Alessia Butera

A Transient Breach in the Epithelial Barrier Leads to Regulatory T-Cell Generation and Resistance to Experimental Colitis

BACKGROUND & AIMS Previous studies have indicated that a defective epithelial barrier leads to inflammation of the underlying lamina propria. Nevertheless, it is likely that physiologic breaks in the barrier must occur for homeostatic regulatory T cells to develop. We determined the effect of agents that disrupt epithelial tight junctions (ethanol and AT1002, a Vibrio cholerae zonula occludens toxin hexapeptide) on regulatory T-cell induction and resistance to induction of colitis by trinitrobenzene sulfonic acid (TNBS). METHODS The effects of ethanol and AT1002 on colon immune function were evaluated by their capacity to induce direct phenotypic or functional changes in effector and regulatory cell populations and their indirect effect on the development of TNBS-induced colitis. The basis of regulatory cell development was evaluated with in vitro studies of isolated dendritic cell populations. The role of innate immunity was evaluated by in vivo gene silencing studies utilizing Toll-like receptor (TLR)-2-specific small interfering RNA (siRNA). RESULTS Both ethanol and AT1002 induced persistent latency-associated peptide-positive CD4(+) regulatory T cells that, as shown in adoptive transfer studies, render mice resistant to the induction of TNBS colitis. The development of these cells requires the presence of an intact microflora and the activity of CD11c(+) dendritic cells. Their induction is also influenced by innate immune factors operating through TLR-2, because attenuation of TLR-2 signaling by in vivo TLR-2 siRNA administration prevents their development. CONCLUSIONS A mild and/or transient breach in epithelial barrier function leads to dominant regulatory T-cell responses that protect the mucosa from inflammation.

Intestinal microflora and immunoregulation

Immune homeostasis at mucosal level results from controlled response to intestinal luminal antigens. Recent insights into the nature of inflammatory bowel diseases, derived mainly from studies of experimental models of colonic inflammation, strongly suggest that they can result from a loss of immune tolerance to antigens in the bacterial microflora. Investigations of the regulatory mechanisms operating at the mucosal level suggest that regulatory cells reactive to the intestinal microflora might play a role in cross-reactive protection toward different antigens. Expansion of microflora-reactive regulatory cells by probiotic administration is able to protect from experimental colitis. Characterization of regulatory cells in response to normal commensal flora, the basis of their development and the role of innate immunity in this process might contribute to the understanding of the development of inflammatory bowel diseases.

Nod2 deficiency is associated with an increased mucosal immunoregulatory response to commensal microorganisms

On the basis of previous studies demonstrating that a breach of the colonic epithelial barrier is associated with a microbiota-dependent increase in lamina propria (LP) regulatory cells, we investigated if the lack of spontaneous intestinal inflammation observed in nucleotide-binding oligomerization domain 2 (Nod2)−/− mice was due to enhanced intestinal regulatory function. We found that the LP CD4+ T-cell population of Nod2−/− mice contains an increased percentage of CD4+ regulatory T cells bearing transforming growth factor -β/latency peptide (LP CD4+LAP (latency-associated peptide) + T cells) both under baseline conditions and following an intentional breach of the colonic barrier induced by ethanol administration. In addition, we found that Nod2−/− mice manifest decreased severity of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis and that TNBS-colitis in Nod2−/− or Nod2+/+ mice is ameliorated by adoptive transfer of LP cells from ethanol-treated mice before, but not after, depletion of LAP+ T cells. This increased regulatory T-cell response in Nod2−/− mice could explain why NOD2 polymorphisms in humans are not in themselves sufficient to establish inflammatory lesions.

The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh−/− mice to oxidative stress. Methodology/Principal Findings The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3+ regulatory T cells were observed only in Mutyh−/− mice. Conclusions The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.

Lamina Propria CD4+LAP+ Regulatory T Cells Are Increased in Active Ulcerative Colitis but Show Increased IL-17 Expression and Reduced Suppressor Activity

BACKGROUND A CD4+CD25- regulatory T cell population expressing the surface TGF-β in its latent form LAP+ [latency associated peptide] cells was proved to be protective in experimental colitis and to be suppressive of human peripheral blood [PB] T proliferation. We investigated the frequency and function of lamina propria [LP] CD4+LAP+ T cells in inflammatory bowel disease [IBD] patients. METHODS Specimens from patients undergoing colonoscopy or bowel resection for IBD and colonic cancer were used as source of lamina propria mononuclear cells [LPMC]. The ulcerative colitis [UC] group was divided according to endoscopic activity evaluated with modified Baron Score. IL-17, IFN-γ, IL-10, LAP, and Foxp3 expression in CD3+CD8- [CD4] or CD3+/CD4+ gated cell population was assessed by immunofluorescence. The ability of FACS-sorted LP CD3+CD8-[CD4] LAP+CD25- to inhibit stimulated autologous PB CD3+CD8-[CD4] LAP- CD25- cells proliferation was assessed. RESULTS LP CD4LAP+ cells were significantly increased, when compared with controls, in active UC patients and not in Crohns disease patients. The majority of LP CD4+LAP+ cells were Foxp3-. The percentage of IL-17+ cells in LP CD3+CD8-[CD4] LAP+ cells was significantly higher in active UC patients when compared with controls. LP CD3+CD8-[CD4]LAP+CD25- isolated from UC patients showed reduced or no ability to inhibit autologous PB CD3+CD8-[CD4]LAP-CD25- cell proliferation when compared with controls. Removal of IL-17+ cells from LP CD3+CD8-[CD4] LAP+ cells increases their suppressive ability. CONCLUSIONS The percentage of LP CD4LAP+ cells is increased in active UC, showing reduced suppressor activity due to their increased proportion of intracellular IL-17 expression.