Selective Autophagy of RIPosomes Maintains Innate Immune Homeostasis during Bacterial Infection (2022)
Figure 1

Figure 1. Pathogenic bacteria trigger the formation of RIPosomes that recruit over the bacteria.

(A) Representative confocal images of RIPosomes in THP-1 cells infected with a red fluorescent protein (RFP) expressing Shigella flexneri (MOI 1:25, 8 h). Scale bar, 10 µm. Zoom panels are digital magnifications.

(B) Representative high content microscopy images (see Appendix figure S1-D for full images; yellow masks represent software algorithms-defined cell boundaries) of RIPosomes in THP-1 cells infected with S. flexneri (MOI 1:25, 8 h). About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well in 3 technical replicates. Right panel, the graph depicts an average number of RIPosomes/cell, which is calculated from 3 biological replicates, Mean ± SD. ***p < 0.005, Student's unpaired t-test.

(C) Western blot analysis of soluble and insoluble fractions of S. flexneri infected THP-1 cells (MOI 1:25, 8 h). Arrow indicates oligomers of RIPK2.

(D) Left panel, representative high content microscopy image of control and RIPK2 knockdown THP-1 cells infected with S. flexneri (MOI 1:25, 8 h). Right panel, the graph depicts an average number of RIPosomes/cell, which is calculated from 3 biological replicates, Mean ± SD. ***p < 0.0005, Student's unpaired t-test.

(E-F) Western blot analysis of soluble and insoluble fractions of S. flexneri infected (E) control siRNA and RIPK2 siRNA transfected THP-1 cells (F) Control CRISPR (RIPK2+/+) cells and RIPK2 CRISPR knockout (RIPK2-/-) HT-29 cells.

(G) Representative confocal images of RIPosomes in mouse BMDMs infected with S. flexneri ( MOI 1:25, 8 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI (pseudo-colored red for better contrast).

(H) Representative confocal images of mouse colon tissues showing recruitment of RIPK2 over the S.flexneri. Scale bar, 1 µm. Zoom panels are super-resolution confocal images. DNA is stained with DAPI (pseudo-colored red for better contrast).

(I) Representative confocal images of RIPosomes in THP-1 cells infected with RFP expressing Salmonella typhimurium (MOI 1:5, 4 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 8 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.

(J) Left panel, representative high-content microscopy images (yellow masks represent software algorithms-defined cell boundaries) of RIPosomes in THP-1 cells infected with S. typhimurium (MOI 1:5, 4 h). The same cell numbers and conditions are used as indicated in Figure 1B. Right panel, the graph depicts an average number of RIPosomes/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test. DNA is stained with DAPI.

(K-M) Representative confocal images of RIPosomes in THP-1 cells infected with (K) E. coli LF82 strain (MOI 1: 10, 8h), (LPS antibody is used to stain LF82) (L) mCherry expressing M. smegmatis (MOI 1:10, 4 h) (M) GFP expressing M. tuberculosis (MOI 1:10, 8 h). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.

(N) Western blot analysis of soluble and insoluble fractions from THP-1 cells, uninfected or infected with E. coli DH5α (MOI 1:10, 8h) or E. coli LF82 strains (MOI 1:10, 8h).

(O) Pictorial representation of data obtained in this figure shows that bacterial infection triggers oligomerization of RIPK2 or RIPosome formation that coats bacteria.