Figure 2. NLRP6 inflammasome signaling is required for IL-18 production upstream of the induction of antimicrobial peptides.

(A, B) Co-immunoprecipitation of FLAG-NLRP6, HA-ASC and MYC-Caspase-1 overexpressed in HEK293T cells. Cell lysates were precipitated using anti-FLAG sepharose beads (A) or anti-HA sepharose beads (B) followed by immunoblotting with anti HA, anti FLAG and anti MYC antibodies.

(C) Immunoblot analysis of pro-caspase-1 (p45) and cleaved caspase-1 (p20) in colon tissue from SPF WT and Nlrp6^−/− mice.

(D) Differential expression between WT and Nlrp6^−/− mice of AMPs versus all other genes. Box = interquartile range (IQR) = 25th to 75th percentile, line - median, star - mean, whiskers - IQR*1.5. Mann-Whitney U-test p<0.05.

(E) Heatmap of AMPs from transcriptome analysis of colon tissue from WT, Asc^−/−, Nlrp6^−/− and Il18^−/− mice.

(F) Colonic Ang4 expression in WT, Asc^−/−, IL18^−/−, Casp1/11^−/−, Nlrp6^−/− and Nlrp3^−/− mice.

(G, H) Targeted mass spectrometry of Ang4 peptides identified in fecal samples obtained from WT or Asc^−/− mice. Colors indicate different fragment ions.

(I–J) Colonic expression of the indicated AMPs in Nlrp6^−/−, Asc^−/−, and Casp1/11^−/− mice following injection of IL-18.

(K–M) Immunoblot quantification of pro-caspase-1 (p45) and cleaved caspase-1 (p20) (K), IL-18 production (L) and Ang4 expression (M) in colon tissue from germ-free and SPF WT and Nlrp6^−/− mice.

(N) Colonic Ang4 expression levels in germ-free Nlrp6^−/− mice, with or without injection of IL-18. Data are expressed as mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.

Pairwise comparison was performed using Student’s t test, unless stated otherwise.

Results shown are representative of two independent repeats (n= 3–14 per group).

See also Figure S2.