Interethnic differences in antigen-presenting cell activation and TLR responses in Malian children during Plasmodium falciparum malaria

Abstract

The Fulani ethnic group from West Africa is relatively better protected against Plasmodium falciparum malaria as compared to other sympatric ethnic groups, such as the Dogon. However, the mechanisms behind this lower susceptibility to malaria are largely unknown, particularly those concerning innate immunity. Antigen-presenting cells (APCs), and in particular dendritic cells (DCs) are important components of the innate and adaptive immune systems. Therefore, in this study we investigated whether APCs obtained from Fulani and Dogon children exhibited differences in terms of activation status and toll-like receptor (TLR) responses during malaria infection. Lower frequency and increased activation was observed in circulating plasmacytoid DCs and BDCA-3+ myeloid DCs of infected Fulani as compared to their uninfected counterparts. Conversely, a higher frequency and reduced activation was observed in the same DC subsets obtained from peripheral blood of P. falciparum-infected Dogon children as compared to their uninfected peers. Moreover, infected individuals of both ethnic groups exhibited higher percentages of both classical and inflammatory monocytes that were less activated as compared to their non-infected counterparts. In line with APC impairment during malaria infection, TLR4, TLR7 and TLR9 responses were strongly inhibited by P. falciparum infection in Dogon children, while no such TLR inhibition was observed in the Fulani children. Strikingly, the TLR-induced IFN-γ release was completely abolished in the Dogon undergoing infection while no difference was seen within infected and non-infected Fulani. Thus, P. falciparum infection is associated with altered activation status of important APC subsets and strongly inhibited TLR responses in peripheral blood of Dogon children. In contrast, P. falciparum induces DC activation and does not affect the innate response to specific TLR ligands in Fulani children. These findings suggest that DCs and TLR signalling may be of importance for the protective immunity against malaria observed in the Fulani.

Figure 1. Interethnic and intraethnic differences in the frequency and activation markers of blood DCs.

PBMCs from the study participants were stained with monoclonal antibodies for subsequent flow cytometric analysis using a FACSCalibur. The samples were categorised as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 13) and infected Fulani (n = 10). An increase was observed of BDCA2⁺ and BDCA3⁺ cells in the circulation of infected Dogon whereas the opposite was seen for the Fulani (Figure 1A). The expression of activation marker HLA-DR (Figure 1B) and CD86 (Figure 1C) was lower in BDCA2⁺ and BDCA3⁺ cells of the Dogon when undergoing infection while it was increased in the Fulani. It is known that DCs travel to the lymph nodes upon activation. Therefore, it is possible that the lower numbers seen in the infected Fulani is a consequence of activation. The boxplots illustrate the medians and the 25^th and 75^th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed by Mann-Whitney rank sum test. *; p≤0.05. **;p<0.01. ***; p<0.001.

Figure 2. Interethnic and intraethnic differences in the frequency and activation markers of circulating monocytes.

PBMCs were stained with monoclonal antibodies and subsequently analyzed using FACSCalibur. The samples were categorized as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 13) and infected Fulani (n = 10). An increase of circulating monocytes was observed for both groups although only statistically significant for the Dogon (Figure 2A). There was a decreased expression of HLA-DR (Figure 2B) in classical monocytes for both ethnic groups. The levels of HLA-DR were also suppressed in the inflammatory monocytes although not statistically significant for the Dogon. The expression of CD86 (Figure 2C) was decreased on both monocytic subsets although only statistically significant for the Dogon classical monocytes from both Dogon and Fulani. Although the levels of activation markers seem similarly regulated in both groups the classical monocytes of the Fulani exhibit a pattern of higher activation when undergoing infection than the Dogon when infected. The boxplots illustrate the medians and the 25^th and 75^th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed by Mann-Whitney rank sum test. *; p≤0.05. ***; p<0.001.

Figure 3. TLR-induced cytokine responses in PBMCs from Dogon and Fulani children with or without P. falciparum infection.

PBMCs (1 million/ml) were stimulated or not with LPS (100 ng/ml), CpG-A (3 µg/ml) or imiquimod (1 µg/ml). After 16 hours, supernatants were collected and analysed for the presence of cytokines. The samples were categorized as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 14) and infected Fulani (n = 23). The levels of IL-1β, IL-6, IL-10, TNF-α and INF-γ were severely supressed in the infected Dogon as compared to their uninfected peers when the cells had been stimulated with either LPS or CpGA. As a result, the levels of the infected Fulani were higher than infected Dogon for all cytokines except for IFN-α when stimulated with LPS and except for IL-1β and IL-10 when stimulated with CpG. Similarly, when stimulated with imiquimod cells of the infected Fulani secreted higher levels of IL-6, IFN-α, TNF-α and INF-γ than the infected Dogon due to the suppressed secretion of Dogon when undergoing infection. Data presented as box plots illustrate the medians and the 25^th and 75^th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed using Mann-Whitney rank sum test. *; p≤0.05. **; p<0.01. ***; p<0.001.

Figure 4. Interethnic and intraethnic differences in the TNF-α/IL-10 and IFN-γ/IL-10 ratios upon TLR stimulation of PBMCs.

PBMCs (1 million/ml) were stimulated or not with LPS (100 ng/ml), CpG-A (3 µg/ml) or imiquimod (1 µg/ml). After 16 hours, supernatants were collected, cytokine levels were analyzed and the TNF-α/IL-10 (Figure 4A) and IFN-γ/IL-10 (Figure 4B) ratios were calculated. The samples from 77 children were categorized as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 23) and infected Fulani (n = 14). The TNF-α/IL-10 ratio (Figure 4A) was significantly lower in the infected Dogon as compared to their uninfected peers when stimulated with CpG or imiquimod. This resulted in significantly higher TNF-α/IL-10 ratio of infected Fulani as compared to the infected Dogon. Due to the severe suppression of IFN-γ the IFN-γ/IL-10 ratios (Figure 4B) were severely suppressed in the LPS and CpG stimulated cells of the Dogon and therefore higher in the infected Fulani than in the infected Dogon. The boxplots illustrate the medians and the 25^th and 75^th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed using Mann-Whitney rank sum test. *; p≤0.05. **; p<0.01. ***; p<0.001.