Heligmosomoides polygyrus bakeri Infection Decreases Smad7 Expression in Intestinal CD4+ T Cells, Which Allows TGF-ß to Induce IL-10-Producing Regulatory T Cells That Block Colitis.

Helminthic infections modulate host immunity and may protect their hosts from developing immunological diseases like inflammatory bowel disease. Induction of regulatory T cells (Tregs) may be an important part of this protective process. Heligmosomoides polygyrus bakeri infection also promotes the production of the regulatory cytokines TGF-ß and IL-10 in the gut. In the intestines, TGF-ß helps induce regulatory T cells. This study used Foxp3/IL-10 double reporter mice to investigate the effect of TGF-ß on the differentiation of colon and mesenteric lymph node-derived murine Foxp3- IL-10- CD4+ T cells into their regulatory phenotypes. Foxp3- IL-10- CD4+ T cells from H. polygyrus bakeri-infected mice, as opposed to T cells from uninfected animals, cultured in vitro with TGF-ß and anti-CD3/CD28 mAb differentiated into Foxp3+ and/or IL-10+ T cells. The IL-10-producing T cells nearly all displayed CD25. Smad7 is a natural inhibitor of TGF-ß signaling. In contrast to gut T cells from uninfected mice, Foxp3- IL10- CD4+ T cells from H. polygyrus bakeri-infected mice displayed reduced Smad7 expression and responded to TGF-ß with Smad2/3 phosphorylation. The TGF-ß-induced Tregs that express IL-10 blocked colitis when transferred into the Rag/CD25- CD4+ T cell transfer model of inflammatory bowel disease. TGF-ß had a greatly diminished capacity to induce Tregs in H. polygyrus bakeri-infected transgenic mice with constitutively high T cell-specific Smad7 expression. Thus, infection with H. polygyrus bakeri causes down-modulation in Smad7 expression in intestinal CD4+ T cells, which allows the TGF-ß produced in response to the infection to induce the Tregs that prevent colitis.

Downregulation of the Syk Signaling Pathway in Intestinal Dendritic Cells Is Sufficient To Induce Dendritic Cells That Inhibit Colitis.

Helminthic infections modulate host immunity and may protect people in less-developed countries from developing immunological diseases. In a murine colitis model, the helminth Heligmosomoides polygyrus bakeri prevents colitis via induction of regulatory dendritic cells (DCs). The mechanism driving the development of these regulatory DCs is unexplored. There is decreased expression of the intracellular signaling pathway spleen tyrosine kinase (Syk) in intestinal DCs from H. polygyrus bakeri-infected mice. To explore the importance of this observation, it was shown that intestinal DCs from DC-specific Syk(-/-) mice were powerful inhibitors of murine colitis, suggesting that loss of Syk was sufficient to convert these cells into their regulatory phenotype. DCs sense gut flora and damaged epithelium via expression of C-type lectin receptors, many of which signal through the Syk signaling pathway. It was observed that gut DCs express mRNA encoding for C-type lectin (CLEC) 7A, CLEC9A, CLEC12A, and CLEC4N. H. polygyrus bakeri infection downmodulated CLEC mRNA expression in these cells. Focusing on CLEC7A, which encodes for the dectin-1 receptor, flow analysis showed that H. polygyrus bakeri decreases dectin-1 expression on the intestinal DC subsets that drive Th1/Th17 development. DCs become unresponsive to the dectin-1 agonist curdlan and fail to phosphorylate Syk after agonist stimulation. Soluble worm products can block CLEC7A and Syk mRNA expression in gut DCs from uninfected mice after a brief in vitro exposure. Thus, downmodulation of Syk expression and phosphorylation in intestinal DCs could be important mechanisms through which helminths induce regulatory DCs that limit colitis.

Heligmosomoides polygyrus bakeri infection activates colonic Foxp3+ T cells enhancing their capacity to prevent colitis.

