Immunomodulating effect of a seaweed extract from Ulva armoricana in pig: Specific IgG and total IgA in colostrum, milk, and blood.

The transfer of passive immunity from sows to piglets can be improved through the administration of immuno-stimulating products before farrowing. This study evaluated the immuno-stimulating effect of an algal sulfated polysaccharide extract (MSP extract) from the green algae Ulva armoricana when administrated orally to sows at the end of gestation. Four diets were tested: Control (no MSP extract), MSP1 (2 g/day of MSP extract), MSP2 (8 g/day), and MSP3 (16 g/day). The experimental diets were provided in two periods: before the last atrophic rhinitis vaccine booster, and a week before farrowing. Anti-Bordetella IgG antibodies were recorded in blood, colostrum, and milk, and total IgA were measured in colostrum and milk. Titer kinetics between the blood sampled before farrowing and colostrum displayed an increase in specific IgG for MSP3. Moreover, the MSP2 diet increased the level of total IgA in milk compared to the control group. Although the immuno-stimulating effect of MSP extract on piglet performance was not concurrent across the different supplementation levels, the present study supports the use of natural algae extract (MSP) as an immunomodulating solution in swine production.

Defining Mononuclear Phagocyte Subset Homology Across Several Distant Warm-Blooded Vertebrates Through Comparative Transcriptomics.

Mononuclear phagocytes are organized in a complex system of ontogenetically and functionally distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic, and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections. We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover, we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey.

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, Peyer's 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.

Development of gut immunoglobulin A production in piglet in response to innate and environmental factors.

The current review focuses on pre- and post-natal development of intestinal immunoglobulin A (IgA) production in pig. IgA production is influenced by intrinsic genetic factors in the foetus as well as extrinsic environmental factors during the post-natal period. At birth, piglets are exposed to new antigens through maternal colostrums/milk as well as exogenous microbiota. This exposure to new antigens is critical for the proper development of the gut mucosal immune system and is characterized mainly by the establishment of IgA response. A second critical period for neonatal intestinal immune system development occurs at weaning time when the gut environment is exposed to new dietary antigens. Neonate needs to establish oral tolerance and in the absence of protective milk need to fight potential new pathogens. To improve knowledge about the immune response in the neonates, it is important to identify intrinsic and extrinsic factors which influence the intestinal immune system development and to elucidate their mechanism of action.

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.

An increase in milk IgA correlates with both pIgR expression and IgA plasma cell accumulation in the lactating mammary gland of PRM/Alf mice.

In mice, during late pregnancy and lactation, maternal precursors of IgA-containing cells (cIgA-cells) are primed in the gut and home to the mammary gland where they secrete IgA. In turn, the ensuing increase in milk IgA mediates immune protection of the newborn gastrointestinal tract. PRM/Alf is an inbred mouse strain which exhibits a substantial post-natal intestinal lengthening which develops throughout the neonatal suckling period, suggesting that the availability of cIg-A cells and the level of protective IgA in milk might also be increased. We confirmed that PRM/Alf milk contains higher amounts of IgA than C57BL/6J throughout lactation, concomitantly with an increase of pIgR on epithelial cells and a higher density of cIgA-cells in the PRM/Alf mammary gland. Furthermore, a search for variations in cellular and humoral factors implicated in regulating cIgA-cell migration towards the mammary gland, including the vascular addressins MAdCAM-1 (mucosal addressin cell adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) as well as the mucosal epithelial chemokine CCL28, did not reveal any quantitative differences in expression between PRM/Alf and C57BL/6J mice strains. Thus our results indicate that these factors are not limiting in the recruitment of cIgA-cells released from the elongated gut of PRM/Alf mice. In the context of intestinal lengthening, these findings strengthen the notion of an entero-mammary gland link, where the neonatal gut is protected by the maternal gut through the immune function of the mammary gland.

ß-Galactomannan and Saccharomyces cerevisiae var. boulardii modulate the immune response against Salmonella enterica serovar Typhimurium in porcine intestinal epithelial and dendritic cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes inflammation, necrosis, and diarrhea in pigs, as well as being an important source of food-borne diseases in humans. Probiotics and prebiotics are promising alternatives to antibiotics to control and prevent intestinal infections. The present work investigated a recently developed ß-galactomannan (ßGM) prebiotic compared to the proven probiotic Saccharomyces cerevisiae var. boulardii on porcine ileum intestinal epithelial cells (IECs) of the IPI-2I line and monocyte-derived dendritic cells (DCs) cocultured in vitro with Salmonella. We observed that both S. cerevisiae var. boulardii and ßGM inhibited the association of Salmonella with IECs in vitro. Our data indicated that ßGM has a higher ability than S. cerevisiae var. boulardii to inhibit Salmonella-induced proinflammatory mRNA (cytokines tumor necrosis factor alpha [TNF-α], interleukin-1α [IL-1α], IL-6, and granulocyte-macrophage colony-stimulating factor [GM-CSF] and chemokines CCL2, CCL20, and CXCL8) and at protein levels (IL-6 and CXCL8). Additionally, ßGM and S. cerevisiae var. boulardii induced some effects on DCs that were not observed on IECs: ßGM and S. cerevisiae var. boulardii showed slight upregulation of mRNA for TNF-α, GM-CSF, and CCR7 receptor on porcine monocyte-derived dendritic cells (DCs). Indeed, the addition of ßGM or S. cerevisiae var. boulardii on DCs cocultured with Salmonella showed higher gene expression (mRNA) for TNF-α, GM-CSF, and CXCL8 compared to that of the control with Salmonella. In conclusion, the addition of ßGM inhibits Salmonella-induced proinflammatory profiles in IECs but may promote DC activation, although associated molecular mechanisms remain to be elucidated.

