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In humans and model species, alterations of sperm DNA methylation patterns have been reported in cases of spermatogenesis defects, male infertility and exposure to toxins or nutritional challenges, suggesting that a memory of environmental or physiological changes is recorded in the sperm methylome. The objective of this study was to ascertain if early life plane of nutrition could have a latent effect on DNA methylation patterns in sperm produced post-puberty. Holstein-Friesian calves were assigned to either a high (H) or moderate (M) plane of nutrition for the first 24 weeks of age, then reassigned to the M diet until puberty, resulting in HM and MM groups. Sperm DNA methylation patterns from contrasted subgroups of bulls in the HM (ejaculates recovered at 15 months of age; n = 9) and in the MM (15 and 16 months of age; n = 7 and 9, respectively) were obtained using Reduced Representation Bisulfite Sequencing. Both 15 and 16 months were selected in the MM treatment as these bulls reached puberty approximately 1 month after the HM bulls. Hierarchical clustering demonstrated that inter-individual variability unrelated to diet or age dominated DNA methylation profiles. While the comparison between 15 and 16 months of age revealed almost no change, 580 differentially methylated CpGs (DMCs) were identified between the HM and MM groups. Differentially methylated CpGs were mostly hypermethylated in the HM group, and enriched in endogenous retrotransposons, introns, intergenic regions, and shores and shelves of CpG islands. Furthermore, genes involved in spermatogenesis, Sertoli cell function, and the hypothalamic-pituitary-gonadal axis were targeted by differential methylation when HM and MM groups were compared at 15 months of age, reflecting the earlier timing of puberty onset in the HM bulls. In contrast, the genes still differentially methylated in MM bulls at 16 months of age were enriched for ATP-binding molecular function, suggesting that changes to the sperm methylome could persist even after the HM and MM bulls reached a similar level of sexual maturity. Together, results demonstrate that enhanced plane of nutrition in pre-pubertal calves associated with advanced puberty induced modest but persistent changes in sperm DNA methylation profiles after puberty., (Copyright © 2020 Perrier, Kenny, Chaulot-Talmon, Byrne, Sellem, Jouneau, Aubert-Frambourg, Schibler, Jammes, Lonergan, Fair and Kiefer.)

Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation.

Foot and mouth disease (FMD) is a highly contagious viral disease with high economic impact, representing a major threat for cloven-hooved mammals worldwide. Vaccines based on adjuvanted inactivated virus (iFMDV) induce effective protective immunity implicating antibody (Ab) responses. To reduce the biosafety constraints of the manufacturing process, a non-replicative human adenovirus type 5 vector encoding FMDV antigens (Ad5-FMDV) has been developed. Here we compared the immunogenicity of iFMDV and Ad5-FMDV with and without the ISA206VG emulsion-type adjuvant in sheep. Contrasted Ab responses were obtained: iFMDV induced the highest Ab levels, Ad5-FMDV the lowest ones, and ISA206VG increased the Ad5-FMDV-induced Ab responses to protective levels. Each vaccine generated heterogeneous Ab responses, with high and low responders, the latter being considered as obstacles to vaccine effectiveness. A transcriptomic study on total blood responses at 24 h post-vaccination revealed several blood gene module activities correlating with long-term Ab responses. Downmodulation of T cell modules' activities correlated with high responses to iFMDV and to Ad5-FMDV+ISA206VG vaccines as also found in other systems vaccinology studies in humans and sheep. The impact of cell cycle activity depended on the vaccine types, as it positively correlated with higher responses to iFMDV but negatively to non-adjuvanted Ad5-FMDV. Finally an elevated B cell activity at 24 h correlated with high Ab responses to the Ad5-FMDV+ISA206VG vaccine. This study provides insights into the early mechanisms driving the Ab response induced by different vaccine regimens including Ad5 vectors and points to T cell modules as early biomarker candidates of different vaccine-type efficacy across species., Competing Interests: Competing interestsN.V. is employed by SEPPIC. P.H. and M.C. are employed by Merial-Boehringer Ingelheim. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The other authors declare no competing interest., (© The Author(s) 2020.)

