Evaluation of the Interaction Between TGF β and Nitric Oxide in the Mechanisms of Progression of Colon Carcinoma

It is recognised that stromal cells determine cancer progression. We have previously shown that active TGFβ produced by rat colon carcinoma cells modulated NO production in rat endothelial cells. To elucidate the role of TGFβ and NO in the mechanisms of interaction of colon carcinoma cells with stromal cells and in cancer progression, we transfected REGb cells, a regressive colon carcinoma clone secreting latent TGFβ, with a cDNA encoding for a constitutively-secreted active TGFβ. Out of 20 injected rats only one tumour progressed, which was resected and sub-cultured (ReBeta cells). ReBeta cells secreted high levels of active TGFβ. The adhesive properties of REGb and Rebeta cells to endothelial cells were similar, showing that the secretion of active TGFβ is not involved in tumour cell adhesion to endothelial cells. ReBeta, but not REGb, cell culture supernatants inhibited cytokine-dependent NO secretion by endothelial cells, but inhibition of NO production was similar in co-cultures of REGb or ReBeta cells with endothelial cells. Therefore, secretion of active TGFβ regulated endothelial NO synthase activity when tumour cells were distant from, but not in direct contact with, endothelial cells. However, only ReBeta cells inhibited cytokine-dependent secretion of NO in coculture with macrophages, indicating that the active-TGFβ-NO axis confers an advantage for tumour cells in their interaction with macrophages rather than endothelial cells in cancer progressio

An optimized protocol for isolating lymphoid stromal cells from the intestinal lamina propria

International audienceMesenchymal stromal cells in lymphoid organs, also called lymphoid stromal cells (LSCs), play a pivotal role in immunity by forming specialized microenvironments that provide signals for leukocyte migration, positioning, and survival. Best characterized in lymphoid organs, LSCs are also abundant in the intestinal mucosa, which harbors a rich repertoire of immune cells. However, the lack of efficient procedures for isolation and purification of LSCs from the intestine has been a major limitation to their characterization. Here we report a new method to efficiently isolate, in addition to immune cells, viable lymphoid stromal cells and other stromal subsets from the intestinal lamina propria for subsequent phenotypic and functional analysis

CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury.

International audienceThe intestinal epithelium is continuously renewed by intestinal epithelial stem cells (IESCs) positioned at the base of each crypt. Mesenchymal-derived factors are essential to maintain IESCs; however, the cellular composition and development of such mesenchymal niche remains unclear. Here, we identify pericryptal CD34(+) Gp38(+) αSMA(-) mesenchymal cells closely associated with Lgr5(+) IESCs. We demonstrate that CD34(+) Gp38(+) cells are the major intestinal producers of the niche factors Wnt2b, Gremlin1, and R-spondin1, and are sufficient to promote maintenance of Lgr5(+) IESCs in intestinal organoids, an effect mainly mediated by Gremlin1. CD34(+) Gp38(+) cells develop after birth in the intestinal submucosa and expand around the crypts during the third week of life in mice, independently of the microbiota. We further show that pericryptal CD34(+)gp38(+) cells are rapidly activated by intestinal injury, up-regulating niche factors Gremlin1 and R-spondin1 as well as chemokines, proinflammatory cytokines, and growth factors with key roles in gut immunity and tissue repair, including IL-7, Ccl2, Ptgs2, and Amphiregulin. Our results indicate that CD34(+) Gp38(+) mesenchymal cells are programmed to develop in the intestine after birth to constitute a specialized microenvironment that maintains IESCs at homeostasis and contribute to intestinal inflammation and repair after injury

Inflammation recapitulates the ontogeny of lymphoid stromal cells.

International audienceStromal cells in lymphoid tissues regulate lymphocyte recruitment and survival through the expression of specific chemokines and cytokines. During inflammation, the same signals recruit lymphocytes to the site of injury; however, the "lymphoid" stromal (LS) cells producing these signals remain poorly characterized. We find that mouse inflammatory lesions and tumors develop gp38(+) LS cells, in recapitulation of the development of LS cells early during the ontogeny of lymphoid organs and the intestine, and express a set of genes that promotes the development of lymphocyte-permissive tissues. These gp38(+) LS cells are induced by a robust pathway that requires myeloid cells but not known Toll- or NOD-like receptors, the inflammasome, or adaptive immunity. Parabiosis and inducible genetic cell fate mapping experiments indicate that local precursors, presumably resident fibroblasts rather that circulating precursors, massively proliferate and give rise to LS cells during inflammation. Our results show that LS cells are both programmed during ontogeny and reinduced during inflammation

In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORgamma t+ T cells

The nuclear hormone receptor retinoic acid receptor-related orphan receptor gamma t (RORgamma t) is required for the generation of T helper 17 cells expressing the proinflammatory cytokine interleukin (IL)-17. In vivo, however, less than half of RORgamma t(+) T cells express IL-17. We report here that RORgamma t(+) T alphabeta cells include Foxp3(+) cells that coexist with IL-17-producing RORgamma t(+) T alphabeta cells in all tissues examined. The Foxp3(+) RORgamma t(+) T alphabeta express IL-10 and CCL20, and function as regulatory T cells. Furthermore, the ratio of Foxp3(+) to IL-17-producing RORgamma t(+) T alphabeta cells remains remarkably constant in mice enduring infection and inflammation. This equilibrium is tuned in favor of IL-10 production by Foxp3 and CCL20, and in favor of IL-17 production by IL-6 and IL-23. In the lung and skin, the largest population of RORgamma t(+) T cells express the gammadelta T cell receptor and produce the highest levels of IL-17 independently of IL-6. Thus, potentially antagonistic proinflammatory IL-17-producing and regulatory Foxp3(+) RORgamma t(+) T cells coexist and are tightly controlled, suggesting that a perturbed equilibrium in RORgamma t(+) T cells might lead to decreased immunoreactivity or, in contrast, to pathological inflammation