Nelly Bernardet

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.

Oral and intraperitoneal administration of phosphorothioate oligodeoxynucleotides leads to control of Cryptosporidium parvum infection in neonatal mice.

BACKGROUND Neonates are particularly vulnerable to infections, in part because of the incomplete development of their immune system. Recent advances in immunostimulatory treatments based on conserved microbial components led us to assess the potential of oligodeoxynucleotides (ODNs) for decreasing the sensitivity of neonates to Cryptosporidium parvum infection. METHODS Neonate mice were treated orally or intraperitoneally (ip) with CpG ODNs or non-CpG ODNs 24 h before C. parvum infection, and parasite load and cytokine up-regulation were evaluated. RESULTS CpG ODN 1668 and non-CpG ODN 1668 administered orally, as well as CpG ODN 1668 administered ip, induced an 80%-95% decrease in intestinal parasite load 6 days after infection. Intraperitoneal and oral pretreatment with CpG ODN 1668 led to a strong initial up-regulation of cytokines and CD69 messenger RNA in the intestine and a decrease in parasite load by a Toll-like receptor 9 (TLR9)-dependent mechanism. By contrast, oral administration of non-CpG ODN 1668 decreased parasite load by a TLR9-independent mechanism. CONCLUSION The control of neonatal C. parvum infection by ip or oral administration of ODNs is feasible by 2 different mechanisms: (1) the well-known interaction involving CpG/TLR9, leading to the production of cytokines and lymphocyte activation, and (2) a new unknown mechanism that is independent of TLR9 and effective orally.

Neonatal goats display a stronger TH1-type cytokine response to TLR ligands than adults

In mammals, Toll-like receptors play a critical role in initiating innate immune responses and modulating adaptive immunity, by recognizing conserved microbial molecular patterns. This study was undertaken to identify specific features of the responses to synthetic toll-like receptor (TLR) agonists in goats, for the definition of tailored immunostimulation strategies. We show here, in contrast to what has been shown in mice, that mesenteric lymph nodes (MLNs) cells and splenocytes from neonatal goats produce much higher levels of TH1-type cytokines than adults in response to various TLR agonists. IL-12 was identified as a critical cytokine for IFNgamma production by CD8(+) neonatal cells. The higher level of IL-12 production by neonatal MLN and spleen cells than by adult cells was not correlated with a higher level of TLR expression or lower levels of production of the regulatory cytokine IL-10. In neonates, two cell populations-class II(+) CD8(+) and class II(+) CD8(-) cells-produce IL-12 in response to R848 and Poly I:C, respectively. Thus, goat kids have characteristics that could be exploited to favor development of the TH1-type responses critical for the control of intracellular pathogens.

Lipopolysaccharide Heterogeneity in the Atypical Group of Novel Emerging Brucella Species

ABSTRACT Recently, novel Brucella strains with phenotypic characteristics that were atypical for strains belonging to the genus Brucella have been reported. Phenotypically many of these strains were initially misidentified as Ochrobactrum spp. Two novel species have been described so far for these strains, i.e., B. microti and B. inopinata, and other strains genetically related to B. inopinata may constitute other novel species as well. In this study, we analyzed the lipopolysaccharides (LPS) (smooth LPS [S-LPS] and rough LPS [R-LPS]) of these atypical strains using different methods and a panel of monoclonal antibodies (MAbs) directed against several epitopes of the Brucella O-polysaccharide (O-PS) and R-LPS. Among the most striking results, Brucella sp. strain BO2, isolated from a patient with chronic destructive pneumonia, showed a completely distinct S-LPS profile in silver stain gels that looked more similar to that of enterobacterial S-LPS. This strain also failed to react with MAbs against Brucella O-PS epitopes and showed weak reactivity with anti-R-LPS MAbs. B. inopinata reference strain BO1 displayed an M-dominant S-LPS type with some heterogeneity relative to the classical M-dominant Brucella S-LPS type. Australian wild rodent strains belonging also to the B. inopinata group showed a classical A-dominant S-LPS but lacked the O-PS common (C) epitopes, as previously reported for B. suis biovar 2 strains. Interestingly, some strains also failed to react with anti-R-LPS MAbs, such as the B. microti reference strain and B. inopinata BO1, suggesting modifications in the core-lipid A moieties of these strains. These results have several implications for serological typing and serological diagnosis and underline the need for novel tools for detection and correct identification of such novel emerging Brucella spp.

