Expression of Tim-3 on neutrophils as a novel indicator to assess disease activity and severity in ankylosing spondylitis

Abstract

Objective: To investigate the expression of Tim-3 on neutrophils in ankylosing spondylitis (AS) patients and its correlation with disease activity, severity, and inflammatory markers.

Methods: Sixty-two AS patients from Guangdong Second Provincial General Hospital and 38 healthy controls (HC) were enrolled. Clinical data, physical exams, and laboratory measurements were recorded. Flow cytometry measured Tim-3 and PD-1 expression on neutrophils, real-time PCR quantified mRNA levels and protein expression of Tim-3 was determined by Western blot. We analyzed the correlation between Tim-3 mean fluorescence intensity (MFI) on neutrophils, inflammatory markers, and AS disease activity and severity.

Results: Tim-3 expression on neutrophils was higher in AS patients than in HC, showing a positive correlation with erythrocyte sedimentation rate (ESR), c-reactive protein (CRP), and Ankylosing Spondylitis Disease Activity Score (ASDAS). Active AS patients (ASDAS ≥ 1.3) had increased Tim-3 MFI compared to inactive ones (ASDAS < 1.3). Regular treatment with non-steroidal anti-inflammatory drugs (NSAIDs), biological disease-modifying anti-rheumatic drugs (bDMARDs), and conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) over a month significantly reduced Tim-3 MFI in AS patients.

Conclusion: Elevated Tim-3 expression on neutrophils correlates with increased inflammatory markers and AS activity. Treatment lowered Tim-3 MFI, suggesting its potential as an indicator for assessing AS disease activity and severity and as a feedback mechanism to reduce tissue damage from inflammation.

Keywords: Tim-3; ankylosing spondylitis; disease activity; immunomodulation; neutrophils.

Figure 1

Elevated the expression of Tim-3 on neutrophils in patients with AS. Flow cytometric analysis was used to detect Tim-3 and PD-1 expression in neutrophils (A–D). (A) Shows the representative scatter plots of Tim-3 expression on neutrophils (left), horizontal bars indicated the percentage of Tim-3-expressing neutrophils in patients with AS and HC (right). (B) Shows the representative scatter plots of PD-1 expression on neutrophils (left), horizontal bars indicated the percentage of PD-1-expressing neutrophils in patients with AS and HC (right). (C) The representative plots and the mean fluorescence intensity (MFI) of Tim-3 expression on neutrophils in patients with AS and HC. (D) The representative plots and MFI of PD-1 on neutrophils in patients with AS and HC. (E) Relative mRNA expression and fold increase of Tim-3 and PD-1 in patients with AS and HC were analyzed by quantitative RT-PCR. *P < 0.05 as compared with HC. (F) Western blot analysis of Tim-3 protein expression in neutrophils from healthy volunteer and AS patient.

Figure 2

The MFI of Tim-3 on neutrophils correlated with markers of inflammation. (A) The MFI of Tim-3 of neutrophils in AS patients correlated significantly with ESR (r = 0.333, P = 0.015). (B) The MFI of Tim-3 of neutrophils in AS patients correlated significantly with CRP (r = 0.360, P = 0.008). (C) The MFI of Tim-3 of neutrophils in AS patients was not associated with WBC (r = 0.06, P = 0.630). (D) The MFI of Tim-3 of neutrophils in AS patients was not associated with neutrophil count (r = −0.010, P = 0.939). ^*P < 0.05, ^**P < 0.01.

Figure 3

The level of Tim-3 MFI on neutrophils was associated with disease activity and severity of AS. (A) The MFI of Tim-3 on neutrophils in AS patients was not associated with BASDAI (r = 0.099, P = 0.534). (B) The MFI of Tim-3 on neutrophils in AS patients was not associated with BASFI (r = 0.002, P = 0.990). (C) The MFI of Tim-3 on neutrophils in AS patients correlated significantly with ASDAS(r = 0.309, P = 0.044). (D) The MFI of Tim-3 on neutrophils in AS patients was significantly increased in active patients compared to those with inactive (P =0.000). ^*P < 0.05, ^***P < 0.001.

Figure 4

Tim-3 on neutrophils had a high diagnostic value for AS by ROC curve analysis. ROC curves of ESR, CRP, and Tim-3 for differentiating AS patients with active disease from inactive patients.

Figure 5

The level of Tim-3 MFI on neutrophils was decreased after treatment. (A) The level of Tim-3 MFI on neutrophils was shown in eight AS patients following regular treatment with NSAIDs, bDMARDs and csDMARDs (p = 0.036). (B) The level of CRP was decreased after treatment. The CRP levels in eight AS patients were measured before and after regular treatment with NSAIDs, bDMARDs, and csDMARDs (p = 0.079). (C) The level of ESR was decreased after treatment. The ESR levels in eight AS patients were measured before and after regular treatment with NSAIDs, bDMARDs, and csDMARDs (p = 0.069). ^*P < 0.05.

Figure 6

The MFI of Tim-3 on neutrophils was not associated with alterations in MRI. The correlation coefficient between the level of Tim-3 on neutrophils and MRI score were analyzed. The inflammation of the hip in MRI was assessed using the hip inflammation MRI scoring system (HIMRISS). The inflammation of the SJ in MRI was assessed by the Spondyloarthritis Research Consortium of Canada (SPARCC) MRI index. (A) The MFI of Tim-3 on neutrophils in AS patients was not associated with HIMRISS. (B) The MFI of Tim-3 on neutrophils in AS patients was not associated with SPARCC.