Benoit Dembele

At Homeostasis Filarial Infections Have Expanded Adaptive T Regulatory but Not Classical Th2 Cells

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.

A Randomized Trial of Doxycycline for Mansonella perstans Infection

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 coinfected with W. bancrofti underwent a second random assignment, to treatment with a single dose of albendazole (400 mg) and ivermectin (150 microg 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 microl 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. (ClinicalTrials.gov number, NCT00340691.)

Patent Filarial Infection Modulates Malaria-Specific Type 1 Cytokine Responses in an IL-10-Dependent Manner in a Filaria/Malaria-Coinfected Population

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 < 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.

Filarial Infection Suppresses Malaria-Specific Multifunctional Th1 and Th17 Responses in Malaria and Filarial Coinfections

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.

Filariasis attenuates anemia and proinflammatory responses associated with clinical malaria: a matched prospective study in children and young adults.

Background Wuchereria bancrofti (Wb) and Mansonella perstans (Mp) are blood-borne filarial parasites that are endemic in many countries of Africa, including Mali. The geographic distribution of Wb and Mp overlaps considerably with that of malaria, and coinfection is common. Although chronic filarial infection has been shown to alter immune responses to malaria parasites, its effect on clinical and immunologic responses in acute malaria is unknown. Methodology/Principal Findings To address this question, 31 filaria-positive (FIL+) and 31 filaria-negative (FIL−) children and young adults, matched for age, gender and hemoglobin type, were followed prospectively through a malaria transmission season. Filarial infection was defined by the presence of Wb or Mp microfilariae on calibrated thick smears performed between 10 pm and 2 am and/or by the presence of circulating filarial antigen in serum. Clinical malaria was defined as axillary temperature ≥37.5°C or another symptom or sign compatible with malaria infection plus the presence of asexual malaria parasites on a thick blood smear. Although the incidence of clinical malaria, time to first episode, clinical signs and symptoms, and malaria parasitemia were comparable between the two groups, geometric mean hemoglobin levels were significantly decreased in FIL− subjects at the height of the transmission season compared to FIL+ subjects (11.4 g/dL vs. 12.5 g/dL, p<0.01). Plasma levels of IL-1ra, IP-10 and IL-8 were significantly decreased in FIL+ subjects at the time of presentation with clinical malaria (99, 2145 and 49 pg/ml, respectively as compared to 474, 5522 and 247 pg/ml in FIL− subjects). Conclusions/Significance These data suggest that pre-existent filarial infection attenuates immune responses associated with severe malaria and protects against anemia, but has little effect on susceptibility to or severity of acute malaria infection. The apparent protective effect of filarial infection against anemia is intriguing and warrants further study in a larger cohort.

Expanded Numbers of Circulating Myeloid Dendritic Cells in Patent Human Filarial Infection Reflect Lower CCR1 Expression

APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infec-tion. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c−CD123lo) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c+CD123lo) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1+ mDCs and a higher percentage of circulating CCR5+ mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites.

Use of High-Dose, Twice-Yearly Albendazole and Ivermectin to Suppress Wuchereria bancrofti Microfilarial Levels

