Pancreastatin Reduces Alternatively Activated Macrophages, Disrupts the Epithelial Homeostasis and Aggravates Colonic Inflammation. A Descriptive Analysis.

Ulcerative colitis (UC) is characterized by modifying alternatively activated macrophages (AAM) and epithelial homeostasis. Chromogranin-A (CHGA), released by enterochromaffin cells, is elevated in UC and is implicated in inflammation progression. CHGA can be cleaved into several derived peptides, including pancreastatin (PST), which is involved in proinflammatory mechanisms. Previously, we showed that the deletion of Chga decreased the onset and severity of colitis correlated with an increase in AAM and epithelial cells' functions. Here, we investigated PST activity in colonic biopsies of participants with active UC and investigated PST treatment in dextran sulfate sodium (DSS)-induced colitis using Chga-/- mice, macrophages, and a human colonic epithelial cells line. We found that the colonic protein expression of PST correlated negatively with mRNA expression of AAM markers and tight junction (TJ) proteins and positively with mRNA expression of interleukin (IL)-8, IL18, and collagen in human. In a preclinical setting, intra-rectal administration of PST aggravated DSS-induced colitis by decreasing AAM's functions, enhancing colonic collagen deposition and disrupting epithelial homeostasis in Chga+/+ and Chga-/- mice. This effect was associated with a significant reduction in AAM markers, increased colonic IL-18 release, and decreased TJ proteins' gene expression. In vitro, PST reduced Chga+/+ and Chga-/- AAM polarization and decreased anti-inflammatory mediators' production. Conditioned medium harvested from PST-treated Chga+/+ and Chga-/- AAM reduced Caco-2 cell migration, viability, proliferation, and mRNA levels of TJ proteins and increased oxidative stress-induced apoptosis and proinflammatory cytokines release. In conclusion, PST is a CHGA proinflammatory peptide that enhances the severity of colitis and the inflammatory process via decreasing AAM functions and disrupting epithelial homeostasis.

Impact of intrarectal chromofungin treatment on dendritic cells-related markers in different immune compartments in colonic inflammatory conditions.

BACKGROUND: Chromofungin (CHR: chromogranin-A 47-66) is a chromogranin-A derived peptide with anti-inflammatory and anti-microbial properties. Ulcerative colitis (UC) is characterized by a colonic decrease of CHR and a dysregulation of dendritic CD11c+ cells. AIM: To investigate the association between CHR treatment and dendritic cells (DCs)-related markers in different immune compartments in colitis. METHODS: A model of acute UC-like colitis using dextran sulphate sodium (DSS) was used in addition to biopsies collected from UC patients. RESULTS: Intrarectal CHR treatment reduced the severity of DSS-induced colitis and was associated with a significant decrease in the expression of CD11c, CD40, CD80, CD86 and interleukin (IL)-12p40 in the inflamed colonic mucosa and CD11c, CD80, CD86 IL-6 and IL-12p40 within the mesenteric lymph nodes and the spleen. Furthermore, CHR treatment decreased CD80 and CD86 expression markers of splenic CD11c+ cells and decreased NF-κB expression in the colon and of splenic CD11c+ cells. In vitro, CHR decreased CD40, CD80, CD86 IL-6 and IL-12p40 expression in naïve bone marrow-derived CD11c+ DCs stimulated with lipopolysaccharide. Pharmacological studies demonstrated an impact of CHR on the NF-κB pathway. In patients with active UC, CHR level was reduced and showed a negative linear relationship with CD11c and CD86. CONCLUSION: CHR has protective properties against intestinal inflammation via the regulation of DC-related markers and CD11c+ cells. CHR could be a potential therapy of UC.

Interdependence between Chromogranin-A, Alternatively Activated Macrophages, Tight Junction Proteins and the Epithelial Functions. A Human and In-Vivo/In-Vitro Descriptive Study.

