Henri Salmon

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.

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.

Membrane markers of the immune cells in swine: an update

Besides their breeding value, swine are increasingly used as biomedical models. As reported in three international swine clusters of differentiation (CD) workshops and in the animal homologue section of the last workshop for the determination of human leukocyte differentiation antigens (HLDA 8), characterisation of leukocyte surface antigens by monoclonal antibodies and other molecular studies have determined the cell lineages and blood leukocyte subsets implicated in the immune response, including cell adhesion molecules involved in cell trafficking. This review focusses on the current state of knowledge of porcine leukocyte differentiation and major histocompatibility complex (SLA) molecules. Examples of porcine particularities such as the double-positive T lymphocytes with the phenotype CD(4+)CD8(low) and CD(4-)CD8(low) alphabeta T cell subsets and the persistence of SLA class II after T-lymphocyte activation are illustrated, as well as the shared characteristics of the Artiodactyla group, such as the high proportion of gammadelta TcR (T cell receptor) T cells in blood and other lymphoid tissues. Furthermore, discrepancies between swine and humans, such as CD16 expression on dendritic cells and CD11b (wCD11R1) tissue distribution are outlined. The rapidly growing information should facilitate manipulation of the swine immune system towards improving disease control, and open new avenues for biomedical research using the pig as a model.

The mammary gland and neonate mucosal immunity.

The passive mucosal protection of neonate mammals is dependent on the continuous supply until weaning of maternally dimeric IgA (monogastric) and IgG1 (ruminants). This lactogenic (humoral) immunity is linked to the gut, the so-called entero-mammary link, because of the translocation of Ig (IgA and IgG1) or the emigration of IgA lymphoblasts from the gut into the mammary gland (MG); on the other hand, studies on the lymphocyte subsets in the MG of artiodactyls sustained the view of a true local immune response, depending on the MG stage development. Accordingly, the increase of the lactogenic immunity may focus on (1) inductor sites (gut and, possibly, the MG), (2) increase in cell traffic from the gut into the MG, and (3) enhancement at the effector site of the Ig production and excretion in milk. A specific mucosal environment (interleukins and hormones) is responsible for IgM/IgA switch, the induction of mucosal homing receptor onto lymphoblasts and mucosal vascular addressins; very few data are available for the mechanism of lymphoblasts recruitment, either IgA or IgG1, although lactogenic hormones have been implicated in the IgA-blasts homing into the mice MG. After weaning, the neonate is able to mount a gut immune response, but the shortage of the suckling period did not seem to be detrimental for its onset. In soyabean allergy, both piglet and calf exhibited gut villus atrophy, gut accumulation of IgA (swine) and IgG1 (cattle) immunocytes, sustaining the view that a specific environment in ruminant is responsible for both IgA and IgG1 production.

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.

Commensal bacteria and expression of two major intestinal chemokines, TECK/CCL25 and MEC/CCL28, and their receptors.

Background CCL25/TECK and CCL28/MEC are CC chemokines primarily expressed in thymic dendritic cells and mucosal epithelial cells. Their receptors, CCR9 and CCR10, are mainly expressed on T and B lymphocytes. In human, mouse, pig and sheep CCL25 and CCL28 play an important role in the segregation and the compartmentalization of the mucosal immune system. As evidenced by early comparisons of germ-free and conventional animals, the intestinal bacterial microflora has a marked effect on host intestinal immune functions. However, little is known about the impact of bacterial colonization on constitutive and induced chemokine expressions as well as on the generation of anti-inflammatory mechanisms. Methodology/Principal Findings Therefore, we decided to focus by qPCR on the mRNA expression of two main gut chemokines, CCL25 and CCL28, their receptors CCR9 and CCR10, the Tregs marker Foxp3 and anti-inflammatory cytokines TGF-β and IL-10 following colonization with different bacterial species within the small intestine. To accomplish this we used an original germ-free neonatal pig model and monoassociated pigs with a representative Gram-negative (Escherichia coli) or Gram-positive (Lactobacillus fermentum) commensal bacteria commonly isolated from the neonatal pig intestine. Our results show a consistent and marked effect of microbial colonization on the mRNA expression of intestinal chemokines, chemokine receptors, Foxp3 and TGF-β. Moreover, as evidenced by in vitro experiments using two different cell lines, the pattern of regulation of CCL25 and CCL28 expression in the gut appears complex and suggests an additional role for in vivo factors. Conclusions/Significance Taken together, the results highlight the key role of bacterial microflora in the development of a functional intestinal immune system in an elegant and relevant model for human immune system development.

