Parasite Expulsion Research Articles

Transmembrane Tumor Necrosis Factor Alpha Is Required for Enteropathy and Is Sufficient To Promote Parasite Expulsion in Gastrointestinal Helminth Infection

To study the specific role of transmembrane tumor necrosis factor (tmTNF) in protective and pathological responses against the gastrointestinal helminth Trichinella spiralis, we compared the immune responses of TNF-alpha/lymphotoxin alpha (LTalpha)(-/-) mice expressing noncleavable transgenic tmTNF to those of TNF-alpha/LTalpha(-/-) and wild-type mice. The susceptibility of TNF-alpha/LTalpha(-/-) mice to T. spiralis infection was associated with impaired induction of a protective Th2 response and the lack of mucosal mastocytosis. Although tmTNF-expressing transgenic (tmTNF-tg) mice also had a reduced Th2 response, the mast cell response was greater than that observed in TNF-alpha/LTalpha(-/-) mice and was sufficient to induce the expulsion of the parasite. T. spiralis infection of tmTNF-tg mice resulted in significant intestinal pathology characterized by villus atrophy and crypt hyperplasia comparable to that induced following the infection of wild-type mice, while pathology in TNF-alpha/LTalpha(-/-) mice was significantly reduced. Our data thus indicate a role for tmTNF in host defense against gastrointestinal helminths and in the accompanying enteropathy. Furthermore, they also demonstrate that TNF-alpha is required for the induction of Th2 immune responses related to infection with gastrointestinal helminth parasites.

Polyclonal and Specific Antibodies Mediate Protective Immunity against Enteric Helminth Infection

Anti-helminth immunity involves CD4+ T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasite that establishes chronic infection. Polyclonal IgG antibodies, present in naive mice and produced following Hp infection, functioned to limit egg production by adult parasites. Comparatively, affinity-matured parasite-specific IgG and IgA antibodies that developed only after multiple infections were required to prevent adult worm development. These data reveal complementary roles for polyclonal and affinity-matured parasite-specific antibodies in preventing enteric helminth infection by limiting parasite fecundity and providing immune protection against reinfection, respectively. We propose that parasite-induced polyclonal antibodies play a dual role, whereby the parasite is allowed to establish chronicity, while parasite load and spread are limited, likely reflecting the long coevolution of helminth parasites with their hosts.

The effectors responsible for gastrointestinal nematode parasites, Trichinella spiralis, expulsion in rats

Helper T (Th2) cells type 2 have a central role in host protective responses to gastrointestinal nematode parasite, Trichinella spiralis infection, but the actual effector mechanisms involved in parasite expulsion are still uncertain. Recent evidences suggest that mast cell recruitment and activation may associate with parasite elimination from host intestines in mice. On the other hand, IgE production may induce defensive responses to primary infection with the helminth in rats. The differences of host effector mechanisms to the same experimental infections might disturb our understanding of the host protective mechanisms to gastrointestinal nematode parasite infection. In order to redefine these differences, we investigated in detail the relationship between intestinal immune responses and worm expulsion following T. spiralis infection among several rat strains including mutants. As a result, there were significant correlations of parasite expulsion with mast cell hyperplasia in addition to serum IgE level. Moreover, mast cell-deficient and dysfunction rats showed delayed worm elimination from their gut. Therefore, the present study suggests that mast cells should also be one of the prominent effector cells involved in T. spiralis parasite expulsion in rats as well as mice.

B cells are required for the host protection to the gastrointestinal nematode parasite Heligmosomoides polygyrus

Previous studies from our laboratory have shown an important role for B cells in the development of Ag‐specific Th2 cells during a polarized Th2‐type response. In this study we examined the role of B cell in host protection to a nematode parasite, Heligmosomoides polygyrus (Hp). Hp is characterized by chronic infection following primary inoculation, but following drug‐induced worm clearance, secondary inoculation triggers a host protective memory Th2‐type response resulting in worm clearance. B cell deficient JhD and control WT mice were orally inoculated with Hp, followed by anti‐helminthic drug treatment on day 14, and one month later challenged with Hp. Compared to WT mice, JhD mice were much more susceptible to Hp infection with high numbers of worms. B cell deficiency did not affect T cell activation, IL‐4 and IL‐13 production by CD4 T cells in mesenteric lymph nodes, and alternatively activated macrophage activation in granulomas surrounding developing larvae in the submucosa. However, immunofluorescent staining of granulomas showed that there were more Gr‐1+ neutrophils adjacent to the parasites, and less Gr‐1+ F4/80+ cells surrounding these Gr‐1+ cells in JhD mice compared to WT mice. These results suggest that B cells are required for the development of an effective memory response culminating in parasite expulsion.

