Thromboangiitis obliterans (TAO), also known as Buerger's disease, is a progressive, non-atherosclerotic, and thrombotic vascular disease of small to medium size arteries of the upper and lower limbs that mostly affects young or middle-aged males [1, 2]. Recurrent acute and chronic inflammation, as well as coagulopathy, trigger irritating ulcers, phalangeal pain, claudication, cold sensitivity or Raynaud's phenomenon, skin discoloration, and gangrene in the extremities [3]. The most potent risk factor for TAO is smoking or using any type of tobacco, and other risk factors include male sex, rickettsia infection, south Asian or Middle Eastern descent, age between 20-45 years, and medical history of Raynaud's disease or autoimmune disease [4].

The precise mechanisms underlying TAO disease remain unclear. Some HLA genotypes (HLA-A9, HLA-B5, and HLA-DRB1), autoantibodies against vascular endothelium and tobacco antigens, anti-elastin and anti-cardiolipin autoantibodies, increased expression of cell adhesion molecules like intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin are involved in the progressive inflammatory response and development of TAO disease [5, 6]. Uncontrolled inflammation induces the secretion of different cytokines and chemokines, which in turn participate in lymphocyte apoptosis, the persistence of circulating immune complexes, endothelial damage, platelet dysfunction, and thereby increasing the thrombotic events and different vascular diseases like TAO, atherosclerosis and autoimmune vasculitis [7, 8].

Previous studies found increased levels of circulating inflammatory, type 1 helper (Th1) cell, and type 17 helper (Th17) cell cytokine profiles in patients with TAO disease [7]. Also, data have shown elevated serum levels of TNF-α in TAO patients, which may be significantly involved in the progression of TAO from the moderate to the severe form of the disease (9). Considering the presence of inflammatory cytokines and mediators in the serum or plasma of TAO patients, the disease is known as systemic inflammation that affects treatment strategies [7, 10, 11].

In addition, further investigations into the role of various cytokines in the pathogenesis of TAO disease can help manage the patients better and clarify the precise immunological mechanisms underlying TAO and other immune system-related vascular diseases. In this study, we evaluated the serum levels of IL-1β, IL-18, IL-6, IL-12, IFN-γ, IL-17, and IL-10 in the TAO patients with amputation, patients with high stenosis atherosclerosis in comparison to non-smoker and smoker healthy controls. Additionally, we investigated the correlation between levels of mentioned cytokines and clinicopathological characteristics in all studied groups.

Subjects

Informed consent was obtained from the participants prior to the study. This study was approved by Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran. The code of ethical approval for this project was IR.SUMS.REC.1402.214.

This case-control study included 20 non-atherosclerotic, non-diabetic, smoker male TAO patients with amputation (aged 40.10 ± 1.30 years) under clinical follow-up. The TAO diagnosis was based on the collaborating specialists following inclusion criteria: ischemic limb disease, onset of symptoms before 50 years of age, infrapopliteal arterial occlusive disease, clinical symptoms of distal venous insufficiency, ischemic involvement of the nerves with pain, absence of atherosclerosis in CT angiography, absence of emboli in CT angiography, lack of calcification in arterial walls, a history of tobacco abuse, arterial wall thickness, high level of collateral vessels under the knee, and corkscrew collateral appearance on angiography. In addition, 20 non-smoker non-diabetic male subjects with 50% or greater stenosis in at least one of the main coronary arteries (aged 43.50 ± 2.16 years) according to angiography criteria [12], 20 non-smoker, non-diabetic healthy male (aged 42.55 ± 1.56 years) and 20 smoker non-diabetic healthy male (aged 44.20 ± 1.43 years) subjects were participated in this study. All TAO and high stenosis patients were selected from individuals referred to hospitals affiliated to Shiraz University of Medical Sciences and demographic characteristics, clinical, and laboratory data were collected during admission. The exclusion criteria were autoimmune diseases, other vasculitis, diabetes, kidney diseases, acute infection, and lack of consent.

Serum Separation

Five milliliters of anticoagulant-free peripheral blood samples were collected from patients and controls. The serum was then separated through density centrifugation (1500 rpm, 5 minutes). Serum samples were finally frozen and stored in a -70 oC freezer until analysis.

