Mucosal-associated invariant T cell is a potential marker to distinguish fibromyalgia syndrome from arthritis

Abstract

Background: Fibromyalgia (FM) is defined as a widely distributed pain. While many rheumatologists and pain physicians have considered it to be a pain disorder, psychiatry, psychology, and general medicine have deemed it to be a syndrome (FMS) or psychosomatic disorder. The lack of concrete structural and/or pathological evidence has made patients suffer prejudice that FMS is a medically unexplained symptom, implying inauthenticity. Furthermore, FMS often exhibits comorbidity with rheumatoid arthritis (RA) or spondyloarthritis (SpA), both of which show similar indications. In this study, disease specific biomarkers were sought in blood samples from patients to facilitate objective diagnoses of FMS, and distinguish it from RA and SpA.

Methods: Peripheral blood mononuclear cells (PBMCs) from patients and healthy donors (HD) were subjected to multicolor flow cytometric analysis. The percentage of mucosal-associated invariant T (MAIT) cells in PBMCs and the mean fluorescent intensity (MFI) of cell surface antigen expression in MAIT cells were analyzed.

Results: There was a decrease in the MAIT cell population in FMS, RA, and SpA compared with HD. Among the cell surface antigens in MAIT cells, three chemokine receptors, CCR4, CCR7, and CXCR1, a natural killer (NK) receptor, NKp80, a signaling lymphocyte associated molecule (SLAM) family, CD150, a degrunulation marker, CD107a, and a coreceptor, CD8β emerged as potential biomarkers for FMS to distinguish from HD. Additionally, a memory marker, CD44 and an inflammatory chemokine receptor, CXCR1 appeared possible markers for RA, while a homeostatic chemokine receptor, CXCR4 deserved for SpA to differentiate from FMS. Furthermore, the drug treatment interruption resulted in alternation of the expression of CCR4, CCR5, CXCR4, CD27, CD28, inducible costimulatory molecule (ICOS), CD127 (IL-7 receptor α), CD94, NKp80, an activation marker, CD69, an integrin family member, CD49d, and a dipeptidase, CD26, in FMS.

Conclusions: Combined with the currently available diagnostic procedures and criteria, analysis of MAIT cells offers a more objective standard for the diagnosis of FMS, RA, and SpA, which exhibit multifaceted and confusingly similar clinical manifestations.

Fig 1. MAIT cells in HD, FMS, RA, and SpA.

A, Representative FACS profile of MAIT cells and NKG2D expression in total, CD8⁺, and DN MAIT cells in PBMC from a FMS patient. The number in the figure shows the percentage of the populations. MAIT cells are defined as Vα7.2⁺CD161^high within CD3⁺ cells. B, The frequency of total MAIT cells in HD (n = 16), FMS (n = 26), RA (n = 21), and SpA (n = 36, missing one sample). The percentage of MAIT cells (Vα7.2⁺CD161^high) within total T cells (CD3⁺) is shown. C, The frequency of CD8⁺ MAIT cells in HD, FMS, RA, and SpA. The percentage of CD8⁺ MAIT cells (Vα7.2⁺CD161^highCD8⁺) within total T cells (CD3⁺) is shown. D, The frequency of DN MAIT cells in HD, FMS, RA, and SpA. The percentage of DN MAIT cells (Vα7.2⁺CD161^highDN) within total T cells (CD3⁺) is shown. E, The frequency of CD4⁺ MAIT cells in HD, FMS, RA, and SpA. The percentage of CD4⁺MAIT cells (Vα7.2⁺CD161^highCD4⁺) within total T cells (CD3⁺) is shown. F, The percentage of CD8⁺ MAIT cells (CD8⁺Vα7.2⁺CD161^high) among total MAIT cells (Vα7.2⁺CD161^high) in HD, FMS, RA, and SpA. G, The percentage of DN MAIT cells (CD8^-CD4^-Vα7.2⁺CD161^high) among total MAIT cells (Vα7.2⁺CD161^high) in HD, FMS, RA, and SpA. H, The percentage of CD4⁺ MAIT cells (CD4⁺Vα7.2⁺CD161^high) among total MAIT cells (Vα7.2⁺CD161^high) in HD, FMS, RA, and SpA. B-H. All data are presented as median. Horizontal line: Median; boxes: 25th percentile and 75th percentile; whiskers: Minimum and Maximum. Asterisk shows the group-pair exhibiting significance. *: P< 0.05, **: P < 0.01 (P value adjusted with the Dunn's multicomponent test after the Kruskal-Wallis test).

