This is the first report showing NF-κB expression in the PBMCs of malaria patients and its correlation with IL-10 and TNF by using sandwich ELISA. The use of ELISA has become a powerful method for measuring protein phosphorylation. ELISA is more quantitative than Western blotting and possesses high specificity and sensitivity due to the use of two antibodies specific for the target protein employed together in the sandwich. NF-κB p65 activation was increased in the PBMCs of
P. vivax and uncomplicated
P. falciparum patients, on both day 0 and day 7, whereas in complicated
P. falciparum patients, elevated NF-κB p65 activity was observed only on day 7 post-treatment. NF-κB activation may be triggered by various ligands or proteins of malaria parasites that induce up-regulation of the NF-κB signaling pathway, leading to nuclear translocation of NF-κB and regulation of gene expression. It is possible that the increased NF-κB p65 levels in the PBMCs with malaria infection are involved in the enhancement of inflammatory cytokines. Consistent with the increased level of phospho-NF-κB p65 in the PBMCs, the immunofluorescence assay confirmed NF-κB p65 immunostaining in PBMC nuclei, indicating the active NF-κB protein state in malaria infection. Data from the literature of experimental
in vitro malaria studies show that the mechanisms induced or involved in the activation of NF-κB p65 include haemozoin (HZ)-induced enhancement of inflammatory cytokines [
11,
16‐
18], activation of matrix metalloproteinase-9 (MMP-9) in human monocytes fed with trophozoites and HZ [
19], and
P. falciparum glycosylphosphatidylinositol (GPI) stimulating monocytes and macrophages, leading to the activation of NF-κB downstream signaling pathways induced expression of pro-inflammatory mediators, such as TNF, IL-6, IL-12, and nitric oxide (NO) [
12,
13]. Recent investigations studied the innate immune response in malaria infection, showing that Toll-like receptor 1 (TLR1), TLR2, and TLR4 were induced in PBMCs from both experimentally [
14,
20] and naturally acquired malaria infections [
14]. These findings suggest that the activation of TLRs by GPI [
21] and HZ [
22] transmit signals in an intracellular pathway leads to the activation of transcription factor NF-κB, which in turn propagates a signal to the nucleus to regulate the expression of pro-inflammatory cytokines. Consequently, these actions could cause increased levels of phospho-NF-κB p65 and nuclear translocation of NF-κB p65 in the PBMCs of malaria patients.
NF-κB p65 activity was decreased in PBMCs from patients with complicated
P. falciparum at admission, consistent with the reduced mean percentage of NF-κB p65 nuclear translocation evidenced by the immunofluorescence study. These findings agree with previous reports which demonstrated that PBMCs from patients with sepsis and major trauma reduced the active form of NF-κB p65 on the day of admission [
23,
24]. The silencing of NF-κB p65 gene expression reported in severe systemic inflammation may also explain the important signaling event in complicated
P. falciparum wherein NF-κB p65 could be repressed by cytokines [
25]. Studies have shown that immunosuppressives such as TGF-β [
26,
27] and IL-10 (also known as anti-inflammatory cytokine) [
28] reportedly alter NF-κB expression and translocation, and contribute to cell desensitization [
26‐
28]. Normally, IL-10 is produced by macrophages as well as T and B lymphocytes, and has been shown to play a significant role in immunoregulation, involving negative feedback on the production of pro-inflammatory cytokine [
29]. It has been reported that increasing plasma IL-10 was detected in cerebral and severe malaria patients at admission, in contrast to the patients with uncomplicated
P. falciparum malaria [
30]. Elevated IL-10 levels have been detected in serum of Thai patients with acute
P. falciparum malaria prior to treatment and the levels were found to return to normal after malaria treatment [
31]. To investigate whether the decrease in nuclear translocation of NF-κB found in complicated
P. falciparum malaria patients is linked to plasma IL-10 levels, IL-10 levels were determined in the malaria groups. In this study, the plasma level of IL-10 was significantly elevated in complicated
P. falciparum malaria infection and had a negative correlation with phospho-NF-κB p65 expression at admission. When the IL-10 levels were high, the phospho-NF-κB p65 levels were low. This correlation was not found in
P. vivax or uncomplicated
P. falciparum infections. At admission, the plasma levels of IL-10 in patients with complicated
P. falciparum malaria were 4.3 times and 3 times higher than in patients with
P. vivax and uncomplicated
P. falciparum, respectively. This observation suggests that decreased levels of NF-κB p65 in the PBMCs of complicated
P. falciparum patients during acute infection could be due to a negative feedback loop mechanism, or the consequence of high levels of IL-10, an important anti-inflammatory cytokine associated with severe disease. It has been shown that IL-10 inhibits NF-κB activation rapidly and in a dose-dependent manner [
28]. At day 7 post-treatment, the plasma levels of IL-10 in complicated
P. falciparum malaria declined 5.7 times from the level on day 0, to the same levels as the
P. vivax and uncomplicated
P. falciparum malaria groups. This trend is similar to results in a previous report [
30]. The lower level of IL-10 could explain the elevated level of activated NF-κB in the PBMCs from complicated
P. falciparum malaria at day 7 post-treatment.
Furthermore, the study investigated whether malaria patient serum could induce NF-κB p65 activation in unstimulated PBMCs. Significantly, transiently increased levels of phospho-NF-κB p65 were found in the healthy PBMCs 30 min after stimulation with malaria serum, consistent with previous studies on endothelial cells [
32], monocytes [
33], and human cardiac myocytes [
34]. However, the transient increased of phospho-NF-κB p65 in malaria sera-induced healthy PBMCS did not concur with the decline of NF-κB p65 at admission in complicated
P. falciparum malaria. The PBMCs from healthy controls are naïve and activation is short-lived. The effect might not be long enough to initiate a complex response to malaria serum. In contrary, the PBMCs collected from complicated
P. falciparum malaria patients was sensitized by prolonged infection and response mechanisms induced by other cell signaling processes. The present study elucidates that sera from malaria patients can induce NF-κB p65 activation in naïve PBMCs. It would be helpful to further analyse the expression of NF-κB in PBMCs from malaria patients after stimulation with malaria sera to determine the event of “desensitization” in malaria. Further work to investigate whether pre-incubation of the malaria sera with an anti-IL-10 neutralizing monoclonal antibody would suppress NF-κB activation in PBMCs from complicated
P. falciparum patients will be of interest.