Male C57BL/6 wild type mice were purchased from the Laboratory Animal Research Center of Shanghai. TLR4 knockout (TLR4−/−) mice, MyD88 knockout (MyD88−/−) mice and TLR3 knockout (TLR3−/−) were obtained from Dr. Billiar’s lab at the University of Pittsburgh. All mice used are on a C57BL/6 background. All experimental protocols involving animals were approved by Institution Animals Care and Use Committee of Tongji and Pittsburgh University. The experiments were performed in accordance with the National Institutes of Health Guidelines for the Use of Laboratory Animals. Mice were 8–12 weeks of age at the time of experiments and were maintained on standard rodent chow and water ad libitum. Animals were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine administered intraperitoneally. Animals were intratracheally administered LPS (5 μg/g body wt; Escherichia coli O111:B4; Sigma, St. Louis, MO) in 50 μl of saline (SAL) or SAL at first time, then after 4 h intratracheally administered Poly I:C (5 μg/g body wt; 31852-29-6; Invivogen) in 50 μl of saline (SAL) or SAL alone at the second time using a MicroSpray syringe. The animals were randomly in one of four groups: SAL/SAL, SAL/Poly I:C, LPS/SAL, and LPS/Poly I:C. At various time points, a 20G sterile catheter was inserted into the trachea to collect bronchoalveolar lavage fluid (BALF) as previously described54. Blood samples were immediately obtained by cardiac puncture and transferred to the laboratory for analysis of cytokines (IL-6, TNF-α), chemokines (MIP-2, MCP-1) in serum and BALF by ELISA. PMN counts in BALF were determined on Wright-Giemsa-stained slides. Briefly, total cell counts were determined on a grid hemocytometer. Then a total of 500 cells were counted in cross-section per sample, and the number of PMNs was calculated as the total cell count times the percentage in the BALF sample. The lungs were rapidly removed from all mice and washed in ice-cold saline. Half of the lung tissues were stored at −80 °C prior to biochemical analyses including MIP-2 expression in lung lysates was measured by Western blotting and RT-PCR, while the other half of the lung tissue was fixed in 4% formalin solution in preparation for histopathological analyses. For histological analysis, lung tissue samples were fixed in 4% paraformaldehyde in PBS overnight at 4 °C. The samples were then dehydrated, embedded in paraffin, and cut into 5 μm sections. After deparaffinization, the tissues were stained with hematoxylin and eosin (H&E) for histological analysis. Lung sections were scored for lung injury, including the following: (1) alveolar and capillary edema, (2) intravascular and peri-bronchial influx of inflammatory cells, (3) thickness of the alveolar wall, and (4) hemorrhage. The items were semiquantitatively scored as none, minimal, light, moderate, or severe (score 0, 1, 2, 3 or 4, respectively) by a pathologist blinded to the experimental group. The lung injury score was obtained by averaging the score from the animals within each group.