Search

Recent developments in multiplex technologies enable the determination of a large nu\mber of soluble proteins such as cytokines in various biological samples. More than a one-by-one determination of the concentration of immune mediators, they permit the establishment of secretion profiles for a more accurate description of conditions related to infectious diseases or vaccination. Cytokine profiling has recently been made available for bovine species with the development of a Luminex® technology-based 15-plex assay. Independently from the manufacturer, we evaluated the bovine cytokine/chemokine multiplex assay for limits of detection, recovery rate, and reproducibility. Furthermore, we assessed cytokine secretion in blood samples from 107 cows upon stimulation with heat-killed bacteria and TLR2/4 ligands compared to a null condition. Secretion patterns were analyzed either using the absolute concentration of cytokines or using their relative concentration with respect to the overall secretion level induced by each stimulus. Using Partial Least Square-Discriminant Analysis, we show that the 15-cytokine profile is different under Escherichia coli, Staphylococcus aureus, and Streptococcus uberis conditions, and that IFN-γ, IL-1β, and TNF-α contribute the most to differentiate these conditions. LPS and E. coli induced largely overlapping biological responses, but S. aureus and S. uberis were associated with distinct cytokine profiles than their respective TLR ligands. Finally, results based on adjusted or absolute cytokine levels yielded similar discriminative power, but led to different stimuli-related signatures.

IntroductionConfronted with the emerging threat of antimicrobial resistance, the development of alternative strategies to limit the use of antibiotics or potentiate their effect through synergy with the immune system is urgently needed. Many natural or synthetic biological response modifiers have been investigated in this context. Among them, β-glucans, a type of soluble or insoluble polysaccharide composed of a linear or branched string of glucose molecules produced by various cereals, bacteria, algae, and inferior (yeast) and superior fungi (mushrooms) have garnered interest in the scientific community, with not less than 10,000 publications over the last two decades. Various biological activities of β-glucans have been reported, such as anticancer, antidiabetic and immune-modulating effects. In vitro, yeast β-glucans are known to markedly increase cytokine secretion of monocytes/macrophages during a secondary challenge, a phenomenon called immune training.MethodsHere, we orally delivered β-glucans derived from the yeast S. cerevisiae to mice that were further challenged with Escherichia coli,Resultsβ-glucan supplementation protected the mice from E. coli intraperitoneal and intra-mammary infections, as shown by a lower bacterial burden and greatly diminished tissue damage. Surprisingly, this was not associated with an increased local immune response. In addition, granulocyte recruitment was transient and limited, as well as local cytokine secretion, arguing for faster resolution of the inflammatory response. Furthermore, ex-vivo evaluation of monocytes/macrophages isolated or differentiated from β-glucan-supplemented mice showed these cells to lack a trained response versus those from control mice.ConclusionIn conclusion, dietary β-glucans can improve the outcome of Escherichia coli infections and dampen tissue damages associated to excessive inflammatory response. The mechanisms associated with such protection are not necessarily linked to immune system hyper-activation or immune training.

Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-α starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-α starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatment is beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease., Author summary Type I interferons are major antiviral effectors produced by the host in response to viral infections. Importantly, delayed or impaired type I IFN signalling response has been shown to correlate with severe COVID-19. These observations provided further impetus to test the administration of exogenous type I IFN as a treatment against SARS-CoV-2 infection in patients. However, studies using MERS-CoV or SARS-CoV infected mice demonstrated that type I interferon treatment was beneficial when administered early, but was ineffective and even caused deleterious immunopathology when administered at later stages of infection. It is therefore crucial to understand how the timing of the type I IFN treatments modulates their efficacy and safety against SARS-CoV-2. In this preclinical study using the SARS-CoV-2-infected Syrian hamster model, we showed that intranasal type I IFN treatment was beneficial only when administered before the onset of symptoms. Importantly, late treatment was ineffective but was not associated with deleterious effects. This study provides important information to interpret clinical trials showing no to modest effects of type I IFNs in COVID-19 patients.

