Upregulation and activation of NLRP6.

(A) Lungs tissue sections from healthy controls and pneumonic patients were stained with antibodies against neutrophils (lipocalin-2+), macrophages (CD68+), and epithelial (CD326+) cells. NLRP6 is shown in red, neutrophils, macrophages, and epithelial cell markers are in green, and DAPI is blue. NLRP6-positive cells are indicated by white arrowheads. (B) Immunofluorescence microscopy was performed on lung sections from healthy controls and S. aureus-infected mice to assess expression of NLRP6. Neutrophils (Ly6G+), macrophages (F4/80+), and epithelial cells (CD 326+) are stained green and NLRP6 is red. NLRP6-positive cells are indicated by white arrowheads. (C) NLRP6 expression was analyzed by western blot in lysates of human healthy control tissue, pneumonic lung tissue, and HL-60 (human neutrophil-like) cells infected with S. aureus (MOI10) for 4 hours. (D) THP-1 (human monocytic) cells were infected with S. aureus (MOI 20) or purified α-hemolysin (hla) for 8 hours and expression of NLRP6 was assessed by western blotting. (E, F) Bone marrow-derived macrophages (BMDMs) were infected with S. aureus (MOI: 20) or hla from S. aureus (50 μg/ml) for 8 hours (E). Expression of NLRP6 protein was analyzed by immunoblotting. (F) Caspase-1 processing by the NLRP6 inflammasome in response to S. aureus or hla in BMDMs. (G) IL-1β secretion by WT and NLRP6 KO BMDMs following S. aureus infection. (H) IL-1β secretion by WT and NLRP6 KO mice after S. aureus (5 X 107 CFU/mouse) infection (N = 6-8/group). (I) BMDMs from WT mice were infected with S. aureus (MOI: 50) and stained for NLRP6 (red) and ASC (green). The white arrowheads demonstrate co-localization of NRP6 and ASC. (J) BMDMs from WT mice were infected with S. aureus (MOI: 50) and stained for NLRP6 (red) and caspase-1 (green). The white arrowheads show co-localization of NRP6 and caspase-1. The graphs show the mean ± SEM of three independent experiments. The images shown are the representative of five different fields from three independent experiments. Magnification: 40X. SA: S. aureus, hla: α-hemolysin. *, p<0.05, **, p<0.01, and *** p<0.001.