Human blood and ethics approval

Human blood apheresis cones were obtained from 35 healthy adult donors with written informed consent, in accordance with guidelines from the Health Sciences Authority of Singapore (IRB No: 2017/2512). Sampling was approved specifically for the collection of samples from healthy donors. For the CHIKV cohort in Singapore, written informed consent was obtained from all participants. The study was approved by the National Healthcare Group’s domain-specific ethics review board (DSRB No. B/08/026).

Patients and plasma collection

The CHIKV cohort study included 30 chikungunya fever patients admitted to the Communicable Disease Centre at Tan Tock Seng Hospital from 1 August through 23 September 2008. The outbreak occurred in an industrial area where the source population comprised predominantly male workers, with only 4 female individuals among the 30 patients in this study. Participants ranged in age from 23 to 67 years, with a median age of 36.5 years. Plasma specimens were collected on the day of admission to the hospital (acute phase; median, 4 days after illness onset). All patients were confirmed to have CHIK fever by reverse-transcription polymerase chain reaction (RT-PCR). Illness was defined as severe if a patient had either a maximum temperature >38.5 °C, a maximum pulse rate >100 beats/min, or a nadir platelet count 9cells/L. Patients who do not fulfil these criteria were classified as having mild illness. The clinical data, viral load data, and systemic cytokine data used for association and correlation analysis were obtained from published datasets⁴³ deposited by the cohort.

Peptides and antagonists

Aedes aegypti sialokinin I peptides were synthesized by solid-phase synthesis using a CEM Liberty Blue automated microwave peptide synthesizer. The purity of the peptides was checked by HPLC and was > 95%. The peptides were dissolved in phosphate-buffered saline (PBS) and stored at a concentration of 5 mM at −20 °C. CP-96345 (NK₁R antagonist) was purchased from Sigma-Aldrich (Darmstadt, Germany) and GR-159897 (NK₂R antagonist) was from Santa Cruz Biotechnology (Texas, USA). The antagonists were prepared in DMSO and stored as 10 mM (CP-96345) and 50 mM (GR-159897) stock solutions.

Cells and virus stocks

Chinese hamster ovary (CHO) cells expressing three distinct types of human NK receptors (NK₁R, NK₂R, and NK₃R) were a generous gift from Dr Shinya Oishi, Faculty of Pharmaceutical Sciences, Kyoto University, Japan. These stably transfected cells were cultured in 10% FBS/Ham’s F12 media supplemented with hygromycin (500 µg/mL) and penicillin-streptomycin (100 U/mL). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO₂.

Human peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood using Ficoll gradient centrifugation. Monocytes were purified through negative selection using an indirect magnetic labeling system (Monocyte Isolation Kit II; Miltenyi Biotec). Isolated monocytes were incubated in complete Iscove’s modified Dulbecco’s medium (IMDM) (HyClone) supplemented with 10% (vol/vol) heat-inactivated human serum (HS) (Sigma-Aldrich) and 1% (vol/vol) penicillin-streptomycin for 6 days to differentiate into monocyte-derived macrophages (MDMs). Medium was changed on day 3 of differentiation.

LR2006 OPY1 (the CHIKV strain originally isolated from a French patient returning from Réunion Island during the 2006 CHIKF outbreak)⁵⁹ and ZsGreen-tagged LR2006 OPY1 were used in this study. Virus stocks used for ex vivo experiments were produced in Vero-E6 cells. For in vivo experiments, viruses were propagated in C6/36 cells. Titers were determined by standard plaque assays using Vero-E6 cells.

Calcium flux assay

Calcium flux assay was carried out on CHO cells stably transfected with one of the human NK receptors (NK₁R, NK₂R, or NK₃R). CHO cells were seeded at a density of 70,000 cells/well in 100 µL of Ham’s F-12 media in a black 96-well clear-bottom plate (Greiner). After overnight incubation, the media was replaced with Calcium 6 assay dye from Molecular Devices (1:1 dilution with buffer solution of 1× HBSS containing 20 mM HEPES) followed by incubation for 2 h at 37 °C for the absorption of dye into the cytoplasm. The intracellular calcium ion flux analysis was performed at different concentrations of sialokinin (tenfold dilution) using a FLIPR Tetra instrument (Molecular Devices). Fluorescence signal was recorded at excitation and emission wavelengths of 485 nm and 535 nm, respectively. The change in fluorescence is presented as the percent change with respect to baseline fluorescence. EC₅₀ values were determined from dose-response curves plotted using Prism 5 (GraphPad).

