Fig 1.
CCR3-dependent tissue eosinophilia is required for immunity to B. malayi.
Enumeration of peritoneal eosinophils (A,B,D,E,G,H) and % recoveries of motile B. malayi in BALB/c WT compared with ΔdblGATA deficient mice (C), in BALB/c WT mice treated with intraperitoneal (ip) rat IgG control or rat anti-CCR3 (αCCR3) (F) or in WT compared with CCR3 deficient mice (I) at indicated time points post-ip infection with 50 BmL3. Data from individual mice with median and interquartile range are plotted. Significant differences between naïve or infected WT controls and experimental groups at a given time point is assessed by Mann-Whitney or Kruskal-Wallis + Dunn’s tests (>2 groups). Data is plotted is either pooled from 2 individual experiments per time-point or from individual experiments with groups of 4–6 mice per group per time-point.
Fig 2.
In situ proliferation of macrophages with an alternatively-activated phenotype develop at the site of B. malayi infection.
Expansion of F4/80 peritoneal Mϕ (A), F4/80 peritoneal Mϕ expression levels of Ki67 (B,C), peritoneal cell (PC) arg1 expression (D) PC arginase activity (E) and F4/80 peritoneal Mϕ expression levels of RELMα (F,G) in WT BALB/c mice at indicated time points post-infection with 50 BmL3. Data from individual mice with median and interquartile range are plotted. Significant differences between naïve or infected WT groups at a given time point is assessed by Mann-Whitney or Kruskal-Wallis + Dunn’s tests (>2 groups). Data is plotted is either pooled from 2–3 individual experiments per time-point or from individual experiments with groups of 4–6 mice per group per time-point.
Fig 3.
Development of AAMϕ in response to B. malayi infection requires adaptive-immune IL-4/IL-4Rα signalling and is associated with resistance to adult parasite establishment.
Expansion of peritoneal Mϕ (A) peritoneal cell (PC) arg1 expression (B) PC arginase activity (C) peritoneal Mϕ arg1 expression (D) Mϕ RELMα expression (E) and recovery of B. malayi (F) at indicated time points post-infection with 50 BmL3 in BALB/c WT, IL-4Rα-/- or SCID mice or in naïve controls. Expansion of peritoneal Mϕ (G,H) or PC arginase activity (I) in BALB/c SCID mice +6d post-treatment with recombinant murine IL-4+rat anti-mouse IL-4 monoclonal antibody complex (rIL-4c) or rat IgG control ip treatments with or without infection with 50 BmL3. Data from individual mice with median and interquartile range are plotted. Significant differences between groups assessed by Mann-Whitney or Kruskal-Wallis + Dunn’s post-hoc tests (>2 groups). Data is from an individual experiment or pooled from 2–3 experiments per time-point using groups of 4–6 mice per group / time-point.
Fig 4.
Eosinophilia does not impact on expansion of arginase-expressing AAMϕ but augments RELMα production.
Expansion of peritoneal Mϕ in BALB/c WT, ΔdblGATA deficient mice or CCR3 deficient mice (A,B) or in WT mice treated ip with rat IgG control or rat αCCR3 (C,D) at indicated time points post-ip infection with 50 BmL3. Arginase activity in PC cells from BALB/c WT, ΔdblGATA deficient or CCR3 deficient mice, WT mice treated ip with rat IgG control or rat αCCR3 (E) and expression of arg1 in purified Mϕ from WT mice treated ip with IgG control or αCCR3 (F) at indicated time points post-ip infection with 50 BmL3. F4/80 peritoneal Mϕ expression levels of RELMα in BALB/c WT or CCR3-/- mice at +14 day post-infection with 50 BmL3 (G,H). Data from individual mice with median and interquartile range are plotted. Significant differences between groups assessed by Mann-Whitney or Kruskal-Wallis + Dunn’s tests (>2 groups). Data is from an individual experiment or pooled from 2–3 individual experiments per time-point using groups of 3–6 mice per group / time-point.
Fig 5.
Temporal ablation of peritoneal Mϕ enhances survival of B. malayi larvae coincident with impaired tissue eosinophilia.
Cytospins of peritoneal cells with macrophages (Mϕ) indicated (A), quantification of macrophages and eosinophils (B,C,E) and recovery of B. malayi larvae (D) at indicated time points post-infection with 50 B. malayi L3 with or without prior treatment with clodronate liposomes (CL) in BALB/c WT mice or naïve controls (d0). Data from individual mice with median and interquartile range are plotted. Significant differences between groups per time point assessed by Mann-Whitney. Data is from an individual experiment or pooled from 2–3 individual experiments per time-point using groups of 4–6 mice per group / time-point.
Fig 6.
BmL3AAMϕ are necessary to sustain a larvicidal tissue eosinophilia.
Cytospins of FACS-sorted BALB/c WT peritoneal SigLecF+ eosinophils (A) or BmL3AAMϕ (B) +14 days post-infection with 50 BmL3. Survival analysis of BmL3 (C) throughout 7-days culture with normal mouse serum (NS) or co-cultured with 106 FACS-sorted eosinophils (Eo), 106 BmL3AAMϕ or combinations of Eo+BmL3AAMϕ, (cells sourced as for A,B). Data is pooled from two individual experiments evaluating motility of 10 BmL3 per condition. Significance of Kaplein-Meir survival analysis vs NS serum control is indicated per condition. Eosinophilia (D,E) +6dpi with 50 BmL3 ip in BALB/c WT mice pre-treated with clodronate liposomes (CL) ip +/- adoptive transfer of 0.75x106 BmL3AAMϕ ip (cells sourced as for B). Time course of peritoneal eosinophilia in BALB/c SCID mice at indicated time points post infection with 50 BmL3 ip (F). Peritoneal eosinophilia (G,H) or recovery of B. malayi larvae (I) at +14 days post-infection with 50 BmL3 in BALB/c SCID and E) and pre-treatment with either rat IgG or rat αCCR3 antibody. Data from individual mice with median and interquartile range are plotted. Significant differences between groups assessed by Kruskal-Wallis + Dunn’s tests. Data is from an individual experiment or pooled from 2–3 individual experiments per time-point using groups of 4–6 mice per group / time-point.
Fig 7.
Exogenous IL-4 bolsters CCR3-dependent eosinophilia and the eosinophilic larvicidal response in SCID mice via BmL3AAMϕ development.
Schematic of experimental approach (A). Flow cytometric assessments of F4/80 Mϕ or SigLecF eosinophil proportions (B), total peritoneal cell number (C), eosinophil number (D) at +6dpi or larval parasite recoveries (E) at +14dpi in BALB/c SCID mice pre-treated ip with either rat IgG, clodronate liposomes (CL) or rat αCCR3 ip, prior to infection with 50 BmL3 and/or up to three doses of rat IgG (25μg), IL-4c (1μg rIL-4 complexed to 5μg rat anti-IL-4) delivered 0d, +2d, +/- +7d with or without daily oral dosing with CCR3 inhibitor. Data from individual mice with median and interquartile range are plotted. Significant differences between test groups and appropriate rat IgG treated controls assessed by Kruskal-Wallis + Dunn’s tests. Data is representative of two individual experiments (B,C) or pooled from 2–3 individual experiments using groups of 3–6 mice per group / time-point.