Cytokine Analysis

Quantitative determinations of IL-1β, IL-18, IL-6, IL-12, IFN-γ, IL-17, and IL-10 were performed on serum samples by multiplex immunoassay method using fluorescence labelled cytokine panel (LEGEND plex, Bio Legend, San Diego, CA) according to the manufacturer’s instructions. For the detection of multiple cytokines, a monoclonal specific antibody for each cytokine was conjugated to a particular set of beads with known internal fluorescence. Multiple cytokines antibody-coated beads were mixed together to allow the cytokines to be measured simultaneously. For analysis of Th1-type and inflammatory cytokines, the sum of cytokine concentrations was allocated to each subset as following: Th1-type: IL-12 + IFN-γ; and inflammatory cytokines: IL-6+ IL-18+ IL-1β, respectively.

All statistical analyses were performed using SPSS version 22 and Graphpad Prism version 8 software, and data were expressed as mean ± SEM. Data Normality was assessed by Kolmogorov-Smirnov test. Mann-Whitney U test was used to evaluate the differences between two groups. Kruskal-Wallis test was used for comparison of variables between more than two groups and Dunn’s test was applied for p-value adjustment in multiple comparisons. Spearman’s rho method was used to evaluate potential correlation between variables. P values less than 0.05 were considered statistically significant. The following symbols were applied to indicate statistically significant findings: *P < 0.05, **P < 0.01***P < 0.001, and ****P < 0.0001.

Demographic, Clinical and Laboratory Characteristics of the Patients and Controls

Table 1: Demographic, clinical and laboratory characteristics of the patients and controls.

Table 1 summarizes the demographic, clinical and laboratory characteristics of patients and controls. The serum level of TG (P = 0.0018), cholesterol (P = 0.0015), LDL (P < 0.0001), PT (P = 0.008), and platelet count (P = 0.0001) were significantly higher in patients with high stenosis compared to other groups. As well, the serum level of HDL (P = 0.009), ESR (P <0.0001), CRP (P = <0.0001), neutrophil count (P = 0.0001) and monocyte count (P = 0.0001) were higher in TAO patients with amputation compared to other groups. Additionally, there were no significant differences in BMI (P = 0.60) and age (P = 0.29) between patients and controls.

Comparison of Inflammatory Parameters between Patients and Controls

Laboratory findings revealed that the mean (± SEM) MLR for TAO patients with amputation was significantly higher than patients with the high stenosis (1.41 ± 0.14 vs.0.85 ± 0.11, P = 0.001), non-smoker (1.41 ± 0.14 vs.0.22 ± 0.06, P < 0.0001), and smoker controls 1.41 ± 0.14vs. 1.32 ± 0.32, P = 0.04, Fig. 1A). Moreover, the mean (± SEM) MLR was lower in the patients with high stenosis than smoker controls (P = 0.03, Fig. 1A). Smoker controls also exhibited significantly elevated MLR compared to non-smokers (P < 0.0001, Fig. 1A).

The mean (± SEM) PLR was higher in TAO patients with amputation than that for the non-smoker controls (305.50 ± 27.96 vs.172 ± 18.75, P < 0.0001, Fig. 1B). Conversely, the mean PLR was lower in TAO patients with amputation than patients with high stenosis and smoker controls (305.50 ± 27.96 vs. 608.80 ± 72.35, P = 0.01 and 305.50 ± 27.96 vs. 562 ± 154.60, P < 0.0001, respectively, Fig. 1B). In addition, higher mean PLR has been reported in high stenosis patients as compared with non-smoker and smoker controls (P < 0.0001 and P = 0.03, respectively, Fig. 1B). Similar to MLR, smoker controls showed higher PLR compared to non-smoker controls (P < 0.0001, Fig. 1B).

