Neutrophil paralysis in Plasmodium vivax malaria

Abstract

Background: The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.

Materials and methods: Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30-45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.

Principal findings: Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).

Conclusion: Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.

Figure 1. High levels IL-1β IL-6, IL-8 and IL-10 in plasma from patients infected with P. vivax.

The cytokines IL-8 (CXCL8), IL-1β, IL-6, IL-10, TNF-α were measured in the plasma of P. vivax-infected subjects (n = 26), before (closed circles) and 30–45 days after treatment (open circles). Dotted lines represent medians of given measurements from healthy donors (HD; n = 13). Levels of cytokines were measured employing the Cytometric Bead Array (CBA). Significant differences are indicated with p-values using Wilcoxon signed rank test when the data failed the normality test.

Figure 2. Systemic activation of monocytes and neutrophils during P. vivax malaria.

Mean fluorescence intensity (MFI) of HLA-DR, TLR2 and TLR4 was evaluated on monocytes (A) and CD62L, CD88, TLR2 and TLR4 on neutrophils (B) in whole blood from P. vivax-infected subjects (n = 12), before (closed circles) and 30–45 days after treatment (open circles). Representative density plots showing the gate strategy for monocytes CD14+ (A) and neutrophils CD66b+CD16+ (B) are shown. Significant differences are indicated with p-values using Wilcoxon signed rank test when the data failed the normality test.

Figure 3. TLR agonists induce production of high IL-1β, IL-6 and TNF-α levels by monocytes from P. vivax-infected subjects.

Purified monocytes (A) or neutrophils (B) from P. vivax-infected subjects before (closed circles; n = 13) and 30–45 days after treatment (open circles; n = 13) were cultured for 48 hours in the absence or presence of LPS or Pam. Levels of IL-1β, IL-6, IL-10, TNF-α, and IL-8 (CXCL8) were measured in supernatant of monocyte (A) and neutrophil (B) cultures. Levels of cytokines were measured employing the Cytometric Bead Array (CBA). Significant differences are indicated p-values using paired t test or Wilcoxon signed rank test when a normality test failed.

Figure 4. Neutrophils from P. vivax-infected patients produce high levels of superoxide and display enhanced phagocytic function.

Neutrophils were isolated from P. vivax-infected patients (closed circles; n = 15) or healthy donors (open circles; n = 15), and the frequencies of neutrophils reducing NBT (left panel) as well as cell containing zymosan (right panel) were quantified. Significant differences are indicated with p-values using unpaired t test or Mann-Whitney test when a normality test failed.

Figure 5. Malaria impairs neutrophils response to CXCR1 and CXCR2 ligand.

Neutrophils were isolated from P. vivax-infected patients (closed circles; n = 15) or healthy donors (open circles; n = 15), and chemotaxis towards IL-8 (CXCL8) and CCL2 was assessed (A). MFI of CXCR1, CXCR2 and CCR2 on neutrophils were evaluated by flow cytometry and representative histograms of CXCR2 expression are shown (B). CXCR2 message was measured by qPCR (C). Significant differences are indicated with p-values using unpaired t test or Mann-Whitney test when a normality test failed.

Figure 6. GRK2 expression is enhanced in neutrophils during acute malaria.

Neutrophils isolated from P. vivax-infected patients (closed circles; n = 11) or healthy donors (closed circles; n = 12) were stained for GRK2 and mean fluorescence intensity (MFI) of GRK2 was quantified (A). Representative fluorescence microscopy illustrating GRK2 expression in neutrophils from a healthy donor and a P. vivax-infected patient (B). Significant difference is indicated with p-values using unpaired t test.