Supplementary MaterialsSupplementary Figures 41598_2017_7724_MOESM1_ESM. In comparison to WT mice, the B2-deficient

Supplementary MaterialsSupplementary Figures 41598_2017_7724_MOESM1_ESM. In comparison to WT mice, the B2-deficient lungs exhibited induction of genes that exhibit surfactant protein, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after seven days, temporal induction of CCAAT/enhancer binding proteins CEBP, , and mRNAs 3C14 times after infections, and differences in alveolar extracellular matrix respiratory and integrity technicians. Stream cytometry and gene expression studies demonstrated robust recovery of alveolar macrophages and recruitment of CD4+ lymphocytes in B2-deficient lungs. Targeting of endophilin B2 alleviates adverse effects of IAV infection on respiratory and immune cells enabling restoration of alveolar homeostasis. Introduction Pathogenic influenza A virus (IAV) strains that arise frequently by mutation or re-assortment of the viral genome have persistently caused seasonal epidemics, regional zoonotic infections, and periodic global pandemics with high morbidity and mortality for centuries1, 2. IAV causes productive infection in airway epithelial cells enabling the buy Tideglusib spread of infection to the lower respiratory tract, disruption of surfactant metabolism, and loss of epithelial barrier integrity3C5; restrictive infection that primes activation of capillary endothelial cells6; and abortive infection in alveolar macrophages and dendritic cells that contributes to induction of innate and adaptive immunity7, 8. The variable life cycle and genetic and antigenic diversity of IAV strains may determine IAV pathogenicity in different host cells9. The respiratory illness caused by severe IAV infection is a poorly resolving post-viral pneumonitis that can progress to acute respiratory distress syndrome (ARDS) and may be fatal depending on the extend of lung injury10, 11. Pathogenic IAV strains disrupt endothelial and epithelial tight junctions4, enhancing microvascular permeability and leakage of fluid into air sacs, and perturb surfactant lipid and protein metabolism3 by alveolar type II epithelial cells increasing surface tension at the air-liquid interface. In addition to functional disruption of the respiratory barrier, IAV infection elicits apoptosis and ER stress that may further aggravate tissue damage and inflammation12C14. The incipient edema, respiratory imbalance between air and fluid-filled alveoli, and the collateral immune response to the viral infection exacerbate respiratory injury that manifest as excessive production of inflammatory mediators, diffuse alveolar damage and hypoxia in terminal ARDS11. Thus, a better understanding of the host factors that contribute to IAV pathogenicity is crucial for the development of therapies that prevent pneumonia and intractable lung injury from IAV infection. Endophilin B2 is a member of the endophilin family of proteins containing an amino-terminal Bin-Amphiphysin-Rvs (N-BAR) domain15, 16. BAR domains are involved in protein-protein dimerization, membrane binding, and curvature formation and sensing16. B2 dimerizes with its better known paralog endophilin B1(B1), which promotes buy Tideglusib mitochondrial apoptosis16, 17, autophagosome formation18, endocytic degradation of growth factor receptors19, and adipose tissue lipid metabolism and insulin resistance20. The B1/B2 heterodimer has been found to be indispensable for degradation buy Tideglusib of the inner mitochondrial membrane and mitophagy for autophagy-dependent clearance of damaged mitochondria21. Interestingly, B2 binding to plectin 1 modulates nuclear cytoskeletal positioning and mechano-transduction22, 23. The present study examines the role of B2 expression buy Tideglusib in pathogenesis of IAV infection using transgenic mice and is based on our recent findings that loss of B2 delays formation of autophagosomes, endosomal acidification, and endosomal trafficking of IAV (B2?/?) mice were generated buy Tideglusib using 129/SvEV ES cells and C57BL/6? J recipients as recently described24, backcrossed for 10 generations to the C57BL/6?J background, and propagated by brother to sister mating24. Control WT C57BL/6?J purchased from The Jackson Laboratory (stock number 000664, Bar Harbor, ME) were bred and maintained locally under identical husbandry conditions. Age matched female mice were used in all experiments. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Pennsylvania State University College of Medicine in accordance to the regulations of the 8th edition of the GUIDE for the Care and Use of Laboratory Animals adopted in the 2015 Public Health Service Policy on Humane Care and Use of Laboratory Animals by the National Institutes of Health Office of Laboratory Animal Welfare, USA. Virus preparation and infections Influenza strain A/8/Puerto Rico/34 (PR8) was grown in the chorioallantoic fluid of ten day old chicken eggs and purified by discontinuous sucrose gradient centrifugation as previously described25. A dose of 1000 fluorescent focus counts (ffc) of IAV MST1R H1N1 PR8 in 40?L of Dulbeccos Phosphate Buffered Saline (DPBS) was instilled intranasally under ketamine/xylazine anesthesia. Mice were monitored for body weight loss and survival as described previously25. Lung and.

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