Also, the mean (± SEM) PNR was significantly lower among TAO patients with amputation as compared with high stenosis patients and both non-smoker and smoker control groups (27.14 ± 0.98 vs. 66.62 ± 2.33, P < 0.0001, 27.14 ± 0.98 vs. 58.72 ± 1.13, P < 0.0001and 27.14 ± 0.98 vs. 50.47 ± 1.65, P = P < 0.0001, respectively, Fig. 1C). The mean PNR was also higher in high stenosis patients than non-smoker and smoker controls (P < 0.0001, and P < 0.0001respectively, Fig. 1C). Similarly, the mean PNR was significantly higher in smoker controls compared to non-smoker (P = 0.0003, Fig. 1C).

The mean (± SEM) NLR in TAO patients with amputation was higher than non-smoker and smoker controls (11.55 ± 1.16 vs. 3.50 ± 0.41, P < 0.0001 and 11.55 ± 1.16 vs. 9.90 ± 2.92., P = 0.01, respectively, Fig. 1D). In addition, higher mean NLR has been reported in high stenosis patients as compared with non-smoker (P < 0.0001, Fig. 1D). Additionally, the mean NLR was higher in smoker controls compared to non-smoker controls (P < 0.0001, Fig. 1D).

Figure 1: The comparison of (A) MLR, (B) PLR, (C) PNR and (D) NLR between patients and controls. TAO, Thromboangiitis obliterans; MLR, monocyte to lymphocyte ratio; PLR, platelet to lymphocyte ratio; PNR, platelet to neutrophil ratio; and NLR, neutrophil to lymphocyte ratio; data are presented as mean ± SEM and analyzed by Kruskal-Wallis test followed by Dunn’s test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Comparison of Serum Cytokines between Controls and Patients

IL-6: As shown in Fig. 2A, the mean (± SEM) serum level of IL-6 was increased in TAO patients with amputation (12166 ± 296.80 pg/mL) as compared with high stenosis patients (3976 ± 147.10 pg/mL; P < 0.0001), smoker controls (368.40 ± 13.96 pg/mL; P < 0.0001) and non-smoker subjects (2.67 ± 0.32 pg/mL; P < 0.0001). In addition, the IL-6 level was higher in high stenosis patients than smoker and non-smoker controls (P < 0.0001 and P < 0.0001, respectively; Fig. 2A). The concentration of IL-6 was higher in smoker controls as compared with non-smoker subjects (P < 0.0001; Fig. 2A).

IFN-γ: As illustrated in Fig. 2B, the mean (± SEM) serum level of IFN-γ was significantly higher in TAO patients with amputation (27460 ± 8627 pg/mL) as compared with high stenosis patients (4196 ± 111.30pg/mL; P < 0.0001), smoker controls (1240 ± 40.16pg/mL; P < 0.0001) and non-smoker subjects (6.01 ± 0.44pg/mL; P < 0.0001). Moreover, the IFN-γ level was higher in high stenosis patients than smoker and non-smoker controls (P < 0.0001 and P < 0.0001, respectively; Fig. 2B). The concentration of IFN-γ was higher in smoker controls as compared with non-smoker subjects (P < 0.0001; Fig. 2B).

IL-1β: Our findings also indicated the mean (± SEM) serum level of IL-1β was significantly higher in TAO patients with amputation (20611 ± 461.50 pg/mL) as compared with high stenosis patients (5846 ± 167.30 pg/mL; P < 0.0001), smoker controls (638.40 ± 20.93 pg/mL; P < 0.0001), and non-smoker subjects (10.97 ± 0.27 pg/mL; P < 0.0001, Fig. 2C). In addition, the IL-1β level was higher in high stenosis patients than smoker and non-smoker controls (P < 0.0001 and P < 0.0001, respectively; Fig. 2C). The concentration of IL-1β was higher in smoker controls as compared with non-smoker subjects (P < 0.0001; Fig. 2C).

IL-12: As illustrated in Fig. 2D, the mean (± SEM) serum level of IL-12 was significantly higher in TAO patients with amputation (16516 ± 4471 pg/mL) as compared with high stenosis patients (4851 ± 140.60 pg/mL; P < 0.0001), smoker controls (664.80 ± 15.16 pg/mL; P < 0.0001) and non-smoker subjects (17.51 ± 0.74 pg/mL; P < 0.0001). Moreover, the IL-12 level was higher in high stenosis patients than smoker and non-smoker controls (P < 0.0001 and P < 0.0001, respectively; Fig. 2D). The concentration of IL-12 was higher in smoker controls as compared with non-smoker subjects (P < 0.0001; Fig. 2D).

