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Enteritis caused by Clostridium difficile toxin (Tx) is a nosocomial disease of increasing clinical concern, but the local mediators of C. difficile TxA inflammation are unknown. The potent vasodilator calcitonin gene-related peptide mediates neurogenic inflammation via the calcitonin receptor-like receptor (CLR). Here we examined the ileum-specific effects of reducing CLR on TxA ileitis by local preinjection of double-stranded RNAs. Treatment with CLR dsRNA for 7 d decreased CLR immunoreactivity, whereas treatment with non-CLR dsRNA did not. Subsequent injection of TxA in the same location increased CLR in rats treated with non-CLR dsRNA but not in rats treated with CLR dsRNA, documenting that local injection of dsRNA is effective in preventing the increase in CLR immunoreactivity in response to local TxA. After non-CLR dsRNA pretreatment, TxA induced robust intestinal secretion, myeloperoxidase activity, and histopathologic indications of inflammation including epithelial damage, congestion, neutrophil infiltration, loss of mucin from goblet cells, and increase in mast cell numbers. After CLR dsRNA pretreatment, TxA-induced changes in intestinal secretion and histopathologic inflammation were improved, including normal mucin staining and fewer resident mast cells. Loss of CLR prevented TxA-mediated activation of NF-κB and concomitant increases in pERK1/2 and TNF-α mRNA. Locally produced CLR plays a proinflammatory role in TxA ileitis via MAPK signaling and TNF-α. The results reported here strongly suggest that a local injection of dsRNA targeting CLR could be an effective local therapeutic approach at the inflammation site in the treatment of a growing, clinically relevant hospital-acquired disease, C. difficile infection.

Mesenteric fat is known to undergo inflammatory changes after 2,4,6,-trinitrobenzensulphonic acid (TNBS)–induced colitis. Neurotensin (NT) and neurotensin receptor 1 (NTR1) have been shown to play a major role in the pathogenesis of intestinal inflammation. This led us to explore whether NT and NTR1 are expressed in the mesenteric fat depots during TNBS-induced colitis and whether NT participates in the increased interleukin (IL)–6 secretion in this inflammatory response. TNBS-induced inflammation in the colon increases NT and NTR1 expression in mesenteric adipose tissues, including mesenteric preadipocytes. Compared with wild-type mice, NT knockout (KO) mice have reduced TNBS-induced colitis accompanied by diminished inflammatory responses in mesenteric adipose tissue. Specifically, IL-6 and p65 phosphorylation levels in mesenteric fat of NT KO mice are also reduced compared with wild-type mice. Mouse 3T3-L1 preadipocytes express NTR1 and its expression is increased after stimulation of preadipocytes with proinflammatory cytokines. NT stimulation of 3T3-L1 preadipocytes overexpressing NTR1 causes PKCδ phosphorylation and IL-6 secretion in a time- and dose-dependent fashion. Moreover, NT-mediated IL-6 expression is nuclear factor–κB and PKCδ dependent. We also found that supernatants from NT-exposed 3T3-L1-NTR1 preadipocytes and mesenteric fat obtained from wild-type mice 2 days after TNBS administration stimulate an IL-6–dependent macrophage migration measured by a macrophage migration assay, whereas this response is reduced when mesenteric fat from NT KO mice is used. These results demonstrate an important role for NT in acute colitis and adipose tissue inflammation associated with experimental colitis that involves direct NT proinflammatory responses in preadipocytes.

Melanin-concentrating hormone (MCH) is expressed primarily in the hypothalamus and has a positive impact on feeding behavior and energy balance. Although MCH is expressed in the gastrointestinal tract, its role in this system remains elusive. We demonstrate that, compared to wild type, mice genetically deficient in MCH had substantially reduced local inflammatory responses in a mouse model of experimental colitis induced by intracolonic administration of 2,4,6 trinitrobenzene sulfonic acid (TNBS). Likewise, mice receiving treatments with an anti-MCH antibody, either prophylactically or after the establishment of colitis, developed attenuated TNBS-associated colonic inflammation and survived longer. Consistent with a potential role of MCH in intestinal pathology, we detected increased colonic expression of MCH and its receptor in patients with inflammatory bowel disease. Moreover, we found that human colonic epithelial cells express functional MCH receptors, the activation of which induces IL-8 expression. Taken together, these results clearly implicate MCH in inflammatory processes in the intestine and perhaps elsewhere.

