Adenosine pathway regulates inflammation during Plasmodium vivax infection

Abstract

Background: Plasmodium spp. infection triggers the production of inflammatory cytokines that are essential for parasite control, and conversely responsible for symptoms of malaria. Monocytes play a role in host defense against Plasmodium vivax infection and represent the main source of inflammatory cytokines and reactive oxygen species. The anti-inflammatory cytokine IL-10 is a key regulator preventing exacerbated inflammatory responses. Studies suggested that different clinical presentations of malaria are strongly associated with an imbalance in the production of inflammatory and anti-inflammatory cytokines.

Methods: A convenience sampling of peripheral blood mononuclear cells from Plasmodium vivax-infected patients and healthy donors were tested for the characterization of cytokine and adenosine production and the expression of ectonucleotidases and purinergic receptors.

Results: Here we show that despite a strong inflammatory response, monocytes also bear a modulatory role during malaria. High levels of IL-10 are produced during P. vivax infection and its production can be triggered in monocytes by P. vivax-infected reticulocytes. Monocytes express high levels of ectonucleotidases, indicating their important role in extracellular ATP modulation and consequently in adenosine production. Plasmatic levels of adenosine are not altered in patients experiencing acute malaria; however, their monocyte subsets displayed an increased expression of P1 purinergic receptors. In addition, adenosine decreases Tumor Necrosis Factor production by monocytes, which was partially abolished with the blockage of the A_2a receptor.

Conclusion: Monocytes have a dual role, attempting to control both the P. vivax infection and the inflammatory response. Purinergic receptor modulators emerge as an untapped approach to ameliorate clinical malaria.

Keywords: Plasmodium vivax; adenosine; ectonucleotidases; malaria; regulation.

Figure 1

IL-10 and ectonucleotidases are upregulated in patients with acute P. vivax infection. (A) Plasma levels of IL-10 were measured using Th1/Th2/Th17 CBA kit in patients during acute malaria (Pv) (n = 23) and healthy donors (HD) (n = 13) (left panel). Intracellular IL-10 production by monocytes with (Pv-RET) and without (-) P. vivax-infected reticulocytes was assessed by flow cytometry (right panel) (n = 14) (right panel). (B) CD39, CD73 and their co-expression were determined in monocytes from Pv (n = 13) and HD (n = 9) using conventional flow cytometry in classical (green, left panel), inflammatory (red, middle panel) and patrolling monocytes (blue, right panel). Scatter plots with bars representing mean ± SEM. *p ≤ 0.05 **p ≤ 0.01 *** or ****p ≤ 0.001.

Figure 2

Ectonucleotidases CD39 and CD73 colocalize within monocyte subsets. CD39, CD73 and their co-expression were determined by imaging flow cytometry on monocyte subsets ex vivo (n = 13) (A) and after culture with (+) or without (-) P. vivax-infected reticulocytes (C) (n = 5). **p ≤ 0.01 ***p ≤ 0.001. Scatter plots with bars representing mean ± SEM. Green, red and blue symbols represent classical (CD14 ⁺ CD16^-), inflammatory (CD14⁺CD16⁺) and patrolling (CD14^loCD16⁺) monocytes (A, C), respectively. Representative images of the expression of CD39 and CD73 (B) and P. vivax-infected reticulocytes and CD39 and CD73 (D) on monocyte subsets.

Figure 3

Plasmodium vivax infection alters the expression of adenosine and purinergic receptors in monocyte subsets. Gene expression was performed in monocyte subsets from HD (n = 8) and 8 Pv (n = 8) by NanoString analysis. (A) The proportion of subjects displaying high mRNA counts for each parameter was assessed using the overall median value as the cut-off, and radar graphs were assembled. Genes evaluated were ADA (purple); ADORA1 (pink); ADORA2a (yellow); ADORA2b (orange); ADORA3 (dark pink); CD210 (blue); P2X1 (green); P2X3 (dark orange); P2X4 (dark green); P2X7 (red); P2Y1 (dark blue); P2Y11 (black); P2Y2 (brown); and P2Y4 (gray). (B) Comparison of mRNA counts of adenosine receptors from monocyte subsets from Pv (circles) and HD (triangles). Scatter plots with bars representing mean ± SEM. *p ≤ 0.05 **p ≤ 0.01. (C) Venn diagrams of genes expressed in 50% or more subjects were built to identify common gene expression between each group. Gene expression among monocyte subsets from Pv (top, left panel) and HD (bottom, left panel). Genes in bold were commonly expressed by patients and HD in the same subset. Gene expression in each monocyte subset between Pv (circle) and HD (triangle) (right panel). Genes in bold were commonly expressed by Pv and HD or intersection group among the monocyte subsets analyzed. Genes in italic were exclusively expressed in a specific monocyte subset by Pv, HD or intersection group.

Figure 4

Adenosine decreases TNF production by monocytes in an adenosine 2a receptor depend-manner. (A) Adenosine levels were measured in the plasma of Pv (n = 30) and HD (n = 11) by UHPLC. (B) Frequencies (top panels) and mean fluorescence intensity (MFI) of TNF-producing monocytes were measured after culture with medium alone (triangles), LPS (circles), and LPS + adenosine (squares) and in the absence (open symbols) or presence of adenosine 2a receptor blocker (ZM241385, solid symbols) or adenosine 2b receptor blocker (MRS1754, gradient symbols) (n = 6–7). Scatter plots with bars representing mean ± SEM. *p ≤ 0.05 **p ≤ 0.01 ****p ≤ 0.001.