Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection.

Introduction: Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Methods: Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Results: Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Discussion: Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.

The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection.

Introduction: Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections. Objective: The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection. Methods: We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi. Results: Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1ß, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFß1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity. Conclusion: These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

Differential Salmonella Typhimurium intracellular replication and host cell responses in caecal and ileal organoids derived from chicken.

Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt-derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell -Salmonella interaction.

Overexpression of Eimeria tenella Rhoptry Kinase 2 Induces Early Production of Schizonts.

Eimeria tenella is an obligate intracellular parasite responsible for avian coccidiosis. Like other apicomplexan parasites, such as Toxoplasma gondii, cell invasion and intracellular development rely on apical organelle content discharge, named micronemes and rhoptries. Some rhoptry (ROP) kinases (ROPK) are key virulence factors in T. gondii. To date, among the 28 ropk genes carried by E. tenella, only two to four were confirmed by proteomic analysis or immunostaining to be expressed at the sporozoite stage. We have previously shown that EtROP1 is implicated in the inhibition of host cell apoptosis by interacting with the cellular p53. This work functionally described the second ROP kinase expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Its overexpression leads to the shortening of the prepatent period and to the early development of first-generation schizonts. Conduction of RNA sequencing analysis and reverse transcriptase quantitative PCR (RT-qPCR) on the host cell allowed us to identify the mitogen-activated protein kinase (MAPK) pathway and the transcription factor cFos to be upregulated by EtROP2. We also showed by immunofluorescence assay that the active kinase EtROP2 is implicated in the p38 MAPK pathway activation. We established here that EtROP2 activates the p38 MAPK pathway through a direct or indirect phosphorylation, leading to the overexpression of the master transcription factor cFos known to be implicated in E. tenella development. IMPORTANCE Rhoptries are specialized secretory organelles found in zoite stages of apicomplexan parasites. In addition to well-conserved rhoptry neck proteins, their protein consists mostly of kinase proteins, highly divergent from eukaryotic kinases. Some of those kinases are described as major virulence factors in Toxoplasma gondii, secreted into the host cell to hijack signaling pathways. Most of those kinases remain to be characterized in Eimeria tenella. Deciphering their cellular function is a prerequisite to supporting their relevance as a druggable target in development of new means of Eimeria tenella control. Secreted divergent kinases that interact with host cell partners to modulate pathways are good candidates, as they coevolve with their host targets to ensure their function within the host and are less prone to mutations that would lead to drug resistance. The absence of any orthologous kinase in host cells makes these parasite kinases a promising drug target candidate.

Cross-Talk Between the Intestinal Epithelium and Salmonella Typhimurium.

Salmonella enterica serovars are invasive gram-negative bacteria, causing a wide range of diseases from gastroenteritis to typhoid fever, representing a public health threat around the world. Salmonella gains access to the intestinal lumen after oral ingestion of contaminated food or water. The crucial initial step to establish infection is the interaction with the intestinal epithelium. Human-adapted serovars such as S. Typhi or S. Paratyphi disseminate to systemic organs and induce life-threatening disease known as typhoid fever, whereas broad-host serovars such as S. Typhimurium usually are limited to the intestine and responsible for gastroenteritis in humans. To overcome intestinal epithelial barrier, Salmonella developed mechanisms to induce cellular invasion, intracellular replication and to face host defence mechanisms. Depending on the serovar and the respective host organism, disease symptoms differ and are linked to the ability of the bacteria to manipulate the epithelial barrier for its own profit and cross the intestinal epithelium. This review will focus on S. Typhimurium (STm). To better understand STm pathogenesis, it is crucial to characterize the crosstalk between STm and the intestinal epithelium and decipher the mechanisms and epithelial cell types involved. Thus, the purpose of this review is to summarize our current knowledge on the molecular dialogue between STm and the various cell types constituting the intestinal epithelium with a focus on the mechanisms developed by STm to cross the intestinal epithelium and access to subepithelial or systemic sites and survive host defense mechanisms.

Establishment of a Newborn Lamb Gut-Loop Model to Evaluate New Methods of Enteric Disease Control and Reduce Experimental Animal Use.

