­Association of Macrophage Activation Syndrome, Natural Killer (NK) Cell Function and Genetic Polymorphisms With Dengue Disease Severity

Background: Severe dengue is characterized by shock, organ dysfunction, and coagulopathy, driven by an excessive host inflammatory response. We investigated the role of hyperinflammation/macrophage activation syndrome (MAS) in dengue pathogenesis including MAS biomarkers, Natural Killer (NK) cell function and genetic polymorphisms. Methods: Samples were included from patients recruited into an observational study in Vietnam and divided into three severity grades (plasma leak severity grades 0,1 and 2). MAS biomarkers, were measured at 4 time-points. The frequencies, activation status and cytotoxic potential of NK cells were analysed by flow cytometry. RNA was extracted from sorted CD56+ NK cells and libraries prepared using SMART-Seq Stranded Kit and sequenced using HiSeq3000 (Illumina). Results: 70 hospitalized dengue patients were included. Patients with severe plasma leakage (grade 2 leakage) had higher MAS biomarkers than patients without plasma leakage, including a higher proportion had ferritin levels of >2000 ng/ml (83.3% vs 45.2%) and H scores (148.5 vs.105.5). Activated CD69+ total NK and CD56 dim cells were decreased in patients with severe leakage compared to grade 1. Perforin expression was reduced in NK CD56 dim and CD56 bright cells in grade 2 patients compared to grade 1, and granzyme B MFI levels were reduced in total NK cells and CD56 dim NK cells. RNA sequencing analysis revealed three previously unreported SNPs in the genes involved in NK cell function to be associated with more severe disease; two in the NK cell lectin-like receptor K1 gene (KLRK1) and one in the PRF1 gene, encoding perforin 1. Conclusions: Features of MAS/hyperinflammation are associated with dengue disease severity, including higher MAS biomarkers, impaired NK cell activation/cytotoxicity and specific SNPs in genes involved in NK cell cytolytic function (KLRK1 and PRF-1). Clinical trials of targeted therapeutic immunomodulation for patients with dengue-associated MAS are warranted. Funding Information: Young Investigator Research Grant: NMRC/ OFYIRG17may066 awarded to S Yacoub; Open Fund - Individual Research Grant, National Medical Research Council, Singapore NMRC/OFIRG/0049/2017 awarded to L.Rivino. OM Sessions was supported by the Singapore Ministry of Education (https://www.moe.gov.sg) with a Startup grant. Declaration of Interests: The authors report no COI. Ethics Approval Statement: Ethical approval was obtained from the Oxford Tropical Research Ethics Committee and the Ethics review Committee at NHTD. Written informed consent was obtained from all participants. Experimental work on PBMCs was performed at Duke-NUS Medical School after approval by the Singapore-NUS Institutional Review Board.