Global characterization of the immune response to inoculation of aluminium hydroxide-based vaccines by RNA sequencing

xix, 195 p.En este trabajo se han analizado muestras correspondientes a un experimento de vacunación de larga duración. Múltiples ovejas fueron expuestas a varias vacunas compuestas de aluminio hidróxido como adyuvante en un periodo de 475 días, con el objetivo de estudiar el mecanismo de acción de dicho adyuvante en el sistema inmune y comprobar si es capaz de llegar a órganos distantes como el cerebro después de su inoculación. Para ello se extrajeron muestras de células mononucleares de sangre periférica y de la corteza del lóbulo parietal y se usaron para la preparación de librerías de secuenciación de ARN y microRNAs (Total RNA-seq y miRNA-seq). Las librerías se analizaron mediante herramientas bioinformáticas y se realizaron multiples análisis: 1. Expresión diferencial tanto para los datos de RNA-seq como para los de miRNA-seq; 2. Anotación de nuevos miRNAs en oveja; 3. Predicción de targets para los miRNAs y análisis de co-expresión con los datos de RNA-seq. Además, como las librerías de Total RNA-seq retienen el ARN no codificante, que esta pobremente anotado en oveja, dichos datos se usaron para la anotación de ARN circulares en oveja y se estudió si dichos ARN no-codificantes pudieran tener algún rol en la actividad del aluminio como adyuvante

Global characterization of the immune response to inoculation of aluminium hydroxide-based vaccines by RNA sequencing

xix, 195 p.En este trabajo se han analizado muestras correspondientes a un experimento de vacunación de larga duración. Múltiples ovejas fueron expuestas a varias vacunas compuestas de aluminio hidróxido como adyuvante en un periodo de 475 días, con el objetivo de estudiar el mecanismo de acción de dicho adyuvante en el sistema inmune y comprobar si es capaz de llegar a órganos distantes como el cerebro después de su inoculación. Para ello se extrajeron muestras de células mononucleares de sangre periférica y de la corteza del lóbulo parietal y se usaron para la preparación de librerías de secuenciación de ARN y microRNAs (Total RNA-seq y miRNA-seq). Las librerías se analizaron mediante herramientas bioinformáticas y se realizaron multiples análisis: 1. Expresión diferencial tanto para los datos de RNA-seq como para los de miRNA-seq; 2. Anotación de nuevos miRNAs en oveja; 3. Predicción de targets para los miRNAs y análisis de co-expresión con los datos de RNA-seq. Además, como las librerías de Total RNA-seq retienen el ARN no codificante, que esta pobremente anotado en oveja, dichos datos se usaron para la anotación de ARN circulares en oveja y se estudió si dichos ARN no-codificantes pudieran tener algún rol en la actividad del aluminio como adyuvante

Novel CircRNA Discovery in Sheep Shows Evidence of High Backsplice Junction Conservation

Circular RNAs (circRNAs) are covalently closed circular non-coding RNAs. Due to their structure, circRNAs are more stable and have longer half-lives than linear RNAs making them good candidates for disease biomarkers. Despite the scientific relevance of these molecules, the study of circRNAs in non-model organisms is still in its infancy. Here, we analyse total RNA-seq data to identify circRNAs in sheep from peripheral blood mononuclear cells (PBMCs) and parietal lobe cortex. Out of 2510 and 3403 circRNAs detected in parietal lobe cortex and in PBMCs, a total of 1379 novel circRNAs were discovered. Remarkably, around 63% of all detected circRNAs were found to be completely homologous to a circRNA annotated in human. Functional enrichment analysis was conducted for both tissues based on GO terms and KEGG pathways. The enriched terms suggest an important role of circRNAs from encephalon in synaptic functions and the involvement of circRNAs from PBMCs in basic immune system functions. In addition to this, we investigated the role of circRNAs in repetitive vaccination experiments via differential expression analysis and did not detect any significant relationship. At last, our results support both the miRNA sponge and the miRNA shuttle functions of CDR1-AS in sheep brain. To our knowledge, this is the first study on circRNA annotation in sheep PBMCs or parietal lobe cortex samples.This work was supported by a Spanish Ministry of Economy grant AGL2013-49137-C3 to BMJ; EV-M is a predoctoral fellow from the University of the Basque Country (UPV/EHU) [PIF15/361] and received a grant for a short-term scientific mission (STSM) from the Functional Annotation of Animal Genomes—European network (FAANG-Europe) [COST Action CA15112

