The efficacy of the benzimidazoles oxfendazole and flubendazole against Litomosoides sigmodontis is dependent on the adaptive and innate immune system

Abstract

Filarial nematodes can cause debilitating diseases such as lymphatic filariasis and onchocerciasis. Oxfendazole (OXF) is one promising macrofilaricidal candidate with improved oral availability compared to flubendazole (FBZ), and OXF is currently under preparation for phase 2 clinical trials in filariasis patients. This study aimed to investigate the immune system's role during treatment with OXF and FBZ and explore the potential to boost the treatment efficacy via stimulation of the immune system. Wild type (WT) BALB/c, eosinophil-deficient ΔdblGata1, IL-4r/IL-5^-/-, antibody-deficient μMT and B-, T-, NK-cell and ILC-deficient Rag2/IL-2rγ^-/- mice were infected with the rodent filaria Litomosoides sigmodontis and treated with an optimal and suboptimal regimen of OXF and FBZ for up to 5 days. In the second part, WT mice were treated for 2-3 days with a combination of OXF and IL-4, IL-5, or IL-33. Treatment of WT mice reduced the adult worm burden by up to 94% (OXF) and 100% (FBZ) compared to vehicle controls. In contrast, treatment efficacy was lower in all immunodeficient strains with a reduction of up to 90% (OXF) and 75% (FBZ) for ΔdblGata1, 50 and 92% for IL-4r/IL-5^-/-, 64 and 78% for μMT or 0% for Rag2/IL-2rγ^-/- mice. The effect of OXF on microfilariae and embryogenesis displayed a similar pattern, while FBZ's ability to prevent microfilaremia was independent of the host's immune status. Furthermore, flow cytometric analysis revealed strain-and treatment-specific immunological changes. The efficacy of a shortened 3-day treatment of OXF (-33% adult worms vs. vehicle) could be boosted to a 91% worm burden reduction via combination with IL-5, but not IL-4 or IL-33. Our results suggest that various components of the immune system support the filaricidal effect of benzimidazoles in vivo and present an opportunity to boost treatment efficacy.

Keywords: Litomosoides sigmodontis; benzimidazole; combination therapy; filariae; flubendazole; helminths; macrofilaricide; oxfendazole.

Figure 1

Reduced treatment efficacy of oxfendazole in immunodeficient mice. (A–H) Indicated mouse strains were naturally infected with Litomosoides sigmodontis and treated orally with 5 or 12.5 mg/kg oxfendazole twice per day for 5 days starting 35 days after the infection. Necropsies were performed 70 days after the infection. (A) Adult worm burden. (B) Microfilariae per 50 μL peripheral blood +1. (C) Representative images of embryonal stages. (D–H) Average number of embryonal stages per female worm in (D) BALB/c (green), (E) ΔdblGata1 (blue), (F) IL-4r/IL-5^−/− (red), (G) Rag2/IL-2rγ^−/− (purple), and (H) μMT mice (black). (A,B) Data shown as median with interquartile range. Numbers show reduction of median in comparison to corresponding vehicle control. (D–H) Data shown as mean ± SEM. (A,B,D–H) Data for BALB/c pooled from 6 experiments, IL-4r/IL-5^−/− pooled from 3 experiments, ΔdblGata1, Rag2/IL-2rγ^−/− and μMT pooled from 2 experiments. Statistical analysis using Kruskal-Wallis with Dunn’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Reduced macrofilaricidal efficacy of flubendazole in immunodeficient mice. (A–G) Indicated mouse strains were naturally infected with Litomosoides sigmodontis and treated subcutaneously with 2 mg/kg flubendazole once per day for 2 or 5 days starting 35 days after the infection. Necropsies were performed 70 days after the infection. (A) Adult worm burden. (B) Microfilariae per 50 μL peripheral blood +1. (C–G) Average number of embryonal stages per female worm in (C) BALB/c (green), (D) ΔdblGata1 (blue), (E) IL-4r/IL-5^−/− (red), (F) Rag2/IL-2rγ^−/− (purple) and (G) μMT mice (black). (A,B) Data shown as median with interquartile range. Numbers show reduction of median in comparison to corresponding vehicle control. (C–G) Data shown as mean ± SEM. (A–G) Data for BALB/c pooled from 5 experiments, ΔdblGata1 and IL-4r/IL-5^−/− pooled from 2 experiments, Rag2/IL-2rγ^−/− and μMT from 1 experiment. Statistical analysis using Kruskal-Wallis with Dunn’s post-hoc test, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 3

