Glucagon-like peptide 2 (GLP-2) is an important intestinal growth factor with anti-inflammatory activity. We hypothesized that GLP-2 decreases mucosal inflammation and the associated increased epithelial proliferation by downregulation of Th1 cytokines attributable to reprogramming of lamina propria immune regulatory cells via an interleukin-10 (IL-10)-independent pathway. The effects of GLP-2 treatment were studied using the IL-10-deficient (IL-10(-/-)) mouse model of colitis. Wild-type and IL-10(-/-) mice received saline or GLP-2 (50 microg/kg sc) treatment for 5 days. GLP-2 treatment resulted in significant amelioration of animal weight loss and reduced intestinal inflammation as assessed by histopathology and myeloperoxidase levels compared with saline-treated animals. In colitis animals, GLP-2 treatment also reduced crypt cell proliferation and crypt cell apoptosis. Proinflammatory (IL-1beta, TNF-alpha, IFN-gamma,) cytokine protein levels were significantly reduced after GLP-2 treatment, whereas IL-4 was significantly increased and IL-6 production was unchanged. Fluorescence-activated cell sorting analysis of lamina propria cells demonstrated a decrease in the CD4(+) T cell population following GLP-2 treatment in colitic mice and an increase in CD11b(+)/F4/80(+) macrophages but no change in CD25(+)FoxP3 T cells or CD11c(+) dendritic cells. In colitis animals, intracellular cytokine analysis demonstrated that GLP-2 decreased lamina propria macrophage TNF-alpha production but increased IGF-1 production, whereas transforming growth factor-beta was unchanged. GLP-2-mediated reduction of crypt cell proliferation was associated with an increase in intestinal epithelial cell suppressor of cytokine signaling (SOCS)-3 expression and reduced STAT-3 signaling. This study shows that the anti-inflammatory effects of GLP-2 are IL-10 independent and that GLP-2 alters the mucosal response of inflamed intestinal epithelial cells and macrophages. In addition, the suggested mechanism of the reduction in inflammation-induced proliferation is attributable to GLP-2 activation of the SOCS-3 pathway, which antagonizes the IL-6-mediated increase in STAT-3 signaling.

Activation of the innate immune system by bacterial DNA and DNA of other invertebrates represents a pathogen recognition mechanism. In this study we investigated macrophage responses to DNA from the intestinal protozoan parasite Entamoeba histolytica. E. histolytica genomic DNA was purified from log-phase trophozoites and tested with the mouse macrophage cell line RAW 264.7. RAW cells treated with E. histolytica DNA demonstrated an increase in levels of tumor necrosis factor alpha (TNF-␣) mRNA and protein production. TNF-␣ production was blocked by pretreatment with chloroquine or monensin. In fact, an NF-B luciferase reporter assay in HEK cells transfected with human TLR9 demonstrated that E. histolytica DNA signaled through Toll-like receptor 9 (TLR9) in a manner similar to that seen with CpG-ODN. Immunofluorescence assays confirmed NF-B activation in RAW cells, as seen by nuclear translocation of the p65 subunit. Western blot analysis demonstrated mitogen-activated protein kinase activation by E. histolytica DNA. E. histolytica DNA effects were abolished in MYD88 ؊/؊ mouse-derived macrophages. In the context of disease, immunization with E. histolytica DNA protected gerbils from an E. histolytica challenge infection. Taken together, these results demonstrate that E. histolytica DNA is recognized by TLR9 to activate macrophages and may provide an innate defense mechanism characterized by the induction of the inflammatory mediator TNF-␣.

Amebiasis is a human disease caused by the protozoan intestinal parasite Entamoeba histolytica. Vaccine development has focused on the parasite's surface galactose-Nacetyl-D-galactosamine inhibitable lectin (Gal-lectin) as a protective antigen. The Gallectin is immunogenic and has been shown to induce Th1 cytokines in vitro and in vivo. The immunological basis of the protective immune response elicited by the Gal-lectin is unknown. In this study, we investigated the response of BALB/c bone marrow-derived DC to E. histolytica Gal-lectin. Incubation of immature DC with Gal-lectin resulted in activation and maturation after 24 h. FACS analysis demonstrated an up-regulation of DC maturation markers CD80, CD86, CD40 and MHC class II upon exposure to Gallectin. The Gal-lectin also induced DC production of IL-12, indicating a Th1 response. Gal-lectin-activated DC were able to stimulate T cell proliferation in an allogeneic mixed leukocyte reaction and adoptive transfer of Gal-lectin-treated DC into naïve mice resulted in IFN-c-producing Gal-lectin-sensitized T cells. The activation of DC by Gallectin was mediated by MAPK and NF-jB. These findings indicate that E. histolytica Gallectin is a potent vaccine antigen capable of directly initiating DC maturation and activation characterized by Th1 cytokine production.