Activation, Impaired Tumor Necrosis Factor-α Production, and Deficiency of Circulating Mucosal-Associated Invariant T Cells in Patients with Scrub Typhus

<div><p>Background</p><p>Mucosal-associated invariant T (MAIT) cells contribute to protection against certain microorganism infections. However, little is known about the role of MAIT cells in <i>Orientia tsutsugamushi</i> infection. Hence, the aims of this study were to examine the level and function of MAIT cells in patients with scrub typhus and to evaluate the clinical relevance of MAIT cell levels.</p><p>Methodology/Principal Findings</p><p>Thirty-eight patients with scrub typhus and 53 health control subjects were enrolled in the study. The patients were further divided into subgroups according to disease severity. MAIT cell level and function in the peripheral blood were measured by flow cytometry. Circulating MAIT cell levels were found to be significantly reduced in scrub typhus patients. MAIT cell deficiency reflects a variety of clinical conditions. In particular, MAT cell levels reflect disease severity. MAIT cells in scrub typhus patients displayed impaired tumor necrosis factor (TNF)-α production, which was restored during the remission phase. In addition, the impaired production of TNF-α by MAIT cells was associated with elevated CD69 expression.</p><p>Conclusions</p><p>This study shows that circulating MAIT cells are activated, numerically deficient, and functionally impaired in TNF-α production in patients with scrub typhus. These abnormalities possibly contribute to immune system dysregulation in scrub typhus infection.</p></div

Regression coefficients of MAIT cell percentages with respect to clinical and laboratory parameters in scrub typhus patients.

<p>Regression coefficients of MAIT cell percentages with respect to clinical and laboratory parameters in scrub typhus patients.</p

Changes in MAIT cell levels and functions in scrub typhus patients.

<p>The percentages of MAIT cells (<u>panel A</u>) in the peripheral blood of 18 scrub typhus patients during active disease and remission were determined by flow cytometry. TNF-α expression (<u>panel B</u>) in the MAIT cell population after stimulation with PMA and IM was determined by intracellular flow cytometry. Data in <u>panel B</u> were obtained from 11 patients with scrub typhus. Symbols represent individual subjects. *p < 0.05 by the Wilcoxon matched-pairs signed rank test.</p

Expression of CD69 and PD-1 and apoptosis of MAIT cells after stimulation with IL-12 and IL-18.

<p>PBMCs (1 × 10⁶/well) were incubated for 24 hours in the presence of IL-12 (50 ng/mL) and IL-18 (50 ng/mL), and then stained with FITC-conjugated anti-CD3, FITC-conjugated annexin V, APC-conjugated anti-TCR Vα7.2, PE-conjugated anti-CD3, PE-conjugated anti-CD69, PE-conjugated anti-PD-1 and PE-Cy5-conjugated anti-CD161 monoclonal antibodies. Percentages of CD69-expressing cells (<u>panel A</u>), annexin V-positive cells (<u>panel C</u>), and PD-1-expressing cells (<u>panel E</u>) among MAIT cells were determined by flow cytometry. Data in <u>panels B, D and F</u> were obtained from 6 HCs. Values are expressed as the mean ± SEM. *p < 0.005 by paired t test.</p

Decreased circulating MAIT cell numbers in the peripheral blood of scrub typhus patients.

<p>Freshly isolated PBMCs from 53 HCs and 38 patients with scrub typhus were stained with APC-Alexa Fluor 750-conjugated anti-CD3, FITC-conjugated anti-TCR γδ, APC-conjugated anti-TCR Vα7.2 and PE-Cy5-conjugated anti-CD161 mAbs, and then analyzed by flow cytometry. Percentages of MAIT cells were calculated using a αβ T cell gate. <u>Panel A</u>: Representative MAIT cell percentages as determined by flow cytometry. <u>Panel B</u>: MAIT cell percentages among peripheral blood αβ T cells. <u>Panel C</u>: Absolute MAIT cell numbers (per microliter of blood). Symbols represent individual subjects and horizontal lines are median values. *p < 0.05, **p < 0.001 by the Mann-Whitney U test.</p

Expression of CD69 and PD-1 and apoptosis of MAIT cells from scrub typhus patients.

<p>Freshly isolated PBMCs were stained with FITC-conjugated anti-CD3, FITC-conjugated annexin V, APC-conjugated anti-TCR Vα7.2, PE-conjugated anti-CD3, PE-conjugated anti-CD69, PE-conjugated anti-PD-1 and PE-Cy5-conjugated anti-CD161 monoclonal antibodies, and then analyzed by flow cytometry. Percentages of CD69-expressing cells (<u>panel A</u>), annexin V-positive cells (<u>panel C</u>) and PD-1-expressing cells (<u>panel E</u>) among MAIT cells were determined by flow cytometry. Data in <u>panels B, D and F</u> were obtained from 14 HCs and 17 patients with scrub typhus. Symbols represent individual subjects and horizontal lines are median values. *p < 0.0001 by the Mann-Whitney U test.</p

Clinical and laboratory characteristics of 38 scrub typhus patients.

<p>Clinical and laboratory characteristics of 38 scrub typhus patients.</p

Expression of IFN-γ, IL-17 and TNF-α in MAIT cells from scrub typhus patients.

<p>Freshly isolated PBMCs (1 × 10⁶/well) were incubated for 4 hours in the presence of PMA (100 ng/ml) and IM (1 μM). Panel A: Representative IFN-γ, IL-17 and TNF-α expression in the MAIT cell population were determined by intracellular flow cytometry after stimulation with PMA and IM. Data in <u>panel B</u> were obtained from 14 HCs and 23 patients with scrub typhus. Symbols represent individual subjects and horizontal lines are median values. *p < 0.05 by the Mann-Whitney U test.</p