Sandrine Melo

Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops

Salmonella enterica subspecies enterica serovar Typhimurium, commonly called S. Typhimurium, can cause intestinal infections in humans and various animal species such as swine. To analyze the host response to Salmonella infection in the pig we used an in vivo gut loop model, which allows the analysis of multiple immune responses within the same animal. Four jejunal gut-loops were each inoculated with 3×108 cfu of S. Typhimurium in 3 one-month-old piglets and mRNA expressions of various cytokines, chemokines, transcription factors, antimicrobial peptides, toll like and chemokine receptors were assessed by quantitative real-time PCR in the Peyer’s patch and the gut wall after 24 h. Several genes such as the newly cloned CCRL1/CCX-CKR were assessed for the first time in the pig at the mRNA level. Pro-inflammatory and T-helper type-1 (Th1) cytokine mRNA were expressed at higher levels in infected compared to non-infected control loops. Similarly, some B cell activation genes, NOD2 and toll like receptor 2 and 4 transcripts were more expressed in both tissues while TLR5 mRNA was down-regulated. Interestingly, CCL25 mRNA expression as well as the mRNA expressions of its receptors CCR9 and CCRL1 were decreased both in the Peyer’s patch and gut wall suggesting a potential Salmonella strategy to reduce lymphocyte homing to the intestine. In conclusion, these results provide insight into the porcine innate mucosal immune response to infection with entero-invasive microorganisms such as S. Typhimurium. In the future, this knowledge should help in the development of improved prophylactic and therapeutic approaches against porcine intestinal S. Typhimurium infections.

New insights into the dual recruitment of IgA+ B cells in the developing mammary gland.

In monogastric mammals, transfer of passive immunity via milk and colostrum plays an important role in protecting the neonate against mucosal infections. Here we analyzed the hypothesis that during gestation/lactation IgA+ plasmablasts leave the intestinal and respiratory surfaces towards the mammary gland (MG). We compared the recruitment of lymphocytes expressing homing receptors alpha4beta1 and alpha4beta7 to expression of their vascular counter-receptors, VCAM-1 and MAdCAM-1. Furthermore, the expression of the chemokines responsible for the recruitment of IgA+ plasmablasts was analyzed. Data confirmed that expressions of CCL28 and MAdCAM-1 in the MG increased during pregnancy and alpha4beta1+ and alpha4beta7+/IgA+ cell recruitment in lactation correlated with increase of CCL28 expression. Interestingly, VCAM-1 expression was found in small blood vessels of the lactating porcine MG, while in mice VCAM-1 was expressed in large blood vessels within the MG. Thus, our results indicate that the recruitment of IgA+ plasmablasts to MG is mediated by VCAM-1/alpha4beta1 and MAdCAM-1/alpha4beta7 in conjunction with CCL28/CCR10. They support the existence of a functional link between entero- and upper respiratory surfaces and MG, thereby, conferring protection against aero-digestive pathogens in the newborn.

Saccharomyces cerevisiae decreases inflammatory responses induced by F4+ enterotoxigenic Escherichia coli in porcine intestinal epithelial cells

Probiotic yeasts may provide protection against intestinal inflammation induced by enteric pathogens. In piglets, infection with F4+ enterotoxigenic Escherichia coli (ETEC) leads to inflammation, diarrhea and intestinal damage. In this study, we investigated whether the yeast strains Saccharomyces cerevisiae (Sc, strain CNCM I-3856) and S. cerevisiae variety boulardii (Sb, strain CNCM I-3799) decreased the expression of pro-inflammatory cytokines and chemokines in intestinal epithelial IPI-2I cells cultured with F4+ ETEC. Results showed that viable Sc inhibited the ETEC-induced TNF-α gene expression whereas Sb did not. In contrast, killed Sc failed to inhibit the expression of pro-inflammatory genes. This inhibition was dependent on secreted soluble factors. Sc culture supernatant decreased the TNF-α, IL-1α, IL-6, IL-8, CXCL2 and CCL20 ETEC-induced mRNA. Furthermore, Sc culture supernatant filtrated fraction < 10 kDa displayed the same effects excepted for TNF-α. Thus, our results extended to Sc (strain CNCM I-3856) the inhibitory effects of some probiotic yeast strains onto inflammation.

