Single-cell analysis of innate cytokine responses to pattern recognition receptor stimulation in children across four continents

Abstract

Innate immunity instructs adaptive immunity, and suppression of innate immunity is associated with an increased risk for infection. We showed previously that whole-blood cellular components from a cohort of South African children secreted significantly lower levels of most cytokines following stimulation of pattern recognition receptors compared with whole blood from cohorts of Ecuadorian, Belgian, or Canadian children. To begin dissecting the responsible molecular mechanisms, we set out to identify the relevant cellular source of these differences. Across the four cohorts represented in our study, we identified significant variation in the cellular composition of whole blood; however, a significant reduction in the intracellular cytokine production on the single-cell level was only detected in South African children's monocytes, conventional dendritic cells, and plasmacytoid dendritic cells. We also uncovered a marked reduction in polyfunctionality for each of these cellular compartments in South African children compared with children from the other continents. Together, our data identify differences in cell composition, as well as profoundly lower functional responses of innate cells, in our cohort of South African children. A possible link between altered innate immunity and increased risk for infection or lower response to vaccines in South African infants needs to be explored.

Figure 1

Gating strategy for the 11-colour panel for flow cytometry.

Figure 2

Cellular composition of whole blood cells at each of the 4 sites (statistical significance p value was *** < 0.005, ** <0.01, * <0.05).

Figure 3

Single-cell cytokine in response to TLR or NLR ligand stimulations show that South African children have a weaker single cell-specific cytokine response. Whole blood obtained from children from 4 different sites were stimulated with R848, LPS, and PGN ligands and measured by flow cytometry for IL6, IL12, IFNα, IFNγ, and TNFα production. A. Monocytes, B. cDCs, C. pDCs. Kruskal-Wallis test was done to look at statistical differences of each cytokine per cell type per stimulation, followed by Dunn’s post-hoc test was applied to each site pairing (statistical significance p value was *** < 0.005, ** <0.01, * <0.05).

Figure 4

Multi-cytokine response of Monocyte-single cell type showed that South Africa is weaker and less diverse in response to TLR and NLR ligands. Whole blood obtained from children from 4 different sites were stimulated with R848, LPS, or PGN ligands and measured by flow cytometry for IL6, IL12, IFNγ, and TNFα levels. The stacked bar graph represents the combination of cytokine contributions per cell type. The line graph indicates the polyfunctional state of the cell type, summarizing the percentage of cell producing a single- (one), double- (two), triple- (three), or quadruple- (four) cytokines in response to stimulation.

Figure 5

Multi-cytokine response of cDC-single cell type showed that South Africa is weaker and less diverse in response to TLR and NLR ligands. Whole blood obtained from children from 4 different sites were stimulated with R848, LPS, or PGN ligands and measured by flow cytometry for IL6, IL12, IFNγ, and TNFα levels. The stacked bar graph represents the combination of cytokine contributions per cell type. The line graph indicates the polyfunctional state of the cell type, summarizing the percentage of cell producing a single- (one), double- (two), triple- (three), or quadruple- (four) cytokines in response to stimulation.

Figure 6

Multi-cytokine response of pDC-single cell type showed that South Africa is weaker and less diverse in response to TLR 7/8 ligand. Whole blood stimulated with R848 ligand and measured by flow cytometry for IL6, IFNα, IFNγ, and TNFα levels. The stacked bar graph represents the combination of cytokine contributions per cell type. The line graph indicates the polyfunctional state of the cell type, summarizing the percentage of cell producing a single- (one), double- (two), triple- (three), or quadruple- (four) cytokines in response to stimulation.

Figure 7

Polyfunctional index (PI) values of children at each site per cell type in response to TLR and NLR ligand stimulation. A. PI numerically evaluated the degree and variation of polyfunctionality within the 4-site cohort. It allows for difference between cytokines produced by the different cell type to defined stimulations (R848, LPS, PGN) to be observed. B. Kruskal-Wallis test was used to compare the 4 sites per cell type and stimulation. Dunn’s post-hoc test was applied to each site paring (statistical significance p value was *** <0.005, ** <0.01, * <0.05).