The mammary gland is unique in female mammals. Mammary tissue undergoes development and remodeling during lactation, a stage associated with high susceptibility to bacterial infections, inducing an inflammatory condition called mastitis. Although the immune response of the mammary gland has been the subject of intense research to improve prevention and treatment efficacy, the precise definition of its immune composition at this particular physiological stage is still missing. We combined single-cell RNA-Seq, flow cytometry, and three-dimensional confocal microscopy techniques to characterize the immune landscape of lactating murine mammary tissue. Macrophages dominated the immune cell repertoire and could be subdivided into at least two subsets: ductal and stromal macrophages. Ductal macrophages represented approximately 80% of the total CD45pos immune cells and co-expressed F4/80 and CD11c, with high levels of MHC class II molecules. They were strategically poised below the alveolar basal cells in contact with the myoepithelial cell network. Adaptive T and B lymphocytes were remarkably less numerous at this stage, which could explain the limited efficacy of vaccination against mastitis. These results support the view that new strategies to increase mammary immunity and prevent mastitis should be devised.

Type I interferon (IFN-I) plays a major role in antiviral and inflammatory processes of the infected host. In the bovine industry, the bovine respiratory disease complex is a major cause of economic and health problems. This disease is caused by interactions of pathogens, together with environmental and host factors. Several pathogens have been identified as causal agents of respiratory diseases in cattle. To better understand how primary infections by viruses predispose animals to further infections by pathogenic bacteria, tools to accurately detect antiviral and immunoregulatory cytokines are needed. To facilitate the detection and quantification of bovine IFN-I, we have established a new specific and sensitive bioassay studies in the bovine host. This assay is based on a Madin-Darby Bovine Kidney (MDBK) cell line that carries a luciferase gene under the control of the IFN-I inducible bovine Mx1 promoter. Specific luciferase activity was measured after stimulation with serial dilutions of recombinant bovine alpha and beta IFNs and human IFN-α. With this novel bioassay we have successfully measured IFN-I production in supernatant from MDBK cells after stimulation of Toll-like receptors (TLR3, TLR7 and TLR8) and RIG-I-like receptors (RIG-I and MDA5), after viral infection with bovine respiratory pathogens, but also in samples from infected calves. Finally, this new bioassay is an easy-to-use and low cost tool to measure the production of bovine Type-I Interferon.

Mastitis is a major problem in dairy farming. Vaccine prevention of mammary bacterial infections is of particular interest in helping to deal with this issue, all the more so as antibacterial drug inputs in dairy farms must be reduced. Unfortunately, the effectiveness of current vaccines is not satisfactory. In this review, we examine the possible reasons for the current shortcomings of mastitis vaccines. Some reasons stem from the peculiarities of the mammary gland immunobiology, others from the pathogens adapted to the mammary gland niche. Infection does not induce sterilizing protection, and recurrence is common. Efficacious vaccines will have to elicit immune mechanisms different from and more effective than those induced by infection. We propose focusing our research on a few points pertaining to either the current immune knowledge or vaccinology approaches to get out of the current deadlock. A possible solution is to focus on the contribution of cell-mediated immunity to udder protection based on the interactions of T cells with the mammary epithelium. On the vaccinology side, studies on the orientation of the immune response by adjuvants, the route of vaccine administration and the delivery systems are among the keys to success.

Type 3 immunity encompasses innate and adaptive immune responses mediated by cells that produce the signature cytokines IL-17A and IL-17F. This class of effector immunity is particularly adept at controlling infections by pyogenic extracellular bacteria at epithelial barriers. Since mastitis results from infections by bacteria such as streptococci, staphylococci and coliform bacteria that cause neutrophilic inflammation, type 3 immunity can be expected to be mobilized at the mammary gland. In effect, the main defenses of this organ are provided by epithelial cells and neutrophils, which are the main terminal effectors of type 3 immunity. In addition to theoretical grounds, there is observational and experimental evidence that supports a role for type 3 immunity in the mammary gland, such as the production of IL-17A, IL-17F, and IL-22 in milk and mammary tissue during infection, although their respective sources remain to be fully identified. Moreover, mouse mastitis models have shown a positive effect of IL-17A on the course of mastitis. A lot remains to be uncovered before we can safely harness type 3 immunity to reinforce mammary gland defenses through innate immune training or vaccination. However, this is a promising way to find new means of improving mammary gland defenses against infection.