Mechanisms of leucocyte recruitment to the inflamed large intestine: redundancy in integrin and addressin usage

The caecal-dwelling nematode Trichuris muris provides a natural model of human whipworm infection. Resistance to T. muris is dependent on a host Th2 response, and CD4+Th2 cells migrate to the gut-associated lymphoid tissue (GALT) to elicit parasite expulsion. Thus, CD4+T cells infiltrate the caecal lamina propria during infection, along with other leucocyte subsets that are not critical for parasite expulsion, such as eosinophils. Trafficking of leucocytes to the GALT has been shown to be dependent on the alpha4beta7/MAdCAM-1 integrin-addressin interaction. However, where inflammation is present, such as during T. muris infection, redundant mechanisms of leucocyte recruitment may also occur in addition to traditional gut-homing interactions. We utilized an anti-integrin/addressin antibody treatment regime to investigate this redundancy in resistant, T. muris-infected C57BL/6 mice. Where only the alpha4beta7/MAdCAM-1 interaction was blocked, mice remained resistant to T. muris infection, making a Th2 response and both CD4+T cells and eosinophils infiltrated the site of infection. However, in the absence of available alpha4beta7 and alpha4beta1, mice became chronically infected with T. muris and mounted a more Th1-biased immune response. Interestingly, CD4+T cells, but not eosinophils, were able to infiltrate the caecum, showing different levels of redundancy between leucocyte subsets during infection.

IL-27 Suppresses Th2 Cell Development and Th2 Cytokines Production from Polarized Th2 Cells: A Novel Therapeutic Way for Th2-Mediated Allergic Inflammation

IL-27 up-regulates Th1 but down-regulates Th2 responses. However, its molecular mechanism and regulatory effects on polarized Th2 cells remain unclear. In this study, we have revealed that IL-27 inhibits Th2 cell development as well as Th2 cytokines production from already polarized Th2 cells by down-regulation of GATA-3 and up-regulation of T-bet expression simultaneously. In vivo daily IL-27 treatment for 1 wk after Leishmania major infection protects BALB/c mice from footpad swelling by diminishing parasite burden via reciprocal regulation of Th1 and Th2 responses. Furthermore, IL-27 stimulation causes marked reduction in the capacity of host mouse to mount a Th2 response against Strongyloides venezuelensis infection. Thus, IL-27-treated mice failed to develop intestinal mastocytosis after S. venezuelensis infection and exhibited a marked delay in parasite expulsion. Finally, intranasal administration of IL-27 inhibits OVA-induced airway hyperresponsiveness and inflammation in OVA-sensitized animals. Thus, IL-27 could provide us with a novel therapeutic way for treating Th2-associated diseases such as bronchial asthma.

The role of IL‐4, IL‐13 and IL‐4Rα in the development of protective and pathological responses to Trichinella spiralis

T helper type 2 (Th2) responses have been shown to be important in protective responses to gastrointestinal (GI) helminth infections and in the development of the intestinal pathology accompanying expulsion of the parasite. Different inbred mouse strains have been shown to develop different cytokine profiles following infection with GI helminths with increased resistance observed in those strains where Th2 cytokines predominate. The aim of this study was to determine the role of IL-4, IL-13 and IL-4Ralpha and the impact of host background on the development of the protective and pathological responses induced by infection with the gastrointestinal helminth Trichinella spiralis. IL-4, IL-13 and IL-4Ralpha were required for the generation of Th2 responses to T. spiralis; however, the role these responses play in the development of protection and enteropathy was less clear. IL-4Ralpha-deficiency mice resulted in substantially reduced parasite expulsion, intestinal pathology and Th2 responses. Similarly, lack of IL-13 resulted in inhibited expulsion and the development of enteropathy. Although Th2 responses were reduced in BALB/c IL-4-/- mice, neither expulsion nor enteropathy were different from wild-type mice. In contrast, C57BL/6 IL-4-/- exhibited delayed expulsion and reduced pathology, suggesting that host genetics are important in the function of individual cytokines. Thus, differences in background genotype may be an important component in the development host protection and the development of intestinal pathology accompanying the loss of GI helminths.

