Previously, it was shown that increased recruitment of NK cells is involved in not only viral clearance but also weight loss (Harker et al., 2010). for which vaccines are not yet available need to be assessed in future studies. = 4/group) except for TLR3-/- TLR7-/- mice (= 3/group). Animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Yonsei University (IACUC-A-201508-412-02, IACUC-A-201508-422-02, IACUC-A-201511-528-01, IACUC-A-201512-543-02, IACUC-A-201512-572-02, and IACUC-A-201611-468-02). Cells and Viruses Madin-Darby canine kidney (MDCK, ATCC, Manassas, VA, United States) cells were cultured in minimal essential medium (MEM, Hyclone, South Logan, UT, United States) containing 10% fetal bovine serum (FBS, Hyclone). X-31ca (H3N2) was propagated in the allantoic cavity of 11-day-old embryonated eggs and titrated by using the plaque assay using MDCK cells at 33C. RSV A2 was propagated in HEp-2 cells (ATCC) as previously described (Lee and Chang, 2017), and the virus titer was determined in HEp-2 cells by using the standard plaque assay. Briefly, a 10-fold serial dilution of RSV A2 stock was made in serum-free MEM, and inoculated to HEp-2 cells in 6-well plates. After incubation for 90 min at 37C, supernatants were discarded and 3 mL of the 1% agarose/growth media mixture was added to each well. After the agar turned solid, plates were incubated for 4 days at 37C. To visualize plaques, 2 mL of 1% agarose containing 50 g/mL neutral red was added and incubated for 24 h at 37C, and then plaques were counted (McKimm-Breschkin, 2004). Immunization and Preparation of Samples Six-week-old female mice were anesthetized by intramuscular infection of alfaxalone (3–hydroxy-5–pregnane-11, 20-dione, Alfaxan; Jurox, Rutherford, NSW, Australia) before intranasal infection with 50 L of virus suspension (106 PFU of X-31ca or RSV A2) or intraperitoneal infection with 100 L of formalin inactivated influenza virus (inactivated X-31ca). Retro-orbital bleeding was performed to collect sera from immunized mice for IgG and IgM antibody titration. Mice were Hydroxyflutamide (Hydroxyniphtholide) euthanized and bronchoalveolar lavage (BAL) fluids were obtained by washing the airway with 1 mL of PBS. BAL cells and supernatants were then separated by centrifugation. BAL fluids were used for the titration of mucosal IgA antibody and cytokine levels, and the collected BAL cells were used to investigate immune cell recruitment. BAL cells were counted by using a hemocytometer. Whole lungs were homogenized, and the lung supernatants and cells were collected by centrifugation. Lung supernatants were used for the viral titration, and the collected lung cells were used Hydroxyflutamide (Hydroxyniphtholide) to investigate immune cell recruitment. BAL and lung cells were counted using a hemocytometer. Lung Viral Titration After RSV A2 challenge, mice were euthanized, and a lung single-cell suspension IL1R2 antibody was obtained by passing lung tissue through a 70-m cell strainer into serum-free MEM. The supernatants were collected by centrifugation and lung viral titration was performed by using a standard plaque assay in HEp-2 cells. The lung viral titer was expressed as PFU/g of lung tissue, and the limit of detection was 100 PFU/g. Cytokines and Antibody Levels The levels of X-31ca, Hydroxyflutamide (Hydroxyniphtholide) RSV G, or F (Sino Biological, Tongzhou Qu, Beijing, China) protein-specific antibodies were determined by ELISA. Plates were coated with 5 105 PFU/well of X-31ca or 50 ng/well of RSV G and F proteins at 4C overnight. After blocking and washing, plates were incubated with twofold dilutions of sera or BAL fluids for 1 h at room temperature (RT). After washing, the plates were incubated with Hydroxyflutamide (Hydroxyniphtholide) HRP-conjugated secondary goat anti-mouse IgA, IgG, or IgM antibody (A90-103P, 116P, or 101P, Bethyl, Montgomery, TX, United States) for 1 h at RT, followed by washing and incubation with TMB substrate solution (BD Biosciences, San Jose, CA, United States) for 30 min at RT in the dark. The colorimetric reaction was stopped by adding 2N H2SO4 solution, and the absorbance was measured at 450 nm using a microplate reader. The concentrations of IFN- and IFN- were measured by using ELISA kits (PBL, Piscataway, NJ, United States), and the levels of IFN-, IL-6, and TNF- in BAL fluids were determined by using a Magnetic Hydroxyflutamide (Hydroxyniphtholide) luminex performance assay multiplex kit (R&D systems, Minneapolis, MN, United States). The experiments were conducted following the manufacturers protocol. Flow Cytometric Analysis To analyze immune cell populations, BAL and lung cells were incubated with rat anti-mouse CD16/CD32 (BD Biosciences) blocking antibody for 10 min at RT..