The high TNF/IL-10 ratio observed in SMA suggests an imbalanced production of inflammatory cytokines that could contribute to anaemia [5, 6]. Whether such an imbalance is an intrinsic characteristic of children with SMA or reflects a specific response pattern to malaria infection involving particular cellular sources has profound implications on the design of intervention strategies to prevent SMA. The data reported here show that SMA patients indeed displayed low spontaneous IL-10 production at admission resulting in higher TNF/IL-10 ratios than CM cases. These findings are consistent with previous studies in Ghana [5, 21], but also more recent studies in Southern Zambia . Interestingly, in response to a monocyte or T cell stimulus IL-10 production readily increased in both CM and SMA patients, but SMA patients were characterized by a much higher amplitude of the IL-10 and TNF monocyte response to LPS compared to CM, possibly reflecting different monocyte priming status. However, the absolute levels of IL-10 reached after PHA-stimulation remained modest, much lower than for CM or any other group. This indicates that children experiencing SMA have no inherent incapacity to produce IL-10 and therefore that the imbalanced cytokine response at admission and upon further stimulation in vitro likely reflects a specific immunological pattern rather than an intrinsic predisposition to a deficient IL10-production.
The data also provide interesting insights into the immune status of children with CM. Although expression levels of surface activation markers on both lymphocytes and monocytes were similar in CM and SMA, CM patients presented a distinct cytokine expression profile, characterized by spontaneous production of high levels of both TNF and IL-10 but limited increase in TNF production after monocyte or T cell stimulation, suggesting an overall relative low-responsiveness to further stimulation. This points to a distinct functional status of circulating T cells and monocytes in SMA and CM, which both differed from the functional status in UM.
UM children had lower lymphocyte counts, limited monocyte deactivation, balanced IL-10 and TNF levels at admission (both lower than CM, IL-10 higher than SMA) and strong responsiveness to monocyte and T cell stimulation. Thus, based on analysis of circulating cells, the three clinical groups had specific response profiles to the ongoing infection and to further monocyte or T cell stimulation. IL-10 and TNF responses to a T cell stimulus were higher in AC than in any of the three clinical malaria groups, suggesting an impaired T cell responsiveness in malaria (regardless of the clinical presentation) as observed by others . Parasite-related factors may explain the specific IL-10 production profile of SMA patients. Some studies [30, 31] but not others  have found that haemozoin phagocytosis triggered the production of IL-10 by monocytes and induced a state of monocyte “anergy/reprogramming” associated with a deregulated production of pro-inflammatory cytokines such as TNF [33, 34]. However, the lack of association of IL-10 plasma levels with the number of circulating haemozoin-containing monocytes observed here, including in CM patients who have the highest IL-10 levels, does not support a direct impact of haemozoin load on IL-10 production by circulating leukocytes.
There was no significant correlation between the number of pigmented monocytes and haemoglobin. This contrast with results from Casals-Pascual et al., although they found a moderately positive correlation (r2 = 0,29) . This reflects the unclear relationship between pigmented leukocytes and the disease manifestation or the parasite biomass. The number of circulating pigmented monocytes depends on a complex clearance kinetics , which may differ depending on whether anaemia is consecutive to an acute infection or results from a protracted infection.
CD36-dependent adhesion of infected erythrocytes to monocytes may modulate the inflammatory cytokine secretion profile, including IL-10 production [37–39] and the low IL-10 plasma levels in SMA patients may reflect the low CD36-binding capacity of their infected erythrocytes . This is supported by the lower proportion of haemozoin-containing monocytes in SMA relative to CM patients, possibly reflecting the reduced phagocytosis of infected erythrocyte subsequent to CD36 binding  but may also merely reflect differences in parasite biomass.
The discrepant IL-10 levels in SMA and CM could result from different types or proportions of IL-10 producing cells. Recent studies suggest that various subsets of CD4+ T cells including Tr1 and Th1 are important contributors [42, 43]. Compared to CM and UM, SMA cases produced lower absolute levels of IL-10 but higher levels of TNF in response to T cell stimulation. This suggests a T cell functional impairment specific for IL-10 production in children with SMA. Whether this reflects an infection-related cytokine expression programming or an effector/regulatory T cell subset imbalance is unclear. Additional work is needed to elucidate this question, especially since depletion of CD4+ T cells significantly alleviates anaemia in a murine model .
Beside T cells, two monocyte subpopulations with different IL-10 producing capacity upon LPS stimulation are now recognized: the regular CD14brightCD16-/dull monocytes producing both TNF and IL-10 and the CD14dim CD16bright monocytes producing high levels of TNF and little or no IL-10 . Although the latter subset was recently found to be enriched in SMA children , comparable CD14/CD16 cytograms and monocyte CD14 MFI (CM: 234.1 ± 262.3; SMA: 162 ± 95) and CD16 MFI (CM: 10.8 ± 12.2; SMA: 13.9 ± 12.1) were observed here.
A significant but transient down-regulation of HLA-DR expression of circulating monocytes was observed in children with severe malaria, irrespective of the clinical form (SMA or CM). HLA-DR down-regulation has been described for dendritic cells in Kenyan children with acute malaria, but was observed in both mild and severe cases . Phagocytosis of haemozoin and exposure to IL-10 both induce down-regulation of monocyte HLA-DR surface expression [25, 48]. However, in the CM and SMA patients studied here, HLA-DR expression was independent from the number of pigmented monocytes and did not correlate with circulating IL-10 levels. The observed HLA-DR down-regulation rather results from the complex integration of multiple anti-inflammatory signals, as observed in severe inflammatory syndromes where it is generally associated with an impaired TNF production capacity in response to further LPS stimulation  reflecting a general cellular reprogramming phenomenon of acute inflammatory injuries [50–52]. The impaired TNF production after LPS stimulation observed in CM, but not in SMA, is reminiscent of this cellular reprogramming and suggests interference between the monocyte signalling pathway involved in the overwhelming cytokine production associated with CM and the LPS-triggered MD2 signalling pathway [53, 54]. Thus, CM appears as an acute inflammatory syndrome with excessive TNF production by monocytes/macrophages rapidly inducing a high counter-regulatory IL-10 production. In contrast, the high TNF levels observed in SMA would result from a more chronic/sustained production of TNF maintained by an impaired IL-10 regulatory feedback reflecting a specific leukocyte polarization/programming state in SMA.