A monocyte-leptin-angiogenesis pathway critical for repair post-infection.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

Genetic Variants of Fatty Acid Amide Hydrolase Modulate Acute Inflammatory Responses to Colitis in Adult Male Mice.

Cannabinoids, including cannabis derived phytocannabinoids and endogenous cannabinoids (endocannabinoids), are typically considered anti-inflammatory. One such endocannabinoid is N-arachidonoylethanolamine (anandamide, AEA), which is metabolized by fatty acid amide hydrolase (FAAH). In humans, there is a loss of function single nucleotide polymorphism (SNP) in the FAAH gene (C385A, rs324420), that leads to increases in the levels of AEA. Using a mouse model with this SNP, we investigated how this SNP affects inflammation in a model of inflammatory bowel disease. We administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) intracolonically, to adult male FAAH SNP mice and examined colonic macroscopic tissue damage and myeloperoxidase activity, as well as levels of plasma and amygdalar cytokines and chemokines 3 days after administration, at the peak of colitis. We found that mice possessing the loss of function alleles (AC and AA), displayed no differences in colonic damage or myeloperoxidase activity compared to mice with wild type alleles (CC). In contrast, in plasma, colitis-induced increases in interleukin (IL)-2, leukemia inhibitory factor (LIF), monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF) were reduced in animals with an A allele. A similar pattern was observed in the amygdala for granulocyte colony stimulating factor (G-CSF) and MCP-1. In the amygdala, the mutant A allele led to lower levels of IL-1α, IL-9, macrophage inflammatory protein (MIP)-1ß, and MIP-2 independent of colitis-providing additional understanding of how FAAH may serve as a regulator of inflammatory responses in the brain. Together, these data provide insights into how FAAH regulates inflammatory processes in disease.

Endogenous prion protein attenuates experimentally induced colitis.

Although the cellular prion protein (PrP(C)) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrP(C) may exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrP(C) expression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)-induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrP(C)), Prnp(-/-) mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp(-/-) animals, PrP(C) overexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp(-/-) mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrP(C) expression from both the endogenous Prnp locus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrP(C) has a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon.

Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors.

The endocannabinoid (EC) system mediates protection against intestinal inflammation. In this study, we investigated the effects of blocking EC degradation or cellular reuptake in experimental colitis in mice. Mice were treated with trinitrobenzene-sulfonic acid in presence and absence of the fatty acid amide hydrolase (FAAH) blocker URB597, the EC membrane transport inhibitor VDM11, and combinations of both. Inflammation was significantly reduced in the presence of URB597, VDM11, or both as evaluated by macroscopic damage score, myeloperoxidase levels, and colon length. These effects were abolished in CB(1)- and CB(2)-receptor-gene-deficient mice. Quantitative reverse transcription polymerase chain reaction after induction of experimental colitis by different pathways showed that expression of FAAH messenger RNA (mRNA) is significantly reduced in different models of inflammation early in the expression of colitis, and these return to control levels as the disease progresses. Genomic DNA from 202 patients with Crohn's disease (CD) and 206 healthy controls was analyzed for the C385A polymorphism in the FAAH gene to address a possible role in humans. In our groups, the C385A polymorphism was equally distributed in patients with CD and healthy controls. In conclusion, drugs targeting EC degradation offer therapeutic potential in the treatment of inflammatory bowel diseases. Furthermore, reduction of FAAH mRNA expression is involved in the pathophysiological response to colitis.

Expression of a functional metabotropic glutamate receptor 5 on enteric glia is altered in states of inflammation.