Epithelial induction of porcine suppressor of cytokine signaling 2 (SOCS2) gene expression in response to Entamoeba histolytica

Suppressor of cytokine signaling (SOCS) proteins are key physiological regulators of both innate and adaptive immunity. These proteins belong to the three major classes of modulators of cytokines signaling. In the following article, we used porcine polarized intestinal cells to study early response to the protozoan, Entamoeba histolytica, and we identified by rapid amplification of cDNA ends (RACE) PCR porcine SOCS1, SOCS4, SOCS5 and SOCS6 encoding sequences. With more than 92% identity predicted porcine SOCS proteins are very similar to their human counterparts. Among SOCS transcripts, only SOCS2 mRNA was significantly induced in epithelial intestinal cells in response to the cytolytic activity of the parasite. The transcriptomic profile obtained after 3h of co-culture of polarized intestinal cells with E. histolytica was clearly oriented toward inflammation and the recruitment of neutrophils. These transcriptomic data have been normalized with accuracy by the utilisation of multiple validated reference genes. The analysis offers a first set of reference genes useful for future studies in porcine intestinal cells. Our data shed light on the understanding of the early response of polarized intestinal cells to E. histolytica and identified a potential involvement of SOCS2 in the parasite regulation of the host response.

Ultra-early weaning in piglets results in low serum IgA concentration and IL17 mRNA expression

In pigs raised for meat production, weaning is a critical period because of related physiological perturbations and negative consequences on performance. Previous studies have shown that early weaning could either impair development of mucosal barrier function or boost intestinal immunologic parameters. In order to obtain further knowledge about the impact of ultra-early weaning on the porcine immune system development, three groups of piglets were weaned at different ages and compared to the unweaned control group. Lower IgA concentrations in ultra-early and early weaned piglets than in other piglets were identified in serum. In the mesenteric lymph node (MLN), significant differences in the mRNA expression of IL17a, TGF beta and FOXP3 were found between specific groups. Indeed, IL17a mRNA was mainly detected in ultra-early weaned piglets while FOXP3 and TGF beta mRNA were associated to both ultra-early weaned and suckling piglets. Reduced serum IgA concentration and MLN induction of a Th17 cytokine in ultra-early weaned piglets could be related to alterations of the mucosal barrier functions consecutive to the milk deprivation. All together, our findings suggest a crucial role for endogenous milk factors onto the onset of IgA synthesis.

Broad early immune response of porcine epithelial jejunal IPI-2I cells to Entamoeba histolytica

Amoebiasis caused by Entamoebahistolytica triggers an acute inflammatory response at early stages of intestinal infection. The patho-physiological study of intestinal amoebiasis requires the development of powerful animal models. Swine provide robust model for human diseases and they could be used to study intestinal amoebiasis. Here, we introduce an in vitro model of swine intestinal epithelial cell (IPI-2I) co-cultured with E. histolytica. Intestinal epithelial cells (IECs) have crucial roles in sensing pathogens and initiating innate immune response, which qualitatively influence adaptive immune response against them. The contact between the two cells induces marked macroscopic lesions of IEC monolayer and striking alteration of the IPI-2I cell phenotype including blebbing, such as loss of attachment before to be phagocyte by the trophozoite. Increase in Lactate Dehydrogenase (LDH) levels in the culture supernatant of IECs was observed when ameba is present and could reflect the cellular cytotoxicity exerted by the parasite. Using quantitative real-time PCR, we identified the up-regulation of cytokines/chemokines implicated in neutrophil chemoattraction and inflammation, such as CCL2, CCL20, CXCL2, CXCL3, GM-CSF, IL1 alpha, IL6 and IL8, in response to the parasite that can further regulate the immunoregulatory functions of the immune cells of the host. The study points a cardinal role of these pro-inflammatory compounds as central mediators in the interaction IECs/ameba and suggests mechanisms by which they coordinate intestinal immune response. This will focus future efforts on delineating the molecular and cellular mechanisms of other cell partners by the way of in vivo infection of swine.