IL-10: As shown in Fig. 2E, the mean (± SEM) serum level of IL-10 was decreased in TAO patients with amputation (3.09 ± 0.22 pg/mL) as compared with high stenosis patients (16.71 ± 0.33 pg/mL; P < 0.0001), smoker controls (36.55 ± 1.47 pg/mL; P < 0.0001) and non-smoker subjects (57.73 ± 1.83 pg/mL; P < 0.0001). In addition, the IL-10 level was lower in high stenosis patients than smoker and non-smoker controls (P < 0.0001 and P < 0.0001, respectively; Fig. 2E). The concentration of IL-10 was lower in smoker controls as compared with non-smoker subjects (P < 0.0001; Fig. 2E).

IL-18: According to our results, the mean (± SEM) serum level of IL-18 was significantly increased in TAO patients with amputation (15166 ± 5071 pg/mL) as compared with high stenosis patients, smoker controls and non-smoker subjects (2146 ± 88.67 pg/mL, P < 0.0001; 364.80 ± 13.35 pg/mL; P < 0.0001; 60.11 ± 1.88 pg/mL, P < 0.0001, Fig. 2F). In addition, the IL-18 level was higher in high stenosis patients than smoker and non-smoker controls (P < 0.0001 and P < 0.0001, respectively; Fig. 2F). The concentration of IL-18 was higher in smoker controls as compared with non-smoker subjects (P < 0.0001; Fig. 2F).

IL-17: As illustrated in Fig. 2G, the mean (± SEM) serum level of IL-17 was significantly higher in TAO patients with amputation (23860 ± 7765 pg/mL) as compared with high stenosis patients (1806 ± 41.32 pg/mL; P < 0.0001), smoker controls (27.92 ± 1.62 pg/mL; P < 0.0001) and non-smoker subjects (2.91 ± 0.33 pg/mL; P < 0.0001). In addition, the IL-17 level was increased in high stenosis patients than smoker and non-smoker controls (P < 0.0001 and P < 0.0001, respectively; Fig. 2G). The concentration of IL-17 was higher in smoker controls as compared with non-smoker subjects (P < 0.0001; Fig. 2G).

Figure 2: Comparison of serum cytokines between controls and patients. Data are presented as mean ± SEM and analyzed by Kruskal-Wallis test followed by Dunn’s test; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Comparison the Inflammatory and Th1-Type Cytokines in Patients and Controls

The levels of IL-1β, IL-6, plus IL-18, as inflammatory cytokines, were significantly higher in TAO patients with amputation (47942 ± 5254) as compared with high stenosis patients, non-smoker controls, and smoker controls (11967 ± 351.60, P < 0.0001; 1372 ± 28.68 P < 0.0001, and 73.75 ± 1.99, P < 0.0001, respectively; Fig. 3A). In addition, the levels of inflammatory cytokines were higher in high stenosis patients than both groups of controls (P < 0.0001, and P < 0.0001, respectively; Fig. 3A). Similarly, the concentration of inflammatory cytokines was higher in smoker controls in comparison to non-smoker controls (P < 0.0001, Fig. 3A).

In respect of Th1-type cytokines, the concentrations of IL-12 plus IFN-γ were higher in TAO patients with amputation (43975 ± 11005.10) as compared with high stenosis patients, non-smoker, and smoker controls (9046 ± 202.40, P < 0.0001; 1905 ± 45.96 P < 0.0001, and 23.52 ± 0.83 P < 0.0001, respectively; Fig. 3B). The levels of Th1-type cytokines were higher in high stenosis patients as compared with both groups of controls (P < 0.0001, and P < 0.0001, respectively; Fig. 3B). Similarly, Th1-type cytokine concentrations were higher in smoker controls than non-smoker controls (P < 0.0001, Fig. 3B).

Figure 3: Comparison of serum inflammatory (IL-1β, IL-6, plus IL-18), and Th1-type (IL-12 plus IFN-γ) in patients and controls; data are presented as mean ± SEM and analyzed by Kruskal-Wallis test followed by Dunn’s test; ****P < 0.0001.