For the dose-response study in the presence of a competitive inhibitor, competitive inhibition was performed using NK₁R antagonist CP-96345 and NK₂R antagonist GR-159897. Following incubation of the cells for 30 min with different concentrations of the antagonist (50, 500, or 5000 nM), the calcium flux assay was performed using sialokinin I [at 8 different concentrations from 0.1 pM to 1 µM (NK₁R and NK₂R) or 1 pM to 10 µM (NK₃R) depending on the receptor type)]. Data were obtained from the average of three repetitions performed in triplicate for each concentration.

Sialokinin, NK₁R/NK₂R antagonist and PI3K inhibitor treatment

Freshly purified human monocytes and six-day-differentiated MDMs (1 × 0⁶ cells/well) were incubated with or without sialokinin peptides in complete IMDM supplemented with 10% (vol/vol) HS (Sigma-Aldrich) and 1% (vol/vol) penicillin-streptomycin. Untreated monocytes and MDMs served as no-treatment controls. NK₁R antagonist CP-96345, NK₂R antagonist GR-159897 and PI3K inhibitor LY-294002 were used to confirm the specific effects of sialokinin. The monocytes and MDMs were pre-treated with CP-96345 or GR-159897 or PI3K inhibitor LY-294002 for 30 min before the addition of sialokinin.

Ex vivo virus infection

CHIKV infections in human whole blood were performed at a multiplicity of infection (MOI) of 10 with CHIKV strain LR2006 OPY1. The whole blood was incubated at 37 °C until harvesting at 24 h post-infection (hpi). The cells were stained for the detection of surface markers via flow cytometry. For CHIKV infection in primary monocytes and MDMs, cells were treated with sialokinin and infected with CHIKV strain LR2006 OPY1 tagged with ZsGreen at MOI = 10. In parallel, cells were also infected with CHIKV in the absence of sialokinin, and these infected but untreated cells served as controls. Cells were then harvested at 24 hpi (monocytes) or 48 hpi (MDMs) for flow cytometry analysis, and viral load was quantified from 140 μL of culture supernatant.

Total RNA extraction and Nanopore cDNA library preparation

Total RNA was isolated from primary human monocytes using a RNeasy Micro Kit (Qiagen), following the manufacturer’s instructions. The RNA samples were further treated with DNase I (Qiagen) and cleaned up using a RNeasy MinElute Cleanup Kit (Qiagen). cDNA libraries were generated from a total of 50 ng RNA according to the Oxford Nanopore Technologies (Oxford Nanopore Technologies Ltd, Oxford, UK) protocol “cDNA-PCR Barcoding Sequencing” with 18 cycles of PCR (4.5 min elongation time). ONT adapters were ligated to the amplified cDNA library.

Nanopore sequencing and differential gene expression analysis

Nanopore libraries were sequenced using a MinION Mk1B with R9.4.1 (PCS108 LR and RNA001 LR) or R9.5 flow cells (TELO LR). The data were generated using MinKNOW 1.11.5 and base called with Guppy GPU base caller. Debarcoding was performed using the SQK-PBK004 Barcoding Kit (Oxford Nanopore Technologies). STAR aligner⁶⁰ was used to map paired-end raw reads to human genome build GRCh38 and counted for genes using featureCounts⁶¹ based on GENCODE v31 gene annotation⁶². Log₂-transformed counts per million mapped reads (log₂CPM), and log₂-transformed reads per kilobase per million mapped reads (log₂RPKM) were computed using the edgeR Bioconductor package⁶³. Data are accessible at NCBI’s Gene Expression Omnibus (GEO) database (GSE291153). Genes with log₂CPM inter-quartile range (IQR) of less than 0.5 across all samples were filtered out from subsequent differentially expressed gene (DEG) analysis. DEG analysis was done using edgeR⁶³.

Flow cytometry for ex vivo experiments

Following harvesting, human monocytes and MDMs were specifically stained for the surface markers: CD45, CD14, CCR2, HLA-DR, CD16, CD11b, and CD169 (for monocytes); or CD45, CD14, HLA-DR, CD64, CD86, CD163, CD206, and CD169 (for MDMs). Dead cells were excluded by staining with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). The stained cells were subsequently incubated with FACS lysing solution (BD Biosciences) to lyse the red blood cells. Data were acquired on a Fortessa flow cytometer (BD Biosciences) with BD FACSDiva software version 9.0. Data analysis was performed using FlowJo version 9.3.2 (Tree Star, Inc.).

Mice

Female wild-type (WT) C57BL/6J mice, aged 4 weeks, were bred and maintained under specific pathogen-free conditions at the Biological Resource Center (BRC) of the Agency for Science, Technology, and Research, Singapore (A*STAR). All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC 241883) of A*STAR, and in compliance with the guidelines of the Agri-Food and Veterinary Authority (AVA) and the National Advisory Committee for Laboratory Animal Research of Singapore (NACLAR). All animal experiments were conducted in an Animal Biosafety Level 2 (ABSL-2) facility.

