Unbalanced Arginine pathway and altered maturation of pleural macrophages in Th2-deficient mice during Litomosoides sigmodontis filarial infection

Abstract

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα^-/-/Il-5^-/- ) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα^-/-/Il-5^-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα^-/-/Il-5^-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80^intermediate macrophages from infected Il-4rα^-/-/Il-5^-/- mice failed to mature into resident F4/80^high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα^-/-/Il-5^-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

Keywords: filariasis; lung; macrophage; microfilaria; nematode; parasite; pleura.

Figure 1

The absence of IL4R/IL5 signaling promotes filarial survival and production of microfilaria. WT and Il-4rα^-/-/Il-5^-/- BALB/c mice were infected with L. sigmodontis and parasitic load was determined at 50 and 70 days post infection (dpi). (A) Number of adult worms in the pleural cavity; results are expressed as mean ± SEM. n=9 WT and n=9 Il-4rα^-/-/Il-5^-/- mice at 50 dpi and n=24 WT and n=24 Il-4rα^-/-/Il-5^-/- mice at 70 dpi, pool of 1-3 experiments. (B) Number of microfilariae (Mfs) in pleural cavity fluid (n=8 per group of mice), in peripheral blood (n=24 per group of mice), in cardiac blood (n=24 per group of mice), in pulmonary flush (n=8 per group of mice) and in the whole lung tissue (n=5 per group of mice). For each location a t-test was performed ^$p<0.05, ^$$p<0.01 and ^$$$p<0.001 represent differences between WT and Il-4rα^-/-/Il-5^-/- mice.

Figure 2

Il-4rα^-/-/Il-5^-/- mice showed decreased pleural pathology and altered balance of the arginine metabolic pathway. WT and Il-4rα^-/-/Il-5^-/- BALB/c mice were infected with L. sigmodontis and pleural cavity pathology and macrophages were investigated 70 days post infection (dpi). (A–F) Scanning Electron Microscopy images of the parietal pleura: (A, B) Smooth pleura in naive mice; (F, I) high density of polyps in 70 dpi WT mice, and (E, F) rare polyps in 70 days-infected Il-4rα^-/-/Il-5^-/- mice. (G) Collagen measurement in the pleural cavity flush. (H) Total number of F4/80⁺ macrophages and Siglec-F⁺ eosinophils as determined by flow cytometry. (I) Expression of prototypical markers of alternative activation Chil3 and Mrc1 in pleural macrophages determined by qRT-PCR. (J) Collagen measurement in macrophage culture supernatant after 24h culture in vitro. (K) Simplified arginine metabolic pathway, adapted from (47, 48). (L) Expression of Arg1 by pleural macrophages determined by qRT-PCR. (M) In vitro arginase activity in pleural macrophages. Arginase activity was measured using QuantiChrom Arginase assay kit. (N) In vitro nitric oxide (NO) production (μM) from pleural macrophages after incubation for 16h with or without IFN-γ or filarial antigen (10µg/ml). (O) Mean fluorescence intensity (MFI) of IFNgR1 expression on F4/80⁺ pleural macrophages. Dots represent individual mice and results are expressed as mean ± SEM. (A–F) Images are representative of n=3 mice per group. (H) Pool of 4–6 independent experiments n=20-27 mice per group. (J) n = 10. (I, L), n=6 per group. Two-way ANOVA: *p<0.05, ***p<0.001 represent differences between group; ^$$p<0.01, ^$$$p<0.001 represent differences between mice strains. (N) n=2-4 mice per group. (O) n = 8.

Figure 3

Pleural macrophages from Th2-deficient mice present higher phagocytic capacity but lower phenotypic diversity. Pleural macrophages from L. sigmodontis infected wild-type (WT) and Il-4rα^-/-/Il-5^-/- BALB/c mice were cultured with E. coli fluorescent bioparticles to address their phagocytic capabilities by confocal multiplex imaging. (A) Diagram of the experimental setup. (B, D) Aspect of the F4/80⁺ and/or MHCII⁺ adherent pleural cells from infected (B) WT and (D) Il-4rα^-/-/Il-5^-/- BALB/c mice. (C, E) Cellpose algorithm (45) cell segmentation outline (while line) and phRodo fluorescence signal (yellow) in infected (B) WT and (D) Il-4rα^-/-/Il-5^-/- BALB/c mice. (F) Phagocytosis expressed as phRodo mean fluorescence intensity (MFI). (G) Measure of the average cell surface. (H) Measure of the average cell roundness. (I, J) Multiplex images of adherent pleural macrophages from (I) WT and (J) Il-4rα^-/-/Il-5^-/- mice showing expression of F4/80 (purple), CD11b (blue), MHCII (green) and CD11c (orange). (K, L) tSNE analysis of single cell fluorescence intensity and shape parameters extracted from cell segmentation of microscopy images. Dots represent individual mice and results are expressed as mean ± SEM of n = 6 mice per group. For the tSNE analysis, at least 600 F4/80⁺ cells per mouse were concatenated. A t-test was performed (data normally distributed) ^$$p<0.01 and ^$$$p<0.001 represent differences between WT and Il-4rα^-/-/Il-5^-/- mice.

