The phytonutrient cinnamaldehyde limits intestinal inflammation and enteric parasite infection.

Phytonutrients such as cinnamaldehyde (CA) have been studied for their effects on metabolic diseases, but their influence on mucosal inflammation and immunity to enteric infection are not well documented. Here, we show that consumption of CA in mice significantly down-regulates transcriptional pathways connected to inflammation in the small intestine, and alters T-cell populations in mesenteric lymph nodes. During infection with the enteric helminth Heligomosomoides polygyrus, CA treatment attenuated infection-induced changes in biological pathways connected to cell cycle and mitotic activity, and tended to reduce worm burdens. Mechanistically, CA did not appear to exert activity through a prebiotic effect, as CA treatment did not significantly change the composition of the gut microbiota. Instead, in vitro experiments showed that CA directly induced xenobiotic metabolizing pathways in intestinal epithelial cells and suppressed endotoxin-induced inflammatory responses in macrophages. Collectively, our results show that CA down-regulates inflammatory pathways in the intestinal mucosa and can limit the pathological response to enteric infection. These properties appear to be largely independent of the gut microbiota, and instead connected to the ability of CA to induce antioxidant pathways in intestinal cells. Our results encourage further investigation into the use of CA and related phytonutrients as functional food components to promote intestinal health in humans and animals.

Hematopoietic cell-derived RELMα regulates hookworm immunity through effects on macrophages.

Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/- ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.

Impact of heat treatment on antigen detection in sera of Angiostrongylus vasorum infected dogs.

BACKGROUND: In the last decade serological tests for detection of circulating Angiostrongylus vasorum antigen and specific antibodies have been developed and adopted for individual diagnosis and epidemiological studies in dogs. Although confirmed positive at necropsy, antigen detection was not possible in single experimentally, as well as naturally infected dogs, possibly due to immune complex formation. The aim of this study was to evaluate the effect of heat treatment on detection of A. vasorum antigen in sera of experimentally (n = 21, 119 follow-up sera) and naturally (n = 18) infected animals. In addition, sera of dogs showing clinical signs consistent with angiostrongylosis (n = 10), of randomly selected dogs (n = 58) and of dogs with other parasitic infections (n = 15) were evaluated. Sera were subjected to heat treatment at 100 °C after addition of 0.5 M EDTA (dilution 1:5) and tested with ELISAs for detection of circulating A. vasorum antigen before and after treatment. RESULTS: Between 5 and 11 weeks post-inoculation (wpi) the percentage of positive untreated samples (experimentally infected dogs) increased over time from 33.3 to 90%. Single samples were still negative between 12 and 15 wpi. Overall, between 5 and 15 wpi, 50.6% (45/89) of the available samples were seropositive. From 3 to 6 wpi EDTA/heat treatment caused a change in 8/34 (23.5%) of the samples, with most (n = 6, 17.6%) converting from positive to negative. In contrast, from 7 to 10 wpi, treatment induced a change in 19/52 (36.5%) samples, with all but one converting from negative to positive. Thirteen of 18 naturally infected dogs were antigen positive before and 15 after EDTA/heat treatment, respectively. Untreated samples of 3 dogs with suspected angiostrongylosis were antigen positive, of which only one remained positive after EDTA/heat treatment. One of 58 untreated random samples was antigen positive; this sample became negative after treatment, while another turned positive. One of 15 dogs infected with other parasites than A. vasorum was positive before but negative after treatment. CONCLUSION: Although heat treatment improves A. vasorum antigen detection between 7 and 10 wpi by immune complex disruption, we do not recommend systematic pretreating sera because of reduced antigen detection between 3 and 6 wpi and impairment of antibody detection, if performed contemporaneously.

An investigation into the effects of maternal supplementation with excess iodine on the mechanisms and impacts of reduced IgG absorption in the lamb postpartum.

