Osteoprotegerin and its ligands RANKL and TRAIL in falciparum, vivax, and knowlesi malaria

Abstract

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of NF-ƙB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL), is increasingly recognized as a marker of poor prognosis in cardiovascular disease. Here, we demonstrate that in adults with falciparum and vivax malaria, OPG is increased, and its ligands TRAIL and RANKL decreased, in association with validated markers of disease severity. Using longitudinal samples from malaria volunteer infection studies, we demonstrate that TRAIL is unexpectedly increased in early infection, suggesting binding of OPG to RANKL prior to TRAIL. Finally, in addition to its known vascular origin, we show that P. falciparum stimulates B cells to produce OPG in vitro, and that B cell OPG production is increased ex vivo in patients with falciparum, vivax, and knowlesi malaria. Our findings provide evidence of the importance of the OPG/RANKL/TRAIL pathway in pathogenesis of acute systemic inflammatory and microvascular diseases, with implications for adjunctive therapies.

Keywords: Disease; Immune response; Molecular biology.

Graphical abstract

Figure 1

Plasma concentrations of osteoprotegerin (OPG) in Malaysian patients with P. falciparum, P. vivax, and P. knowlesi malaria Pf = P. falciparum; Pv = P. vivax; Pk = P. knowlesi. Errors bars represent median and inter-quartile range. Data for patients with knowlesi malaria have been previously reported, and are included here for comparison with falciparum and vivax malaria.

Figure 2

Plasma concentrations of RANKL and TRAIL in Malaysian patients with P. falciparum, P. vivax, and P. knowlesi malaria Errors bars represent median and inter-quartile range.

Figure 3

Longitudinal concentrations of OPG, RANKL and TRAIL in participants inoculated with P. falciparum (left column) and P. vivax (right column) Black line represents median. Gray lines represent individual participants. Comparisons by Wilcoxon matched pair signed rank test for baseline (day of controlled malaria infection) and day of antimalarial treatment, and baseline and end of study measurements. Data for OPG has already been reported, and is included here for context.

Figure 4

Malaria parasites can induce OPG expression in B cells (A) PBMCs from buffy coats from Australian donors (n = 10) were stimulated with uninfected RBCs (uRBCs) or P, falciparum-infected RBCs (pRBCs) and OPG production from B cells assessed by flow cytometry. Representative gating figure of OPG from B cells following pRBC or uRBC stimulation. (B) OPG B cell expression from malaria-naive donors, after stimulation with uRBCs or pRBCs. The p value was calculated using Wilcoxon signed-rank test. The boxplots represent the median and inter-quartile range. (C) Analysis of B cell subsets that produce OPG. Representative gating figure of IgD expression from OPG positive cells B cells. (D) IgD+ and IgD− B cell subsets as a proportion of all OPG+ B cells following parasite stimulation of PBMCs from malaria-naïve donors (n = 4). The p value was calculated using Wilcoxon signed-rank test. The boxplots represent the median and inter-quartile range. See also Figure S3.

Figure 5

B cell production of OPG during clinical malaria (A) OPG expression from B cells at enrollment (day 0) and convalescence (day 28), gating example. (B) Proportion of B cells expressing OPG in Malaysian patients with P. falciparum (n = 6 day 0 and n = 5 day 28), P. vivax (n = 8) and P. knowlesi (n = 7), compared to Malaysian healthy controls (n = 5). p values between healthy controls and day 0 malaria infection were calculated using Mann Whitney. p values between day 0 and day 28 were calculated using Wilcox signed-rank test. The boxplots represent the median and inter-quartile range. (C) IgD expression in OPG+ B cells, gating example. (D) IgD+ and IgD- B cell subsets as a proportion of all OPG+ B cells at enrollment in patients with malaria. p values were calculated using Kruskal-Wallis (KW). The boxplots represent the median and inter-quartile range. (E) CD27 and CD21 expression in OPG+ IgD− B cells, gating example. (F) CD27 and CD21 B cell subsets as a proportion of all IgD− OPG+ B cells at enrollment in patients with malaria. p values were calculated using Kruskal-Wallis (KW). The boxplots represent the median and inter-quartile range. See also Figure S3.