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The lumen-dwelling protozoan Giardia is an important parasitic cause of diarrheal disease worldwide. Infection can persist over extended periods with minimal intestinal inflammation, suggesting that Giardia may attenuate host responses to ensure its survival, although clearance eventually occurs in most cases. IL-10 is an anti-inflammatory regulator critical for intestinal homeostasis and controlling host responses to bacterial exposure, yet its potential role in coordinating antiprotozoal host defense in the intestine is not known. In this study, we found that murine infection with the natural enteric pathogen Giardia muris induced a transient IL-10 response after 2-4 wk at the primary site of infection in the upper small intestine, but parasite colonization and eradication were not affected by the absence of the cytokine in gene-targeted mice. However, IL-10 was critical for controlling infection-associated immunological sequelae in the colon because severe and persistent diarrhea and colitis were observed in IL-10-deficient mice within 1-2 wk postinfection but not in uninfected littermate controls. Inflammation was characterized by epithelial hyperplasia, neutrophil and macrophage expansion, and Th1 induction and could be prevented by blockade of IL-12/IL-23 p40 but not depletion of CD11c + dendritic cells. Furthermore, the intestinal microbiota underwent characteristic shifts in composition and was required for disease because antibiotics and loss of TLR signaling in MyD88-deficient mice protected against colitis. Together, our data suggest that transient infection by a luminal and seemingly noninflammatory pathogen can trigger sustained colitis in genetically susceptible hosts, which has broader implications for understanding postinfectious syndromes and other chronic intestinal inflammatory conditions., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The function of mucosal dendritic cell (DC) subsets in immunity and inflammation is not well understood. In this study, we define four DC subsets present within the lamina propria and mesenteric lymph node compartments based on expression of CD103 and CD11b. Using IL-12p40 YFP (Yet40) reporter mice, we show that CD103(+)CD11b(-) mucosal DCs are primary in vivo sources of IL-12p40; we also identified CD103(-)CD11b(-) mucosal DCs as a novel population producing this cytokine. Infection was preferentially found in CD11b(+) DCs that were negative for CD103. Lamina propria DCs containing parasites were negative for IL-12p40. Instead, production of the cytokine was strictly a property of noninfected cells. We also show that vitamin A metabolism, as measured by ALDH activity, was preferentially found in CD103(+)CD11b(+) DC and was strongly downregulated in all mucosal DC subsets during infection. Finally, overall apoptosis of lamina propria DC subsets was increased during infection. Combined, these results highlight the ability of intestinal Toxoplasma infection to alter mucosal DC activity at both the whole population level and at the level of individual subsets., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Macrophages (Mφs) accumulate at sites of inflammation, and, because they can assume several functionally distinct states of activation, they can either drive or restrain inflammatory responses. Once believed to depend on the recruitment of blood monocytes, it is now clear that the accumulation of Mφs in some tissues can result from the proliferation of resident Mφs in situ. However, little is known about the proliferation and activation state of Mφ subsets in the gut during the development and resolution of intestinal inflammation. We show that inflammatory Mφs accumulate in the large intestine of mice during the local inflammatory response to infection with the gastrointestinal nematode parasite Trichuris muris. Classically activated Mφs predominate initially (as the inflammation develops) and then, following worm expulsion (as the inflammation resolves), both the resident and inflammatory populations of Mφs become alternatively activated. A small but significant increase in the proliferation of inflammatory Mφs is seen but only during the resolution phase of the inflammatory response following both worm expulsion and the peak in Mφ accumulation. In contrast to recent studies in the pleural and peritoneal cavities, the proliferation of resident and alternatively activated Mφs does not increase during the inflammatory response. Furthermore, in CCR2(-/-) mice, monocyte recruitment to the gut is impeded, and the accumulation of alternatively activated Mφs is greatly reduced. In conclusion, the recruitment of blood monocytes is the principle mechanism of Mφ accumulation in the large intestine. This study provides a novel insight into the phenotype and behavior of intestinal Mφ during infection-driven inflammation., (Copyright © 2014 The Authors.)

