Wuchereria bancrofti-infected individuals harbor distinct IL-10-producing regulatory B and T cell subsets which are affected by anti-filarial treatment

Abstract

Despite worldwide mass drug administration, it is estimated that 68 million individuals are still infected with lymphatic filariasis with 19 million hydrocele and 17 million lymphedema reported cases. Despite the staggering number of pathology cases, the majority of LF-infected individuals do not develop clinical symptoms and present a tightly regulated immune system characterized by higher frequencies of regulatory T cells (Treg), suppressed proliferation and Th2 cytokine responses accompanied with increased secretion of IL-10, TGF-β and infection-specific IgG4. Nevertheless, the filarial-induced modulation of the host`s immune system and especially the role of regulatory immune cells like regulatory B (Breg) and Treg during an ongoing LF infection remains unknown. Thus, we analysed Breg and Treg frequencies in peripheral blood from Ghanaian uninfected endemic normals (EN), lymphedema (LE), asymptomatic patent (CFA+MF+) and latent (CFA+MF-) W. bancrofti-infected individuals as well as individuals who were previously infected with W. bancrofti (PI) but had cleared the infection due to the administration of ivermectin (IVM) and albendazole (ALB). In summary, we observed that IL-10-producing CD19+CD24highCD38dhigh Breg were specifically increased in patently infected (CFA+MF+) individuals. In addition, CD19+CD24highCD5+CD1dhigh and CD19+CD5+CD1dhighIL-10+ Breg as well as CD4+CD127-FOXP3+ Treg frequencies were significantly increased in both W. bancrofti-infected cohorts (CFA+MF+ and CFA+MF-). Interestingly, the PI cohort presented frequency levels of all studied regulatory immune cell populations comparable with the EN group. In conclusion, the results from this study show that an ongoing W. bancrofti infection induces distinct Breg and Treg populations in peripheral blood from Ghanaian volunteers. Those regulatory immune cell populations might contribute to the regulated state of the host immune system and are probably important for the survival and fertility (microfilaria release) of the helminth.

Fig 1. Increased CD19⁺CD24^highCD5⁺CD1d^high Breg frequencies in peripheral blood of W. bancrofti-infected and LE individuals.

Using flow cytometry, peripheral whole blood cells from endemic normals (EN; n = 54), latent (CFA+MF-; n = 41) and patent (CFA+MF+; n = 13) Wuchereria bancrofti-infected, lymphedema (LE, n = 50) and previously infected individuals (PI; n = 65) were analyzed for frequencies (%) of (A) CD19⁺CD24^high regulatory B cells expressing (B) CD5⁺CD1d^high. Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after a Kruskal-Wallis-test followed by a Dunn`s multiple comparison post hoc analysis. In addition, Spearman correlations were performed between MF counts and (C) CD19⁺CD24^high or (D) CD19⁺CD24^highCD5⁺CD1d^high frequencies.

Fig 2. Increased CD19⁺CD5⁺CD1d^highIL-10⁺ Breg frequencies in peripheral blood of W. bancrofti-infected individuals.

Freshly isolated peripheral whole blood cells (100μl/well) from endemic normals (EN; n = 54), latent (CFA+MF-; n = 41) and patent (CFA+MF+; n = 13) Wuchereria bancrofti-infected, lymphedema (LE, n = 50) and previously infected individuals (PI; n = 65) were cultivated in 10% FCS/RPMI-1640 medium (100μl/well) and left either (A-C) un-stimulated or (D-F) cultured with eBioscience^™ cell stimulation cocktail (PMA) for 4 hours at room temperature. Thereafter, peripheral blood cells were analyzed for frequencies (%) of CD19⁺ B cells expressing (A, D) CD5⁺CD1d^high and CD19⁺CD5⁺CD1d^high regulatory B cells expressing (B, E) IL-10. Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after a Kruskal-Wallis-test followed by a Dunn`s multiple comparison post hoc analysis. In addition, Spearman correlations were performed between MF counts and frequencies of CD19⁺CD24^highCD38^highIL-10⁺ which were either (C) un-stimulated or (F) PMA stimulated.

Fig 3. Increased CD19⁺CD24^highCD38d^highIL-10⁺ Breg frequencies in peripheral blood of patent W. bancrofti-infected individuals.

Freshly isolated peripheral whole blood cells (100μl/well) from endemic normals (EN; n = 54), latent (CFA+MF-; n = 41) and patent (CFA+MF+; n = 13) Wuchereria bancrofti-infected, lymphedema (LE, n = 50) and previously infected individuals (PI; n = 65) were cultivated in 10% FCS/RPMI-1640 medium (100μl/well) and left either (A-C) un-stimulated or (D-F) cultured with eBioscience^™ cell stimulation cocktail (PMA) for 4 hours at room temperature. Thereafter, peripheral blood cells were analyzed for frequencies (%) of CD19⁺ B cells expressing (A, D) CD24^highCD38^high and CD19⁺CD24^highCD38^high immature B cells expressing (B, E) IL-10. Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after a Kruskal-Wallis-test followed by a Dunn`s multiple comparison post hoc analysis. In addition, Spearman correlations were performed between MF counts and frequencies of CD19⁺CD24^highCD38^highIL-10⁺ which were either (C) un-stimulated or (F) PMA stimulated.

Fig 4. Increased CD4⁺CD127^-FOXP3⁺ Treg frequencies in peripheral blood of W. bancrofti-infected and LE individuals.

Using flow cytometry, peripheral whole blood cells from endemic normals (EN; n = 54), latent (CFA+MF-; n = 41) and patent (CFA+MF+; n = 13) Wuchereria bancrofti-infected, lymphedema (LE, n = 50) and previously infected individuals (PI; n = 65) were analyzed for frequencies (%) of (A) CD4⁺CD127^-FOXP3⁺ regulatory T cells expressing (C) Neuropiln-1 and (D) HELIOS. Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after a Kruskal-Wallis-test followed by a Dunn`s multiple comparison post hoc analysis. (B) Spearman correlation was performed between MF counts and CD4⁺CD127^-FOXP3⁺ regulatory T cell frequencies.