The neurokinin 1 receptor (NK1R), a G protein-coupled receptor involved in diverse functions including pain and inflammation, has two putative N-linked glycosylation sites, Asn-14 and Asn-18. We studied the role of N-linked glycosylation in the functioning of the NK1R by constructing three receptor mutants: two single mutants (Asn → Gln-14 and Asn → Gln-18) and a double mutant, lacking both glycosylation sites. Using a lentiviral transfection system, the mutants were stably transfected into NCM 460 cells, a nontransformed human colonic epithelial cell line. We observed that the magnitude of glycosylation as estimated by changes in gel migration depends on the number of glycosylation sites available, with the wild-type receptor containing the greatest amount of glycosylation. All mutant receptors were able to bind to substance P and neurokinin A ligand with similar affinities; however, the double mutant, nonglycosylated NK1R showed only half the B max of the wild-type NK1R. In terms of receptor function, the ablation of both N-linked glycosylation sites did not have a profound effect on the receptors' abilities to activate the MAP kinase families (p42/p44, JNK, and p38), but did affect SP-induced IL-8 secretion. All mutants were able to internalize, but the kinetics of internalization of the double mutant receptor was more rapid, when compared with wild-type NK1R. Therefore, glycosylation of NK1R may stabilize the receptor in the plasma membrane. These results contribute to the ongoing elucidation of the role of glycosylation in G protein-coupled receptors and the study of the neurokinin receptors in particular.

Intracolonic administration of trinitrobenzene sulfonic acid in mice causes inflammation in the colon that is accompanied by increased expression of proinflammatory cytokines and of the substance P (SP), neurokinin 1 receptor (NK-1R) in the proximal mesenteric fat depot. We also investigated whether human mesenteric preadipocytes contain NK-1R and examined the functional consequences of exposure of these cells to SP as it relates to proinflammatory signaling. We found that human mesenteric preadipocytes express NK-1R both at the mRNA and protein levels. Exposure of human mesenteric preadipocytes to SP increased NK-1R mRNA and protein expression by 3-fold, and stimulated IL-8 mRNA expression and protein secretion. This effect was abolished when these cells were pretreated with the specific NK-1R antagonist CJ 012,255. Moreover, human mesenteric preadipocytes transfected with a luciferase promoter/reporter system containing the IL-8 promoter with a mutated NF-κB site lost their ability to respond to SP, indicating that SP-induced IL-8 expression is NF-κB-dependent. This report indicates that human mesenteric preadipocytes contain functional SP receptors that are linked to proinflammatory pathways, and that SP can directly increase NK-1R expression. We speculate that mesenteric fat depots may participate in intestinal inflammatory responses via SP–NK-1R-related pathways, as well as other systemic responses to the presence of an ongoing inflammation of the colon.

Commensal and enteroinvasive microbes in the human gut release bacterial flagellin, a specific microbial ligand of Toll-like receptor 5 (TLR5). However, the pathophysiological role of bacterial flagellin in gastrointestinal inflammation has not been determined. Here we evaluated the role of bacterial flagellin using native human colonic mucosa and the mouse colitis model of dextran sulfate sodium (DSS). We demonstrate that, in intact human colonic mucosa, the flagellin/TLR5 response occurs only after exposure to the basolateral, not the apical, surface, implying a basolaterally polarized TLR5 response in human colonic mucosa. In this context, flagellin exposure to injured colonic mucosa due to DSS administration in mice resulted in a TLR5-associated response evaluated by in vivo activation of mitogen-activated protein kinase/extracellular signal-related kinase 1/2 (MEK1/2) and elevated IL-6, TNF-α, and keratinocyte-derived chemokine production, whereas intact colonic mucosa did not respond to flagellin. Moreover, flagellin exposure to injured mouse colon in vivo , but not to intact colon, also significantly aggravated colonic inflammation, increased mouse mortality, and enhanced histopathological damage in the colonic mucosa. However, the TLR2-specific agonist, peptidoglycan or lipoteichoic acid, did not cause an inflammatory response in intact or DSS-injured mouse colon. Furthermore, intracolonic flagellin administration in mice causes severe apoptosis in colonic epithelium disrupted by DSS administration. These data suggest that intracolonic flagellin via TLR5 engagement is able to elicit inflammatory responses in disrupted colon, whereas the normal colon is not responsive to bacterial flagellin. These results demonstrate that bacterial flagellin plays an important role in the development and progress of colitis.

