RESUMEN
Mayaro virus (MAYV) is an emerging arbovirus. Previous studies have shown antibody Fc effector functions are critical for optimal monoclonal antibody-mediated protection against alphaviruses; however, the requirement of Fc gamma receptors (FcγRs) for protection during natural infection has not been evaluated. Here, we showed mice lacking activating FcγRs (FcRγ-/-) developed prolonged clinical disease with more virus in joint-associated tissues. Viral clearance was associated with anti-MAYV cell surface binding rather than neutralizing antibodies. Lack of Fc-FcγR engagement increased the number of monocytes through chronic timepoints. Single cell RNA sequencing showed elevated levels of pro-inflammatory monocytes in joint-associated tissue with increased MAYV RNA present in FcRγ-/- monocytes and macrophages. Transfer of FcRγ-/- monocytes into wild type animals was sufficient to increase virus in joint-associated tissue. Overall, this study suggests that engagement of antibody Fc with activating FcγRs promotes protective responses during MAYV infection and prevents monocytes from being potential targets of infection.
RESUMEN
The plant cuticle is a complex extracellular lipid barrier that has multiple protective functions. We investigated cuticle deposition by integrating metabolomics and transcriptomics data gathered from six different maize seedling organs of four genotypes, the inbred lines B73 and Mo17, and their reciprocal hybrids. These datasets captured the developmental transition of the seedling from heterotrophic skotomorphogenic growth to autotrophic photomorphogenic growth, which is a transition that is highly vulnerable to environmental stresses. Statistical interrogation of these data reveals that the predominant determinant of cuticle composition is seedling organ type, whereas the seedling genotype has a smaller effect on this phenotype. Gene-to-metabolite associations assessed by integrated statistical analyses identified three gene networks connected with the deposition of different elements of the cuticle: a) cuticular waxes; b) monomers of lipidized cell wall biopolymers, including cutin and suberin; and c) both of these elements. These gene networks reveal three metabolic programs that appear to support cuticle deposition, including processes of chloroplast biogenesis, lipid metabolism, and molecular regulation (e.g., transcription factors, post-translational regulators and phytohormones). This study demonstrates the wider physiological metabolic context that can determine cuticle deposition and lays the groundwork for new targets for modulating properties of this protective barrier.
RESUMEN
BACKGROUND: Cancer neoantigens arise from protein-altering somatic mutations in tumor and rank among the most promising next-generation immuno-oncology agents when used in combination with immune checkpoint inhibitors. We previously developed a computational framework, REAL-neo, for identification, quality control, and prioritization of both class-I and class-II human leucocyte antigen (HLA)-presented neoantigens resulting from somatic single-nucleotide mutations, small insertions and deletions, and gene fusions. In this study, we developed a new module, SPLICE-neo, to identify neoantigens from aberrant RNA transcripts from two distinct sources: (1) DNA mutations within splice sites and (2) de novo RNA aberrant splicings. METHODS: First, SPLICE-neo was used to profile all DNA splice-site mutations in 11,892 tumors from The Cancer Genome Atlas (TCGA) and identified 11 profiles of splicing donor or acceptor site gains or losses. Transcript isoforms resulting from the top seven most frequent profiles were computed using novel logic models. Second, SPLICE-neo identified de novo RNA splicing events using RNA sequencing reads mapped to novel exon junctions from either single, double, or multiple exon-skipping events. The aberrant transcripts from both sources were then ranked based on isoform expression levels and z-scores assuming that individual aberrant splicing events are rare. Finally, top-ranked novel isoforms were translated into protein, and the resulting neoepitopes were evaluated for neoantigen potential using REAL-neo. The top splicing neoantigen candidates binding to HLA-A*02:01 were validated using in vitro T2 binding assays. RESULTS: We identified abundant splicing neoantigens in four representative TCGA cancers: BRCA, LUAD, LUSC, and LIHC. In addition to their substantial contribution to neoantigen load, several splicing neoantigens were potent tumor antigens with stronger bindings to HLA compared with the positive control of antigens from influenza virus. CONCLUSIONS: SPLICE-neo is the first tool to comprehensively identify and prioritize splicing neoantigens from both DNA splice-site mutations and de novo RNA aberrant splicings. There are two major advances of SPLICE-neo. First, we developed novel logic models that assemble and prioritize full-length aberrant transcripts from DNA splice-site mutations. Second, SPLICE-neo can identify exon-skipping events involving more than two exons, which account for a quarter to one-third of all skipping events.
Asunto(s)
Antígenos de Neoplasias , Neoplasias , Empalme del ARN , Humanos , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/genética , Neoplasias/inmunología , Neoplasias/genéticaRESUMEN
Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.
Asunto(s)
Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Humanos , Animales , Conejos , Bevacizumab/farmacología , Losartán/farmacología , Tuberculosis/microbiología , Antituberculosos/farmacología , Granuloma , Tuberculosis Latente/microbiologíaRESUMEN
BACKGROUND: Hemocytes are immune cells that patrol the mosquito hemocoel and mediate critical cellular defense responses against pathogens. However, despite their importance, a comprehensive transcriptome of these cells was lacking because they constitute a very small fraction of the total cells in the insect, limiting the study of hemocyte differentiation and immune function. RESULTS: In this study, an in-depth hemocyte transcriptome was built by extensive bulk RNA sequencing and assembly of hemocyte RNAs from adult A. gambiae female mosquitoes, based on approximately 2.4 billion short Illumina and about 9.4 million long PacBio high-quality reads that mapped to the A. gambiae PEST genome (P4.14 version). A total of 34,939 transcripts were annotated including 4,020 transcripts from novel genes and 20,008 novel isoforms that result from extensive differential splicing of transcripts from previously annotated genes. Most hemocyte transcripts identified (89.8%) are protein-coding while 10.2% are non-coding RNAs. The number of transcripts identified in the novel hemocyte transcriptome is twice the number in the current annotation of the A. gambiae genome (P4.14 version). Furthermore, we were able to refine the analysis of a previously published single-cell transcriptome (scRNAseq) data set by using the novel hemocyte transcriptome as a reference to re-define the hemocyte clusters and determine the path of hemocyte differentiation. Unsupervised pseudo-temporal ordering using the Tools for Single Cell Analysis software uncovered a novel putative prohemocyte precursor cell type that gives rise to prohemocytes. Pseudo-temporal ordering with the Monocle 3 software, which analyses changes in gene expression during dynamic biological processes, determined that oenocytoids derive from prohemocytes, a cell population that also gives rise to the granulocyte lineage. CONCLUSION: A high number of mRNA splice variants are expressed in hemocytes, and they may account for the plasticity required to mount efficient responses to many different pathogens. This study highlights the importance of a comprehensive set of reference transcripts to perform robust single-cell transcriptomic data analysis of cells present in low abundance. The detailed annotation of the hemocyte transcriptome will uncover new facets of hemocyte development and function in adult dipterans and is a valuable community resource for future studies on mosquito cellular immunity.