International audience; The objective was to assess effects of experimentally induced undernutrition on responses to an intramammary lipopolysaccharide (LPS) challenge in early-lactation cows. Starting at 24 +/- 3 d in milk, multiparous Holstein cows either received a ration containing 48% straw for 96 h to restrict nutrient intake (REST, n = 8) or were allowed ad libitum intake of a lactation diet (CONT, n = 9). After 72 h on diet or after an equivalent period for CONT, 50 mu g of LPS (Escherichia coli 0111:B4) was injected into one healthy rear mammary quarter to induce an acute inflammation response. Blood samples were collected weekly until 7 wk of lactation, daily during feed restriction (or control), before and at 1, 2, 4, 6, 10, and 24 h relative to LPS injection. Foremilk quarter samples were collected before and at 4, 6, 10, and 24 h after LPS injection. Dry matter intake, milk yield, energy balance, plasma glucose, nonesterifled fatty acids (NEFA), and beta-hydroxybutyrate (BHB) concentrations did not differ between CONT and REST immediately before nutrient restriction in REST (least squares means at d -1 were 21.8, 39.0 kg/d, -2.5 MJ/d, and 3.78, 0.415, 0.66 mM, respectively) but were significantly altered at 72 h of nutrient restriction (9.8, 28.3 kg/d, -81.6 MJ/d, and 2.77, 1.672, and 2.98 mM, respectively), when the LPS challenge was performed. The rectal temperature increment from baseline values in response to LPS did not differ, but cortisol increment was greater and cortisol response area under the curve (AUC) tended to be greater [202 vs. 122 (ng/mL) x 10 h] for REST than CONT. No treatment differences were observed in foremilk IL-8, IL-1 beta, tumor necrosis factor-alpha, and chemokine (C-X-C motif) ligand 3 concentrations in response to LPS injection. Composite milk somatic cell count per milliliter (6.919 x 10(6) vs. 1.956 x 10(6) cells/mL) and total number of somatic cells secreted in milk per day were greater for REST than CONT during the day following LPS. Plasma glucose, urea, and insulin concentrations increased after the LPS challenge, suggesting establishment of insulin resistance and modifications of glucose metabolism to support acute inflammation in both CONT and REST. Nonetheless, nutrient-restricted cows had delayed plasma insulin and glucose responses to LPS, smaller insulin AUC but greater glucose AUC compared with CONT, despite the limited nutrient availability to sustain an inflammation response. Undernutrition altered peripheral metabolic responses to an intramammary LPS challenge but had limited effects on selected indicators of inflammation response in early-lactation cows.

International audience; An enduring controversy exists about low milk cell counts and susceptibility to mastitis. The concentration of milk leukocytes, or somatic cell count (SCC), is a well-established direct indicator of mammary gland inflammation that is highly correlated with the presence of a mammary infection. The SCC is also used as a trait for the selection of dairy ruminants less prone to mastitis. As selection programs favor animals with less SCC, and as milk cells contribute to the defense of the mammary gland, the idea that susceptibility to mastitis could possibly be increased in the long term has been put forward and is still widely debated. Epidemiological and experimental studies aimed at relating SCC to susceptibility to mastitis have yielded results that seem contradictory at first sight. Nevertheless, by taking into account the immunobiology of milk and mammary tissue cells and their role in the defense against infection, along with recent studies on SCC-based divergent selection of animals, the issue can be settled. Apparent SCC-linked susceptibility to mastitis is a phenotypic trait that may be linked to immunomodulation but not to selection.

International audience; The urge to reduce antimicrobials use in dairy farming has prompted a search for alternative solutions. As infections of the mammary gland is a major reason for antibiotic administration to dairy ruminants, mammary probiotics have recently been presented as a possible alternative for the treatment of mastitis. To assess the validity of this proposal, we performed a general appraisal of the knowledge related to probiotics for mammary health by examining their potential modes of action and assessing the compatibility of these mechanisms with the immunobiology of mammary gland infections. Then we analyzed the literature published on the subject, taking into account the preliminary in vitro experiments and the in vivo trials. Preliminary experiments aimed essentially at exploring in vitro the capacity of putative probiotics, mainly lactic acid bacteria (LABs), to interfere with mastitis-associated bacteria or to interact with mammary epithelial cells. A few studies used LABs selected on the basis of bacteriocin production or the capacity to adhere to epithelial cells to perform in vivo experiments. Intramammary infusion of LABs showed that LABs are pro-inflammatory for the mammary gland, inducing an intense influx of neutrophils into milk during lactation and at drying-off. Yet, their capacity to cure mastitis remains to be established. A few preliminary studies tackle the possibility of using probiotics to interfere with the teat apex microbiota or to prevent the colonization of the teat canal by pathogenic bacteria. From the analysis of the published literature, it appears that currently there is no sound scientific foundation for the use of probiotics to prevent or treat mastitis. We conclude that the prospects for oral probiotics are not promising for ruminants, those for intramammary probiotics should be considered with caution, but that teat apex probiotics deserve further research.

