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Substance P (SP) is a proinflammatory mediator implicated in inflammatory bowel disease (IBD) and other inflammatory states. SP acts by stimulating the neurokinin-1 receptor (NK-1R) on T lymphocytes and other cell types, and regulates these cells in a complex interplay with multiple cytokines. The mechanisms of interaction among these inflammatory mediators are not yet fully understood. Here, we demonstrate that function of the NK-1R, a member of the G protein-coupled receptor (GPCR) superfamily, is modulated by TGF-beta. The latter acts not on a GPCR but via serine-threonine kinase-class receptors. By flow confocal image analysis, we demonstrate that TGF-beta delays SP-induced NK-1R internalization on mucosal T cells isolated from a mouse model of IBD and on granuloma T cells in murine schistosomiasis. Furthermore, luciferase reporter-gene assays revealed that NK-1R stimulation activates the nuclear factor of activated T cell- and activator protein-1-dependent signaling pathways, which are known triggers of effector T-cell cytokine production. TGF-beta markedly increases SP-induced activation of these signaling cascades, suggesting that delayed NK-1R internalization results in enhanced signaling. Providing a link to amplified immune function, SP and TGF-beta, when applied in combination, trigger a strong release of the proinflammatory cytokines IFN-gamma and IL17 from intestinal inflammatory T cells, whereas either agonist alone shows no effect. These observations establish precedent that members of two distinct receptor superfamilies can interact via a previously unrecognized mechanism, and reveal a paradigm of GPCR transregulation that is relevant to IBD and possibly other disease processes.

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL-10 or TGF-beta, that are important in suppressing colitis. Helminths induce mucosal T cell IL-10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL-10-dependent manner in WT mice. Helminths also stimulate mucosal TGF-beta release. As TGF-beta exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF-beta signaling in helminthic modulation of intestinal immunity. T cell TGF-beta signaling is interrupted in TGF-beta receptor II dominant negative (TGF-betaRII DN) mice by T-cell-specific over-expression of a TGF-betaRII DN. We studied LPMC responses in WT and TGF-betaRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF-beta signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL-10 secretion requires intact T cell TGF-beta-signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF-betaRII DN mice. Thus, T cell TGF-beta signaling is essential for helminthic stimulation of mucosal IL-10 production, helminthic modulation of intestinal IFN-gamma generation and H. polygyrus-mediated suppression of chronic colitis.

Substance P is a tachykinin that enhances pathways of inflammation. Leukocytes at sites of intestinal inflammation make substance P. This study explored the role of interleukin-12 (IL-12), IL-23, and the regulatory cytokines IL-10 and transforming growth factor beta (TGF-beta) in controlling leukocyte substance P production. In murine schistosomiasis, it was found that IL-12 and IL-23 drive substance P gene expression and peptide synthesis in murine splenic T cells and macrophages, respectively. Cytokine induction of substance P synthesis both in T cells and in macrophages depends on intracellular NF-kappaB activation and is Stat4 independent. IL-10 inhibits T-cell substance P production, while TGF-beta blocks macrophage substance P expression. Intestinal macrophages also produce substance P, subject mostly to IL-23 and TGF-beta regulation. Hemokinin is another tachykinin with homology to substance P. Macrophages and T cells make hemokinin, but hemokinin production is not subject to IL-12 or IL-23 regulation.

Helminths are immune modulators that down-regulate colitis in inflammatory bowel disease. In animal models, intestinal bacteria drive colitis and in humans certain alleles of the LPS receptor protein TLR4 increase inflammatory bowel disease susceptibility. To understand helminthic immune modulation in the gut, we studied the influence of intestinal Heligmosomoides polygyrus colonization on LPS-induced lamina propria mononuclear cell (LPMC) cytokine responses in mice. LPS did not stimulate TGFbeta production from LPMC of uninfected mice. LPS strongly induced LPMC from worm-infected animals to secrete TGFbeta, but not TNF-alpha or IL-12. The TGFbeta derived from mucosal T cells. Helminth infection up-regulated TLR4 expression only in lamina propria T cells. LPMC from worm-infected TLR4 mutant animals did not respond to LPS, suggesting that LPS required TLR4 to stimulate TGFbeta secretion. Thus, during helminth infection, LPS challenge induces mucosal T cells to make TGFbeta through a TLR4-dependent process without promoting synthesis of proinflammatory cytokines.