Figure 4

Pleural macrophages from Il-4rα^-/-/Il-5^-/- mice fail to mature into resident macrophages. Pleural cells were isolated from L. sigmodontis infected WT and Il-4rα^-/-/Il-5^-/- BALB/c mice at 70 dpi and analyzed by flow cytometry for F4/80, Ly6C, MHCII and Siglec-F. (A) Experimental setup and gating strategy. (B) Samples were concatenated and a tSNE was performed on F4/80⁺ cells. (C) Representative plots and gating strategy for F4/80^intermediate and F4/80^high macrophages and for naïve and infected WT and Il-4rα^-/-/Il-5^-/- BALB/c mice. An alternative gating strategy for F4/80^intermediate and F4/80^high including a CD11b and CD115 pre-gating is displayed in Supplementary Figure 3 . (D) Representative plots and gating strategy for F4/80^-MHCII^-Ly6C⁺ monocytes. (E) Absolute numbers of F4/80^high macrophages. (F) Absolute numbers of F4/80^intermediate macrophages. (G) Absolute numbers of F4/80^-MHCII^-Ly6C⁺ monocytes. Dots represent individual mice and results are expressed as mean ± SEM of n = 8 mice per group. Two-way ANOVA: ^$p<0.01, ^$$$p<0.001 represent differences between mice strains, n = 8. ns, not significant.

Figure 5

Monocyte-derived macrophages replenish the alveolar niche. Bronchoalveolar cells were isolated from naive or L. sigmodontis infected WT and Il-4rα^-/-/Il-5^-/- BALB/c mice at 70 dpi. CD11c⁺F4/80⁺ alveolar macrophages were analyzed by flow cytometry. (A) Experimental setup. (B) Representative plots and gating strategy for Siglec-F⁺ eosinophils. Average frequency +/- SEM of the different population is displayed. (C) Absolute number of CD11c⁺ F4/80⁺ alveolar macrophages. (D) Representative plots and gating strategy for CD11c⁺F4/80⁺ alveolar macrophages. Average frequency +/- SEM of the different population is displayed. (E) Mean fluorescence intensity (MFI) of RELMα expression on CD11c⁺ F4/80⁺ alveolar macrophages. RELMα expression was normalized by subtracting the Fluorescence Minus One (FMO) control MFI. (F) Representative expression of RELMα by CD11c⁺ F4/80⁺ alveolar macrophages. Shaded histograms represent FMO controls. (G) Average frequency of Siglec-F^low MHC⁺, Siglec-F⁺MHC⁺ and Siglec-F⁺MHC^- cells among CD11c⁺ F4/80⁺ alveolar macrophages. (H) Representative plots and gating strategy for Siglec-F^low MHC⁺, Siglec-F⁺ MHC⁺ and Siglec-F⁺ MHC^- cells among CD11c⁺ F4/80⁺ alveolar macrophages. Dots represent individual mice and results are expressed as mean ± SEM. (C) n=30-34 naive and n=20 infected mice per group, pool of 6 independent experiments. (E) n=6-8 mice per group, pool of 2 independent experiments. (G) n=10-16 mice per group, pool of 3 independent experiments. Two-way ANOVA: ^$$$p<0.001 represent differences between mice strains.

Figure 6

Potential effects of altered Th2-immune responses on pleural and alveolar macrophages dynamics and functions during patent Litomosoides sigmodontis infection. (A) In the pleural cavity, adult worms and microfilariae induce a strong recruitment of F4/80^intermediate Monocyte-derived macrophages (MoMac). Some of them will then mature into F4/80^high resident macrophage (ResMac) in an IL4R-dependent manner. Macrophages acquire an IL4R-driven alternative activation state and increase Arginase1 (Arg1) expression and activity. This might lead to increased proline availability and collagen production in the cavity. On the other hand, pleural macrophages upregulate IFNgR1 and in the presence of IFNγ, macrophages produce nitric oxide (NO). NO could then favor pathogen killing but also tissue damage. Together, tissue damage and increased collagen could favor tissue fibrosis. In the absence of IL4-receptor and IL5, MoMac are still recruited but fail to mature into ResMac. IFNgR1 expression is not upregulated leading to an unresponsiveness to IFNγ impairing NO production. Arg1 expression is not affected and its activity is increased compared to WT mice, but, maybe because of the absence of tissue damage, this is not sufficient to initiate tissue fibrosis. (B) In the bronchoalveolar space, monocyte-derived Siglec-F^low/+ MHC⁺ alveolar macrophages seem to repopulate the Siglec-F⁺ MHC^- bona fide alveolar macrophage population. In the absence of Th2-immune responses and eosinophils, the maturation of these transient macrophages could be faster.