Specific elisas

After 5 h of the indicated treatments, mice were sacrificed, and air pouches were washed with 2 mL of ice-cold saline. The lavage fluid was cooled on ice; the cells were then recovered and counted by the manufacturer. Supernatants were harvested and stored at −80 °C for further cytokine IL-6 detection using specific ELISAs (BD Pharmingen and R&D Systems, San Jose, USA) [28 (link)]. Standard curves for the cytokines were obtained using the reference concentrations supplied. Our results are the mean of three independent experiments.

Mouse infection assays were performed as previously reported [29 (link)]. Mice were intraperitoneally injected with HA9801 3 × 10⁸ CFU/mouse with or without PTX3 (5 μg, 10 μg and 20 μg, respectively). Every 2 h, three infected mice were sacrificed by cervical dislocation and their blood, lungs and livers were harvested, weighed and homogenized in PBS. Subsequently, the homogenates and bloods were serially 10-fold diluted and plated on THA medium to enumerate CFU. At the same time, the cytokine IL-6 in the blood of infected mice was detected by specific ELISAs (BD Pharmingen and R&D Systems).

HEp-2 cells were infected with RSV as described above in Section 2.4 and supernatants were collected at 24 h of minocycline treatment and RSV infection. The supernatants were centrifuged at 14,000 × g for 10 min to get rid of cellular debris and used for quantification of IL-6, IL-8, CXCL-10, IL-12p40 and TNF-α by specific ELISAs (BD-Biosciences, San Jose, CA, USA).