Análisis del potencial epizoótico de cepas de Mycobacterium avium subsp. paratuberculosis aisladas de diversos hospedadores empleando modelos de infección in vitro

299 p.Map es el agente causal de la paratuberculosis en una gran variedad de rumiantes domésticos y silvestres. Una vez que Map entra en el hospedador, generalmente a través de la vía fecal-oral, atraviesa el epitelio intestinal para ser posteriormente fagocitado por los macrófagos subepiteliales. Generalmente, los macrófagos responden frente al ataque de cualquier microorganismo induciendo una respuesta inmune dirigida a su eliminación. Sin embargo, las cepas virulentas de Map han desarrollado ciertas estrategias que les permiten evadir la respuesta inmune del hospedador y sobrevivir intracelularmente. La interacción Map-macrófagos del hospedador que se produce en los estadios iniciales de la infección es un indicador de la virulencia de Map y determina el riesgo de progresión de la enfermedad. El objetivo general de esta tesis doctoral es el de comparar la interacción de aislados de Map procedentes de rumiantes domésticos y silvestres y con distintos genotipos con macrófagos bovinos y ovinos, al ser éstas las dos especies animales en las que la paratuberculosis causa mayores pérdidas económicas. Este análisis permitirá determinar la virulencia, patogenicidad y adaptación a un determinado hospedador de distintos aislados de Map y sentará las bases para tratar de mejorar las estrategias de manejo y control de la paratuberculosis en granjas donde el ganado doméstico comparte pastos con animales silvestres potencialmente infectados con Map. Con objeto de determinar diferencias en la virulencia en los primeros estadios de la infección, se comparó la entrada, multiplicación y supervivencia de 11 aislados de Map de diversos genotipos (C, S y B) y procedentes de distintas localizaciones geográficas y hospedadores (bovino, ovino, caprino, jabalí, bisonte, ciervo y gamo) en modelos de infección in vitro bovinos y ovinos. Así mismo, se analizó la respuesta de los macrófagos a la infección con los aislados de Map seleccionados mediante el análisis de los perfiles de expresión de determinadas citoquinas y proteínas implicadas en apoptosis y destrucción tisular. Ambos parámetros, capacidad de supervivencia de los distintos aislados de Map y respuesta de los macrófagos a la infección, se correlacionaron con objeto de determinar la virulencia de cada aislado en los hospedadores bovino y ovino. Así mismo, y con el fin de comparar la virulencia de diversos aislados de Map en estadios más avanzados de la infección se determinó su capacidad para generar microgranulomas en condiciones in vitro. Por último y con objeto de adaptar los modelos de macrófagos desarrollos en este tesis a otros objetivos, como por ejemplo para su utilización en la evaluación de actividad fagocítica inducida por prototipos vacunales, se desarrolló un método inmunomagnético de enriquecimiento de monocitos CD14 + de bovino, ovino y caprino utilizando nanopartículas magnéticas funcionalizadas con un anticuerpo monoclonal anti-CD14 humano en combinación con la tecnología MACS.NEIKER-Tecnalia, Instituto Vasco de Investigación y Desarrollo Agrari

Identification and characterization of miRNAs in spleens of sheep subjected to repetitive vaccination

Accumulative evidence has shown that short non-coding RNAs such as miRNAs can regulate the innate and adaptive immune responses. Aluminium hydroxide is a commonly used adjuvant in human and veterinary vaccines. Despite its extended use, its mechanism of action is not fully understood and very few in vivo studies have been done to enhance understanding at the molecular level. In this work, we took advantage of a previous long-term experiment in which lambs were exposed to three different treatments by parallel subcutaneous inoculations with aluminium-containing commercial vaccines, an equivalent dose of aluminium or mock injections. Spleen samples were used for miRNA-seq. A total of 46 and 16 miRNAs were found differentially expressed when animals inoculated with commercial vaccines or the adjuvant alone were compared with control animals, respectively. Some miRNAs previously related to macrophage polarization were found dysregulated exclusively by the commercial vaccine treatment but not in the aluminium inoculated animals. The dysregulated miRNAs in vaccine group let-7b-5p, miR-29a-3p, miR-27a and miR-101-3p are candidates for further research, since they may play key roles in the immune response induced by aluminium adjuvants added to vaccines. Finally, protein–protein interaction network analysis points towards leucocyte transendothelial migration as a specific mechanism in animals receiving adjuvant only

