Acute gut inflammation reduces neural activity and spine maturity in hippocampus but not basolateral amygdala.

Gastrointestinal tract (gut) inflammation increases stress and threat-coping behaviors, which are associated with altered activity in fear-related neural circuits, such as the basolateral amygdala and hippocampus. It remains to be determined whether inflammation from the gut affects neural activity by altering dendritic spines. We hypothesized that acute inflammation alters dendritic spines in a brain region-specific manner. Here we show that acute gut inflammation (colitis) evoked by dextran sodium sulfate (DSS) did not affect the overall spine density in the CA1 region of hippocampus, but increased the relative proportion of immature spines to mature spines on basal dendrites of pyramidal neurons. In contrast, in animals with colitis, no changes in spine density or composition on dendrites of pyramidal cells was observed in the basolateral amygdala. Rather, we observed decreased spine density on dendrites of stellate neurons, but not the relative proportions of mature vs immature spines. We used cFos expression evoked by the forced swim task as a measure of neural activity during stress and found no effect of DSS on the density of cFos immunoreactive neurons in basolateral amygdala. In contrast, fewer CA1 neurons expressed cFos in mice with colitis, relative to controls. Furthermore, CA1 cFos expression negatively correlated with active stress-coping in the swim task and was negatively correlated with gut inflammation. These data reveal that the effects of acute gut inflammation on synaptic remodeling depend on brain region, neuronal phenotype, and dendrite location. In the hippocampus, a shift to immature spines and hypoactivity are more strongly related to colitis-evoked behavioral changes than is remodeling in basolateral amygdala.

Behavioral adaptations in a relapsing mouse model of colitis.

Inflammatory bowel disease (IBD) is characterized by relapsing periods of gut inflammation, and is comorbid with depression, anxiety, and cognitive deficits. Animal models of IBD that explore the behavioral consequences almost exclusively use acute models of gut inflammation, which fails to recapitulate the cyclic, chronic nature of IBD. This study sought to identify behavioral differences in digging, memory, and stress-coping strategies in mice exposed to one (acute) or three (chronic) cycles of gut inflammation, using the dextran sodium sulfate (DSS) model of colitis. Similar levels of gut pathology were observed between acute and chronically exposed mice, although mice in the chronic treatment had significantly shorter colons, suggesting more severe disease. Behavioral measures revealed an unexpected pattern in which chronic treatment evoked fewer deficits than acute treatment. Specifically, acutely-treated mice showed alterations in measures of object burying, novel object recognition, object location memory, and stress-coping (forced swim task). Chronically-treated animals, however, showed similar alterations in object burying, but not the other measures. These data suggest an adaptive or tolerizing effect of repeated cycles of peripheral gut inflammation on mnemonic function and stress-coping, whereas some other behaviors continue to be affected by gut inflammation. We speculate that the normalization of some functions may involve the reversion to the baseline state of the hypothalamic-pituitary-adrenal axis and/or levels of neuroinflammation, which are both activated by the first exposure to the colitic agent.

Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Macrophages play central roles in immunity as early effectors and modulating adaptive immune reponses; we implicated macrophages in the anticolitic effect of infection with the tapeworm Hymenolepis diminuta. Here, gene arrays revealed that H. diminuta antigen (HdAg) evoked a program in murine macrophages distinct from that elicited by IL-4. Further, HdAg suppressed LPS-evoked release of TNF-α and IL-1ß from macrophages via autocrine IL-10 signaling. In assessing the ability of macrophages treated in vitro with an extract of H. diminuta [M(HdAg)] to affect disease, intravenous, but not peritoneal, injection of M(HdAg) protected wild-type but not RAG1-/- mice from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Administration of splenic CD4+ T cells from in vitro cocultures with M(HdAg), but not those cocultured with M(IL-4) cells, inhibited DNBS-induced colitis; fractionation of the T-cell population indicated that the CD4+CD25+ T cells from cocultures with M(HdAg) drove the suppression of DNBS-induced colitis. Use of IL-4-/- or IL-10-/- CD4+ T cells revealed that neither cytokine alone from the donor cells was essential for the anticolitic effect. These data illustrate that HdAg evokes a unique regulatory program in macrophages, identifies HdAg-evoked IL-10 suppression of macrophage activation, and reveals the ability of HdAg-treated macrophages to educate ( i.e., condition) and mobilize CD4+CD25+ T cells, which could be deployed to treat colonic inflammation.-Reyes, J. L., Lopes, F., Leung, G., Jayme, T. S., Matisz, C. E., Shute, A., Burkhard, R., Carneiro, M., Workentine, M. L., Wang, A., Petri, B., Beck, P. L., Geuking, M. B., McKay, D. M., Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Helminth Antigen-Conditioned Dendritic Cells Generate Anti-Inflammatory Cd4 T Cells Independent of Antigen Presentation via Major Histocompatibility Complex Class II.

