Despite the well-documented immune suppression associated with human helminth infections, studies characterizing the immune response at the single-cell level are scanty. We used multiparameter flow cytometry to characterize the type of effector (Th1, Th2, and Th17) and regulatory (natural T regulatory cells [nTregs] and adaptive Treg cells [aTreg/type 1 regulatory cells (Tr1s)]) CD4+ and CD8+ T cells in filaria-infected (Fil+) and -uninfected (Fil−) individuals at homeostasis (in the absence of stimulation). Frequencies of CD4+ lymphocytes spontaneously producing IL-4, IL-10, and IL-17A were significantly higher in Fil+, as were those of IL-10+/IL-4+ double-producing CD4+ cells. Interestingly, frequencies of Th17 and aTreg/Tr1s but not classical Th1 or Th2 cells were significantly increased in Fil+ compared to Fil− individuals. Although the frequency of nTreg was increased in Fil+, IL-10 was overwhelmingly produced by CD4+CD25− cells. Moreover, the concentration of IL-10 produced spontaneously in vitro strongly correlated with the integrated geometric mean fluorescence intensity of IL-10–producing aTreg/Tr1s in Fil+. Together, these data show that at steady state, IL-10–producing aTreg/Tr1 as well as nTreg and effector Th17 CD4+ cells are expanded in vivo in human filarial infections. Moreover, we have established baseline ex vivo frequencies of effector and Tregs at homeostasis at a population level.

Background Mansonella perstans infection is common in areas of Africa where Wuchereria bancrofti, a causative agent of lymphatic filariasis, is endemic. M. perstans is refractory to standard antifilarial therapies. The recent discovery of bacterial endosymbionts (e.g., wolbachia) in most filarial species, including M. perstans, provides new therapeutic options for reducing microfilaremia. Methods In an open-label, randomized trial, we recruited subjects with M. perstans microfilaremia, with or without concomitant W. bancrofti infection, from four villages in Mali and randomly assigned them to receive doxycycline, at a dose of 200 mg daily for 6 weeks (106 subjects), or no treatment (110). At 6 months, subjects who were co-infected with W. bancrofti underwent a second random assignment, to treatment with a single dose of albendazole (400 mg) and ivermectin (150 μg per kilogram of body weight) or no treatment. Subjects were monitored daily during the first 6-week study period for adverse events. M. perstans and W. bancrofti microfilarial levels were assessed at 6, 12, and 36 months. Results At 12 months, 67 of 69 subjects who had received treatment with doxycycline only (97%) had no detectable M. perstans microfilariae per 60 μl of blood, as compared with 10 of 63 subjects who had received no treatment (16%) (relative risk, 6.18; 95% confidence interval, 3.63 to 11.89; P<0.001). At 36 months, M. perstans microfilaremia remained suppressed in 48 of 64 subjects who had received treatment with doxycycline only (75%), a finding that was consistent with a macrofilaricidal effect of doxycycline. Vomiting was more frequent in the doxycycline-treated group than in the untreated group (17% vs. 4%). Conclusions These results are consistent with previous findings that M. perstans harbors the intracellular endosymbiont, wolbachia, and suggest that doxycycline is an effective therapy for M. perstans infection.