Quantitative isolation and in vivo imaging of malaria parasite liver stages

The liver stages of Plasmodium, the causative agent of malaria, are the least explored forms in the parasite’s life cycle despite their recognition as key vaccine and drug targets. In vivo experimental access to liver stages of human malaria parasites is practically prohibited and therefore rodent model malaria parasites have been used for in vivo studies. However, even in rodent models progress in the analysis of liver stages has been limited, mainly due to their low abundance and associated difficulties in visualisation and isolation. Here, we present green fluorescent protein (GFP)-tagged Plasmodium yoelii rodent malaria parasite liver infections in BALB/c mice as an excellent quantitative model for the live visualisation and isolation of the so far elusive liver stages. We believe P. yoelii GFP-tagged liver stages allow, for the first time, the efficient quantitative isolation of intact early and late liver stage-infected hepatocyte units by fluorescence activated cell sorting. GFP-tagged liver stages are also well suited for intravital imaging, allowing us for the first time to visualise them in real time. We identify previously unrecognised features of liver stages including vigorous parasite movement and expulsion of ‘extrusomes’. Intravital imaging thus reveals new, important information on the malaria parasite’s transition from tissue to blood stage.

OX40 interactions in gastrointestinal nematode infection

The immune expulsion of gastrointestinal nematode parasites is usually associated with T helper type 2 (Th2) responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. The intestinal inflammatory response accompanying infection with gastrointestinal helminths is thought to be a contributory factor leading to the expulsion of the parasite. However, we have shown that the intestinal inflammation, which is controlled by interleukin (IL)-4, is not required for parasite expulsion. OX40-OX40 ligand (L) signals have been shown to be important for the development of Th2 immune responses but are also involved in a number of inflammatory diseases including those of the intestine. Here, we have investigated the effect of OX40 and OX40L fusion protein treatment on the induction of protective Th2 responses and enteropathy following infection with the gastrointestinal nematode Trichinella spiralis. Treatment with an OX40-immunoglobulin (Ig) blocking fusion protein resulted in enhanced expulsion of the parasite and an increase in the accompanying mastocytosis, despite unaltered levels of Th2 cytokines. Furthermore, there was a delay in the villus atrophy and crypt hyperplasia usually associated with this infection. In contrast, levels of Th2 cytokines were greatly up-regulated in mice treated with an OX40L-Ig activating fusion protein, yet the expulsion of the parasite and the enteropathy were unaffected. Therefore, OX40 ligation potentiates the Th2 response without enhancing host protective immune responses, whereas blocking the OX40-OX40L interaction enhances host protection without promoting Th2 cytokine responses during Trichinella spiralis infection.

Rapid improvement of immunity to Teladorsagia circumcincta is achieved through a reduction in the demand for protein in lactating ewes

Protein supplementation can improve the resistance to parasites of periparturient ewes, as indicated by reduced nematode egg excretion and worm burdens. However, the rate at which this improvement can occur is largely unknown. We investigated the rate of improvement by assessing temporal changes in faecal egg counts after we experimentally reduced nutrient demand. Three groups of nine pregnant ewes each were trickle infected with Teladorsagia circumcincta from day −70 to day 16 (parturition is day 0). Two groups of twin-rearing ewes were fed at 0.8 (L22) or 1.2 (H22) times their assumed metabolizable protein requirements, and a third group was fed the same daily food allowances as L22 ewes, but one of their lambs was removed on day 10 (L21). Ewes were slaughtered on day 21 to assess worm burdens, in vitro larval establishment on abomasal explants, and mucosal inflammatory cells. Faecal egg counts of L22 ewes were higher than H22 ewes throughout lactation. After the removal of one lamb, faecal egg counts of L21 ewes decreased within 5 days to levels similar to H22 ewes. Relative to L22 ewes, L21 and H22 ewes had lower worm burdens, parasite per capita fecundity and in vitro establishment rates of both T. circumcincta and Haemonchus contortus. Mucosal mast cell and eosinophil counts were similar for all ewes, but H22 ewes had higher globule leukocyte counts than L22 and L21 ewes. The data suggest that a reduction in protein demand can rapidly improve periparturient immunity to T. circumcincta. This may be associated with increased parasite expulsion, reduced fecundity and non-parasite specific reduction of in vitro larval establishment.

