CD4 T-cell expression of IFN-γ and IL-17 in pediatric malarial anemia

Abstract

In Plasmodium falciparum holoendemic transmission regions of western Kenya, life-threatening pediatric malaria manifests primarily as severe malarial anemia (SMA, Hb≤6.0 g/dL with any density parasitemia). To determine the role that CD4+ T-cell-driven inflammatory responses have in the pathogenesis of SMA, peripheral CD4+ T-cell populations and their intracellular production of pro-inflammatory cytokines (IFN-γ and IL-17) were characterized in children aged 12-36 months of age stratified into two groups: non-severe malarial anemia (non-SMA, Hb≥6.0 g/dL, n = 50) and SMA (n = 39). In addition, circulating IFN-γ and IL-17 were measured as part of a Cytokine 25-plex Antibody Bead Kit, Human (BioSource™ International). Children with SMA had higher overall proportions of circulating lymphocytes (P = 0.003) and elevated proportions of lymphocytes expressing IFN-γ (P = 0.014) and comparable IL-17 (P = 0.101). In addition, SMA was characterized by decreased memory-like T-cells (CD4+CD45RA-) expressing IL-17 (P = 0.009) and lower mean fluorescence intensity in memory-like CD4+ T-cells for both IFN-γ (P = 0.063) and IL-17 (P = 0.006). Circulating concentrations of IFN-γ were higher in children with SMA (P = 0.009), while IL-17 levels were comparable between the groups (P = 0.164). Furthermore, circulating levels of IFN-γ were negatively correlated with IL-17 levels in both groups of children (SMA: r = -0.610, P = 0.007; and non-SMA: r = -0.516, P = 0.001), while production of both cytokines by lymphocytes were positively correlated (SMA: r = 0.349, P = 0.037; and non-SMA: r = 0.475, P = 0.001). In addition, this correlation was only maintained by the memory-like CD4+ T cells (r = 0.365, P = 0.002) but not the naïve-like CD4+ T cells. However, circulating levels of IFN-γ were only associated with naïve-like CD4+ T cells producing IFN-γ (r = 0.547, P = 0.028), while circulating levels of IL-17 were not associated with any of the cell populations. Taken together, these results suggest that enhanced severity of malarial anemia is associated with higher overall levels of circulating lymphocytes, enhanced intracellular production of IFN-γ by peripheral lymphocytes and high circulating IFN-γ levels. In addition, the observed inverse relationship between the circulating levels of IFN-γ and IL-17 together with the reduction in the levels of memory-like CD4+ T cells expressing IL-17 in children with SMA may suggest possible relocation of these cells in the deeper tissues for their pathological effect.

Fig 1. Characterization of CD4+ T-cell sub-populations.

(A) Initial gating was performed on lymphocytes in the overall cell population during acquisition. (B) The CD4+ T-cell population was then selected by gating on CD4+ cells in the lymphocyte cell population during analyses. (C) Due to the limitation of the four-color cytometer, the populations of CD4+ cells used in subsequent analyses were CD4+ cells from the lymphocyte gate. To determine the proportion of CD4+ cells that were T-cells (CD3+CD4+), expression of CD3 by the CD4+ cells (gated in B) was determined. The median proportion of CD3+CD4+ (T-cells) among the CD4+ cells was 98.9%, suggesting that the CD4+ gating selected an enriched population of CD4+ T-cells.

Fig 2. Lymphocytes expressing IFN-γ and IL-17 in acute malaria.

Data are presented as proportions (%). The proportions were determined by flow cytometry immediately after staining. The proportions of different lymphocyte populations were determined using FlowJo Software (TreeStar, Ashland, OR, USA). The line in the middle represents the median value. Between groups comparisons were performed by Mann-Whitney U test. (A) The proportion of lymphocytes was higher in the SMA group relative to non-SMA group. (B) The proportion of lymphocytes expressing IFN-γ was higher in the SMA group relative to non-SMA group. (C) The proportion of lymphocytes expressing IL-17 was higher in the SMA group relative to non-SMA group.

Fig 3. Memory-like CD4+ T-cells expressing IFN-γ and IL-17 in acute malaria.

Data are presented as proportions (%). The proportions were determined by flow cytometry immediately after staining. The proportions of different lymphocyte populations were determined using Flowjo Software (TreeStar, Ashland, OR, USA). The line in the middle represents the median value. Between groups comparisons were performed by Mann-Whitney U test. The proportion of CD4+ T-cells was comparable between the groups (data not shown). (A) The proportion of naïve-like CD4+ T-cells expressing IFN-γ was comparable between the groups. (B) The proportion of naïve-like CD4+ T-cells expressing IL-17 was comparable between the groups. (C) The proportion of memory-like CD4+ T-cells expressing IFN-γ was comparable between the groups. (D) The proportion of memory-like CD4+ T-cells expressing IL-17 was decreased in SMA relative to non-SMA group.

Fig 4. Intensity of IFN-γ and IL-17 production by memory-like CD4+ T-cells.

Data are presented as mean fluorescence intensity (MFI). The MFI was determined by FlowJo Software (TreeStar, Ashland, OR, USA). The line in the middle represents the median value. Between groups comparisons in the clinical categories were performed by Mann-Whitney U tests. (A) MFI of IFN-γ in CD4+CD45RA- cells. (B) MFI of IL-17 in CD4+CD45RA- cells.

Fig 5. Circulating levels of IFN-γ and IL-17 in children with differing malarial anemia severity.

Box-plots depict the data where the box represents the interquartile range, the line through the box is the median, and whiskers illustrate the 10th and 90th percentiles. Circulating IFN-γ (n = 64; non-SMA = 42; SMA = 22) and IL-17 (n = 64; non-SMA = 42; SMA = 22) concentrations were determined in plasma using the human Cytokine 25-plex Ab Bead Kit, (BioSource™ International) according to the manufacturer’s protocol. Plates were read on a Luminex 100™ system (Luminex Corporation) and analyzed using the Bio-Plex Manager Software (Bio-Rad Laboratories). Between groups comparisons in the clinical categories were performed by Mann-Whitney U tests. (A) Circulating IFN-γ levels between non-SMA and SMA. (B) Circulating IL-17 levels between non-SMA and SMA.