Tumor suppressor p53 regulates intestinal type 2 immunity.

The role of p53 in tumor suppression has been extensively studied and well-established. However, the role of p53 in parasitic infections and the intestinal type 2 immunity is unclear. Here, we report that p53 is crucial for intestinal type 2 immunity in response to the infection of parasites, such as Tritrichomonas muris and Nippostrongylus brasiliensis. Mechanistically, p53 plays a critical role in the activation of the tuft cell-IL-25-type 2 innate lymphoid cell circuit, partly via transcriptional regulation of Lrmp in tuft cells. Lrmp modulates Ca2+ influx and IL-25 release, which are critical triggers of type 2 innate lymphoid cell response. Our results thus reveal a previously unrecognized function of p53 in regulating intestinal type 2 immunity to protect against parasitic infections, highlighting the role of p53 as a guardian of immune integrity.

The Taste Receptor TAS1R3 Regulates Small Intestinal Tuft Cell Homeostasis.

Tuft cells are an epithelial cell type critical for initiating type 2 immune responses to parasites and protozoa in the small intestine. To respond to these stimuli, intestinal tuft cells use taste chemosensory signaling pathways, but the role of taste receptors in type 2 immunity is poorly understood. In this study, we show that the taste receptor TAS1R3, which detects sweet and umami in the tongue, also regulates tuft cell responses in the distal small intestine. BALB/c mice, which have an inactive form of TAS1R3, as well as Tas1r3-deficient C57BL6/J mice both have severely impaired responses to tuft cell-inducing signals in the ileum, including the protozoa Tritrichomonas muris and succinate. In contrast, TAS1R3 is not required to mount an immune response to the helminth Heligmosomoides polygyrus, which infects the proximal small intestine. Examination of uninfected Tas1r3-/- mice revealed a modest reduction in the number of tuft cells in the proximal small intestine but a severe decrease in the distal small intestine at homeostasis. Together, these results suggest that TAS1R3 influences intestinal immunity by shaping the epithelial cell landscape at steady-state.

Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit.

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.

Epithelial cell specific Raptor is required for initiation of type 2 mucosal immunity in small intestine.

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25, a critical initiator of the type 2 immune response to parasite infection. When Raptor, a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1), was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice, tuft cells, IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced, but Tm burden was not affected. When Tm infected mice were treated with rapamycin, DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection, tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT, but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4, tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary, our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity.

The common mouse protozoa Tritrichomonas muris alters mucosal T cell homeostasis and colitis susceptibility.

The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths, and viruses. Through coevolution, mammals and these microbes have developed a symbiosis that is sustained through the host's continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing T cell-driven colitis in nonlittermate mouse strains, we serendipitously identified that a nongenetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naive wild-type mice and accelerated colitis in Rag1-/- mice after T cell transfer. Overall, we describe a novel cross-kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research.

Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut.

The intestinal epithelium forms an essential barrier between a host and its microbiota. Protozoa and helminths are members of the gut microbiota of mammals, including humans, yet the many ways that gut epithelial cells orchestrate responses to these eukaryotes remain unclear. Here we show that tuft cells, which are taste-chemosensory epithelial cells, accumulate during parasite colonization and infection. Disruption of chemosensory signaling through the loss of TRMP5 abrogates the expansion of tuft cells, goblet cells, eosinophils, and type 2 innate lymphoid cells during parasite colonization. Tuft cells are the primary source of the parasite-induced cytokine interleukin-25, which indirectly induces tuft cell expansion by promoting interleukin-13 production by innate lymphoid cells. Our results identify intestinal tuft cells as critical sentinels in the gut epithelium that promote type 2 immunity in response to intestinal parasites.

A critical review and meta-analysis of the efficacy of whole-cell killed Tritrichomonas foetus vaccines in beef cattle.

This review assesses the efficacy of whole cell Tritrichomonas foetus vaccine to prevent and treat trichomoniasis in beef cattle. Three databases were searched in June 2012. Eligible studies compared infection risk, open risk, and abortion risk in heifers or infection risk in bulls that received vaccine compared with no vaccine. Study results were extracted, summary effect measures were calculated, and the quality of the evidence was assessed. From 334 citations identified, 10 were relevant to the review. For heifers, there was limited evidence of moderate quality to assess the impact of vaccination on infection risk (RR, 0.89; P = .16; 95% CI, 0.76-1.05; 6 randomized and 4 nonrandomized studies; 251 animals) and open risk (RR, 0.80; P = .06; 95% CI, 0.63-1.01; 6 randomized and 5 nonrandomized studies; 570 animals). The quality of the body of work describing the impact of vaccination on abortion risk was low (summary RR, 0.57; P = .0003; 95% CI, 0.42-0.78; 3 randomized and 2 nonrandomized studies; 176 animals). The quality of evidence was very low for duration of infection (mean difference, -23.42; P = .003; 95% CI, -38.36 to -7.85; 2 randomized and 3 nonrandomized studies; 163 animals). Although the summary effect measures suggest a benefit to vaccination, due to publication bias the effect reported here is likely an over estimate of efficacy. For bull-associated outcomes, the evidence base was low or very low quality.