A recently identified feature of the host response to infection with helminth parasites is suppression of concomitant disease. Dendritic cells (DCs) exposed to antigens from the tapeworm Hymenolepis diminuta significantly reduce the severity of dinitrobenzene sulfonic acid-induced colitis in mice. Here we elucidate mechanisms underlying this cellular immunotherapy. We show a requirement for Ccr7 expression on transferred H. diminuta antigen-treated (HD)-DCs, suggesting that homing to secondary lymphoid tissues is important for suppression of colitis. Furthermore, sodium metaperiodate-sensitive helminth-derived glycans are required to drive the anti-colitic response in recipient mice. Induction of Th2-type cytokines and Gata-3+Cd4+ cells in secondary lymphoid tissues is dependent on major histocompatibility complex class II (MHC II) protein expression on transferred DCs, although remarkably, transfer of MHC II-/- HD-DCs still attenuated dinitrobenzene sulfonic acid-induced colitis in recipient mice. Moreover, transfer of Cd4+ splenic T cells retrieved from mice administered MHC II-/- HD-DCs suppressed dinitrobenzene sulfonic acid-induced colitis in recipient mice. Our studies reveal that HD-DCs can suppress colitis via an alternative MHC II-independent pathway that involves, in part, mobilization of T-cell responses. These data support the utility of HD-DCs in blocking colitis, revealing a requirement for Ccr7 and providing for HD-DC autologous immunotherapy for disease in which MHC II expression and/or function is compromised.

Treatment with Cestode Parasite Antigens Results in Recruitment of CCR2+ Myeloid Cells, the Adoptive Transfer of Which Ameliorates Colitis.

Awareness of the immunological underpinnings of host-parasite interactions may reveal immune signaling pathways that could be used to treat inflammatory disease in humans. Previously we showed that infection with the rat tapeworm, Hymenolepis diminuta, used as a model helminth, or systemic delivery of worm antigen (HdAg) significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Extending these analyses, intraperitoneal injection of HdAg dose-dependently suppressed dextran sodium sulfate (DSS)-induced colitis, and this was paralleled by reduced gamma interferon (IFN-γ), interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-α) production and increased IL-10 production from mitogen-activated splenocytes. Treatment with HdAg resulted in a CCR2-dependent recruitment of CDllb+ F4/80+ Ly6Chi Gr-1lo monocyte-like cells into the peritoneum 24 h later that were predominantly programmed death ligand 1 (PD-L1) positive and CXCR2 negative. In vitro assays indicated that these cells were unable to suppress T cell proliferation but enhanced IL-10 and IL-4 production from activated T cells. Adoptive transfer of the HdAg-recruited monocytic cells into naive mice blocked DSS-induced colitis. These findings add to the variety of means by which treatment with parasitic helminth-derived antigens can ameliorate concomitant disease. A precise understanding of the mechanism(s) of action of HdAg and other helminth-derived antigens (and a parallel consideration of putative side effects) may lead to the development of novel therapies for human idiopathic disorders such as inflammatory bowel disease.

IL-22 Restrains Tapeworm-Mediated Protection against Experimental Colitis via Regulation of IL-25 Expression.

