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. Author manuscript; available in PMC: 2010 Apr 1.
Published in final edited form as: Immunity. 2009 Apr 9;30(4):556–565. doi: 10.1016/j.immuni.2009.02.005

Figure 5. NLRP3 inflammasome activation in response to influenza virus is dependent upon lysosomal maturation and ROS production in human cells.

Figure 5

(A) Depiction of the primary ciliated human airway epithelial (HAE) cultures which were infected with the human pathogenic influenza virus A/Victoria/3/75 (H3N2; MOI = 1). Supernatants are collected from the apical and basolateral surfaces. (B) Robust viral replication was observed over the course of the HAE infection. (C) IL-1β levels were detected in the apical, but not basolateral, compartment 48 hours post-infection. (D) Human nasal airway epithelial cell lines (JME) were infected with A/Victoria/3/75 (MOI=1) and increased NLRP3 and ASC mRNA expression was observed. Data was normalized to 18s and compared to the expression in similarly treated human type II alveolar epithelial-like cell lines (A549). (E) Increased IL-1β protein was observed 24 hours post-infection in the cell free supernatant from the JMEs. (F) Human THP-1 monocyte cell lines were infected with A/Victoria/3/75 (MOI=5) and increased IL-1β levels were observed in the supernatant over a 24hr time course. (G–I) Influenza mediated IL-1β generation is dependent upon the NLRP3 inflammasome in human monocytes. (G) Human THP-1 cells were infected with lentivirus containing shRNA for ASC, NLRP3 or TUCAN (shASC, shNLRP3 or shTUCAN) or a mutated sh target sequence (shmut). Cell free supernatants were collected 24 hours post-infection (MOI=1). Influenza induced NLRP3- and ASC- dependent increase in IL-1β (*p<0.05), which was independent of TUCAN. (H) The general caspase inhibitor ZVAD-CHO and (I) the caspase-1 specific inhibitor Ac-YVAD-CHO, both inhibited IL-1β release in a dose-dependent manner (*p<0.05). (J–L) Influenza mediated IL-1β maturation requires lysosomal maturation and ROS production in THP-1. IL-1β release during influenza infection (MOI=5) was attenuated following treatment with (J) the lysosome inhibitor bafilomycin A (100–250nM), (K) the cathepsin B inhibitor CA-074-Me (50μM), and (L) the ROS inhibitors APDC (100μM) and NAc (50μM)(*p<0.05). (M) Treatment with the ROS inhibitor NAc (250mg/kg) inhibits influenza mediated IL-1β release in vivo (*p<0.05). NAc Mock, n=1; NAc Influenza, n=5, Vehicle Influenza, n=3.