Sialic acid-specific lectin-mediated adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis.

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.

Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences.

Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples.

The heat shock response and major heat shock proteins of Tritrichomonas mobilensis and Tritrichomonas augusta.

The responses to heat shock in Tritrichomonas mobilensis, a squirrel monkey parasite and Tritrichomonas augusta, an amphibian trichomonad, were evaluated by means of metabolic labeling with [35S]methionine. Electrophoretically separated trichomonad proteins synthesized at different temperatures were visualized by autoradiography and the label incorporation quantitated by a trichloroacetic acid precipitation procedure. A considerable difference in thermotolerance between the two species was found as the protein synthesis reached a maximum at 41 C in T. mobilensis and 37 C in T. augusta. The latter tolerated temperature increases 13 C above normal cultivation temperatures as compared to only 4 C thermotolerance range above normal in T. mobilensis. Major heat shock proteins (Hsps) were expressed in both T. mobilensis (with apparent Mr 94, 72, and 58 kDa) and T. augusta (Mr 94, 70, and 56 kDa) as revealed by autoradiography. Western blot analysis with polyclonal antibody against DnaK of Escherichia coli showed the presence of antigenic Hsp70 homologs in both trichomonads. Similarly, a polyclonal antibody against Hsp60 with broad interspecies cross-reactivity detected Hsp60 homologs in both T. mobilensis and T. augusta. The anti-DnaK antibody cross-reacted with a T. mobilensis protein localized in Golgi apparatus as demonstrated by immunoelectron microscopy. Immunocytochemistry on trichomonad frozen sections revealed the presence of the Hsp60 homolog in light-microscopic granules corresponding to hydrogenosomes.

A reduction in the duration of infection with Tritrichomonas foetus following vaccination in heifers and the failure to demonstrate a curative effect in infected bulls.

Seven batches of 25% water-phase, oil-in-water vaccine were prepared from whole cultures of Tritrichomonas foetus. Two inoculations were given, spaced 6 weeks apart, to virgin heifers and infected bulls. A significant reduction (P less than 0.01) in the duration of infection in vaccinated heifers was seen when they were challenged by being bred to a bull infected with the same isolate as that contained in the vaccine. Only 1/12 vaccinated heifers were pregnant 4.5 months after the end of the breeding season compared to 2/12 in the control group. The vaccine, therefore, has no practical advantage. Vaccine was supplied to 2,724 bulls on properties where the infection was present. From these bulls, 110 reliable results were obtained, where bulls had been infected, been inoculated and tested 1 month later. No curative effect was demonstrable with 69/110 (62.7%) bulls, remaining infected after the course of inoculations. There was also no difference between vaccine batches or between bulls of different ages. Further work on improving the vaccine is indicated. Three media suitable for the culture of T. foetus are described in detail.

Characterization of Tritrichomonas foetus antigens by use of monoclonal antibodies.

The specificity for and function of monoclonal antibodies against Tritrichomonas foetus were characterized. Four monoclonal antibodies generated by immunization of mice with live T. foetus were selected on the basis of enzyme-linked immunosorbent assay reactions. The approximate molecular masses of the predominant proteins were determined by Western blotting (immunoblotting). Monoclonal antibody TF3.8 recognized a predominant band at approximately 155 kilodaltons, whereas TF3.2 reacted with several bands. Monoclonal antibodies TF1.17 and TF1.15 recognized broad bands between 45 and 75 kilodaltons. The first two antibodies (TF3.8 and TF3.2) did not react with the surface of T. foetus, as determined by live-cell immunofluorescence, agglutination, and immobilization, whereas two other monoclonal antibodies (TF1.17 and TF1.15) did react with surface epitopes, as determined by these criteria. The latter two monoclonal antibodies also mediated complement-dependent killing of T. foetus and prevented of adherence of organisms to bovine vaginal epithelial cells. One antibody, TF1.15, also killed in the absence of complement. Since these functions are in vitro correlates of protection, the antigens recognized by these monoclonal antibodies may induce protective immunity.

Measurement of antibody in serum and genital fluids of bulls by ELISA after vaccination and challenge with Tritrichomonas foetus.

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to Tritrichomonas foetus using both whole cell antigen (WCA) and membrane protein antigen (MPA). The test was used to detect specific antibody in serum, preputial washings and seminal plasma samples from 7 adult bulls which were vaccinated subcutaneously on 3 occasions with a membrane protein vaccine against T. foetus var brisbane in an oil adjuvant, and from 4 unvaccinated control animals. One month after administration of the third dose of vaccine, vaccinated and control bulls were repeatedly challenged with the live vaccine strain of the T. foetus. A steady increase in serum antibody titre was detected after each inoculation of vaccine when both antigens were used in the ELISA. However, MPA was more sensitive. After challenge, vaccinated bulls developed an increased titre. No specific antibody was detected in control bulls, except in one bull after challenge in which seroconversion was detected. The serum antibody titres of both groups of animals were also measured with the microagglutination test which proved less sensitive than the ELISA. Antibody titres to both antigens, although lower than in serum, were detected in the seminal plasma of vaccinated animals. The control bulls remained non-responsive. No antibody was detected by ELISA in preputial washings from either control or vaccinated bulls prior to challenge. Post-challenge, some of the vaccinated bulls were responsive with both antigens whereas the control bulls remained negative.

