Leveraging large-scale datasets and single cell omics data to develop a polygenic score for cisplatin-induced ototoxicity

Abstract Background Cisplatin-induced ototoxicity (CIO), characterized by irreversible and progressive bilateral hearing loss, is a prevalent adverse effect of cisplatin chemotherapy. Alongside clinical risk factors, genetic variants contribute to CIO and genome-wide association studies (GWAS) have highlighted the polygenicity of this adverse drug reaction. Polygenic scores (PGS), which integrate information from multiple genetic variants across the genome, offer a promising tool for the identification of individuals who are at higher risk for CIO. Integrating large-scale hearing loss GWAS data with single cell omics data holds potential to overcome limitations related to small sample sizes associated with CIO studies, enabling the creation of PGSs to predict CIO risk. Results We utilized a large-scale hearing loss GWAS and murine inner ear single nuclei RNA-sequencing (snRNA-seq) data to develop two polygenic scores: a hearing loss PGS (PGSHL) and a biologically informed PGS for CIO (PGSCIO). The PGSCIO included only variants which mapped to genes that were differentially expressed within cochlear cells that showed differential abundance in the murine snRNA-seq data post-cisplatin treatment. Evaluation of the association of these PGSs with CIO in our target CIO cohort revealed that PGSCIO demonstrated superior performance (P = 5.54 × 10− 5) relative to PGSHL (P = 2.93 × 10− 3). PGSCIO was also associated with CIO in our test cohort (P = 0.04), while the PGSHL did not show a significant association with CIO (P = 0.52). Conclusion This study developed the first PGS for CIO using a large-scale hearing loss dataset and a biologically informed filter generated from cisplatin-treated murine inner ear snRNA-seq data. This innovative approach offers new avenues for developing PGSs for pharmacogenomic traits, which could contribute to the implementation of tailored therapeutic interventions. Further, our approach facilitated the identification of specific cochlear cells that may play critical roles in CIO. These novel insights will guide future research aimed at developing targeted therapeutic strategies to prevent CIO

Development and application of a vaccinia virus based system to study viral proteins modulating interferon expression and interferon induced antiviral activities

The interferon (IFN) system is integral to antiviral innate immunity in vertebrate hosts. Inside a cell, viral pathogen associated molecular patterns (PAMPs) trigger the IFN response, comprised of IFN induction and an IFN-induced antiviral state. However, viruses have evolved strategies to counteract the IFN system. The E3 protein of vaccinia virus (VV), encoded by the E3L gene, impedes cytokine expression and suppresses the activation and function of antiviral proteins. Deletion of the E3L gene (VVΔE3L) produces an IFN sensitive mutant virus that is replication defective in most human cell lines. Due to the limited human cell lines available to support VVΔE3L replication, the capacity of E3 inhibition of human IFN-induced antiviral activities is not well defined. In this study, VVΔE3L was generated and characterized to facilitate the study of other viral IFN antagonists at modulating human IFN-induced antiviral responses. A human liver carcinoma cell line, Huh7, was found to support VVΔE3L replication. A comprehensive analysis of VVΔE3L IFN sensitivity revealed E3 inhibits all human type I and type II IFN-induced antiviral activities by modulation of the protein kinase R (PKR) pathway. Influenza non-structural protein 1 (NS1) is well-known to mediate the suppression of IFN induction and IFN action in influenza virus infections. However, the IFN antagonizing potential of influenza NS1 may be virus subtype and/or isolate specific. VVΔE3L was next applied as an expression vector to study influenza NS1 function in modulating IFN-induced antiviral activities and IFN induction in human cells. Recombinant viruses were generated to express influenza NS1 (from avian H5N1 and pandemic viruses 1918 pH1N1, 1968 pH3N2, and 2009 pH1N1) in replacement of E3. It was found that influenza NS1 inhibits human IFN-induced antiviral activity in a subtype and isolate specific manner. Moreover, influenza NS1 differentially regulates human IFN expression in a virus isolate-dependent manner. Altogether, this work highlights the potential of VVΔE3L as an excellent virus model system to study viral proteins modulating IFN expression and IFN-induced antiviral activities in human cells.February 201

Specification of CD8+ T lymphocyte fates during adaptive immunity revealed by single cell gene expression analyses (P1448)

