Ly6G deficiency alters the dynamics of neutrophil recruitment and pathogen capture during Leishmania major skin infection

Abstract

Neutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils' ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system.

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.

Figure 2

Uptake of neutrophil-derived material by monocyte-derived phagocytes is not altered by Ly6G-deficiency. (a,b) Confocal microscopy of Ly6G^cre/+ × Rosa-tdTomato reporter mice infected with EGFP-expressing L. major 2 days p.i., demonstrating concomitant content of L. major parasites along with neutrophil-derived material in CD54⁺ (a) and CD11c⁺ cells (b) and thus suggesting the uptake of L. major together with neutrophil material. Parasites are pictured in green and the neutrophil-derived material in red. Scale bar: 10 µm. (c) Flow cytometry analysis of infected cells from Ly6G^cre/+ × Rosa-tdTomato reporter mice (left panel), Ly6G-deficient Ly6G^cre/cre × Rosa-tdTomato (middle panel) and non-fluorescent Ly6G^+/+ littermate control mice (right panel) are depicted. The upper row shows the histograms for quantification of the neutrophil-derived tdTomato signal (omitting the tdTomato^high neutrophils) in mo-DCs (CD11b⁺CD11c⁺MHCII⁺) and mo-macro (CD11b⁺CD11c^-MHCII⁺). Middle row: Gating on infected (EGFP⁺) and non-infected (EGFP^-) population of phagocytes. Lower row: Exclusive uptake of neutrophil-derived tdTomato material in the L. major infected (EGFP⁺), but not the uninfected cell population. Ly6G^+/+ littermate control mice not expressing tdTomato certify that spectral overlap of EGFP and tdTomato is not responsible for the higher tdTomato-signal in infected cells. (d,e) Analysis of the mean fluorescence intensity (MFI) of neutrophil-derived tdTomato signal in control and Ly6G deficient mice for mo-macro (d) and mo-DCs (e) revealed that tdTomato content is Ly6G independent, Thus, the uptake of neutrophil material by recruited monocyte-derived phagocytes is not altered due to Ly6G deficiency. Infected and non-infected cells are shown from Ly6G^cre/+ × Rosa-tdTomato (black symbols), Ly6G^cre/cre × Rosa-tdTomato mice (white symbols) and Ly6G^+/+ littermates (grey symbols). Each dot represents one infected ear. Horizontal bars represent mean; ***p < 0.001; ns not significant as determined by one-way ANOVA with Bonferroni post test for multiple comparisons.

Figure 3

Different kinetics in the early recruitment of Ly6G-deficient neutrophils. (a) Intravital 2-photon microscopy of L. major-EGFP infected Ly6G^cre/+ × Rosa-tdTomato (upper panel) and Ly6G^cre/cre × Rosa-tdTomato (lower panel) mice 60, 90 and 120 min p.i. shows that neutrophils are recruited to the site of infection within less than 2 h. Red tracks show the accumulated neutrophil migration paths over time. Scale bar 100 µm. (b) Numbers of neutrophil shapes present in the imaged volume over time. The curves for the corresponding images in (a) are shown in black, the other curves are shown in grey for comparison. (c) Left panel: Increase in neutrophil number in the imaged volume per minute, shown separatedly for the time period between 60 and 120, or 120 and 180 min. Data from Ly6G^cre/+ × Rosa-tdTomato (closed symbols) and Ly6G^cre/cre × Rosa-tdTomato (open symbols) is shown. Each symbol represents one infected ear analyzed by intravital 2-photon microscopy. *p > 0.05; ns not significant as determined by one-way ANOVA with pairwise Bonferroni post test. Right panel: comparison of the neutrophil increase rate between 60 and 120 versus 120 to 180 min p.i. in Ly6G^cre/+ × Rosa-tdTomato (closed symbols) and Ly6G^cre/cre × Rosa-tdTomato (open symbols) animals. *p < 0.05 as determined by unpaired t test. The initial lower neutrophil recruitment in Ly6Gcre/cre mice was compensated by higher entry rate 2–3 h p.i. (d) Left panel: Neutrophil instantaneous speeds, shown separatedly for the time period between 60 and 120, or 120 and 180 min. Data from Ly6G^cre/+ × Rosa-tdTomato (closed symbols) and Ly6G^cre/cre × Rosa-tdTomato (open symbols) is shown. Each symbol represents one infected ear analyzed by intravital 2-photon microscopy. *p > 0.05 as determined by one-way ANOVA with pairwise Bonferroni post test. Right panel: comparison of the neutrophil instantaneous speeds between 60 to 120 versus 120 to 180 min p.i. in Ly6G^cre/+ × Rosa-tdTomato (closed symbols) and Ly6G^cre/cre × Rosa-tdTomato (open symbols) animals. ns not significant as determined by unpaired t test. (e) Distribution of neutrophil instantaneous speeds in proximity (< 5 µm) and at larger distance from EGFP-expressing L. major. Data are shown separately for the time periods between 60 and 120 versus 120 and 180 min p.i. for Ly6G^cre/+ × Rosa-tdTomato (left panels) and Ly6G^cre/cre × Rosa-tdTomato (right panels). (f) Analysis of the mean neutrophil distance to EGFP-expressing L. major. Data are shown separately for the time periods between 60 and 120 versus 120 and 180 min p.i. for Ly6G^cre/+ × Rosa-tdTomato (closed symbols) and Ly6G^cre/cre × Rosa-tdTomato (open symbols). Horizontal bars denote the median; ns, not significant as determined by one-way ANOVA with Bonferroni post test for multiple comparisons. (g) Analysis of neutrophil instantaneous speeds in proximity (< 5 µm) and at larger distance from EGFP-expressing L. major. Data are shown separately for the time periods between 60 and 120 versus 120 and 180 min. p.i. for Ly6G^cre/+ × Rosa-tdTomato (closed symbols) and Ly6G^cre/cre × Rosa-tdTomato (open symbols). Horizontal bars denote the median. Data were analyzed using a two-way ANOVA yielding a significant contribution of L. major distance and time point of analysis to neutrophil speed, but not of the Ly6G genotype.