Enteric infectious diseases are not all well controlled, which leads to animal suffering and sometimes death in the most severe cases, in addition to economic losses for farmers. Typical symptoms of enteric infections include watery diarrhea, stomach cramps or pain, dehydration, nausea, vomiting, fever and weight loss. Evaluation of new control methods against enteric infections requires the use of many animals. We aimed to develop a new method for an initial in vivo screen of promising compounds against neonatal diseases such as cryptosporidiosis while limiting experimental animal use. We therefore adapted an in vivo method of multiple consecutive but independent intestinal loops to newborn lambs delivered by cesarean section, in which endotoxin responsiveness is retained. This new method allowed for the screening of natural yeast fractions for their ability to stimulate immune responses and to limit early Cryptosporidium parvum development. This model may also be used to investigate host-pathogen interactions and immune responses in a neonatal controlled environment.

Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle.

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host-pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7-9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella.

Intestinal organoids in farm animals.

In livestock species, the monolayer of epithelial cells covering the digestive mucosa plays an essential role for nutrition and gut barrier function. However, research on farm animal intestinal epithelium has been hampered by the lack of appropriate in vitro models. Over the past decade, methods to culture livestock intestinal organoids have been developed in pig, bovine, rabbit, horse, sheep and chicken. Gut organoids from farm animals are obtained by seeding tissue-derived intestinal epithelial stem cells in a 3-dimensional culture environment reproducing in vitro the stem cell niche. These organoids can be generated rapidly within days and are formed by a monolayer of polarized epithelial cells containing the diverse differentiated epithelial progeny, recapitulating the original structure and function of the native epithelium. The phenotype of intestinal organoids is stable in long-term culture and reflects characteristics of the digestive segment of origin. Farm animal intestinal organoids can be amplified in vitro, cryopreserved and used for multiple experiments, allowing an efficient reduction of the use of live animals for experimentation. Most of the studies using livestock intestinal organoids were used to investigate host-microbe interactions at the epithelial surface, mainly focused on enteric infections with viruses, bacteria or parasites. Numerous other applications of farm animal intestinal organoids include studies on nutrient absorption, genome editing and bioactive compounds screening relevant for agricultural, veterinary and biomedical sciences. Further improvements of the methods used to culture intestinal organoids from farm animals are required to replicate more closely the intestinal tissue complexity, including the presence of non-epithelial cell types and of the gut microbiota. Harmonization of the methods used to culture livestock intestinal organoids will also be required to increase the reproducibility of the results obtained in these models. In this review, we summarize the methods used to generate and cryopreserve intestinal organoids in farm animals, present their phenotypes and discuss current and future applications of this innovative culture system of the digestive epithelium.

Cryptosporidium parvum Subverts Antimicrobial Activity of CRAMP by Reducing Its Expression in Neonatal Mice.

Cryptosporidium parvum causes diarrhea in infants under 5 years, in immunosuppressed individuals or in young ruminants. This parasite infects the apical side of ileal epithelial cells where it develops itself and induces inflammation. Antimicrobial peptides (AMPs) are part of the innate immune response, playing a major role in the control of the acute phase of C. parvum infection in neonates. Intestinal AMP production in neonates is characterized by high expressions of Cathelicidin Related Antimicrobial Peptide (CRAMP), the unique cathelicidin in mice known to fight bacterial infections. In this study, we investigated the role of CRAMP during cryptosporidiosis in neonates. We demonstrated that sporozoites are sensitive to CRAMP antimicrobial activity. However, during C. parvum infection the intestinal expression of CRAMP was significantly and selectively reduced, while other AMPs were upregulated. Moreover, despite high CRAMP expression in the intestine of neonates at homeostasis, the depletion of CRAMP did not worsen C. parvum infection. This result might be explained by the rapid downregulation of CRAMP induced by infection. However, the exogenous administration of CRAMP dampened the parasite burden in neonates. Taken together these results suggest that C. parvum impairs the production of CRAMP to subvert the host response, and highlight exogenous cathelicidin supplements as a potential treatment strategy.

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies.

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines. The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry. Eggs were quickly collected after laying at a broiler breeder farm. They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator. The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Update on Cryptosporidium spp.: highlights from the Seventh International Giardia and Cryptosporidium Conference.

