Highlights d Eosinophils release extracellular DNA traps and inhibit microfilariae motility d Microfilariae-induced eosinophil extracellular DNA traps are a conserved mechanism d Microfilariae trigger DNA release by eosinophils through the dectin-1 receptor d Microfilariae coated with DNA traps are cleared more efficiently in vivo
Group 2 innate lymphoid cells (ILC2s) are inducers of type 2 immune responses, but their role during filarial infection remains unclear. In the present study, we used the Litomosoides sigmodontis rodent model of filariasis to analyze ILC2s during infection in susceptible BALB/c mice that develop a chronic infection with microfilaremia and semi-susceptible C57BL/6 mice that eliminate the filariae shortly after the molt into adult worms and thus do not develop microfilaremia. ILC2s (CD45+ Lineage- TCRβ- CD90.2+ Sca-1+ IL-33R+ GATA-3+) were analyzed in the pleural cavity, the site of L. sigmodontis infection, after the infective L3 larvae reached the pleural cavity (9 days post infection, dpi), after the molt into adult worms (30dpi) and during the peak of microfilaremia (70dpi). C57BL/6 mice had significantly increased ILC2 numbers compared to BALB/c mice at 30dpi, accompanied by substantially higher IL-5 and IL-13 levels, indicating a stronger type 2 immune response in C57BL/6 mice upon L. sigmodontis infection. At this time point the ILC2 numbers positively correlated with the worm burden in both mouse strains. ILC2s and GATA-3+ CD4+ T cells were the dominant source of IL-5 in L. sigmodontis-infected C57BL/6 mice with ILC2s showing a significantly higher IL-5 expression than CD4+ T cells. To investigate the importance of ILC2s during L. sigmodontis infection, ILC2s were depleted with anti-CD90.2 antibodies in T and B cell-deficient Rag2-/- C57BL/6 mice on 26-28dpi and the outcome of infection was compared to isotype controls. Rag2-/- mice were per se susceptible to L. sigmodontis infection with significantly higher worm burden than C57BL/6 mice and developed microfilaremia. Depletion of ILC2s did not result in an increased worm burden in Rag2-/- mice, but led to significantly higher microfilariae numbers compared to isotype controls. In conclusion, our data demonstrate that ILC2s are essentially involved in the control of microfilaremia in Rag2-/- C57BL/6 mice.
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.
Infections with helminths affect more than one billion people worldwide. Despite an urgent need there is no vaccine available that would confer long lasting protection against helminth infections. Previous studies indicated that a vaccination with irradiated infective L3 reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as adjuvant improves the efficacy of a vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in increased neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels at the site of injection. To investigate the in vivo impact on parasite clearance, BALB/c mice received 3 subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Serum analysis before the challenge infection confirmed the induction of L. sigmodontis-specific antibodies in response to the immunization and serum from immunized mice significantly reduced larval motility in vitro with naïve cells. 63 days after the challenge infection, vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a significantly greater reduction in adult worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). Further, the treatment of L. sigmodontis infection with 3pRNA alone, but not poly(I:C), resulted in a reduced worm burden, supporting the therapeutic potential for the activation of RIG-I with 3pRNA. In conclusion, our data demonstrate that the additional activation of nucleic acid sensing immune receptors boosts the immune response and provides better protection against L. sigmodontis. Thus, the use of nucleic acid receptor agonists as vaccine adjuvants represents a promising novel strategy to improve the efficacy of vaccines against filariae and potentially of other helminths.
Oxfendazole is one of the lead macrofilaricidal candidates for the treatment of onchocerciasis and lymphatic filariasis. Originally, oxfendazole was developed for the veterinary market, where it is mainly used to treat intestinal helminth infections. In humans, oxfendazole was proven to be safe in multiple ascending dose studies. Furthermore, previous experimental studies demonstrated that the benzimidazoles class is active in animals and humans against filarial nematodes. In the present study, we have compared the efficacy of oxfendazole isomers with the commercially available racemic mixture Dolthene against the rodent filaria Litomosoides sigmodontis in female BALB/c mice. Treatment with either the isomers or Dolthene led to a reduction of the adult worm burden by 94-98% following the ten-day treatment and by 72% (oxfendazole (-)), 85% (oxfendazole (+)) and 91% (Dolthene) following the five-day treatment. No statistically significant differences in the macrofilaricidal efficacy against L. sigmodontis were observed for both isomers and Dolthene. Metabolites of oxfendazole are fenbendazole and fenbendazole sulfone. Two hours after treatment with Dolthene and both oxfendazole isomers, fenbendazole sulfone, but rarely fenbendazole, was detected. The oxfendazole (-) isomer was metabolised at the highest rate to fenbendazole sulfone. Furthermore, oxfendazole isomers have a comparable pharmacokinetic profile in dogs. In conclusion, our data does not point at the development of a single isomer for future use in humans.
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