The relationship between Staphylococcus aureus and innate immunity is highly complex and requires further investigation to be deciphered. i.p. challenge of C57BL/6 and DBA/2 mice, resistant and susceptible to the infection, respectively, resulted in different patterns of cytokine production and neutrophil recruitment. Staphylococcus aureus infection induced macrophage pyroptosis, an inflammasome-dependent cell death program, whose rates significantly differed between C57BL/6 and DBA/2 mice. Fast rate pyroptosis of C57BL/6 macrophages released high levels of IL-1β but limited the synthesis of other cytokines such as TNF-α, IL-6, CXCL1, and CXCL2. Conversely, the extended survival of DBA/2 macrophages allowed substantial production of these NF-κB-related cytokines. Phenotyping of resting macrophages in different mouse strains revealed differential predisposition toward specific macrophage phenotypes that modulate S. aureus-mediated inflammasome activation. Treatment of DBA/2 susceptible mice with inflammasome inducers (i.e. nigericin and ATP) artificially increased pyroptosis and lowered the levels of NF-κB-related inflammatory cytokines, but restored IL-1β to levels similar to those in C57BL/6 mice. Collectively, this study promotes the concept that, in association with host genetics, the basal phenotype of resident macrophages influences the early inflammatory response and possibly participates in S. aureus infection outcome via the inflammasome pathway and subsequent pyroptosis.

β-glucans are glucose polymers present in the fungal cell wall and, as such, are recognized by innate immune cells as microbial-associated pattern through Dectin-1 receptor. Recent studies have highlighted the ability of the pathogenic yeast Candida albicans or its cell wall-derived β(1,3) (1,6)-glucans to increase human monocytes cytokine secretion upon secondary stimulation, a phenomenon now referred as immune training. This ability of monocytes programming confers β-glucans an undeniable immunotherapeutic potential. Our objective was to determine whether β-glucans from S. cerevisiae, a non-pathogenic yeast, are endowed with such a property. For this purpose we have developed a short-term training model based on LPS re-stimulation of mouse bone marrow-derived macrophages primed with S. cerevisiae β-glucans. Through a transcriptome analysis, we demonstrated that β-glucans induced a specific gene expression signature involving the PI3K/AKT signaling pathway as in human monocytes. Moreover, we showed that over-expression of Csf2 (that encodes for GM-CSF) was a Dectin-1-dependent feature of β-glucans-induced priming of macrophages. Further experiments confirmed that GM-CSF up-regulated Dectin-1 cell surface expression and amplified macrophages response along β-glucans-mediated training. However, the blockade of GM-CSFR demonstrated that GM-CSF was not primarily required for β-glucans-induced training of macrophages although it can substantially improve it. In addition, we found that mouse macrophages trained with β-glucans up-regulated their expression of the four and a half LIM-only protein 2 (Fhl2) in a Dectin-1-dependent manner. Consistently, we observed that intracellular levels of FHL2 increased after stimulation of macrophages with β-glucans. In conclusion, our experiments provide new insights on GM-CSF contribution to the training of cells from the monocytic lineage and highlights FHL2 as a possible regulator of β-glucans associated signaling.

International audience; Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.

Fetal/neonatal alloimmune thrombocytopenia is a frequent disease in humans where alloantibodies against platelet Ags lead to platelet destruction and hemorrhage. Although a role in the disease for Abs against MHC has been suspected, this has not been formally demonstrated. Since 2007, a hemorrhagic syndrome due to thrombocytopenia and designated as bovine neonatal pancytopenia (BNP) has been recognized in calves in several European countries. An inactivated antiviral vaccine is strongly suspected to be involved in this syndrome because of its highly frequent use in the dams of affected calves. In this study, we show that BNP is an alloimmune disease, as we reproduced the signs by transferring serum Abs from vaccinated BNP dams into healthy neonatal calves. Ab specificity was strongly associated with the presence of allogeneic MHC class I Abs in the dams. MHC class I staining was also observed on Madin–Darby bovine kidney cells, a cell line related to the one used to produce the vaccine Ag. Our report emphatically demonstrates that alloimmunization against MHC class I is associated with a substantial risk of developing cytopenia-associated syndromes in neonates when a cell line of the same species is used to produce an inactivated vaccine injected into the mother.