Clostridium difficile, the causative agent of antibiotic-associated colitis, mediates inflammatory diarrhea by releasing toxin A, a potent 308-kDa enterotoxin. Toxin A-induced inflammatory diarrhea involves many steps, including mucosal release of substance P (SP) corticotropin-releasing hormone (CRH) and neutrophil transmigration. Here we demonstrate that, compared with wild type, mice genetically deficient in CRH (Crh-/-) have dramatically reduced ileal fluid secretion, epithelial cell damage, and neutrophil transmigration 4 h after intraluminal toxin A administration. This response is associated with diminished mucosal activity of the neutrophil enzyme myeloperoxidase compared with that of wildtype mice. In wild-type mice, toxin A stimulates an increase in intestinal SP content compared with buffer administration. In contrast, toxin A administration inCrh-/-mice fails to result in an increased SP content. Moreover, immunohistochemical experiments showed that CRH and SP are colocalized in some enteric nerves of wild-type mice, and this colocalization is more evident after toxin A administration. These results provide direct evidence for a major proinflammatory role for CRH in the pathophysiology of enterotoxin-mediated inflammatory diarrhea and indicate a SP-linked pathway.

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R−/− mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R−/− animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.

Previously we reported that pretreatment of rats with the substance P (SP) antagonist CP-96,345 inhibits the enterotoxic responses following administration of toxin A fromClostridium difficileinto ileal loops, indicating that SP participates in the intestinal responses to this toxin. We now report that injection of toxin A into rat ileum causes a rapid increase in SP content in lumbar dorsal root ganglia (DRG) and mucosal scrapings 30–60 min after toxin A administration. Toxin A-mediated fluid secretion, mannitol permeability, and ileal histologic damage is significantly increased only after 2 hr. Toxin A also causes an increase in the abundance of SP mRNA in lumbar DRG and ileal mucosa as measured by reverse transcription–PCR. Lamina propria macrophages (LPMs) obtained from toxin A-injected loops release greater amounts of tumor necrosis factor α (TNFα) and SP as compared with LPMs isolated from buffer-injected loops (P< 0.01). Pretreatment of rats with the SP antagonist CP-96,345 inhibits toxin A-mediated TNFα release from isolated LPMs, whereas an inactive enantiomer (CP-96,344) of the SP antagonist has no effect. LPMs obtained from toxin A-injected ileal loops incubatedin vitrowith SP (10−8to 10−9M) show enhanced TNFα secretion, whereas LPMs isolated from buffer-injected loops do not respond to SP. In addition, LPMs obtained from toxin A-injected ileal loops incubatedin vitrowith CP-96,345 showed a diminished TNFα release. Our results indicate that activated LPMs secrete SP during toxin A enteritis that can lead to secretion of cytokines, suggesting an autocrine/paracrine regulation of cytokine secretion by SP from LPMs during intestinal inflammation.

We previously reported that short-term immobilization stress of rats causes increased colonic mucin release, goblet cell depletion, prostaglandin E2 secretion, and colonic mast cell activation, as well as increased colonic motility. The purpose of this study was to investigate whether neurotensin (NT), a peptide expressed in both brain and digestive tract, participates in these responses. Rats were pretreated with SR 48692 (1 mg/kg, i.p.), an NT antagonist, 15 min before immobilization (30 min). The administration of the antagonist significantly inhibited stress-mediated secretion of colonic mucin, prostaglandin E2, and a product of rat mast cells, rat mast cell protease II (P < 0.05), but did not alter the increase in fecal pellet output caused by immobilization stress. Immobilization stress also resulted in a quantifiable decrease in the abundance of NT receptor mRNA in rat colon compared with that in colonic tissues from nonimmobilized rats as measured by densitometric analysis of in situ hybridization studies (P < 0.03). We conclude that the peptide NT is involved in colonic goblet cell release and mucosal mast cell activation after immobilization stress.