Figure 3. cxcr4b is not required for the establishment of macrophage parasitism, but macrophages are necessary to mediate cxcr4b-dependent angiogenesis.

(A,B) Recruitment of macrophages (A) and neutrophils (B) 3 hours post local hindbrain (HB) infection at 2 dpf with Mm was unaffected by cxcr4b mutation. Each data point in (A,B) represents the recruitment measured in 1 embryo. Experiments were performed in 2 replicates (cumulated in the graphs), each with 17–35 embryos per group. Total number of embryos analysed: 38 (Mock-cxcr4b^+/+), 41 (Mock-cxcr4b^−/−), 53 (Mm-cxcr4b^+/+), 49 (Mm-cxcr4b^−/−). C–D cxcr4b is dispensable for the capability of macrophages to counteract intracellular replication of Mm Δerp. Infected macrophages (mpeg1:mCherry-F⁺) were classified into three phenotypic classes, according to the severity of intracellular infection (1–5, 6–10, or >10 bacteria). (C) represents the percentage of macrophages per larva that populates each class. (D) represents the overall distribution of macrophage phenotypes (macrophages from all larvae cumulated). Experiments were performed in 2 replicates (cumulated in the graphs), each with 16–17 larvae per group. Total number of larvae analysed in C: 32 (cxcr4b^+/+), 33 (cxcr4b^−/−). Total number of macrophages analysed in D: 387 (cxcr4b^+/+), 334 (cxcr4b^−/−). (E–G) % of mpeg1:mCherry-F signal overlapping with Mm-GFP signal in cxcr4b^−/− and cxcr4b^+/+. No significant deviation in macrophage composition of the trunk granuloma lesions was detected. Each data point represents 1 granuloma. Experiment was performed in 1 replicate. Total number of granuloma analysed: 37 (cxcr4b^+/+), 23 (cxcr4b^−/−). Representative example images are shown in (F and G). (H) Expression of cxcr4b in macrophages (mpeg1⁺) and neutrophils (mpx⁺). Both cell subsets express cxcr4b at significantly higher levels than the negative (non-fluorescent) cell population. Data represent fold changes relative to the negative cell fraction. Cells were sorted at 2 dpf, from at least 100 embryos. Experiment was performed in 3 replicates. I-J. Macrophages are indispensable to mediate granuloma vascularisation (I) and an increased number of neutrophils (by irf8 morpholino knockdown) cannot compensate for macrophage deficiency, neither in cxcr4b^−/− nor in cxcr4b^+/+. Deficient bacterial expansion in irf8 knockdown condition (irf8-MO) compared to standard control morpholino treatment (Stctr-MO) can be attributed to the lack of vascularisation as suggested by non-significant differences in burden between the knockdowns and control cxcr4b^−/− (J). Data are analysed as in Fig. 2A,B, but infections were performed at 33 hpf, instead of 2 dpf. Experiments were performed in 1 replicate. Total number of granuloma analysed: 47 (Stctr-MO-cxcr4b^+/+), 47 (Stctr-MO-cxcr4b^−/−), 61 (irf8-MO-cxcr4b^+/+), 73 (irf8-MO-cxcr4b^−/−).