An experiment was conducted to determine: (1) the effect of excess maternal I supplementation on the thyroid hormone status of the ewe and her progeny; (2) potential mechanisms underpinning the failure of passive transfer associated with excess I and (3) the growing lambs' response to natural gastrointestinal infection. Twin-bearing ewes received one of two treatments (n 32/treatment group): basal diet (C) or C plus 26·6 mg of iodine/ewe per d (I), supplied as calcium iodate. Ewes were individually fed from day 119 of gestation to parturition. Progeny of I ewes had lower (P<0·01) serum IgG concentrations from 24 h to 28 d postpartum but higher serum IgG concentrations at day 70 postpartum (P<0·05). I supplementation increased the relative expression of Fc receptor, IgA, IgM high affinity and polymeric Ig receptor in the ileum of the lamb at 24 h postpartum; however, thyroid hormone receptor-ß (THRB) and ß-2-microglobulin (B2M) expression declined (P<0·05). Progeny of I ewes had higher growth rates to weaning (P<0·05) and lower faecal egg count (FEC) for Nematodirus battus (P<0·05) between weeks 6 and 10 postpartum. In conclusion, excess maternal I supplementation negatively affected the thyroid hormone status, serum IgG concentration, ileal morphology and the gene expression of THRB and B2M in the ileum and ras-related protein (RAB) RAB25 and the mucin gene (MUC) MUC1 in the duodenum of the lamb postpartum. These effects were followed by an enhancement of average daily gain and lower N. battus FEC in the pre-weaning period of I-supplemented lambs.

Selenoprotein Expression in Macrophages Is Critical for Optimal Clearance of Parasitic Helminth Nippostrongylus brasiliensis.

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trsp(fl/fl)Cre(WT) mice that express selenoproteins driven by tRNA(Sec) (Trsp), whereas N. brasiliensis-infected Trsp(fl/fl)Cre(LysM) selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ(12)-PGJ2 and 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses.

Changes in Heligmosomoides polygyrus glycoprotein pattern by saponins impact the BALB/c mice immune response.

Saponins of marigold (Calendula officinalis), in particular derivatives of 3-O-monoglucuronide of oleanolic acid, are able to reduce infectivity of Heligmosomoides polygyrus in mice. The purpose of this study was to understand the immune activation provoked by third-stage larvae exposed to marigold glucuronides. We also examined the pattern of glycosylation of larval antigens which appeared to be crucial for induction of cytokine production in BALB/c mice; higher concentrations of IL-6, IFN-γ, IL-10 and TNF-α were observed in serum or intestine one week post infection. Three weeks later, in the chronic phase of infection, cells in culture were able to produce IL-6, IFN-γ, TNF-α and IL-17. Restimulation of cells with H. polygyrus antigen resulted in reduced production of IL-6, and TNF-α. The pattern of cytokine production co-existed with reduced expression of terminal glucose, α-linked mannose, N-acetyl-galactosamine, ß-galactose, N-acetyl-glucosamine and α-fucose in several protein bands. Galactose, as a new terminal carbohydrate residue appeared in 20-24kDa protein bands. The number of immunogenic epitopes in parasitic antigens was reduced; only three protein bands of 56, 26 and 12kDa were recognized by IgG1. These studies provide a model system to find the glycosylated molecules expressed on nematodes that improve establishment and survival and characterize cytokine production in mice infected with larvae exposed to saponin. Identification of these molecules is the first step in the recognition of key antigenic epitopes able to induce protective or tolerogenic immune responses.

Selenium status alters the immune response and expulsion of adult Heligmosomoides bakeri worms in mice.

Heligmosomoides bakeri is a nematode with parasitic development exclusively in the small intestine of infected mice that induces a potent STAT6-dependent Th2 immune response. We previously demonstrated that host protective expulsion of adult H. bakeri worms from a challenge infection was delayed in selenium (Se)-deficient mice. In order to explore mechanisms associated with the delayed expulsion, 3-week-old female BALB/c mice were placed on a torula yeast-based diet with or without 0.2 ppm Se, and after 5 weeks, they were inoculated with H. bakeri infective third-stage larvae (L3s). Two weeks after inoculation, the mice were treated with an anthelmintic and then rested, reinoculated with L3s, and evaluated at various times after reinoculation. Analysis of gene expression in parasite-induced cysts and surrounding tissue isolated from the intestine of infected mice showed that the local-tissue Th2 response was decreased in Se-deficient mice compared to that in Se-adequate mice. In addition, adult worms recovered from Se-deficient mice had higher ATP levels than worms from Se-adequate mice, indicating greater metabolic activity in the face of a suboptimal Se-dependent local immune response. Notably, the process of worm expulsion was restored within 2 to 4 days after feeding a Se-adequate diet to Se-deficient mice. Expulsion was associated with an increased local expression of Th2-associated genes in the small intestine, intestinal glutathione peroxidase activity, secreted Relm-ß protein, anti-H. bakeri IgG1 production, and reduced worm fecundity and ATP-dependent metabolic activity.

