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.

Corticotropin-Releasing Hormone Receptor 2 Signaling Promotes Mucosal Repair Responses after Colitis.

The corticotropin-releasing hormone family mediates functional responses in many organs, including the intestine. Activation of corticotropin-releasing hormone receptor 2 (CRHR2) in the colonic mucosa promotes inflammation during acute colitis but inhibits inflammation during chronic colitis. We hypothesized that specific modulation of CRHR2 signaling in the colonic mucosa can promote restoration of the epithelium through stimulation of cell proliferative, migratory, and wound healing responses. Mucosal repair was assessed after dextran sodium sulfate (DSS)-induced colitis in mice receiving intracolonic injections of a CRHR2 antagonist or vehicle and in Crhr2(-/-) mice. Histologic damage, cytokine expression, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and Ki-67 immunoreactivity were evaluated. Cell viability, proliferation, and migration were compared between parental and CRHR2-overexpressing colonic epithelial cells. Protein lysates were processed for phosphoprotein assays and a wound healing assay performed in vitro. Administration of a CRHR2 antagonist after DSS-induced colitis increased disease activity, delayed healing, and decreased epithelial cell proliferation in vivo. Colons from these mice also showed increased apoptosis and proinflammatory cytokine expression. Compared with controls, Crhr2(-/-) mice showed increased mortality in the DSS healing protocol. CRHR2-overexpressing cells had increased proliferation and migration compared with parental cells. Wound healing and signal transducer and activator of transcription 3 activity were elevated in CRHR2-overexpressing cells after urocortin 2 and IL-6 treatment, suggesting advanced healing progression. Our results suggest that selective CRHR2 activation may provide a targeted approach to enhance mucosal repair pathways after colitis.

Identification of a novel substance P (SP)-neurokinin-1 receptor (NK-1R) 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 non-coding RNAs that negatively regulate gene expression. However, whether SP modulates expression of microRNAs in human colonic epithelial cells remains unknown. 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 TNBS and 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, while overexpression of miR-221-5p reduced IL-6R expression. NF-κB and JNK inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and colonic biopsies from Ulcerative Colitis, but not Crohn's Disease subjects, compared to controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor, exacerbated TNBS-and DSS-induced colitis, and increased colonic TNF-α, Cxcl10, and Col2 α 1 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.

Substance P mediates pro-inflammatory cytokine release form 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 signalling 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 to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate 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 (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients. RESULTS: Tacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies 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 receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that 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-1ß, 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.