Effect of Saccharomyces cerevisiae var. Boulardii and ß-galactomannan oligosaccharide on porcine intestinal epithelial and dendritic cells challenged in vitro with Escherichia coli F4 (K88).

Probiotic and prebiotics, often called "immune-enhancing" feed additives, are believed to deal with pathogens, preventing the need of an immune response and reducing tissue damage. In this study, we investigated if a recently developed ß-galactomannan (ßGM) had a similar protective role compared to Saccharomyces cerevisiae var. Boulardii (Scb), a proven probiotic, in the context of enterotoxigenic Escherichia coli (ETEC) infection. ETEC causes inflammation, diarrhea and intestinal damage in piglets, resulting in large economic loses worldwide. We observed that Scb and ßGM products inhibited in vitro adhesion of ETEC on cell surface of porcine intestinal IPI-2I cells. Our data showed that Scb and ßGM decreased the mRNA ETEC-induced gene expression of pro-inflammatory cytokines TNF-α, IL-6, GM-CSF and chemokines CCL2, CCL20 and CXCL8 on intestinal IPI-2I. Furthermore, we investigated the putative immunomodulatory role of Scb and ßGM on porcine monocyte-derived dendritic cells (DCs) per se and under infection conditions. We observed a slight up-regulation of mRNA for TNF-α and CCR7 receptor after co-incubation of DC with Scb and ßGM. However, no differences were found in DC activation upon ETEC infection and Scb or ßGM co-culture. Therefore, our results indicate that, similar to probiotic Scb, prebiotic ßGM may protect intestinal epithelial cells against intestinal pathogens. Finally, although these products may modulate DC activation, their effect under ETEC challenge conditions remains to be elucidated.

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.

Saccharomyces cerevisiae modulates immune gene expressions and inhibits ETEC-mediated ERK1/2 and p38 signaling pathways in intestinal epithelial cells.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. ETEC infections cause pro-inflammatory responses in intestinal epithelial cells and subsequent diarrhea in pigs, leading to reduced growth rate and mortality. Administration of probiotics as feed additives displayed health benefits against intestinal infections. Saccharomyces cerevisiae (Sc) is non-commensal and non-pathogenic yeast used as probiotic in gastrointestinal diseases. However, the immuno-modulatory effects of Sc in differentiated porcine intestinal epithelial cells exposed to ETEC were not investigated. METHODOLOGY/PRINCIPAL FINDINGS: We reported that the yeast Sc (strain CNCM I-3856) modulates transcript and protein expressions involved in inflammation, recruitment and activation of immune cells in differentiated porcine intestinal epithelial IPEC-1 cells. We demonstrated that viable Sc inhibits the ETEC-induced expression of pro-inflammatory transcripts (IL-6, IL-8, CCL20, CXCL2, CXCL10) and proteins (IL-6, IL-8). This inhibition was associated to a decrease of ERK1/2 and p38 MAPK phosphorylation, an agglutination of ETEC by Sc and an increase of the anti-inflammatory PPAR-γ nuclear receptor mRNA level. In addition, Sc up-regulates the mRNA levels of both IL-12p35 and CCL25. However, measurement of transepithelial electrical resistance displayed that Sc failed to maintain the barrier integrity in monolayer exposed to ETEC suggesting that Sc does not inhibit ETEC enterotoxin activity. CONCLUSIONS: Sc (strain CNCM I-3856) displays multiple immuno-modulatory effects at the molecular level in IPEC-1 cells suggesting that Sc may influence intestinal inflammatory reaction.

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.

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.

Expression and immunogenicity of the mycobacterial Ag85B/ESAT-6 antigens produced in transgenic plants by elastin-like peptide fusion strategy.

This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

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.

Differences in transcriptomic profile and IgA repertoire between jejunal and ileal Peyer's patches.