Helminthic infections protect mice from colitis in murine models of inflammatory bowel disease and also may protect people. Helminths like Heligmosomoides polygyrus bakeri can induce regulatory T cells (Treg). Experiments explored whether H. polygyrus bakeri infection could protect mice from colitis through activation of colonic Treg and examined mechanisms of action. We showed that H. polygyrus bakeri infection increased the number of T cells expressing Foxp3 in the colon. More importantly, Foxp3(+)/IL-10(-) and Foxp3(+)/IL-10(+) T cell subsets isolated from the colon of H. polygyrus bakeri-infected mice prevented colitis when adoptively transferred into a murine model of inflammatory bowel disease, whereas Treg from uninfected mice could not provide protection. Only the transferred colonic Foxp3(+)/IL-10(-) T cells from H. polygyrus bakeri-infected mice readily accumulated in the colon and mesenteric lymph nodes of recipient mice, and they reconstituted the Foxp3(+)/IL-10(-) and Foxp3(+)/IL-10(+) T cell subsets. However, transferred Foxp3(+)/IL-10(+) T cells disappeared. IL-10 expression by Foxp3(+) T cells was necessary for colitis prevention. Thus, H. polygyrus bakeri infection activates colonic Foxp3(+) T cells, making them highly regulatory. The Foxp3(+) T cells that fail to express IL-10 may be critical for populating the colon with the Foxp3(+)/IL-10(+) T cells, which are required to control colitis.

Heligmosomoides polygyrus bakeri induces tolerogenic dendritic cells that block colitis and prevent antigen-specific gut T cell responses.

Immunological diseases such as inflammatory bowel disease (IBD) are infrequent in less developed countries, possibly because helminths provide protection by modulating host immunity. In IBD murine models, the helminth Heligmosomoides polygyrus bakeri prevents colitis. It was determined whether H. polygyrus bakeri mediated IBD protection by altering dendritic cell (DC) function. We used a Rag IBD model where animals were reconstituted with IL10⁻/⁻ T cells, making them susceptible to IBD and with OVA Ag-responsive OT2 T cells, allowing study of a gut antigenic response. Intestinal DC from H. polygyrus bakeri-infected Rag mice added to lamina propria mononuclear cells (LPMC) isolated from colitic animals blocked OVA IFN-γ/IL-17 responses in vitro through direct contact with the inflammatory LPMC. DC from uninfected Rag mice displayed no regulatory activity. Transfer of DC from H. polygyrus bakeri-infected mice into Rag mice reconstituted with IL10⁻/⁻ T cells protected animals from IBD, and LPMC from these mice lost OVA responsiveness. After DC transfer, OT2 T cells populated the intestines normally. However, the OT2 T cells were rendered Ag nonresponsive through regulatory action of LPMC non-T cells. The process of regulation appeared to be regulatory T cell independent. Thus, H. polygyrus bakeri modulates intestinal DC function, rendering them tolerogenic. This appears to be an important mechanism through which H. polygyrus bakeri suppresses colitis. IFN-γ and IL-17 are colitogenic. The capacity of these DC to block a gut Ag-specific IFN-γ/IL-17 T cell response also is significant.

TGF-beta regulates T-cell neurokinin-1 receptor internalization and function.

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.

Heligmosomoides polygyrus infection can inhibit colitis through direct interaction with innate immunity.

Less developed countries have a low incidence of immunological diseases like inflammatory bowel disease (IBD), perhaps prevented by the high prevalence of helminth infections in their populations. In the Rag IL-10(-/-) T cell transfer model of colitis, Heligmosomoides polygyrus, an intestinal helminth, prevents and reverses intestinal inflammation. This model of colitis was used to explore the importance of innate immunity in H. polygyrus protection from IBD. Rag mice briefly exposed to H. polygyrus before reconstitution with IL-10(-/-) colitogenic T cells are protected from colitis. Exposure to H. polygyrus before introduction of IL-10(-/-) and OT2 T cells reduced the capacity of the intestinal mucosa to make IFN-gamma and IL-17 after either anti-CD3 mAb or OVA stimulation. This depressed cytokine response was evident even in the absence of colitis, suggesting that the downmodulation in proinflammatory cytokine secretion was not just secondary to improvement in intestinal inflammation. Following H. polygyrus infection, dendritic cells (DCs) from the lamina propria of Rag mice displayed decreased expression of CD80 and CD86, and heightened expression of plasmacytoid dendritic cell Ag-1 and CD40. They were also less responsive to lamina proprias, producing less IL-12p40 and IL-10. Also diminished was their capacity to present OVA to OT2 T cells. These experiments infer that H. polygyrus does not require direct interactions with T or B cells to render animals resistant to colitis. DCs have an important role in driving both murine and human IBD. Data suggest that phenotypic alternations in mucosal DC function are part of the regulatory process.

