Insights into deregulated TNF and IL-10 production in malaria: implications for understanding severe malarial anaemia

Abstract

Background: Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients.

Methods: The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined.

Results: Monocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001).

Conclusions: These data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.

Figure 1

Monocyte and T cell activation status in the different clinical groups at admission. A. Monocytes were identified based on their forward (FSC) and side scatter profiles as well as their CD14 positivity (green events). Representative cytograms of monocytes from a UM and a CM patient are shown. B. The percentages of CD14⁺ cells (monocytes) positive for HLA-DR were similar between CM and SMA cases and between UM and AC. However, both CM and SMA cases had higher percentages of HLA-DR⁺ monocytes than UM or AC children. C. A similar profile was found for the monocyte HLA-DR mean fluorescence intensity (MFI). D. The percentages of CD3⁺ cells (T cells) positive for CD69 or HLA-DR, early and late T cell activation markers, respectively were similar between clinical cases but significantly lower in the AC children. Percentages were determined at admission for each group. Data are presented as box plots: the box shows the interquartile range, the line through the box is the median and whiskers indicate the 5^th and 95^th percentiles. CM, SMA, UM and AC refer to cerebral malaria, severe malarial anemia, uncomplicated malaria and asymptomatic controls, respectively. * denotes P ≤ .05; ** denotes P ≤ .01; # denotes P > .05.

Figure 2

IL-10 and TNF production capacity in malaria patients. The IL-10 and TNF production capacity were measured after LPS and PHA stimulation as described in Methods. Fold increases from spontaneous cytokine secretion i.e. un-stimulated samples are presented as box plots: the box shows the interquartile range, the line through the box is the median and whiskers indicate the 5^th and 95^th percentiles. Statistical significance was determined by Mann–Whitney test. * denotes P ≤ .05; ** denotes P ≤ .01; # denotes P > .05.