The effect of filarial infections on malaria-specific immune responses was investigated in Malian villages coendemic for filariasis (Fil) and malaria. Cytokines were measured from plasma and Ag-stimulated whole blood from individuals with Wuchereria bancrofti and/or Mansonella perstans infections (Fil+; n = 19) and those without evidence of filarial infection (Fil−; n = 19). Plasma levels of IL-10 (geometric mean [GM], 22.8 vs 10.4) were higher in Fil+ compared with Fil−, whereas levels of IFN-inducible protein (IP)-10 were lower in Fil+ (GM, 66.3 vs 110.0). Fil+ had higher levels of spontaneously secreted IL-10 (GM, 59.3 vs 6.8 pg/ml) and lower levels of IL-2 (1.0 vs 1.2 pg/ml) than did Fil−. Although there were no differences in levels of Staphylococcus aureus enterotoxin B-induced cytokines between the two groups, Fil+ mounted lower IL-12p70 (GM, 1.11 vs 3.83 pg/ml; p = 0.007), IFN-γ (GM, 5.44 vs 23.41 pg/ml; p = 0.009), and IP-10 (GM, 29.43 vs 281.7 pg/ml; p = 0.007) responses following malaria Ag (MalAg) stimulation compared with Fil−. In contrast, Fil+ individuals had a higher MalAg-specific IL-10 response (GM, 7318 pg/ml vs 3029 pg/ml; p = 0.006) compared with those without filarial infection. Neutralizing Ab to IL-10 (but not to TGFβ) reversed the down-regulated MalAg-specific IFN-γ and IP-10 (p &lt; 0.001) responses in Fil+. Together, these data demonstrate that filarial infections modulate the Plasmodium falciparum-specific IL-12p70/IFN-γ secretion pathways known to play a key role in resistance to malaria and that they do so in an IL-10-dependent manner.

Wolbachiae are bacterial endosymbionts of insects and many filarial nematodes whose products trigger inflammation in filarial infections. The dependence of the parasites on their endosymbionts has also led to the use of antibiotics directed against the Wolbachiae, therapy that has been demonstrated to have a profound salutary effect on filarial infections. The identification of Wolbachiae in Mansonella species has been conclusively shown for Mansonella ozzardi (Mo), but not for Mansonella perstans (Mp). Using primers known to amplify the 16S ribosomal DNA of other filarial Wolbachiae, an identical 1393bp band was found in all samples tested. Sequence analysis of these samples demonstrated a single consensus sequence for Mp Wolbachia 16S rDNA that was most similar to Wolbachia sequences from other filarial nematodes. When aligned with the only other Mansonella Wolbachia sequence (Mo) there were only 8 nucleotide differences in the 1369bp overlapping sequence. Phylogenetic dendrograms, examining the relationship of the Mp Wolbachia to other Wolbachia 16S rDNA, showed that the Wolbachia tracked almost identically to the 5S rRNA of their parasite host. Wolbachia surface protein (WSP) was also demonstrated in protein extracted from Mp-containing whole blood. In advance of a treatment trial of Mp, a method for the quantitation of Mp Wolbachia was developed and used to demonstrate not only a relationship between microfilarial numbers and Wolbachia copy numbers, but also to demonstrate the effect of antibiotic on ridding Mp of Wolbachia.

The mechanisms underlying the modulation of both the malaria-specific immune response and the course of clinical malaria in the context of concomitant helminth infection are poorly understood. We used multiparameter flow cytometry to characterize the quality and the magnitude of malaria-specific T cell responses in filaria-infected and -uninfected individuals with concomitant asymptomatic Plasmodium falciparum malaria in Mali. In comparison with filarial-uninfected subjects, filarial infection was associated with higher ex vivo frequencies of CD4+ cells producing IL-4, IL-10, and IL-17A (p = 0.01, p = 0.001, and p = 0.03, respectively). In response to malaria Ag stimulation, however, filarial infection was associated with lower frequencies of CD4+ T cells producing IFN-γ, TNF-α, and IL-17A (p < 0.001, p = 0.04, and p = 0.04, respectively) and with higher frequencies of CD4+IL10+T cells (p = 0.0005). Importantly, filarial infection was associated with markedly lower frequencies of malaria Ag-specific Th1 (p < 0.0001), Th17 (p = 0.012), and “TNF-α” (p = 0.0008) cells, and a complete absence of malaria-specific multifunctional Th1 cells. Filarial infection was also associated with a marked increase in the frequency of malaria-specific adaptive regulatory T/Tr1 cells (p = 0.024), and the addition of neutralizing anti–IL-10 Ab augmented the amount of Th1-associated cytokine produced per cell. Thus, among malaria-infected individuals, concomitant filarial infection diminishes dramatically the frequencies of malaria-specific Th1 and Th17 T cells, and alters the quality and magnitude of malaria-specific T cell responses.