Abstract Although it is well established that heterogeneity of lymphocyte fate is an essential feature of adaptive immune responses, how and when these divergent cellular fates are specified remains unknown. It has been previously shown that a T lymphocyte responding to a microbial infection can undergo asymmetric division to yield two daughter cells that are differentially fated from inception. Such a model suggests that the progeny arising from each of the two differentially fated daughter cells might exhibit distinct patterns of gene expression. Because strategies analyzing bulk cell populations cannot distinguish differences among individual cells, we applied high-throughput single cell gene expression profiling to interrogate single CD8+ T lymphocytes throughout the course of an immune response to a bacterial infection. We demonstrate that terminally differentiated effector cells and self-renewing memory cells exhibit unique gene expression profiles. Moreover, we are able to identify cells early during the immune response, beginning with the first cell division, that exhibit distinct gene expression patterns characteristic of effector or memory cells. Finally, we derive a network model that allows us to predict the sequential activation of genes that orchestrate each cellular fate, providing a comprehensive view of CD8+ T lymphocyte differentiation. Together, these results provide novel insights into the specification of lymphocyte fates during an immune response.</jats:p

Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons▿

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L−C7L−) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L−C7L− at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L−C7L− fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells

Vaccinia Virus E3 Suppresses Expression of Diverse Cytokines through Inhibition of the PKR, NF-κB, and IRF3 Pathways▿

The vaccinia virus double-stranded RNA binding protein E3 has been demonstrated to inhibit the expression of cytokines, including beta interferon (IFN-β) and tumor necrosis factor alpha (TNF-α). However, few details regarding the molecular mechanisms of this inhibition have been described. Using real-time PCR arrays, we found that E3 suppressed the induction of a diverse array of cytokines representing members of the IFN, interleukin (IL), TNF, and transforming growth factor cytokine families. We discovered that the factor(s) responsible for the induction of IL-6, TNF-α, and inhibin beta A (INHBA) was associated with the early and late phases of virus infection. In contrast, the factor(s) which regulates IFN-β induction was associated with the late phase of replication. We have found that expression of these cytokines can be induced by transfection of cells with RNA isolated from vaccinia virus-infected cells. Moreover, we provide evidence that E3 antagonizes both PKR-dependent and PKR-independent pathways to regulate cytokine expression. PKR-dependent activation of p38 and NF-κB was required for vaccinia virus-induced INHBA expression, whereas induction of TNF-α required only PKR-dependent NF-κB activation. In contrast, induction of IL-6 and IFN-β was largely PKR independent. IL-6 induction is regulated by NF-κB, while IFN-β induction is mediated by IFN-β promoter stimulator 1 and IFN regulatory factor 3/NF-κB. Collectively, these results indicate that E3 suppresses distinct but interlinked host signaling pathways to inhibit the expression of a diverse array of cytokines

RNA species generated in vaccinia virus infected cells activate cell type-specific MDA5 or RIG-I dependent interferon gene transcription and PKR dependent apoptosis

AbstractRNA species produced during virus replication are pathogen-associated molecular patterns (PAMPs) triggering cellular innate immune responses including induction of type I interferon expression and apoptosis. Pattern recognition receptors (PRRs) for these RNAs include the retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation associated gene 5 (MDA5) and the dsRNA dependent protein kinase (PKR). Currently, poxvirus PAMPs and their associated PRRs are not well characterized. We report that RNA species generated in vaccinia infected cells can activate MDA5 or RIG-I dependent interferon-β (IFN-β) gene transcription in a cell type-specific manner. These RNA species also induce the activation of apoptosis in a PKR dependent, but MDA5 and RIG-I independent, manner. Collectively our results demonstrate that RNA species generated during vaccinia virus replication are major PAMPs activating apoptosis and IFN-β gene transcription. Moreover, our results delineate the signaling pathways involved in the recognition of RNA-based poxvirus PAMPs

Regulation of Asymmetric Division and CD8+ T Lymphocyte Fate Specification by Protein Kinase Cζ and Protein Kinase Cλ/ι

Abstract During an immune response against a microbial pathogen, activated naive T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. In this study, we show that the absence of the atypical protein kinase C (PKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8+ T lymphocyte division. These alterations were associated with aberrant acquisition of a pre-effector transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for atypical PKC in regulating asymmetric division and the specification of divergent CD8+ T lymphocyte fates early during an immune response.</jats:p