Fig 2. Comparison of the cell surface antigen expression level in MAIT cells between HD and FMS.

A, Chemokine receptor expression in total, CD8⁺, and DN MAIT cells. B, Co-stimulatory molecule expression in total, CD8⁺, and DN MAIT cells. C, Cytokine receptor expression in total, CD8⁺, and DN MAIT cells. D, SLAM family, memory, and activation marker expression in total, CD8⁺, and DN MAIT cells. E, NK receptor expression in total, CD8⁺, and DN MAIT cells. F, CD95 (Fas) expression in total, CD8⁺, and DN MAIT cells. G, Integrin family expression in total, CD8⁺, and DN MAIT cells. H, Miscellaneous molecule expression in total, CD8⁺, and DN MAIT cells. A-H. MFI is shown with median. The dotted line indicates MFI for the isotype control. Horizontal line: Median; boxes: 25th percentile and 75th percentile; whiskers: Minimum and Maximum. Asterisk shows the group-pair exhibiting significance. *: P< 0.05, **: P < 0.01, ***: P<0.001 (the nonparametric Mann-Whitney U test)

Fig 3. Potential biomarkers distinguishing HD, FMS, RA and SpA.

MFI is shown with median for the indicated cell surface antigen. The dotted line indicates MFI for the isotype control. Horizontal line: Median; boxes: 25th percentile and 75th percentile; whiskers: Minimum and Maximum. The number in figure shows a P value after the Kruskal-Wallis test. Asterisk shows the group-pairs exhibiting significance. *: P< 0.05, **: P < 0.01, ***:P<0.001 (P value adjusted with the Dunn's multicomponent test). total: total MAIT cells; CD8⁺; CD8⁺ MAIT cells; DN: DN MAIT cells.

Fig 4. Biochemical and physical parameters in FMS, RA, and SpA.

A: Serum CRP concentrations in FMS, RA, and SpA (left panel).: Serum MMP-3 concentrations in FMS, RA, and SpA (right panel). B: PVAS in FMS, RA, and SpA (left panel).: FVAS in FMS, RA, and SpA (right panel). A-B. Horizontal line: Median; boxes: 25th percentile and 75th percentile; whiskers: Minimum and Maximum. Asterisk shows the group-pairs exhibiting significance. *: P< 0.05, **: P < 0.01, ***: P<0.001 (P value adjusted after the Kruskal-Wallis test with the Dunn's multicomponent test). C: Correlation between PVAS/FVAS and MAIT cell percentage (% of Vα7.2⁺CD161^high cells among CD3⁺ cells) in a cohort of 26 FMS (left panels), of 21 RA (middle panels), and of 36 SpA (right panels) patients. The correlation was analyzed with the Spearman rank correlation test. *: P< 0.05, r: correlation coefficient.

Fig 5. Effect of the daily drug treatment on MAIT cell frequency in FMS.

The percentage of total, CD8⁺, DN, and CD4⁺ MAIT cells (Vα7.2⁺CD161^high) within total T cells (CD3⁺) from the same individuals (n = 9) before and after the drug treatment interruption is shown. The statistical significance and P value were with the Wilcoxon matched-pairs signed rank test. Asterisk shows significance. *: P< 0.05

Fig 6. Effect of the daily drug treatment on MAIT cell surface antigens in FMS.

MFI of the indicated antigen in total, CD8⁺, and DN MAIT cells from the same individual (n = 9) before and after the drug treatment interruption is shown. The statistical significance was assessed with the Wilcoxon matched-pairs signed rank test. Asterisk shows significance. *: P< 0.05, **: P < 0.01