Correlation between Different Type of Cytokines and Demographic Characteristics

We evaluated the association between different cytokines profiles and demographic characteristics in all study groups.

Non-Smoker Controls: There were no significant correlations between cytokine concentrations and demographic characteristics in non-smoker controls (Fig. 4A).

Figure 4: Correlation analysis of the different cytokine types with demographic characteristics in all study groups. (A) Heat map correlation of cytokine types with demographic characteristics in the non-smoker controls, (B) Heat map correlation of cytokine types with demographic characteristics in the smoker controls, (C) Heat map correlation of cytokine types with demographic characteristics in high stenosis patients, (D) Heat map correlation of cytokine types with demographic characteristics in TAO patients with amputation. (Red: up-regulated, blue: down-regulated). The P-value and R determined according to Spearman’s rank correlation test.

Smoker Controls: As shown in Fig. 4B, we indicated that inflammatory cytokines positively correlated with TG (P = 0.31 ; r = 0.72), cholesterol (P = 0.33; r = 0.72), LDL (P = 0.21; r = 0.78), MPV (P = 0.23; r = 0.71), ESR (P = 0.03; r = 0.66), CRP (P = 0.02; r = 0.61), NLR (P = 0.04; r = 0.74), PLR (P = 0.02; r = 0.72), MLR (P = 0.02; r = 0.68), neutrophil count (P = 0.01; r = 0.75), and platelet count (P = 0.03; r = 0.70). Conversely, the level of inflammatory cytokines negatively correlated with HDL (P = 0.02; r = - 0.70) and lymphocyte count (P = 0.01; r = - 0.60) (Fig. 4B). The Th1-type cytokines positively associated with TG (P = 0.02; r = 0.69), cholesterol (P = 0.02; r = 0.75), LDL (P = 0.02; r = 0.73), MPV (P = 0.02.; r = 0.70), ESR (P = 0.03; r = 0.61), CRP (P = 0.04; r = 0.64), NLR (P = 0.02; r = 0.72), PLR (P = 0.02; r = 0.76), MLR (P = 0.02; r = 0.71), neutrophil count (P = 0.01; r = 0.71), and platelet count (P = 0.03; r = 0.71) (Fig 4B). However, there was negative association between Th1-type cytokines and HDL (P = 0.01; r = - 0.75) as well as lymphocyte count (P = 0.04; r = - 0.62) (Fig. 4B). As shown in Fig. 4B, we found that Th17-type cytokines positively correlated with TG (P = 0.02; r = 0.81), cholesterol (P = 0.02; r = 0.73), LDL (P = 0.02; r = 0.72), MPV (P = 0.03; r = 0.69), ESR (P = 0.02; r = 0.62), CRP (P = 0.03; r = 0.65), NLR (P = 0.02; r = 0.76.), PLR (P = 0.03; r = 0.71), MLR (P = 0.03; r = 0.72), neutrophil count (P = 0.03; r = 0.70), and platelet count (P = 0.02; r = 0.72). Conversely, the level of Th17-type cytokines negatively correlated with HDL (P = 0.01; r = - 0.72) and lymphocyte count (P = 0.03; r = - 0.63) (Fig. 4B). The anti-inflammatory cytokine negatively associated with TG (P = 0.02; r = - 0.73), cholesterol (P = 0.02.; r = -0.71.), LDL (P = 0.03; r =- 0.67), MPV (P = 0.01; r = - 0.73), ESR (P = 0.03; r = - 0.68), CRP (P = 0.02; r = - 0.67.), NLR (P = 0.01; r = - 0.78), PLR (P = 0.02; r = - 0.75), MLR (P = 0.03; r = - 0.69), neutrophil count (P = 0.02; r = - 0.74), and platelet count (P = 0.01; r = - 0.73) (Fig 4B). However, there was positive association between anti-inflammatory cytokine and HDL (P = 0.02; r = 0.74) as well as lymphocyte count (P = 0.03; r = 0.61) (Fig. 4B).

