Vaccine immunomodulation and protection mechanism against. Mycobacterium avium subsp. paratuberculosis: Focus on innate responses.

294 p.La paratuberculosis (PTB) es una enteritis granulomatosa crónica causada por Mycobacterium aviumsubsp. paratuberculosis (Map) que afecta principalmente a rumiantes domésticos. A pesar de que existenvacunas contra la PTB con efectividad demostrada, su aplicación en el ganado bovino está prohibidadebido a la interferencia que produce con las pruebas de diagnóstico de la tuberculosis bovina (bTB) quese realizan durante los controles oficiales. En el presente trabajo de Tesis se evaluan las vías intradérmicay oral como alternativas a la subcutánea y varios prototipos de vacunas orales en el modelo de infecciónde Map en conejo. Algunas de las estrategias de vacunación testadas han mostrado eficacia frente a lainfección aparte de no producir interferencias con la prueba de diagnóstico de la bTB. Además de evaluarla protección ejercida por las vacunas, también se ha analizado la modulación de la respuesta inmunitariaasociada a la vacunación mediante el estudio de la polarización de los macrófagos locales del intestino,efectos en la expresión de citoquinas de células mononucleares de sangre periférica, funcionalidad deneutrófilos de sangre periférica y niveles de la respuesta humoral, entre otras. Adicionalmente, se haestudiado la interacción de neutrófilos y macrófagos bovinos ex vivo en su respuesta frente a Map encomparación con otra micobacteria atenuada menos patógena. En conjunto, los resultados de esta tesis revelan que los neutrófilos pueden jugar un papel más importante de lo que se ha pensado hasta ahora en las etapas iniciales de la infección con Map y que la estimulación de estas células inmunitarias mediantela vacunación puede aumentar las posibilidades de éxito del hospedador en su defensa ante la infecciónNeike

Bovine Neutrophils Release Extracellular Traps and Cooperate With Macrophages in Mycobacterium Avium Subsp. Paratuberculosis Clearance In Vitro

Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guerin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1 beta and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1 beta and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection siteFunding was provided by Spanish central government and Basque research project PROBAK (RTA 2017-00089-00-00) and by the Departamento de Economia e Infraestructuras of the Basque Government. IL-A held a predoctoral grant from Departamento de Economia e Infraestructuras of the Basque Government (2017) and was granted an EMBO short-term fellowship (8407) and a FEMS research and training grant (FEMS-GO-2019-507). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publicatio

Oral vaccination stimulates neutrophil functionality and exerts protection in a Mycobacterium avium subsp. paratuberculosis infection model

Abstract Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis (PTB), a granulomatous enteritis in ruminants that exerts high economic impact on the dairy industry worldwide. Current vaccines have shown to be cost-effective against Map and in some cases confer beneficial non-specific effects against other pathogens suggesting the existence of trained immunity. Although Map infection is mainly transmitted by the fecal-oral route, oral vaccination has not been deeply studied. Therefore, the aim of this study was to compare the oral route with a set of mycobacterial and non-mycobacterial vaccines with a subcutaneously administered commercially available vaccine. Training effects on polymorphonuclear neutrophils (PMNs) and homologous and heterologous in vivo protection against Map were investigated in the rabbit infection model. Oral vaccination with inactivated or live vaccines was able to activate mucosal immunity as seen by elevation of serum IgA and the expression of IL4 in peripheral blood mononuclear cells (PBMCs). In addition, peripheral PMN phagocytosis against Map was enhanced by vaccination and extracellular trap release against Map and non-related pathogens was modified by both, vaccination and Map-challenge, indicating trained immunity. Finally, PBMCs from vaccinated animals stimulated in vitro with Map antigens showed a rapid innate activation cytokine profile. In conclusion, our data show that oral vaccination against PTB can stimulate neutrophil activity and both innate and adaptive immune responses that correlate with protection.The research was funded by the Department of Economy, Sustainability and Environment of the Basque Government and by grant RTA 2017-00089-00-00 of the National Institute for Agronomic Research (INIA) to N.E. I.L.-A. and M.O. both held predoctoral fellowships from the DEI of the Basque Government. This research was also partly supported by the Agriculture Funding Consortium members Alberta Agriculture and Forestry and Alberta Milk (2018F019R) to J.D.B. CIC bioGUNE thanks the Ministry of Science and Innovation for the Severo Ochoa excellence award (SEV-2016-0644

Early response of monocyte-derived macrophages from vaccinated and non-vaccinated goats against in vitro infection with Mycobacterium avium subsp. paratuberculosis

[EN]Paratuberculosis is a disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Vaccination is the most cost-effective control method. However, despite the fact that macrophages are the main target cells for this pathogen, the precise mechanisms behind the response of the macrophage to Map infection and how it is modified by vaccination are yet poorly understood. The aim of this study was to investigate the effect of Silirum® vaccination in the early immune response of caprine monocyte-derived macrophages (CaMØs). Peripheral blood mononuclear cells (PBMCs) were obtained from vaccinated and non-vaccinated goats, cultured in vitro until differentiation to macrophages and infected with Map. After a 24 h incubation, Map viability and DNA were assessed in culture by viable colony count and real time quantitative polymerase chain reaction (qPCR). In addition, Map phagocytosis and expression of IL-10, IL-12, IFN-γ, TNF-α, IL-17A, IL-1β, iNOS, IL-6 and MIP-1β were also evaluated through immunofluorescence labelling and reverse transcriptase qPCR (RT-qPCR), respectively. A significant reduction of Map viability was observed in both supernatants (P < 0.05) and CaMØs (P < 0.001) from the vaccinated group. Similarly, the percentage of infected CaMØs and the number of internalized Map by CaMØs (P < 0.0001) was higher in the vaccinated group. Finally, iNOS (P < 0.01) and IL-10 were significantly up-regulated in CaMØs from vaccinated goats, whereas only MIP-1β was up-regulated in non-vaccinated animals (P < 0.05). These results show that vaccination modifies the immune response of CaMØs, suggesting that the phagocytosis and microbiocidal activity of macrophages against Map is enhanced after vaccination.SIThe Spanish Ministry of Science and Innovation (Projects AGL2015-66540-C2-1-R and RTI2018-099496-B-I00) and Junta de Castilla y León (Project LE259P18) financed this work. This work was also financially supported by the University of León. Noive Arteche Villasol has been financially supported by a predoctoral contract (BES-2016-076513) from the Spanish Ministry of Science and Innovation. Daniel Gutiérrez-Expósito and José Espinosa are the recipients of a postdoctoral contract from the Ministry of Science and Innovation (Grants No. FJCI-2017-32020 and FJC2019-042422-I, respectively). Iraia Ladero-Auñon holds a pre-doctoral grant from “Departamento de Economía e Infraestructuras” of the Basque Government