TLR2 and TLR9 modulate enteric nervous system inflammatory responses to lipopolysaccharide.

BACKGROUND: Accumulating evidence suggest that the enteric nervous system (ENS) plays important roles in gastrointestinal inflammatory responses, which could be in part mediated by Toll-like receptor (TLR) activation. The aim of this study was to characterise the expression and functionality of TLR2/4/9 in the ENS. METHODS: TLR2/4/9 expression was assessed in the plexuses of adult rats and embryonic ENS cultures by immunofluorescence and quantitative PCR. Following stimulation with TLR2/4/9 ligands or their combinations, activation of NF-kB, production of TNF-α, IL-6 and MCP-1 and chemoattraction of RAW264.7 macrophages were evaluated by means of Western blot, ELISA, immunofluorescence and migration assays in transwell inserts. RESULTS: TLR2/4/9 staining colocalised with enteric neuronal markers, whereas their presence in enteroglial processes was low to inexistent. Stimulation of ENS cultures with selective ligands induced NF-kB activation and release of cytokines and chemokines by neurons and resident immunocytes. TLR2 neutralisation before lipopolysaccharide (LPS) challenge reduced production of inflammatory mediators, whereas combination of TLR2/4 ligands promoted macrophage migration. Combined stimulation of cultures with LPS and the CpG oligonucleotide 1826 (TLR4/9 ligands) caused a synergic increase in chemoattraction and cytokine production. CONCLUSIONS: Our results suggest that the ENS, and particularly enteric neurons, can integrate a variety of microbial signals and respond in a relatively selective fashion, depending on the particular TLRs stimulated. These findings additionally suggest that the ENS is capable of initiating a defensive response against pathogens and expanding inflammation.

Short-fiber protein of ad40 confers enteric tropism and protection against acidic gastrointestinal conditions.

The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter ß-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases.

Functional inclusion bodies produced in bacteria as naturally occurring nanopills for advanced cell therapies.

Inclusion bodies (50-500 nm in diameter) produced in recombinant bacteria can be engineered to contain functional proteins with therapeutic potential. Upon exposure, these protein particles are efficiently internalized by mammalian cells and promote recovery from diverse stresses. Being fully biocompatible, inclusion bodies are a novel platform, as tailored nanopills, for sustained drug release in advanced cell therapies.

Functionally enhanced siRNA targeting TNFα attenuates DSS-induced colitis and TLR-mediated immunostimulation in mice.

Tumor necrosis factor (TNFα) is a proinflammatory cytokine involved in the pathogenesis of inflammatory bowel disease (IBD). Although TNFα has been extensively targeted using systemic drugs, the use of antisense and small interfering RNA (siRNA) to drive down its expression at the site of inflammation should provide important advantages. In this study, native and chemically modified siRNA against TNFα was developed and characterized using a murine model of IBD. siRNA with 2'-O-methyl and propanediol modifications (siTNF-OMe-P) were resistant to nuclease degradation and provided better silencing efficacy in vitro as compared to unmodified siRNA. Every modification reduced nonspecific Toll-like receptor (TLR)-mediated immunomodulation in human peripheral blood mononuclear cells (PBMC) cells. Intrarectal administration of siTNF-OMe-P significantly ameliorated the clinical endpoints and histopathological severity in 5% dextran sulphate sodium (DSS)-treated mice as compared to unmodified and other chemically modified siRNAs. Differential gene expression assessed in siTNF-OMe-P-treated animals correlated with improved colon integrity and reduced TLR activation as compared to all treatment groups. All in all, this study demonstrates that propanediol and 2'-O-methyl modifications have profound functional consequences for siRNA efficacy in vivo. Consequently, this strategy has potential implications for therapeutic intervention in IBD and other diseases.

Neuromuscular changes in a rat model of colitis.

Intracolonic administration of Trichinella spiralis larvae in rats causes colitis with features similar to ulcerative colitis, notably with inflammation predominantly limited to the colonic mucosa. Our aim was to characterize the functional and neurochemical changes occurring within the myenteric (MP) and submucosal plexuses (SMP) during T. spiralis-induced colitis. Infected rats had decreased body weight, altered stool consistency and elevated myeloperoxidase activity, 6 and 14 days post-infection (PI). Responses to acetylcholine and KCl in circular muscle strips were reduced in infected tissues, demonstrating an impairment of contractility. In addition, there was a decrease in spontaneous motor activity and reduced sensitivity to the nitric oxide synthase (NOS) inhibitor L-NOArg, corresponding with a significant reduction in NOS immunoreactive neurons in the MP of infected animals. T. spiralis did not alter the total number of myenteric or submucosal neurons. Substance P innervation of submucosal blood vessels was reduced after infection, as were submucosal calretinin and calbindin immunoreactive neurons. No changes in choline acetyltransferase and calcitonin gene-related peptide immunoreactivity were observed. T. spiralis-induced colitis causes profound neuromuscular adaptations. The reduction in NOS neurons appears to underlie changes in motility.