SerpinB2, a member of the serine protease inhibitor family, is expressed by macrophages and is significantly upregulated by inflammation. Recent studies implicated a role for SerpinB2 in the control of Th1 and Th2 immune responses, but the mechanisms of these effects are unknown. In this study, we used mice deficient in SerpinB2 (SerpinB2(-/-)) to investigate its role in the host response to the enteric nematode, Heligmosomoides bakeri. Nematode infection induced a STAT6-dependent increase in intestinal SerpinB2 expression. The H. bakeri-induced upregulation of IL-4 and IL-13 expression was attenuated in SerpinB2(-/-) mice coincident with an impaired worm clearance. In addition, lack of SerpinB2 in mice resulted in a loss of the H. bakeri-induced smooth muscle hypercontractility and a significant delay in infection-induced increase in mucosal permeability. Th2 immunity is generally linked to a CCL2-mediated increase in the infiltration of macrophages that develop into the alternatively activated phenotype (M2). In H. bakeri-infected SerpinB2(-/-) mice, there was an impaired infiltration and alternative activation of macrophages accompanied by a decrease in the intestinal CCL2 expression. Studies in macrophages isolated from SerpinB2(-/-) mice showed a reduced CCL2 expression, but normal M2 development, in response to stimulation of Th2 cytokines. These data demonstrate that the immune regulation of SerpinB2 expression plays a critical role in the development of Th2-mediated protective immunity against nematode infection by a mechanism involving CCL2 production and macrophage infiltration.

CD4(+) Th2 cytokine responses promote the development of allergic inflammation and are critical for immunity to parasitic helminth infection. Recent studies highlighted that basophils can promote Th2 cytokine-mediated inflammation and that phenotypic and functional heterogeneity exists between classical IL-3-elicited basophils and thymic stromal lymphopoietin (TSLP)-elicited basophils. However, whether distinct basophil populations develop after helminth infection and their relative contributions to anti-helminth immune responses remain to be defined. After Trichinella spiralis infection of mice, we show that basophil responses are rapidly induced in multiple tissue compartments, including intestinal-draining lymph nodes. Trichinella-induced basophil responses were IL-3-IL-3R independent but critically dependent on TSLP-TSLPR interactions. Selective depletion of basophils after Trichinella infection impaired infection-induced CD4(+) Th2 cytokine responses, suggesting that TSLP-dependent basophils augment Th2 cytokine responses after helminth infection. The identification and functional classification of TSLP-dependent basophils in a helminth infection model, coupled with their recently described role in promoting atopic dermatitis, suggests that these cells may be a critical population in promoting Th2 cytokine-associated inflammation in a variety of inflammatory or infectious settings. Collectively, these data suggest that the TSLP-basophil pathway may represent a new target in the design of therapeutic intervention strategies to promote or limit Th2 cytokine-dependent immunity and inflammation.

IL-12-mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2(-/-)) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2(-/-) mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2(-/-) mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2(-/-)CD8+ DC compared with WT CD8+ DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2(-/-) animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.

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.

The ATP-gated P2X(7) receptor (P2X(7)R) was shown to be an important mediator of inflammation and inflammatory pain through its regulation of IL-1β processing and release. Trichinella spiralis-infected mice develop a postinflammatory visceral hypersensitivity that is reminiscent of the clinical features associated with postinfectious irritable bowel syndrome. In this study, we used P2X(7)R knockout mice (P2X(7)R(-/-)) to investigate the role of P2X(7)R activation in the in vivo production of IL-1β and the development of postinflammatory visceral hypersensitivity in the T. spiralis-infected mouse. During acute nematode infection, IL-1β-containing cells and P2X(7)R expression were increased in the jejunum of wild-type (WT) mice. Peritoneal and serum IL-1β levels were also increased, which was indicative of elevated IL-1β release. However, in the P2X(7)R(-/-) animals, we found that infection had no effect upon intracellular, plasma, or peritoneal IL-1β levels. Conversely, infection augmented peritoneal TNF-α levels in both WT and P2X(7)R(-/-) animals. Infection was also associated with a P2X(7)R-dependent increase in extracellular peritoneal lactate dehydrogenase, and it triggered immunological changes in both strains. Jejunal afferent fiber mechanosensitivity was assessed in uninfected and postinfected WT and P2X(7)R(-/-) animals. Postinfected WT animals developed an augmented afferent fiber response to mechanical stimuli; however, this did not develop in postinfected P2X(7)R(-/-) animals. Therefore, our results demonstrated that P2X(7)Rs play a pivotal role in intestinal inflammation and are a trigger for the development of visceral hypersensitivity.

