Publications by authors named "Gilles Foucras"

43 Publications

Th17-related mammary immunity, but not a high systemic Th1 immune response is associated with protection against E. coli mastitis.

NPJ Vaccines 2020 Nov 24;5(1):108. Epub 2020 Nov 24.

IHAP, Université de Toulouse, ENVT, INRAE, 31076, Toulouse, France.

Vaccination against bovine mastitis lags behind despite high demand from the dairy industry and margin for efficacy improvement. We previously compared two immunization protocols against E. coli using either only the intramuscular route or a combination of intramuscular and mammary ductal routes, also known as 'prime and pull' strategy. A homologous mammary challenge during the memory phase showed that immunization favorably modified the mastitis course, notably in locally immunized cows in comparison to intramuscular and control adjuvant-only groups. Here, we performed whole-blood profiling through RNA-seq transcriptome and plasma cytokine 15-plex analyses at time points of the E. coli mastitis that showed significant clinical and laboratory differences among the groups. Diminished production of inflammatory cytokines and increased IFNγ were detected in the blood of immunized cows, where a T lymphocyte activation profile was evidenced at 12-h post infection. Acute phase neutropenia was less severe in these cows, and pathways related to neutrophil diapedesis and monocyte activation were also present. Furthermore, three intramammary-immunized cows showing faster healing and shorter mastitis duration had gene profiles that differed from their counterparts, but without any clue for the mastitis susceptibility difference. Inasmuch, when gene expression of CD4 T cells was assessed in mammary tissue, enrichment of IL-17-associated pathways was identified in the quarters of intramammary-immunized cows not only after challenge but also in the control quarters that were not infected. These findings indicate that local immunization mobilizes protective mechanisms that rely on the settlement of type 3 immunity-related CD4 T cells prior to infection.
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http://dx.doi.org/10.1038/s41541-020-00258-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686320PMC
November 2020

Type 3 immunity: a perspective for the defense of the mammary gland against infections.

Vet Res 2020 Oct 15;51(1):129. Epub 2020 Oct 15.

IHAP, Université de Toulouse, INRAE, ENVT, Toulouse, France.

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.
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http://dx.doi.org/10.1186/s13567-020-00852-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559147PMC
October 2020

Flock sensitivity and specificity of pooled fecal qPCR and pooled serum ELISA for screening ovine paratuberculosis.

PLoS One 2019 26;14(12):e0226246. Epub 2019 Dec 26.

UMR INRA ENVT 1225 IHAP, Ecole Nationale Vétérinaire de Toulouse, Toulouse Cedex, France.

The aim of our study was to evaluate the flock sensitivity and specificity of fecal qPCR and serum ELISA using pooled samples for screening paratuberculosis in French sheep. Using individual feces with low or high qPCR Ct values from ewes sampled in 14 infected flocks, a total of 555 pools of size 5, 10 and 20 were created by diluting individual materials in negative feces and analysed using a commercial IS900 qPCR kit. The relative performances of pooled serum ELISA analysis were evaluated based on the analysis of 181 different pools of size 5 and 10, composed of individual serum samples of various individual S/P values. Results showed that for pools of size 5, 10 or 20, individual fecal samples with low Ct values were invariably detected. Conversely fecal samples with high Ct values were associated with a lower detection rate in both pools of size 5 (87.0% to 90.0%), 10 (63.0% to 70.7%) and 20 (46.7% to 60.0%). After lowering the decision threshold to 25% and 15% for serum pools of size 5 and 10 respectively, the pooled serum ELISA relative sensitivity ranged between 62.2% and 100.0% depending on the composition of the pools. Finally, a simulation study was carried out to evaluate the performances of 16 screening strategies at flock level, with varying pool size (5 to 20) and number (5 to 60). The use of pooled serum ELISA led to very false positive detection rate ranging between 37.6% and 91.8% in paratuberculosis free flocks and prevents its further use in that context. For infection prevalence ≤ 5%, the flock sensitivity based on pooled fecal qPCR ranged between 39.0% (5 pools of size 10) and 99.9% (300 sampled individuals, with pools of size 5,10 or20), and was always above 93% when the infection prevalence was greater or equal to 15%. We conclude that pooled-fecal qPCR but not pooled-serum ELISA could be a useful tool to detect sheep flocks infected with paratuberculosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226246PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932769PMC
March 2020

Expansion, isolation and first characterization of bovine Th17 lymphocytes.