Antibody enhances killing of Tritrichomonas foetus by the alternative bovine complement pathway.

The role of bovine antibody and complement in host defense against Tritrichomonas foetus was measured by using an assay of trichomonad viability based on protozoal uptake of tritiated adenine. Moderate killing was measured in the absence of antibody only with high concentrations of complement-preserved hypogammaglobulinemic bovine serum. However, very low concentrations of hyperimmune serum promoted significant enhancement (P less than 0.05) of killing by complement. Heat inactivation of complement (56 degrees C for 30 min) eliminated antibody-dependent and -independent killing. Similarly, depletion of bovine factor B in serum by heat treatment (50 degrees C for 45 min) abolished antibody-dependent and -independent killing. However, selective inactivation of the classical complement pathway with magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not affect antibody-dependent or -independent killing by complement. These findings demonstrate antibody enhancement of complement-mediated killing of T. foetus by the alternative pathway of bovine complement.

Identification of Tritrichomonas foetus in sections of bovine placental tissue with monoclonal antibodies.

Sections of bovine placenta from cases of bovine trichomoniasis were examined for the presence of Tritrichomonas foetus by standard histological methods using phase-contrast microscopy and by indirect immunofluorescence assay (IFA) employing monoclonal antibodies (mAbs) specific for T. foetus. Parasites were identified readily in deparaffinized tissue up to 4 yr old by IFA with 2 mAbs previously shown to bind to the surface of living T. foetus. These results indicated that the IFA provided a rapid and specific method of identifying T. foetus in tissue sections as compared to standard histological methods.

The immune response in cattle infected with Tritrichomonas foetus.

Holando-Argentina calves (males and females) were experimentally infected with Tritrichomonas foetus var. Belfast (T. foetus) by introducing 10(7) protozoa into the preputial and vaginal cavities, in order to analyse the course of the immune response to infection. Samples of serum, vaginal mucus and preputial secretion were taken periodically and assayed by means of microagglutination of living protozoa. The serum antibody titre, which averaged 32 before infection and was equivalent to titres in a non-infected group, increased to 512 in the heifers 11 weeks later and to 128 in the bulls 4 months post-infection. Agglutinating antibodies were not detected in the preputial cavity, but heifers showed antibodies in the vaginal mucus and became trichomoniasis free after 4 months. Conversely, genital secretions from the bulls gave rise to positive cultures during the whole period of experimentation. The intradermal sensitivity was checked using a soluble antigen from T. foetus. The diameter of the papula increased up to three times in heifers, while in bulls the results were no different than those from the non-infected group. Serum antibodies were of the IgG2 subclass, while those isolated from vaginal mucus were characterized as IgG1, an opsonizing antibody. Heifers were refractory to challenge infection after 1 year. The poor immune response in bulls is consistent with their role as carriers of T. foetus.

Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective.

Antigenic relationship among field isolates of Tritrichomonas foetus from cattle.

Analysis of protein and antigen profiles of Tritrichomonas foetus isolates from cattle from 5 western states was accomplished by sodium dodecyl sulfate polyacrylamide-gel electrophoresis, immunoblot, immunoprecipitation, and fluorography techniques. Total protein profiles of all isolates were compared by Coomassie brilliant blue staining of T foetus protein samples prepared by 4 protein-extraction methods. Antigenic tritrichomonas proteins were identified by immunoblot assay with polyclonal bovine or rabbit anti-T foetus serum. Additionally, [14C]glucosamine-labeled T foetus was used for total and antigenic glycoprotein analyses. Detectable differences in the composition of total proteins or antigenic tritrichomonal proteins were not observed among all isolates. However, intensity differences in some antigenic protein bands were apparent. Bovine and rabbit sera from immunized animals possessed antibodies to the same antigenic tritrichomonal proteins. Each T foetus isolate contained 4 to 7 molecular weight size classes of glycoprotein, which were labeled by [14C]glucosamine; however, only 3 to 4 glycoproteins were identified as antigens by bovine or rabbit antiserum.

Development and preliminary assessment of a polyclonal antibody-based enzyme immunoassay for the detection of Tritrichomonas foetus antigen in breeding cattle.

More sensitive tests are required for the diagnosis of Tritrichomonas foetus infection in cattle and an antigen-detecting enzyme immunoassay (EIA) has been applied to this purpose. An affinity purified immunoglobulin fraction obtained from rabbits immunised with cultured T. foetus served as both capture antibody and as biotinylated indicator antibody. While highly sensitive in the detection of antigen derived from cultured organisms, the assay showed poor sensitivity in the detection of antigen in the cervico-vaginal mucus of artificially infected heifers, with only 75% of culture-positive samples being considered positive for antigen. In a direct comparison, 23/122 samples from a naturally infected dairy herd gave positive cultures, while only 10/122 samples were considered antigen positive by EIA.