Mononuclear phagocytes are organized in a complex system of ontogenically 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, indicating conservation of the genetic support for mononuclear phagocyte organization throughout jawed vertebrates but likely not in agnathans. Altogether this work provides molecular clues to the definition and functions of mononuclear phagocyte subsets across vertebrates which shall be useful to rigorously identify these cells and to design universal strategies to manipulate them in many target species towards the goal to reach and maintain global health.

Remerciements au LEGTA de Vendôme, Loir-et-Cher; Intramammary infusion of the antigen used to sensitize cows by the systemic route induces a local inflammation associated with neutrophil recruitment. We hypothesize that this form of delayed type hypersensitivity, which may occur naturally during infections or could be induced intentionally by vaccination, can impact the outcome of mammary gland infections. We immunized cows with ovalbumin to identify immunological correlates of antigen-specific mammary inflammation. Intraluminal injection of ovalbumin induced a mastitis characterized by a prompt tissue reaction (increase in teat wall thickness) and an intense influx of leukocytes into milk of 10 responder cows out of 14 immunized animals. The magnitude of the local inflammatory reaction, assessed through milk leukocytosis, correlated with antibody titers, skin thickness test, and production of IL-17A and IFN-γ in a whole-blood antigen stimulation assay (WBA). The production of these two cytokines significantly correlated with the magnitude of the milk leukocytosis following the ovalbumin intramammary challenge. The IL-17A and IFN-γ production in the WBA was dependent on the presence of CD4+ cells in blood samples. In vitro stimulation of peripheral blood lymphocytes with ovalbumin followed by stimulation with PMA/ionomycin allowed the identification by flow cytometry of CD4+ T cells producing either IL-17A, IFN-γ, or both cytokines. The results indicate that the antigen-specific WBA, and specifically IL-17A and IFN-γ production by circulating CD4+ cells, can be used as a predictor of mammary hypersensitivity to protein antigens. This prompts further studies aiming at determining how Th17 and/or Th1 lymphocytes modulate the immune response of the mammary gland to infection.

Classical studies have demonstrated that in vitro priming of naive CD4 T cells to become T helper (Th)2 cells is strikingly dependent on interleukin (IL)-4, whereas priming for interferon (IFN)gamma production is IL-12/IFNgamma-dependent. Therefore, it was quite surprising when we noted that priming of naive C57BL/6 CD4(+) cells to become IL-4 producers was substantially inhibited by the addition of anti-IFNgamma antibodies. This was true using immobilized anti-CD3 and anti-CD28 antibodies or soluble anti-CD3/anti-CD28 and antigen-presenting cells in the presence or absence of added IL-4. Priming of CD4 T cells from IFNgamma(-/-) C57BL/6 mice with immobilized anti-CD3 and anti-CD28 resulted in limited production of IL-4, even with the addition of 1,000 U/ml of IL-4. Titrating IFNgamma into such cultures showed a striking increase in the proportion of T cells that secreted IL-4 upon challenge; this effect was completely IL-4-dependent in that it was blocked with anti-IL-4 antibody. Thus, IFNgamma plays an unanticipated but substantial role in Th2 priming, although it is an important Th1 cytokine, and under certain circumstances a Th1 inducer.

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vβ repertoire, different from the TCR Vβ profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IAd-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.

International audience; Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R-0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R-0 and SG33-VP7 stimulated an antigen-specific CD4(+) response and Cav-VP7 R-0 stimulated substantial proliferation of antigen-specific CD8(+) lymphocytes. Encouraged by the results obtained with the Cav-VP7 R-0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R-0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.

Th1 and Th2 cells produce different cytokines and have distinct functions. Th1/Th2 cell differentiation is influenced, among other factors, by the nature of TCR-MHC interactions. However, how the TCR transduces a signal resulting in IFN-gamma or IL-4 production is a matter of debate. For example, some authors reported a loss of calcium signaling pathway in Th2 cells. We used a T cell hybridoma producing IL-4 upon weak TCR stimulation and both IL-4 and IFN-gamma for strong TCR engagement as a model to study how TCR signaling pathways are differentially activated in both conditions of stimulation and how this influences the production of cytokines. We show that: (1) the calcium response is identical following weak and strong TCR stimulation; (2) mitogen-activated protein kinase(MAPK) activation is a gradual phenomenon depending upon the strength of TCR activation; (3) a calcium response, even weak, triggers IL-4 expression; (4) IFN-gamma synthesis requires not only a calcium response but also MAPK activation. The MAPK pathway is dispensable for IL-4 production, although it amplifies IL-4 synthesis upon strong TCR stimulation; (5) TCR-induced IL-4 production also depends on calcium signaling in Th2 cells, while IFN-gamma synthesis is dependent, in addition, on MAPK activation in Th1 cells.