Sci Rep 2019 11 6;9(1):16115. Epub 2019 Nov 6.

ISP, INRA, Université de Tours, UMR1282, Nouzilly, France.

Interleukin 17A-producing T helper cells (Th17) are CD4+ T cells that are crucial to immunity to extracellular bacteria. The roles of these cells in the bovine species are poorly defined, because the characterization of bovine Th17 cells lags behind for want of straightforward cultivation and isolation procedures. We have developed procedures to differentiate, expand, and isolate bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation with antibodies to CD3 and CD28, we expanded IL-17A-positive CD4+ T cells in a serum-free cell culture medium supplemented with TGF-β1, IL-6 and IL-2. Populations of CD4+ T cells producing IL-17A or IFN-γ or both cytokines were obtained. Isolation of IL-17A-secreting CD4+ T cells was performed by labelling surface IL-17A, followed by flow cytometry cell sorting. The sorted Th17 cells were restimulated and could be expanded for several weeks. These cells were further characterized by cytokine profiling at transcriptomic and protein levels. They produced high amounts of IL-17A and IL-17F, and moderate amounts of IL-22 and IFN-γ. The techniques developed will be useful to characterize the phenotypic and functional properties of bovine Th17 cells.
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http://dx.doi.org/10.1038/s41598-019-52562-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834651PMC
November 2019

A validation study of loci associated with mastitis resistance in two French dairy sheep breeds.

Genet Sel Evol 2019 Feb 13;51(1). Epub 2019 Feb 13.

GenPhySE, Université de Toulouse, INRA, ENVT, Castanet-Tolosan, France.

Background: The identification of loci associated with resistance to mastitis or of the causative mutations may be helpful in breeding programs for dairy sheep as it is for cattle worldwide. Seven genomic regions that control milk somatic cell counts, an indirect indicator of udder infection, have already been identified in sheep (Spanish Churra, French Lacaune and Italian Sardinian-Lacaune backcross populations). In this study, we used a 960 custom-designed ovine single nucleotide polymorphism (SNP) chip in Lacaune and Manech Tête Rousse dairy sheep to validate these seven genomic regions associated with mastitis.

Results: The most significant SNP (rs868996547) on Ovis aries chromosome (OAR) 3 was a previously described mutation in the suppressor of cytokine signalling 2 (SOCS2) gene. An antagonist effect of this causal candidate between health and growth in Lacaune sheep was confirmed. Effects of the mutation on the infectious status of the udder, i.e. increases in milk somatic cell counts and bacteria shedding, were also identified. This SNP was not present in the data available on Manech Tête Rousse. Three other regions associated with mastitis were also confirmed on OAR16 (Manech Tête Rousse), 19 (Lacaune) and 2 (both breeds). For the OAR2 region, we validated previously detected SNPs in several other breeds (Sarda, Churra, and Chios). For significant SNPs in the four mastitis regions, the effect varied from 0.24 to 0.67 phenotypic standard deviation of the traits. Two of the mastitis quantitative trait loci (QTL) regions (OAR2 and 16) that we validated here were also associated in opposite ways with milk production traits in both populations.

Conclusions: These results indicate, at least in part, a genomic basis for the trade-off between milk production and mastitis resistance. Four of the seven mastitis QTL regions that were previously identified in independent populations, were confirmed in this study, which demonstrates partial sharing of mastitis-related genetic mechanisms between different distant dairy sheep populations.
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http://dx.doi.org/10.1186/s12711-019-0448-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375205PMC
February 2019

A Critical Appraisal of Probiotics for Mastitis Control.