Role of T cell TGF-beta signaling in intestinal cytokine responses and helminthic immune modulation.

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.

Colonization with Heligmosomoides polygyrus suppresses mucosal IL-17 production.

Helminth exposure appears to protect hosts from inappropriate inflammatory responses, such as those causing inflammatory bowel disease. A recently identified, strongly proinflammatory limb of the immune response is characterized by T cell IL-17 production. Many autoimmune type inflammatory diseases are associated with IL-17 release. Because helminths protect from these diseases, we examined IL-17 production in helminth-colonized mice. We colonized mice with Heligmosomoides polygyrus, an intestinal helminth, and analyzed IL-17 production by lamina propria mononuclear cells (LPMC) and mesenteric lymph node (MLN) cells. Colonization with H. polygyrus reduces IL-17A mRNA by MLN cells and inhibits IL-17 production by cultured LPMC and MLN cells. Helminth exposure augments IL-4 and IL-10 production. Blocking both IL-4 and IL-10, but not IL-10 alone, restores IL-17 production in vitro. Colonization of colitic IL-10-deficient mice with H. polygyrus suppresses LPMC IL-17 production and improves colitis. Ab-mediated blockade of IL-17 improves colitis in IL-10-deficient mice. Thus, helminth-associated inhibition of IL-17 production is most likely an important mechanism mediating protection from inappropriate intestinal inflammation.

Interleukin-12 (IL-12) and IL-23 induction of substance p synthesis in murine T cells and macrophages is subject to IL-10 and transforming growth factor beta regulation.

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.

Coinfection with the intestinal nematode Heligmosomoides polygyrus markedly reduces hepatic egg-induced immunopathology and proinflammatory cytokines in mouse models of severe schistosomiasis.

Infection with the trematode helminth Schistosoma mansoni results in a parasite egg-induced, CD4 T-cell-mediated, hepatointestinal granulomatous and fibrosing inflammation that varies greatly in severity, with a higher frequency of milder forms typically occurring in regions where the disease is endemic. One possible explanation for this is that in these regions the degree of inflammation is lessened by widespread concurrent infection with gastrointestinal nematodes. We tested this hypothesis by establishing a murine coinfection model in which mice were infected with the intestinal nematode parasite Heligmosomoides polygyrus prior to infection with S. mansoni. In CBA mice that naturally display a severe form of schistosomiasis, preinfection with H. polygyrus resulted in a marked reduction in schistosome egg-induced hepatic immunopathology, which was associated with significant decreases in the levels of interleukin-17 (IL-17), gamma interferon, tumor necrosis factor alpha, IL-23, IL-6, and IL-1beta and with increases in the levels of IL-4, IL-5, IL-10, and transforming growth factor beta in mesenteric lymph node cells, purified CD4 T cells, and isolated liver granuloma cells. There also were increases in liver Ym1 and forkhead box P3 transcription factor expression. In another model of high-pathology schistosomiasis induced in C57BL/6 mice by immunization with schistosome egg antigens in complete Freund's adjuvant, coinfection with the nematodes also resulted in a marked inhibition of hepatic immunopathology accompanied by similar shifts in cytokine production. These findings demonstrate that intestinal nematodes prevent Th1- and Th17-cell-mediated inflammation by promoting a strong Th2-polarized environment associated with increases in the levels of alternatively activated macrophages and T regulatory cells, which result in significant amelioration of schistosome-induced immunopathology.

Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut.

