Supplementary Materials [Online Supplement] ajrccm_177_7_771__index. (HV68) infection augments fluorescein isothiocyanate (FITC)Cinduced pulmonary fibrosis. Wild-type mice were given FITC or saline or HV68 intratracheally on Day 0. On Day 14, FITC-treated mice received HV68 or mock infection intranasally. Lungs were harvested for collagen determination on Day 21. Data represent n = 10C12 mice per group collected in two independent experiments. We looked at lung histology to Quercetin kinase activity assay confirm the results of our biochemical measurements of collagen content. Using the same protocol as above, lungs were harvested on Day 21, and inflated, fixed, and embedded in paraffin. Sections were then stained with hematoxylin and eosin or Quercetin kinase activity assay Masson’s trichrome. Representative sections shown in Figure 2 (of Figure 2. There is evidence of interstitial edema, intraalveolar hemorrhage, alveolar epithelial denudation, and sloughing off of injured/dead epithelial cells. At the higher power, the mononuclear infiltrate is also evident. These histologic findings are consistent with evidence of diffuse alveolar damage and are similar to findings noted in cases of acute exacerbation in human IPF. However, FITC alone is also capable of producing similar pathologic patterns. As such, whereas these changes are consistent with diffuse alveolar damage and acute lung injury, they likely represent quantitative changes in response to the viral infection rather than qualitative changes. Open in a separate window Figure 2. Histologic analysis confirms that Prkwnk1 murine gammaherpesvirus-68 (HV68) infection leads to increased fibrotic response to fluorescein isothiocyanate (FITC). at 1,000 original magnification). The point to examples of interstitial edema. Chronic inflammatory cells and intraalveolar hemorrhage are also evident. The highlights the evidence of alveolar epithelial denudation as indicated by the = 0.04). Virus gene expression was measured using real-time RT-PCR in both groups of mice. We analyzed expression of the glycoprotein B (gB) gene, which Quercetin kinase activity assay encodes part of the virus capsid, and the viral DNA polymerase gene, both of which are expressed during lytic infection (Figure 4B). There is expression of both of these genes on Day 21 post-FITC (7 d after viral infection), and there is an approximately 3.5-fold increase in expression of both genes in mice that were given FITC and HV68 versus those given saline followed by HV68 infection. Open in a separate window Figure 4. There is active lytic viral replication at Day 7 after murine gammaherpesvirus-68 (HV68) infection, and previous fibrotic insult leads to increased viral load. Wild-type mice were given either saline or fluorescein isothiocyanate (FITC) intratracheally on Day 0. On Day 14, they were given HV68 (5 104 pfu) intranasally. Lungs were harvested on Day 21. (= 0.04). ( 0.001). However, the UVHV68 was unable to augment fibrosis (FITC + mock = 125.7 7.5 vs. FITC + UVHV68 = 125.1 5.2 g/ml collagen). Therefore, lytic viral replication, not just viral antigen, is necessary for viral exacerbation of FITC-induced fibrosis. Open in a separate window Figure 5. Lytic viral replication is necessary to augment the fluorescein isothiocyanate (FITC)Cinduced fibrotic response. Wild-type mice were given FITC intratracheally on Day 0. On Day 14, FITC-treated mice received 5 104 pfu murine gammaherpesvirus-68 (HV68) ( 0.05), and mRNA expression was elevated 2.75-fold. TNF- was produced at even higher levels in HV68 versus mock-treated mice (2,736 192 vs. 1,078 176 pg/ml). IL-13 was also elevated, but the overall amounts of IL-13 produced were much lower than the levels of IFN-. Open in a separate window Figure 6. Murine gammaherpesvirus-68 (HV68) augments the fibrotic response to fluorescein isothiocyanate (FITC) in the presence of a significant Th1 immune response. Wild-type mice were given FITC intratracheally on Day 0 and then given either mock ( 0.05 compared with FITC + mock infection. TNF = tumor necrosis factor-. To determine whether Th2 cytokines were critical for the exacerbation of fibrosis, we tested the ability of HV68 to exacerbate FITC-induced fibrosis in the Th2-deficient IL-4/13?/? mice. BALB/c Quercetin kinase activity assay and IL-4/13?/? mice were injected with FITC on Day 0. The mice were then given 5 104 pfu HV68 or mock infection on Day 14, and lungs were harvested on Day 21 for collagen measurement via Sircol assay. Figure 7 demonstrates that HV68 can exacerbate FITC-induced fibrosis in IL-4/13?/? mice (52.68 4.43 vs. 77.73 Quercetin kinase activity assay 11.67 g/ml, = 0.01). We have seen similar results in IL-13?/? mice with a 40% increase in collagen after HV68 infection (data not shown). Therefore,.