Front Vet Sci 2018 10;5:251. Epub 2018 Oct 10.

IHAP, Université de Toulouse, ENVT, INRA, UMR1225, Toulouse, France.

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 experiments and the trials. Preliminary experiments aimed essentially at exploring 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 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.
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http://dx.doi.org/10.3389/fvets.2018.00251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191464PMC
October 2018

Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response.

Front Immunol 2017 11;8:1089. Epub 2017 Sep 11.

Université de Toulouse, INRA, INP, ENVT, IHAP, Toulouse, France.

β-Glucans (BGs) are glucose polymers present in the fungal cell wall (CW) 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 or its CW-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 BGs an undeniable immunotherapeutic potential. Our objective was to determine whether BGs from , a non-pathogenic yeast, are endowed with such a property. For this purpose, we have developed a short-term training model based on lipopolysaccharide re-stimulation of mouse bone marrow-derived macrophages primed with BGs. Through a transcriptome analysis, we demonstrated that BGs induced a specific gene expression signature involving the PI3K/AKT signaling pathway as in human monocytes. Moreover, we showed that over-expression of (that encodes for GM-CSF) was a Dectin-1-dependent feature of BG-induced priming of macrophages. Further experiments confirmed that GM-CSF up-regulated Dectin-1 cell surface expression and amplified macrophages response along BG-mediated training. However, the blockade of GM-CSFR demonstrated that GM-CSF was not primarily required for BG-induced training of macrophages although it can substantially improve it. In addition, we found that mouse macrophages trained with BGs upregulated their expression of the four and a half LIM-only protein 2 () in a Dectin-1-dependent manner. Consistently, we observed that intracellular levels of FHL2 increased after stimulation of macrophages with BGs. 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 BG-associated signaling.
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http://dx.doi.org/10.3389/fimmu.2017.01089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601002PMC
September 2017

Estimation of the sensitivity and specificity of two serum ELISAs and one fecal qPCR for diagnosis of paratuberculosis in sub-clinically infected young-adult French sheep using latent class Bayesian modeling.

BMC Vet Res 2017 Aug 3;13(1):230. Epub 2017 Aug 3.

IHAP, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076, Toulouse Cedex 3, France.

Background: The objective was to evaluate the diagnostic accuracy of two serum ELISAs and one quantitative PCR on feces for the diagnosis of paratuberculosis in sub-clinically infected young-adult sheep. A cross-sectional study was performed to collect 1197 individual blood and fecal samples from 2- to 3-year-old sub-clinically infected ewes in 14 closed meat sheep flocks in France. Fecal excretion was determined using qPCR based on IS900 sequence detection, and serology was performed on serum samples using two commercial ELISAs. Data were analyzed in a 3-test multiple-population Bayesian latent class model accounting for potential dependence between the three tests fitted in OpenBUGS. Separate analyses were performed according to whether doubtful ELISA results were handled as positive or negative and based on two thresholds for fecal qPCR (Ct ≤ 42 or Ct ≤ 40).

Results: The best fit to the data was provided by accounting for a pairwise dependence between the two ELISAs on sensitivity and pairwise dependence between the three tests on specificity. Under this model, the estimated ELISA sensitivities were 17.4% (95% PCI: 10.6 - 25.9) and 17.9% (95% PCI 11.4 - 25.6), with estimated specificities of 94.8% (95% PCI: 93.1 - 96.3) and 94.0% (95% PCI: 92.2 - 95.7). Fecal qPCR demonstrated significantly higher sensitivity (47.5%; 95% PCI: 29.3 - 69.9) and specificity (99.0%; 95% PCI: 97.9 - 99.9) than the ELISAs. Assumptions regarding doubtful ELISA results and qPCR thresholds had only a slight impact on test accuracy estimates. Models not accounting for pairwise dependence between ELISA and fecal qPCR results yielded higher sensitivity and specificity estimates but always provided a worse fit to the data.