BACKGROUND Annual mass treatment with albendazole and ivermectin is the mainstay of current strategies to interrupt transmission of Wuchereria bancrofti in Africa. More-effective microfilarial suppression could potentially reduce the time necessary to interrupt transmission, easing the economic burden of mass treatment programs in countries with limited resources. METHODS To determine the effect of increased dose and frequency of albendazole-ivermectin treatment on microfilarial clearance, 51 W. bancrofti microfilaremic residents of an area of W. bancrofti endemicity in Mali were randomized to receive 2 doses of annual, standard-dose albendazole-ivermectin therapy (400 mg and 150 μg/kg; n = 26) or 4 doses of twice-yearly, increased-dose albendazole-ivermectin therapy (800 mg and 400 μg/kg; n = 25). RESULTS Although microfilarial levels decreased significantly after therapy in both groups, levels were significantly lower in the high-dose, twice-yearly group at 12, 18, and 24 months. Furthermore, there was complete clearance of detectable microfilariae at 12 months in the 19 patients in the twice-yearly therapy group with data available at 12 months, compared with 9 of 21 patients in the annual therapy group (P < .001, by Fishers exact test). This difference between the 2 groups was sustained at 18 and 24 months, with no detectable microfilariae in the patients receiving twice-yearly treatment. Worm nests detectable by ultrasonography and W. bancrofti circulating antigen levels, as measured by enzyme-linked immunosorbent assay, were decreased to the same degree in both groups at 24 months, compared with baseline. CONCLUSIONS These findings suggest that increasing the dosage and frequency of albendazole-ivermectin treatment enhances suppression of microfilariae but that this effect may not be attributable to improved adulticidal activity.

Interferon regulatory factor modulation underlies the bystander suppression of malaria antigen‐driven IL‐12 and IFN‐γ in filaria‐malaria co‐infection

In areas where polyparasitism is highly prevalent, the impact of multiple parasites on the host response is underestimated. In particular, the presence of helminth infection coincident with malaria profoundly alters the production of malaria‐specific IFN‐γ, IL‐12p70, CXCL9, CXCL10 and CXCL11, cytokines/chemokines known to be critical in mediating malaria‐specific immunity. In order to elucidate the mechanisms underlying the suppression of malaria‐specific cytokines/chemokines, we assessed the expression of malaria‐specific IL‐12Rβ1, IL‐12Rβ2 and interferon regulatory factor (IRF)‐1 in blood obtained from 18 filaria‐infected (Fil+) and 17 filaria‐uninfected (Fil−) individuals in a filaria‐malaria co‐endemic region of Mali. We found that Fil+ individuals had significantly lower RNA expression of IRF‐1 but not IL‐12Rβ1 or IL‐12Rβ2 in response to malaria antigen stimulation. We also measured the frequency of IL‐12‐producing DCs from these subjects and found that Fil+ subjects had lower frequencies of IL‐12+ mDCs after malaria antigen stimulation than did the Fil− subjects. Modeling these data in vitro, we found that mDCs pre‐exposed to live microfilariae not only produced significantly lower levels of CXCL‐9, CXCL‐10, IL‐12p35, IL‐12p40, IL‐12p19 and CXCL‐11 following stimulation with malaria antigen but also markedly downregulated the expression of IRF‐1, IRF‐2 and IRF‐3 compared with microfilaria‐unexposed mDCs. siRNA‐inhibition of irf‐1 in mDCs downregulated the production of IL‐12p70 through repression of IL‐12p35. Our data demonstrate that the modulation of IRFs seen in filarial (and presumably other tissue‐invasive helminths) infection underlies the suppression of malaria‐specific cytokines/chemokines that play a crucial role in immunity to malaria.

The Impact of Six Annual Rounds of Mass Drug Administration on Wuchereria bancrofti Infections in Humans and in Mosquitoes in Mali

Wuchereria bancrofti prevalence and transmission were assessed in six endemic villages in Sikasso, Mali prior to and yearly during mass drug administration (MDA) with albendazole and ivermectin from 2002 to 2007. Microfilaremia was determined by calibrated thick smear of night blood in adult volunteers and circulating filarial antigen was measured using immunochromatographic card test in children < 5 years of age. Mosquitoes were collected by human landing catch from July to December. None of the 686 subjects tested were microfilaremic 12 months after the sixth MDA round. More importantly, circulating antigen was not detected in any of the 120 children tested, as compared with 53% (103/194) before the institution of MDA. The number of infective bites/human/year decreased from 4.8 in 2002 to 0.04 in 2007, and only one mosquito containing a single infective larva was observed 12 months after the final MDA round. Whether this dramatic reduction in transmission will be sustained following cessation of MDA remains to be seen.