Background: Ulcerative colitis (UC) is characterized by altered chromogranin-A (CHGA), alternatively activated macrophages (M2) and intestinal epithelial cells (IECs). We previously demonstrated that CHGA is implicated in colitis progression by regulating the macrophages. Here, we investigated the interplay between CHGA, M2, tight junctions (TJ) and IECs in an inflammatory environment. Methods: Correlations between CHGA mRNA expression of and TJ proteins mRNA expressions of (Occludin [OCLN], zonula occludens-1 [ZO1], Claudin-1 [CLDN1]), epithelial associated cytokines (interleukin [IL]-8, IL-18), and collagen (COL1A2) were determined in human colonic mucosal biopsies isolated from active UC and healthy patients. Acute UC-like colitis (5% dextran sulphate sodium [DSS], five days) was induced in Chga-C57BL/6-deficient (Chga-/-) and wild type (Chga+/+) mice. Col1a2 TJ proteins, Il-18 mRNA expression and collagen deposition were determined in whole colonic sections. Naïve Chga-/- and Chga+/+ peritoneal macrophages were isolated and exposed six hours to IL-4/IL-13 (20 ng/mL) to promote M2 and generate M2-conditioned supernatant. Caco-2 epithelial cells were cultured in the presence of Chga-/- and Chga+/+ non- or M2-conditioned supernatant for 24 h then exposed to 5% DSS for 24 h, and their functional properties were assessed. Results: In humans, CHGA mRNA correlated positively with COL1A2, IL-8 and IL-18, and negatively with TJ proteins mRNA markers. In the experimental model, the deletion of Chga reduced IL-18 mRNA and its release, COL1A2 mRNA and colonic collagen deposition, and maintained colonic TJ proteins. Chga-/- M2-conditioned supernatant protected caco-2 cells from DSS and oxidative stress injuries by improving caco-2 cells functions (proliferation, viability, wound healing) and by decreasing the release of IL-8 and IL-18 and by maintaining the levels of TJ proteins, and when compared with Chga+/+ M2-conditioned supernatant. Conclusions: CHGA contributes to the development of intestinal inflammation through the regulation of M2 and epithelial cells. Targeting CHGA may lead to novel biomarkers and therapeutic strategies in UC.

A Gene Expression Analysis of M1 and M2 Polarized Macrophages.

Macrophages are professional innate immune cells that are broadly disseminated throughout the body, shape various innate and adaptive immune responses, and play crucial roles in inflammation, homeostasis, wound healing, and tissue remodelling. According to their surrounding microenvironments, macrophages can differentiate themselves in different phenotypes. Over the last two decades, gene expression profiling has been used to decipher new transcripts associated with macrophage phenotypes. This chapter outlines protocols used to isolate and culture murine macrophages and how they can be "polarized" to obtain a specific phenotype. Furthermore, we describe a protocol for gene expression profiling using a quantitative real-time polymerase chain reaction (qPCR), a high-standard technology in the field of gene expression.

The immune-sleep crosstalk in inflammatory bowel disease.

Sleep disorders are progressively common and sometimes are associated with aberrant regulation of the adaptive and innate immune responses. Sleep interruption can increase the inflammatory burden by enhancing the pro-inflammatory cytokines particularly in patients with chronic diseases such as inflammatory bowel disease (IBD). IBD is a chronic inflammatory disease characterized by immune dysregulation, dysbiosis of gut microbiome, and poor-quality life. Therefore, this review highlights the crosstalk between sleep and immune responses during the progression of IBD.

Denosumab Regulates Gut Microbiota Composition and Cytokines in Dinitrobenzene Sulfonic Acid (DNBS)-Experimental Colitis.

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-1ß, 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.

Semaphorin 3E regulates apoptosis in the intestinal epithelium during the development of colitis.