IL-25 (IL-17E) is a member of the IL-17 cytokine family. IL-25-deficient mice exhibit impaired Th2 immunity against nematode infection, implicating IL-25 as a key component in mucosal immunity. The sources of IL-25 and mechanisms responsible for the induction of Th2 immunity by IL-25 in the gastrointestinal tract remain poorly understood. There is also little information on the regulation of IL-25 during inflammation or its role in gut function. In the current study, we investigated the regulation of IL-25 during Nippostrongylus brasiliensis infection and the contribution of IL-25 to the infection-induced alterations in intestinal function. We found that epithelial cells, but not immune cells, are the major source of IL-25 in the small intestine. N. brasiliensis infection-induced upregulation of IL-25 depends upon IL-13 activation of STAT6. IL-25(-/-) mice had diminished intestinal smooth muscle and epithelial responses to N. brasiliensis infection that were associated with an impaired Th2 protective immunity. Exogenous IL-25 induced characteristic changes similar to those after nematode infection but was unable to restore the impaired host immunity against N. brasiliensis infection in IL-13(-/-) mice. These data show that IL-25 plays a critical role in nematode infection-induced alterations in intestinal function that are important for host protective immunity, and IL-13 is the major downstream Th2 cytokine responsible for the IL-25 effects.

Our aim was to elucidate the contribution of mucosal mast cells to the effector phase of a secondary immune response to Trichinella spiralis. During secondary infection, rats expel 90-99% of T. spiralis first-stage larvae from the intestine in a matter of hours. This phenomenon appears to be unique to rats and has been called rapid expulsion. Primary intestinal infection by T. spiralis induces mastocytosis, and mast cell degranulation occurs when challenged rats exhibit rapid expulsion. These observations have engendered the view that mast cells mediate rapid expulsion. In this study, we report that immunization of adult Albino Oxford rats by an infection limited to the muscle phase did not induce intestinal mastocytosis, yet such rats exhibited rapid expulsion when challenged orally. Although mastocytosis was absent, the protease unique to mucosal mast cells, rat mast cell protease II (RMCPII), was detected in sera at the time of expulsion. We further evaluated mast cell activity in neonatal rats that display rapid expulsion. Pups born to infected dams displayed rapid expulsion, and RMCPII was detected in their sera. By feeding pups parasite-specific mAbs or polyclonal Abs before challenge infection, it was possible to dissociate mast cell degranulation from parasite expulsion. These results indicate that rapid expulsion can occur in the absence of either intestinal mastocytosis or RMCPII release. Furthermore, release of RMCPII is not sufficient to cause expulsion. The data argue against a role for mast cells in the mechanism underlying the effector phase of protective immunity against T. spiralis in rats.

TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88(-/-) mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-gamma production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4(+) T cell IFN-gamma reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88(-/-) mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-gamma responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule.

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.

The distribution of several pathogenic helminth infections coincides geographically with many devastating microbial diseases, including enteric bacterial infections. To dissect the mechanisms by which helminths modulate the host's response to enteric bacteria and bacteria-mediated intestinal inflammation, we have recently established a coinfection model and shown that coinfection with the helminth Heligmosomoides polygyrus exacerbates colitis induced by infection with the gram-negative bacterial pathogen Citrobacter rodentium. The disease severity of the coinfected mice was correlated with high Citrobacter loads in the gut, translocation of the bacteria into mucosal and systemic immune compartments, delayed bacterial clearance, and a significantly enhanced colonic TNF-alpha response. In the present study, using our in vivo coinfection model as well as in vitro approaches, we test the hypothesis that the phenotypic and functional alterations in macrophages induced by the helminth-driven T cell response may contribute to the observed alterations in the response to C. rodentium. We show that via a STAT6-dependent mechanism H. polygyrus coinfection results in a marked infiltration into the colonic lamina propria of F4/80+ cells that have the phenotype of alternatively activated macrophages. Functional analysis of these macrophages further shows that they are impaired in their killing of internalized bacteria. Yet, these cells produce an enhanced amount of TNF-alpha in response to C. rodentium infection. These results demonstrate that helminth infection can impair host protection against concurrent enteric bacterial infection and promote bacteria-induced intestinal injury through a mechanism that involves the induction of alternatively activated macrophages.

