Figure 3. Nlrp3 inflammasome components are highly expressed in myeloid cells and in the lungs during influenza A virus infection.

(A–B) ASC and Nlrp3 gene expression was assessed in a panel of cells considered important for viral infection. Primary mouse bone marrow derived macrophages (BM), B cells, T cells, mouse airway epithelial cells (MAECs) and mouse embryonic fibroblasts (MEFs) were assessed by real-time (rt) quantitative PCR with samples normalized to 18s and standardized to expression in MEFs. Each cell line was assessed in triplicate and data are representative of 3 independent experiments. (C–D) Primary mouse BM s and MAECs were differentiated and challenged with mouse adapted influenza A/PR/8/34 (MOI=10). Nlrp3, Nlrc4 and IL-1β transcripts were assessed by rtPCR normalized to 18s and standardized to naive levels. (C) A significant increase in IL-1β mRNA expression was observed in BM following influenza challenge (MOI=10); however, no significant change was observed in Nlrp3 or Nlrc4 expression (*p<0.05). (D) Influenza challenge (MOI=1) resulted in a significant increase in Nlrp3 expression and a modest decrease in Nlrc4 and IL-1β expression in the MAECs. (E) Whole lungs were removed from wild type mice either 3 dpi. or 7 dpi. and homogenized. Total RNA was extracted from tissue pellets and expression levels of Nlrp3, ASC, Il-1β and Caspase-1 (Casp1) were assessed by rtPCR normalized to the 18S house keeping gene and compared to the mock-infected lungs. Transcription levels are significantly elevated by day 3 following influenza inoculation and begin to decline by day 7 (*p<0.05). All experiments are representative of at least 2 independent experiments with 3–5 mice per group.