While cryptosporidiosis is recognized as being among the most common causes of human parasitic diarrhea in the world, there is currently limited knowledge on Cryptosporidium infection mechanisms, incomplete codification of diagnostic methods, and a need for additional therapeutic options. In response, the Seventh International Giardia and Cryptosporidium Conference (IGCC 2019) was hosted from 23 to 26 June 2019, at the Rouen Normandy University, France. This trusted event brought together an international delegation of researchers to synthesize recent advances and identify key research questions and knowledge gaps. The program of the interdisciplinary conference included all aspects of host-parasite relationships from basic research to applications to human and veterinary medicine, and environmental issues associated with waterborne parasites and their epidemiological consequences. In relation to Cryptosporidium and cryptosporidiosis, the primary research areas for which novel findings and the most impressive communications were presented and discussed included: Cryptosporidium in environmental waters, seafood, and fresh produce; Animal epidemiology; Human cryptosporidiosis and epidemiology; Genomes and genomic evolution encompassing: Comparative genomics of Cryptosporidium spp., Genomic insights into biology, Acquiring and utilizing genome sequences, Genetic manipulation; Host-parasite interaction (immunology, microbiome); and Diagnosis and treatment. High quality presentations discussed at the conference reflected decisive progress and identified new opportunities that will engage investigators and funding agencies to spur future research in a "one health" approach to improve basic knowledge and the clinical and public health management of zoonotic cryptosporidiosis.

TITLE: Mise à jour sur Cryptosporidium spp.: Faits saillants de la Septième Conférence Internationale sur Giardia et Cryptosporidium. ABSTRACT: Bien que la cryptosporidiose soit reconnue comme l'une des premières causes de diarrhée parasitaire humaine dans le monde, la connaissance des mécanismes de l'infection par Cryptosporidium est limitée, la codification des méthodes diagnostiques est incomplète et des options thérapeutiques supplémentaires sont requises. En réponse à cette situation, la Septième Conférence Internationale sur Giardia and Cryptosporidium (IGCC 2019) s'est tenue du 23 au 26 juin 2019, à l'Université de Rouen-Normandie, France. Cet événement renommé a rassemblé une délégation internationale de chercheurs pour faire la synthèse des avancées récentes et identifier les principaux thèmes de recherche et les lacunes dans les connaissances. Le programme de cette conférence interdisciplinaire comprenait tous les aspects des relations hôte-parasite, de la recherche fondamentale aux applications à la médecine humaine et vétérinaire, ainsi que les questions environnementales liées aux parasites d'origine hydrique et leurs conséquences épidémiologiques. En ce qui concerne Cryptosporidium et la cryptosporidiose, les principaux domaines de recherche pour lesquels de nouvelles découvertes et les communications les plus impressionnantes ont été présentées et discutées comprenaient : Cryptosporidium dans les eaux environnementales, les fruits de mer et les produits frais ; Épidémiologie animale ; Cryptosporidiose et épidémiologie humaine ; Génomes et évolution génomique englobant : Génomique comparative des Cryptosporidium spp., Perspectives génomiques en biologie, Acquisition et utilisation des séquences du génome, Manipulation génétique ; Interaction hôte-parasite (immunologie, microbiome) ; Diagnostic et traitement. Les présentations de grande qualité discutées à la conférence ont fait état de progrès décisifs et ont permis de cerner de nouvelles possibilités qui inciteront les chercheurs et les organismes de financement à stimuler la recherche future dans une approche « une seule santé ¼ afin d'améliorer les connaissances de base et la gestion clinique et de santé publique de la cryptosporidiose zoonotique.

The Absence of Gut Microbiota Alters the Development of the Apicomplexan Parasite Eimeria tenella.

Coccidiosis is a widespread intestinal disease of poultry caused by a parasite of the genus Eimeria. Eimeria tenella, is one of the most virulent species that specifically colonizes the caeca, an organ which harbors a rich and complex microbiota. Our objective was to study the impact of the intestinal microbiota on parasite infection and development using an original model of germ-free broilers. We observed that germ-free chickens presented significantly much lower load of oocysts in caecal contents than conventional chickens. This decrease in parasite load was measurable in caecal tissue by RT-qPCR at early time points. Histological analysis revealed the presence of much less first (day 2pi) and second generation schizonts (day 3.5pi) in germ-free chickens than conventional chickens. Indeed, at day 3.5pi, second generation schizonts were respectively immature only in germ-free chickens suggesting a lengthening of the asexual phase of the parasite in the absence of microbiota. Accordingly to the consequence of this lengthening, a delay in specific gamete gene expressions, and a reduction of gamete detection by histological analysis in caeca of germ-free chickens were observed. These differences in parasite load might result from an initial reduction of the excystation efficiency of the parasite in the gut of germ-free chickens. However, as bile salts involved in the excystation step led to an even higher excystation efficiency in germ-free compared to conventional chickens, this result could not explain the difference in parasite load. Interestingly, when we shunted the excystation step in vivo by infecting chickens with sporozoites using the cloacal route of inoculation, parasite invasion was similar in germ-free and in conventional chickens but still resulted in significantly lower parasite load in germ-free chickens at day 7pi. Overall, these data highlighted that the absence of intestinal microbiota alters E. tenella replication. Strategies to modulate the microbiota and/or its metabolites could therefore be an alternative approach to limit the negative impact of coccidiosis in poultry.