Conclusions: Although the overall sensitivity of serum ELISAs and fecal qPCR remains low, the higher diagnostic performances of fecal qPCR make it more suitable for paratuberculosis diagnosis in sub-clinically infected sheep. Our results also illustrate that all dependence structures should be investigated when evaluating diagnostic test accuracy and selection based on a rigorous statistical approach.
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http://dx.doi.org/10.1186/s12917-017-1145-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543559PMC
August 2017

Behavioral and patho-physiological response as possible signs of pain in dairy cows during Escherichia coli mastitis: A pilot study.

J Dairy Sci 2017 Oct 26;100(10):8385-8397. Epub 2017 Jul 26.

Université de Toulouse, Ecole Nationale Vétérinaire de Toulouse (ENVT), INRA, Interactions Hôtes-Agents Pathogènes (IHAP), F-31076 Toulouse, France.

Bovine mastitis is one of the most common diseases in the dairy industry and it is a major welfare problem. Pain during mastitis is generally assessed through behavior but a combination of indicators would increase the chances of detecting pain and assessing its intensity. The aim of this study was to assess behavioral and patho-physiological responses as possible signs of pain experienced by cows after experimental intramammary challenge (mastitis) with Escherichia coli. Six Holstein-Friesian cows received an inoculation of E. coli P4 in one healthy quarter. Evolution of the disease was assessed using bacteriological growth and somatic cell counts (SCC). Cows' response to the challenge was monitored by direct behavioral and clinical observations, data loggers, rumen temperature sensors, and indicators of inflammation, stress, and oxidative status. From all data recorded, the variables that contributed most to the discrimination of mastitis phases were obtained by factorial discriminant analysis. Baseline levels of all indicators corresponded to values before challenge. Specifically, we weighted data relating to lying behavior by the observations at the same hour of the day before challenge to eliminate the circadian rhythm effect. We identified 3 phases that were discriminated by factorial discriminant analysis with good performance. Nine indicators varied according to the phase of the disease: cows' attitude toward their surroundings, tail position, clinical signs, ear position, variation of postural changes, concentrations of haptoglobin and serum amyloid A (SAA), cortisol blood levels, and rumen temperature (as a surrogate for body temperature). In phase 1 (4 to 8 h postinoculation), E. coli proliferated exponentially in milk but inflammation indicators remained at baseline levels. Cows were less attentive toward their surroundings (median score, 0.63), and postural changes (lying/standing) were less frequent (0.75 times from baseline). In phase 2 (12 to 24 h postinoculation), bacterial concentrations peaked around 12 h and then began to decrease concomitantly with a sharp SCC increase. Cows were less attentive toward their surroundings (score, 0.54), had high plasma cortisol (31.3 ng/mL) and SAA (100.3 µg/mL) concentrations, and rumen temperature was increased (40.3°C). In phase 3 (32 to 80 h postinoculation), bacterial concentrations decreased concomitantly with high SCC levels. Cows had high levels of haptoglobin (0.57 mg/mL) and SAA (269 µg/mL) but showed no behavioral changes. Dairy cows displayed changes of behavioral, inflammatory, and stress parameters after E. coli mammary inoculation. Our results suggest that cows may have experienced discomfort in the preclinical phase (phase 1) and pain in the acute phase (phase 2) but neither discomfort nor pain in the remission phase (phase 3). Although larger controlled studies are needed to confirm our findings, this knowledge could be useful for early detection of E. coli mastitis and for decision-making regarding the initiation of pain-relief treatment during mastitis in dairy cows. This would improve animal welfare and potentially faster disease remission.
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http://dx.doi.org/10.3168/jds.2017-12796DOI Listing
October 2017

Local immunization impacts the response of dairy cows to Escherichia coli mastitis.

Sci Rep 2017 06 13;7(1):3441. Epub 2017 Jun 13.

ISP, INRA, Université de Tours, UMR1282, Nouzilly, France.

Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action has not been documented, and immune responses protecting the mammary gland against E. coli are not completely understood. To improve our knowledge of mammary gland immune protection, cows immunized either intramuscularly or intramammarily with the E. coli P4 were submitted to a homologous mastitis challenge. A third group of mock-immunized cows serve as challenge controls. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical signs and reduction in milk secretion were also contained. This occurred with a modification of the cytokine profile, such as an increase in IFN-γ and a reduction in TNF-α concentrations in milk. Concentrations of IL-17A and IL-22 increased in milk at the onset of the inflammatory response and remained high up to the elimination of bacteria, but concentrations did not differ between groups. Accelerated bacteriological cure was not linked to an increase in the initial efficiency of phagocytosis in milk. Results support the idea that antibodies did not play a major role in the improvement, and that cell-mediated immunity is the key to understanding E. coli vaccine-induced protection of the mammary gland.
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http://dx.doi.org/10.1038/s41598-017-03724-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469773PMC
June 2017

Whole blood transcriptome analysis reveals potential competition in metabolic pathways between negative energy balance and response to inflammatory challenge.

Sci Rep 2017 05 24;7(1):2379. Epub 2017 May 24.

Université de Toulouse, École Nationale Vétérinaire de Toulouse (ENVT), INRA, Interactions Hôtes - Agents Pathogènes (IHAP), F-31076, Toulouse, France.

Negative Energy Balance (NEB) is considered to increase susceptibility to mastitis. The objective of this study was to improve our understanding of the underlying mechanisms by comparing transcriptomic profiles following NEB and a concomitant mammary inflammation. Accordingly, we performed RNA-seq analysis of blood cells in energy-restricted ewes and control-diet ewes at four different time points before and after intra mammary challenge with phlogogenic ligands. Blood leucocytes responded to NEB by shutting down lipid-generating processes, including cholesterol and fatty acid synthesis, probably under transcriptional control of SREBF 1. Furthermore, fatty acid oxidation was activated and glucose oxidation and transport inhibited in response to energy restriction. Among the differentially expressed genes (DEGs) in response to energy restriction, 64 genes were also differential in response to the inflammatory challenge. Opposite response included the activation of cholesterol and fatty acid synthesis during the inflammatory challenge. Moreover, activation of glucose oxidation and transport coupled with the increase of plasma glucose concentration in response to the inflammatory stimuli suggested a preferential utilization of glucose as the energy source during this stress. Leucocyte metabolism therefore undergoes strong metabolic changes during an inflammatory challenge, which could be in competition with those induced by energy restriction.
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http://dx.doi.org/10.1038/s41598-017-02391-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443788PMC
May 2017

High somatic cell counts and changes in milk fat and protein contents around insemination are negatively associated with conception in dairy cows.

Theriogenology 2017 Jan 1;88:18-27. Epub 2016 Oct 1.

IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France. Electronic address:

The fertility of dairy cows has decreased dramatically worldwide over the last few decades, and several causes of this trend have been reported. Several studies have associated compromised udder health with deteriorating reproduction performance. Subclinical ketosis (SCK) has also been reported to be a risk factor for decreased conception. The objective of the present study was to describe how SCK might interact with the reported association between udder health and conception in dairy cows. Data from the French Milk Control Program and data on 8,549,667 instances of artificial insemination (AI) and their corresponding preceding and subsequent test-days from 5,979,701 Holstein cows were examined over a 5-year period (2008-2012). The effect of udder health was evaluated through a low (L) or high (H) somatic cell count (SCC) before and after AI using a threshold of 200,000 cells/mL, and transformed into four groups (LL, LH, HL, and HH). Three proxies for defining SCK were proposed based on the milk fat and protein content (or their ratio) before AI. Statistical analysis first included a generalized additive model to help define the optimal threshold values. Next, a logistic regression with a Poisson correction was performed. On average, the risk of conception at first AI was reduced by 14% for LH or HH cows (relative risk [and 95% CI] = 0.86 [0.85-0.87]) when the SCC increased or remained high within 40 days before and after AI, relative to LL group. The reduction of conception success associated with SCK (fat and protein contents changes) varied from 3% to 17% depending on the used SCK proxy. Including the interaction term SCCSCK clearly showed that the association of increased SCC around AI with conception success was modified by the presence of SCK. A cow that already has SCK and experiences an increase in SCC around or after AI exhibits up to 2 times further decrease in conception success compared with a cow with a high SCC and no SCK. In conclusion, this study reinforces the previously described association between intramammary inflammation around or after AI and a decreased rate of conception. These findings highlight how SCK interacts with the above-mentioned relationship by strengthening the negative association between mastitis and conception success. In addition, the present work supports the theory that local inflammation may affect the whole-body response and alter the functions of other organs, such as the reproductive tract.
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http://dx.doi.org/10.1016/j.theriogenology.2016.09.043DOI Listing
January 2017

Single-cell analysis reveals new subset markers of murine peritoneal macrophages and highlights macrophage dynamics upon Staphylococcus aureus peritonitis.

Innate Immun 2016 07 24;22(5):382-92. Epub 2016 May 24.

Université de Toulouse, INP, ENVT, UMR1225, IHAP, F-31076 Toulouse, France INRA, UMR1225, IHAP, F-31076 Toulouse, France

Resident macrophages play a central role in maintaining tissue homeostasis and immune surveillance. Here, we used single cell-based qPCR coupled with flow cytometry analysis to further define the phenotypes of large and small resident peritoneal macrophages (LPMs and SPMs, respectively) in mice. We demonstrated that the expression of Cxcl13, IfngR1, Fizz-1 and Mrc-1 clearly distinguished between LPMs and SPMs subsets. Using these markers, the dynamics of peritoneal macrophages in a Staphylococcus aureus-induced peritonitis model were analyzed. We found that S. aureus infection triggers a massive macrophage disappearance reaction in both subsets. Thereafter, inflammatory monocytes rapidly infiltrated the cavity and differentiated to replenish the SPMs. Although phenotypically indistinguishable from resident SPMs by flow cytometry, newly recruited SPMs had a different pattern of gene expression dominated by M2 markers combined with M1 associated features (inos expression). Interestingly, S. aureus elicited SPMs showed a robust expression of Cxcl13, suggesting that these cells may endorse the role of depleted LPMs and contribute to restoring peritoneal homeostasis. These data provide information on both resident and recruited macrophages dynamics upon S. aureus infection and demonstrate that single-cell phenotyping is a promising and highly valuable approach to unraveling macrophage diversity and plasticity.
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http://dx.doi.org/10.1177/1753425916651330DOI Listing
July 2016

Triggering Dectin-1-Pathway Alone Is Not Sufficient to Induce Cytokine Production by Murine Macrophages.

PLoS One 2016 3;11(2):e0148464. Epub 2016 Feb 3.

Université de Toulouse, INP-ENVT, UMR 1225, IHAP, Toulouse, France.

β-glucans (BG) are abundant polysaccharides of the Saccharomyces cerevisiae cell wall (Sc CW), an industry byproduct. They have immuno-stimulatory properties upon engagement of dectin-1 (Clec7a), their main receptor on particular immune cells, and they actually become of great interest because of their preventive or therapeutic potentials. Zymosan, a crude extract of Sc CW was studied as a prototypic BG, despite its miscellaneous PAMPs content. Here, we examined the response of murine wild type or Clec7a-/- bone marrow-derived macrophages (BMDM) to products with increasing BG content (15, 65 or 75%) and compared their effects with those of other dectin-1 ligands. The enrichment process removed TLR ligands while preserving dectin-1 activity. The most enriched extracts have very low NFκB activity and triggered low amounts of cytokine production in contrast with crude products like zymosan and BG15. Furthermore, MyD88-/- BMDM did not produce TNFα in response to crude Sc CW extracts, whereas their response to BG-enriched extracts was unaffected, suggesting that BG alone are not able to initiate cytokine secretion. Although Sc CW-derived BG stimulated the late and strong expression of Csf2 in a dectin-1-dependent manner, they remain poor inducers of chemokine and cytokine production in murine macrophages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148464PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4739705PMC
July 2016

A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model.

PLoS Genet 2015 Dec 11;11(12):e1005629. Epub 2015 Dec 11.

INRA, UMR 1388 Génétique, Physiologie et Systèmes d'Elevage, Castanet-Tolosan, France.

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host's inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.
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http://dx.doi.org/10.1371/journal.pgen.1005629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4676722PMC
December 2015

Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes.

PLoS One 2015 16;10(9):e0137755. Epub 2015 Sep 16.

INP, ENVT, Université de Toulouse, Toulouse, France; UMR1225, Interactions Hôte Agents Pathogènes, INRA, Toulouse, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573518PMC
May 2016

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

Front Immunol 2015 19;6:299. Epub 2015 Jun 19.

UR892, Virologie et Immunologie Moléculaires, INRA, Domaine de Vilvert , Jouy-en-Josas , France.

Mononuclear phagocytes are organized in a complex system of ontogenetically and functionally distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic, and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections. We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover, we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey.
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http://dx.doi.org/10.3389/fimmu.2015.00299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473062PMC
July 2015

Pyroptosis of resident macrophages differentially orchestrates inflammatory responses to Staphylococcus aureus in resistant and susceptible mice.

Eur J Immunol 2015 Mar 21;45(3):794-806. Epub 2015 Jan 21.

Université de Toulouse, INP, ENVT, Toulouse, France; INRA, IHAP, Toulouse, France.

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.
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http://dx.doi.org/10.1002/eji.201445098DOI Listing
March 2015

Expression of VP7, a Bluetongue virus group specific antigen by viral vectors: analysis of the induced immune responses and evaluation of protective potential in sheep.

PLoS One 2014 3;9(11):e111605. Epub 2014 Nov 3.

UPE, ANSES, INRA, ENVA, UMR 1161 ANSES/INRA/ENVA, Maisons-Alfort, France.

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 R0) 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 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 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 R0 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111605PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4218782PMC
August 2015

Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system.

Vet Res 2013 Jun 11;44:40. Epub 2013 Jun 11.

INRA, UMR1282, ISP, F 37380, Nouzilly, France.

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1β was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.
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http://dx.doi.org/10.1186/1297-9716-44-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686618PMC
June 2013

Bluetongue virus serotype 1 in wild ruminants, France, 2008-10.

J Wildl Dis 2012 Oct;48(4):1047-51

Institut National de Recherche Agronomique, Unité Mixte Recherche 1225 IHAP, 23 chemin des Capelles, 31076 Toulouse Cedex, France.

The persistence of Bluetongue virus serotype 1 (BTV-1) circulation was evaluated in red deer (Cervus elaphus), roe deer (Capreolus capreolus), mouflons (Ovis ammon), and Pyrenean chamois (Rupicapra pyrenaica pyrenaica) sampled during two hunting seasons between September 2008 and February 2010 in the East Pyrenean Mountains, France. The prevalence of BTV antibody in red deer was high and not significantly different between the two hunting seasons (50.9% and 49.6%, respectively). The prevalence of BTV-1 RNA in red deer was 50.3% in 2008. Conversely, only 10.8% of samples from red deer were BTV-1 RNA-positive in 2010, and most of them showed only weak positive results. In other investigated species, the prevalence of infection was low. High elevation was associated with reduced infection rates and could explain the low prevalence observed in mouflons and Pyrenean chamois. These results support the hypothesis that, apart from red deer, wild ungulates are unlikely to be involved in the maintenance or circulation of BTV in the investigated region. Mass vaccination in livestock might have reduced BTV-1 circulation in red deer, although annual variation due to acquired immunity or fluctuations in vector abundance should also be considered.
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http://dx.doi.org/10.7589/2011-12-359DOI Listing
October 2012

Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant.