Distinct immunological changes in different immunodeficient strains after anti-filarial treatment. (A–H) Indicated mouse strains were naturally infected with Litomosoides sigmodontis and treated with (A,B,E,F) oxfendazole or (C,D,G,H) flubendazole 35 days after the infection. Necropsies were performed 70 days after the infection and immune cell populations in (A–D) spleen and (E–H) thoracic cavity were analyzed via flow cytometry. Heat maps show fold change of (A,C,E,G) mean of immune cell frequencies and (B,D,F,H) mean of total cell counts after treatment in comparison to corresponding vehicle controls. (A,B,E,F) Data for BALB/c (green) pooled from 6 experiments, IL-4r/IL-5^−/− (red) pooled from 3 experiments, ΔdblGata1 (blue), Rag2/IL-2rγ^−/− (purple) and μMT (black) pooled from 2 experiments. (C,D,G,H) Data for BALB/c pooled from 5 experiments, ΔdblGata1 and IL-4r/IL-5^−/− pooled from 2 experiments, Rag2/IL-2rγ^−/− and μMT from 1 experiment.

Figure 4

Combination of oxfendazole with interleukin-5 improves macrofilaricidal treatment efficacy in shortened treatment regimen. (A–G) Six-week old female BALB/c mice were naturally infected with Litomosoides sigmodontis and treated with 12.5 mg/kg oxfendazole twice per day for 5 days (positive control) or 3 days (shortened treatment) with or without addition of intranasal application of 2 μg IL-4, IL-5, or IL-33 once per day. Necropsies were performed 70 days after the infection. (A) Adult worm burden. (B) Microfilariae per 50 μL peripheral blood +1. (C) Average number of embryonal stages per female worm. (D) Frequency of eosinophils [CD8⁻, CD11b⁺, Ly6G⁻, Siglec-F⁺] in spleen. (E) Frequency of eosinophils [CD8⁻, CD11b⁺, Siglec-F⁺] in thoracic cavity. (F) Frequency of monocytes [CD8⁻, CD11b⁺, Siglec-F⁻, RELMα⁻, Ly6G⁻, I-ab^(lo)] in thoracic cavity. (G) Total cell count of monocytes in thoracic cavity. (A,B,D–G) Data shown as median with interquartile range. Numbers show reduction of median in comparison to vehicle control. (C) Data shown as mean ± SEM. (A–C) Data for IL-4, IL-5, IL-33 from 1 experiment, data for other groups pooled from 2 experiments (n = 6–7 per group per experiment). (D–G) Representative data for two experiments. (A-C,E) Statistical analysis using Kruskal-Wallis with Dunn’s post-hoc test, (D,F,G) Statistical analysis using One-Way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Histological changes in the lung after combination therapy. (A–H) Six-week old female BALB/c mice were naturally infected with Litomosoides sigmodontis and treated with 12.5 mg/kg oxfendazole twice per day for 5 days (positive control) or 3 days (shortened treatment) with or without addition of intranasal application of 2 μg IL-4, IL-5, or IL-33 once per day. Necropsies were performed 70 days after the infection and lungs were processed for histological analysis. (A) H&E staining of perivascular spaces (PVS). (B) Quantification of Hematoxylin positive nuclei per mm² in the PVS. (C) H&E staining of bronchial arteries. Arrows indicate the artery boundaries. (D) Quantification of bronchial vein thickness. (E) Alcian blue staining of mucus in bronchial epithelium. (F) Quantification of mucus on bronchiole surface. (G) Luxol fast blue staining of eosinophils. Left image displays a Luxol blue positive eosinophil and an alveolar macrophage. The corner zoom on the right image highlights the eosinophil lobed nucleus. (H) Quantification of eosinophil infiltration in the lung. (A–H) Results are expressed as median with interquartile of n = 3–7 mice per group (1 experiment). Numbers show reduction of median in comparison to vehicle control. Statistical analysis using One-way ANOVA followed by Dunnett’s Multiple Comparison Tests, *p < 0.05, **p < 0.01, ***p < 0.001.