Aristea Sideri

Substance P Mediates Proinflammatory Cytokine Release From Mesenteric Adipocytes in Inflammatory Bowel Disease Patients

Background & Aims Substance P (SP) neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes, and SP binding activates proinflammatory signaling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with inflammatory bowel disease (IBD) and examined their responsiveness to SP compared with control human mesenteric preadipocytes. We investigated the effect of the neuropeptide SP on cytokine expression in preadipocytes of IBD versus control patients and evaluated the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions. Methods Mesenteric fat was collected from control, ulcerative colitis (UC) and Crohn’s disease patients (n = 10–11 per group). Preadipocytes were isolated, expanded in culture, and exposed to substance P. Colon biopsy samples were obtained from control and IBD patients. Results Tacr-1 and -2 mRNA were increased in IBD preadipocytes compared with controls, but Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared with controls. Disease-dependent responses to SP were also observed between Crohn’s disease and UC preadipocytes. Interleukin 17A (IL-17A) mRNA expression and release increased after SP treatment in both Crohn’s disease and UC preadipocytes; IL-17RA mRNA increased in colon biopsies samples from IBD patients. Conclusions Preadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptors in the IBD colon suggests that a fat-colonic mucosa inflammatory loop may be active during IBD.

Effects of obesity on severity of colitis and cytokine expression in mouse mesenteric fat. Potential role of adiponectin receptor 1

In inflammatory bowel disease (IBD), obesity is associated with worsening of the course of disease. Here, we examined the role of obesity in the development of colitis and studied mesenteric fat-epithelial cell interactions in patients with IBD. We combined the diet-induce obesity with the trinitrobenzene sulfonic acid (TNBS) colitis mouse model to create groups with obesity, colitis, and their combination. Changes in the mesenteric fat and intestine were assessed by histology, myeloperoxidase assay, and cytokine mRNA expression by real-time PCR. Medium from human mesenteric fat and cultured preadipocytes was obtained from obese patients and those with IBD. Histological analysis showed inflammatory cell infiltrate and increased histological damage in the intestine and mesenteric fat of obese mice with colitis compared with all other groups. Obesity also increased the expression of proinflammatory cytokines including IL-1β, TNF-α, monocyte chemoattractant protein 1, and keratinocyte-derived chemokine, while it decreased the TNBS-induced increases in IL-2 and IFN-γ in mesenteric adipose and intestinal tissues. Human mesenteric fat isolated from obese patients and those with and IBD demonstrated differential release of adipokines and growth factors compared with controls. Fat-conditioned media reduced adiponectin receptor 1 (AdipoR1) expression in human NCM460 colonic epithelial cells. AdipoR1 intracolonic silencing in mice exacerbated TNBS-induced colitis. In conclusion, obesity worsens the outcome of experimental colitis, and obesity- and IBD-associated changes in adipose tissue promote differential mediator release in mesenteric fat that modulates colonocyte responses and may affect the course of colitis. Our results also suggest an important role for AdipoR1 for the fat-intestinal axis in the regulation of inflammation during colitis.

Chronic unpredictable stress regulates visceral adipocyte‐mediated glucose metabolism and inflammatory circuits in male rats

Chronic psychological stress is a prominent risk factor involved in the pathogenesis of many complex diseases, including major depression, obesity, and type II diabetes. Visceral adipose tissue is a key endocrine organ involved in the regulation of insulin action and an important component in the development of insulin resistance. Here, we examined for the first time the changes on visceral adipose tissue physiology and on adipocyte‐associated insulin sensitivity and function after chronic unpredictable stress in rats. Male rats were subjected to chronic unpredictable stress for 35 days. Total body and visceral fat was measured. Cytokines and activated intracellular kinase levels were determined using high‐throughput multiplex assays. Adipocyte function was assessed via tritiated glucose uptake assay. Stressed rats showed no weight gain, and their fat/lean mass ratio increased dramatically compared to control animals. Stressed rats had significantly higher mesenteric fat content and epididymal fat pad weight and demonstrated reduced serum glucose clearing capacity following glucose challenge. Alterations in fat depot size were mainly due to changes in adipocyte numbers and not size. High‐throughput molecular screening in adipocytes isolated from stressed rats revealed activation of intracellular inflammatory, glucose metabolism, and MAPK networks compared to controls, as well as significantly reduced glucose uptake capacity in response to insulin stimulation. Our study identifies the adipocyte as a key regulator of the effects of chronic stress on insulin resistance, and glucose metabolism, with important ramifications in the pathophysiology of several stress‐related disease states.

