These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive scarring of the lung parenchyma, resulting in a steady decline of lung function and ultimately respiratory failure. The disease course of IPF is extremely variable, with some patients exhibiting stability of symptoms for prolonged periods of time, whereas others exhibit rapid progression and loss of lung function. Viral infections have been implicated in IPF and linked to disease severity; however, whether they directly contribute to progression is unclear. We previously classified patients as rapid and slow progressors on the basis of clinical features and expression of the pathogen recognition receptor, Toll-like receptor 9 (TLR9). Activation of TLR9 in vivo exacerbated IPF in mice and induced differentiation of myofibroblasts in vitro, but the mechanism of TLR9 up-regulation and progression of fibrosis are unknown. Herein, we investigate whether transforming growth factor (TGF)-β, a pleiotropic cytokine central to IPF pathogenesis, regulates TLR9 in lung myofibroblasts. Results showed induction of TLR9 expression by TGF-β in lung myofibroblasts and a distinct profibrotic myofibroblast phenotype driven by stimulation with the TLR9 agonist, CpG-DNA. Chronic TLR9 stimulation resulted in stably differentiated α-smooth muscle actin(+)/platelet-derived growth factor receptor α(+)/CD44(+)/matrix metalloproteinase-14(+)/matrix metalloproteinase-2(+) myofibroblasts, which secrete inflammatory cytokines, invade Matrigel toward platelet-derived growth factor, and resist hypoxia-induced apoptosis. These results suggest a mechanism by which TGF-β and TLR9 responses in myofibroblasts collaborate to drive rapid progression of IPF.

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA.

Lytic replication of Kaposi's Sarcoma Associated Herpes Virus (KSHV) exacerbates Kaposi's sarcoma (KS) progression, an angiogenic spindle‐cell sarcoma associated with AIDS. Current antiviral therapy targeting lytic replication can prevent KS in seropositive patients. KSHV lytic infection is not manifested in vitro, and currently there are no animal models that display pathogenic phenotypes. In this study, we employed a biological and genetically mouse pathogen similar to KSHV, the murine gammaherpes virus (gHV) 68 (MHV68), that readily infects laboratory mice providing a valuable small animal model. Moreover, MHV68 in vitro infection exhibits a default lytic infection. Several studies have shown that during infection KSHV employs several viral proteins to de‐regulate the host transcription factor hypoxia inducible factor 1 alpha (HIF1α) and KSHV‐induced activation of HIF1α is necessary for viral persistence and KS progression. However, little is known about the role of HIF1 α in gHV replication and pathogenesis. To further investigate the role of this host factor, we induced transcriptional inactivation of HIF1α in MHV68 virus‐specific cells in vivo. Conditional knock out of HIF1α was achieved by infecting transgenic mice with Cre‐recombinase (Cre) LoxP specific site within the functional domain of HIF1α gene using a recombinant MHV68 that expressing a CMV driven Cre expression. Our data demonstrated for the first time that HIF1α inactivation during gammaherpes virus infection in vivo affects viral gene transcription resulting in impaired virus expansion and early clearance of acute infection in a mouse model. In addition, our results from in vitro MHV68 lytic infection of mouse fibroblasts lacking HIF1α showed that lytic virus production and transcription is decreased. During latency establishment in vivo, the frequency of MHV68 latent splenocytes undergoing latent to lytic replication was largely decreased as well. Our data suggest that HIF1α is required for sustained acute infection since it's deletion in virus‐infected cells resulted in early clearance of productive infection and impairment in reactivation. Moreover, HIF1α is required for mRNA upregulation of glycolytic enzymes during MHV68 lytic infection suggesting a role for HIF1a as a modulator of cell energetics and viral gene expression. We conclude that gammaherpes viruses required the function of the cellular host factor HIF1α in order to effectively replicate and establish latency within its host. Support or Funding Information National Cancer Institute 3R01CA136387‐08S1 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The COVID-19 pandemic has claimed millions of lives worldwide. In this report, we used a preclinical mouse model to investigate the prophylactic and therapeutic value of intranasal IFN-λ for this acute respiratory disease.