High Stenosis Patients

As shown in Fig. 4C, we observed that inflammatory cytokines positively correlated with TG (P = 0.01; r = 0.79), cholesterol (P = 0.01; r = 0.82), LDL (P = 0.02; r = 0.78), MPV (P = 0.02; r = 0.76), ESR (P = 0.02; r = 0.76), CRP (P = 0.02; r = 0.76), NLR (P = 0.01; r = 0.81), PLR (P = 0.03; r = 0.92), MLR (P = 0.02; r = 0.87), neutrophil count (P = 0.02; r = 0.77), and platelet count (P = 0.03; r = 0.76). Conversely, the level of inflammatory cytokines negatively correlated with HDL (P = 0.02; r = - 0.78) and lymphocyte count (P = 0.01; r = - 0.67) (Fig. 4C). The Th1-type cytokines positively associated to TG (P = 0.02; r = 0.72), cholesterol (P = 0.01; r = 0.85), LDL (P = 0.02; r = 0.83), MPV (P = 0.02; r = 0.72), ESR (P = 0.02; r = 0.71), CRP (P = 0.02; r = 0.74), NLR (P = 0.02; r = 0.73), PLR (P = 0.01; r = 0.96), MLR (P = 0.02; r = 0.79), neutrophil count (P = 0.02; r = 0.71), and platelet count (P = 0.02; r = 0.74) (Fig 4B). However, there was negative association between Th1-type cytokines and HDL (P = 0.02; r = - 0.75) as well as lymphocyte count (P = 0.04; r = - 0.60) (Fig. 4C). As shown in Fig. 4C, we found that Th-17 type cytokines positively correlated with TG (P = 0.01; r = 0.88), cholesterol (P = 0.01; r = 0.83), LDL (P = 0.01; r = 0.82), MPV (P = 0.73; r = 0.73), ESR (P = 0.02; r = 0.71), CRP (P = 0.02; r = 0.75), NLR (P = 0.01; r = 0.74), PLR (P = 0.01; r = 0.90), MLR (P = 0.02; r = 0.86), neutrophil count (P = 0.02; r = 0.73), and platelet count (P = 0.01; r = 0.78). Conversely, the level of inflammatory cytokines negatively correlated with HDL (P = 0.02; r = - 0.88) and lymphocyte count (P = 0.04; r = - 0.68) (Fig. 4C). The anti-inflammatory cytokines negatively associated with TG (P = 0.01; r = - 0.78), cholesterol (P = 0.03; r = - 0.65), LDL (P = 0.03; r = - 0.68), MPV (P = 0.02; r = 0.79), ESR (P = 0.02; r = - 0.67), CRP (P = 0.02; r = - 0.67), NLR (P = 0.01; r = - 0.75), PLR (P = 0.01; r = - 0.85), MLR (P = 0.02; r = - 0.84), neutrophil count (P = 0.02; r = - 0.76), and platelet count (P = 0.04; r = - 0.66) (Fig 4C). However, there was positive association between anti-inflammatory cytokines and HDL (P = 0.01; r = 0.84) as well as lymphocyte count (P = 0.03; r = 0.63) (Fig. 4C).