Semaphorin 3E (SEMA3E) has emerged as an axon-guiding molecule that regulates various biological processes including the immune responses and apoptosis. However, its role in the pathophysiology of colitis remains elusive. We investigated the role of SEMA3E in intestinal epithelial cells (IECs) activation, using biopsies from patients with active ulcerative colitis (UC), a mouse model of UC, and an in-vitro model of intestinal mucosal healing. In this study, we confirmed that the mRNA level of SEMA3E is reduced significantly in patients with UC and demonstrated a negative linear association between SEMA3E mRNA and p53-associated genes. In mice, genetic deletion of Sema3e resulted in an increase onset and severity of colitis, p53-associated genes, apoptosis, and IL-1beta production. Recombinant SEMA3E treatment protected against colitis and decreased these effects. Furthermore, in stimulated epithelial cells, recombinant SEMA3E treatment enhanced wound healing, resistance to oxidative stress and decreased apoptosis and p53-associated genes. Together, these findings identify SEMA3E as a novel regulator in intestinal inflammation that regulates IECs apoptosis and suggest a potential novel approach to treat UC.

Semaphorin-3E attenuates intestinal inflammation through the regulation of the communication between splenic CD11C+ and CD4+ CD25- T-cells.

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.

Mucosal immunity and gut microbiota in dogs with chronic enteropathy.

Chronic enteropathy (CE) in dogs is a chronic and relapsing immunopathology, of unknown etiology, that usually manifests with a plethora of clinical signs reflecting the underlying heterogeneity in its pathogenesis. Alterations of the mucosal immune responses and the gut microbiota composition are thought to play an essential role in CE. Similar to humans, it is hypothesized that the breakdown in mucosal tolerance leads to aberrant and pathological immune responses toward the gut microbiota, that in turn, may contribute to the severity of disease, at least for certain CE subsets. Therefore, in this review, we discuss some of the most relevant and recent insights microbiological and immunological aspects characterizing CE in dogs.

Effects of Temperature on the Expression of Two Ovarian Differentiation-Related Genes foxl2 and cyp19a1a.

Exposure to stress induces a series of responses and influences a wide range of biological processes including sex differentiation in fish. The present work investigated the molecular and physiological response to thermal stress throughout the early development stage covering the whole period of sex differentiation of bluegill, Lepomis macrochirus. Larvae were treated using three temperatures, 17, 24, and 32°C from 6 to 90 days posthatching (dph) in 30-L round tanks. There is no significant difference of the sex ratio and survival among the three temperature groups in the geographic population used in this study. Two ovarian differentiation-related genes foxl2 and cyp19a1a were detected at 7 dph suggesting that these genes have already played a role prior to sex differentiation. The expression of foxl2 reached the peak and was thermosensitive just prior to the onset of ovarian differentiation at 27 dph. Histological examination displayed that the proliferation of germ cells and ovarian differentiation were delayed at the low-temperature treatment (17°C) at 97 dph compared with higher temperatures. In conclusion, the water temperature regulates the sex differentiation of bluegill through modulation of the expression of foxl2 and cyp19a1a. A comparative study of the expression profile of sex differentiation-related genes in species will shed light on the evolution of sex-determination mechanisms and the impact of stress on sex differentiation.

Catestatin Regulates Epithelial Cell Dynamics to Improve Intestinal Inflammation.

Ulcerative colitis (UC) is characterized by aberrant regulation of tight junctions (TJ), signal transducer and activator of transcription 3 (STAT3), and interleukin (IL)-8/18, which lead to intestinal barrier defects. Catestatin (CST), an enterochromaffin-derived peptide, regulates immune communication and STAT-3 in the inflamed intestine. Here, we investigated the effects of CST during the development of inflammation using human biopsies from patients with active UC, human colonic epithelial cells (Caco2), and an experimental model of UC (dextran sulfate sodium [DSS]-colitis). In UC patients, the protein and mRNA level of CST was significantly decreased. Colonic expression of CST showed a strong positive linear relationship with TJ proteins and STAT3, and a strong negative correlation with IL-8 and IL-18. Intra-rectal administration of CST reduced the severity of experimental colitis, IL-18 colonic levels, maintained TJ proteins and enhanced the phosphorylation of STAT3. CST administration increased proliferation, viability, migration, TJ proteins, and p-STAT3 levels, and reduced IL-8 & IL-18 in LPS- & DSS-induced Caco2 cell epithelial injury, and the presence of STAT-3 inhibitor abolished the beneficial effect of CST. In inflammatory conditions, we conclude that CST could regulate intestinal mucosal dynamic via a potential STAT3-dependent pathway that needs to be further defined. Targeting CST in intestinal epithelial cells (IECs) should be a promising therapeutic approach such as when intestinal epithelial cell homeostasis is compromised in UC patients.