Tissue total RNA isolation

For total RNA extraction from tissues, mice were anesthetized with ketamine (150 mg/kg) and xylazine (10 mg/kg), followed by perfusion with PBS. Joint footpads (including the ankle joint and footpad) and spleens were collected and stored in TRIzol reagent (Invitrogen) at −80 °C. Tissues were homogenized using a rotor-stator homogenizer (Xiril Dispomix) at 4000 rpm for 15 s. The homogenized samples were mixed with 230 μL of chloroform and centrifuged at 12,000 rpm for 10 min at 4 °C. The aqueous phase was collected and used for total RNA isolation with the RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions. The concentration of extracted total RNA was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific).

Gene expression study

Total RNA was extracted from harvested cells using the RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using the QuantiNova SYBR Green PCR Kit (Qiagen) in a 12.5 μL reaction volume, according to the manufacturer’s recommendations. All reactions were run on a Bio-Rad CFX384 Real-Time PCR System. Thermal cycling conditions were as follows: 50 °C for 10 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 10 s. Fold changes in gene expression were calculated relative to the uninfected control group and presented using the 2^−ΔΔCT method.

Viral RNA extraction and viral load analysis

Viral RNA was extracted from the culture supernatant using a QIAamp Viral RNA Mini Kit (Qiagen), following the manufacturer’s instructions. TaqMan probe quantitative real-time PCR (qRT-PCR)⁶⁴ was performed to quantify CHIKV NS1 RNA using a QuantiTect Probe RT-PCR kit (Qiagen).

Western blotting

To detect the phosphorylation status of PI3K/Akt kinases following sialokinin stimulation, cells were lysed with lysis buffer (Cell Signaling Technology) containing 2 mM PMSF. Lysates were separated by SDS-PAGE and subjected to western blotting using the following antibodies: P-PI3K (p85; Tyr458/p55; Tyr199, Cell Signaling Technology, 1:2000), PI3K (p85, Cell Signaling Technology, 1:2000), P-Akt (Ser473, Cell Signaling Technology, 1:2000), and Akt (Cell Signaling Technology, 1:2000). Signal intensities were visualized using a ChemiDoc imaging system (Bio-Rad) and quantified using Image Lab software version 6.0.1 (Bio-Rad).

In vivo virus infection

Mice were subcutaneously inoculated with 1 × 10⁶ pfu of CHIKV in 30 µL PBS at the ventral side of the right hind footpad. Disease progression in terms of joint swelling and viremia was monitored daily until 10 dpi, as previously described⁶⁵. Joint swelling was determined by measuring the height and width of the footpad with an electronic calliper (Mitutoyo) and calculated as the relative increase compared to pre-infection (0 dpi) with the following formula: [(x–day 0)/day 0], where x is the footpad size measurement for a given dpi. To track viremia, 10 µL of blood from the tail vein were collected and mixed in 10 µL of citrate-phosphate-dextrose solution (Sigma-Aldrich) with 120 µL PBS.

Mouse spleen and joint cell isolation

Mice were infected with CHIKV and euthanized by cervical dislocation at 6 dpi. The footpads and ankles were removed, de-skinned, and placed immediately in a 4 mL digestion medium containing dispase (2 U/mL; Invitrogen), collagenase IV (20 μg/mL; Sigma-Aldrich), and DNase I mix (50 μg/mL; Roche Applied Science) in complete Roswell Park Memorial Institute (RPMI) medium. Spleens were dissociated in RPMI 1640 medium containing 10% FBS (complete RPMI). The cells were then isolated as previously described⁴⁰.

Phenotyping of leukocytes

Isolated joint cells were first blocked with 1% mouse/rat serum (Sigma-Aldrich) blocking buffer for 10 min. The cells were then stained for 20 min with the following antibodies: BUV395-conjugated anti-mouse CD45 (clone 30-F11; BD Biosciences), Pacific Blue-conjugated anti-mouse CD4 (clone RM4-5; BioLegend), CF594-conjugated anti-mouse CD8 (clone 53-6.7; BD Biosciences), PE-Cy7-conjugated anti-mouse CD3 (clone 17A2; BioLegend), APC-Cy7-conjugated anti-mouse Ly6C (clone HK1.4; BioLegend), Alexa Fluor 700-conjugated anti-mouse MHC-II (clone M5/114.15.2; BioLegend), PerCP-Cy5.5-conjugated anti-mouse LFA-1 (clone H155-78; BioLegend), BV650-conjugated anti-mouse CD11b (clone M1/70; BioLegend), BV605-conjugated anti-mouse CD11c (clone N418; BioLegend), CF594-conjugated anti-mouse Ly6G (clone 1A8; BD Biosciences), eFluor450-conjugated anti-mouse B220 (clone RA3-6B2; eBioscience), PE-conjugated anti-mouse MerTK (clone DS5MMER; eBioscience), APC-conjugated anti-mouse CD64 (clone X54-5/7.1; BioLegend), biotin-conjugated anti-mouse NK1.1 (clone PK136; eBioscience), and BUV737 streptavidin (BD Biosciences). Samples were acquired on a LSR II flow cytometer (BD Biosciences) with FACSDiva software and analysed using FlowJo software.