Aluminiodun txerto-laguntzaileen eraginaren azterketa sistemen biologiaren bidez

Hamarkadak dira aluminio konposatuak txertoen laguntzaile gisa erabiltzen direla albaitaritzan eta giza txertoetan. Laguntzaile horien erabilera oso hedatua egon arren, ez da ongi ezagutzen zein mekanismoren bidez eragiten dituzten ondorio onuragarriak eta, aldizka, kontrako erreakzioak eragin ditzakete. Sistemen biologiako tekniken bidez laguntzaile hauen sinadura molekularra ikertu da, gizakietan zein etxabereetan. Hainbat eragin-mekanismo proposatu dira aluminioaren sistema immunea pizteko ahalmena azaltzeko eta analisi transkriptomikoetan erlazionatutako geneak aurkitu dira. Batzuetan emaitzak kontraesanezkoak dira, aktibatzen den erantzun immunea desberdina izan daitekeelako egoeraren arabera. Aluminiogatzen tamainak, formak, propietate fisiko-kimikoek eta antigenoen absortzioak eragina dutela ematen du era berean. Laguntzaile hauek ez dira antigeno-garraiatzaile soilak, sistema immunea kitzikatzen duten arriskuseinale endogenoak pizten baitituzte. Argitzeke dago oraindik haien eragin-mekanismo zehatza eta epe luzeko aktibazio immunitarioak sistema immunitarioaren gainestimulazioa eragin dezakeen.; Aluminium compounds have been used as adjuvants for years in veterinary and human vaccines, but, despite their widespread use, the mechanism of how aluminium-based adjuvants exert their beneficial effects is still not fully understood and they occasionally can cause adverse reactions. The molecular signature of these adjuvants has been studied by system biology techniques in human and livestock. Transcriptomic analyses have found dysregulated genes linked to some of the proposed mechanisms of action of aluminium. Sometimes the results are contradictory because the immune reaction that is activated may be different depending on the situation. The shape, size, physicochemical properties or antigen absorption of aluminium salts can also have an effect. It seems that aluminium-containing adjuvants are not simple delivery vehicles for antigens, but also induce endogenous danger signals that stimulate the immune system. Whether this contributes to long-lasting immune activation or to the overstimulation of the immune system remains to be elucidated

Whole Transcriptome Approach to Evaluate the Effect of Aluminium Hydroxide in Ovine Encephalon

Aluminium hydroxide adjuvants are crucial for livestock and human vaccines. Few studies have analysed their effect on the central nervous system in vivo. In this work, lambs received three different treatments of parallel subcutaneous inoculations during 16 months with aluminium-containing commercial vaccines, an equivalent dose of aluminium hydroxide or mock injections. Brain samples were sequenced by RNA-seq and miRNA-seq for the expression analysis of mRNAs, long non-coding RNAs and microRNAs and three expression comparisons were made. Although few differentially expressed genes were identified, some dysregulated genes by aluminium hydroxide alone were linked to neurological functions, the lncRNA TUNA among them, or were enriched in mitochondrial energy metabolism related functions. In the same way, the miRNA expression was mainly disrupted by the adjuvant alone treatment. Some differentially expressed miRNAs had been previously linked to neurological diseases, oxidative stress and apoptosis. In brief, in this study aluminium hydroxide alone altered the transcriptome of the encephalon to a higher degree than commercial vaccines that present a milder effect. The expression changes in the animals inoculated with aluminium hydroxide suggest mitochondrial disfunction. Further research is needed to elucidate to which extent these changes could have pathological consequences.This work was supported by the Spanish Ministry of Economy grant [MINECO project AGL2013-49137-C3 to BMJ, LL and DA]; University of the Basque Country (UPV/EHU) predoctoral fellowships [PIF15/361 to EV-M and PIF17/306 to MB-A]; and University of the Basque Country (UPV/EHU) postdoctoral fellowship [ESPDOC16/43 to NA]. Thanks to M. Ortega for technical help

Molecular Signature of Aluminum Hydroxide Adjuvant in Ovine PBMCs by Integrated mRNA and microRNA Transcriptome Sequencing

There have been few in vivo studies on the effect of aluminum hydroxide adjuvant and its influence on the immune response to vaccination. In this study, lambs received a parallel subcutaneous treatment with either commercial vaccines containing aluminum hydroxide or an equivalent dose of this compound only with the aim of identifying the activated molecular signature. Blood samples were taken from each animal at the beginning and at the end of the experiment and PBMCs isolated. Total RNA and miRNA libraries were prepared and sequenced. After alignment to the Oar3.1 reference genome and differential expression with 3 programs, gene enrichment modeling was performed. For miRNAs, miRBase and RNAcentral databases were used for detection and characterization. Three expression comparisons were made: vaccinated animals at the beginning and at the end of the treatment, adjuvanted animals at the same times, and animals of both treatments at the end of the experiment. After exposure to both treatments, a total of 2,473; 2,980 and 429 differentially expressed genes were identified in vaccinated animals, adjuvanted animals and animals at the end of both treatments, respectively. In both adjuvant and vaccine treated animals the NF-kappa B signaling pathway was enriched. On the other hand, it can be observed a downregulation of cytokines and cytokine receptors in the adjuvanted group compared to the vaccinated group at the final time, suggesting a milder induction of the immune response when the adjuvant is alone. As for the miRNA analysis, 95 miRNAs were detected: 64 previously annotated in Ovis aries, 11 annotated in Bos taurus and 20 newly described. Interestingly, 6 miRNAs were differentially expressed in adjuvant treated animals, and 3 and 1 in the other two comparisons. Lastly, an integrated miRNA-mRNA expression profile was developed, in which a miRNA-mediated regulation of genes related to DNA damage stimulus was observed. In brief, it seems that aluminum-containing adjuvants are not simple delivery vehicles for antigens, but also induce endogenous danger signals that can stimulate the immune system. Whether this contributes to long-lasting immune activation or to the overstimulation of the immune system remains to be elucidated.This work was supported by a MINECO project grant (AGL2013-49137-C3-3-R to BJ and AGL2013-49137-C3-2-R to LL and AGL2013-49137-C3-1 to DdA), a predoctoral fellowship from the UPV/EHU to EV-M (PIF15/361) and a postdoctoral fellowship from the UPV/EHU to NA (ESPDOC16/43)