Mixed Bacillus Species Enhance the Innate Immune Response and Stress Tolerance in Yellow Perch Subjected to Hypoxia and Air-Exposure Stress.

Stress enhances the disease susceptibility in fish by altering the innate immune responses, which are essential defense mechanisms. The use of probiotics is increasingly popular in the aquaculture industry. Yellow perch is a promising candidate for aquaculture. We investigated the efficiency of a mixed Bacillus species in minimizing the potential problems resulting from husbandry practices such as hypoxia and exposure to air in yellow perch. We showed that hypoxia and air exposure conditions induced a significant reduction in the early innate immune response (lysozyme activity, interferon-induced-GTP-binding protein-Mx1 [mx], interleukin-1ß [il1ß], serum amyloid-A [saa]), and a substantial increase in cortisol, heat shock protein (Hsp70), glutathione peroxidase (Gpx), superoxide dismutase (Sod1) that associated with a decline in insulin-like growth factor-1 (Igf1). Mixed Bacillus species administration improved the early innate responses, reduced cortisol, Hsp70, Gpx and Sod1, and elevated Igf1 levels. Bacillus species treated group showed faster recovery to reach the baseline levels during 24 h compared to untreated group. Therefore, mixed Bacillus species may enhance yellow perch welfare by improving the stress tolerance and early innate immune response to counterbalance the various husbandry stressors. Further studies are warranted to investigate the correlations between the aquaculture practices and disease resistance in yellow perch.

Chromogranin-A and its derived peptides and their pharmacological effects during intestinal inflammation.

The gastrointestinal tract is the largest endocrine organ that produces a broad range of active peptides. Mucosal changes during inflammation alter the distribution and products of enteroendocrine cells (EECs) that play a role in immune activation and regulation of gut homeostasis by mediating communication between the nervous, endocrine and immune systems. Patients with inflammatory bowel disease (IBD) typically have altered expression of chromogranin (CHG)-A (CHGA), a major soluble protein secreted by EECs that functions as a pro-hormone. CHGA gives rise to several bioactive peptides that have direct or indirect effects on intestinal inflammation. In IBD, CHGA and its derived peptides are correlated with the disease activity. In this review we describe the potential immunomodulatory roles of CHGA and its derived peptides and their clinical relevance during the progression of intestinal inflammation. Targeting CHGA and its derived peptides could be of benefit for the diagnosis and clinical management of IBD patients.

Distinct roles for phosphoinositide 3-kinases γ and δ in malignant B cell migration.

The PI 3-kinases (PI3K) are essential mediators of chemokine receptor signaling necessary for migration of chronic lymphocytic leukemia (CLL) cells and their interaction with tissue-resident stromal cells. While the PI3Kδ-specific inhibitor idelalisib shows efficacy in treatment of CLL and other B cell malignancies, the function of PI3Kγ has not been extensively studied in B cells. Here, we assess whether PI3Kγ has non-redundant functions in CLL migration and adhesion to stromal cells. We observed that pharmaceutical PI3Kγ inhibition with CZC24832 significantly impaired CLL cell migration, while dual PI3Kδ/γ inhibitor duvelisib had a greater impact than single isoform-selective inhibitors. Knockdown of PI3Kγ reduced migration of CLL cells and cell lines. Expression of the PI3Kγ subunits increased in CLL cells in response to CD40L/IL-4, whereas BCR cross-linking had no effect. Overexpression of PI3Kγ subunits enhanced cell migration in response to SDF1α/CXCL12, with the strongest effect observed within ZAP70 + CLL samples. Microscopic tracking of cell migration within chemokine gradients revealed that PI3Kγ functions in gradient sensing and impacts cell morphology and F-actin polarization. PI3Kγ inhibition also reduced CLL adhesion to stromal cells to a similar extent as idelalisib. These findings provide the first evidence that PI3Kγ has unique functions in malignant B cells.

