Aedes aegypti saliva modulates inflammasome activation and facilitates flavivirus infection in vitro

Abstract

Mosquito borne flaviviruses such as dengue and Zika represent a major public health problem due to globalization and propagation of susceptible vectors worldwide. Vertebrate host responses to dengue and Zika infections include the processing and release of pro-inflammatory cytokines through the activation of inflammasomes, resulting in disease severity and fatality. Mosquito saliva can facilitate pathogen infection by downregulating the host's immune response. However, the role of mosquito saliva in modulating host innate immune responses remains largely unknown. Here, we show that mosquito salivary gland extract (SGE) inhibits dengue and Zika virus-induced inflammasome activation by reducing NLRP3 expression, Caspase-1 activation, and 1L-1β secretion in cultured human and mice macrophages. As a result, we observe that SGE inhibits virus detection in the early phase of infection. This study provides important insights into how mosquito saliva modulates host innate immunity during viral infection.

Keywords: Biological sciences; Natural sciences; Virology.

Graphical abstract

Figure 1

Aedes aegypti salivary gland extract (SGE) inhibits DENV or ZIKV-induced inflammasome activation in human macrophages PMA-primed THP-1 cells were infected with DENV (5 MOI), DENV (5 MOI) + Ae. aegypti SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + Ae. aegypti SGE (2 pairs), Ae. aegypti SGE (2 pairs) or left untreated for 36 h. (A) Secretion of mature IL-1β and cleaved caspase-1 in the supernatants and cell lysate expression of NLRP3 were assessed by Western blot. (B–F) (B) Active caspase-1 was analyzed by caspase-1 Glo assay. IL-1β (C), IL-6 (D), IL-8 (E), and IL-18 (F) secretions were measured by ELISA. One-way ANOVA with Tukey’s multiple comparisons were performed. ∗∗∗∗p < 0.0001., ∗∗∗p < 0.001., ∗∗p < 0.01. Molecular weights are indicated in kDa. Data from three independent experiments performed in triplicate are plotted.

Figure 2

Aedes aegypti salivary gland extract (SGE) inhibits DENV or ZIKV-induced inflammasome activation in murine macrophages RAW-ASC cells (ASC-expressing murine macrophages) were primed with Pam3CSK4 and infected with DENV (5 MOI), DENV (5 MOI) + SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + SGE (2 pairs), SGE (2 pairs), or were left untreated for 36 h. As a positive control, cells were treated with LPS (100 ng/mL) for 6 h followed by Nigericin (10 μM) for 24 h. (A) Active caspase-1 was analyzed by caspase-1 Glo assay. (B) IL-1β secretions were measured by ELISA. Data from three independent experiments performed in triplicate are plotted. One-way ANOVA with Tukey’s multiple comparisons were performed. ∗∗∗∗p < 0.0001.

Figure 3

Aedes aegypti salivary gland extract (SGE) inhibits DENV or ZIKV-triggered mitochondrial ROS in human macrophages PMA primed THP-1 cells were infected with DENV (5 MOI), DENV (5 MOI) + SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + SGE (2 pairs), SGE (2 pairs) or were left untreated for 36 h in 96 well black wall/clear bottom plate. Cells were treated with rotenone (positive control, 10 μM) for 3 h or Mitotempo (negative control, 500 μM) for 60 min at 37°C. Cells were then incubated with MitoROS 580 at 37°C for 1 h. The fluorescence signal was monitored at Ex/Em = 485/535 nm with bottom read mode using a microplate reader. Data from three independent experiments performed in triplicate are plotted. One-way ANOVA with Tukey’s multiple comparisons were performed. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Figure 4

NLRP3 plays a crucial role in Ae aegypti salivary gland extract (SGE)-mediated reduction of inflammasome activation (A–D) PMA-primed THP-1 and NLRP3 KO THP-1 cells were infected with DENV (5 MOI), DENV (5 MOI) + SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + SGE (2 pairs), SGE (2 pairs), or were left untreated for 36 h. Subsequently, we assessed the activation of caspase-1 using the caspase-1 Glo assay (A and B). Secretion of mature IL-1β was assessed by ELISA (C and D). Data from three independent experiments performed in triplicate are plotted. One-way ANOVA with Tukey’s multiple comparisons were performed. ∗∗∗∗p < 0.0001., ∗∗p < 0.01., ns, not significant.