The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, alpha-enolase, and alpha- and beta-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.

Following an abrupt transition at birth from the sterile uterus to an environment with abundant commensal and pathogenic microbes, neonatal mammals are protected by maternal Abs at mucosal surfaces. We show in mice that different Ab isotypes work in distinct ways to protect the neonatal mucosal surface. Secretory IgA acts to limit penetration of commensal intestinal bacteria through the neonatal intestinal epithelium: an apparently primitive process that does not require diversification of the primary natural Ab repertoire. In contrast, neonatal protection against the exclusively luminal parasite Heligmosomoides polygyrus required IgG from primed females. This immune IgG could either be delivered directly in milk or retrotransported via neonatal Fc receptor from the neonatal serum into the intestinal lumen to exert its protective effect.

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.

The Peyer's patches (PP) and mesenteric lymph nodes (MLN) are structural components of the gut-associated lymphoid tissues and contribute to the induction of immune responses toward infection in the gastrointestinal tract. These secondary lymphoid organs provide structural organization for efficient cellular interactions and the initiation of primary adaptive immune responses against infection. Immunity against primary infection with the enteric apicomplexan parasite, Eimeria vermiformis, depends on the rapid induction of local Th1 responses. Lymphotoxin (LT)-deficient mice which have various defects in secondary lymphoid organs were infected with E. vermiformis. The relative susceptibility of LTalpha(-/-), LTbeta(-/-), LTalpha(+/-)beta(+/-) mice and bone marrow chimeras, indicated that rapid protective Th1 responses required both PP and MLN. Moreover, the timing of Th1 induction in both MLN and gut was dependent on the presence of PP suggesting a level of cooperation between immune responses induced in these distinct lymphoid structures. The delay in Th1 induction was attributable to the delayed arrival of a broad range of dendritic cell subsets in the MLN and a substantial reduction of CD8alpha(-)CD11b(high) B220(-) dendritic cells in PP-deficient mice.

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.

We demonstrated in this study the critical role of NKT cells in the lethal ileitis induced in C57BL/6 mice after infection with Toxoplasma gondii. This intestinal inflammation is caused by overproduction of IFN-gamma in the lamina propria. The implication of NKT cells was confirmed by the observation that NKT cell-deficient mice (Jalpha281(-/-)) are more resistant than C57BL/6 mice to the development of lethal ileitis. Jalpha281(-/-) mice failed to overexpress IFN-gamma in the intestine early after infection. This detrimental effect of NKT cells is blocked by treatment with alpha-galactosylceramide, which prevents death in C57BL/6, but not in Jalpha281(-/-), mice. This protective effect is characterized by a shift in cytokine production by NKT cells toward a Th2 profile and correlates with an increased number of mesenteric Foxp3 lymphocytes. Using chimeric mice in which only NKT cells are deficient in the IL-10 gene and mice treated with anti-CD25 mAb, we identified regulatory T cells as the source of the IL-10 required for manifestation of the protective effect of alpha-galactosylceramide treatment. Our results highlight the participation of NKT cells in the parasite clearance by shifting the cytokine profile toward a Th1 pattern and simultaneously to immunopathological manifestation when this Th1 immune response remains uncontrolled.

Memory peripheral Th2 immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. We have examined the localized immune response to a nematode parasite that encysts in the small intestine. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4(+), TCR-alphabeta(+) T cells surrounding the cyst. Laser capture microdissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host:parasite interface.

Th2 immune responses to a number of infectious pathogens are dependent on B7-1/B7-2 costimulatory molecule interactions. We have now examined the Th2 immune response to Nippostrongylus brasiliensis (Nb) in B7-1/B7-2(-/-) mice and show that Th2 effector cells develop that can mediate worm expulsion and produce substantial Th2 cytokines comparable with wild-type infected mice; however, in marked contrast, B cell Ag-specific Ab production is abrogated after B7 blockade. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of Nb plus OVA or either alone. Only the combination of Nb plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that Nb acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells. Furthermore, using the DO11.10 TCR-transgenic T cell adoptive transfer model, we show that blocking B7-1/B7-2 interactions does not impair nonparasite Ag-specific DO11.10 Th2 cell differentiation; however, DO11.10 T cell cycle progression and migration to the B cell zone are inhibited.

