Reduced T regulatory cell response during acute Plasmodium falciparum infection in Malian children co-infected with Schistosoma haematobium

Abstract

Background: Regulatory T cells (Tregs) suppress host immune responses and participate in immune homeostasis. In co-infection, secondary parasite infections may disrupt the immunologic responses induced by a pre-existing parasitic infection. We previously demonstrated that schistosomiasis-positive (SP) Malian children, aged 4-8 years, are protected against the acquisition of malaria compared to matched schistosomiasis-negative (SN) children.

Methods and findings: To determine if Tregs contribute to this protection, we performed immunologic and Treg depletion in vitro studies using PBMC acquired from children with and without S. haematobium infection followed longitudinally for the acquisition of malaria. Levels of Tregs were lower in children with dual infections compared to children with malaria alone (0.49 versus 1.37%, respectively, P = 0.004) but were similar months later, during a period with negligible malaria transmission. The increased levels of Tregs in SN subjects were associated with suppressed serum Th1 cytokine levels, as well as elevated parasitemia compared to co-infected counterparts.

Conclusions: These results suggest that lower levels of Tregs in helminth-infected children correlate with altered circulating cytokine and parasitologic results which may play a partial role in mediating protection against falciparum malaria.

Figure 1. T regulatory cell detection.

Quantification of T regulatory cells gated on CD3⁺ViViD⁻CD19⁻CD4⁺ with representative plots depicting the percentage of CD25^hi and FOXP3⁺ expression (closed histogram) in (a) a child with S. haematobium and concomitant P. falciparum malaria (SP Mal) and (b) a child with P. falciparum malaria (SN Mal) alone. The percentages of gated populations are denoted in each filled histogram (a, b). Region placement was aided by fluorescence minus one (FMO) determinations for CD69⁺ and FOXP3⁺ cells (open histogram). Dot plots of percentage of CD3⁺ViViD⁻CD19⁻CD4⁺CD69⁻CD25^hiFOXP3⁺ expression and segregated by group and season are depicted in children (c) aged 4–14 years, (d) aged 4–8 years and (e) aged 9–14 years. Each dot represents one individual. Bars represent the median and asterisks depict statistically significant differences between the indicated subset of volunteers and SP children during the transmission season (paired student T test with level of significance set at P<0.05).

Figure 2. Intracellular cytokine production to malaria and schistosoma antigens.

Representative example of intracellular cytokine (IL-2 and IFN-γ) detection in PBMC, from a child with concomitant S. haematobium and P. falciparum infection, in response to both malaria and schistosoma pooled antigen stimulation and media control for comparison (panel a). Mean intracellular cytokine production from PBMC stimulated with pooled (b) malaria and (c) schistosoma antigens collected in children aged 4–14 years with (SP Mal) and without S. haematobium (SN Mal) measured during the transmission and follow-up dry season. Cytokine-producing cells were found to be predominantly CD3⁺CD19⁻CD4⁺CD8⁻ T cells. Cytokine expression is reported as net % cytokine producing cells (stimulant minus media control) and standard deviations.

Figure 3. Intracellular cytokine production to malaria and schistosoma antigens after T regulatory cell depletion.

Representative example of intracellular cytokine detection in PBMC, from a child with concomitant S. haematobium and P. falciparum infection, mock or anti-CD25^hi depleted prior to stimulation with malaria or schistosoma antigenic pools. Removal of regulatory T cells appears to reverse suppressed immunologic responses resulting in enhanced intracellular IL-2 and IFN-γ in the anti-CD25hi depleted cell population. Media controls are included for comparison.

Figure 4. Proliferative responses to malaria and schistosoma antigens.

Shown are the geometric mean lymphocyte proliferative responses as determined by [³H]-thymidine incorporation to malaria and schistosoma antigen pool stimulation (4a and b) in PBMC collected during the malaria transmission season and again in the dry season in children with (SP Mal, n = 20) and children without (SN Mal, n = 17) S. haematobium. Responders refer to those individuals with significant proliferative responses to malaria antigen stimulation (n = 8 SP Mal and n = 6 SN Mal, Figure 4a) or to schistosoma antigen stimulation during the transmission season (n = 13 SP Mal and n = 1 SN Mal, Figure 4b), with their subsequent dry season values depicted. The dotted line depicts the minimally significant SI of 2.0. The asterix depicts significant differences between SP Mal and SN Mal (P<0.001). No differences were noted between mock or anti-CD25^hi depletion experiments (not shown).