Fig 1.
Parasitic success is dramatically reduced in Cxcr4+/1013 mutant mice.
L. sigmodontis (40 larvae) were subcutaneously injected into wt and Cxcr4+/1013 C57BL/6 mice. Worms were harvested in the pleural cavity of the mice 20 days p.i. and counted (number of L3 filariae, nF). Results are expressed as mean +/- SEM (9.75 +/- 0.65 and 3 +/- 0.26 for wt and Cxcr4+/1013 C57BL/6 mice respectively), n = 20 and each mouse is represented by a dot (pool of 4 independent experiments with 5 mice per group), t-test ***: p < 0.001.
Fig 2.
Transient reversion of neutropenia in infected Cxcr4+/1013 mice.
A: Total numbers of lymphocytes, eosinophils and neutrophils were determined on blood smears from non-infected mice at day 0 (non inf. D0) and from subcutaneously infected mice 20 days p.i. (post inf. D20). Open dots and black dots are representing wt and Cxcr4+/1013 mice, respectively (n = 8, each mouse is represented by a dot). Means +/- SEM were at D0 for wt and Cxcr4+/1013, respectively: for lymphocytes 8.21 +/- 0.77 and 2.02 +/- 0.39; for eosinophils, 0.49 +/- 0.09 and 0.11 +/- 0.03 and for neutrophils 1.24 +/- 0.17 and 0.42 +/- 0.1. Differences between wt and Cxcr4+/1013 mice were analyzed by t-test. *: p < 0.05, **: p < 0.01, ***: p <0.001. B: Total numbers of blood neutrophils throughout the course of the filarial infection in infected wt and Cxcr4+/1013 mice (n = 8). Differences between wt and Cxcr4+/1013 mice were analyzed by one-way ANOVA with repeated measures then Bonferroni. *: p < 0.05, **: p < 0.01. C: Blood neutrophils counts in Cxcr4+/1013 mice at different time points upon SC inoculation with either 40 infective larvae (SC L3, ○, plain line) or 10μg L3-derived whole body crude extracts (SC WBE, ●, dotted line) or upon IV inoculation with 40 infective larvae (IVL3, Ƴ, plain line) or 10μg L3-derived whole body crude extracts (IVWBE, ■, dotted line). Neutrophil counts were done over a 20 days’ time period following inoculation and were expressed as ratio to wt mice levels. Ratios close to 1 depict a trend toward normalization of the blood neutrophils counts in the Cxcr4+/1013 mice. Raw data are presented in S2 Table. Results are expressed as mean + SEM, n = 4 to 5 and 8.
Fig 3.
Reversion of the parasitic success in Cxcr4+/1013 mice upon intravenous inoculation.
Both wt (white bars) and Cxcr4+/1013 (black bars) mice were injected with 40 infective larvae either subcutaneously (SC) or intravenously (IV) and worms were recovered in the pleural cavity at 20 days p.i. Filarial loads (means +/- SEM) were 5.89 +/- 0.53 and 2 +/- 0.21 for the wt and Cxcr4+/1013 SC-inoculated mice, respectively; and 13.1 +/- 1.1 and 12.25 +/- 1.1 for the wt and Cxcr4+/1013 IV-inoculated mice, respectively. The parasitic success represents for each mouse the larvae counts recovered in the pleural cavity expressed as percent of the inoculum. Results are expressed as mean +/- SEM, n = 7 to 12, One-way ANOVA then Bonferroni *: p < 0.05; ***: p < 0.001; ns = not significant.
Fig 4.
Steady state levels and recruitment of neutrophils in the skin upon filariae inoculation.
Skin sections were collected before inoculation (control mice, Ctrl) or excised from the inoculation site 6 hours p.i. (Infected mice, Inf), embedded in paraffin and stained with anti-NIMP-R14 for the visualization of neutrophils. Representative H&E stained skin sections for each group (wt and Cxcr4+/1013 control (Ctrl) and infected mice (Inf)) are displayed and the total number of neutrophils per mm2 skin in the dermis-hypodermis and the SLCT layers are given in the graph below. Representative sections: scale bar = 100μm, magnification x40. Representative x100 magnifications: scale bar = 40μm. n = 7 to 8 per group. One-way ANOVA then Bonferroni *: p < 0.05; **: p < 0.01, ***: p < 0.001.
Fig 5.
Effect of neutrophil depletion on parasitic burden.
