colons. Butyrate inhibited EGC proliferation in vivo and in vitro but had no effect on glial marker expression. These results indicate that the EGC myenteric network continues to develop after birth, and luminal factors such as butyrate endogenously produced in the colon may affect this development., (Copyright © 2016 the American Physiological Society.)

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/., (© The Author(s) 2016. Published by Oxford University Press.)

Foetal pig neuroblasts are interesting candidates as a cell source for transplantation, but xenotransplantation in the brain requires the development of adapted immunosuppressive treatments. As systemic administration of high doses of cyclosporine A has side effects and does not protect xenotransplants forever, we focused our work on local control of the host immune responses. We studied the advantage of cotransplanting syngenic mesenchymal stem cells (MSC) with porcine neuroblasts (pNb) in immunocompetent rat striata. Two groups of animals were transplanted, either with pNb alone or with both MSC and pNb. At day 63, no porcine neurons were detected in the striata that received only pNb, while four of six rats transplanted with both pNb and MSC exhibited healthy porcine neurons. Interestingly, 50% of the cotransplanted rats displayed healthy grafts with pNF70+ and TH+ neurons at 120 days post-transplantation. qPCR analyses revealed a general dwindling of pro- and anti-inflammatory cytokines in the striata that received the cotransplants. Motor recovery was also observed following the transplantation of pNb and MSC in a rat model of Parkinson's disease. Taken together, the present data indicate that the immunosuppressive properties of MSC are of great interest for the long-term survival of xenogeneic neurons in the brain., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

DNA barcoding, being developed for biomonitoring, requires a database of reference sequences and knowledge of how much sequences can deviate before they are assigned to separate species. The molecular hunt for hidden species also raises the question of species definitions. We examined whether there are objective criteria for sequence-based species delimitation in diatoms, using Nitzschia palea, an important monophyletic indicator species already known to contain cryptic diversity. Strains from a wide geographical range were sequenced for 28S rRNA, COI and rbcL. Homogeneity indices and the Chao index failed to objectively select a precise number of species existing in N. palea as well as an evolutionary method based on coalescence theory. COI always gave higher diversity estimations than 28S rRNA or rbcL. Mating data did not provide a precise calibration of molecular species thresholds. Rarefaction curves indicated that further MOTUs would be detected with more isolates than we sampled (81 clones, 42 localities). Although some genotypes had intercontinental distributions, there was a positive relationship between genetic and geographical distance, suggesting even higher richness than we assessed, given that many regions were not sampled. Overall, no objective criteria were found for species separation; instead barcoding will need a consensual approach to molecular species limits., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The Gomphonema parvulum complex includes species displaying considerable morphological variability and a wide geographical distribution. These characteristics make them difficult to identify by microscopy and raise the question of their taxonomic validity and of the possibility of biogeographical differentiation between them. In this context, we isolated 39 G. parvulum s.l. strains from rivers located in a tropical island (Mayotte) and in mainland Europe. By sequencing three DNA fragments (ITS, rbcL and cox1), four clades (A, B, C and D) were clearly identified, and an additional one (B') was distinguishable only on the rbcL sequence. The main four clades can be separated by their morphological criteria, in particular by the shape of the central area, but some overlaps were found between them. We therefore consider that the G. parvulum complex contains at least four semi-cryptic species corresponding to the four main clades. One of them (A) was found only on Mayotte, while two others (C and D) were found only in Europe. The last clade (B) contained strains from both Europe and Mayotte. Pyrosequencing data confirmed the geographical differences in the distribution of these species, suggesting that the G. parvulum complex displays biogeographic structure., (Copyright © 2013 Elsevier GmbH. All rights reserved.)