Method: 1) WT, caspase-1 KO, NLRP3 KO and ASC KO Bone Marrow Derived Macrophages (BMDM) were infected with RSV for 12h and 24h.Supernatants were collected and IL1b levels analyzed via ELISA. 2) Cell pellets from WT, NLRP3 KO and ASC KO BMDM were collected and lysates used to analyze caspase-1 activation via western blotting. 3) WT BMDM were pretreated with NFkB inhibitor then infected with RSV. Supernatants were collected for IL1b analysis and RNA isolated for RT-PCR of pro-Il1b, NLRP3 and ASC. 4) WT, TLR2 KO and MyD88 KO BMDM were infected with RSV for 12h and 24h. Supernatants were collected for IL1b analysis and RNA isolated to examine gene expression, 5) WT BMDM were pretreated with reactive oxygen species (ROS) inhibitor, NaCl or KCl then infected with RSV. IL1b levels were analyzed via ELISA.
Results: 1) During RSV infection NLRP3, ASC and caspase-1 are required for IL1β secretion. 2) NLRP3 and ASC are required for caspase 1 activation during RSV infection. 3)TLR2/MyD88/NFκB is required for pro-IL1β and NLRP3 expression during RSV infection. 4) ROS and K+ efflux are required for IL1β secretion during RSV infection
Conclusion: We have identified the mechanism of inflammation during RSV infection. TLR2/MyD88/NFkB provides the first signal while K+ and ROS provide the second signal. This mechanism may provide insight into the involvement of other viruses such as EBV in periodontitis. Furthermore, such pathway could be targeted as potential therapy to reduce IL1b secretion thus reducing periodontitis
Keywords: Cytokine, Immunology, Infection and Inflammation