Filarial parasites develop faster and reproduce earlier in response to host immune effectors that determine filarial life expectancy

Abstract

Humans and other mammals mount vigorous immune assaults against helminth parasites, yet there are intriguing reports that the immune response can enhance rather than impair parasite development. It has been hypothesized that helminths, like many free-living organisms, should optimize their development and reproduction in response to cues predicting future life expectancy. However, immune-dependent development by helminth parasites has so far eluded such evolutionary explanation. By manipulating various arms of the immune response of experimental hosts, we show that filarial nematodes, the parasites responsible for debilitating diseases in humans like river blindness and elephantiasis, accelerate their development in response to the IL-5 driven eosinophilia they encounter when infecting a host. Consequently they produce microfilariae, their transmission stages, earlier and in greater numbers. Eosinophilia is a primary host determinant of filarial life expectancy, operating both at larval and at late adult stages in anatomically and temporally separate locations, and is implicated in vaccine-mediated protection. Filarial nematodes are therefore able to adjust their reproductive schedules in response to an environmental predictor of their probability of survival, as proposed by evolutionary theory, thereby mitigating the effects of the immune attack to which helminths are most susceptible. Enhancing protective immunity against filarial nematodes, for example through vaccination, may be less effective at reducing transmission than would be expected and may, at worst, lead to increased transmission and, hence, pathology.

Figure 1. Filarial nematodes developed faster when IL-5 driven eosinophils were present.

Litomosoides sigmodontis filarial nematodes developed slower during their larval stages in IL-5 deficient (IL-5^−/−) mice than in C57BL/6 wild type controls as measured (A) by their shorter lengths (** p = 0.015, ANOVA; n = 50 larvae nested in 5 mice per group) and (B) by their delayed moulting to the 4^th larval stage at D10 p.i. (** p = 0.0007, Chi² test; n = 5 mice). (C) At D30 p.i., however, no differences in the moulting rate to the adult stage were observed between IL-5⁻/⁻ mice and wild type controls (n = 5 mice). The constitutive absence of eosinophils in PHIL mice resulted in slower larval development as judged by (D) their lengths in both male and female mice (*, p = 0.04 for the effect of mouse strain when variation due to mouse sex is accounted for, GLM; n = 57 to 59 in 7 mice) and by (E) their moulting rates (p = 0.02, Fisher Exact Test; n = 7 mice) at D12 p.i. as compared to C57BL/6 wild type controls. None of the treatments affected larval survival (see Figure S2A and S2B). Error bars depict s.e.m.

Figure 2. Filarial nematodes responded to the presence of IL-5 driven eosinophils at the outset of infection.

(A) Topical injection of recombinant IL-5 (rIL5) resulted in a local subcutaneous increase (p = 0.05, Wilcoxon rank-sum test, n = 5) but no systemic increase in eosinophil recruitment relative to other lymphocyte populations. (B) The addition of rIL5 upon inoculation of infective larvae to BALB/c mice accelerated their growth before their 3rd larval moult, at D7 p.i. (* p = 0.019, unpaired two-tailed t-test; n = 30, no significant effect of mouse). This occurred independently of mouse genetic background as similar data were obtained in BALB/c and in C57BL/6 mice. (C) The depletion of eosinophils by α-CCR3 antibody treatment 24 h before infection resulted in a prolonged reduction of eosinophilia and (D) in a slower larval development that were not rescued by the addition of rIL-5 (p = 0.003, ANOVA and Dunn's multiple comparison post test: ** p<0.01; * p<0.05; n = 19 to 23). None of the treatments affected larval survival (see Figure S2C). Error bars depict s.e.m.

Figure 3. Adaptive immunity accelerates early larval development of L. sigmodontis despite impeding its sexual maturation.

(A) Cell recruitment increased significantly over time during infection when filariae were inoculated into rag ^−/−, il4 ^−/− and double deficient (rag ^−/− il-4 ^−/−) mice that lack T cells, B cells, and IL-4, but was severely lower overall in rag ^−/− il-4 ^−/− than in C57BL/6 wild type controls. (B) Filarial larvae in rag ^−/− il-4 ^−/− mice developed slowest unless rIL-5 was added upon their delivery to the host. (C) However, by D30 p.i. the parasites in rag ^−/− il-4 ^−/− mice had compensated for their slower development and reached the adult stage earlier than in wild type controls, likely due to the continuous attack by eosinophils and other inflammatory cells in the control mice (see Figure S3A). (D) This resulted in the release of more offspring in rag ^−/− il-4 ^−/− mice than what is observed in susceptible immunocompetent BALB/c mice. * p<0.05; ** p<0.01, Wilcoxon rank-sum test; n = 5–10 mice per group. Error bars represent s.e.m.

Figure 4. Early eosinophilia enhances L. sigmodontis reproductive output.

(A) When co-inoculated with eosinophilia-inducing rIL-5 and L3 parasites, BALB/c mice became microfilaraemic sooner than in control infections as suggested by the proportion of mice presenting blood circulating microfilariae by D55 p.i. (p = 0.08, Fisher's exact test, n = 17, analysis restricted to mice that became microfilaraemic). (B) Early rIL-5-induced eosinophilia resulted in increased microfilaraemia throughout patency (effect of treatment on microfilaraemia p = 0.0001, negative binomial glm; n = 12, data points represent means ± s.e.m.) and a marginally earlier peak in microfilaraemia (occurring on day 68.5±1 and 72.8±2 in treated mice and controls, respectively, p = 0.09).