Mosquito salivary sialokinin reduces monocyte activation and chikungunya virus-induced inflammation via neurokinin receptors

Abstract

Global warming is expanding mosquito habitats and increasing mosquito-borne diseases. In tropical and sub-tropical regions, chikungunya virus (CHIKV) transmitted by Aedes mosquitoes has become a major concern due to the debilitating chronic joint disease it causes. Mosquito saliva contains bioactive factors that enhance viral infection, with sialokinin identified as a key contributor to vascular leakage and viral spread in mice. Here, we demonstrate that sialokinin binds to neurokinin receptors and restricts the activation of human myeloid cells. Mechanistically, sialokinin facilitates early viral dissemination, as evidenced by increased viral load in the contralateral footpad at 1 day post-infection, and significantly reduces circulating CD169+ monocytes while suppressing IFN-γ-producing T-cell-driven inflammation, as reflected by reduced joint footpad swelling in female CHIKV-infected mice. Clinically, patients with severe CHIKV disease exhibited higher levels of IgG antibodies against sialokinin, which correlated with higher viral loads and systemic inflammatory markers. Our findings highlight the multifaceted role of sialokinin in facilitating early viral dissemination and modulating host immunity during CHIKV infection. Given the growing threat of mosquito-borne diseases in a warming, disease-burdened world, targeting mosquito salivary factors like sialokinin could offer a novel therapeutic strategy to mitigate viral-induced inflammation and improve clinical outcomes.

Fig. 1. Sialokinin modulates monocyte activation via NK receptors and the PI3K/Akt signaling pathway.

a RNA-Seq profiling of primary human monocytes (n = 3) at 24 h post-treatment. Volcano plot indicates differentially expressed genes (DEGs) between sialokinin-treated vs. non-treated monocytes. DEG analysis was performed using edgeR, with multiple testing corrected via the Benjamini–Hochberg FDR method. Significantly upregulated and downregulated genes are shown in red and blue, respectively. b Flow cytometry shows reduced surface expression of Siglec-1/CD169 in sialokinin-treated monocytes compared with untreated monocytes (n = 10 biological replicates) at 24 h post-treatment. Data were analyzed by paired two-tailed t-test. c Pearson correlation matrix for the expressions of 12 significant DEGs from RNA-Seq data (n = 3). d Western blot analysis shows that sialokinin stimulation activated the PI3K-Akt pathway in human monocytes, with increased levels of phosphorylated PI3K (P-PI3K) and Akt (P-Akt) at 20, 40, and 60 min. Pre-treatment with either the NK₁R inhibitor CP-96345 or the NK₂R inhibitor GR-159897 led to inhibition of PI3K and Akt as early as 20 min post-stimulation. Relative expression levels of P-PI3K/PI3K (n = 4 biological replicates) and P-Akt/Akt (n = 5 biological replicates) in monocytes at the 20-min time point were quantified with ImageJ. Levels are expressed as fold change relative to the non-treated controls. Bar graphs represent the mean ± standard deviation (SD). Statistical comparisons between sialokinin-treated sample and untreated control were performed using paired two-tailed t-test. e Schematic representation of CD169 downregulation in response to sialokinin via NK receptor/PI3K/Akt activation. f The monocytes were pre-treated with the NK₁R antagonist (CP-96345; n = 5 biological replicates), NK₂R antagonist (GR-159897; n = 5 biological replicates), both antagonists in combination (n = 4 biological replicates), or the PI3K inhibitor (LY-294002; n = 5 biological replicates) prior to sialokinin stimulation. CD169 levels at 24 h post-treatment were measured by mean fluorescence intensity (MFI) and expressed as fold change relative to non-treated controls. Statistical comparisons were performed using paired two-tailed t-test or Wilcoxon signed-rank test, depending on data distribution.

Fig. 2. Sialokinin reduces activation and CHIKV infection in primary human myeloid cells.

Human monocytes and monocyte-derived macrophages (MDMs) from ten healthy donors were infected with CHIKV in the presence or absence of sialokinin (10 μM). a Activation profiles of monocytes and MDMs were assessed by flow cytometry, focusing on CD169 expression. Sialokinin treatment significantly reduced CD169 expression was lower in both monocytes and MDMs following CHIKV infection (MOI = 10) at 24 (monocytes) or 48 (MDMs) hours post-infection. b CHIKV infection was determined using FACS analysis and qRT-PCR on the viral supernatant. Viral load was quantified by qRT-PCR targeting CHIKV nsP1 viral copies. Data were log₁₀-transformed prior to statistical analysis. CHIKV infectivity toward monocytes and MDMs was reduced in the presence of sialokinin. Statistical analyses were performed using either a paired two-tailed t-test or a Wilcoxon signed-rank test, depending on data distribution.

Fig. 3. Sialokinin reduces circulating CD169⁺ monocytes and joint inflammation in CHIKV-infected animals.