Interleukin (IL)-22, an immune cell-derived cytokine whose receptor expression is restricted to non-immune cells (e.g. epithelial cells), can be anti-inflammatory and pro-inflammatory. Mice infected with the tapeworm Hymenolepis diminuta are protected from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Here we assessed expulsion of H. diminuta, the concomitant immune response and the outcome of DNBS-induced colitis in wild-type (WT) and IL-22 deficient mice (IL-22-/-) ± infection. Interleukin-22-/- mice had a mildly impaired ability to expel the worm and this correlated with reduced or delayed induction of TH2 immunity as measured by splenic and mesenteric lymph node production of IL-4, IL-5 and IL-13 and intestinal Muc-2 mRNA and goblet cell hyperplasia; in contrast, IL-25 increased in the small intestine of IL-22-/- mice 8 and 12 days post-infection compared to WT mice. In vitro experiments revealed that H. diminuta directly evoked epithelial production of IL-25 that was inhibited by recombinant IL-22. Also, IL-10 and markers of regulatory T cells were increased in IL-22-/- mice that displayed less DNBS (3 mg, ir. 72h)-induced colitis. Wild-type mice infected with H. diminuta were protected from colitis, as were infected IL-22-/- mice and the latter to a degree that they were almost indistinguishable from control, non-DNBS treated mice. Finally, treatment with anti-IL-25 antibodies exaggerated DNBS-induced colitis in IL-22-/- mice and blocked the anti-colitic effect of infection with H. diminuta. Thus, IL-22 is identified as an endogenous brake on helminth-elicited TH2 immunity, reducing the efficacy of expulsion of H. diminuta and limiting the effectiveness of the anti-colitic events mobilized following infection with H. diminuta in a non-permissive host.

Adoptive transfer of helminth antigen-pulsed dendritic cells protects against the development of experimental colitis in mice.

Infection with helminth parasites and treatment with worm extracts can suppress inflammatory disease, including colitis. Postulating that dendritic cells (DCs) participated in the suppression of inflammation and seeking to move beyond the use of helminths per se, we tested the ability of Hymenolepis diminuta antigen-pulsed DCs to suppress colitis as a novel cell-based immunotherapy. Bone marrow derived DCs pulsed with H. diminuta antigen (HD-DCs), or PBS-, BSA-, or LPS-DCs as controls, were transferred into wild-type (WT), interleukin-10 (IL-10) knock-out (KO), and RAG-1 KO mice, and the impact on dinitrobenzene sulphonic acid (DNBS)-induced colitis and splenic cytokine production assessed 72 h later. Mice receiving HD-DCs were significantly protected from DNBS-induced colitis and of the experimental groups only these mice displayed increased Th2 cytokines and IL-10 production. Adoptive transfer of HD-DCs protected neither RAG-1 nor IL-10 KO mice from DNBS-colitis. Furthermore, the transfer of CD4(+) splenocytes from recipients of HD-DCs protected naïve mice against DNBS-colitis, in an IL-10 dependent manner. Thus, HD-DCs are a novel anti-colitic immunotherapy that can educate anti-colitic CD4(+) T cells: mechanistically, the anti-colitic effect of HD-DCs requires that the host has an adaptive immune response and the ability to mobilize IL-10.

Helminth parasites and the modulation of joint inflammation.

There is an urgent need to develop better therapeutics for autoimmune and autoinflammatory diseases, of which musculoskeletal disorders such as rheumatoid arthritis are particularly prevalent and debilitating. Helminth parasites are accomplished masters at modifying their hosts' immune activity, and so attention has focused on rodent-helminth model systems to uncover the workings of the mammalian immune response to metazoan parasites, with the hope of revealing molecules and/or mechanisms that can be translated into better treatments for human autoimmune and idiopathic disorders. Substantial proof-of-principal data supporting the concept that infection with helminth parasites can reduce the severity of concomitant disease has been amassed from models of mucosal inflammation. Indeed, infection with helminth parasites has been tried as a therapy in inflammatory bowel disease, and there are case reports relating to other conditions (e.g., autism); however, the impact of infection with parasitic helminths on musculoskeletal diseases has not been extensively studied. Here, we present the view that such a strategy should be applied to the amelioration of joint inflammation and review the literature that supports this contention.

Post-arrival screening for malaria in asymptomatic refugees using real-time PCR.

Malaria is a significant health risk to refugee populations originating from endemic areas, but there is little consensus on screening and/or treatment approaches for malaria in this population. Furthermore, detection of malaria in semi-immune asymptomatic refugees is limited by the sensitivity of diagnostic tests used for screening. We determined the prevalence of malaria by microscopy and real-time polymerase chain reaction (PCR) in a consecutive population of 324 asymptomatic refugees examined in Edmonton, Canada, during 2009-2010. Although all thick and thin blood smear results were negative, 10 subjects (3.1%) tested PCR positive for Plasmodium DNA. Interestingly, 6 of 10 PCR positive subjects are at risk of malaria relapse by P. vivax or P. ovale infections. These results suggest that appropriate guidelines for malaria screening should consider the risk of relapsing infections, and they highlight the potential usefulness of real-time PCR in the diagnosis of asymptomatic malaria.