Amebic colitis is an important worldwide parasitic disease for which there is not a well-established animal model. In this work we show that intracecal inoculation of Entamoeba histolytica trophozoites led to established infection in 60% of C3H mice, while C57BL/6 or BALB/c mice were resistant, including mice genetically deficient for IL-12, IFN-gamma, or inducible NO synthase. Infection was a chronic and nonhealing cecitis that pathologically mirrored human disease. Characterization of the inflammation by gene chip analysis revealed abundant mast cell activity. Parasite-specific Ab and cellular proliferative responses were robust and marked by IL-4 and IL-13 production. Depletion of CD4(+) cells significantly diminished both parasite burden and inflammation and correlated with decreased IL-4 and IL-13 production and loss of mast cell infiltration. This model reveals important immune factors that influence susceptibility to infection and demonstrates for the first time the pathologic contribution of the host immune response in amebiasis.

Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with pronounced mastocytosis mediated by a Th2-type response involving IL-4, IL-10, and IL-13. Here we demonstrate that IL-18 is a key negative regulator of protective immune responses against T. spiralis in vivo. IL-18 knockout mice are highly resistant to T. spiralis infection, expel the worms rapidly and subsequently develop low levels of encysted muscle larvae. The increased speed of expulsion is correlated with high numbers of mucosal mast cells and an increase in IL-13 and IL-10 secretion. When normal mice were treated with rIL-18 in vivo, worm expulsion was notably delayed, and the development of mastocytosis and Th2 cytokine production was significantly reduced. The treatment had no effect on intestinal eosinophilia or goblet cell hyperplasia but specifically inhibited the development of mastocytosis. Addition of rIL-18 to in vitro cultures of bone marrow-derived mast cells resulted in a significant reduction in cell yields as well as in the number of IL-4-secreting mast cells. In vivo treatment of T. spiralis-infected IFN-gamma knockout mice with rIL-18 demonstrated that the inhibitory effect of IL-18 on mastocytosis and Th2 cytokine secretion is independent of IFN-gamma. Hence, IL-18 plays a significant biological role as a negative regulator of intestinal mast cell responses and may promote the survival of intestinal parasites in vivo.

Acute and lethal ileitis can be elicited in certain strains of inbred mice after oral infection with the intracellular protozoan parasite Toxoplasma gondii. The development of this inflammatory process is dependent upon the induction of a robust Th1 response, including overproduction of IFN-gamma, TNF-alpha, and NO, as has been reported in other experimental models of human inflammatory bowel disease. In this study we have investigated the role of CD4(+) T cells from the lamina propria (LP) in the early inflammatory events after T. gondii infection using isolated and primary cultured intestinal cells from infected mice and immortalized mouse mIC(cl2) intestinal epithelial cells. Primed LP CD4(+) T cells isolated from parasite-infected mice produce substantial quantities of both IFN-gamma and TNF-alpha. IFN-gamma- and TNF-alpha-producing LP CD4(+) T cells synergize with infected mIC(cl2) and enhance the production of several inflammatory chemokines including macrophage-inflammatory protein-2, monocyte chemoattractant protein-1, monocyte chemoattractant protein-3, macrophage-inflammatory protein-1alphabeta, and IFN-gamma-inducible protein-10. Furthermore, primed LP CD4(+) T cells cocultured with infected mIC(cl2) inhibited replication of the parasite in the intestinal epithelial cells. Thus, LP CD4(+) T cells can interact with parasite-infected intestinal epithelial cells and alter the expression of several proinflammatory products that have been associated with the development of intestinal inflammation. The interaction between these two components of the gut mucosal compartment (CD4(+) T cells and enterocytes) may play a role in the immunopathogenesis of this pathogen-driven experimental inflammatory bowel disease model.