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

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

RNA-Seq analysis of ileocecal valve and peripheral blood from Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis revealed dysregulation of the CXCL8/IL8 signaling pathway

17 páginas, 5 tablas, 5 figuras.Paratuberculosis is chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Whole RNA-sequencing (RNA-Seq) is a promising source of novel biomarkers for early MAP infection and disease progression in cattle. Since the blood transcriptome is widely used as a source of biomarkers, we analyzed whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. Total RNA was prepared from peripheral blood (PB) and ICV samples, and RNA-Seq was used to compare gene expression between animals with focal or diffuse histopathological lesions in gut tissues versus control animals with no detectable signs of infection. Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural MAP infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IF127). The biological processes (BP) enriched in the PB gene expression profiles from the cows with diffuse lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched. Some of the identified DE genes, BP and metabolic pathways will be studied further to develop novel diagnostic tools, vaccines and immunotherapeutics.This work was supported by grants from the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA) and by European Funds for Regional Development (FEDER) (INIA RTA2014-00009-C02 and RTA2018-094192). The study is partially funded by the Principado de Asturias (PCTI 2018-2020, GRUPIN: IDI2018-000237). Maria Canive and Cristina Blanco-Vazquez are recipients of INIA fellowships. The authors thank ASTEGA Veterinary services for their assistance on sample collection. We are also grateful to Kyle Hearn for the careful editing of the manuscript

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)

Methionine adenosyltransferase 1a antisense oligonucleotides activate the liver-brown adipose tissue axis preventing obesity and associated hepatosteatosis

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis. High methionine and S-adenosylmethionine serum levels are related with obesity. Here the authors show that knockdown of methionine adenosyltransferase by using antisense oligonucleotides provides beneficial effects in obesity and comorbidities.This work was supported by Ayudas para apoyar grupos de investigacion del sistema Universitario Vasco (IT971-16) and MCIU/AEI/FEDER, UE (RTI2018-095134-B-100) (to P.A.), (RTI2018-099413-B-I00 and RED2018-102379-T) (to R.N.), PID2020119486RB-100 (to M.V.R.) and (RTI2018-096759-A-100) (to T.C.D). EFSD/Lilly European Diabetes Research Program, MICIU (PID2019-104399RB-I00), Fundacion AECC PROYE19047SABI, and Comunidad de Madrid IMMUNOTHERCAN-CM B2017/BMD-3733 (to G.S.). La CAIXA Foundation LCF/PR/HP17/52190004, MINECO-FEDER SAF2017-87301-R, AYUDAS FUNDACION BBVA A EQUIPOS DE INVESTIGACION CIENTIFICA UMBRELLA 2018 and AECC Scientific Foundation, grant name: Rare Cancers 2017 (to M.L.M.-C.). AECC Scientific Foundation (to T.C.D.). Xunta de Galicia 2020-PG015 (to R.N.) Gilead Sciences International Research Scholars Program in Liver Disease (to M.V.R.). Personal fellows: E.P.F. was awarded with Juan de la Cierva-Formacion, FJC2018-035449-I. C.F. was awarded with Sara Borrell (CD19/00078). CIC bioGUNE thanks MCIU for the Severo Ochoa Excellence Accreditation (SEV-2016-0644). The authors thank Dr. Manuel Lafitas laboratory (Getxo, Bizkaia, Spain) for his valuable help in the analysis of biochemical parameters