Cathelicidin suppresses lipid accumulation and hepatic steatosis by inhibition of the CD36 receptor

Background and objectives:Obesity is a global epidemic which increases the risk of the metabolic syndrome. Cathelicidin (LL-37 and mCRAMP) is an antimicrobial peptide with an unknown role in obesity. We hypothesize that cathelicidin expression correlates with obesity and modulates fat mass and hepatic steatosis.Materials and methods:Male C57BL/6 J mice were fed a high-fat diet. Streptozotocin was injected into mice to induce diabetes. Experimental groups were injected with cathelicidin and CD36 overexpressing lentiviruses. Human mesenteric fat adipocytes, mouse 3T3-L1 differentiated adipocytes and human HepG2 hepatocytes were used in the in vitro experiments. Cathelicidin levels in non-diabetic, prediabetic and type II diabetic patients were measured by enzyme-linked immunosorbent assay.Results:Lentiviral cathelicidin overexpression reduced hepatic steatosis and decreased the fat mass of high-fat diet-treated diabetic mice. Cathelicidin overexpression reduced mesenteric fat and hepatic fatty acid translocase (CD36) expression that was reversed by lentiviral CD36 overexpression. Exposure of adipocytes and hepatocytes to cathelicidin significantly inhibited CD36 expression and reduced lipid accumulation. Serum cathelicidin protein levels were significantly increased in non-diabetic and prediabetic patients with obesity, compared with non-diabetic patients with normal body mass index (BMI) values. Prediabetic patients had lower serum cathelicidin protein levels than non-diabetic subjects.Conclusions:Cathelicidin inhibits the CD36 fat receptor and lipid accumulation in adipocytes and hepatocytes, leading to a reduction of fat mass and hepatic steatosis in vivo. Circulating cathelicidin levels are associated with increased BMI. Our results demonstrate that cathelicidin modulates the development of obesity.

Identification of a Novel Substance P-Neurokinin-1 Receptor MicroRNA-221-5p Inflammatory Network in Human Colonic Epithelial Cells

Background & Aims Substance P (SP), a neuropeptide member of the tachykinin family, plays a critical role in colitis. MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. We examined whether SP modulates expression of microRNAs in human colonic epithelial cells. Methods We performed microRNA profiling analysis of SP-stimulated human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NCM460-NK-1R). Targets of SP-regulated microRNAs were validated by real-time polymerase chain reaction (RT-PCR). Functions of miRNAs were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS) models of colitis. Results SP stimulated differential expression of 29 microRNAs, including miR-221-5p, the highest up-regulated miR (by 12.6-fold) upon SP stimulation. Bioinformatic and luciferase reporter analyses identified interleukin-6 receptor (IL-6R) mRNA as a direct target of miR-221-5p in NCM460 cells. Accordingly, SP exposure of NCM460-NK-1R cells increased IL-6R mRNA expression, and overexpression of miR-221-5p reduced IL-6R expression. Nuclear factor κB and c-Jun N-terminal kinase inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and in colonic biopsy samples from ulcerative colitis but not Crohn’s disease patients compared with controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic tumor necrosis factor-α, C-X-C motif chemokine 10 (Cxcl10), and collagen, type II, α 1 (Col2α1) mRNA expression. In situ hybridization in TNBS- and DSS-exposed colons revealed increased miR-221-5p expression primarily in colonocytes. Conclusions Our results reveal a novel NK-1R-miR-221-5p-IL-6R network that protects from colitis. The use of miR-221-5p mimics may be a promising approach for colitis treatment.

Mesenteric Adipose-derived Stromal Cells From Crohn’s Disease Patients Induce Protective Effects in Colonic Epithelial Cells and Mice With Colitis

Mesenteric adipose tissue hyperplasia is a hallmark of Crohn’s disease (CD). Recently, we showed that mesenteric adipose-derived stromal cells (ADSCs) from CD, ulcerative colitis, and control patients synthesize and release adipokines in a disease-dependent manner. Here we examined the expression profiles of CD and control patient-derived mesenteric ADSCs and studied the effects of their extracellular mediators on colonocyte signaling in vitro and experimental colitis in vivo. ADSCs were isolated from mesenteric fat of control and CD patients. Microarray profiling and network analysis were performed in ADSCs and human colonocytes treated with conditioned media from cultured ADSCs. Mice with acute colitis received daily injections of conditioned media from patient-derived ADSCs, vehicle, or apolactoferrin. Proliferative responses were evaluated in conditioned media–treated colonocytes and mouse colonic epithelium. Total protein was isolated from cultured colonocytes after treatment with apolactoferrin for Western blot analysis of phosphorylated intracellular signaling kinases. Microarray profiling revealed differential mRNA expression in CD patient-derived ADSCs compared with controls, including lactoferrin. Administration of CD patient-derived medium or apolactoferrin increased colonocyte proliferation compared with controls. Conditioned media from CD patient-derived ADSCs or apolactoferrin attenuated colitis severity in mice and enhanced colonocyte proliferation in vivo. ADSCs from control and CD patients show disease-dependent inflammatory responses and alter colonic epithelial cell signaling in vitro and in vivo. Furthermore, we demonstrate lactoferrin production by adipose tissue, specifically mesenteric ADSCs. We suggest that mesenteric ADSC-derived lactoferrin may mediate protective effects and participate in the pathophysiology of CD by promoting colonocyte proliferation and the resolution of inflammation.