Increased lytic efficiency of bovine macrophages trained with killed mycobacteria

Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response.Funding for these studies was provided by the EU project WildTBVac (Contract #613799) and by grants from the Instituto Nacional de Investigación y Tecnología Agraria y alimentaria (INIA, RTA2011-00049) and the Ministry of Science (MINECO, AGL2014-56305) and European Funds Regional Development (FEDER).Peer Reviewe

Análisis del potencial epizoótico de cepas de Mycobacterium avium subsp. paratuberculosis aisladas de diversos hospedadores empleando modelos de infección in vitro

299 p.Map es el agente causal de la paratuberculosis en una gran variedad de rumiantes domésticos y silvestres. Una vez que Map entra en el hospedador, generalmente a través de la vía fecal-oral, atraviesa el epitelio intestinal para ser posteriormente fagocitado por los macrófagos subepiteliales. Generalmente, los macrófagos responden frente al ataque de cualquier microorganismo induciendo una respuesta inmune dirigida a su eliminación. Sin embargo, las cepas virulentas de Map han desarrollado ciertas estrategias que les permiten evadir la respuesta inmune del hospedador y sobrevivir intracelularmente. La interacción Map-macrófagos del hospedador que se produce en los estadios iniciales de la infección es un indicador de la virulencia de Map y determina el riesgo de progresión de la enfermedad. El objetivo general de esta tesis doctoral es el de comparar la interacción de aislados de Map procedentes de rumiantes domésticos y silvestres y con distintos genotipos con macrófagos bovinos y ovinos, al ser éstas las dos especies animales en las que la paratuberculosis causa mayores pérdidas económicas. Este análisis permitirá determinar la virulencia, patogenicidad y adaptación a un determinado hospedador de distintos aislados de Map y sentará las bases para tratar de mejorar las estrategias de manejo y control de la paratuberculosis en granjas donde el ganado doméstico comparte pastos con animales silvestres potencialmente infectados con Map. Con objeto de determinar diferencias en la virulencia en los primeros estadios de la infección, se comparó la entrada, multiplicación y supervivencia de 11 aislados de Map de diversos genotipos (C, S y B) y procedentes de distintas localizaciones geográficas y hospedadores (bovino, ovino, caprino, jabalí, bisonte, ciervo y gamo) en modelos de infección in vitro bovinos y ovinos. Así mismo, se analizó la respuesta de los macrófagos a la infección con los aislados de Map seleccionados mediante el análisis de los perfiles de expresión de determinadas citoquinas y proteínas implicadas en apoptosis y destrucción tisular. Ambos parámetros, capacidad de supervivencia de los distintos aislados de Map y respuesta de los macrófagos a la infección, se correlacionaron con objeto de determinar la virulencia de cada aislado en los hospedadores bovino y ovino. Así mismo, y con el fin de comparar la virulencia de diversos aislados de Map en estadios más avanzados de la infección se determinó su capacidad para generar microgranulomas en condiciones in vitro. Por último y con objeto de adaptar los modelos de macrófagos desarrollos en este tesis a otros objetivos, como por ejemplo para su utilización en la evaluación de actividad fagocítica inducida por prototipos vacunales, se desarrolló un método inmunomagnético de enriquecimiento de monocitos CD14 + de bovino, ovino y caprino utilizando nanopartículas magnéticas funcionalizadas con un anticuerpo monoclonal anti-CD14 humano en combinación con la tecnología MACS.NEIKER-Tecnalia, Instituto Vasco de Investigación y Desarrollo Agrari

Anti-Inflammatory and Antiapoptotic Responses to Infection: A Common Denominator of Human and Bovine Macrophages Infected with Mycobacterium avium Subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of a chronic intestinal inflammation in ruminants named Johne's disease or paratuberculosis and a possible etiopathological agent of human Crohn's disease (CD). Analysis of macrophage transcriptomes in response to Map infection is expected to provide key missing information in the understanding of the role of this pathogen in establishing an inappropriate and persistent infection in a susceptible host and of the molecular mechanisms that might underlie the early phases of CD. In this paper we summarize transcriptomic studies of human and bovine peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDMs), and macrophages-like cell lines in vitro infected with Map. Most studies included in this paper consistently reported common gene expression signatures of bovine and human macrophages in response to Map such as enhanced expression of the anti-inflammatory cytokines IL-10 and IL-6, which promote bacterial survival. Overexpression of IL-10 could be responsible for the Map-associated reduction in the expression of the proapoptotic TNF-α gene observed in bovine and human macrophages