The intestinal epithelium forms an essential barrier between a host and its microbiota. Protozoa and helminths are members of the gut microbiota of mammals, including humans, yet the many ways that gut epithelial cells orchestrate responses to these eukaryotes remain unclear. Here we show that tuft cells, which are taste-chemosensory epithelial cells, accumulate during parasite colonization and infection. Disruption of chemosensory signaling through the loss of TRMP5 abrogates the expansion of tuft cells, goblet cells, eosinophils, and type 2 innate lymphoid cells during parasite colonization. Tuft cells are the primary source of the parasite-induced cytokine interleukin-25, which indirectly induces tuft cell expansion by promoting interleukin-13 production by innate lymphoid cells. Our results identify intestinal tuft cells as critical sentinels in the gut epithelium that promote type 2 immunity in response to intestinal parasites.

Heligmosomoides polygyrus abrogates antigen-specific gut injury in a murine model of inflammatory bowel disease.

BACKGROUND: Developing countries have a low incidence of inflammatory bowel disease (IBD), perhaps prevented by the high prevalence of helminth infections and other alterations in intestinal flora and fauna. Helminth infections prevent colitis in various murine models of IBD. IBD may be driven by an aberrant immune response to luminal antigen(s). METHODS: We developed a murine model of IBD in which gut injury was induced by a specific antigen to better simulate the IBD disease process and to determine if helminth infections could abolish gut injury induced by an orally administered antigen. The model features pan-enterocolitis triggered by feeding ovalbumin (OVA). RESULTS: The intestinal inflammation is antigen-specific and generates interleukin (IL)-17 and interferon-gamma (IFN-γ), but not IL-4. Full expression of the disease required T cells with defective capacity to make IL-10 and treatment with a noninjurious, low dose of a nonsteroidal antiinflammatory drug. Exposure to Heligmosomoides polygyrus abrogated this antigen-induced gut injury. H. polygyrus colonization induced Foxp3(+) T regulatory cells (Tregs) and mucosal production of IL-10 from non-T cells. Lamina propria mononuclear cells from H. polygyrus-infected mice released less IL-17 and IFN-γ constitutively and when stimulated with OVA or anti-CD3/CD28 monoclonal antibodies. CONCLUSIONS: We developed a murine IBD model featuring antigen-specific enterocolitis and demonstrate for the first time that gut inflammation induced by an antigen could be abrogated by H. polygyrus infection. Protection was associated with suppressed IL-17 and IFN-γ production, induction of Foxp3(+) Tregs, and elevated secretion of non-T-cell-derived IL-10, all of which could be part of the protective processes.

Alteration of the murine gut microbiota during infection with the parasitic helminth Heligmosomoides polygyrus.

BACKGROUND: In a murine model of inflammatory bowel disease (IBD), treatment of colitis in IL-10 gene-deficient mice with the parasitic helminth Heligmosomoides polygyrus ameliorates colonic inflammation. The cellular and molecular mechanisms driving this therapeutic host response are being studied vigorously. One proposed mechanism is that H. polygyrus infection favors the outgrowth or suppression of certain bacteria, which in turn help modulate host immunity. METHODS: To quantify the effect of H. polygyrus infection on the composition of the gastrointestinal (GI) tract microbiota, we conducted two independent microbial ecology analyses of C57BL/6 mice. We obtained and analyzed 3,353 bacterial 16S rRNA encoding gene sequences from the ileum and cecum of infected and uninfected mice as well as incective H. polygyrus larvae at the outset of the second experiment and adult worms taken directly from the mouse duodenum at the end of the second experiment. RESULTS: We found that a significant shift in the abundance and relative distribution of bacterial species in the ileum of mice is associated with H. polygyrus infection. Members of the bacterial family Lactobacillaceae significantly increased in abundance in the ileum of infected mice reproducibly in two independent experiments despite having different microbiotas present at the outset of each experiment. CONCLUSIONS: These data support the concept that helminth infection shifts the composition of intestinal bacteria. The clinical consequences of these shifts in intestinal flora are yet to be explored.

Heligmosomoides polygyrus promotes regulatory T-cell cytokine production in the murine normal distal intestine.