The mechanisms that influence the polarization of CD4 T cells specific for allogeneic MHC class II molecules in vivo are still poorly understood. We have examined the pathway of alloreactive CD4 T cell differentiation in a situation in which only CD4 T cells could be activated in vivo. In this report we show that priming of adult mice with allogeneic APC, in the absence of MHC class I-T cell interactions, induces a strong expansion of type 2 cytokine-producing allohelper T cells. These alloantigen-specific CD4 T cells directly recognize native allogeneic MHC class II molecules on APC and secrete, in addition to the prototypic Th2 cytokines IL-4, IL-5, and IL-10, large amounts of TGF-β. The default Th2-phenotype acquisition is not genetically controlled and occurred both in BALB/c and C57BL/6 mice. CD8 T cells are the principal cell type that controls CD4 T cell differentiation in vivo. Furthermore, we demonstrate that strong Th2 priming can be induced not only with allogeneic splenocytes but also with a low number of bone marrow-derived dendritic cells. Finally, using a passive transfer system, we provide direct evidence that CD8 T cell expansion in situ promotes alloreactive Th1 cell development principally by preventing their default development to the Th2 pathway in a mechanism that is largely IFN-γ independent. Therefore, this work demonstrates that type 2 cytokine production represents a dominant pathway of alloreactive CD4 T cell differentiation in adult mice, a phenomenon that was initially thought to occur only during the neonatal period.

We have analyzed the requirement for β2-microglobulin (β2m)-dependent T cells in the generation of allogeneic Th2 responses in vivo. A neonatal injection of semiallogeneic cells in BALB/c mice induces a state of chimerism that promotes the differentiation of donor-specific CD4+ T cells toward the Th2 phenotype. Polyclonal T-B cell interactions occur in this model between host Th2 and donor B cells, resulting in the production of IgE Abs. IgE production and Th2-priming are critically dependent upon the early production of IL-4. Our data in the present paper demonstrate that: 1) IgE synthesis and the up-regulation of MHC class II and CD23 molecules on B cells are independent of β2m expression in the host, 2) no difference in the induction of CD4 alloreactive Th2 cells could be observed between β2m−/− and their wild-type control littermates when Th2-priming was measured in adult mice, and 3) the Th2 response and IgE production is induced in the complete absence of β2m-dependent T cells both in the host and in the inoculum. Therefore, using a variety of assays, we could not demonstrate diminished responses in mice with a disrupted β2m gene in this model of Th2-mediated allogeneic interaction, indicating that β2m-dependent NK1.1+ and CD8+ T cells are not required for the generation of alloreactive Th2 responses in vivo.

International audience; Within lymphopenic recipients, naïve T cells undergo proliferation that is induced by homeostatic mechanisms. Earlier studies have demonstrated that commensal antigens play a key role in inducing the proliferation. However, a relative contribution of endogenous self antigens in this process has not been formally investigated. In this study, we utilized a pharmacologic inhibitor that blocks T cell egress from the lymphoid tissues, antibiotics, and germ-free animals to examine the role of commensal and self antigens. The results suggest that T cell proliferation under lymphopenic conditions is a heterogeneous process triggered by both exogenous commensal and endogenous self antigens.

Recombinant myxoma virus (MYXV) can be produced without a loss of infectivity, and its highly specific host range makes it an ideal vaccine vector candidate, although careful examination of its interaction with the immune system is necessary. Similar to rabbit bone marrow-derived dendritic cells (BM-DCs), ovine dendritic cells can be infected by SG33, a MYXV vaccine strain, and support recombinant antigen expression. The frequency of infected cells in the nonhost was lower and the virus cycle was abortive in these cell types. Among BM-DC subpopulations, Langerhans cell-like DCs were preferentially infected at low multiplicities of infection. Interestingly, ovine BM-DCs remained susceptible to MYXV after maturation, although apoptosis occurred shortly after infection as a function of the virus titer. When gene expression was assessed in infected BM-DC cultures, type I interferon (IFN)-related and inflammatory genes were strongly upregulated. DC gene expression profiles were compared with the profiles produced by other poxviruses in interaction with DCs, but very few commonalities were found, although genes that were previously shown to predict vaccine efficacy were present. Collectively, these data support the idea that MYXV permits efficient priming of adaptive immune responses and should be considered a promising vaccine vector along with other poxviruses.