Improving resilience against natural gastrointestinal nematode infections in browsing kids during the dry season in tropical Mexico.

The objective was to determine the effect of supplementary feeding on the resilience and resistance of Criollo kids against natural gastrointestinal nematode (GIN) infections, when browsing native vegetation during the dry season in tropical Mexico. Thirty-three two-month-old Criollo kids, raised nematode free, were included at weaning in a 20-week trial. The kids were placed into four groups. Two groups of eight kids were offered 100g/day soybean and sorghum meal (26%:74% respectively fresh basis) (treated/supplemented (T-S) and infected/supplemented (I-S)). Two groups remained with no supplement for the duration of the trial (infected/non-supplemented (I-NS) (n=9) and treated/non-supplemented (T-NS) (n=8)). Kids in groups T-S and T-NS were drenched with 0.2mg of moxidectin/kg body weight orally (Cydectin, Fort Dodge) every 28 days. Groups I-S and I-NS were naturally infected with GIN. The animals browsed native vegetation for an average of 7h/day together with a herd of 120 naturally infected adult goats. Cumulative live weight gain (CLWG), packed cell volume (PCV), haemoglobin (Hb), total plasma protein and plasma albumin were recorded every 14 days as measurements of resilience. Resistance parameters (faecal egg counts (FEC) and peripheral eosinophil counts (PEC)) were also measured. Bulk faecal cultures were made for each group every 28 days. Every month a new pair of initially worm-free tracer kids assessed the infectivity of the vegetation browsed by the animals. Tracer kids and faecal cultures showed that kids faced low mixed infections (Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum). Under conditions of scarce vegetation, such as those in the present study, supplemented groups (I-S and T-S) had higher growth rates compared to the non-supplemented groups independently of the control of GIN infection with anthelmintic (AH) treatment (P<0.001). Supplementary feeding did not affect FEC or PEC. In the absence of supplementation, lack of AH treatment may lead to outbreaks of clinical nematodosis. The supplementary feeding was economically feasible.

Deficiencies in selenium and/or vitamin E lower the resistance of mice to Heligmosomoides polygyrus infections.

Previous studies have shown that deficiencies in selenium (Se) and/or vitamin E (VE) can exacerbate the infectivity and pathogenesis of coxsackievirus B3 and influenza. Both Se and VE play a role in immune function and antioxidant defense. To determine whether these deficiencies would affect the normal course of infection with a metazoan parasite, mice were made deficient in Se and/or VE and inoculated with the gastrointestinal nematode parasite Heligmosomoides polygyrus. Both primary and secondary infections were assessed. Although the course of a primary infection with H. polygyrus was unaffected by diet, diets deficient in Se, VE, and both Se and VE (Se/VE double-deficiency) all caused delayed adult worm expulsion and increased fecundity during a secondary infection; suggesting an impaired intestinal response. H. polygyrus-induced IL-4 levels were diet-independent; but Se/VE double-deficiency blocked the H. polygyrus-induced IL-4 receptor-associated decrease in sodium-dependent glucose absorption in the jejunum that contributes to worm expulsion. In contrast, Se/VE double-deficiency had no effect on the infection-induced, IL-4R-associated increase in epithelial cell permeability that accompanies the infection. These results suggest that both Se and VE are required for specific IL-4-related changes in intestinal physiology that promote host protection against H. polygyrus.

Impact of vitamin E or selenium deficiency on nematode-induced alterations in murine intestinal function.

