The results showed that local inhibition of TNF- could significantly reduce the high mortality in mice induced by lethal influenza infection, which indicated that TNF- in lung tissue might play a pathologic role in severe virus infection. of TNF- can elicit protection against influenza contamination. Methods In this study, the commercially available TNF- inhibitor etanercept was used to inhibit TNF- induced by lethal A/FM/1/47 (H1N1) influenza virus contamination of mice. The effects of TNF- inhibition on mouse survival, pathologic changes, immune cell infiltration, inflammatory cytokine secretion, Toll-like receptor expression, and activation of ABCC4 the NF-B (nuclear factor kappa B) signaling pathway were evaluated. Results The intranasal delivery of etanercept provided significant protection against mortality (30% of mice survived up to 14 days after contamination) in mice treated with etanercept. In contrast, no survivors were found beyond 6 days in mice treated with saline after lethal challenge with H1N1 influenza virus. It was observed that etanercept significantly Selonsertib reduced inflammatory cell infiltration (for example, macrophages and neutrophils), inflammatory cytokine secretion (for example, interleukin-6, TNF-, and interferon gamma), and expression of Toll-like receptors (and and type IV (Sigma, USA), 50 U/ml DNase I (Sigma), and 1 mg/ml trypsin inhibitor type II-s (Sigma) for 1 hour at 37C. The suspension was then crushed through a 40-m basket filter, and unwanted red blood cells were lysed by using red blood cell lysis buffer made up of 0.02 TrisCHCl (pH 7.4) and 0.14 NH4Cl. Inflammatory cells were purified by centrifugation in 35% (vol/vol) PBS-buffered Percoll (GE Healthcare Life Sciences, USA) at 500 for 15 minutes. Cell pellets were resuspended in staining buffer (RPMI-1640 medium), and Fc receptors were blocked by using 25 g/ml anti-mouse CD16/32. Cells were stained with fluorescently labeled antibodies against the following mouse proteins: CD11b+, F480-, Ly6G+ (neutrophils), CD11b+, F480+, and Ly6G- (macrophage/monocytes), CD3e-, CD49b+ [natural killer (NK) cells], CD3e+, CD19+ (B cells), CD3e+, CD4+ (T-helper cells), CD3e+, and CD8a+ (cytotoxic T cells) [22,23]. All antibodies were purchased from BD Biosciences (USA). The average Selonsertib counting of immune cells was calculated from three individual experiments. Inflammatory signaling pathways (Toll-like receptors and NF-B) and influenza virus replication On days 2 and 4 after contamination, mice (geneand glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were as described in Additional file 1. By using cDNAs as templates, quantitative real-time PCR was carried out by using the SYBR Green PCR Grasp Mix (Applied Biosystems) in a StepOne Plus Real-Time PCR Detection System (Applied Biosystems), according to the manufacturers instructions and with the following thermocycling parameters: 94C for 5 minutes; followed by 94C for 5 seconds, 60C for 30 seconds for 40 cycles, with a final melting curve analysis of 60C to 95C. The mRNA expression levels Selonsertib were normalized to the corresponding expression level of the housekeeping gene. The results of qPCR were from three separated impartial experiments. The remaining right-lung lobes were used for immunohistochemistry. Tissue sections (10 m) were cut and processed as described earlier. The primary antibody, phospho-NF-B p65 (Ser536) (93H1) rabbit monoclonal antibody (Cell Signaling Technology, Inc., USA) was used to evaluate the activation of the inflammatory NF-B signaling pathway. Statistical analyses All statistical analyses were performed by using GraphPad Prism for Windows (Version 6.0). The Gehan-Breslow-Wilcoxon test was used to analyze the survival of mice, whereas the one-way ANOVA was used to analyze other experimental data. In all cases, probability values less than 0.05 (Saline or 2.5 mg/kg of etanercept was administered i.n. to mice 2 hours after i.n. contamination with 10 TCID50 of influenza virus A/FM/1/47 (H1N1). (A, B) Mice were monitored for survival and body-weight loss. Data were derived from three individual experiments, with a total of 30 mice per group (10 mice per group each time). Survival curves show data until day 14 after contamination, because further mortality was not observed at later time points. (C, D) Lung/body weight and inflammation score were evaluated on day 4 after contamination. Mice were weighed (grams) and euthanized. Whole lungs were harvested, weighed (grams), and the corresponding lung/body index was calculated. The scores were calculated as follows: none, 0; 25%, 1; 26% to 50%, 2; 51% to 75%,.