IL-4 and IL-13 promote gastrointestinal worm expulsion, at least in part, through effects on nonlymphoid cells, such as intestinal epithelial cells. The role of IL-4/IL-13 in the regulation of intestinal epithelial function during Heligmosomoides polygyrus (Hp) infection was investigated in BALB/c mice infected with Hp or treated with a long-lasting formulation of recombinant mouse IL-4/alphaIL-4 complexes (IL-4C) for 7 days. Separate groups of BALB/c mice were drug-cured of initial infection and later reinfected and treated with anti-IL-4R mAb, an antagonist of IL-4 and IL-13 receptor binding, or with a control mAb. Segments of jejunum were mounted in Ussing chambers, and short circuit current responses to acetylcholine, histamine, serotonin, PGE2, and glucose were determined. Although only modest changes in epithelial cell function were observed during primary Hp infection, IL-4C or a secondary Hp infection each induced more dramatic changes, including increased mucosal permeability, reduced sodium-linked glucose absorption, and increased Cl- secretory response to PGE2. Some, but not all, effects of IL-4C and Hp infection were dependent on enteric nerves. Hp-induced changes in epithelial function were attenuated or prevented by anti-IL-4R mAb. Thus, IL-4/IL-13 mediate many of the effects of Hp infection on intestinal epithelial cell function and do so both through direct effects on epithelial cells and through indirect, enteric nerve-mediated prosecretory effects. These immune system-independent effector functions of IL-4/IL-13 may be important for host protection against gastrointestinal nematodes.

B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma.

We examined effects of mast cell glycosaminoglycans on the establishment of the intestinal nematode, Strongyloides venezuelensis, in the mouse small intestine. When intestinal mastocytosis occurred, surgically implanted adult worms could not invade and establish in the intestinal mucosa. In mast cell-deficient W/Wv mice, inhibition of adult worm invasion was not evident as compared with littermate +/+ control mice. Mucosal mastocytosis and inhibition of S. venezuelensis adult worm mucosal invasion was tightly correlated. To determine effector molecules for the invasion inhibition, adult worms were implanted with various sulfated carbohydrates including mast cell glycosaminoglycans. Among sulfated carbohydrates tested, chondroitin sulfate (ChS)-A, ChS-E, heparin, and dextran sulfate inhibited invasion of adult worms into intestinal mucosa in vivo. No significant inhibition was observed with ChS-C, desulfated chondroitin, and dextran. ChS-E, heparin, and dextran sulfate inhibited adhesion of S. venezuelensis adult worms to plastic surfaces in vitro. Furthermore, binding of intestinal epithelial cells to adhesion substances of S. venezuelensis, which have been implicated in mucosal invasion, was inhibited by ChS-E, heparin, and dextran sulfate. Because adult worms of S. venezuelensis were actively moving in the intestinal mucosa, probably exiting and reentering during infection, the possible expulsion mechanism for S. venezuelensis is inhibition by mast cell glycosaminoglycans of attachment and subsequent invasion of adult worms into intestinal epithelium.

Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i. e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.

Resistance of adult C57BL/6 mice to severe Cryptosporidium parvum infection is dependent on CD4+alpha beta+ TCR lymphocytes. In this study, we demonstrated that treatment with anti-IFN-gamma mAb extended oocyst excretion 18 days longer, and anti-IL-4 mAb extended oocyst excretion at least 11 days longer than isotype control mAb treatment. Analysis of the specific activity of anti-IFN-gamma mAb present in treated mouse sera suggested that IFN-gamma may have a limited role in the resolution phase of infection. Changes were also documented in numbers of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in Peyer's patches and intraepithelium of adult C57BL/6 mice during resolution of C. parvum infection. Resistance to initial severe infection was associated with CD4+alpha beta+IFN-gamma+ lymphocytes, and eventual resolution of infection was associated with CD4+alpha beta+IL-4+ lymphocytes. Analysis of cytokine expression following in vitro stimulation with C. parvum Ags during resolution of infection demonstrated consistent increases in CD4+alpha beta+IL-4+ lymphocytes, but not CD4+alpha beta+IFN-gamma+ lymphocytes. The relevance of CD4+alpha beta+IL-4+ lymphocytes in protection against C. parvum was then evaluated in C57BL/6 IL-4 gene knockout mice (IL-4(-/-)). Adult IL-4(-/-) mice excreted oocysts in feces approximately 23 days longer than IL-4(+/+) mice. Further, anti-IFN-gamma mAb treatment increased the severity and the duration of infection in IL-4(-/-) mice compared with those in IL-4(+/+) mice. Together, the data demonstrated that IFN-gamma was important in the control of severity of infection, and either IFN-gamma or IL-4 accelerated termination of infection. However, neither IL-4 nor IFN-gamma was required for the final clearance of infection from the intestinal tract of adult mice.

