Fig. 4

Impaired neutrophil recruitment and survival in Sindbis virus (SINV)- > local Shigella co-infection. (A) 72 hpf mpx:GFP larvae were sequentially injected with SINV-GFP in the bloodstream then subcutaneously with Shigella-DsRed one day later (96 hpf). As control, mpx:GFP larvae were injected subcutaneously with Shigella only at 96 hpf. The infected larvae were imaged with a fluorescent stereomicroscope over time at 100 hpf (4 h post Shigella infection) and at 120 hpf (24 h post Shigella infection), to monitor neutrophil recruitment to the locally injected bacteria. Overlay of green (SINV and neutrophil) and red (Shigella) fluorescence is shown. The white box indicates the region chosen to count the recruited neutrophils. (B) Neutrophil recruitment quantification upon sublethal Shigella-DsRed (red symbol) injection or sequential SINV + Shigella (blue symbols) injection. Neutrophils were counted from images taken on live infected larvae [white box delimitated the region chosen to count the recruited neutrophils in (A)] using ImageJ software, and plotted as specified in Section “Materials and Methods.” Data are from one experiment (n = 12 larvae scored for each condition). Mean ± SEM are also shown (horizontal bars). (C) Frames extracted from maximum intensity projection of in vivo time-lapse confocal imaging sessions of 96 hpf mpx:GFP larvae injected subcutaneously with Shigella-DsRed alone (top panel) or of SINV + Shigella co-infected larvae that had been injected one day before with SINV-GFP in the bloodstream (at 72 hpf) (bottom panel). Overlay of green (SINV and neutrophils) and red (Shigella) fluorescence of the caudal area of the larvae is shown. Time indicated on the frames is upon subcutaneously Shigella injection. See also Video S1 in Supplementary Material. Scale bar: 50 µm. (D) Neutrophil recruitment quantification upon subcutaneous Shigella-DsRed (red symbol) injection or sequential bloodstream SINV-GFP injection followed the day after by subcutaneous Shigella-DsRed (blue symbols) injection. Neutrophils were manually counted at 30 min, 2 and 3 h post Shigella injection from maximum intensity projections frames of confocal acquisitions of live infected larvae (to count the recruited neutrophils the region taken into consideration is shown in (B) and plotted as specified in Section “Materials and Methods.” Data plotted are from n = 4 to 5 larvae scored for each condition. Mean ± SEM are also shown (horizontal bars). (E) Frames extracted from maximum intensity projection of confocal acquisition of SINV + Shigella mpx:GFP co-infected larvae. SINV-GFP was injected in the bloodstream at 72 hpf and Shigella-DsRed was subcutaneously injected the day after, at 96 hpf. The acquisition of the infected larvae was started about 30 min after Shigella injection. Three dying Shigella engulfing neutrophils are shown (annotated as 1, 2, and 3 on the frames). Overlay of green (SINV and neutrophils) and red (Shigella) fluorescence of the caudal area of the larvae is shown. Time indicated on the frames is upon subcutaneously Shigella injection. See also Video S2 in Supplementary Material. Scale bar: 20 µm. (F) Dying neutrophils quantitation upon subcutaneous Shigella-DsRed (red symbol) injection or sequential bloodstream SINV-GFP injection followed the day after by subcutaneous Shigella-DsRed (blue symbols) injection. Dying neutrophils were manually tracked and quantified from maximum intensity projections of confocal acquisitions and plotted as specified in Section “Materials and Methods.” Data plotted are from n = 4 Shigella-infected larvae and n = 6 SINV + Shigella-infected larvae scored. Mean ± SEM are also shown (horizontal bars).