Exploiting worm and allergy models to understand Th2 cytokine biology

Helminthic parasites and many allergens trigger highly polarized Th2-type immune responses. In most helminth infections, the Th2 response often leads to parasite expulsion or sequestration. During murine Schistosoma mansoni infection, however, the parasites persist and the chronic Th2 response induces severe pathological changes in the gut and liver. Thus, the study of schistosome infections in mice has become a popular model to study the pathogenesis of Th2 cytokine-mediated disease. This review will discuss recent findings from the schistosomiasis model that may be relevant to the understanding of allergic inflammation, asthma and Th2 cytokine biology in general. Evidence is accumulating that the Th2 pathway is not a 'default pathway' but one that is actively instructed by mechanisms that are only beginning to be understood. Other areas of intensive investigation include studies on alternatively activated macrophages, the role of dendritic cells in Th2 response development, the inhibitory function of IL-10, regulatory T-cells and decoy receptors on chronic Th2-mediated inflammation, and the role of chitinases in mediating Th2 disease. Finally, the development of novel eosinophil-deficient mice has also accelerated our understanding of the contribution of this important cell type to Th2 immunity. Many findings from the schistosomiasis model have been subsequently demonstrated in models of allergic disease, illustrating the utility of this model to dissect basic mechanisms of Th2-mediated inflammation. Further study of helminth-induced Th2 responses may expedite the discovery of new therapeutics for a wide range of Th2-associated diseases.

IL-18 with IL-2 protects against Strongyloides venezuelensis infection by activating mucosal mast cell–dependent type 2 innate immunity

C57BL/6 (B6) and B6 background STAT6−/− mice pretreated with IL-18 plus IL-2 showed prominent intestinal mastocytosis and rapidly expelled implanted adult worms of the gastrointestinal nematode Strongyloides venezuelensis. In contrast, identically pretreated mast cell–deficient W/Wv mice failed to do so. Thus, activated mucosal mast cells (MMC) are crucial for parasite expulsion. B6 mice infected with S. venezuelensis third-stage larvae (L3) completed parasite expulsion by day 12 after infection, whereas IL-18−/− or IL-18Rα −/− B6 mice exhibited marked impairment in parasite expulsion, suggesting a substantial contribution of IL-18–dependent MMC activation to parasite expulsion. Compared with IL-18−/− or IL-18Rα −/− mice, S. venezuelensis L3–infected STAT6−/− mice have poorly activated MMC and sustained infection; although their IL-18 production is normal. Neutralization of IL-18 and IL-2 further reduces expulsion in infected STAT6−/− mice. These results suggest that collaboration between IL-18–dependent and Th2 cell–dependent mastocytosis is important for prompt parasite expulsion.

Cells, cytokines and other molecules associated with rejection of gastrointestinal nematode parasites

Infections with gastrointestinal nematodes represent a major problem for human health and animal health and production. The physical size of the parasite, its changing life-cycle stages and the relative inaccessibility to host cells pose unique challenges to the immune system, which has evolved specialized strategies for parasite control. This paper reviews the work performed in the authors’ laboratories to identify components that are involved in the natural rejection response against ruminant gastrointestinal nematode parasites, in particular Haemonchus contortus in sheep. The results of these studies indicate that stage-specific antibodies act in concert with effector cells, in particular globular leukocytes (intraepithelial mast cells) and eosinophils, appropriately activated/primed by type 2 (T2) cytokines, to initiate different mechanisms of parasite expulsion and killing. In addition, other molecules, in particular carbohydrate binding galectins, may be involved in strengthening the final effector phase of the rejection response.

Interleukin-13 (IL-13) Pathway

Interleukin-13 (IL-13), like IL-4, is a cytokine produced by T(H)2 type helper T cells in response to signaling through the T cell antigen receptor and by mast cells and basophils upon cross-linkage of the high-affinity receptor for immunoglobulin E (IgE). It is also produced by activated eosinophils. IL-13 induces many of the same responses as IL-4 and shares a receptor subunit with IL-4. IL-13 has been implicated in airway hypersensitivity and mucus hypersecretion, inflammatory bowel disease, and parasitic nematode expulsion.

Accelerated Intestinal Epithelial Cell Turnover: A New Mechanism of Parasite Expulsion

The functional integrity of the intestinal epithelial barrier forms a major defense against invading pathogens, including gastrointestinal-dwelling nematodes, which are ubiquitous in their distribution worldwide. Here, we show that an increase in the rate of epithelial cell turnover in the large intestine acts like an "epithelial escalator" to expel Trichuris and that the rate of epithelial cell movement is under immune control by the cytokine interleukin-13 and the chemokine CXCL10. This host protective mechanism against intestinal pathogens has implications for our wider understanding of the multifunctional role played by intestinal epithelium in mucosal defense.

