Hyperreactive onchocerciasis is characterized by a combination of Th17-Th2 immune responses and reduced regulatory T cells

Abstract

Clinical manifestations in onchocerciasis range from generalized onchocerciasis (GEO) to the rare but severe hyperreactive (HO)/sowda form. Since disease pathogenesis is associated with host inflammatory reactions, we investigated whether Th17 responses could be related to aggravated pathology in HO. Using flow cytometry, filarial-specific cytokine responses and PCR arrays, we compared the immune cell profiles, including Th subsets, in individuals presenting the two polar forms of infection and endemic normals (EN). In addition to elevated frequencies of memory CD4+ T cells, individuals with HO showed accentuated Th17 and Th2 profiles but decreased CD4+CD25hiFoxp3+ regulatory T cells. These profiles included increased IL-17A+, IL-4+, RORC2+ and GATA3+CD4+ T cell populations. Flow cytometry data was further confirmed using a PCR array since Th17-related genes (IL-17 family members, IL-6, IL-1β and IL-22) and Th2-related (IL-4, IL-13, STAT6) genes were all significantly up-regulated in HO individuals. In addition, stronger Onchocerca volvulus-specific Th2 responses, especially IL-13, were observed in vitro in hyperreactive individuals when compared to GEO or EN groups. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network instigating the development of severe onchocerciasis.

Figure 1. Higher frequencies of monocytes and memory T cells in HO individuals.

Isolated PBMCs from EN and individuals presenting either generalized (GEO) or hyperreactive onchocerciasis (HO) were stained with a combination of antibodies to determine the frequencies of CD8⁺ T cells (A); NK cells [CD3^-CD16^brightCD56^dim (B) and CD3^-CD16^dimCD56^bright (C)]; NKT cells [CD3⁺CD16⁺ (D) and CD3⁺CD56⁺ (E)]; CD19⁺CD27⁺ memory B cells (F); CD14⁺ monocytes (G); memory CD4⁺ T cells (H) and naive CD4⁺ T cells (I). Graphs show box whiskers (tukey) with outliers from EN n = 10, GEO n = 10 and HO n = 6. Asterisks show statistical differences (Kruskal-Wallis and Mann Whitney test) between the groups indicated by the brackets (*p<0.05, **p<0.01).

Figure 2. Hyperreactive individuals exhibit a dominant IL-4 and IL-17 phenotype.

Isolated PBMCs from EN (n = 10) and O. volvulus-infected individuals presenting either GEO (n =  10) or HO (n = 6) were activated with a cell stimulation cocktail for 6 hours. Thereafter, cells were stained with an anti-CD4 antibody and after fixation and permeabilization further stained with anti-human antibodies specific for IFN-γ (A), IL-4 (B), IL-17A (C) and IL-10 (D). Intracellular cytokine expression was determined on the CD4⁺ T cell population by flow cytometry. Graphs show percentages as box whiskers (tukey) with outliers. Asterisks show statistical differences (Kruskal-Wallis and Mann Whitney test) between the groups indicated by the brackets (*p<0.05, **p<0.01, ***p<0.001).

Figure 3. Hyperreactive onchocerciasis individuals present higher frequencies of CD4⁺Foxp3⁺ but not CD4⁺CD25^hiFoxp3⁺ T cells.

PBMCs from EN and O. volvulus-infected GEO or HO individuals were activated with a cell stimulation cocktail for 6 hours. Thereafter, cells were stained for CD4 and the transcription factors T-bet (A) or Foxp3 (B). PBMCs fractions were also stained with CD25 to assess the numbers of CD4⁺CD25^hi (C) and CD4⁺CD25^hiFoxp3⁺ (D) T cells in each individual (EN n = 16, GEO n =  16, HO n =  6). Cell population frequencies were determined via flow cytometry. Graphs show percentages as box whiskers (tukey) with outliers. Asterisks show statistical differences (Kruskal-Wallis and Mann Whitney test) between the groups indicated by the brackets (*p<0.05, **p<0.01, ***p<0.001).

Figure 4. Immune profiles of hyperreactive onchocerciasis individuals are associated with elevated Th2 and Th17 factors.

PBMCs isolated from EN (n = 16) and O. volvulus-infected GEO (n = 16) or HO (n = 6) individuals were activated with a cell stimulation cocktail for 6 hours. Thereafter, cells were stained for CD4 and the transcription factors GATA3 (A) or RORC2 (B). Ratios of CD4⁺lL-17A⁺ vs. CD4⁺IL-4⁺ T cells (C) and CD4⁺RORC2⁺ vs. CD4⁺CD25^hiFoxp3⁺ (D) were determined for each individual. Cell population frequencies were determined using flow cytometry. Graphs show percentages as box whiskers (tukey) with outliers. Asterisks show statistical differences (Kruskal-Wallis and Mann Whitney test) between the groups indicated by the brackets (*p<0.05, **p<0.01, ***p<0.001). (E–G) After PBMCs were stimulated with αCD3/αCD28 (40,000 beads/ml) for 3h, RNA was extracted and transcribed into cDNA. Thereafter, PCR Arrays were performed using the RT² Profiler PCR Array Human Th17 kit. Bars show fold change ± SEM increase in the indicated gene expression between four age-matched males presenting either GEO or HO forms of infection.

Figure 5. Hyperreactive individuals present stronger filarial-specific Th2 responses in vitro.

Isolated PBMCs (1×10⁵/well) from EN (n = 16) and O. volvulus-infected GEO (n = 16) or HO (n = 6) individuals were left either unstimulated (Cont.) or activated with either O. volvulus antigen extract (OvAg, 20 µg/ml) or αCD3/αCD28 (40,000 beads/ml) for 7 days. Thereafter, levels of IL-5 (A, B), IL-13 (C, D), IL-10 (E, F) and IFN-γ (G, H) were measured in the culture supernatants using a FlowCytomix™ Multiplex kit via flow cytometry. Graphs show data as box whiskers (tukey) with outliers. Asterisks show statistical differences (Kruskal-Wallis and Mann Whitney test) between the groups indicated by the brackets (*p<0.05, **p<0.01, ***p<0.001).

Figure 6. PBMCs from hyperreactive onchocerciasis individuals exhibit strong IL-6 responses following filarial specific activation in vitro.

Isolated PBMCs (1×10⁵/well) from EN (n = 16) and O. volvulus-infected GEO (n = 16) or HO (n = 6) individuals were left either unstimulated (Cont.) or activated with either O. volvulus antigen extract (OvAg, 20 µg/ml) or αCD3/αCD28 (40,000 beads/ml) for 7 days. Thereafter, levels of IL-17A (A, B), IL-6 (C, D), IL-22 (E,F) and IL-1β (G,H) were measured in the culture supernatants using a FlowCytomix™ Multiplex kit via flow cytometry. Graphs show data as box whiskers(tukey) with median, interquartile ranges and outliers. Asterisks show statistical differences (Kruskal-Wallis and Mann Whitney test) between the groups indicated by the brackets (*p<0.05, **p<0.01, ***p<0.001).