Migration, site selection, and development of Ornithodiplostomum sp. metacercariae (Digenea: Strigeoidea) in fathead minnows (Pimephales promelas).

The metacercarial stage of trematodes is typically considered an encysted, developmentally quiescent, resting stage. Yet the metacercariae of some species of strigeoid trematode undergo extravagant development within specific tissues of their second intermediate host. Our understanding of patterns of migration, site selection and development of these types of metacercariae is known for only a few species. In this study, we characterize the invasion and development of Ornithodiplostomum sp. metacercariae in their second intermediate host, the fathead minnow, Pimephales promelas. Diplostomules completed their migration into the abdominal cavity between 15 min and 48 h p.i. Most diplostomules migrated along muscular and connective tissue then penetrated the peritoneal lining of the abdominal cavity en route to the liver or pancreas. Alternatively, some diplostomules migrated within the host's circulatory system, including the heart and arteries of the hepatic portal system. Metacercarial development in the liver and pancreas involved distinct growth, encystment and consolidation phases. Metacercarial volume increased 15-fold between 48 h and 4 weeks p.i., presumably due to absorptive and/or ingestive feeding activities within host tissues. By 2 weeks p.i., metacercariae were enveloped within a cyst wall and they were found loosely attached to the surfaces of internal tissues or unattached within the body cavity. These results emphasize the complex nature of metacercarial migration and growth and demonstrate that their growth and encystment phases occur within different habitats within their intermediate hosts.

Migration and site selection of Ornithodiplostomum ptychocheilus (Trematoda: Digenea) metacercariae in the brain of fathead minnows ( Pimephales promelas).

The migration of subadult parasites to preferred sites within final hosts is well characterized. In contrast, the migration of larval stages of trematodes to specific sites within their second intermediate hosts is poorly understood. We used a serial necropsy approach to characterize the migration of Ornithodiplostomum ptychocheilus diplostomules from the point of cercarial penetration, to encystment within the outermost tissues of the brain of fathead minnows. Diplostomules utilized peripheral nerves to access the central nerve cord, or they used specific cranial nerves to directly access the brain. Within 3 h of exposure to cercariae, 46% of all diplostomules were observed within the medulla of the brain. Diplostomules subsequently utilized specific neural tracts to reach lateral regions of the outermost tissue layer of the optic lobes, the stratum marginale. Diplostomules remained in this layer during their 4-week growth phase, then shifted site to the adjacent meninges for encystment. Characterization of a habitat shift for developing versus encysted metacercariae helps explain the results of previous ecological studies that document transient changes in the effects of metacercariae on the surivival, behaviour, and anti-parasite defences of infected fish.

Density and maturation of rodlet cells in brain tissue of fathead minnows (Pimephales promelas) exposed to trematode cercariae.

Evidence for the presumed linkage between the enigmatic rodlet cells of fish and exposure to helminths is anecdotal and indirect. We evaluated the proliferation and development of rodlet cells in the optic lobes of fathead minnows exposed to cercariae of Ornithodiplostomum ptychocheilus. Mean rodlet cell densities (ca. 10/mm(2)) in the optic lobes were similar between unexposed controls and minnows with 1- and 2-week old infections. Rodlet cell densities increased at 4 weeks p.i., reaching maxima (ca. 200/mm(2)) at 6 weeks p.i., followed by a decline at 9 weeks. This temporal pattern of proliferation and maturation paralleled the development of metacercariae within the optic lobes. Unencysted metacercariae develop rapidly within tissues of the optic lobes for approximately 4 weeks after penetration by cercariae, then shift to the adjacent meninges to encyst. The former stage is associated with tissue damage, the latter with massive inflammation of the meninges. Thus, peak densities and maturation of rodlet cells correspond to the period when inflammation of the meninges caused by the large metacercarial cysts is at a maximum. Our results support recent contentions that rodlet cells comprise part of the host inflammatory defence response.