Yves Le Vern

The ERK-dependent signalling is stage-specifically modulated by FSH, during primary Sertoli cell maturation

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent differentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA- and also Src-dependent manner. This activation is required for FSH-induced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic effect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their differentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite effects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells.

Similar pattern of iNOS expression, NO production and cytokine response in genetic and vaccination-acquired resistance to Marek's disease.

NO is produced by macrophages through activation of the inducible enzyme NOS and its production is triggered as an antiviral and antitumoral immune mechanism. Replication of Mareks disease herpes virus (MDV) is inhibited by NO in vitro. MDV induces T-lymphomas in the chicken and a genetic resistance to tumor development has been linked to the B21 major histocompatibility complex. During the first initial week of viral replication after inoculation of the highly virulent RB-1B MDV strain, histocompatible B21/B21 chickens developed strong iNOS expression and NO production capacity in the spleen, in parallel with strong systemic NO production in the serum. Comparable NO response was not seen with the vaccinal strain HVT. In contrast, reduction in spleen macrophage number and delay in iNOS gene expression was observed in genetically susceptible B13/B13 chickens after MDV infection, in addition to suppression of IFN-gamma-inducible NO production. However, vaccination with HVT 3 days before RB-1B inoculation restored strong iNOS gene expression in the spleen 1 week later and inducible NO production 3 weeks later. Following the pattern of iNOS gene expression, early strong expression of cytokines with powerful iNOS-inducing activity such as IFN-gamma and CC chemokines from the MIP family (MIP-1beta, K203) was observed in genetic resistance and resistance acquired after vaccination with HVT. In conclusion, resistance to MDV appeared preferentially linked in both types of resistance to the early establishment of cytokine induction characteristic of a Th1 immune response, thus favoring the development of an early and strong NO response.

Flow cytometry analysis of drug transport mechanisms in Haemonchus contortus susceptible or resistant to anthelmintics

Abstract The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamil-bodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

Intestinal CD103+ dendritic cells are key players in the innate immune control of Cryptosporidium parvum infection in neonatal mice.

Cryptosporidium parvum is a zoonotic protozoan parasite found worldwide, that develops only in the gastrointestinal epithelium and causes profuse diarrhea. Using a mouse model of C. parvum infection, we demonstrated by conditional depletion of CD11c+ cells that these cells are essential for the control of the infection both in neonates and adults. Neonates are highly susceptible to C. parvum but the infection is self-limited, whereas adults are resistant unless immunocompromised. We investigated the contribution of DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in intestinal CD103+ DC during the first weeks of life, before weaning, due to weak production of chemokines by neonatal intestinal epithelial cells (IEC). Increasing the number of intestinal CD103+ DC in neonates by administering FLT3-L significantly reduced susceptibility to the infection. During infections in neonates, the clearance of the parasite was preceded by a rapid recruitment of CD103+ DC mediated by CXCR3-binding chemokines produced by IEC in response to IFNγ. In addition to this key role in CD103+ DC recruitment, IFNγ is known to inhibit intracellular parasite development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFNγ in the lamina propria and the draining lymph nodes. Thus, CD103+DC are key players in the innate immune control of C. parvum infection in the intestinal epithelium. The relative paucity of CD103+ DC in the neonatal intestine contributes to the high susceptibility to intestinal infection.

Differential modulation of β-defensin gene expression by Salmonella Enteritidis in intestinal epithelial cells from resistant and susceptible chicken inbred lines.

beta-Defensins are important components of innate immunity in mucosal tissue, a major entry site for several pathogens. These small cationic peptides possess antimicrobial activity against various microorganisms including Salmonella. Two chicken inbred lines, 6 and 15I, diverge phenotypically with respect to levels of Salmonella Enteritidis intestinal carriage and to level of gene expression of two beta-defensins, AvBD1 and AvBD2. The cellular source of these two defensins in the intestinal tissue has not previously been explored. Therefore embryonic intestinal cells were isolated from both chicken lines. Primary intestinal cell cultures expressed epithelial specific markers (villin and E-cadherin) and differentially expressed two beta-defensin genes AvBD1 and AvBD2 according to chicken line. Furthermore, S. Enteritidis interfered with AvBD2 expression only in the cells from the susceptible line 15I. Our embryonic cell culture model demonstrated that intestinal epithelium express beta-defensin antimicrobial peptides that may play a role in immunoprotection against Salmonella Enteritidis.