Helminths down-regulate inflammation and may prevent development of several autoimmune illnesses, such as inflammatory bowel disease. We determined if exposure to the duodenal helminth Heligmosomoides polygyrus establishes cytokine pathways in the distal intestine that may protect from intestinal inflammation. Mice received 200 H. polygyrus larvae and were studied 2 weeks later. Lamina propria mononuclear cells (LPMC) were isolated from the terminal ileum for analysis and in vitro experiments. Mice with H. polygyrus were resistant to trinitrobenzenesulfonic acid (TNBS)-induced colitis, a Th1 cytokine-dependent inflammation. Heligmosomoides polygyrus did not change the normal microscopic appearance of the terminal ileum and colon and minimally affected LPMC composition. However, colonization altered LPMC cytokine profiles, blocking gamma interferon (IFN-gamma) and interleukin 12 (IL-12) p40 release but promoting IL-4, IL-5, IL-13, and IL-10 secretion. IL-10 blockade in vitro with anti-IL-10 receptor (IL-10R) monoclonal antibody restored LPMC IFN-gamma and IL-12 p40 secretion. IL-10 blockade in vivo worsened TNBS colitis in H. polygyrus-colonized mice. Lamina propria CD4(+) T cells isolated from colonized mice inhibited IFN-gamma production by splenic T cells from worm-free mice. This inhibition did not require cell contact and was dependent on IL-10. Heligmosomoides polygyrus colonization inhibits Th1 and promotes Th2 and regulatory cytokine production in distant intestinal regions without changing histology or LPMC composition. IL-10 is particularly important for limiting the Th1 response. The T-cell origin of these cytokines demonstrates mucosal regulatory T-cell induction.

Heligmosomoides polygyrus induces TLR4 on murine mucosal T cells that produce TGFbeta after lipopolysaccharide stimulation.

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.

Induction of CD8+ regulatory T cells in the intestine by Heligmosomoides polygyrus infection.

This study determined whether Heligmosomoides polygyrus induces intestinal regulatory T cells. Splenic T cells proliferate strongly when cultured with anti-CD3 and antigen-presenting cells (APC). Lamina propria T cells from mice with H. polygyrus mixed with normal splenic T cells from uninfected mice inhibited proliferation over 90%. Lamina propria T cells from mice without H. polygyrus only modestly affected T cell proliferation. The worm-induced regulatory T cell was CD8+ and required splenic T cell contact to inhibit proliferation. The regulation also was IL-10 independent, but TAP-dependent, suggesting that it requires major histocompatibility complex (MHC) class I interaction. Additional studies employed mice with transgenic T cells that did not express functional TGF-beta receptors. The lamina propria T regulator inhibited proliferation of these transgenic T cells nearly 100%, suggesting that TGF-beta signaling via the T cell was not required. CD8+ T cells were needed for worms to reverse piroxicam-induced colitis in Rag mice (T and B cell deficient) reconstituted with IL-10-/- T cells. Thus H. polygyrus induces a regulatory CD8+ lamina propria T cell that inhibits T cell proliferation and that appears to have a role in control of colitis.

IL-12 induction of mRNA encoding substance P in murine macrophages from the spleen and sites of inflammation.

Substance P (SP), a neuropeptide, interacts with the neurokinin 1 receptor (NK-1R) on immune cells to help control IFN-gamma production. In murine schistosomiasis mansoni, schistosome worms produce ova that incite focal Th2-type granulomatous inflammation within the liver and intestines. Normal gut is characterized by a controlled state of inflammation. IL-10 knockout mice develop chronic Th1-type colitis spontaneously. Both schistosome granulomas and gut mucosa display an SP immune regulatory circuit. However, the origin and regulation of SP production at these sites of inflammation are poorly understood. Macrophages are a potential source of SP. We therefore studied macrophages (F4/80(+)) from these models of inflammation. SP mRNA (preprotachykinin A (PPT A)) was detected within the schistosome granuloma, spleen, and lamina propria macrophages. Compared with those from wild-type mice, granuloma macrophages from STAT6(-/-) mice had 10-fold higher PPT A mRNA expression, whereas in STAT4(-/-) animals, PPT A mRNA expression was nearly abolished. IL-12 signals via STAT4 to induce Th1-type inflammation. It was demonstrated that IL-12, but not IL-18, induces SP mRNA expression in resting splenic macrophages from Schistosoma-infected mice and in wild-type lamina propria mononuclear cells. Thus, macrophages are a source for SP at these sites of chronic inflammation, and IL-12 and STAT4 are regulators of macrophage SP mRNA expression.