In many species such as sheep and pig, there are two types of Peyer's patches (PP): several discrete patches in the jejunum and a long and continuous patch in the ileum. Most of the immunoglobulin A in the gut is generated by B-cells in the PP germinal centers. Moreover, swine like ovine ileal PP might be important for antigen independent B-cell repertoire diversification. We examined, by quantitative real-time PCR, the expression of 36 transcripts of antimicrobial peptides, chemokines, interleukines, Toll-like receptors and transcription factors from both PP and we highlighted the differences by a principal component analysis. Ileal PP was characterized by a higher mRNA expression of CCL28, IL5, IL10, TLR2 and TLR4 while jejunal PP showed higher mRNA expression of antimicrobial peptides, CCL25, FOXP3, IL4, T-Bet, TSLP and SOCS2. Then, we analyzed some VDJ rearrangements to assess immunoglobulin repertoire diversity in jejunal and ileal PP from weaned piglets. The IgA and IgM repertoires were more diverse in ileal than in jejunal piglet PP. All these results could be related to the rarefaction of interfollicular T-cell zone and the presence in ileal versus jejunal lumen of a more diversified microflora. These findings shed a light on the functional differences between both PP.

Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts.

BACKGROUND: Normal mammary gland contains an extravascular population of B lymphoblasts, precursors of the immunoglobulin plasma cells that play a key role in the passive protection of neonates by secreting immunoglobulins to colostrum and milk. We investigated the presence of chemoattractants in the milk by analysing the chemoattractant activity of various fractions of this secretion. Milk chemoattractants are potentially involved in the recruitment of lymphocytes from the maternal bloodstream in lactating mammary glands. RESULTS: The dilution-related lymphoid cell chemoattraction of whey was associated with a < 10 kDa ultrafiltrate. Active fractions were purified by reverse-phase high performance liquid chromatography. Two peptides of 2.7 kDa (DMREANYKNSDKYFHARGNYDAA) and 1 kDa (RPPGLPDKY) were identified as fragments of the SAA protein family, tentatively identified as SAA2. Only the 2.7 kDa synthetic peptide displayed chemotactic activity, at two different optimal concentrations. At the lower concentration (3.7 nM), it attracted B-cell lymphoblasts, whereas at the higher (3.7 microM), it attracted B lymphocytes. Then, the SAA mRNA expression was analysed and we observed more SAA transcripts during lactation than gestation. CONCLUSION: These data are consistent with the SAA23-45 fragment being involved in preplasma B-cell recruitment to the mammary gland and resultant benefit to the neonate.

Saccharomyces boulardii effects on gastrointestinal diseases.

Health benefits attributed to probiotics have been described for decades. They include the treatment and the prevention of gastrointestinal diseases, vaginal and urinary infections and allergies. Saccharomyces boulardii, a species of yeast widely distributed, has been described as a biotherapeutic agent since several clinical trials displayed its beneficial effects in the prevention and the treatment of intestinal infections and in the maintenance of inflammatory bowel disease. All these diseases are characterized by acute diarrhoea. Administration of the yeast in combination or not with an antibiotherapy has shown to decrease significantly the duration and the frequency of diarrhoea. Experimental studies elucidated partially the molecular mechanisms triggered to improve the host health. The discovery of its anti-inflammatory and immuno-modulatory activities in correlation with the advances in the understanding of mucosal immunology opens a new field of perspectives in S. boulardii therapeutic applications.

Humoral and cellular factors of maternal immunity in swine.

Immunoglobulins cannot cross the placenta in pregnant sows. Neonatal pigs are therefore agammaglobulinemic at birth and, although immunocompetent, they cannot mount rapid immune responses at systemic and mucosal sites. Their survival depends directly on the acquisition of maternal immunity via colostrum and milk. Protection by maternal immunity is mediated by a number of factors, including specific systemic humoral immunity, involving mostly maternal IgG transferred from blood to colostrum and typically absorbed within the first 36 h of life. Passive mucosal immunity involves local humoral immunity, including the production of secretory IgA (sIgA), which is transferred principally via milk until weaning. The mammary gland (MG) produces sIgA, which is, then secreted into the milk via the poly-Ig receptor (pIgR) of epithelial cells. These antibodies are produced in response to intestinal and respiratory antigens, including pathogens and commensal organisms. Protection is also mediated by cellular immunity, which is transferred via maternal cells present in mammary secretions. The mechanisms underlying the various immunological links between MG and the mucosal surfaces involve hormonally regulated addressins and chemokines specific to these compartments. The enhancement of colostrogenic immunity depends on the stimulation of systemic immunity, whereas the enhancement of lactogenic immunity depends on appropriate stimulation at induction sites, an increase in cell trafficking from the gut and upper respiratory tract to the MG and, possibly, enhanced immunoglobulin production at the effector site and secretion in milk. In addition, mammary secretions provide factors other than immunoglobulins that protect the neonate and regulate the development of mucosal immunity--a key element of postnatal adaptation to environmental antigens.

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.

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 x 10(8) 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.