TLR2 deficiency impairs early macrophage accumulation at the gut after robotic MAC 101 and Mma20 gut microinjection. (A) Schematic of the experimental workflow. (B,C) Representative fluorescence images showing bacterial burden and macrophage number at the gut region after 0.5 and 2.5 hours of gut microinjection of MAC 101 (B) and Mma20 (C). Representative images are tlr2 wild-type larvae under germ-free (GF) condition. Bacteria are shown in magenta. Scale bar: 100 µm for full-body images; 25 µm for zooms. (D,E) Quantification of bacterial burden after 0.5 (D) and 2.5 (E) hours of gut microinjection of MAC 101. No significant differences of bacterial burden were found among groups. (F,G) Quantification of the number of macrophages at the gut region after 0.5 (F) and 2.5 (G) hours of gut microinjection of MAC 101. The number of macrophages at the gut region was higher in the tlr2 wild-type larvae compared to the tlr2 mutants after 0.5 and 2.5 hours of MAC 101 gut microinjection in both GF and CONVD conditions. For MAC 101 infection, the data from GF tlr2+/+ (n=103) group, GF tlr2-/- (n=79) group, CONVD tlr2+/+ (n=92) group and CONVD tlr2-/- (n=110) group are based on three independent experiments. (H,I) Quantification of bacterial burden after 0.5 (H) and 2.5 (I) hours of gut microinjection of Mma20. No significant differences of bacterial burden were found among groups at 0.5 hpi. At 2.5 hpi, tlr2 mutant larvae had higher bacterial burden than the wild-type control in the CONVD condition. For tlr2 wild-type larvae, GF larvae had a higher bacterial burden than CONVD larvae. (J,K) Quantification of the number of macrophages at the gut region after 0.5 (J) and 2.5 (K) hours of gut microinjection of Mma20. The number of macrophages at the gut region was higher in tlr2 wild-type larvae compared to the tlr2 mutants after 0.5 and 2.5 hours of Mma20 gut microinjection in both GF and CONVD conditions. For Mma20 infection, the data from GF tlr2+/+ (n=79) group, GF tlr2-/- (n=72) group, CONVD tlr2+/+ (n=59) group and CONVD tlr2-/- (n=62) group are based on three independent experiments. Statistical significant difference was determined by one-way ANOVA, red arrows point to the median, ns, non-significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001.
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