Figure 1

Equal neutrophil recruitment efficiency under Ly6G-deficiency, but decreased uptake of L. major parasites during early stage of infection. (a) Infection of Ly6G deficient Ly6G^cre/cre × Rosa-tdTomato mice and Ly6G proficient Ly6G^cre/+ × Rosa-tdTomato control mice with EGFP expressing L. major. Gating strategy to identify neutrophils lacking the Ly6G marker based on the low MHC class II expression and intermediate expression of Ly6C (CD11b^hiMHCII^loLy6C^int) in Ly6G^cre/+ × Rosa-tdTomato control and Ly6G-deficient Ly6G^cre/cre × Rosa-tdTomato mice (left and middle panel). Histogram illustrates the quantification of the tdTomato- and Ly6G-signal on gated neutrophils (CD11b^hiLy6C^int) in Ly6G^cre/+ × Rosa-tdTomato and Ly6G-deficient Ly6G^cre/cre × Rosa-tdTomato mice (right panel). (b) Proportion of neutrophils among CD45⁺ cells 2, 7 and 28 days p.i. with 10⁶ metacyclic L. major-EGFP parasites in the infected tissue analyzed by flow cytometry. Each dot represents one infected ear. Horizontal bars represent the mean; ns, not significant as determined by two-way ANOVA (time, genotype) with Bonferroni post test for the genotype. (c) Recruitment of monocyte-derived phagocytes to the site of infection. Gating strategy for monocyte-derived dendritic cells (moDC, CD45⁺CD11b⁺ CD11c⁺MHCII⁺) and monocyte-derived macrophages (mo-macro, CD45⁺CD11b⁺ CD11c^-MHCII⁺) in heterozygeous Ly6G^cre/+ and homozygous Ly6G^cre/cre mice. (d,e) Percentage of infected (EGFP containing) mo-macro (d) and mo-DCs (e) among CD45⁺ cells after quantitative evaluation of flow cytometry. Significant decrease of mo-DCs at late infection phase 28 days p.i. Each dot represents one infected ear. Horizontal bars represent the median; ***p < 0.001; ns not significant as determined by two-way ANOVA (time, genotype) with pairwise Bonferroni post test. (f) Unchanged parasite burden in L. major infected ear determined by limiting dilution 7 and 28 days p.i. Horizontal bars represent mean; ns not significant as determined by two-way ANOVA (time, genotype) with Bonferroni post test for the genotype. (g) Histograms depicting the infection rate of infected neutrophils (left panel), mo-macro (middle panel) and mo-DCs (right panel) after infection with EGFP-expressing L. major 2, 7 and 28 days p.i. in Ly6G^cre/+ and Ly6G^cre/cre mice. (h–j) Quantitative evaluation of histograms shown in (g). Proportion of infected neutrophils (h), mo-macro (i) and mo-DCs (j) analyzed via EGFP fluorescence of the parasite within the cells at day 2, 7 and 28 p.i. in Ly6G^cre/+ and Ly6G^cre/cre mice. Analysis revealed that Ly6G-deficient neutrophils phagocytose less parasites at early infection phase. Each dot represents one infected ear. Horizontal bars represent the mean; **p < 0.01; ns not significant as determined by two-way ANOVA (time, genotype) with Bonferroni post test for the genotype. Data are pooled from at least three independent experiments. (k) Mean Ly6C and CD11b fluorescence in tdTomato-expressing neutrophils from infected Ly6G^cre/+ × Rosa-tdTomato and Ly6G^cre/cre × Rosa-tdTomato mice. The expression of Ly6C surface protein is increased in Ly6G-deficient animals. Each dot represents one infected ear. Horizontal bars represent mean; *p < 0.05; ns not significant as determined by one-way ANOVA with pairwise Bonferroni post test. Data are pooled from at least three independent experiments.