Mouse Memory CD8 T Cell Subsets Defined by Tissue-Resident Memory Integrin Expression Exhibit Distinct Metabolic Profiles.

Tissue-resident memory CD8 T cells (TRM) principally reside in peripheral nonlymphoid tissues, such as lung and skin, and confer protection against a variety of illnesses ranging from infections to cancers. The functions of different memory CD8 T cell subsets have been linked with distinct metabolic pathways and differ from other CD8 T cell subsets. For example, skin-derived memory T cells undergo fatty acid oxidation and oxidative phosphorylation to a greater degree than circulating memory and naive cells. Lung TRMs defined by the cell-surface expression of integrins exist as distinct subsets that differ in gene expression and function. We hypothesize that TRM subsets with different integrin profiles will use unique metabolic programs. To test this, differential expression and pathway analysis were conducted on RNA sequencing datasets from mouse lung TRMs yielding significant differences related to metabolism. Next, metabolic models were constructed, and the predictions were interrogated using functional metabolite uptake assays. The levels of oxidative phosphorylation, mitochondrial mass, and neutral lipids were measured. Furthermore, to investigate the potential relationships to TRM development, T cell differentiation studies were conducted in vitro with varying concentrations of metabolites. These demonstrated that lipid conditions impact T cell survival, and that glucose concentration impacts the expression of canonical TRM marker CD49a, with no effect on central memory-like T cell marker CCR7. In summary, it is demonstrated that mouse resident memory T cell subsets defined by integrin expression in the lung have unique metabolic profiles, and that nutrient abundance can alter differentiation.

Cell-to-Cell Variation in Defective Virus Expression and Effects on Host Responses during Influenza Virus Infection.

Virus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the variability at the single-cell level and how it impacts virus-host interactions at a system level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability in viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show (i) a correlation between DVGs and virus-induced variation of the host transcriptional program and (ii) an association between differential inductions of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single-cell level improve our understanding of the complex virus-host interplay during influenza virus infection.IMPORTANCE Defective influenza virus particles generated during viral replication carry incomplete viral genomes and can interfere with the replication of competent viruses. These defective genomes are thought to modulate the disease severity and pathogenicity of an influenza virus infection. Different defective viral genomes also introduce another source of variation across a heterogeneous cell population. Evaluating the impact of defective virus genomes on host cell responses cannot be fully resolved at the population level, requiring single-cell transcriptional profiling. Here, we characterized virus and host transcriptomes in individual influenza virus-infected cells, including those of defective viruses that arise during influenza A virus infection. We established an association between defective virus transcription and host responses and validated interfering and immunostimulatory functions of identified dominant defective viral genome species in vitro This study demonstrates the intricate effects of defective viral genomes on host transcriptional responses and highlights the importance of capturing host-virus interactions at the single-cell level.

Profiling the airway in the macaque model of tuberculosis reveals variable microbial dysbiosis and alteration of community structure.

BACKGROUND: The specific interactions of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), and the lung microbiota in infection are entirely unexplored. Studies in cancer and other infectious diseases suggest that there are important exchanges occurring between host and microbiota that influence the immunological landscape. This can result in alterations in immune regulation and inflammation both locally and systemically. To assess whether Mtb infection modifies the lung microbiome, and identify changes in microbial abundance and diversity as a function of pulmonary inflammation, we compared infected and uninfected lung lobe washes collected serially from 26 macaques by bronchoalveolar lavage over the course of infection. RESULTS: We found that Mtb induced an initial increase in lung microbial diversity at 1 month post infection that normalized by 5 months of infection across all macaques. Several core genera showed global shifts from baseline and throughout infection. Moreover, we identified several specific taxa normally associated with the oral microbiome that increased in relative abundance in the lung following Mtb infection, including SR1, Aggregatibacter, Leptotrichia, Prevotella, and Campylobacter. On an individual macaque level, we found significant heterogeneity in both the magnitude and duration of change within the lung microbial community that was unrelated to lung inflammation and lobe involvement as seen by positron emission tomography/computed tomography (PET/CT) imaging. By comparing microbial interaction networks pre- and post-infection using the predictive algorithm SPIEC-EASI, we observe that extra connections are gained by Actinomycetales, the order containing Mtb, in spite of an overall reduction in the number of interactions of the whole community post-infection, implicating Mtb-driven ecological reorganization within the lung. CONCLUSIONS: This study is the first to probe the dynamic interplay between Mtb and host microbiota longitudinally and in the macaque lung. Our findings suggest that Mtb can alter the microbial landscape of infected lung lobes and that these interactions induce dysbiosis that can disrupt oral-airway boundaries, shift overall lung diversity, and modulate specific microbial relationships. We also provide evidence that this effect is heterogeneous across different macaques. Overall, however, the changes to the airway microbiota after Mtb infection were surprisingly modest, despite a range of Mtb-induced pulmonary inflammation in this cohort of macaques.

Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia.

The co-evolution of myxoma virus (MYXV) and the European rabbit occurred independently in Australia and Europe from different progenitor viruses. Although this is the canonical study of the evolution of virulence, whether the genomic and phenotypic outcomes of MYXV evolution in Europe mirror those observed in Australia is unknown. We addressed this question using viruses isolated in the United Kingdom early in the MYXV epizootic (1954-1955) and between 2008-2013. The later UK viruses fell into three distinct lineages indicative of a long period of separation and independent evolution. Although rates of evolutionary change were almost identical to those previously described for MYXV in Australia and strongly clock-like, genome evolution in the UK and Australia showed little convergence. The phenotypes of eight UK viruses from three lineages were characterized in laboratory rabbits and compared to the progenitor (release) Lausanne strain. Inferred virulence ranged from highly virulent (grade 1) to highly attenuated (grade 5). Two broad disease types were seen: cutaneous nodular myxomatosis characterized by multiple raised secondary cutaneous lesions, or an amyxomatous phenotype with few or no secondary lesions. A novel clinical outcome was acute death with pulmonary oedema and haemorrhage, often associated with bacteria in many tissues but an absence of inflammatory cells. Notably, reading frame disruptions in genes defined as essential for virulence in the progenitor Lausanne strain were compatible with the acquisition of high virulence. Combined, these data support a model of ongoing host-pathogen co-evolution in which multiple genetic pathways can produce successful outcomes in the field that involve both different virulence grades and disease phenotypes, with alterations in tissue tropism and disease mechanisms.

Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration.

Selective neuronal loss is the hallmark of neurodegenerative diseases. In patients with amyotrophic lateral sclerosis (ALS), most motor neurons die but those innervating extraocular, pelvic sphincter, and slow limb muscles exhibit selective resistance. We identified 18 genes that show >10-fold differential expression between resistant and vulnerable motor neurons. One of these, matrix metalloproteinase-9 (MMP-9), is expressed only by fast motor neurons, which are selectively vulnerable. In ALS model mice expressing mutant superoxide dismutase (SOD1), reduction of MMP-9 function using gene ablation, viral gene therapy, or pharmacological inhibition significantly delayed muscle denervation. In the presence of mutant SOD1, MMP-9 expressed by fast motor neurons themselves enhances activation of ER stress and is sufficient to trigger axonal die-back. These findings define MMP-9 as a candidate therapeutic target for ALS. The molecular basis of neuronal diversity thus provides significant insights into mechanisms of selective vulnerability to neurodegeneration.

Glioblastoma stem-like cells give rise to tumour endothelium.

Glioblastoma (GBM) is among the most aggressive of human cancers. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133(+) fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133(+) stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133(+)/CD144(+) progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133(+) tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab or to a γ-secretase inhibitor as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133(+) cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.