Uric acid is a mediator of the Plasmodium falciparum-induced inflammatory response

Abstract

Background: Malaria triggers a high inflammatory response in the host that mediates most of the associated pathologies and contributes to death. The identification of pro-inflammatory molecules derived from Plasmodium is essential to understand the mechanisms of pathogenesis and to develop targeted interventions. Uric acid derived from hypoxanthine accumulated in infected erythrocytes has been recently proposed as a mediator of inflammation in rodent malaria.

Methods and findings: We found that human erythrocytes infected with Plasmodium falciparum gradually accumulate hypoxanthine in their late stages of development. To analyze the role of hypoxanthine-derived uric acid induced by P. falciparum on the inflammatory cytokine response from human blood mononuclear cells, cultures were treated with allopurinol, to inhibit uric acid formation from hypoxanthine, or with uricase, to degrade uric acid. Both treatments significantly reduce the secretion of TNF, IL-6, IL-1beta and IL-10 from human cells.

Conclusions and significance: Uric acid is a major contributor of the inflammatory response triggered by P. falciparum in human peripheral blood mononuclear cells. Since the inflammatory reaction induced by P. falciparum is considered a major cause of malaria pathogenesis, identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease.

Figure 1. Mature P. falciparum infected erythrocytes accumulate high levels of hypoxanthine.

Hypoxanthine was analyzed in the soluble fraction of lysates of human erythrocytes infected with P. falciparum at different times after infection in a synchronized culture. Lysates of uninfected erythrocytes cultured for the same times were used as controls. Shown are GC - selected reaction monitoring MS ion plots using the m/z 365.2 to m/z 251.2 product ion MS/MS transition. Hypoxanthine accumulated in cultured uninfected erythrocytes (red lines) and P. falciparum infected erythrocytes (black lines) is shown. Infected erythrocytes at 33 h (mature trophozoites, thick lines) and 40 h (schizonts, think lines) were purified from synchronized cultures of 2% parasitemia.

Figure 2. Mature P. falciparum infected erythrocytes induce TNF, IL-6 and IL-1β from PBMCs.

(A–C) PBMCs were incubated with mature P. falciparum infected erythrocytes (squares) or uninfected erythrocytes (circles) at the indicated ratio of erythrocyte to PBMC for 6 h. Data represent the average of triplicated samples with standard deviations. Incubation media were collected and TNF (A), IL-6 (B), or IL-1β (C) concentrations were determined by flow cytometry using cytometric bead array. (D) P. falciparum infected erythrocytes were cultivated alone or in the presence of 2 mM allopurinol. Synchronized cultures were seeded at 0.5% rings and the culture media was changed daily. The percentage of infected erythrocytes was calculated after 0 h (black bars), 24 h (white bars) and 48 h (grey bars) of culture.

Figure 3. P. falciparum-derived uric acid induces TNF, IL-6 and IL-1β release from PBMCs.

PBMCs were incubated with media alone (control), uninfected erythrocytes (RBCs) or P. falciparum-infected erythrocytes (iRBCs) at a ratio of (5∶1; erythrocyte∶PBMC) for 6 h (24 h for IL-10) in the absence (black bars) or presence (white bars) of 2 mM allopurinol (A–D) or 0.1 mg/ml uricase (E–H). Incubation media were collected and TNF (A,E), IL-6 (B,F), IL-1β (C,G) and IL-10 (D,H) concentrations were determined by flow cytometry using cytometric bead array. Data represent the average of triplicated samples with standard deviations. *, indicates significant differences (p<0.05) in cytokine release by PBMCs incubated with iRBCs when compared to IRBCs in the presence of allopurinol or uricase.

Figure 4. Time course of P. falciparum-induced TNF, IL-6 and IL-1β release from PBMCs in the presence of allopurinol.

PBMCs were incubated with mature P. falciparum infected erythrocytes (squares), uninfected erythrocytes (circles) or media alone (diamonds) at a ratio of (5∶1; erythrocyte∶PBMC) for the indicated time points in the presence (white symbols) or absence (black symbols) of 2 mM allopurinol. Incubation media were collected and TNF (A), IL-6 (B), IL-1β (C) or IL-10 (D) concentrations were determined by ELISA. Data represent the average of triplicated samples with standard deviations. *, indicates significant differences (p<0.05) in cytokine release by PBMCs incubated with iRBCs when compared to IRBCs in the presence of allopurinol.