Estradiol-17␤ (E 2 ) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E 2 elicits epithelial mitogenesis through epithelial ER versus indirectly via ERpositive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB͞c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and͞or stroma (S), or lacking ER altogether: wt-S ؉ wt-E, wt-S ؉ ko-E, ko-S ؉ ko-E, and ko-S ؉ wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E 2 or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [ 3 H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S ؉ wt-E, wt-S ؉ ko-E), E 2 induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S ؉ ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S ؉ ko-E and ko-S ؉ wt-E), epithelial labeling index was low and not stimulated by E 2 despite epithelial ER expression in ko-S ؉ wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E 2 -induced uterine epithelial proliferation. Instead, E 2 induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.

Inflammatory bowel disease (IBD) is prevalent in industrialized countries, but rare in less‐developed countries. Helminths, common in less‐developed countries, may induce immunoregulatory circuits protective against IBD. IL‐10–/– mice given piroxicam develop severe and persistent colitis. Lamina propria mononuclear cells from colitic IL‐10–/– mice released IFN‐γ and IL‐12. The ongoing piroxicam‐induced colitis could be partially blocked with anti‐IL‐12 monoclonal antibody suggesting that the inflammation was at least partly IL‐12 dependent. Colonization of piroxicam‐treated colitic IL‐10–/– mice with Heligmosomoides polygyrus (an intestinal helminth) suppressed established inflammation and inhibited mucosal IL‐12 and IFN‐γ production. H. polygyrus augmented mucosal IL‐13, but not IL‐4 or IL‐5 production. Transfer of mesenteric lymph node (MLN) T cells from IL‐10–/– animals harboring H. polygyrus into colitic IL‐10–/– recipients inhibited colitis. MLN T cells from worm‐free mice did not. Foxp3 (scurfin) drives regulatory T cell function. H. polygyrus enhanced Foxp3 mRNA expression in MLN T cells that had regulatory activity. This suggests that H. polygyrus inhibits ongoing IL‐10–/– colitis in part through blocking mucosal Th1 cytokine production. Resolution of inflammation is associated with increased IL‐13 production and can be adoptively transferred by MLN T cells.

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Helminth exposure appears to protect hosts from inappropriate inflammatory responses such as those causing inflammatory bowel disease. A recently identified, strongly pro-inflammatory limb of the immune response is characterized by T cell IL17 production. Many autoimmune type inflammatory diseases are associated with IL17 release. Because helminths protect from these diseases, we examined IL17 production in helminth-colonized mice. We colonized mice with Heligmosomoides polygyrus, an intestinal helminth, and analyzed IL17 production by lamina propria mononuclear (LPMC) and mesenteric lymph node (MLN) cells. Colonization with H. polygyrus reduces IL17A mRNA by MLN cells and inhibits IL17 production by cultured LPMC and MLN cells. Helminth exposure augments IL4 and IL10 production. Blocking both IL4 and IL10, but not IL10 alone restores IL17 production in vitro. Colonization of colitic IL10-deficient mice with H. polygyrus suppresses LPMC IL17 production and improves colitis. Antibody-mediated blockade of IL17 improves colitis in IL10-deficient mice. Thus, helminth-associated inhibition of IL17 production is likely an important mechanism mediating protection from inappropriate intestinal inflammation.

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-γ 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.

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL-10 or TGF-b, 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-b release. As TGF-b exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF-b signaling in helminthic modulation of intestinal immunity. T cell TGF-b signaling is interrupted in TGF-b receptor II dominant negative (TGF-bRII DN) mice by T-cellspecific over-expression of a TGF-bRII DN. We studied LPMC responses in WT and TGF-bRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF-b 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-b-signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF-bRII DN mice. Thus, T cell TGF-b signaling is essential for helminthic stimulation of mucosal IL-10 production, helminthic modulation of intestinal IFN-c generation and H. polygyrus-mediated suppression of chronic colitis. IntroductionHelminth exposure is associated with immune modulation in the human or murine host and decreased immune reactivity to antigens unrelated to the parasite [1]. This response may be useful in the treatment of autoimmune and immunological diseases, like inflammatory bowel disease [2,3]. Helminths stimulate the host to produce Th2 (IL-4, IL-5, IL-9, IL-13) or regulatory (IL-10, TGF-b) cytokines, while blocking Th1 cytokine responses [4,5].Helminthic parasites limit disease activity in various animal models of inflammatory bowel disease. For example, rectal trinitrobenzene sulfonic acid administration causes a T cell cytokinedriven colitis that is prevented by systemic administration of 1870Schistosoma mansoni eggs or duodenal colonization with Heligmosomoides polygyrus larvae [6,7]. H. polygyrus-mediated regulation of IFN-g, IL-12/23 (p40) production and protection from colitis is in part blocked by inhibiting IL-10 signaling in this trinitrobenzene sulfonic acid model. IL-10 is a major immune regulatory cytokine [8] that helps prevent intestinal inflammation. Accelerated, severe colitis is triggered in IL-10 deficient and not WT mice by Helicobacter hepaticus infection or treatment of the animals with non-steroidal antinflammatory drugs [9][10][11]. Although different cells can produce IL-10 and regulate immune responses, animal studies implicate CD4 T lymphocyte derived IL-10 as a non-redundant regulator of intestinal immune balance [12]. IL-10 is involved in helminthic regulation of mucosal Th1 cytokine responses where T cells constitute the major source of intestinal 14]. The mechanism of how helminths induce IL-10-producing T cells is unknown. ...

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.

Helminthic infections protect mice from colitis in murine models of inflammatory bowel disease and also may protect people. Helminths like H. bakeri (Hpb) can induce Tregs. Experiments explored if Hpb infection could protect mice from colitis through activation of colonic Treg and examined mechanisms of action. We showed that Hpb infection increased the number of T cells expressing Foxp3 in the colon. More importantly, Foxp3+/IL10- and Foxp3+/IL10+ T cell subsets isolated from the colon of Hpb infected mice prevented colitis when adoptively transferred into a murine model of inflammatory bowel disease, while Tregs from uninfected mice could not provide protection. Only the transferred colonic Foxp3+/IL10- T cells from Hpb infected mice readily accumulated in the colon and MLN of recipient mice, and they reconstituted the Foxp3+/IL10- and Foxp3+/IL10+ T cell subsets. However, transferred Foxp3+/IL10+ T cells disappeared. IL10 expression by Foxp3+ T cells was necessary for colitis prevention. Thus, Hpb infection activates colonic Foxp3+ T cells making them highly regulatory. The Foxp3+ T cells that fail to express IL10 may be critical for populating the colon with the Foxp3+/IL10+ T cells, which are required to control colitis.