Protective effect of adipose tissue-derived mesenchymal stromal cells in an experimental model of high-risk colonic anastomosis.

BACKGROUND: Dehiscence of intestinal anastomosis results in high morbidity and mortality. The aim of this study was to investigate the effects of locally administered adipose tissue-derived mesenchymal stromal cells in a model of high-risk colonic anastomosis in rats. METHODS: Seven days after induction of colitis with 2,4,6-trinitrobenzene sulfonic acid, Wistar rats were submitted to a transection of the descending colon followed by end-to-end anastomosis and were then treated with 2×106 adipose tissue-derived mesenchymal stromal cells (from the preperitoneal fat) or an acellular culture solution instilled onto the surface of the anastomosis. At day 14, after macroscopic survey of the abdominal cavity, the anastomotic area was submitted to histologic and immunohistochemical analysis, evaluation of myeloperoxidase activity, fibrosis, epithelial integrity, NF-κ B activation, expression of inflammatory cytokines, and extracellular matrix-related genes. RESULTS: Anastomotic leakage and mortality associated with high-risk anastomosis decreased with treatment with adipose tissue-derived mesenchymal stromal cells (P < .03). Application of adipose tissue-derived mesenchymal stromal cells resulted in lower histologic scores (P = .011), decreased deposition of collagen fibers (P = .003), preservation of goblet cells (P = .033), decreased myeloperoxidase activity (P = .012), decreased accumulation of CD4+ T-cells (P = .014) and macrophages (P = .011) in the lamina propria, a decrease in the number of apoptotic cells (P = .008), and the activation of NF-κ B (P = .036). Overexpression of IL-17, TNF-α , IFN-γ, and metalloproteinases in the acellular culture solution-treated, high-risk anastomosis group decreased (P < .05) to near normal values with adipose tissue-derived mesenchymal stromal cells treatment. CONCLUSION: Improvements in outcomes of a high-risk colonic anastomosis with adipose tissue-derived mesenchymal stromal cells therapy reflect the immunomodulatory activity and healing effect of these cells, even after just topical administration and reinforces their use in future translational research.

Schistosoma mansoni Coinfection Attenuates Murine Toxoplasma gondii-Induced Crohn's-Like Ileitis by Preserving the Epithelial Barrier and Downregulating the Inflammatory Response.

Background and aims: Mice orally infected with T. gondii develop Crohn's disease (CD)-like enteritis associated with severe mucosal damage and a systemic inflammatory response, resulting in high morbidity and mortality. Previously, helminthic infections have shown therapeutic potential in experimental colitis. However, the role of S. mansoni in T. gondii-induced CD-like enteritis has not been elucidated. Our study investigated the mechanisms underlying T. gondii-induced ileitis and the potential therapeutic effect of S. mansoni coinfection. Methods: C57BL/6 mice were infected by subcutaneous injection of cercariae of the BH strain of S. mansoni, and 7-9 weeks later, they were orally infected with cysts of the ME49 strain of T. gondii. After euthanasia, the ileum was removed for histopathological analysis; staining for goblet cells; immunohistochemistry characterizing mononuclear cells, lysozyme expression, apoptotic cells, and intracellular pathway activation; and measuring gene expression levels by real-time PCR. Cytokine concentrations were measured in the serial serum samples and culture supernatants of the ileal explants, in addition to myeloperoxidase (MPO) activity. Results:T. gondii-monoinfected mice presented dense inflammatory cell infiltrates and ulcerations in the terminal ileum, with abundant cell extrusion, apoptotic bodies, and necrosis; these effects were absent in S. mansoni-infected or coinfected animals. Coinfection preserved goblet cells and Paneth cells, remarkably depleted in T. gondii-infected mice. Densities of CD4- and CD11b-positive cells were increased in T. gondii- compared to S. mansoni-infected mice and controls. MPO was significantly increased among T. gondii-mice, while attenuated in coinfected animals. In T. gondii-infected mice, the culture supernatants of the explants showed increased concentrations of TNF-alpha, IFN-gamma, and IL-17, and the ileal tissue revealed increased expression of the mRNA transcripts for IL-1 beta, NOS2, HMOX1, MMP3, and MMP9 and activation of NF-kappa B and p38 MAPK signaling, all of which were counterregulated by S. mansoni coinfection. Conclusion:S. mansoni coinfection attenuates T. gondii-induced ileitis by preserving mucosal integrity and downregulating the local inflammatory response based on the activation of NF-kappa B and MAPK. The protective function of prior S. mansoni infection suggests the involvement of innate immune mechanisms and supports a conceptually new approach to the treatment of chronic inflammatory diseases, including CD.