Differential Recruitment of T- and IgA B-lymphocytes in the Developing Mammary Gland in Relation to Homing Receptors and Vascular Addressins

The mammary gland (MG) develops new vasculature and is colonized by lymphocytes, primarily T-cells, during pregnancy. In contrast, during lactation it is colonized primarily by IgA-containing B-cells (c-IgA cells). To explain this difference, we analyzed the spatiotemporal relationships between lymphocytes that expressed peripheral or mucosal homing receptors (HR) and the location of their vascular counterreceptors using quantitative immunohistochemical techniques. We observed that the density of β7+/CD3+ T-cells varied with the amount of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-stained area. Both increased during pregnancy to peak at delivery, decreased rapidly in early lactation to a steady level in mid- and late lactation, and returned to resting values after weaning. Although 60% of these β7 +/CD3+ T-cells scattered in the epithelium co-expressed αEβ7, whereas the remaining 40% in association with blood vessels were α4β7, these results are consistent with a role of MAdCAM-1 in the localization of α4β7 + T-cells. In contrast to T-cells, β7 +/c-IgA+ B plasmablasts (∼ 30% of total c-IgA cells) were located at the alveolar confluence, and their numbers increased in mid- and late lactation when MAdCAM-1 density plateaued. However, both T-and B-cells decreased after weaning. These results show an association between MAdCAM-1 expression level and recruitment of T-cells that does not hold for c-IgA B cells. Furthermore, the recruitment and accumulation of α4β7 + c-IgA cells are reminiscent of locally produced chemoattractants.

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.

Analyses of mAb reactive with porcine CD8

Among all mAb submitted to the first porcine CD workshop, based on FCM analyses six mAb could be identified to recognize the porcine CD8 analogue (workshop Nos. 004, 051, 052, 053, 108 and 109). In immunoprecipitation studies three mAb (Nos. 004, 108 and 109) recognized an antigen with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. The molecular masses of the antigens recognized by the three other mAb (Nos. 051, 052 and 053) are still unknown. Epitope analyses performed by blocking experiments led to the determination of two CD8 epitopes: CD8a and CD8b. CD8a is recognized by mAb Nos. 004, 051 and 052, and CD8b by Nos. 053, 108 and 109.

Humoral defence improvement and haematopoiesis stimulation in sows and offspring by oral supply of shark-liver oil to mothers during gestation and lactation

Shark-liver oil (SLO) contains two bioactive lipids: alkylglycerols and n-3 PUFA. Alkylglycerols have immunostimulating and haematopoietic properties, while n-3 PUFA are essential for optimal neonatal development. We investigated the beneficial effects of dietary supplementation with 32 g SLO/d to twelve pregnant and then lactating sows (from day 80 of pregnancy to weaning) on the growth and immune status of their offspring, compared with a control group. Sows were vaccinated against Aujeszkys disease 21 d before term. Blood samples were collected from sows before treatment, on delivery and 14 d later, and from five piglets per litter on days 2, 21 and 36 after birth; colostrum and milk samples were collected 12 h, 14 and 28 d postpartum. Compared with controls, supplemented sows had higher levels of both erythrocytes and Hb in their blood, and higher concentrations of IgG, alkylglycerols and n-3 PUFA in their mammary secretions. In piglets from supplemented sows, leucocytes and IgG were higher. Supplementation with SLO resulted in an increase in Aujeszky antibodies in both blood and colostrum of sows after vaccination, together with an increase in Aujeszky antibodies in piglet blood. Our findings demonstrate that improvement of both passive and active immune status in piglets is related to the consumption of alkylglycerols associated with n-3 PUFA in the sow diet. The overall improvement in offspring health status by SLO supplementation to the mother could be of interest for optimisation of the lipid diet during and after pregnancy.