Multi-species annotation of transcriptome and chromatin structure in domesticated animals.

BACKGROUND: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells). RESULTS: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species. CONCLUSIONS: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.

Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis.

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N-terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co-immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.

Batf3-Dependent Intestinal Dendritic Cells Play a Critical Role in the Control of Cryptosporidium parvum Infection.

Understanding the protective immune response to Cryptosporidium parvum infection is of critical importance to reduce the widespread impact caused by this disease in young individuals. Here, we analyzed the various subsets of CD103+ and CD103- intestinal dendritic cells (DCs) of wild-type and Batf3-/- neonatal mice at homoeostasis and investigated their role during infection. Neonatal Batf3-/- mice had a low CD103+/CD103- DC ratio, resulting in higher susceptibility to the acute phase of the infection and they could not cure the infection. Early during infection, CD103- DCs of Batf3-/- neonates had a lower ability to produce interleukin-12 than their wild-type littermates and lower levels of interferon-gamma mRNA were detected in the infected mucosa. Amplification of CD103+ DCs in Batf3-/- neonates prior to infectious challenge reduced their susceptibility to infection. CD103+ DCs thus outperform CD103- DCs in controlling C. parvum infections and represent a primary target of host-directed immunotherapies dedicated to neonates.

Innate immune responses play a key role in controlling infection of the intestinal epithelium by Cryptosporidium.

Cryptosporidium infection leads to acute diarrhea worldwide. The development of cryptosporidiosis is closely related to the immune status of its host, affecting primarily young ruminants, infants, and immunocompromised individuals. In recent years, several studies have improved our knowledge on the immune mechanisms responsible for the control of the acute phase of the infection and have highlighted the importance of innate immunity. The parasite develops in the apical side of intestinal epithelial cells, giving these cells a central role, as they are both the exclusive host cell for replication of the parasite and participate in the protective immune response. Epithelial cells signal the infection by producing chemokines, attracting immune cells to the infected area. They also actively participate in host defense by inducing apoptosis and releasing antimicrobial peptides, free or incorporated into luminal exosomes, with parasiticidal activity. The parasite has developed several escape mechanisms to slow down these protective mechanisms. Recent development of several three-dimensional culture models and the ability to genetically manipulate Cryptosporidium will greatly help to further investigate host-pathogen interactions and identify virulence factors. Intestinal epithelial cells require the help of immune cells to clear the infection. Intestinal dendritic cells, well known for their ability to induce and orchestrate adaptive immunity, play a key role in controlling the very early steps of Cryptosporidium parvum infection by acting as immunological sentinels and active effectors. However, inflammatory monocytes, which are quickly and massively recruited to the infected mucosa, seem to participate in the loss of epithelial integrity. In addition to new promising chemotherapies, we must consider stimulating the innate immunity of neonates to strengthen their ability to control Cryptosporidium development. The microbiota plays a fundamental role in the development of intestinal immunity and may be considered to be a third actor in host-pathogen interactions. There is an urgent need to reduce the incidence of this yet poorly controlled disease in the populations of developing countries, and decrease economic losses due to infected livestock.

Paneth Cell Defects Induce Microbiota Dysbiosis in Mice and Promote Visceral Hypersensitivity.