Cutting edge: hemokinin has substance P-like function and expression in inflammation.

Substance P (SP) belongs to the tachykinin family of molecules. SP, cleaved from preprotachykinin A, is a neuropeptide and a proinflammatory leukocyte product. SP engages neurokinin 1 receptor (NK-1R) to stimulate cells. Hemokinin (HK) is another tachykinin that binds NK-1R. HK comes from preprotachykinin C, which is distinct from preprotachykinin A. We determined whether HK functions like SP at inflammatory sites. Preprotachykinin C mRNA was in murine schistosome granulomas and intestinal lamina propria mononuclear cells. Granuloma T cells and macrophages expressed preprotachykinin C mRNA. HK bound granuloma T cell NK-1R with high affinity. SP and HK stimulated IFN-gamma production with equal potency. NK-1R antagonist blocked the effect of SP and HK on IFN-gamma secretion. Thus, both HK and SP are expressed at sites of chronic inflammation and share cell origin, receptor, and immunoregulatory function. Two distinct but functionally overlapping tachykinins govern inflammation through NK-1R at sites of chronic inflammation.

IL-18 and IL-12 signal through the NF-kappa B pathway to induce NK-1R expression on T cells.

Substance P engages the T cell neurokinin 1 receptor (NK-1R) to enhance IFN-gamma production. NK-1R on T cells is inducible. We studied mechanisms regulating T cell NK-1R expression. Murine splenocytes were cultured for 4 h with or without rIL-12 or rIL-18. Both IL-12 and IL-18 induced splenic T cells to express NK-1R transcripts. Induction was blocked by actinomycin D, but not cycloheximide, suggesting that protein synthesis was not required for initiation of NK-1R gene transcription. Inhibition of T cell NF-kappa B activation or NF-kappa B nuclear translocation also blocked NK-1R transcription. IL-12 and IL-18 strongly induce NK-1R mRNA expression in splenocytes from Stat4(-/-) mice, suggesting that the Stat4 pathway was not required for the induction of NK-1R transcription. Splenic T cells exposed to IL-12 or IL-18 in the presence of IL-10 expressed no NK-1R mRNA. However, TGF beta did not prevent NK-1R mRNA expression. Thus, IL-12 and IL-18 induce T cells to express NK-1R through NF-kappa B activation. IL-10, a regulator of the Th1 response, blocks this activation. These data further suggest that SP and NK-1R, which promote IFN-gamma synthesis, are part of the Th1 pathway of immunity.

Substance P regulates Th1-type colitis in IL-10 knockout mice.

Substance P (SP) is a proinflammatory molecule that interacts with a neurokinin 1 receptor (NK-1R), which is on T cells and helps control IFN-gamma production. IL-10(-/-) mice given a nonsteroidal anti-inflammatory drug (NSAID) develop Th1 colitis. We studied the importance of SP and NK-1R in this colitis model. LP T cells were isolated to study their NK-1R expression. LP T cells from IL-10(-/-) mice expressed NK-1R and produced IFN-gamma only after NSAID treatment and induction of colitis. LP T cells from NSAID-treated wild-type controls or from age-matched untreated IL-10(-/-) animals did not express NK-1R or produce IFN-gamma. Experiments showed that IL-12 induced NK-1R transcription in CD4(+) T cells cultured in vitro. However, T cells cultured with IL-12 and IL-10 did not express NK-1R. IL-10 also down-modulated ongoing NK-1R expression. Mice given NK-1R antagonist after NSAID induction of severe colitis showed nearly complete reversal of inflammation, and LP T cells ceased IFN-gamma secretion. Thus, intestinal inflammation in IL-10(-/-) mice is associated with the appearance of NK-1R in mucosal T cells, and an interplay between IL-12 and IL-10 regulates T cell NK-1R transcription. NK-1R antagonist reverses ongoing intestinal inflammation attesting to the importance of SP and its receptor in mucosal inflammation.