Expression of Toll-Like Receptor 4 and Downstream Effectors in Selected Cecal Cell Subpopulations of Chicks Resistant or Susceptible to Salmonella Carrier State

ABSTRACT Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1β (IL-1β), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1β, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

CD40 engagement strongly induces CD25 expression on porcine dendritic cells and polarizes the T cell immune response toward Th1.

Orientation of the immune response toward Th1, Th2, Th17 or Treg plays an important role in self-tolerance and defence against pathogens and tumors. However, this orientation has not been fully characterised in the pig and little is known about the influence of maturation stimulus on the capacity of dendritic cells selectively to direct different types of Th cell responses. Dendritic cell (DC) maturation can be induced by different agents such as inflammatory cytokines, TLR ligands and CD40L. However, the role of the latter in the maturation of pig DC has never been reported. In this study we analysed how different maturation agents influence the capacity of DC to skew the immune response. Monocyte-derived porcine DCs were matured with human CD40L-transfected L-cells, Lipopolysaccharide (LPS) alone or LPS in combination with Tumor necrosis factor-alpha (TNFalpha) and interferon-alpha (IFNalpha). We found that human CD40L induced DC maturation characterised by increased expression of co-stimulatory CD80/86 molecules, high production of IL-12p40 in DC and induction of IFNgamma and t-bet mRNA in T cells, suggesting a Th1 orientation. Moreover we report for the first time the appearance of CD25 after activation of porcine DC. Furthermore, DC activated with TNF+LPS+IFN showed the highest allo-stimulatory capacity of allogeneic lymphocytes and induced IL-17 mRNA in T lymphocytes, suggesting a Th17 orientation that has never been previously reported in the pig. We also showed that immature DCs did not produce any IL-10 or IL-12 and induced both GATA-3 and IL-13 transcription in allogeneic MLR suggesting a Th2 orientation. This study therefore underlines that the nature of the stimulus strongly influences the capacity of DC to steer the immune response in the pig.

Nramp1 Is Not a Major Determinant in the Control of Brucella melitensis Infection in Mice

ABSTRACT Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1s) and C.CB congenic mice (Nramp1r) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by Brucella. However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene appears to be of limited importance for the natural resistance of mice to Brucella.

Enzymatic effect of hypodermin A, a parasite protease, on bovine lymphocyte membrane antigens

The protease hypodermin A (HA) is produced by the parasitic warble‐fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti‐lymphocyte receptor antibodies tested. Anti‐BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti‐MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 μg/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)‐induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

Ovine CD16+/CD14- blood lymphocytes present all the major characteristics of natural killer cells.

Natural killer (NK) cells have a key role in the innate immune response against pathogens because of their cytotoxic properties and production of interferon-gamma (IFN-γ). Some insight into ruminant NK cell biology has been gained through the characterization of bovine NK cells as NKp46+/CD3− cells. However, ovine NK cells have been little studied because of the lack of specific antibodies. Most NK cells in humans and cattle express CD16. We found that an antibody against human CD16 that cross-reacts with bovine NK cells also recognizes cell populations in ovine peripheral blood mononuclear cells. Using double labelling with CD14 revealed the same profile as described in other species, and we identified a putative NK cell population. We therefore sorted this ovine CD16+/CD14− cell population and tested it for NK cell characteristics. More than 80% of sorted CD16+/CD14− cells expressed perforin. After a week of culture in the presence of IL-2 and IL-15, ovine CD16+/CD14− cells had become large cells with intra-cytoplasmic granules containing perforin, and the vast majority displayed an activated CD2−/low/CD25+/CD8+ phenotype, as observed for bovine NKp46+/CD3− cells. Moreover, these cells expressed transcripts for the NKp46 receptor, and were cytotoxic in a CD16-mediated redirected lysis assay against a murine cell line, P815, and in a direct lysis assay against the ovine cell line, IDO5. Finally, ovine CD16+/CD14− cells having expanded for 7 days in culture secreted IFN-γ in response to IL-12 in a dose-dependent manner. Taken together, these findings led us to conclude that the ovine CD16+/CD14− lymphocyte sub-population displays the phenotype and functional characteristics of NK cells.