BACKGROUND & AIMS: Separation of newborn rats from their mothers induces visceral hypersensitivity and impaired epithelial secretory cell lineages when they are adults. Little is known about the mechanisms by which maternal separation causes visceral hypersensitivity or its relationship with defects in epithelial secretory cell lineages. METHODS: We performed studies with C3H/HeN mice separated from their mothers as newborns and mice genetically engineered (Sox9flox/flox-vil-cre on C57BL/6 background) to have deficiencies in Paneth cells. Paneth cell deficiency was assessed by lysozyme staining of ileum tissues and lysozyme activity in fecal samples. When mice were 50 days old, their abdominal response to colorectal distension was assessed by electromyography. Fecal samples were collected and microbiota were analyzed using Gut Low-Density Array quantitative polymerase chain reaction. RESULTS: Mice with maternal separation developed visceral hypersensitivity and defects in Paneth cells, as reported from rats, compared with mice without maternal separation. Sox9flox/flox-vil-Cre mice also had increased visceral hypersensitivity compared with control littermate Sox9flox/flox mice. Fecal samples from mice with maternal separation and from Sox9flox/flox-vil-cre mice had evidence for intestinal dysbiosis of the microbiota, characterized by expansion of Escherichia coli. Daily gavage of conventional C3H/HeN adult mice with 109 commensal E coli induced visceral hypersensitivity. Conversely, daily oral administration of lysozyme prevented expansion of E coli during maternal separation and visceral hypersensitivity. CONCLUSIONS: Mice with defects in Paneth cells (induced by maternal separation or genetically engineered) have intestinal expansion of E coli leading to visceral hypersensitivity. These findings provide evidence that Paneth cell function and intestinal dysbiosis are involved in visceral sensitivity.

Cryptosporidium parvum increases intestinal permeability through interaction with epithelial cells and IL-1ß and TNFα released by inflammatory monocytes.

Intestinal epithelial cells form a single layer separating the intestinal lumen containing nutriments and microbiota from the underlying sterile tissue and therefore play a key role in maintaining homeostasis. We investigated the factors contributing to the alteration of the epithelial barrier function during Cryptosporidium parvum infection. Infected polarized epithelial cell monolayers exhibit a drop in transepithelial resistance associated with a delocalization of E-cadherin and ß-catenin from their intercellular area of contact, the adherens junction complex. In neonatal mice infected by C. parvum, the increased permeability is correlated with parasite development and with an important recruitment of Ly6c+ inflammatory monocytes to the subepithelial space. TNFα and IL-1ß produced by inflammatory monocytes play a key role in the loss of barrier function. Our findings demonstrate for the first time that both the parasite and inflammatory monocytes contribute to the loss of intestinal barrier function during cryptosporidiosis.

The early intestinal immune response in experimental neonatal ovine cryptosporidiosis is characterized by an increased frequency of perforin expressing NCR1(+) NK cells and by NCR1(-) CD8(+) cell recruitment.

Cryptosporidium parvum, a zoonotic protozoan parasite, causes important losses in neonatal ruminants. Innate immunity plays a key role in controlling the acute phase of this infection. The participation of NCR1+ Natural Killer (NK) cells in the early intestinal innate immune response to the parasite was investigated in neonatal lambs inoculated at birth. The observed increase in the lymphocyte infiltration was further studied by immunohistology and flow cytometry with focus on distribution, density, cellular phenotype related to cytotoxic function and activation status. The frequency of NCR1+ cells did not change with infection, while their absolute number slightly increased in the jejunum and the CD8+/NCR1- T cell density increased markedly. The frequency of perforin+ cells increased significantly with infection in the NCR1+ population (in both NCR1+/CD16+ and NCR1+/CD16- populations) but not in the NCR1-/CD8+ population. The proportion of NCR1+ cells co-expressing CD16+ also increased. The fraction of cells expressing IL2 receptor (CD25), higher in the NCR1+/CD8+ population than among the CD8+/NCR1- cells in jejunal Peyer's patches, remained unchanged during infection. However, contrary to CD8+/NCR1- lymphocytes, the intensity of CD25 expressed by NCR1+ lymphocytes increased in infected lambs. Altogether, the data demonstrating that NK cells are highly activated and possess a high cytotoxic potential very early during infection, concomitant with an up-regulation of the interferon gamma gene in the gut segments, support the hypothesis that they are involved in the innate immune response against C. parvum. The early significant recruitment of CD8+/NCR1- T cells in the small intestine suggests that they could rapidly drive the establishment of the acquired immune response.

CCL20 Displays Antimicrobial Activity Against Cryptosporidium parvum, but Its Expression Is Reduced During Infection in the Intestine of Neonatal Mice.

CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance.