Analyse intégrative des données multi-dimensionnelles pour l'étude de la vaccination vis-à-vis des infections mammaires et pulmonaires chez les bovins

Dans le but de prévenir la sélection de bactéries résistantes aux antibiotiques, la volonté de réduire leur usage en médecine vétérinaire est un choix sociétal largement affiché. Le développement de méthodes alternatives telles que la vaccination est nécessaire. Dans l’espèce bovine, les infections mammaires et pulmonaires restent très présentes, et de nombreuses zones d’ombre subsistent sur la compréhension des mécanismes immunitaires de défense pour les prévenir. Cette thèse s’inscrit dans le cadre de l’étude de la vaccination à l’aide d’approches multidimensionnelles dans le double objectif de comprendre les mécanismes qu’elles mobilisent et d’élaborer de nouvelles méthodes vaccinales plus performantes. Les techniques dites de haut débit telles que la transcriptomique ou encore la protéomique ciblée sont de plus en plus utilisées dans les questions de recherche. En effet, leur utilisation permet d’identifier et d’étudier de façon large et sans a priori des mécanismes encore méconnus notamment dans le domaine de l’infectiologie. La stratégie vaccinale associant une immunisation par les voies systémique et locale permet une meilleure protection des vaches laitières vis-à-vis d’une infection expérimentale par E. coli, comparée à l’immunisation systémique seule. Pour la compréhension des mécanismes immunitaires protecteurs induits par la vaccination locale, une étude transcriptomique haut débit à l’aide du séquençage de l’ARN a été réalisée sur les cellules du sang, puis sur les lymphocytes CD4 extraits du tissu mammaire. En parallèle, une étude protéomique moyen débit via le dosage de cytokines par une méthode multiplexe utilisant des billes magnétiques a été conduite en parallèle. Une intégration de l’ensemble des données couplées à des informations sur l’état clinique après l’épreuve infectieuse a permis d’attribuer la protection induite par la vaccination à l’activité des lymphocytes producteurs d’interleukine 17 dans le tissu mammaire. La deuxième étude s’inscrit dans le cadre de l’étude de la vaccination contre les maladies respiratoires des bovins. Les effets d’un protocole de préparation des animaux incluant la vaccination ont été mesurés chez des jeunes bovins et un suivi de la réponse immunitaire, en parallèle des performances zootechniques et de la morbidité a été réalisé. Via l’utilisation de méthodes multidimensionnelles, les résultats montrent que la prévalence des signes cliniques et des lésions pulmonaires n’ont pas été efficacement prévenus par les interventions vaccinales. Des conditions d’hébergement défavorables ont un impact négatif sur la santé respiratoire malgré la vaccination. Ces travaux, outre la collection d’informations nouvelles sur la réponse immunitaire induite par la vaccination, ouvrent des perspectives sur de nouvelles modalités de prévention des deux principales maladies justifiant l’utilisation d’antibiotiques en élevage bovin

Analyse intégrative des données multi-dimensionnelles pour l'étude de la vaccination vis-à-vis des infections mammaires et pulmonaires chez les bovins

In order to prevent selection of multi-resistant bacteria to antibiotics, reduction of their use in veterinary medicine is expected by the society in general. The development of alternative methods such as vaccination seems necessary to help reducing the disease prevalence. However, in the bovine species, and in particular with regard to mammary and pulmonary infections, while understanding of the complex immune mechanisms is not optimal and many grey areas remain, the development of innovative strategies is necessary. The topic of my Doctoral thesis is related to the study of vaccination using multidimensional approaches with the double purpose of deciphering associated mechanisms and improve development of new vaccine methods with improved efficacy. High throughput technics such as transcriptomics or targeted proteomics are increasingly present in the research arsenal. Indeed, their implementation allows the discovery of mechanisms that are still poorly known, particularly in the field of infectious diseases. A prime and pull vaccine strategy against mastitis caused by E. coli was more efficient at preventing clinical signs than the systemic vaccination alone. To understand the protective immune mechanisms associated with this protocol of immunization, a high-throughput transcriptomic study by RNA sequencing was carried out in the blood and mammary tissues. In parallel, a medium throughput proteomic study via the determination of cytokines in plasma, milk and mammary tissue extract was carried out using a multiplex method. An integrative analysis of these data coupled with information on clinical records after an experimental infectious challenge was performed. This study made it possible to attribute the protection induced by the vaccination to the activity of CD4 lymphocytes producing IL-17 in the mammary tissue. The second study was carried out on vaccination against respiratory diseases. Within the frame of a study on the benefits of a preconditioning programme, a vaccine strategy was evaluated in fattening young bulls and immune monitoring, in parallel to recording of growth and clinical signs was carried out. Using multivariate methods, a lack of protective effect even after multiple vaccine injections was observed. Preconditioning in poor housing conditions has a negative impact on respiratory health and growth performances of the animals despite multiple vaccinations against the main respiratory pathogens. Our study paves the way to novel vaccine strategies in addition to providing important information on the mechanisms provided by vaccination against two of the most frequent infectious diseases that motivate antibiotic treatment in cattle.Dans le but de prévenir la sélection de bactéries résistantes aux antibiotiques, la volonté de réduire leur usage en médecine vétérinaire est un choix sociétal largement affiché. Le développement de méthodes alternatives telles que la vaccination est nécessaire. Dans l’espèce bovine, les infections mammaires et pulmonaires restent très présentes, et de nombreuses zones d’ombre subsistent sur la compréhension des mécanismes immunitaires de défense pour les prévenir. Cette thèse s’inscrit dans le cadre de l’étude de la vaccination à l’aide d’approches multidimensionnelles dans le double objectif de comprendre les mécanismes qu’elles mobilisent et d’élaborer de nouvelles méthodes vaccinales plus performantes. Les techniques dites de haut débit telles que la transcriptomique ou encore la protéomique ciblée sont de plus en plus utilisées dans les questions de recherche. En effet, leur utilisation permet d’identifier et d’étudier de façon large et sans a priori des mécanismes encore méconnus notamment dans le domaine de l’infectiologie. La stratégie vaccinale associant une immunisation par les voies systémique et locale permet une meilleure protection des vaches laitières vis-à-vis d’une infection expérimentale par E. coli, comparée à l’immunisation systémique seule. Pour la compréhension des mécanismes immunitaires protecteurs induits par la vaccination locale, une étude transcriptomique haut débit à l’aide du séquençage de l’ARN a été réalisée sur les cellules du sang, puis sur les lymphocytes CD4 extraits du tissu mammaire. En parallèle, une étude protéomique moyen débit via le dosage de cytokines par une méthode multiplexe utilisant des billes magnétiques a été conduite en parallèle. Une intégration de l’ensemble des données couplées à des informations sur l’état clinique après l’épreuve infectieuse a permis d’attribuer la protection induite par la vaccination à l’activité des lymphocytes producteurs d’interleukine 17 dans le tissu mammaire. La deuxième étude s’inscrit dans le cadre de l’étude de la vaccination contre les maladies respiratoires des bovins. Les effets d’un protocole de préparation des animaux incluant la vaccination ont été mesurés chez des jeunes bovins et un suivi de la réponse immunitaire, en parallèle des performances zootechniques et de la morbidité a été réalisé. Via l’utilisation de méthodes multidimensionnelles, les résultats montrent que la prévalence des signes cliniques et des lésions pulmonaires n’ont pas été efficacement prévenus par les interventions vaccinales. Des conditions d’hébergement défavorables ont un impact négatif sur la santé respiratoire malgré la vaccination. Ces travaux, outre la collection d’informations nouvelles sur la réponse immunitaire induite par la vaccination, ouvrent des perspectives sur de nouvelles modalités de prévention des deux principales maladies justifiant l’utilisation d’antibiotiques en élevage bovin

Integrative analysis of multidimensionnal data for the study of vaccination against bovine mammary and lung infections

Dans le but de prévenir la sélection de bactéries résistantes aux antibiotiques, la volonté de réduire leur usage en médecine vétérinaire est un choix sociétal largement affiché. Le développement de méthodes alternatives telles que la vaccination est nécessaire. Dans l’espèce bovine, les infections mammaires et pulmonaires restent très présentes, et de nombreuses zones d’ombre subsistent sur la compréhension des mécanismes immunitaires de défense pour les prévenir. Cette thèse s’inscrit dans le cadre de l’étude de la vaccination à l’aide d’approches multidimensionnelles dans le double objectif de comprendre les mécanismes qu’elles mobilisent et d’élaborer de nouvelles méthodes vaccinales plus performantes. Les techniques dites de haut débit telles que la transcriptomique ou encore la protéomique ciblée sont de plus en plus utilisées dans les questions de recherche. En effet, leur utilisation permet d’identifier et d’étudier de façon large et sans a priori des mécanismes encore méconnus notamment dans le domaine de l’infectiologie. La stratégie vaccinale associant une immunisation par les voies systémique et locale permet une meilleure protection des vaches laitières vis-à-vis d’une infection expérimentale par E. coli, comparée à l’immunisation systémique seule. Pour la compréhension des mécanismes immunitaires protecteurs induits par la vaccination locale, une étude transcriptomique haut débit à l’aide du séquençage de l’ARN a été réalisée sur les cellules du sang, puis sur les lymphocytes CD4 extraits du tissu mammaire. En parallèle, une étude protéomique moyen débit via le dosage de cytokines par une méthode multiplexe utilisant des billes magnétiques a été conduite en parallèle. Une intégration de l’ensemble des données couplées à des informations sur l’état clinique après l’épreuve infectieuse a permis d’attribuer la protection induite par la vaccination à l’activité des lymphocytes producteurs d’interleukine 17 dans le tissu mammaire. La deuxième étude s’inscrit dans le cadre de l’étude de la vaccination contre les maladies respiratoires des bovins. Les effets d’un protocole de préparation des animaux incluant la vaccination ont été mesurés chez des jeunes bovins et un suivi de la réponse immunitaire, en parallèle des performances zootechniques et de la morbidité a été réalisé. Via l’utilisation de méthodes multidimensionnelles, les résultats montrent que la prévalence des signes cliniques et des lésions pulmonaires n’ont pas été efficacement prévenus par les interventions vaccinales. Des conditions d’hébergement défavorables ont un impact négatif sur la santé respiratoire malgré la vaccination. Ces travaux, outre la collection d’informations nouvelles sur la réponse immunitaire induite par la vaccination, ouvrent des perspectives sur de nouvelles modalités de prévention des deux principales maladies justifiant l’utilisation d’antibiotiques en élevage bovin.In order to prevent selection of multi-resistant bacteria to antibiotics, reduction of their use in veterinary medicine is expected by the society in general. The development of alternative methods such as vaccination seems necessary to help reducing the disease prevalence. However, in the bovine species, and in particular with regard to mammary and pulmonary infections, while understanding of the complex immune mechanisms is not optimal and many grey areas remain, the development of innovative strategies is necessary. The topic of my Doctoral thesis is related to the study of vaccination using multidimensional approaches with the double purpose of deciphering associated mechanisms and improve development of new vaccine methods with improved efficacy. High throughput technics such as transcriptomics or targeted proteomics are increasingly present in the research arsenal. Indeed, their implementation allows the discovery of mechanisms that are still poorly known, particularly in the field of infectious diseases. A prime and pull vaccine strategy against mastitis caused by E. coli was more efficient at preventing clinical signs than the systemic vaccination alone. To understand the protective immune mechanisms associated with this protocol of immunization, a high-throughput transcriptomic study by RNA sequencing was carried out in the blood and mammary tissues. In parallel, a medium throughput proteomic study via the determination of cytokines in plasma, milk and mammary tissue extract was carried out using a multiplex method. An integrative analysis of these data coupled with information on clinical records after an experimental infectious challenge was performed. This study made it possible to attribute the protection induced by the vaccination to the activity of CD4 lymphocytes producing IL-17 in the mammary tissue. The second study was carried out on vaccination against respiratory diseases. Within the frame of a study on the benefits of a preconditioning programme, a vaccine strategy was evaluated in fattening young bulls and immune monitoring, in parallel to recording of growth and clinical signs was carried out. Using multivariate methods, a lack of protective effect even after multiple vaccine injections was observed. Preconditioning in poor housing conditions has a negative impact on respiratory health and growth performances of the animals despite multiple vaccinations against the main respiratory pathogens. Our study paves the way to novel vaccine strategies in addition to providing important information on the mechanisms provided by vaccination against two of the most frequent infectious diseases that motivate antibiotic treatment in cattle

Local Th17 immunity upon mammary immunization is protective against E. coli mastitis

Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action is poorly documented and the immune response protecting the mammary gland against E. coli is not completely understood. To improve our knowledge of mammary gland immune defenses, we compared the response of three groups of six cows that received either an intramuscular or an intramammary protocol of immunizationagainst E. coli P4 before a homologous challenge. The control group received adjuvant only. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical score was also reduced and mammary secretion was preserved (Herry, 2017). High-throughput profiling using a newly developed cytokine 15-plex assay indicated a diminished TNFα production while increased IFNγ was detected in the immunized groups. Antibody response did not correlate with protection, but cellular immunity better related to protection of the mammary gland. Indeed, a transcriptome analysis performed using RNA sequencing on blood samples collected during immunization and infection phases shows that the lymphocyte response was activated in all groups 12 hours after inoculation and correlated with lower clinical scores as shown by weighted correlation network analysis. At the same time, neutrophils were produced and recruited with a moderate neutropenia in immunized groups. Type I interferon response correlated with intensity and persistence of inflammation during the late phase of mastitis. Furthermore, to assess the local T cell response in mammary tissues of locally immunized and control cows, CD4pos T cells were isolated by fluorescence cell sorting upon mammary tissue digestion of challenged and control glands, and their transcriptome determined by RNA sequencing. Results show that IL-17 expression was increased during E. coli infection and Th17 response was significantly enriched in immunized glands. These findings indicate that protective mechanisms linked to local immunization rely on IL-17-mediated immunity

Local Th17 immunity upon mammary immunization is protective against <em>E. coli</em> mastitis

International audienceCurrent vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action is poorly documented and the immune response protecting the mammary gland against E. coli is not completely understood. To improve our knowledge of mammary gland immune defenses, we compared the response of three groups of six cows that received either an intramuscular or an intramammary protocol of immunizationagainst E. coli P4 before a homologous challenge. The control group received adjuvant only. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical score was also reduced and mammary secretion was preserved (Herry, 2017). High-throughput profiling using a newly developed cytokine 15-plex assay indicated a diminished TNFα production while increased IFNγ was detected in the immunized groups. Antibody response did not correlate with protection, but cellular immunity better related to protection of the mammary gland. Indeed, a transcriptome analysis performed using RNA sequencing on blood samples collected during immunization and infection phases shows that the lymphocyte response was activated in all groups 12 hours after inoculation and correlated with lower clinical scores as shown by weighted correlation network analysis. At the same time, neutrophils were produced and recruited with a moderate neutropenia in immunized groups. Type I interferon response correlated with intensity and persistence of inflammation during the late phase of mastitis. Furthermore, to assess the local T cell response in mammary tissues of locally immunized and control cows, CD4pos T cells were isolated by fluorescence cell sorting upon mammary tissue digestion of challenged and control glands, and their transcriptome determined by RNA sequencing. Results show that IL-17 expression was increased during E. coli infection and Th17 response was significantly enriched in immunized glands. These findings indicate that protective mechanisms linked to local immunization rely on IL-17-mediated immunity

Looking for correlates of protection against bovine <em>Escherichia coli</em> mastitis: the local recruitment of Th17 cells upon mammary immunization

International audienc

Functional and RNA-seq analyses provide insights into the protective response mechanisms upon immunization against bovine <em>E. coli</em> mastitis

National audienc

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

International audienceVaccination 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 IFN gamma 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

Development of a bovine/ovine cytokine 15-plex assay for immunoprofiling of the cellular response in ruminants

International audienceEvaluation of the immune status must be assessed in highly controlled conditions to be amenable for reproducibility. Furthermore, whereas the cellular response plays a major role, immunity has been measured for a long time through antibody production. For the purpose of assessing immune cell responsiveness in ruminants, we developed and validated a whole blood assay coupled to a high-throughput multiplex cytokine assay using the Luminex technology. A custom Milliplex assay was developed with MERCKMillipore company for the measurement of 15 bovine/ovine cytokines of both the innate and adaptive immunity, and chemokines. Whole blood samples were collected from 110 dairy cows in an INRA experimental unit, and immediately stimulated during 24 hours with heat-killed bacteria (Escherichia coli, Staphylococcus aureus, and Streptococcus uberis), Toll-like receptor ligands (FSL-1 for TLR2, LPS for TLR4, and Gardiquimod for TLR7-8), or soluble anti-CD3/CD28 monoclonal antibodies. Cytokine secretion was determined and compared with a sample prepared in the same condition but without any stimulant. Using ANOVA and data mining methods (PCA, clustering…), we showed that the protocol is able to detect differences between bacteria, with S. uberis being the most potent to induce a response, compared to S. aureus and E. coli that were distinct but more closely related to each other. Similarly, TLR ligands could be distinguished, and Gardiquimod was significantly different from the MyD88- associated TLR2/4-activating ligands. LPS and E. coli provided very similar response profiles confirming previous data indicating that a large part of the E. coli response is mediated through LPS signaling. A high variability of response was detected amongst cattle suggesting environmental and genetic variations of the cytokine response. Altogether, results indicate that cytokine profiling in cattle is achievable and can be used in further work to delineate more precisely the responsiveness of cattle in various situations, including variability of the genetic background

Standardized Whole Blood Assay and Bead-Based Cytokine Profiling Reveal Commonalities and Diversity of the Response to Bacteria and TLR Ligands in Cattle

International audienceRecent 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-g, IL-1b, and TNF-a 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