IFN Receptor 2 Regulates TNF-α-Mediated Damaging Inflammation during Aspergillus Pulmonary Infection.

The increased incidence of invasive pulmonary aspergillosis, caused by Aspergillus fumigatus, occurring in patients infected with severe influenza or SARS-CoV-2, suggests that antiviral immune responses create an environment permissive to fungal infection. Our recent evidence suggests that absence of the type I IFN receptor 2 subunit (IFNAR2) of the heterodimeric IFNAR1/2 receptor is allowing for this permissive immune environment of the lung through regulation of damage responses. Because damage is associated with poor outcome to invasive pulmonary aspergillosis, this suggested that IFNAR2 may be involved in A. fumigatus susceptibility. In this study, we determined that absence of IFNAR2 resulted in increased inflammation, morbidity, and damage in the lungs in response to A. fumigatus challenge, whereas absence of IFNAR1 did not. Although the Ifnar2-/- mice had increased morbidity, we found that the Ifnar2-/- mice cleared more conidia compared with both wild-type and Ifnar1-/- mice. However, this early clearance did not prevent invasive disease from developing in the Ifnar2-/- mice as infection progressed. Importantly, by altering the inflamed environment of the Ifnar2-/- mice early during A. fumigatus infection, by neutralizing TNF-α, we were able to reduce the morbidity and fungal clearance in these mice back to wild-type levels. Together, our results establish a distinct role for IFNAR2 in regulating host damage responses to A. fumigatus and contributing to an A. fumigatus-permissive environment through regulation of inflammation. Specifically, our data reveal a role for IFNAR2 in regulating TNF-α-mediated damage and morbidity during A. fumigatus infection.

Diet-induced changes in metabolism influence immune response and viral shedding dynamics in Jamaican fruit bats.

Land-use change may drive viral spillover from bats into humans, partly through dietary shifts caused by decreased availability of native foods and increased availability of cultivated foods. We manipulated diets of Jamaican fruit bats to investigate whether diet influences shedding of a virus they naturally host. To reflect dietary changes experienced by wild bats during periods of nutritional stress, bats were fed either standard or putative suboptimal diets which were deprived of protein (suboptimal-sugar) and/or supplemented with fat (suboptimal-fat). Upon H18N11 influenza A-virus infection, bats fed the suboptimal-sugar diet shed the most viral RNA for the longest period, but bats fed the suboptimal-fat diet shed the least viral RNA for the shortest period. Unlike mice and humans, bats fed the suboptimal-fat diet displayed higher pre-infection levels of metabolic markers associated with gut health. Diet-driven heterogeneity in viral shedding may influence population-level viral dynamics in wild bats and alter risk of shedding and spillover to humans.

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells.

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.

Striated muscle gene therapy for the treatment of lipoprotein lipase deficiency.

Excessive circulating triglycerides due to reduction or loss of lipoprotein lipase activity contribute to hypertriglyceridemia and increased risk for pancreatitis. The only gene therapy treatment for lipoprotein lipase deficiency decreases pancreatitis but minimally reduces hypertriglyceridemia. Synthesized in multiple tissues including striated muscle and adipose tissue, lipoprotein lipase is trafficked to blood vessel endothelial cells where it is anchored at the plasma membrane and hydrolyzes triglycerides into free fatty acids. We conditionally knocked out lipoprotein lipase in differentiated striated muscle tissue lowering striated muscle lipoprotein lipase activity causing hypertriglyceridemia. We then crossed lipoprotein lipase striated muscle knockout mice with mice possessing a conditional avian retroviral receptor gene and injected mice with either a human lipoprotein lipase retrovirus or an mCherry control retrovirus. Post-heparin plasma lipoprotein lipase activity increased for three weeks following human lipoprotein lipase retroviral infection compared to mCherry infected mice. Human lipoprotein lipase infected mice had significantly lower blood triglycerides compared to mCherry controls and were comparable to wild-type blood triglyceride levels. Thus, targeted delivery of human lipoprotein lipase into striated muscle tissue identifies a potential therapeutic target for lipoprotein lipase deficiency.

Transplantation of Skeletal Muscle Stem Cells.

Transplanting adult stem cells provides a stringent test for self-renewal and the assessment of comparative engraftment in competitive transplant assays. Transplantation of satellite cells into mammalian skeletal muscle provided the first critical evidence that satellite cells function as adult muscle stem cells. Transplantation of a single satellite cell confirmed and extended this hypothesis, providing proof that the satellite cell is a bona fide adult skeletal muscle stem cell as reported by Sacco et al. (Nature 456(7221):502-506). Satellite cell transplantation has been further leveraged to identify culture conditions that maintain engraftment and to identify self-renewal deficits in satellite cells from aged mice. Conversion of iPSCs (induced pluripotent stem cells) to a satellite cell-like state, followed by transplantation, demonstrated that these cells possess adult muscle stem cell properties as reported by Darabi et al. (Stem Cell Rev Rep 7(4):948-957) and Mizuno et al. (FASEB J 24(7):2245-2253). Thus, transplantation strategies involving either satellite cells derived from adult muscles or derived from iPSCs may eventually be exploited as a therapy for treating patients with diseased or failing skeletal muscle. Here, we describe methods for isolating dispersed adult mouse satellite cells and satellite cells on intact myofibers for transplantation into recipient mice to study muscle stem cell function and behavior following engraftment .

Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay.

Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3' untranslated region. Upon satellite cell activation, p38α/ß MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis.

Regulation of nucleocytoplasmic transport in skeletal muscle.

Proper skeletal muscle function is dependent on spatial and temporal control of gene expression in multinucleated myofibers. In addition, satellite cells, which are tissue-specific stem cells that contribute critically to repair and maintenance of skeletal muscle, are also required for normal muscle physiology. Gene expression in both myofibers and satellite cells is dependent upon nuclear proteins that require facilitated nuclear transport. A unique challenge for myofibers is controlling the transcriptional activity of hundreds of nuclei in a common cytoplasm yet achieving nuclear selectivity in transcription at specific locations such as neuromuscular synapses and myotendinous junctions. Nucleocytoplasmic transport of macromolecular cargoes is regulated by a complex interplay among various components of the nuclear transport machinery, namely nuclear pore complexes, nuclear envelope proteins, and various soluble transport receptors. The focus of this review is to highlight what is known about the nuclear transport machinery and its regulation in skeletal muscle and to consider the unique challenges that multinucleated muscle cells as well as satellite cells encounter in regulating nucleocytoplasmic transport during cell differentiation and tissue adaptation. Understanding how regulated nucleocytoplasmic transport controls gene expression in skeletal muscle may lead to further insights into the mechanisms contributing to muscle growth and maintenance throughout the lifespan of an individual.