Figure 4

Figure 4

nlrc3-like deficiency promotes the activation of macrophage inflammasome. (A) Schematic view of FACS of mfap4-GFP⁺ macrophages with phagocytosed Tdtomato⁺M. marinum and subsequent detection of the expression of inflammation-related genes by RT-PCR. (B) A representative image of zebrafish embryos infected with M. marinum:Tdtomato at 10 hpi. i uninfected Tg (mfap4-GFP) embryo; ii. Tg (mfap4-GFP) embryo infected with M. marinum:Tdtomato; iii. Enlarged view of caudal hematopoietic tissue (CHT) region in ii. white arrows indicate macrophages with phagocytosed bacteria; white triangles indicate macrophages without phagocytosed bacteria; white asterisks, free M. marinum. (C) Gating strategy for FACS. i: Sample: Tg(mfap4: GFP) embryos infected with M. marinum:Tdtomato; ii. uninfected wild-type zebrafish; iii. Tdtomato single color control, wild-type zebrafish infected with M. marinum:Tdtomato; iv. GFP single color control, uninfected Tg(mfap4: GFP). P1, free M. marinum:Tdtomato; P2, mfap4-GFP+ macrophages; P3, mfap4-GFP+ macrophages with phagocytosed M. marinum:Tdtomato. (D) Expression of pro-inflammatory genes in macrophages at 10 hpi. The values represent the average of three independent biological repeats, 25 embryos per group in each repeat. (E) The expression of pro-inflammatory genes in embryos with overexpressing nlrc3-like in myeloid cells. (F) Representative images of M. marinum:wasabi proliferation in nlrc3-like -/-, asc -/- nlrc3-like -/-, and their nonmutant siblings. (G) Graph of green fluorescence intensity in nlrc3-like -/-, asc -/- nlrc3-like -/-, and their nonmutant siblings at the indicated time points. The values shown are the combined results of two independent experimental replicates. (B, F) scale bar = 200 μm *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, not statistically significant (p>0.05).