T cell IFN-γ ELISpot assay

The ELISpot assay was performed according to the manufacturer’s instructions (Mabtech) using the ELISpot Plus: Mouse IFN‑γ (ALP) kit. Mice were sacrificed at 6 days post-infection (dpi), and joint footpad tissues and spleens were harvested and processed as described above. Ex vivo CD4⁺ T cell enrichment was carried out using the Mouse CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec), following the manufacturer’s protocol. CHIKV-specific T cell stimulation was conducted in complete RPMI medium supplemented with 30 U/ml IL-2 and 1.5 × 10⁶ CHIKV virions per well, using 5000 isolated T cells. Complete RPMI containing 30 U/ml IL-2 was used as a negative control. For all wells containing joint footpad cells, 1.5 × 10⁵ splenocytes from non-infected mice were added as antigen-presenting cells (APCs). Plates were incubated at 37 °C with 5% CO₂ for 15 h. After incubation, cells were removed, and wells were developed according to the manufacturer’s instructions. Spot counting and imaging were performed using the MABTECH IRIS system.

Cytokine profiling in the joints

Mice were anesthetized by intraperitoneal injection of 150 mg of ketamine and 10 mg/kg xylazine cocktail, followed by intracardial perfusion with PBS at the indicated time points. Subsequently, the footpads were collected and homogenized in 1.5 mL RIPA buffer (50 mM Tris-HCl pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA) with 1× protease inhibitors (Roche) using a gentleMACS M tube with a gentleMACS Dissociator (Miltenyi Biotec). Cell lysates were then sonicated at 70% intensity for 15 s (Branson Ultrasonics Sonifier™ S-450), and the supernatants were collected for cytokine/chemokine quantification as described below. Data are expressed as pg/mL in footpad lysate.

Multiplex microbead immunoassay for cytokine quantification

Cytokine and chemokine concentrations in mice serum and footpad lysates were quantified simultaneously using a multiplex microbead-based immunoassay, the Cytokine & Chemokine 36-Plex Mouse ProcartaPlex Panel 1 A (EPX360-26092-901; Thermo Scientific), following the manufacturer’s protocol. Data were acquired using a Luminex FlexMap 3D instrument (Millipore) and analysed with Bio-Plex Manager™ 6.0 software (Bio-Rad) based on standard curves plotted through a five-parameter logistic curve setting. The cytokines and chemokines assayed included: IFN-γ, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF-α, GM-CSF, IL-18, IL-10, IL-17A, IL-22, IL-23, IL-27, IL-9, GRO-α, IP-10, MCP-1, MCP-3, MIP-1α, MIP-1β, MIP-2, RANTES, eotaxin, IFN-α, IL-15/IL-15R, IL-28, IL-31, IL-1α, IL-3, G-CSF, LIF, ENA-78/CXCL5, and M-CSF.

Evaluation of the level of antibodies against sialokinin

To test the level of antibodies against sialokinin, 96-well ELISA plates (Nunc Maxisorp) were coated with 50 μL/well of sialokinin peptides (2 μg/mL) prepared in PBS and were incubated overnight at 4 °C. Plates were then blocked for 1.5 h with 5% milk powder in 1× PBST (blocking buffer) at 37 °C and incubated with 100 μL/well of a 1/100 dilution of human sera in sample diluent (2.5% milk powder in 1× PBST) at 37 °C for 1 h. The plates were then incubated with 100 μL/well of horseradish peroxidase (HRP)-labeled goat anti-human IgG (1:1000) antibodies (Jackson ImmunoResearch) at 37 °C for 30 min. Readout was detected with TMB substrate solution (Sigma-Aldrich) and terminated with sulfuric acid (Sigma-Aldrich). Absorbance was measured at 450 nm in a microplate autoreader (Tecan). Peptides are considered positive if absorbance values are at least twofold higher than the mean values of pooled healthy donor samples.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 9.0.0; GraphPad Software). Parametric or non-parametric tests were used to compare between groups and controls, as appropriate. p-values less than 0.05 were considered statistically significant. Plots were generated using GraphPad Prism version 9.0.0.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.