Novel IS711 Chromosomal Location Useful for Identification of Marine Mammal Brucella Genotype ST27, Which Is Associated with Zoonotic Infection

ABSTRACT We report a novel IS711 chromosomal location that is specific for the Brucella genotype ST27 previously associated with Pacific marine mammals and human zoonotic infection in New Zealand and Peru. Our data support the previous observation that this peculiar genotype is distinct from those commonly isolated from the Atlantic and currently classified within the species B. ceti and B. pinnipedialis.

Novel IS711-specific chromosomal locations useful for identification and classification of marine mammal Brucella strains.

ABSTRACT We report five new IS711 chromosomal locations that are specific for marine mammal Brucella groups of strains and useful for their identification and classification. Our data support their current classification into two species, Brucella ceti and B. pinnipedialis, with subgroups in each, but also the possibility of additional species.

Cellular and Molecular Mechanisms Underlying the Strong Neonatal IL-12 Response of Lamb Mesenteric Lymph Node Cells to R-848

Background Comparative studies on the response of neonates and adults to TLR stimulation have been almost exclusively limited to comparisons of human neonatal cord blood cells with peripheral blood from adults, and analyses of spleen cell responses in mice. We need to extend these studies and gain further information regarding such responses at mucosal sites. Methodology/Principal Findings We used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth. In response to the imidazoquinoline compound R-848, neonatal mesenteric lymph node (MLN) and spleen cells produced more IL-12 and, consequently, more IFNγ than their adult counterparts. This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards. Intracellular assays and depletion experiments identified CD14+CD11b+CD40+ cells as the main producer of IL-12. These cells accounted for a greater proportion of neonatal than of adult MLN cells, and also produced, in direct response to R-848, more IL-12 after isolation. This strong IL-12 response in neonates occurred despite the production of larger amounts of the regulatory cytokine IL-10 and the stronger upregulation of SOCS-1 and SOCS-3 mRNA levels than in adult cells, and was correlated with an increase in p38/MAPK phosphorylation. Conclusions/Significance This is the first attempt to decipher the mechanism by which neonatal MLN cells produce more IL-12 than adult cells in response to the TLR8 agonist R-848. CD14+CD11b+CD40+ IL-12-producing cells were more numerous in neonate than in adult MLN cells and displayed higher intracellular responsiveness upon R-848 stimulation. This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.

Mesenteric lymph node cells from neonates present a prominent IL-12 response to CpG oligodeoxynucleotide via an IL-15 feedback loop of amplification

At birth, the immune system is still in development making neonates more susceptible to infections. The recognition of microbial ligands is a key step in the initiation of immune responses. It can be mimicked to stimulate the immune system by the use of synthetic ligands recognising pattern recognition receptors. In human and mouse, it has been found that neonatal cytokine responses to toll-like receptor (TLR) ligands differ in many ways from those of adults but the relevant studies have been limited to cord blood and spleen cells. In this study, we compared the responses in neonate and adult sheep to CpG oligodeoxynucleotides (ODN), a TLR9 ligand, in both a mucosal and a systemic organ. We observed that in response to CpG-ODN more IL-12 was produced by neonatal than adult sheep cells from mesenteric lymph nodes (MLN) and spleen. This higher IL-12 response was limited to the first 20 days after birth for MLN cells but persisted for a longer period for spleen cells. The major IL-12-producing cells were identified as CD14+CD11b+. These cells were poor producers of IL-12 in response to direct stimulation with CpG-ODN and required the cooperation of other MLN cells. The difference in response to CpG-ODN between neonates and adults can be attributed to both a higher proportion of CD14+CD11b+ cells in neonate lambs and their higher capacity to produce IL-15. The IL-15 increases IL-12 production by an amplifying feedback loop involving CD40.

CCR5 is involved in controlling the early stage of Cryptosporidium parvum infection in neonates but is dispensable for parasite elimination

Chemokines play a critical role in immune cell trafficking and the transition from an innate to an acquired immune response. We analyzed host response in neonatal mice deficient in chemokine receptor CCR5 following infection with the intracellular protozoan parasite Cryptosporidium parvum. CCR5 neonatal mice had a higher parasite burden at the early stage of infection but eliminated the parasite as efficiently as their wild-type counterparts. The higher sensitivity of neonates at the beginning of infection was not due to an altered IFNgamma response. An increased CCR2-attracting chemokine response associated with the recruitment of CCR2-positive cells in the infected mucosa may have compensated for the absence of CCR5. A lack of CCR5 thus has an impact in the early stage of C. parvum infection in neonates, but this receptor is dispensable for subsequent parasite elimination.