Chromogranin-A Regulates Macrophage Function and the Apoptotic Pathway in Murine DSS colitis.

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.

Reactivation of Intestinal Inflammation Is Suppressed by Catestatin in a Murine Model of Colitis via M1 Macrophages and Not the Gut Microbiota.

While there is growing awareness of a relationship between chromogranin-A (CHGA) and susceptibility to inflammatory conditions, the role of human catestatin [(hCTS); CHGA352-67] 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-1ß, and TNF- α], anti-inflammatory cytokines (IL-10, TGF- ß), 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-1ß, 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-ß, 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.

Expression of Hsp70, Igf1, and Three Oxidative Stress Biomarkers in Response to Handling and Salt Treatment at Different Water Temperatures in Yellow Perch, Perca flavescens.

Stress is a major factor that causes diseases and mortality in the aquaculture industry. The goal was to analyze the expression of stress-related biomarkers in response to different stressors in yellow perch, which is an important aquaculture candidate in North America and highly sensitive to handling in captivity. Three fish groups were established, each having four replicates, and subjected to water temperatures of 14, 20, and 26°C and acute handling stress was performed followed by a salt treatment for 144h at a salinity of 5 ppt. Serum and hepatic mRNA levels of heat shock protein (hsp70), insulin-like growth factor 1 (Igf1), glutathione peroxidase (Gpx), superoxide dismutase 1 (Sod1), and glutathione reductase (Gsr) were quantified at seven times interval over 144 h using ELISA and RT-qPCR. Handling stress caused a significant down-regulation in Hsp70, Gpx, Sod1, and Gsr at a water temperature of 20°C compared to 14 and 26°C. Igf1 was significantly upregulated at 20°C and down-regulated at 14 and 26°C. Salt treatment had a transient reverse effect on the targeted biomarkers in all groups at 72 h, then caused an upregulation after 144 h, compared to the control groups. The data showed a negative strong regulatory linear relationship between igf1 with hsp70 and anti-oxidative gene expressions. These findings could provide valuable new insights into the stress responses that affect fish health and could be used to monitor the stress.

Chromofungin Ameliorates the Progression of Colitis by Regulating Alternatively Activated Macrophages.

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: CHGA47-66) 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.

Chromofungin (CHR: CHGA47-66) is downregulated in persons with active ulcerative colitis and suppresses pro-inflammatory macrophage function through the inhibition of NF-κB signaling.

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: CHGA47-66) 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.

Retinoic acid-mediated anti-inflammatory responses in equine immune cells stimulated by LPS and allogeneic mesenchymal stem cells.

Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. Allogeneic mesenchymal stem cells (MSCs)-based therapy is an effective approach to induce tissue healing and regeneration in many equine orthopedic conditions. However, MSCs-based therapies induced inflammatory responses in vivo. This study aimed to: 1. Determine the effect of RA cell culture treatment on inflammatory responses of lipopolysaccharides (LPS)- and allogeneic MSCs-stimulated peripheral blood mononuclear cells (PBMCs). 2. Determine the effect of RA on stimulated MSCs viability and morphology. Allogeneic MSCs-stimulated PBMCs had significant decreases in the anti-inflammatory cytokines (IL-10, IL-1ra, TGF-ß1), increases in the pro-inflammatory mediators (IL-1ß, IL-6, TNF-α, SAA), and increases of CD14 and MHC II percent positive cells compared to LPS- and non-stimulated PBMCs. Retinoic acid treatment of LPS- and allogeneic MSCs-stimulated PBMCs counterbalanced the induced inflammatory responses. Moreover, RA significantly improved the viability and morphology of stimulated MSCs. These findings highlighted the potential complications of equine allogeneic MSCs-based therapies and the immuno-modulatory effect of RA on equine stimulated cells. In conclusion, the use of RA to ameliorate allogeneic MSCs therapy associated inflammation may offer advantages that would require further investigations.