During blood-stage malaria, the innate immune system initiates the production of pro-inflammatory cytokines, including IFN-γ, that are critical to host defense and responsible for severe disease. Nonetheless, the innate immune pathways activated during this process in human malaria remain poorly understood. Here, we identify TLR8 as an essential sensor of Plasmodium falciparum -infected red blood cells (iRBC). In human immune cells, iRBC and RNA purified from iRBC were detected by TLR8 but not TLR7 leading to IFN-γ induction in NK cells. While TLR7 and 9 have been shown to lead to IFN-γ in mice, our data demonstrate that TLR8 was the only TLR capable of inducing IFN-γ release in human immune cells. This unique capacity was mediated by the release of IL-12p70 and bioactive IL-18 from monocytes, the latter via a hitherto undescribed pathway. Altogether, our data are the first reported activation of TLR8 by protozoan RNA and demonstrate both the critical role of TLR8 in human blood-stage malaria and its unique functionality in the human immune system. Moreover, our study offers important evidence that mouse models alone may not be sufficient to describe the human innate immune response to malaria.

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.

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1-/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1-/- mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected Ifnar1-/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in PbAAma1OVA-infected Ifnar1-/- mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during PbAAma1OVA-infection in Ifnar1-/- mice and thereby are contributing to the protection from ECM.

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.