Figure 5

Aedes aegypti salivary gland extract (SGE) rescues cell death without effecting DENV or ZIKV replication (A) PMA-primed THP-1 cells were infected with DENV (5 MOI), DENV (5 MOI) + SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + SGE (2 pairs), SGE (2 pairs), or left untreated for 36 h in 96-well black wall/clear bottom plate. As a positive control for inflammasome activation, cells were treated with LPS (100 ng/mL) for 6 h followed by ATP (5 mM) for 1 h. As a positive control for cell death, cells were treated with Triton X-100 for 10 min. All treatments were performed in a medium containing SYTOX Green Nucleic Acid Stain (50 nM). The fluorescence signal was monitored at Ex/Em = 480/530 nm with bottom read mode using a microplate reader. The signals were normalized with controls (wells without cells) and compared with the values of cells treated with Triton X-100, which was considered as 100% cell death. (B) PMA-primed THP-1 cells were infected with DENV (5 MOI), DENV (5 MOI) + SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + SGE (2 pairs), SGE (2 pairs), or were left untreated for 24 h in a 6 well plate. Infected cell supernatants were analyzed by plaque assay to quantify infectious viral particles. Data from three independent experiments performed in triplicate are plotted. One-way ANOVA with Tukey’s multiple comparisons were performed. ∗∗∗∗p < 0.0001., ns - not significant.

Figure 6

Aedes aegypti salivary gland extract (SGE) downregulates mRNA expression of innate immune and antiviral genes during DENV or ZIKV infection PMA-primed THP-1 cells were infected with DENV (5 MOI), DENV (5 MOI) + SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + SGE (2 pairs), SGE (2 pairs), or were left untreated for 24 h. Gene expression was analyzed using RT-qPCR. (A and B) Heatmap showing the expression patterns of commonly affected genes by DENV and ZIKV infection. Pink and purple represent the upregulated and down regulated genes, respectively. Data from three independent experiments performed in triplicate were normalized and plotted.

Figure 7

Aedes aegypti salivary gland extract (SGE) inhibits early innate immune sensing of DENV and ZIKV infection PMA-primed THP-1 cells were infected with DENV (5 MOI), DENV (5 MOI) + SGE (2 pairs), ZIKV (5 MOI), ZIKV (5 MOI) + SGE (2 pairs), SGE (2 pairs), or were left untreated for 3 h. Protein expression of total NF-kB-p65, Syk, Stat 1, and IRF3 were assessed along with the phosphorylated forms P-NF-kB-p65, P-NF-kB-p50, P-Syk, P -Stat-1, Stat-1, Phospho (P)- IRF3 in cell lysates by Western blot. GAPDH expression was used as a protein loading control.

Figure 8

Schematic representation of Ae aegypti salivary gland extract (SGE) effects on innate immune responses during DENV and ZIKV infection In macrophages, DENV and ZIKV infection induce inflammasome activation along with the expression of genes that modulate the antiviral response. However, during a blood meal, mosquito injects salivary proteins and viral particles into the vertebrate host’s skin. Mosquito saliva from Ae. aegypti interferes with innate immune responses and changes the host antiviral response against DENV and ZIKV. As the viral particles enter into the host skin, mosquito saliva downregulates SYK, NF-kB, IRF3, and STAT-1 protein expression in the early stages of viral infection in macrophages (3 h). Subsequently, it proceeds to inhibit the activation of the inflammasome, effectively suppressing NLRP3 protein expression, IL-1β secretion, caspase-1 activation, and mitigating mitochondrial ROS generation. These actions collectively contribute to a reduction in cell death triggered by DENV and ZIKV infection.