Neutrophil depletion was performed via intraperitoneal inoculation of anti-Ly6G antibody 6 hours (A, arrow) prior SC inoculation with filariae (Day (D) 0) and analyses were carried out at the indicated time throughout the course of the filarial infection up to 20 days. A and B: Effect of the neutrophil-depleting treatment on circulating neutrophil levels (A) and skin-resident neutrophils (B). A: Blood levels of neutrophils from the onset of the inoculation of the anti-Ly6G (clone 1A8) antibody 6 hours prior SC infection up to 20 days p.i. in wt (open triangles) and Cxcr4+/1013 (black triangles) mice. Control wt (open dots) and Cxcr4+/1013 mice (black dots) were injected with PBS. Results are expressed as mean +/- SEM, n = 6 to 8, One-way ANOVA p < 0.05, Bonferroni *: p < 0.05, **p < 0.01, ***: p < 0.001. B: Effect of the anti-Ly6G (clone 1A8) antibody on skin-infiltrating neutrophils 6 hours (H6) and 12 ous (H12) after antibody injection in non-infected mice. Skin sections were stained with anti-NIMP-R14 antibody to count neutrophils that were reported as number per mm2 of dermis-hypodermis and SLCT layers. Doted lines indicated steady state neutrophil levels in the dermis-hypodermis layer of wt and Cxcr4+/1013 mice. C: Filarial load in the pleural cavity 20 days p.i. in wt (open dots) and Cxcr4+/1013 (black dots) mice injected with PBS (Ctrl) or with anti-Ly6G antibody clone 1A8 (left panel, Ab + Inf) or clone NIMP-R14 (right panel, Ab + Inf). Recovered worms were counted (nF). Results are expressed as mean +/- SEM, n = 4 to 8 (clone 1A8) and n = 4 to 6 (clone NIMP-R14). One-way ANOVA then Bonferroni *: p < 0.05; ***: p < 0.001; ns: not significant.
Fig 6.
Neutrophils from Cxcr4+/1013 mice display higher responses to filariae exposure.
A: Left panel: microscopic nitroblue tetrazolium (NBT) assay in wt and Cxcr4+/1013 mice. Production of superoxide anion (O2-) by BM-neutrophils-derived mice is revealed by the black NBT deposits (arrows) within the cells (lower panel). Scale bar = 20μm. Representative pictures of an unstained neutrophil (above) and NBT stained neutrophils (below) reporting > 90% of cells with NBT deposits. Experiment has been performed twice with wt (n = 3–4 mice) and mutant mice-derived neutrophils (n = 3–4 mice). Right panel: Relative reactive oxygen species (ROS) content measured via a Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay in wt (white bars) or Cxcr4+/1013 (black bars) mice neutrophils activated or not with L3 (20 L3 for 15 minutes). ROS content was expressed as the ratio of DCFH-DA signal of L3-actived cells relative to unstimulated cells (Activation Ratio). Experiment has been performed twice with wt (n = 5 and 4 mice) and mutant mice-derived neutrophils (n = 5 and 4 mice). T-test: *: p < 0.05. DCFH-DA signal of unstimulated cells were not statistically different (p = 0.5421) between wt (MFI = 7923 +/- 909 SEM) and Cxcr4+/1013 (MFI = 8811 +/- 1058 SEM) mice. B: Left panel: MPO content in wt (white bars) or Cxcr4+/1013 (black bars) derived neutrophils left unstimulated (Un) or after incubation with L3 (20 L3 for 1 hour). Samples were permeabilized and stained with anti-MPO-FITC antibodies and assessed by flow cytometry. Results expressed as MFI are from one representative experiment out of two (mice number: n = 5). Two-way ANOVA then Bonferroni *: p < 0.05, **: p < 0.01. Right panel: extracellular myeloperoxidase contents in culture supernatants measured using an MPO ELISA kit. Results are expressed as mean +/- SEM of two independent experiments pooled together (mice number: n = 3). Two-way ANOVA then Bonferroni. *: p < 0.05 when comparing stimulation factor (RPMI or L3), #: p < 0.05 when comparing mouse strains for a given time point and treatment. C: Quantification of necrotic and NETs releasing neutrophils left unstimulated or incubated with L3 (20 L3 for 4 hours) followed by staining of the extracellular DNA with SYTOX. Left panel: The top panels illustrated the different states of neutrophil activation and were from wt neutrophils stimulated with L3. Living cells were not fluorescent and among Sytox+ cells, NETs-releasing neutrophils (NET, arrows) are harboring a Sytox+ halo that distinguished them from necrotic neutrophils (nec, arrows). Scale bar: 50μm. Other panels are representative of neutrophils of wt mice either left unstimulated (Un) or stimulated with L3. Scale bar: 500μm. Right panel: cells were analyzed (200 events) and the number of Sytox positive cells (Sytox +, stripped bars) and of Sytox negative cells (Sytox -, plain bars) for wt (white bars) or Cxcr4+/1013 (black bars) neutrophils were reported for each condition. Number of mice: n = 4. D: Mouse neutrophils were incubated with L3 (10 larvae) or 100 nM PMA for 4, 24 and 36 hours. Culture supernatants were analyzed for extracellular DNA using the PicoGreen assay. Results are expressed as mean +/- SEM of two independent experiments pooled together (number of mice: n = 3). Two-way ANOVA then Bonferroni. *: p < 0.05 when comparing stimulation factor (RPMI, PMA or L3), #: p < 0.05 when comparing mouse strains for a given time point and treatment.