International audience; Interleukin-17A (IL-17A) and IL-17F have been shown to mediate a crucial crosstalk between the immune system and various epithelial tissues, stimulating various defensive mechanisms to bacterial infections. A number of studies have characterized the response to IL-17A and IL-17F of epithelial cells from airways, intestine, and skin, but not from the mammary gland. To evaluate the potential contribution of IL-17 to the immune defense of the mammary gland, we analyzed the effects of recombinant bovine IL-17A and IL-17F on primary bovine mammary epithelial cells (MEC) by quantitative PCR and ELISA. We found expression (mRNA) of the two components of the IL-17 receptor complex, IL-17RA and IL-17RC, in mammary tissue and MEC in vitro. The expression of a number of genes encoding cytokines, chemokines and proteins endowed with antibacterial activities was increased by IL-17A, and to a lesser extent by IL-17F, but the magnitude of responses was modest. As expected, responses were augmented by the combination of IL-17A or IL-17F with TNF-a. Interestingly, responses of a few of the tested genes, such as IL8, CCL20, iNOS, and CfB, were augmented by the combination of IL-17A with staphylococcal lipoteichoic acid or muramyl dipeptide, bacterial agonists of the innate immune system. This can be interpreted as indicating that IL-17A and IL-17F are tailored to exert their full potential in a septic environment. MEC responses were characterized by the expression of chemokines targeting not only neutrophils (CXCL3 and CXCL8) but also mononuclear leucocytes (CCL2, CCL20). Production of IL-6 was low and the inflammatory cytokines TNF-a and IL-1b were expressed (mRNA) but proteins were not secreted. Altogether, our results suggest that IL-17A and IL-17F have a potential to modulate the mammary gland immune response to mastitis-causing pathogens.

Background The existence of a genetic basis for host responses to bacterial intramammary infections has been widely documented, but the underlying mechanisms and the genes are still largely unknown. Previously, two divergent lines of sheep selected for high/low milk somatic cell scores have been shown to be respectively susceptible and resistant to intramammary infections by Staphylococcus spp. Transcriptional profiling with an 15K ovine-specific microarray of the milk somatic cells of susceptible and resistant sheep infected successively by S. epidermidis and S. aureus was performed in order to enhance our understanding of the molecular and cellular events associated with mastitis resistance. Results The bacteriological titre was lower in the resistant than in the susceptible animals in the 48 hours following inoculation, although milk somatic cell concentration was similar. Gene expression was analysed in milk somatic cells, mainly represented by neutrophils, collected 12 hours post-challenge. A high number of differentially expressed genes between the two challenges indicated that more T cells are recruited upon inoculation by S. aureus than S. epidermidis. A total of 52 genes were significantly differentially expressed between the resistant and susceptible animals. Further Gene Ontology analysis indicated that differentially expressed genes were associated with immune and inflammatory responses, leukocyte adhesion, cell migration, and signal transduction. Close biological relationships could be established between most genes using gene network analysis. Furthermore, gene expression suggests that the cell turn-over, as a consequence of apoptosis/granulopoiesis, may be enhanced in the resistant line when compared to the susceptible line. Conclusions Gene profiling in resistant and susceptible lines has provided good candidates for mapping the biological pathways and genes underlying genetically determined resistance and susceptibility towards Staphylococcus infections, and opens new fields for further investigation.

Background Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. Results Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1. The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. Conclusions This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11c-positive cells were generated after 6-7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class II molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b(int/hi) cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b(dull) cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines.

T cell numbers are maintained within narrow ranges in vivo . Introduction of naïve cells into lymphopenic environments results in proliferation and differentiation driven by the recognition of peptide/MHC complexes and by cytokine signaling. This process, often described as homeostatic proliferation, is here referred to as spontaneous proliferation. We show that, although the presence of memory CD4 T cells of broad repertoire efficiently inhibits proliferation/differentiation of naïve CD4 T cells, a memory population of similar size comprised of cells with a repertoire of limited diversity fails to do so, implying that cells of a given specificity prevent responses of cells of the same or related specificity. This finding suggests that the immune system has evolved mechanisms to attain a memory cell repertoire of great diversity independently of foreign antigens.

