Yaya I. Coulibaly

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.

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.

Molecular identification of Wolbachia from the filarial nematode Mansonella perstans

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.

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.

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.

Genetics of low spinal muscular atrophy carrier frequency in sub‐Saharan Africa

Spinal muscular atrophy (SMA) is one of the most common severe hereditary diseases of infancy and early childhood in North America, Europe, and Asia. SMA is usually caused by deletions of the survival motor neuron 1 (SMN1) gene. A closely related gene, SMN2, modifies the disease severity. SMA carriers have only 1 copy of SMN1 and are relatively common (1 in 30–50) in populations of European and Asian descent. SMN copy numbers and SMA carrier frequencies have not been reliably estimated in Malians and other sub‐Saharan Africans.

Highly heterogeneous, activated and short-lived regulatory T cells during chronic filarial infection

The mechanisms underlying the increase in the numbers of regulatory T (Treg) cells in chronic infection settings remain unclear. Here we have delineated the phenotype and transcriptional profiles of Treg cells from 18 filarial‐infected (Fil+) and 19 filarial‐uninfected (Fil−) subjects. We found that the frequencies of Foxp3+ Treg cells expressing CTLA‐4, GITR, LAG‐3, and IL‐10 were significantly higher in Fil+ subjects compared with that in Fil− subjects. Foxp3‐expressing Treg‐cell populations in Fil+ subjects were also more heterogeneous and had higher expression of IL‐10, CCL‐4, IL‐29, CTLA‐4, and TGF‐β than Fil− subjects, each of these cytokines having been implicated in immune suppression. Moreover, Foxp3‐expressing Treg cells from Fil+ subjects had markedly upregulated expression of activation‐induced apoptotic genes with concomitant downregulation of those involved in cell survival. To determine whether the expression of apoptotic genes was due to Treg‐cell activation, we found that the expression of CTLA‐4, CDk8, RAD50, TNFRSF1A, FOXO3, and RHOA were significantly upregulated in stimulated cells compared with unstimulated cells. Taken together, our results suggest that in patent filarial infection, the expanded Treg‐cell populations are heterogeneous, short‐lived, activated, and express higher levels of molecules known to modulate immune responsiveness, suggesting that filarial infection is associated with high Treg‐cell turnover.

Human Migration and the Spread of the Nematode Parasite Wuchereria bancrofti

The human disease lymphatic filariasis causes the debilitating effects of elephantiasis and hydrocele. Lymphatic filariasis currently affects the lives of 90 million people in 52 countries. There are three nematodes that cause lymphatic filariasis, Brugia malayi, B. timori, and Wuchereria bancrofti, but 90% of all cases of lymphatic filariasis are caused solely by W. bancrofti. Here we use population genomics to identify the geographic origin of W. bancrofti and reconstruct its spread. Previous genomic sequencing efforts have suffered from difficulties in obtaining Wb DNA. We used selective whole genome amplification to enrich W. bancrofti DNA from infected blood samples and were able to analyze 47 whole genomes of W. bancrofti from endemic locations in Haiti, Mali, Kenya, and Papua New Guinea. Our results are consistent with a Southeast Asia or East Asia origin for W. bancrofti spread around the globe by infecting migrating populations of humans. Austronesians probably introduced W. ban-crofti to Madagascar where later migrations moved it to continental Africa. From Africa, W. bancrofti spread to the New World during the transatlantic slave trade. The greater genetic diversity of W. bancrofti populations from Haiti are also consistent with genetic admixture from multiple source populations. Genome scans for locally adapted haplotypes identified genes associated with human immune suppression and insecticide sensitivity. Locally adapted haplotypes may provide a foundation to understand the distribution of W. bancrofti compared to that of other filarial nematodes and how populations may differ in response to eradication efforts.