Protective role of RIPK1 scaffolding against HDV-induced hepatocyte cell death and the significance of cytokines in mice.

Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNα/ßR KO and wild type mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.

Identification of sheep lncRNAs related to the immune response to vaccines and aluminium adjuvants.

BACKGROUND: Long non-coding RNAs (lncRNAs) are involved in several immune processes, including the immune response to vaccination, but most of them remain uncharacterised in livestock species. The mechanism of action of aluminium adjuvants as vaccine components is neither not fully understood. RESULTS: We built a transcriptome from sheep PBMCs RNA-seq data in order to identify unannotated lncRNAs and analysed their expression patterns along protein coding genes. We found 2284 novel lncRNAs and assessed their conservation in terms of sequence and synteny. Differential expression analysis performed between animals inoculated with commercial vaccines or aluminium adjuvant alone and the co-expression analysis revealed lncRNAs related to the immune response to vaccines and adjuvants. A group of co-expressed genes enriched in cytokine signalling and production highlighted the differences between different treatments. A number of differentially expressed lncRNAs were correlated with a divergently located protein-coding gene, such as the OSM cytokine. Other lncRNAs were predicted to act as sponges of miRNAs involved in immune response regulation. CONCLUSIONS: This work enlarges the lncRNA catalogue in sheep and puts an accent on their involvement in the immune response to repetitive vaccination, providing a basis for further characterisation of the non-coding sheep transcriptome within different immune cells.

Expression analysis of lung miRNAs responding to ovine VM virus infection by RNA-seq.

BACKGROUND: MicroRNAs (miRNAs) are short endogenous, single-stranded, noncoding small RNA molecules of approximately 22 nucleotides in length. They regulate gene expression posttranscriptionally by silencing mRNA expression, thus orchestrating many physiological processes. The Small Ruminant Lentiviruses (SRLV) group includes the Visna Maedi Virus (VMV) and Caprine Arthritis Encephalitis (CAEV) viruses, which cause a disease in sheep and goats characterized by pneumonia, mastitis, arthritis and encephalitis. Their main target cells are from the monocyte/macrophage lineage. To date, there are no studies on the role of miRNAs in this viral disease. RESULTS: Using RNA-seq technology and bioinformatics analysis, the expression levels of miRNAs during different clinical stages of infection were studied. A total of 212 miRNAs were identified, of which 46 were conserved sequences in other species but found for the first time in sheep, and 12 were completely novel. Differential expression analysis comparing the uninfected and seropositive groups showed changes in several miRNAs; however, no significant differences were detected between seropositive asymptomatic and diseased sheep. The robust increase in the expression level of oar-miR-21 is consistent with its increased expression in other viral diseases. Furthermore, the target prediction of the dysregulated miRNAs revealed that they control genes involved in proliferation-related signalling pathways, such as the PI3K-Akt, AMPK and ErbB pathways. CONCLUSIONS: To the best of our knowledge, this is the first study reporting miRNA profiling in sheep in response to SRLV infection. The known functions of oar-miR-21 as a regulator of inflammation and proliferation appear to be a possible cause of the lesions caused in the sheep's lungs. This miRNA could be an indicator for the severity of the lung lesions, or a putative target for therapeutic intervention.

Identifying transcriptomic profiles in ovine spleen after repetitive vaccination.

Aluminum hydroxide has long been employed as a vaccine adjuvant for its safety profile, although its precise mechanism of action remains elusive. In this study, we investigated the transcriptomic responses in sheep spleen following repetitive vaccination with aluminum adjuvanted vaccines and aluminum hydroxide alone. Notably, this work represents the first exploration of the sheep spleen transcriptome in such conditions. Animals were splitted in 3 treatment groups: vaccine group, adjuvant alone group and control group. A total of 18 high-depth RNA-seq libraries were sequenced, resulting in a rich dataset which also allowed isoform-level analysis. The comparisons between vaccine-treated and control groups (V vs C) as well as between vaccine-treated and adjuvant-alone groups (V vs A) revealed significant alterations in gene expression profiles, including protein coding genes and long non-coding RNAs. Among the differentially expressed genes, many were associated with processes such as endoplasmic reticulum (ER) stress, immune response and cell cycle. The analysis of co-expression modules further indicated a correlation between vaccine treatment and genes related to ER stress and unfolded protein response. Surprisingly, adjuvant-alone treatment had little impact on the spleen transcriptome. Additionally, the role of alternative splicing in the immune response was explored. We identified isoform switches in genes associated with immune regulation and inflammation, potentially influencing protein function. In conclusion, this study provides valuable insights into the transcriptomic changes in sheep spleen following vaccination with aluminum adjuvanted vaccines and aluminum hydroxide alone. These findings shed light on the molecular mechanisms underlying vaccine-induced immune responses and emphasize the significance of antigenic components in aluminum adjuvant mechanism of action. Furthermore, the analysis of alternative splicing revealed an additional layer of complexity in the immune response to vaccination in a livestock species.

Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

The sheep miRNAome: Characterization and distribution of miRNAs in 21 tissues.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate gene expression. In livestock species, miRNAs also show great potential as biomarkers for animal health and product quality. In sheep, few miRNAs have been described in comparison with other livestock species or model organisms. We uniformly analyzed 172 public ovine small RNA sequencing datasets from 21 different tissues in order to predict conserved and novel miRNA precursors and profile their expression patterns. In addition to the 106 annotated sheep miRNAs, 1047 previously unannotated miRNA precursor sequences were detected and 41 % of them were assigned an orthologue from other close species. Considering expression levels, a set of miRNAs with high sequence conservation were detected in all tissues, while 733 mature miRNAs were robustly expressed in at least one tissue. 270 miRNAs showed high tissue specificity index values. Brain, male reproductive tissues and PBMCs showed the most distinct expression patterns. Strikingly, over one hundred precursors from the ruminant specific family of mir-2284/mir-2285 miRNAs were found, which were enriched in immune related tissues. This work supports the known high conservation of many miRNAs, but also highlights the potential of clade-specific innovations in ruminant evolution.

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.

Transcriptomic meta-analysis reveals unannotated long non-coding RNAs related to the immune response in sheep.

Long non-coding RNAs (lncRNAs) are involved in several biological processes, including the immune system response to pathogens and vaccines. The annotation and functional characterization of lncRNAs is more advanced in humans than in livestock species. Here, we take advantage of the increasing number of high-throughput functional experiments deposited in public databases in order to uniformly analyse, profile unannotated lncRNAs and integrate 422 ovine RNA-seq samples from the ovine immune system. We identified 12302 unannotated lncRNA genes with support from independent CAGE-seq and histone modification ChIP-seq assays. Unannotated lncRNAs showed low expression levels and sequence conservation across other mammal species. There were differences in expression levels depending on the genomic location-based lncRNA classification. Differential expression analyses between unstimulated and samples stimulated with pathogen infection or vaccination resulted in hundreds of lncRNAs with changed expression. Gene co-expression analyses revealed immune gene-enriched clusters associated with immune system activation and related to interferon signalling, antiviral response or endoplasmic reticulum stress. Besides, differential co-expression networks were constructed in order to find condition-specific relationships between coding genes and lncRNAs. Overall, using a diverse set of immune system samples and bioinformatic approaches we identify several ovine lncRNAs associated with the response to an external stimulus. These findings help in the improvement of the ovine lncRNA catalogue and provide sheep-specific evidence for the implication in the general immune response for several lncRNAs.

The dystrophia myotonica WD repeat-containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase.

Despite its potential clinical relevance, the product of the DMWD (dystrophia myotonica, WD repeat containing) gene is a largely uncharacterized protein. The DMWD amino acid sequence is similar to that of WDR20, a known regulator of the USP12 and USP46 deubiquitinases (DUBs). Here, we apply a combination of in silico and experimental methods to investigate several aspects of DMWD biology. Molecular evolution and phylogenetic analyses reveal that WDR20 and DMWD, similar to USP12 and USP46, arose by duplication of a common ancestor during the whole genome duplication event in the vertebrate ancestor lineage. The analysis of public human gene expression datasets suggests that DMWD expression is positively correlated with USP12 expression in normal tissues and negatively correlated with WDR20 expression in tumors. Strikingly, a survey of the annotated interactome for DMWD and WDR20 reveals a largely nonoverlapping set of interactors for these proteins. Experimentally, we first confirmed that DMWD binds both USP12 and USP46 through direct coimmunoprecipitation of epitope-tagged proteins. We found that DMWD and WDR20 share the same binding interface in USP12, suggesting that their interaction with the DUB may be mutually exclusive. Finally, we show that both DMWD and WDR20 promote USP12 enzymatic activity, but they differentially modulate the subcellular localization of the DUB. Altogether, our findings suggest a model whereby mutually exclusive binding of DMWD and WDR20 to USP12 may lead to formation of deubiquitinase complexes with distinct subcellular localization, potentially targeting different substrate repertoires.

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.