Expression of SOCS1-7 and CIS mRNA in porcine tissues

The Suppressor Of Cytokine Signaling (SOCS) proteins are key physiological regulators of the immune system. Little is known about tissue expression of SOCS and data in pigs are extremely scarce. In order to further study SOCS in pigs, preliminary data must be collected. In the current report, we first identified the three most suitable reference genes in ten porcine tissues. The beta-2-microglobulin (B2MI) reference gene was most often particularly suitable in our conditions. Then, using three reference genes we determined the mRNA expression of SOCS1-7 and CIS in every selected tissue. Constitutive mRNA expression was identified for all the members of the SOCS family in the ten tissues. Interestingly, the constitutive mRNA expression of SOCS1, SOCS3, SOCS7 and CIS was rather heterogeneous between tissues while for SOCS2, SOCS4, SOCS5 and SOCS6 differences of expression were less obvious. Highest CIS and SOCS mRNA expressions were observed in large intestine (SOCS1, SOCS3, SOCS4, SOCS6, and CIS), small intestine (SOCS1, SOCS4, SOCS6, and CIS), spleen (SOCS2, SOCS3, SOCS5, SOCS7, and CIS), trachea (SOCS3) and thymus (SOCS1, SOCS2, SOCS4, SOCS7, and CIS). These data will help for further studies about the role of SOCS proteins in the control of porcine innate and adaptive responses.

Effects of dietary yeast strains on immunoglobulin in colostrum and milk of sows

The ban of antibiotic growth promoters in pig diet required the development of alternative strategies and reinforced the importance of maternal immunity to protect neonates from intestinal disorders. Milk from sows fed active dry yeasts during gestation and lactation exhibited higher immunoglobulin (Ig) and protein content in milk at day 21 of lactation. In this study, we investigated whether the administration of Saccharomyces cerevisiae strains of various origins (Sc01, Sc02, Sb03) to sows during late gestation and lactation could induce higher Ig content in colostrum and milk. Results show that yeast supplementation did not increase significantly sow body weight at days 112 of gestation and 18 of lactation as well as piglet body weight gain from birth to weaning. In contrast, the IgG level in colostrum was increased in comparison with the control group when sows were supplemented with Sc01 at both 0.05 and 0.5% (p<0.05) and Sb03 at 0.5% (p<0.01). During the lactation, the level of milk IgG remained significantly higher in comparison with the control group when sows were supplemented with Sc02 at 0.05% and 0.5% and with Sb03 at 0.5%. Furthermore, in comparison with the control sows, the level of milk IgA was significantly maintained in sows supplemented with the 3 yeast strains at 0.05%. The incidence of piglet diarrhoea was decreased in groups Sc01 at both 0.05% and 0.5% and Sc02 at 0.05%. Thus, these results show that the 3 yeast strains display immunostimulatory effects on maternal immunity, but only Sc01 supplementation at 0.05% allowed jointly the increase of IgG level in colostrum, the maintenance of IgA level in milk and the decrease of piglet diarrhoea incidence. This stimulation of maternal immunity could be associated with a better systemic (colostrum IgG) and local (milk IgA) protection of neonates and suggests that dietary yeasts may have stimulated the local gut immune system of sows.

Towards the establishment of a porcine model to study human amebiasis.

Background Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. Methodology/Principal Findings We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. Conclusions The pig model could help with simultaneously studying intestinal and extraintestinal lesion development.

CCL28 involvement in mucosal tissues protection as a chemokine and as an antibacterial peptide

CCL28 chemokine is expressed by epithelial cells of various mucosal tissues. This chemokine binds to CCR3 and CCR10 receptors and plays an essential role in the IgA antibody secreting cells (IgA-ASC) homing to mucosal surfaces and to lactating mammary gland as well. In addition, CCL28 has been shown to exert a potent antimicrobial activity against both Gram-negative and Gram-positive bacteria and fungi. Using the pig model, we investigated the expression of both CCR10 and CCR3 receptors in a large panel of mucosal tissues. RT-PCR analysis revealed the expression of CCR3 and CCR10 mRNA in salivary glands, nasal mucosae, Peyers patches, small and large intestine, suggesting the presence of leucocytes expressing these receptors within these tissues. CCR10 mRNA was observed in sow mammary gland at late gestation with an increasing level during lactation. Recombinant porcine CCL28 protein was produced and mass spectrometry analysis revealed antimicrobial chemokines features such as a high pI value (10.2) and a C-terminal highly positively-charged region. Using a viable count assay, we showed that CCL28 displayed antimicrobial activity against enteric pathogens and was effective in killing Salmonella serotypes Dublin and Choleraesuis, enteroinvasive Escherichia coli K88 and non-pathogenic E. Coli K12. The potent antimicrobial function of CCL28 combined with its wide distribution in mucosal tissues and secretions suggest that this protein plays an important role in innate immune protection of the epithelial surfaces.