Increased level and interferon-γ production of circulating natural killer cells in patients with scrub typhus

Abstract

Background: Natural killer (NK) cells are essential immune cells against several pathogens. Not much is known regarding the roll of NK cells in Orientia tsutsugamushi infection. Thus, this study aims to determine the level, function, and clinical relevance of NK cells in patients with scrub typhus.

Methodology/principal findings: This study enrolled fifty-six scrub typhus patients and 56 health controls (HCs). The patients were divided into subgroups according to their disease severity. A flow cytometry measured NK cell level and function in peripheral blood. Circulating NK cell levels and CD69 expressions were significantly increased in scrub typhus patients. Increased NK cell levels reflected disease severity. In scrub typhus patients, tests showed their NK cells produced higher amounts of interferon (IFN)-γ after stimulation with interleukin (IL)-12 and IL-18 relative to those of HCs. Meanwhile, between scrub typhus patients and HCs, the cytotoxicity and degranulation of NK cells against K562 were comparable. CD69 expressions were recovered to the normal levels in the remission phase.

Conclusions: This study shows that circulating NK cells are activated and numerically increased, and they produced more IFN-γ in scrub typhus patients.

Fig 1. Increased circulating NK cell numbers in the peripheral blood of scrub typhus patients.

Freshly isolated PBMCs from 56 HCs and 56 patients with scrub typhus were stained with FITC-conjugated anti-CD56, PE-conjugated anti-CD3, and PerCP-conjugated anti-CD45 mAbs, and then analyzed by flow cytometry. Percentages of NK cells were calculated using a CD45/SSC gate. Panel A: Representative NK cell percentages as determined by flow cytometry. Panel B: NK cell percentages among peripheral blood lymphocytes. Panel C: Absolute NK cell numbers (per microliter of blood). Symbols represent individual subjects and horizontal lines indicate median values. Panel D: The ratios of CD3-CD56^bright/CD3-CD56^dim NK cell percentages among peripheral blood lymphocytes. Data are shown as box plots. Each box represents the 25th and 75th percentiles. The line inside the boxes represent the median. Whiskers represent the 10th and 90th percentiles. *p < 0.05, **p < 0.0005 by the ANCOVA test.

Fig 2. Expression of CD69 in circulating NK cells obtained from scrub typhus patients.

Freshly isolated PBMCs from 18 HCs and 31 patients with scrub typhus were stained with FITC-conjugated anti-CD56, PE-conjugated anti-CD69, APC-conjugated anti-CD3, and PerCP-conjugated anti-CD45 mAbs, and then analyzed by flow cytometry. Panel A: Representative CD69-expressing NK cell percentages as determined by flow cytometry. Panel B: Percentages of CD69-expressing cells among NK cells. Panel C: Absolute CD69-expressing NK cell numbers (per microliter of blood). Symbols represent individual subjects and horizontal lines indicate median values. *p < 0.005, **p < 0.0001 by the ANCOVA test.

Fig 3. Plasma levels of IFN-γ, IL-12, IL-17, IL-18 and TNF-α in patients with scrub typhus.

Plasma samples of the patients were collected before specific treatment on admission. Plasma levels of IFN-γ (panel A), IL-17 (panel B), IL-12 (panel C), IL-18 (panel D), and TNF-α (panel E) were determined by Luminex and ELISA. Data were obtained from 15 age- and sex-matched HCs and 25 patients with scrub typhus. Symbols represent individual subjects and horizontal lines indicate median values. *p < 0.005, **p < 0.001, ***p < 0.0001 by the Mann-Whitney U test.

Fig 4. Cytokine-mediated CD69 and IFN-γ expression in NK cells.

Freshly isolated PBMCs (1 × 10⁶/well) were incubated for 24 hours in the presence of IL-12 (50 ng/mL) and IL-18 (50 ng/mL), or PBS as a control. Expression levels of CD69 and IFN-γ in NK cells were determined by flow cytometry after stimulation with IL-12 and IL-18. Panel A: Representative CD69 expression in NK cells. Black line and dotted line represent CD69 expression in NK cells in the presence of IL-12 and IL-18 or PBS, respectively. Shaded regions indicate isotype-matched control mAbs. Panel B: Representative IFN-γ percentages in CD69+ and CD69- NK cell subsets. Data in panels A and B were obtained from 3 HCs. Panel C: Representative IFN-γ percentages in NK cells as determined by intracellular flow cytometry. Data in panel D were obtained from 10 HCs and 5 patients with scrub typhus. Values are expressed as the mean ± SEM. *p < 0.05 by unpaired t-test.

Fig 5. Cytotoxicity of NK cells in scrub typhus patients.

Panel A and B: NK cytotoxicity. Freshly isolated PBMCs (panel A) or purified NK cells (panel B) from 30 HCs and 20 patients with scrub typhus were cocultured with K562 cells for 4 hours, and then stained with FITC-conjugated anti-CD45 mAb and PI. Cytotoxicity was determined as the percentage of apoptotic K562 cells by flow cytometry. Panel C: CD107a expression in NK cells. PBMCs obtained from 15 HCs and 15 patients with scrub typhus patients were stained with FITC-conjugated anti-CD107a or isotype control mAbs and then incubated with K562 cells. After 1 hour, monensin was added and the cells were incubated for an additional 4 hours. The cells were then stained with PerCP-conjugated anti-CD3 and PE-conjugated anti-CD56 mAbs, and then CD107a expression in NK cells was analyzed by flow cytometry. Symbols represent individual subjects and horizontal lines indicate median values. ns = not significant by the ANCOVA test. Panel D: NK cell cytotoxicity of purified CD69- and CD69+ NK cell subsets in scrub typhus patients. Results are representative of 3 independent experiments.

Fig 6. Changes in NK cell levels and CD 69 expression in scrub typhus patients.

The percentages of NK cells (panel A) and CD69-expressing NK cells (panel B) in the peripheral blood of 11 scrub typhus patients during active disease and remission were determined by flow cytometry. Symbols represent individual subjects. *p < 0.005 by the Wilcoxon matched-pairs signed rank test.