The evolution of the IFN system, the major innate antiviral mechanism of vertebrates, remains poorly understood. According to the detection of type I IFN genes in cartilaginous fish genomes, the system appeared 500 My ago. However, the IFN system integrates many other components, most of which are encoded by IFN-stimulated genes (ISGs). To shed light on its evolution, we have used deep RNA sequencing to generate a comprehensive list of ISGs of zebrafish, taking advantage of the high-quality genome annotation in this species. We analyzed larvae after inoculation of recombinant zebrafish type I IFN, or infection with chikungunya virus, a potent IFN inducer. We identified more than 400 zebrafish ISGs, defined as being either directly induced by IFN or induced by the virus in an IFNR-dependent manner. Their human orthologs were highly enriched in ISGs, particularly for highly inducible genes. We identified 72 orthology groups containing ISGs in both zebrafish and humans, revealing a core ancestral ISG repertoire that includes most of the known signaling components of the IFN system. Many downstream effectors were also already present 450 My ago in the common ancestor of tetrapods and bony fish and diversified as multigene families independently in the two lineages. A large proportion of the ISG repertoire is lineage specific; around 40% of protein-coding zebrafish ISGs had no human ortholog. We identified 14 fish-specific gene families containing multiple ISGs, including finTRIMs. This work illuminates the evolution of the IFN system and provides a rich resource to explore new antiviral mechanisms., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Long-term immunity is of great importance for protection against pathogens and has been extensively studied in mammals. Successive heterologous infections can affect the maintenance of immune memory, inducing attrition of T memory cells and diminishing B cell mediated protection. In fish, the basis of immune memory and the mechanisms of immunization to heterologous pathogens remain poorly understood. We sequentially immunized isogenic rainbow trout with two immunologically distinct viruses, VHSV and IPNV, either with one virus only or in combination, and analyzed the antibody responses and repertoires. Neutralizing antibodies and ELISPOT did not reveal an effect of heterologous immunization. Using a consensus read sequencing approach that incorporates unique barcodes to each cDNA molecule, we focused on the diversity expressed by selected responding VH/C combinations. We identified both public and private responses against VHSV and/or IPNV in all groups of fish. In fish immunized with two viruses, we registered no significant reduction in the persistence of the response toward the primary immunization. Similarly, the response to the second immunization was not affected by a prior vaccination to the other virus. Our data suggest that heterologous immunization does not enforce attrition of pre-existing antibody producing cells, which may impair the protection afforded by multiple successive vaccinations. These observations are potentially important to improve vaccination strategies practiced in aquaculture., (Copyright © 2019 Navelsaker, Magadan, Jouneau, Quillet, Olesen, Munang'andu, Boudinot and Evensen.)

Vaccination induces "public" antibody clonotypes common to all individuals of a species, that may mediate universal protection against pathogens. Only few studies tried to trace back the origin of these public B-cell clones. Here we used Illumina sequencing and computational modeling to unveil the mechanisms shaping the structure of the fish memory antibody response against an attenuated Viral Hemorrhagic Septicemia rhabdovirus. After vaccination, a persistent memory response with a public VH5JH5 IgM component was composed of dominant antibodies shared among all individuals. The rearrangement model showed that these public junctions occurred with high probability indicating that they were already favored before vaccination due to the recombination process, as shown in mammals. In addition, these clonotypes were in the naïve repertoire associated with larger similarity classes, composed of junctions differing only at one or two positions by amino acids with comparable properties. The model showed that this property was due to selective processes exerted between the recombination and the naive repertoire. Finally, our results showed that public clonotypes greatly expanded after vaccination displayed several VDJ junctions differing only by one or two amino acids with similar properties, highlighting a convergent response. The fish public memory antibody response to a virus is therefore shaped at three levels: by recombination biases, by selection acting on the formation of the pre-vaccination repertoire, and by convergent selection of functionally similar clonotypes during the response. We also show that naive repertoires of IgM and IgT have different structures and sharing between individuals, due to selection biases. In sum, our comparative approach identifies three conserved features of the antibody repertoire associated with public memory responses. These features were already present in the last common ancestors of fish and mammals, while other characteristics may represent species-specific solutions.

Lung inflammation is frequently involved in respiratory conditions and it is strongly controlled by mononuclear phagocytes (MNP). We previously studied porcine lung MNP and described a new population of cells presenting all the features of alveolar macrophages (AM) except for their parenchymal location, that we named AM-like cells. Herein we showed that AM-like cells are macrophages phagocytosing blood-borne particles, in agreement with a pulmonary intravascular macrophages (PIM) identity. PIM have been described microscopically long time ago in species from the Laurasiatheria superorder such as bovine, swine, cats or cetaceans. We observed that PIM were more inflammatory than AM upon infection with the porcine reproductive and respiratory syndrome virus (PRRSV), a major swine pathogen. Moreover, whereas PRRSV was thought to mainly target AM, we observed that PIM were a major producer of virus. The PIM infection was more correlated with viremia in vivo than AM infection. Finally like AM, PIM-expressed genes were characteristic of an embryonic monocyte-derived macrophage population, whose turnover is independent of bone marrow-derived hematopoietic precursors. This last observation raised the interesting possibility that AM and PIM originate from the same lung precursor.

Rationale: In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understood., Objectives: To document the role of B cells in TB in an unbiased manner., Methods: We generated the transcriptome of B cells isolated from Mycobacterium tuberculosis (Mtb)-infected mice and validated the identified key pathways using in vitro and in vivo assays. The obtained data were substantiated using B cells from pleural effusion of patients with TB., Measurements and Main Results: B cells isolated from Mtb-infected mice displayed a STAT1 (signal transducer and activator of transcription 1)-centered signature, suggesting a role for IFNs in B-cell response to infection. B cells stimulated in vitro with Mtb produced type I IFN, via a mechanism involving the innate sensor STING (stimulator of interferon genes), and antagonized by MyD88 (myeloid differentiation primary response 88) signaling. In vivo, B cells expressed type I IFN in the lungs of Mtb-infected mice and, of clinical relevance, in pleural fluid from patients with TB. Type I IFN expression by B cells induced an altered polarization of macrophages toward a regulatory/antiinflammatory profile in vitro. In vivo, increased provision of type I IFN by B cells in a murine model of B cell-restricted Myd88 deficiency correlated with an enhanced accumulation of regulatory/antiinflammatory macrophages in Mtb-infected lungs., Conclusions: Type I IFN produced by Mtb-stimulated B cells favors macrophage polarization toward a regulatory/antiinflammatory phenotype during Mtb infection.

Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)