On the cytokine/chemokine network during Plasmodium vivax malaria: new insights to understand the disease

Abstract

Background: The clinical outcome of malaria depends on the delicate balance between pro-inflammatory and immunomodulatory cytokine responses triggered during infection. Despite the numerous reports on characterization of plasma levels of cytokines/chemokines, there is no consensus on the profile of these mediators during blood stage malaria. The identification of acute phase biomarkers might contribute to a better understanding of the disease, allowing the use of more effective therapeutic approaches to prevent the progression towards severe disease. In the present study, the plasma levels of cytokines and chemokines and their association with parasitaemia and number of previous malaria episodes were evaluated in Plasmodium vivax-infected patients during acute and convalescence phase, as well as in healthy donors.

Methods: Samples of plasma were obtained from peripheral blood samples from four different groups: P. vivax-infected, P. vivax-treated, endemic control and malaria-naïve control. The cytokine (IL-6, IL-10, IL-17, IL-27, TGF-β, IFN-γ and TNF) and chemokine (MCP-1/CCL2, IP-10/CXCL10 and RANTES/CCL5) plasma levels were measured by CBA or ELISA. The network analysis was performed using Spearman correlation coefficient.

Results: Plasmodium vivax infection induced a pro-inflammatory response driven by IL-6 and IL-17 associated with an immunomodulatory profile mediated by IL-10 and TGF-β. In addition, a reduction was observed of IFN-γ plasma levels in P. vivax group. A lower level of IL-27 was observed in endemic control group in comparison to malaria-naïve control group. No significant results were found for IL-12p40 and TNF. It was also observed that P. vivax infection promoted higher levels of MCP-1/CCL2 and IP-10/CXCL10 and lower levels of RANTES/CCL5. The plasma level of IL-10 was elevated in patients with high parasitaemia and with more than five previous malaria episodes. Furthermore, association profile between cytokine and chemokine levels were observed by correlation network analysis indicating signature patterns associated with different parasitaemia levels.

Conclusions: The P. vivax infection triggers a balanced immune response mediated by IL-6 and MCP-1/CCL2, which is modulated by IL-10. In addition, the results indicated that IL-10 plasma levels are influenced by parasitaemia and number of previous malaria episodes.

Keywords: Chemokines; Cytokines; Malaria; Plasmodium vivax.

Fig. 1

Inflammatory cytokine plasma levels. Comparative analysis of IL-6 (a), IL-17 (b), IL-12p40 (c), TNF (d) and IFN-γ production among malaria-naïve control group (n = 15), endemic control group (n = 10), P. vivax group (n = 75) and P. vivax-treated group (n = 10) were performed using Kruskal–Wallis test followed by Dunn Post-hoc. The dotted lines (—) represent the detection limit of the assay. IL-6 levels in acute-phase and convalescence period from P. vivax-infected patients (e) was compared between P. vivax group (n = 10) and P. vivax-treated group using Paired t-test or Wilcoxon test, according to data distribution. A p value <0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Regulatory cytokine plasma levels. Comparative analysis of IL-10 (a total production; b paired analysis between infected and treated individuals) and TGF-β among malaria-naïve control group (n = 15), endemic control group (n = 10), P. vivax group (n = 75) and P. vivax-treated group (n = 10) were performed using Kruskal–Wallis test followed by Dunn Post-hoc. A p value <0.05 was considered significant. The dotted lines (—) represent the detection limit of the assay. Paired analysis was performed between P. vivax group (n = 10) and P. vivax-treated group using Paired t-test or Wilcoxon test, according to data distribution. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Plasma levels of IL-27 during vivax malaria. Comparative analysis of IL-27 among malaria-naïve control group (n = 15), endemic control group (n = 10), P. vivax group (n = 75) and P. vivax-treated group (n = 10) were performed using Kruskal–Wallis test followed by Dunn Post-hoc. A p value <0.05 (*) was considered significant. The dotted lines (—) represent the detection limit of the assay

Fig. 4

Chemokine plasma levels. Comparative analysis of MCP-1/CCL2 (a), IP-10/CXCL10 (b), and RANTES/CCL5 (c) production among malaria-naïve control group (n = 15), endemic control group (n = 10), P. vivax group (n = 75) and P. vivax-treated group (n = 10) were performed using Kruskal–Wallis test followed by Dunn Post-hoc. The dotted lines (—) represent the detection limit of the assay. Levels of MCP-1/CCL2 (D), IP-10/CXCL10 (e) and RANTES/CCL5 (f) in acute-phase and convalescence period from P. vivax infected patients were determined from P. vivax group (n = 10) and P. vivax-treated group using Paired t-test or Wilcoxon test, according to data distribution. A p value <0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

IL-10 plasma levels and their associations. a Parasitaemia: high vs low number of parasites. Comparison between high (>500 parasites/cu mm, n = 33) and low (≤500 parasites/cu mm, n = 32) number of parasites was performed using Mann–Whitney test. b Number of previous malaria episodes. Comparisons among first malaria (n = 10), ≤5 episodes (n = 24) and >5 episodes (n = 32) were performed using Kruskal–Wallis test followed by Dunn Post-hoc. A p value <0.05 was considered significant. *p < 0.05. The dotted lines (—) represent the detection limit of the assay

Fig. 6

Plasma mediators network. Correlations among 11 mediators during P. vivax infection were plotted in network graphs. Each circle represents a cytokine or chemokine and the connecting lines represent significant correlations between two mediators. Solid and dotted lines, respectively, represent positive and negative correlations. The line thickness represents the significance degree. a P. vivax group (n = 73). b P. vivax sub-group with low parasitaemia (≤500 parasites/cu mm) (n = 34). c P. vivax sub-group with high parasitaemia (> 500 parasites/cu mm) (n = 34). The correlation analyses were evaluated by Spearman correlation test. A p value <0.05 was considered significant