It is widely accepted that females have superior immune responses than males, but the ways by which sex hormones may enhance T cell responses are still poorly understood. In the present study, we analyzed the effect of estrogens on CD4 T cell activation and differentiation after immunization with exogenous antigens. We show that administration of low doses of 17beta-estradiol (E2) to castrated female mice results in a striking increase of antigen-specific CD4 T cell responses and in the selective development of IFN-gamma-producing cells. Quantitative assessment of the frequency of T cells bearing a public TCR beta chain CDR3 motif demonstrated that the clonal size of primary antigen-specific CD4 T cells was dramatically increased in immune lymph nodes from E2-treated mice. By using mice with disrupted estrogen receptor (ER) alpha or beta genes, we show that ERalpha, but not ERbeta, was necessary for the enhanced E2-driven Th1 cell responsiveness. Furthermore, ERalpha expression in hematopoietic cells was essential, since E2 effects on Th1 responses were only observed in mice reconstituted with bone marrow cells from ERalpha+/+, but not ERalpha-deficient mice. These results demonstrate that estrogen administration promotes strong antigen-specific Th1 cell responses in a mechanism that requires functional expression of ERalpha in hematopoietic cells.

The interactions between CD28 and its ligand CD86 are critical for the regulation of T cell responses. However, it is not clear whether CD4+ T cells expressing low and high avidity TCR are equally dependent on CD28 costimulation for their activation and expansion. To address this issue, we have used multimers of I-Ad molecules linked to a peptide derived from the Leishmania major homolog for the receptor of activated C kinase (LACK) antigen to compare the fate of LACK-specific CD4+ T cells in Leishmania-infected BALB/c mice which have been treated or not with anti-CD86 mAb. Although the administration of anti-CD86 mAb did not completely prevent the expansion of LACK-specific T cells, their frequency and number were markedly reduced. In mice treated with anti-CD86 mAb as well as in control animals, L. major induced the clonal expansion of LACK-specific T cells which expressed a canonical low avidity Valpha8/Vbeta4 TCR. Taken together, our results suggest that the molecular interactions between CD28 on T cells and CD86 on APC serve to amplify and modulate T cell responses without promoting breadth in the TCR repertoire.

The T cell receptor (TCR) alpha and beta chains are encoded by a series of stochastic rearrangements between variable (V), diversity (D) for TCR beta chain only, and joining (J) gene segments, creating hypervariable complementarity-determining region 3 (CDR3) regions that contact the peptide/MHC complex and confer specificity. In the present paper, we applied the recently developed real-time quantitative RT-PCR technique to the detection of rearranged TCR beta chain mRNA transcripts. We designed BV- and BJ-specific primers together with TaqMan probes specific for the CDR3 regions of the clones of interest. As an external reference, we used plasmids containing the entire TCR beta chains, making it possible to normalize the number of specific rearranged BV-J mRNA copies among the total number of TCR beta chains. Here, we present data validating this fluorogenic PCR-based method for the quantification of several TCR clonotypes characteristic of the CD4 T cell response to hen egg white lysozyme (HEL) in mice of the H-2d haplotype. This accurate and sensitive procedure permits the precise determination of T cell clone frequencies ranging from 10(-2) to less than 10(-5) in normal biological samples; it may provide an alternative approach when frequencies are too low to be assessed by flow cytometry.

Using multimers of MHC class II molecules linked to a peptide derived from the Leishmania LACK antigen, we have compared the fate of parasite-specific CD4+ T cells in resistant and susceptible mice transgenic for the β chain of a LACK-specific TCR. Activated T cells were readily detected in the draining lymph nodes of infected animals. Although the kinetics of activation and expansion were similar in both strains, T cells from susceptible and resistant mice expressed low- and high-affinity TCR, respectively. As T cells from resistant mice produced more IFN-γ and less IL-4 than those from susceptible animals, our results suggest that differences in TCR usage between MHC-matched animals may influence the development of the antiparasite immune response.

T cells expand without intentional antigen stimulation when transferred into adult lymphopenic environments. In this study, we show that the physiologic lymphopenic environment existing in neonatal mice also supports CD4 T cell proliferation. Strikingly, naive CD4 T cells that proliferate within neonates acquire the phenotypic and functional characteristics of memory cells. Such proliferation is inhibited by the presence of both memory and naive CD4 T cells, is enhanced by 3-day thymectomy, is independent of IL-7, and requires a class II MHC-TCR interaction and a CD28-mediated signal. CD44 bright CD4 T cells in neonates have a wide repertoire as judged by the distribution of Vβ expression. Thus, lymphopenia-induced T cell proliferation is a physiologic process that occurs during the early postnatal period.