Plasma IP-10, apoptotic and angiogenic factors associated with fatal cerebral malaria in India

Abstract

Background: Plasmodium falciparum in a subset of patients can lead to cerebral malaria (CM), a major contributor to malaria-associated mortality. Despite treatment, CM mortality can be as high as 30%, while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM is mediated by alterations in cytokine and chemokine homeostasis, inflammation as well as vascular injury and repair processes although their roles are not fully understood. The hypothesis for this study is that CM-induced changes in inflammatory, apoptotic and angiogenic factors mediate severity of CM and that their identification will enable development of new prognostic markers and adjunctive therapies for preventing CM mortalities.

Methods: Plasma samples (133) were obtained from healthy controls (HC, 25), mild malaria (MM, 48), cerebral malaria survivors (CMS, 48), and cerebral malaria non-survivors (CMNS, 12) at admission to the hospital in Jabalpur, India. Plasma levels of 30 biomarkers ((IL-1beta, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, G-CSF, GM-CSF, IFN-gamma, IP-10, MCP-1 (MCAF), MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, Fas-ligand (Fas-L), soluble Fas (sFas), soluble TNF receptor 1 (sTNF-R1) and soluble TNF receptor 2 (sTNFR-2), PDGF bb and VEGF)) were simultaneously measured in an initial subset of ten samples from each group. Only those biomarkers which showed significant differences in the pilot analysis were chosen for testing on all remaining samples. The results were then compared between the four groups to determine their role in CM severity.

Results: IP-10, sTNF-R2 and sFas were independently associated with increased risk of CM associated mortality. CMNS patients had a significantly lower level of the neuroprotective factor VEGF when compared to other groups (P < 0.0045). The ratios of VEGF to IP-10, sTNF-R2, and sFas distinguished CM survivors from non survivors (P < 0.0001).

Conclusion: The results suggest that plasma levels of IP-10, sTNF-R2 and sFas may be potential biomarkers of CM severity and mortality. VEGF was found to be protective against CM associated mortality and may be considered for adjunctive therapy to improve the treatment outcome in CM patients.

Figure 1

Log plasma biomarker levels and disease severity. Log median levels (pg/ml) of twelve (12) biomarkers [sTNF-R1, sTNF-R2 IL-1ra, sFas, Fas-L, IP-10, MIP-1β, IL-8, IL-10, VEGF, PDGFbb and FGF basic protein]. With the exception of sFas, Fas-L, sTNF-R1, and sTNF-R2 that were measured by ELISA, all other biomarkers were quantified via Luminex™ from blood samples collected from patients at time of admission. HC – healthy controls, MM – mild malaria, CMS – cerebral malaria survivors, CMNS – cerebral malaria non-survivors. Box plots represent medians and 25^th and 75^th percentiles. Bars mark the 10^th and 90^th percentiles, and outliers are plotted as points. HC versus MM, CMS, and CMNS: *** p < 0.05, $$$ p < 0.0045 bonferroni adjustment (alpha = 0.05/12)

Figure 2

Log plasma biomarker levels and disease severity. Log median levels (pg/ml) of eighteen (18) biomarkers [IFN-γ, TNF-α, MIP-1α, IL-1β, IL-2, RANTES, IL-4, IL-5, G-CSF, IL-6, IL-9, Eotaxin, IL-12 (p70), IL-13, GM-CSF, IL-15, IL-17 and MCP-1 (MCAF)]. Biomarkers were quantified via Luminex™ from blood samples collected from patients at time of admission. HC – healthy controls, MM – mild malaria, CMS – cerebral malaria survivors, CMNS – cerebral malaria non-survivors. Box plots represent medians and 25^th and 75^th percentiles. Bars mark the 10^th and 90^th percentiles, and outliers are plotted as points. HC versus MM, CMS, and CMNS: *** p < 0.05, $$$ p < 0.0045 bonferroni adjustment (alpha = 0.05/18)

Figure 3

Biomarkers with significant differences between disease severity groups as determined by multivariate analyses. HC – healthy controls, MM – mild malaria, CMS – cerebral malaria survivors, CMNS – cerebral malaria non-survivors. Box plots represent medians and 25^th and 75^th percentiles. Bars mark the 10^th and 90^th percentiles, and outliers are plotted as points. MM versus CMS and CMNS: *** p < 0.05, $$$ p < 0.0045 bonferroni adjustment (alpha = 0.05/11), CMS versus CMNS: +++ p < 0.05, ### p < 0.0045 bonferroni adjustment (alpha = 0.05/11).

Figure 4

Relationship of the mean ratios of IP-10, sTNF-R2, and sFas to angiogenic protective factors VEGF and PDGF bb with disease severity. Line represents best fit with 95% confidence intervals in shaded regions.