Since Familial Pulmonary Fibrosis (FPF) manifests in older adults and telomere attrition is common in FPF and sporadic Idiopathic Pulmonary Fibrosis (IPF), we postulated that accelerated aging, as determined by epigenetic clock (DNA methylation) measurements, could occur in FPF. We measured DNAge from blood of patients with FPF and a group of first-degree relatives of FPF patients without disease (termed “at-risk” for FPF) with or without genetic rare variants (RVs) in telomerase pathway genes. We observed accelerated epigenetic aging with increased DNAge compared to chronological age in individuals at-risk for FPF and FPF patients compared to healthy controls. We found that increased DNAge manifests independently of the presence of RVs in telomerase pathway genes or telomere length in peripheral blood cells. These findings suggest that increased DNAge could be an independent risk factor for the development of FPF. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement 5K12HD043483-12 P01HL172729 ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: These studies were approved by the Vanderbilt University Institutional Review Board (IRB# 020343, 080780). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

RATIONALE: BALB/c and STAT1−/− mice provide models of Th1 and enhanced Th2 inflammation in respiratory syncytial virus (RSV) infection, respectively. We analyzed the cellular source of IL-4 in RSV-infected BALB/c and STAT1−/− mice. METHODS: Mice were infected with RSV. Lungs were harvested at time points and homogenized. IL-4+ cells were identified by intracellular cytokine staining and flow cytometry, sorted, and stained with Wright-Giemsa. IL-4+ cells were also identified using BALB/c IL4 GPF reporter mice (4get) and STAT1−/−4get mice. Natural killer T (NKT) cells as a source of IL-4 was investigated using CD1d tetramer analyses and by using NKT cell-deficient CD1d−/−STAT1−/− mice. RESULTS: CD3-CD49b+ cells, not T cells, were the predominant source of IL-4 in the lungs of RSV-infected BALB/c and STAT1−/− mice. CD3-CD49b+IL-4+ cells sorted from the lungs of RSV-infected STAT1−/− mice and stained with Wright-Giemsa were granulocytes. Using 4get and STAT1−/−4get mice, we identified these cells as basophils (CD3−CD49b+FcɛRI+c-kit−). RSV infection resulted in more IL-4-expressing basophils in the lungs of STAT1−/− mice than in wild-type BALB/c mice.We ruled out NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. We also ruled out NKT cells as IL-4 expressors in RSV-infected STAT1−/− mice using CD1d−/−STAT1−/− mice. Basophils and, to a lesser extent, T cells, expressed IL-4 in RSV-infected STAT1−/− mice. Basophils were the only cells that expressed IL-4 in the lungs of RSV-infected BALB/c mice. CONCLUSIONS: Basophils were the major source of IL-4 in the lung in primary RSV infection. STAT1 modulated RSV-induced basophilia.

IL-4 contributes to immunopathology induced in mice by primary respiratory syncytial virus (RSV) infection. However, the cellular source of IL-4 in RSV infection is unknown. We identified CD3−CD49b+ cells as the predominant source of IL-4 in the lungs of RSV-infected BALB/c mice. We ruled out T cells, NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. Using IL4 GFP reporter mice (4get) mice, we identified the IL-4-expressing cells in RSV infection as basophils (CD3−CD49b+FcεRI+c-kit−). Because STAT1−/− mice have an enhanced Th2-type response to RSV infection, we also sought to determine the cellular source and role of IL-4 in RSV-infected STAT1−/− mice. RSV infection resulted in significantly more IL-4-expressing CD3−CD49b+ cells in the lungs of STAT1−/− mice than in BALB/c mice. CD49b+IL-4+ cells sorted from the lungs of RSV-infected STAT1−/− mice and stained with Wright-Giemsa had basophil characteristics. As in wild-type BALB/c mice, IL-4 contributed to lung histopathology in RSV-infected STAT1−/− mice. Depletion of basophils in RSV-infected STAT1−/− mice reduced lung IL-4 expression. Thus, we show for the first time that a respiratory virus (RSV) induced basophil accumulation in vivo. Basophils were the primary source of IL-4 in the lung in RSV infection, and STAT1 was a negative regulator of virus-induced basophil IL-4 expression.

We developed a novel mouse model of malignant pleural effusion (MPE) by injecting Lewis lung cancer (LLC) cells directly into the pleural space of syngeneic C57B/6 mice. The pleural effusions in this model share common cellular and biochemical features with human MPEs. Implantation and growth of pleural tumors triggers a host inflammatory response characterized by a mixed inflammatory cell influx into the pleural fluid. LLC cells exhibited high basal nuclear factor (NF)-kappaB activity in vitro and in vivo, which we used to drive expression of a NF-kappaB-dependent green fluorescent protein-firefly luciferase fusion reporter construct. NF-kappaB-dependent reporter expression allowed intravital tracing of pleural tumors. Inhibition of NF-kappaB in LLC cells did not affect cell viability in culture; however, injection of LLC cells expressing a dominant NF-kappaB inhibitor resulted in decreased tumor burden, decreased pleural effusion volume, and decreased pleural effusion TNF-alpha levels. These studies indicate that tumor NF-kappaB activity regulates pleural tumor progression. This reproducible model of MPE can be used to further study the influence of specific host and tumor factors on the pathogenesis of MPE and evaluate new therapeutic strategies.