Neutrophil Extracellular Traps Effectively Control Acute Chikungunya Virus Infection

Abstract

The Chikungunya virus (CHIKV) is a re-emerging arbovirus, in which its infection causes a febrile illness also commonly associated with severe joint pain and myalgia. Although the immune response to CHIKV has been studied, a better understanding of the virus-host interaction mechanisms may lead to more effective therapeutic interventions. In this context, neutrophil extracellular traps (NETs) have been described as a key mediator involved in the control of many pathogens, including several bacteria and viruses, but no reports of this important protective mechanism were documented during CHIKV infection. Here we demonstrate that the experimental infection of mouse-isolated neutrophils with CHIKV resulted in NETosis (NETs release) through a mechanism dependent on TLR7 activation and reactive oxygen species generation. In vitro, mouse-isolated neutrophils stimulated with phorbol 12-myristate 13-acetate release NETs that once incubated with CHIKV, resulting in further virus capture and neutralization. In vivo, NETs inhibition by the treatment of the mice with DNase resulted in the enhanced susceptibility of IFNAR^-/- mice to CHIKV experimental acute infection. Lastly, by accessing the levels of MPO-DNA complex on the acutely CHIKV-infected patients, we found a correlation between the levels of NETs and the viral load in the blood, suggesting that NETs are also released in natural human infection cases. Altogether our findings characterize NETosis as a contributing natural process to control CHIKV acute infection, presenting an antiviral effect that helps to control systemic virus levels.

Keywords: Chikungunya; NETs; innate response; neutrophils; viral infection.

Figure 1

Isolated neutrophils release NET following CHIKV, but not ZIKV or DENV infection. (A) Quantification of NETs by MPO–DNA PicoGreen in the supernatant of isolated mouse neutrophils incubated with CHIKV at different MOIs for 1, 2, 4, and 8 h. (B) Representative image of immunofluorescence of mouse neutrophils incubated with CHIKV for 4 h. Bars = 50 μm. Magnification ×40. Cells were stained with DAPI (blue), anti-Ly6G (red), and anti-H3 citrulline (green). (C) Quantification of NET by MPO–DNA PicoGreen in the supernatant of mouse neutrophils incubated with PMA (100 nM), CHIKV, ZIKV, or DENV (MOI = 5) for 4 h. (D) Representative immunofluorescence image of mouse neutrophils incubated with CHIKV, ZIKV, or DENV (MOI = 5) for 4 h. Bars = 50 μm. Magnification ×40. Cells were stained with DAPI (blue), anti-Ly6G (red), and anti-H3 citrulline (green). (E) Percentage of live neutrophils incubated with MOCK control, CHIKV, or ZIKV for 4 h. Data are presented as mean ± SD (n = 3 per group); *p < 0.05 and **p < 0.01, assessed by two-way (A) or one-way ANOVA (C,E) with Bonferroni's comparisons test. Representative results of two experiments performed independently.

Figure 2

CHIKV triggers TLR7- and ROS-dependent NETosis. (A) Production of ROS by mouse neutrophils incubated with CHIKV (MOI = 5) and treated or not with apocynin (300 μm) for 30 min prior to CHIKV incubation. (B) Representative image of an immunofluorescence assay of mouse neutrophils incubated with CHIKV (MOI = 5) and treated or not with apocynin (300 μM) for 30 min prior to CHIKV stimulation. Bars = 50 μm. Magnification ×40. Samples were stained with DAPI (blue) and anti-H3 citrulline (green). (C) Quantification of NETs by MPO–DNA PicoGreen in the supernatant of TLR3^−/−, TLR3/7/9^−/−, TLR9^−/−, and IFNAR^−/− mouse-isolated neutrophils after incubation with CHIKV (MOI = 5 for 4 h). (D) RT-qPCR for CHIKV from neutrophils incubated with virus stocks after 1, 2, and 4 h. Data are presented as mean ± SD (n = 4 per group); *p < 0.05, ***p < 0.001, and ****p < 0.0001, assessed by one-way (A,D) or two-way ANOVA (C) with Bonferroni's comparisons test. Representative results of two experiments performed independently.

Figure 3

NETs release contributes to virus neutralization. (A) Quantification of NETs by MPO–DNA PicoGreen in the supernatant of mouse neutrophils incubated with medium, CHIKV, PMA (100 nM), and DNase (5 mg/ml) for 4 h. (B) Viral load quantification by PFU assay of CHIKV virus stocks incubated with medium, NETs, or NET predigested with DNase for 2 h. (C) Representative immunofluorescence image of mouse neutrophils incubated with CHIKV (MOI = 5 for 4 h). Bars = 50 μm. Magnification ×40. Cells were stained with DAPI (blue), anti-H3 citrulline (green), and anti-CHIKV (red). Data are presented as mean ± SD (n = 4 per group); *p < 0.05, **p < 0.01, and ****p < 0.0001, assessed by one-way ANOVA with Bonferroni's comparisons test. Representative results of two experiments performed independently.

Figure 4

DNase treatment increases viral load in vivo. (A) Survival of IFNAR^−/− mice infected i.p. with 30 PFU of CHIKV and treated with saline or DNase (10 mg/kg, s.c., every 12 h). (B) Quantification of NETs by MPO–DNA PicoGreen and (C) viral load in the plasma of infected IFNAR^−/− treated with saline or DNase. Data are mean ± SD (n = 10 per group); *p < 0.05, ***p < 0.001, and ****p < 0.0001, assessed by Mantel–Cox log–rank test (A) or two-way ANOVA with Bonferroni's comparisons test (B,C). Representative results of three experiments performed independently.

Figure 5

CHIKV induces NETosis in human neutrophils, which is correlated with viral load in clinical samples. (A) Quantification of NETs by MPO–DNA PicoGreen of human neutrophils incubated with CHIKV (MOI = 5 for 4 h). (B) Representative immunofluorescence image of human neutrophils incubated with CHIKV (MOI 5 for 4 h). Bar = 50 μm. Magnification ×20. Samples were stained with DAPI (blue) and anti-H3 citrulline (green). (C) Viral load in the serum of acute CHIKV patients assessed by rRT-PCR. (D) Spearman's correlation between NETs concentration and viral load in the serum of acute CHIKV patients. Data are presented as mean ± SD (n = 3 per group); ***p < 0.001 and ****p < 0.0001, assessed by one-way ANOVA with Bonferroni's comparisons test (A), Mann–Whitney test (C), and Spearman's correlation (D).