C57BL/6 WT 4-week-old (CHIKV, n = 8; CHIKV + sialokinin, n = 10) mice were infected with 1 × 10⁶pfu of CHIKV subcutaneously at the joint footpad. Sialokinin (1 μg) was administered concurrently with CHIKV in the treatment group. a CD11b⁺ Ly6C⁺ CD169⁺ monocytes in blood of CHIKV-infected mice were monitored daily by flow cytometry. b Representative images at 6 dpi show reduced joint footpad swelling in sialokinin-treated mice. Arrows indicate the area of persistent swelling. Joint swelling was monitored over 14 dpi. c Viremia was assessed from tail vein blood (1 to 10 dpi) via qRT-PCR targeting CHIKV nsP1. Data were collated from two independent experiments and are shown as mean ± SEM, analyzed using two-tailed t-test. d At 1 dpi, tissue viral loads were quantified by qRT-PCR of CHIKV nsP1 viral copies in the contralateral left joint footpad, the right joint footpad and the spleen of CHIKV-infected mice (CHIKV, n = 7; CHIKV + sialokinin, n = 7). Statistical comparisons used unpaired two-tailed t-test or Mann–Whitney U test, depending on data distribution. e At 6 dpi, viral loads in the infected right joint footpad were measured by qRT-PCR of CHIKV nsP1 viral copies (CHIKV, n = 5; CHIKV + sialokinin, n = 5) and analyzed using unpaired two-tailed t-test. f CD4⁺ T cell cytokine profiles (IFN-γ, TNF-α, IL-4, IL-10, and IL-17A) were assessed at 6 dpi in joint footpads of CHIKV-infected (CHIKV, n = 6; CHIKV + sialokinin, n = 7) and mock (n = 6) mice after PMA/ionomycin stimulation. Data are presented as mean ± SD, and analyzed using one-way ANOVA with Tukey’s post-hoc test. g Representative images of ELISpot wells depicting the number of IFN-γ-producing cells in enriched CD4+ T cells from joint footpads at 6 dpi. Quantitative plots show the total numbers of IFN-γ-producing CD4+ T cells per infected joint footpad. Statistical comparison between the two groups was performed using unpaired two-tailed t-test.

Fig. 4. Sialokinin treatment reduces joint footpad inflammation in CHIKV-infected mice.

a C57BL/6 WT 4-week-old mice were infected with 1 × 10⁶ pfu of CHIKV subcutaneously at the joint footpad. Mice were treated with 1 mg/kg of sialokinin or PBS (control) via intraperitoneal injection from days 2 to 7 post-infection (dpi). Created in BioRender. Ng, L. (2025) https://BioRender.com/fj6toz3. b Circulating CD11b⁺Ly6G⁺ neutrophils and CD11b⁺ Ly6C⁺CD169⁺ monocytes were quantified by flow cytometry. Data are presented as mean ± SD (n = 5 per group). Statistical comparisons used unpaired two-tailed t-test. c Joint swelling was monitored to 14 dpi. Viremia was assessed from tail vein blood (1 to 10 dpi) via qRT-PCR targeting CHIKV nsP1. Data are presented as mean ± SD (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired two-tailed t-test. Tissue viral loads were measured at 6 dpi in the right joint footpad (CHIKV, n = 5; CHIKV + sialokinin, n = 5). Statistical comparisons were performed using unpaired two-tailed t-test. d Immune cell subsets in the joint footpad were analyzed at 6 dpi (PBS, n = 7; sialokinin, n = 7; mock, n = 5). Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. e CD4⁺ T cell cytokine profiles (IFN-γ, TNF-α, IL-4, IL-10, and IL-17A) were assessed at 6 dpi in the joint footpads of CHIKV-infected (PBS, n = 7; sialokinin, n = 7) and mock (n = 5) mice after PMA/ionomycin stimulation. Data are presented as mean ± SD and analyzed using one-way ANOVA with Tukey’s post-hoc test. CD4+ T cells isolated from right joint footpads at 6 dpi (n = 5 per group) were stimulated with CHIKV for ELISpot assay. Representative images and quantitative plots show reduced CHIKV-specific IFN-γ-producing CD4+ T cells in the joint footpads of treated mice at 6 dpi. Statistical comparison used unpaired two-tailed t-test. f Cell lysates from the joints of CHIKV-infected (PBS, n = 7; sialokinin, n = 7) and mock (n = 5) mice at 6 dpi were analyzed using a 36-plex microbead-based immunoassay. Immune mediators are grouped based on function. Heatmap colors indicate relative concentration (blue = low, red = high).

Fig. 5. Sialokinin-specific IgG response in CHIKV patient cohort.

a IgG levels against sialokinin were measured via ELISA in acute samples (median 4 days after illness onset) from 30 patients with confirmed CHIKV infection. Data are presented as mean ± SD. The pie chart illustrates the proportion of patients exhibiting different fold increases in sialokinin IgG compared to the healthy pooled sample. b The associations between sialokinin IgG levels and disease severity, viral load, and systemic inflammatory CRP levels in acute CHIKV patient samples. Statistical analyses were conducted using the two-tailed Mann–Whitney U test and Spearman rank correlation analysis. c The correlation between sialokinin IgG levels and the acute systemic cytokine response in CHIKV patients. Immune mediator concentrations were quantified using microbead-based immunoassay. The heatmap displays Spearman’s rank correlation coefficient values for each immune mediator and the sialokinin IgG response. Statistical analyses were performed using two-tailed Spearman rank correlation analysis, with significance indicated by *p < 0.05.