The effects of deficiencies in the antioxidant nutrients, vitamin E and selenium, on the host response to gastrointestinal nematode infection are unknown. The aim of the study was to determine the effect of antioxidant deficiencies on nematode-induced alterations in intestinal function in mice. BALB/c mice were fed control diets or diets deficient in selenium or vitamin E and the response to a secondary challenge inoculation with Heligmosomoides polygyrus was determined. Egg and worm counts were assessed to determine host resistance. Sections of jejunum were mounted in Ussing chambers to measure changes in permeability, absorption, and secretion, or suspended in organ baths to determine smooth muscle contraction. Both selenium and vitamin E deficient diets reduced resistance to helminth infection. Vitamin E, but not selenium, deficiency prevented nematode-induced decreases in glucose absorption and hyper-contractility of smooth muscle. Thus, vitamin E status is an important factor in the physiological response to intestinal nematode infection and may contribute to antioxidant-dependent protective mechanisms in the small intestine.

The influence of zinc in mice on infection with Angiostrongylus cantonensis.

BALB/cByJ mice were divided into zinc-deficient (ZD), high zinc-supplemented (ZH), adequately zinc-replete (ZA) and normal control groups by daily dietary control. The body weight in ZD and ZH mice became significantly less than that of normal control mice from 4 weeks (P < 0.002) until 7 weeks (P < 0.0001) after consecutive zinc control. The kinetics of change in body weight of ZD and ZH mice after infection with Angiostrongylus cantonensis were similar to that of uninfected groups mentioned above. The worms recovered at days 7, 14 and 21 after infection from ZD and ZH mice were significantly more in number than that in control mice (P < 0.01). However, the worms from day 21 after infection were shorter in size than those in control mice (P < 0.01). The production of interleukin (IL)-5 was significantly depressed in cultured spleen cells from uninfected and infected ZD and ZH mice, compared with that from respective control group (P < or = 0.02). Furthermore, significantly lower eosinophil counts were observed in the cerebral spinal fluid(CSF) of ZD and ZH mice 3 weeks after infection than in the CSF of control mice (P < 0.001). The levels of immunoglobulins IgG, IgM, IgA and IgE in the supernatant of cultivated spleen cells and serum from ZD and ZH mice were all lower than those of control mice. Thus, the level of zinc correlated with the defence against infection by A. cantonensis, due to the influence of zinc on the growth of mice and their production of IL-5, eosinophils and immunoglobulins.

Enteric infection acts as an adjuvant for the response to a model food antigen.

Oral administration of soluble protein Ags typically induces Ag-specific systemic nonresponsiveness. However, we have found that feeding a model food protein, OVA, to helminth-infected mice primes for a systemic OVA-specific Th2 response. In this report we show that, in addition to creating a Th2-priming cytokine environment, helminth infection up-regulates costimulatory molecule expression on mucosal, but not peripheral, APCs. To examine the consequences of mucosal infection for the T cell response to orally administered Ag, we adoptively transferred transgenic, OVA-specific, T cells into normal mice. We found that helminth infection enhances the expansion and survival of transgenic T cells induced by Ag feeding. Transfer of 5,6-carboxyfluorescein diacetate succinimidyl ester-labeled donor cells showed that T cell proliferation in response to Ag feeding takes place primarily in the mesenteric lymph nodes. Upon subsequent peripheral exposure to Ag in adjuvant, the proliferative capacity of the transferred transgenic T cells was reduced in noninfected mice that had been fed OVA. Helminth infection abrogated this reduction in proliferative capacity. Our data suggests that enteric infection can act as an adjuvant for the response to dietary Ags and has implications for allergic responses to food and the efficacy of oral vaccination.

Heligmosomoides polygyrus immunomodulatory factor (IMF), targets T-lymphocytes.

Experiments were performed to investigate the immunological site of action of an immunomodulatory factor (IMF), isolated from the gastrointestinal nematode Heligmosomoides polygyrus. IMF inhibited antibody production in murine and human 'T-helper (Th-2) driven' immunoassays. The effects were mediated via T lymphocytes as T cell-depleted cultures failed to respond to IMF, a result confirmed by prepulsing discrete cell subsets with the immunomodulant. Although the molecular nature and mode of action of IMF has yet to be determined, it would appear to be a relatively small non-proteinaceous molecule. From this data, we suggest that H. polygyrus secretes a systemically-active IMF from the intestinal lumen, to down-regulate Th-2 cell development in order to promote its survival in a potentially immunologically hostile environment.