Hyperinflammatory Syndrome, Natural Killer Cell Function, and Genetic Polymorphisms in the Pathogenesis of Severe Dengue

Abstract

Background: Severe dengue, characterized by shock and organ dysfunction, is driven by an excessive host immune response. We investigated the role of hyperinflammation in dengue pathogenesis.

Methods: Patients recruited into an observational study were divided into 3 plasma leak severity grades. Hyperinflammatory biomarkers were measured at 4 time points. Frequencies, activation, and cytotoxic potential of natural killer (NK) cells were analyzed by flow cytometry. RNA was extracted from sorted CD56+ NK cells and libraries were prepared using SMART-Seq and sequenced using HiSeq3000 (Illumina).

Results: Sixty-nine patients were included (grade 0, 42 patients; grade 1, 19 patients; grade 2, 8 patients). Patients with grade 2 leakage had higher biomarkers than grade 0, including higher peak ferritin levels (83.3% vs 45.2%) and H-scores (median, 148.5 vs 105.5). NK cells from grade 2 patients exhibited decreased expression of perforin and granzyme B and activation markers. RNA sequencing revealed 3 single-nucleotide polymorphisms in NK cell functional genes associated with more severe leakage-NK cell lectin-like receptor K1 gene (KLRK1) and perforin 1 (PRF1).

Conclusions: Features of hyperinflammation are associated with dengue severity, including higher biomarkers, impaired NK cell function, and polymorphisms in NK cell cytolytic function genes (KLRK1 and PRF1). Trials of immunomodulatory therapy in these patients is now warranted.

Keywords: dengue; hyperinflammation; immunomodulation; macrophage activation syndrome; single-nucleotide polymorphisms.

Figure 1.

Kinetics of biomarkers over illness course, by disease severity. The middle line in each box represents the median. The upper and lower edges of each box represent the interquartile range. The points are the actual values of the biomarker levels and are colored by leakage grade. The y-axis is transformed using log-transformation. Abbreviations: IL, interleukin; sCD25, soluble CD25.

Figure 2.

Natural killer (NK) cell phenotype. A, Identification of total CD56⁺, CD56^dim, and CD56^bright NK cells based on CD56 and CD16 expression shown for a representative patient. Left plot is gated on live, CD14^–CD3^– cells; right plot is gated on total CD56⁺ NK cells. B, Plots showing granzyme B and perforin (left) or CD69 (right) expression in 3 representative patients with grade 0, 1, and 2. C–E, Granzyme B expression depicted as mean fluorescence intensity (MFI) in CD56⁺ (C), CD56^dim (D), and CD56^bright NK cells (E) from grade 0, 1, and 2 patients. F–H, Perforin expression depicted as MFI in CD56⁺ (F), CD56^dim (G), and CD56^bright NK cells (H). I–K, Percentages of CD69⁺ cells were expressed in CD56⁺ (I), CD56^dim (J), and CD56^bright NK cells (K). L–N, Live CD14^–CD3^– cells were concatenated after downsampling and analyzed simultaneously by t-distributed stochastic neighbor embedding (tSNE) in all 50 patients (L); levels of expression of granzyme B, perforin, CD69, CD16, and CD56 are shown in N from blue to orange (low to high, respectively). tSNE maps from patients with grade 0, 1, and 2 are shown separately (left to right, respectively). A cluster of NK cells with high levels of expression of granzyme B and perforin is highlighted with a black line. All analysis included in this figure were performed at days 5–8 from illness onset (grade 0, 5.68 ± 1.05 days; grade 1, 5.33 ± 0.72 days; grade 2, 6.14 ± 1.34 days) for 50 patients (grade 0, n = 28; grade 1, n = 15; grade 2, n = 7). Statistics were calculated by Kruskal–Wallis test followed by Dunn multiple comparison test. *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant.

Figure 3.

Detection of novel single-nucleotide polymorphisms (SNPs) from sorted natural killer (NK) cells in genes known to be involved in NK cell cytotoxic pathway. Presence of SNPs in the cohort is shown by clinical grade: grade 0 (blue), grade 1 (orange), and grade 2 (gray). Asterisks indicate positions that varied significantly between clinical grades.