MicroRNA-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis

Substance P (SP) mediates colitis. SP signaling regulates the expression of several miRNAs, including miR-31-3p, in human colonocytes. However, the role of miR-31-3p in colitis and the underlying mechanisms has not been elucidated. We performed real-time PCR analysis of miR-31-3p expression in human colonic epithelial cells overexpressing neurokinin-1 receptor (NCM460 NK-1R) in response to SP stimulation and in NCM460 cells after IL-6, IL8, tumor necrosis factor (TNF)-α, and interferon-γ exposure. Functions of miR-31-3p were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis. Targets of miRNA-31-3p were confirmed by Western blot analysis and luciferase reporter assay. Jun N-terminal kinase inhibition decreased SP-induced miR-31-3p expression. miR-31-3p expression was increased in both TNBS- and DSS-induced colitis and human colonic biopsies from ulcerative colitis, compared with controls. Intracolonic administration of a miR-31-3p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic TNF-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) mRNA expression. Conversely, overexpression of miR-31-3p ameliorated the severity of DSS-induced colitis. Bioinformatic, luciferase reporter assay, and Western blot analyses identified RhoA as a target of miR-31-3p in NCM460 cells. Constitutive activation of RhoA led to increased expression of CCL2, IL6, TNF-α, and CXCL10 in NCM460-NK-1R cells on SP stimulation. Our results reveal a novel SP-miR-31-3p-RhoA pathway that protects from colitis. The use of miR-31-3p mimics may be a promising approach for colitis treatment.

547 Effects of Substance P on Pro and Anti-Inflammatory Responses of Human Mesenteric Preadipocytes Isolated From IBD Patients

Oxytocin (OT) is best known for its hormonal roles in milk let down and parturition. It has also been found in neurons that project within the brain and plays roles in mediating the central effects of nurturing stimuli. We have recently demonstrated that a subset of enteric neurons expresses OT and that about 70% of adult enteric neurons express the oxytocin receptor (OTR). OTR expression, which is developmentally regulated, also occurs in crypt epithelial cells where it concentrates at junctional complexes, particularly at the crypt-villus boundary. We tested hypotheses that endogenous enteric OT signaling affects intestinal motility, inflammation, and mucosal homeostasis. Dry stool mass and fecal water content were found to be greater in mice lacking OTR (OTRKO) than in WT mice. Total gastrointestinal transit time in OTRKO animals was also faster than that of WT animals; however, the time OTRKO mice required to expel glass beads inserted into their rectum was greater than that of WT animals. The severity of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in OTRKO mice was more severe compared to that in WT animals. Clinical scores (aggregating weight loss, stool blood and consistency), histological damage scores, and abundance of transcripts encoding pro-inflammatory cytokines and chemokines, were all significantly greater in OTRKO than in WT mice. Administration of exogenous OT, moreover, ameliorated TNBS-induced colitis in WT mice. Absorption of FITC-dextran was greater in OTRKO than in WT, suggesting that intestinal macromolecular permeability is increased in the absence of OT signaling. Exudative enteropathy, however, did not occur in OTRKO mice and following its intravenous infusion, horseradish peroxidase was found to be trapped at tight junctions between enterocytes. Villus height and crypt depth were each smaller in the intestines of OTRKO than WT mice, suggesting that signaling by crypt epithelial OTR plays a role in mucosal homeostasis. The current study supports the idea that enteric oxytocinergic signaling is physiologically significant; it slows intestinal propulsion, modulates inflammation, dampens macromolecular permeability, and contributes to the maintenance mucosal homeostasis. Observations are consistent with an overarching hypothesis that oxytocinergic signaling counterbalances effects of other molecules, such as corticotrophin releasing hormone, which mediate effects of stress. If so, then congenital or acquired defects in enteric oxytocinergic signaling may decrease resistance to stress and thus contribute to the pathophysiology of intestinal disorders, such as irritable bowel syndrome or inflammatory bowel disease. Development of means of initiating or enhancing oxytocinergic signaling may also be useful in treating stress-related disorders.