Damage-associated molecular patterns in inflammatory bowel disease: From biomarkers to therapeutic targets.

The chronic inflammatory process underlying inflammatory bowel disease (IBD), comprising Crohn's disease and ulcerative colitis, derives from the interplay of several components in a genetically susceptible host. These components include environmental elements and gut microbiota a dysbiosis. For decades, immune abnormalities have been investigated as critically important in IBD pathogenesis, and attempts to develop effective therapies have predominantly targeted the immune system. Nevertheless, immune events represent only one of the constituents contributing to IBD pathogenesis within the context of the complex cellular and molecular network underlying chronic intestinal inflammation. These factors need to be appreciated within the milieu of non-immune components. Damage-associated molecular patterns (DAMPs), which are essentially endogenous stress proteins expressed or released as a result of cell or tissue damage, have been shown to act as direct pro-inflammatory mediators. Excessive or persistent signalling mediated by such molecules can underlie several chronic inflammatory disorders, including IBD. The release of endogenous DAMPs amplifies the inflammatory response driven by immune and non-immune cells and promotes epigenetic reprogramming in IBD. The effects determine pathologic changes, which may sustain chronic intestinal inflammation and also underlie specific disease phenotypes. In addition to highlighting the potential use of DAMPs such as calprotectin as biomarkers, research on DAMPs may reveal novel mechanistic associations in IBD pathogenesis and is expected to uncover putative therapeutic targets.

Sulfate-reducing bacteria stimulate gut immune responses and contribute to inflammation in experimental colitis.

The intestinal microbiota is critical for mammalian immune system development and homeostasis. Sulfate-reducing bacteria (SRB) are part of the normal gut microbiota, but their increased levels may contribute to colitis development, likely in association with hydrogen sulfide (H2S) production. Here, we investigated the effects of SRB in the gut immune response in germ-free mice, and in experimental colitis. After 7days of colonization with Desulfovibrio indonesiensis or with a human SRB consortium (from patients with colitis), germ-free mice exhibited alterations in the colonic architecture, with increased cell infiltration in the lamina propria. SRB colonization upregulated the Th17 and Treg profiles of cytokine production/cell activation, in T cells from mesenteric lymph nodes. These alterations were more pronounced in mice colonized with the human SRB consortium, although D. indonesiensis colonization produced higher levels of H2S. Importantly, the colon of C57BL/6 mice with colitis induced by TNBS or oxazolone had increased SRB colonization, and the administration of D. indonesiensis to mice with TNBS-induced colitis clearly exacerbated the alterations in colonic architecture observed in the established disease, and also increased mouse weight loss. We conclude that SRB contribute to immune response activation in the gut and play an important role in colitis development.

Diet and microbiota in inflammatory bowel disease: The gut in disharmony.

Bacterial colonization of the gut shapes both the local and the systemic immune response and is implicated in the modulation of immunity in both healthy and disease states. Recently, quantitative and qualitative changes in the composition of the gut microbiota have been detected in Crohn's disease and ulcerative colitis, reinforcing the hypothesis of dysbiosis as a relevant mechanism underlying inflammatory bowel disease (IBD) pathogenesis. Humans and microbes have co-existed and co-evolved for a long time in a mutually beneficial symbiotic association essential for maintaining homeostasis. However, the microbiome is dynamic, changing with age and in response to environmental modifications. Among such environmental factors, food and alimentary habits, progressively altered in modern societies, appear to be critical modulators of the microbiota, contributing to or co-participating in dysbiosis. In addition, food constituents such as micronutrients are important regulators of mucosal immunity, with direct or indirect effects on the gut microbiota. Moreover, food constituents have recently been shown to modulate epigenetic mechanisms, which can result in increased risk for the development and progression of IBD. Therefore, it is likely that a better understanding of the role of different food components in intestinal homeostasis and the resident microbiota will be essential for unravelling the complex molecular basis of the epigenetic, genetic and environment interactions underlying IBD pathogenesis as well as for offering dietary interventions with minimal side effects.

P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells.

P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RBlow. Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.

Disruption of the Hedgehog signaling pathway in inflammatory bowel disease fosters chronic intestinal inflammation.