A fascinating feature of the intestinal mucosal immune system is its ability to guard against invasion by pathogens while avoiding a response to the many potential Ags present in food. The phenomenon of systemic tolerance after oral administration of protein Ags is well documented, but the cellular and molecular basis for the observed nonresponsiveness is not fully understood. To gain insight into the role of the mucosal microenvironment in the induction of orally induced nonresponsiveness, we attempted to induce tolerance to OVA in mice primed for a Th2-biased mucosal immune response by infection with the nematode parasite Heligmosomoides polygyrus. We found that oral tolerance for Th1-type responses to OVA is maintained when OVA is fed during the peak of the mucosal immune response to H. polygyrus. Tolerance for Th2 cytokine responses or a Th2-dependent isotype of IgG is not induced in this Th2-biased mucosal environment. Treatment of infected mice with rIL-12 to reverse the Th2 polarity of the parasite-specific immune response restores tolerance of both Th1 and Th2 responses to OVA. We conclude that the polarized Th2 response induced by this enteric infection plays a central role in determining whether or not systemic tolerance is induced. Our results imply that attempts to use oral administration of Ag to suppress systemic immune responses will be influenced strongly by the presence of mucosal infection.

The first line of defense of the host after primary infection by Toxoplasma gondii is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IEL) (mainly of CD8 phenotype), mucus, and secretory Igs. T. gondii cysts were administered orally to CBA/J mice to determine whether parasite-specific cytotoxic IEL can be elicited within the intestine. We found that oral infection led to an increase in the relative percentages of the CD8 beta + and Thy-1+ IEL populations between day 9 and day 13 after infection. At these times, T. gondii-primed, but not control mice, generated parasite-specific cytotoxic effector IEL for toxoplasma-infected macrophages. This cytotoxic activity was genetically restricted. By using magnetically activated cell sorting, the effector IEL were shown to be CD8 alpha/beta + Thy-1+ T lymphocytes and FACScan analysis revealed that they mainly express TCR-alpha beta. A significant level of cytotoxicity was also observed against T. gondii-infected epithelial cells from the MODE-K cell line, suggesting that parasite-specific IEL may ensure epithelial integrity by rapidly killing infected enterocytes. Finally, toxoplasma stimulation in vitro led to IFN-gamma production by T. gondii-primed IEL. Taken together, these data suggest that antigen-specific IEL, bearing the CD8 alpha/beta + Thy-1+, TCR-alpha beta + phenotype, can act directly as effector cells through a specific CTL activity at the intestinal level and may indirectly activate other mucosal effector mechanisms through IFN-gamma secretion.

Enteric administration of antigen can induce systemic tolerance. In murine schistosomiasis mansoni, blood flukes produce eggs which enter the intestine. An immunologic phenomenon associated with this disease is a spontaneous diminution in the intensity of the granulomatous response in the liver, lungs, and colonic mucosa with chronic infection, which is termed modulation. It was determined whether modulation of liver granulomas could be induced by enteric immunization with schistosome eggs. Mice infected for 4 wk were immunized by injection of 25,000 eggs into cecal pouches. This induced modulation of liver granulomas by the eighth week of infection. Neither cecal injection of normal saline nor i.p. or subcutaneous injection of eggs could induce the modulatory process. Modulation could be adoptively transferred from enterically immunized donors by injection of spleen cells into infected recipients or into uninfected recipients with synchronous liver granulomas induced by the hepatic embolization of schistosome eggs. Spleen cells treated with anti-Thy-1.2 or anti-Lyt-1.1 and complement could no longer adoptively transfer modulation. These data show that enteric immunization with schistosome eggs can induce modulation of the liver granuloma by a cellular mechanism similar to that described for the natural infection.