Comparative dynamics and phenotype of the murine immune response to Trichinella spiralis and Trichinella pseudospiralis

Infection of NIH mice with Trichinella spiralis and Trichinella pseudospiralis results in qualitatively comparable immune responses. Antigen-specific proliferation by mesenteric lymph node cells was transient and temporally associated with intestinal infection, but in contrast was sustained throughout infection by splenocytes. Early cytokine production by mesenteric lymph node cells was dominated by interleukin 10, but also IL-5 and IL-4, with rapid resolution following parasite expulsion from the gut. Splenocytes showed a mixed profile of cytokine production, although again dominated by IL-10 and sustained over 60 days of infection. All antibody classes were evident, with early production of IgA and IgG1, and subsequent secretion of other subclasses including IgG2a. Granulocytic infiltration of the spleen was significantly greater in T. spiralis infection. The concentration of serum corticosterone generally remained within normal boundaries, although was raised by day 60 in T. spiralis-infected mice. We conclude that the systemic suppression of inflammation reported for T. pseudospiralis does not result from selective induction of regulatory cytokines, or a major difference in the immune response to infection with T. spiralis.

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils

Neutrophils, eosinophils and macrophages are cells that interact with invading parasites and naive hosts have been shown to have anti-parasitic activity. The initial reaction of these leukocytes is the generation of reactive oxygen species (ROS) to play in parasite expulsion. The present work was carried out to study the effect of total extract, scolex and membrane fractions from Cysticercus cellulosae on respiratory burst by pig neutrophils. Hydrogen peroxide (H2O2) production by neutrophils incubated with metacestode fractions from C. cellulosae showed an increase of: 190% (total extract), 120% (scolex) and 44% (membrane). High antioxidant catalatic activity (33%, 28%, 28% by total extract, scolex and membrane, respectively) was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. Scolex and membrane fractions increased the phagocytic capacity of neutrophils (44% and 28%, respectively). On the other hand, total cysticerci did not alter the phagocytosis, possibly due to modifications in membrane function, caused by high ROS production from neutrophils in the presence of total cysticerci. Total fraction from C. cellulosae is toxic for neutrophils as shown by the decrease in phagocytic capacity, probably caused by high levels of ROS formation. The difference in toxicity of total extract, scolex and membrane fractions on neutrophils can be explained by the presence of an antigenic effect of the vesicular fluid in the total extract of C. cellulosae.

Notch Signaling-Dependant Expulsion of Parasites through Mast Cell-Mediated Immunity.

[Background] Notch signaling is known to be important in hematopoiesis, but very little information is available about its significance in mast cells. Here we provide direct evidence that notch signaling is critical for both development and function of mast cells in vitro and in vivo.[Methods] A Lin− fraction of mouse bone marrow cells was cultured on immobilized Delta1 in the presence of SCF and IL-3, and emerging Lin−FcεRI+c-Kit+ mast cells were characterized. Next, production of mouse mast cell protease-1 (mMCP-1), which is specific for nematode infection through locally expressed TGF-β1 in vivo, by bone marrow-derived mast cells (BMMC) was analyzed after the stimulation with Delta1 in the presence of TGF-β1. Finally, mice were infected with Strongyloides venezuelensis after pre-treatment with Delta1, and expulsion of the worms was examined.[Results] Lin−FcεRI+c-Kit+ mast cells developed remarkably earlier if stimulated with Delta1 (at one week, 15% vs. 3%). DAPT, a γ-secretase inhibitor, blocked the Delta1 effect, while it did not affect the regular time-course mast cell generation by SCF and IL-3. SB431542, a selective inhibitor of TGF-β1 signaling, also blocked early mast cell generation by Delta1. Delta1 augmented mMCP-1 expression and secretion from BMMC by 50 fold. Both DAPT and SB431542 showed a dose-dependent inhibition of Delta1 effect on mMCP-1 expression and secretion. Pre-treatment of the hosts with Delta1 promoted the expulsion of S. venezuelensis, (left/inoculated ratios of worms, 3% vs. 40%) while Delta1 had no effect in the mast cell-deficient W/Wv mice.[Discussion] Our observations reveal that notch signaling regulates both development and function of mast cells in vitro in conjunction with TGF-β1 signaling. In vivo, it is also likely that Delta1 facilitates the functional maturation of intestinal mast cells to eradicate parasites. More precise mechanism of Delta1 action on mast cells in vivo is under a study. [Display omitted]

The IL-27 Receptor (WSX-1) Is an Inhibitor of Innate and Adaptive Elements of Type 2 Immunity

Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1-/- mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.

Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis.

Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule beta (RELMbeta) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmbeta and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmbeta).