The main feature of this study is the comparison of total and functional P. falciparum -specific IgE responses in two populations of low and high malaria transmission levels, India and Gabon respectively and their relationship with disease severity. Several clinical groups were compared: endemic non-infected controls, asymptomatics and different clinical manifestations including uncomplicated, severe non-cerebral and cerebral malaria.
Indian and Gabonese individuals exhibited a different range of plasma levels of circulating IgE These cohorts had a different age range, with the Gabonese groups being children from 0 to 6 years of age, whereas Indian patients had a mean age of 30 years. In the two populations, irrespective of its concentration and also consistent with previously published data, the IgE distribution tended to increase upon parasite stimulation [8, 10, 11]. In the Indian population, circulating IgE levels were seven times higher in endemic controls than in non-endemic controls. This suggests that exposure to the parasite strongly influences the production of IgE, although this difference may also be due to other endemic factors . Nevertheless, it was reported that IgE levels were greatest in patients developing severe disease than in CM group (Figure 1A and 1B). When considering the percentage of patients that produce high levels of IgE per group, it was shown that IgE levels are higher in UM and SM (Figure 1C and 1D) than in CM patients who had values similar to that of the controls [8, 24]. In addition, the median levels of circulating IgE in the ExCM group were close to that of the CM group. Also, no significant correlation was found between both IgE and TNF levels in the CM group. These observations are contrary to published data describing increased IgE levels that correlate with high concentrations of circulating TNF, a cytokine associated with malaria severity and also with pRBC adherence on brain capillary endothelial cells [9, 10, 25, 26]. The results suggest that either IgE does not play an important role in CM pathogenesis, or that these antibodies may participate in the parasite sequestration into the brain or other organ capillaries .
There was a significant increase in IgE levels with age in Gabonese children independent of the disease group (Figure 2). This increase in IgE production between 0 and six years of age may also reflect an increase in the capacity of the immune system to respond to parasite infections [7, 27]. Such a correlation was not found in Indian groups. This would be expected because the Indian groups comprised mainly adults, with the few children being older than five years. Although most of individuals in the Gabonese endemic control group had already been in contact with the parasite, as demonstrated by the high titres of specific antibody to P. falciparum -infected red blood cells observed in these children, the median plasma IgE concentrations were compared with those of the Indian NEC group. Although the median total IgE levels in the Indian NEC groups was higher than that of the Gabonese EC group, suggesting that age could be an important factor in IgE production, the higher IgE levels in the Indian population may also be interpreted as the result of either environmental factors, such as predominance of food allergies, of the genetic background, which may predispose to developing IgE responses [13, 27, 29–32] or of a co-infection with other parasites, such as helminths affecting the IgE responses in these patients [33, 34]. In the Gabonese cohorts, IgE levels tended to correlate negatively to parasitaemia except in UM whereas all patient groups from India showed a positive correlation.
The pool of circulating IgE comprises both monomeric and complexed immunoglobulins [35, 36]. A functional test was performed, based on the ability of the circulating IgE from the sera of different patient groups to induce degranulation of mast cells in the presence of pRBC antigens to better estimate the P. falciparum specific IgE response. This test does not provide specific IgE concentration within total IgE. It is based on the specific IgE induced percentage of mast cells degranulation. Degranulation was measured by quantifying β-hexosaminidase release. Functional P. falciparum -specific IgEs were detected in randomly chosen patients from all groups in both the Gabonese and Indian populations, except for ExCM Indian patients. The highest percentage of patients with functional anti-parasite IgEs was found in the Gabonese AI and UM and Indian EC and UM groups, which decreased in the SM and CM groups (Figure 3A and 3B). This suggests a protective role for P. falciparum -specific IgE, and is consistent with previous published data [13, 14].
Although the CM group had a low percentage of patients able to induce degranulation, it was the only group where there was one patient serum inducing a mast cell degranulation above 30%. This intense response may be associated with the presence of IgE with higher affinity for P. falciparum antigens, as previously reported by Gonzalez-Espinosa et al. . However, the level of degranulation can also be enhanced by the number of receptors involved in recognizing the antigen-IgE complex, which can strongly affect the size of the secretory response [37, 38]. There was no evident correlation between the level of functional P. falciparum -specific IgE (percentage of enzyme release) and the level of total IgE per group within each population. This is unsurprising, given that both the monomeric and complexed forms of circulating parasite-specific IgE can affect the level of degranulation [39–41]. It is also acceptable that total IgE levels were not directly correlated to FcåRI upregulation levels [40, 42]. The minimum level of receptors occupied by the parasite antigen-specific IgE complex required to induce a degranulation response is 10%. Consequently, this response will be independent of the total IgE levels . An increase in specific IgE levels has been also seen in other parasitic infections, such as helminthiasis, in which the specific IgEs usually help to eliminate the pathogens either through hypersensitivity reactions resulting from mast cell degranulation or by inducing antibody-dependent cell-mediated responses .
High levels of IgE in P. falciparum -infected individuals have been shown to be due to an underlying imbalance in favour of IL-4 in the ratio of CD4+ T cell producers, which are responsible for the IgG/IgM isotype switching to IgE . High levels of circulating IL-4 have also been associated with a greater parasite antigen-specific production of IgE in individuals less susceptible to malaria . Also, Th1-type pro-inflammatory cytokines, such as IFN-γ and TNF are thought to play an important role in the both protecting against and increasing the pathogenesis of cerebral malaria [3, 4, 22]. Plasma TNF, IFN-γ and IL-10 levels were measured in both the Indian and Gabonese groups. In the Gabonese groups, no direct correlation was found between IgE and IFN-γ or TNF levels in the UM, SM and CM patients. However, the level of IgE production was correlated to IL-10 levels in UM and AI patients (Figure 4). In this group, there was a positive association between IL-10 levels and parasite load, but not between IgE levels and parasitaemia. In the Indian groups, IFN-γ, TNF and IL-10 levels were all significantly correlated with IgE levels independent of the group (Figures 5A, B and 5C). When looking for a correlation per group, no significant statistic value was found. IL-4 is the main cytokine responsible for IgE production, inducing antibody isotype switching from IgG and IgM to IgE . Previous studies have shown an association between the IL-4/IFN-γ levels and IgE levels, suggesting an induced Th2-type switched response . As IL-4 levels were different between groups only in Indians, a correlation was expected between this ratio and IgE levels, and surprisingly, the results showed an opposite correlation (p = 0,012). The levels of IL-4 remained almost constant across all groups as IgE levels increased. Therefore, this opposite correlation arises due to a parallel increase in both IFN-γ and IgE levels. This suggests that, in the Indian population, IL-4 does not seem to directly influence IgE levels. It has been shown experimentally that there are conditions under which alternative mechanisms may induce IgE production independent of IL-4 . Regarding other cytokine ratios, like TNF/IL-10, no significant correlation was found with total IgE levels.
In conclusion, these results showed that total IgE levels increased in infected patients, mainly in UM and SM patients but not in CM patients. The decrease in total IgE levels in the CM group was associated with a higher intensity of mast cell degranulation induced in vitro by P. falciparum -specific IgE. In addition, no correlation was found between total and functional P. falciparum -specific IgE levels.
The results reported here show the high activity of functional circulating P. falciparum- specific IgE in asymptomatic malaria patients. However, no correlation was observed between plasma levels of total IgE, the disease phenotype and the cytokines pattern in the different groups of patients studied. The opposite association between pro- and anti-inflammatory cytokine ratios and IgE levels reveals the complexity of immune response disruption occurring during malaria in patients from low and high malaria endemic region.