The metabotropic glutamate receptor 5 (mGluR5) is expressed by astrocytes and its expression is modulated by inflammation. Enteric glia have many similarities to astrocytes and are the most numerous cell in the enteric nervous system (ENS). We investigated whether enteric glia express a functional mGluR5 and whether expression of this receptor was altered in colitis. In both enteric plexuses of the ileum and colon of guinea pigs and mice, we observed widespread glial mGluR5 expression. Incubation of isolated segments of the guinea pig ileum with the mGluR5 specific agonist RS-2-chloro-5-hydroxyphenylglycine (CHPG) caused a dose-dependent increase in the glial expression of c-Fos and the phosphorylated form of the extracellular signal-regulated kinase 1/2. Preincubation of tissues with the group I metabotropic glutamate receptor antagonist, S-4-carboxyphenylglycine, abolished the effects of CHPG. We examined mGluR5 expression in the guinea pig trinitrobenzene sulfonic acid and the IL-10 gene-deficient (IL-10(-/-)) mouse models of colitis. In guinea pigs, mGluR5 immunoreactivity became diffusely localized over the colonic myenteric ganglia, suggesting a change in receptor distribution. In contrast, glial mGluR5 expression was significantly reduced in the colonic myenteric plexus of IL-10(-/-) mice, as assessed with both real-time quantitative RT-PCR as well as immunohistochemistry and image analysis. These changes occurred without concomitant changes to enteric ganglia or glial fibrillary acidic protein expression in the IL-10(-/-) mouse. Our data suggest that enteric glia are a functional target of the glutamatergic neurotransmitter system in the ENS and that changes in mGluR5 expression may be of physiological significance during colitis.

Inducible nitric oxide synthase from bone marrow-derived cells plays a critical role in regulating colonic inflammation.

BACKGROUND & AIMS: Nitric oxide (NO) is an important mediator of intestinal inflammation. Inducible NO synthase (iNOS) is the main source of NO in inflammation. Because iNOS is ubiquitously expressed, our aim was to determine which cellular source(s) of iNOS plays the central role in mediating intestinal inflammation. METHODS: Chimeric lines were produced via bone marrow (BM) transplantation following irradiation. Colitis was induced with dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS). The severity of colitis and markers of inflammation were assessed in standard fashion. Leukocyte recruitment was assessed by intravital microscopy. RESULTS: The irradiated chimeric lines with iNOS-/- BM-derived cells were markedly more resistant to both DSS- and TNBS-induced injury. Resistance to DSS-induced colitis was lost when wild-type (wt) BM was used to reconstitute iNOS-/- mice. Neutrophils were the main source of iNOS in DSS-induced colitis. iNOS-/- chimeric lines had decreased colonic macrophage inflammatory protein 1beta and tumor necrosis factor alpha expression and increased levels of the protective growth factor, keratinocyte growth factor. LPS-mediated leukocyte recruitment was reduced in iNOS-/- mice, and there were marked changes in the inflammatory cell infiltrates between the chimeric lines with iNOS-/- vs wt BM-derived cells. Furthermore, the lamina propria CD4 +ve cells from chimeric lines with iNOS-/- BM-derived cells had reduced intracellular cytokine expression. CONCLUSIONS: iNOS produced by BM-derived cells plays a critical role in mediating the inflammatory response during colitis. Cell-specific regulation of iNOS may represent a novel form of therapy for patients with inflammatory bowel disease.

Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro.

The proteinase-activated thrombin receptor-1 (PAR-1) belongs to a unique family of G protein-coupled receptors activated by proteolytic cleavage. We studied the effect of PAR-1 activation in the regulation of ion transport in mouse colon in vitro. Expression of PAR-1 in mouse colon was assessed by RT-PCR and immunohistochemistry. To study the role of PAR-1 activation in chloride secretion, mouse colon was mounted in Ussing chambers. Changes in short-circuit current (Isc) were measured in tissues exposed to either thrombin, saline, the PAR-1-activating peptide TFLLR-NH2, or the inactive reverse peptide RLLFT-NH2, before electrical field stimulation (EFS). Experiments were repeated in the presence of either a PAR-1 antagonist or in PAR-1-deficient mice to assess receptor specificity. In addition, studies were conducted in the presence of chloride-free buffer or the muscarinic antagonist atropine to assess chloride dependency and the role of cholinergic neurons in the PAR-1-induced effect. PAR-1 mRNA was expressed in full-thickness specimens and mucosal scrapings of mouse colon. PAR-1 immunoreactivity was found on epithelial cells and on neurons in submucosal ganglia where it was colocalized with both VIP and neuropeptide Y. After PAR-1 activation by thrombin or TFLLR-NH2, secretory responses to EFS but not those to forskolin or carbachol were significantly reduced. The reduction in the response to EFS was not observed in the presence of the PAR-1 antagonist, in PAR-1-deficient mice, when chloride was excluded from the bathing medium, or when atropine was present. PAR-1 is expressed in submucosal ganglia in the mouse colon and its activation leads to a decrease in neurally evoked epithelial chloride secretion.

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon.

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.