Hedgehog (Hh) signaling is essential for intestinal homeostasis and has been associated with inflammation and tissue repair. We hypothesized that Hh signaling could affect the inflammatory process in inflammatory bowel disease (IBD). For this purpose, colon specimens from the inflamed and non-inflamed mucosa of 15 patients with Crohn's disease (CD), 15 with ulcerative colitis, and 15 controls were analyzed by immunohistochemistry and real-time PCR. The production and modulation of cytokines were measured by ELISA from culture explants. Apoptosis was assessed by TUNEL and caspase-3 activity assays. Chemotaxis was evaluated using a transwell system. Primary human intestinal and skin fibroblasts were used for analyzing migration and BrdU incorporation. Hh proteins were generally expressed at the superficial epithelium, and a marked reduction was observed in CD. In the lamina propria, Gli-1 predominantly co-localized with vimentin- and alpha-smooth muscle actin-positive cells, with lower levels observed in CD. In colon explants, Hh stimulation resulted in reduction, while blockade increased, TNF α, IL-17, and TGF ß levels. Apoptotic rates were higher in inflamed samples, and they increased after Hh blockade. Levels of Gli-1 mRNA were negatively correlated with caspase-3 activity. Hh blockade increased chemoattraction of monocytes. Primary fibroblasts incorporated more BrdU, but migrated less after Hh blockade. These results suggest that Hh signaling provides a negative feedback to the lamina propria, down-regulating inflammatory cytokines, and inhibiting leukocyte migration and fibroblast proliferation, while favoring fibroblast migration. Therefore, Hh signaling is strongly implicated in the pathogenesis of intestinal inflammation, and it may represent a novel therapeutic target for IBD.

Neuroimmunomodulation in the Gut: Focus on Inflammatory Bowel Disease.

Intestinal immunity is finely regulated by several concomitant and overlapping mechanisms, in order to efficiently sense external stimuli and mount an adequate response of either tolerance or defense. In this context, a complex interplay between immune and nonimmune cells is responsible for the maintenance of normal homeostasis. However, in certain conditions, the disruption of such an intricate network may result in intestinal inflammation, including inflammatory bowel disease (IBD). IBD is believed to result from a combination of genetic and environmental factors acting in concert with an inappropriate immune response, which in turn interacts with nonimmune cells, including nervous system components. Currently, evidence shows that the interaction between the immune and the nervous system is bidirectional and plays a critical role in the regulation of intestinal inflammation. Recently, the maintenance of intestinal homeostasis has been shown to be under the reciprocal control of the microbiota by immune mechanisms, whereas intestinal microorganisms can modulate mucosal immunity. Therefore, in addition to presenting the mechanisms underlying the interaction between immune and nervous systems in the gut, here we discuss the role of the microbiota also in the regulation of neuroimmune crosstalk involved in intestinal homeostasis and inflammation, with potential implications to IBD pathogenesis.

The role of innate immunity receptors in the pathogenesis of inflammatory bowel disease.

Innate immunity constitutes the first line of defense, fundamental for the recognition and the initiation of an inflammatory response against microorganisms. The innate immune response relies on the sensing of microbial-associated molecular patterns through specialized structures such as toll-like receptors (TLRs) and the nucleotide oligomerization domain- (NOD-) like receptors (NLRs). In the gut, these tasks are performed by the epithelial barrier and the presence of adaptive and innate immune mechanisms. TLRs and NLRs are distributed throughout the gastrointestinal mucosa, being more expressed in the epithelium, and in lamina propria immune and nonimmune cells. These innate immunity receptors exhibit complementary biological functions, with evidence for pathways overlapping. However, as tolerance is the predominant physiological response in the gastrointestinal mucosa, it appears that the TLRs are relatively downregulated, while NLRs play a critical role in mucosal defense in the gut. Over the past two decades, genetic polymorphisms have been associated with several diseases including inflammatory bowel disease. Special emphasis has been given to the susceptibility to Crohn's disease, in association with abnormalities in the NOD2 and in the NLRP3/inflammasome. Nevertheless, the mechanisms underlying innate immune receptors dysfunction that result in the persistent inflammation in inflammatory bowel disease remain to be clarified.

Hedgehog pathway signaling regulates human colon carcinoma HT-29 epithelial cell line apoptosis and cytokine secretion.

The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted ß-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.