Physiol Genomics 2012 Apr 14;44(7):403-16. Epub 2012 Feb 14.

Université de Toulouse, Institut National Polytechnique (INP), École Nationale Vétérinaire de Toulouse (ENVT), Unité Mixte de Recherche (UMR)1225, Interactions Hôtes - Agents Pathogènes (IHAP), Toulouse, France.

Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune response-related genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria.
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http://dx.doi.org/10.1152/physiolgenomics.00155.2011DOI Listing
April 2012

Myxomavirus as a vector for the immunisation of sheep: protection study against challenge with bluetongue virus.

Vaccine 2012 Feb 13;30(9):1609-16. Epub 2012 Jan 13.

INRA, UMR1225, IHAP, F-31076 Toulouse, France.

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.
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http://dx.doi.org/10.1016/j.vaccine.2011.12.108DOI Listing
February 2012

Both exogenous commensal and endogenous self antigens stimulate T cell proliferation under lymphopenic conditions.

Cell Immunol 2012 25;272(2):117-23. Epub 2011 Nov 25.

Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, United States.

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.
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http://dx.doi.org/10.1016/j.cellimm.2011.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244518PMC
April 2012

Alloantibodies against MHC class I: a novel mechanism of neonatal pancytopenia linked to vaccination.

J Immunol 2011 Dec 14;187(12):6564-70. Epub 2011 Nov 14.

Université de Toulouse, Institut National Polytechnique de Toulouse, Ecole Nationale Vétérinaire de Toulouse, F-31076 Toulouse, France.

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.
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http://dx.doi.org/10.4049/jimmunol.1102533DOI Listing
December 2011

Staphylococcal-associated molecular patterns enhance expression of immune defense genes induced by IL-17 in mammary epithelial cells.

Cytokine 2011 Dec 17;56(3):749-59. Epub 2011 Oct 17.

INRA, UR1282 Infectiologie Animale et Santé Publique (IASP), F-37380 Nouzilly, France.

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-α. 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-α and IL-1β 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.
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http://dx.doi.org/10.1016/j.cyto.2011.09.020DOI Listing
December 2011

Gene expression profiling of dendritic cells reveals important mechanisms associated with predisposition to Staphylococcus infections.

PLoS One 2011 12;6(8):e22147. Epub 2011 Aug 12.

Université de Toulouse, INP, ENVT; UMR 1225, IHAP, Toulouse, France.

Background: Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics.

Methodology/principal Findings: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs) by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line.

Conclusion/significance: We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment. The distinct transcriptional profiles of dendritic cells obtained from resistant and susceptible animals may explain susceptibility towards S. aureus infections in a broader context.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022147PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155527PMC
February 2012

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources.

BMC Genomics 2011 May 11;12(1):225. Epub 2011 May 11.

Parco Tecnologico Padano - CERSA, Via Einstein, 26900 Lodi, Italy.

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.
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http://dx.doi.org/10.1186/1471-2164-12-225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3118214PMC
May 2011

Transcriptomic analysis of milk somatic cells in mastitis resistant and susceptible sheep upon challenge with Staphylococcus epidermidis and Staphylococcus aureus.

BMC Genomics 2011 Apr 28;12:208. Epub 2011 Apr 28.

INRA, UR631, SAGA, F-31326 Castanet-Tolosan, France.

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.
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http://dx.doi.org/10.1186/1471-2164-12-208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096985PMC
April 2011

Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression.

J Immunol Methods 2008 Nov 15;339(1):1-10. Epub 2008 Aug 15.

INRA, Toulouse, France.

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.
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http://dx.doi.org/10.1016/j.jim.2008.07.012DOI Listing
November 2008