TAO Patients with Amputation

As shown in Fig. 4D, we found that inflammatory cytokines positively correlated with ESR (P = 0.01; r = 0.96), CRP (P = 0.02; r = 0.86), NLR (P = 0.02; r = 0.71), PLR (P = 0.03; r = 0.72), MLR (P = 0.02; r = 0.71), neutrophil count (P = 0.02; r = 0.87), and platelet count (P = 0.01; r = 0.96). Conversely, the level of inflammatory cytokines negatively correlated with MPV (P = 0.01; r = - 0.86) and lymphocyte count (P = 0.02; r = - 0.87) (Fig. 4D). The Th1-type cytokines positively associated to ESR (P = 0.01; r = 0.91), CRP (P = 0.02; r = 0.94), NLR (P = 0.02; r = 0.83), PLR (P = 0.02; r = 0.76), MLR (P = 0.03; r = 0.68), neutrophil count (P = 0.02; r = 0.91), and platelet count (P = 0.02; r = 0.94) (Fig 4B). However, there was negative association between Th1-type cytokines and MPV (P = 0.01; r = - 0.92) as well as lymphocyte count (P = 0.01.; r = - 0.90) (Fig. 4D). As shown in Fig. 4D, we found that Th-17 type cytokines positively correlated with ESR (P = 0.02; r = 0.91), CRP (P = 0.02; r = 0.95), NLR (P = 0.01; r = 0.94), PLR (P = 0.02.; r = 0.88), MLR (P = 0.03; r = 0.63), neutrophil count (P = 0.02; r = 0.93), and platelet count (P = 0.01; r = 0.88). Conversely, the level of inflammatory cytokines negatively correlated with MPV (P = 0.01; r = - 0.93) and lymphocyte count (P = 0.01; r = - 0.88) (Fig. 4D). The anti-inflammatory cytokines negatively associated with ESR (P = 0.02; r = - 0.87), CRP (P = 0.02; r = - 0.97), NLR (P = 0.01; r = - 0.85), PLR (P = 0.01; r = - 0.73), MLR (P = 0.04; r = - 0.59), neutrophil count (P = 0.02; r = - 0.86), and platelet count (P = 0.02; r = - 0.76) (Fig 4D). However, there was positive association between anti-inflammatory cytokines and MPV (P = 0.01; r = 0.90) as well as lymphocyte count (P = 0.01; r = 0.73) (Fig. 4D).

The main etiology of TAO is not yet elucidated. TAO is regarded as an inflammatory peripheral vascular disease because different inflammatory mediators like cytokines may contribute to proliferative lesions or thrombus formation in the peripheral vessels as well as vascular inflammation. It is possible that the immune system plays a critical role in the etiology of TAO through progression of vascular tissue inflammation, i.e., the formation of immune complexes and activation of cell-mediated phagocytosis. In the current study, we investigated the concentrations of different cytokines in patients with TAO in comparison with high stenosis patients, non-smokers, and smoker controls.

At first, we observed significantly higher levels of inflammatory (IL-1β, IL-6, IL-18), Th1-type (IL-12, IFN-γ), and Th17-type (IL-17) cytokines and lower levels of anti-inflammatory (IL-10) cytokine in the patients with TAO. Furthermore, we showed a positive correlation between the concentration of inflammatory, Th1-type and Th17 type cytokines and ESR, CRP, NLR, PLR and MLR in TAO patients. This finding is in line with previous studies, which reported higher levels of various cytokines in TAO patients [7, 13-15]. It has been shown that serum levels of Th17-associated cytokines such as IL-17, IL-22, and IL-23 were significantly higher in the TAO patients compared to the smoker control group (5). Further analysis of different cytokine levels also showed that TNF-α, IL-6, and HMGB1 concentrations were higher in TAO patients than in non-smoker healthy controls [16]. Moreover, a significant increase in the serum level of IL-6 was found in smokers TAO patients as compared to non-smokers TAO patients [17]. On the other hand, we observed that the serum level of anti-inflammatory cytokine IL-10 was significantly lower in the patient with TAO, and similarly, Joviliano et al, also have supported our findings [7]. The role of inflammatory cytokines in the initiation and progression of vascular diseases has been clarified [18, 19]. Accordingly, they stimulate the expression of adhesion molecules on the endothelium and facilitate the recruitment of inflammatory immune cells such as neutrophils and monocytes. Furthermore, they regulate the expression of Platelet-derived growth factor (PDGF), vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF), and ECM degrading MMPs (matrix metalloproteinases), which affect the vascular smooth muscle cell (VSMC) growth and migration [20]. In vascular disease, Th1 and Th17 cells are considered the most harmful T cells due to their secretion of inflammatory cytokines, activation of macrophages, and influence on endothelial cells [21, 22].

We then observed higher serum levels of lipid profile (including cholesterol, triglycerides, and high-density lipoprotein) in the patients with high stenosis (Table 1). In contrast to the patients with high stenosis, the serum lipid profile levels in TAO patients were similar to non-smoker controls. In addition, the serum level of HDL in TAO patients and non-smoker controls was higher than two other groups. It has been reported that TAO can be distinguished from atherosclerosis based on lipid profile in rat models [23]. Moreover, previous studies have been reported the serum cholesterol level in TAO patients ranged from 157 mg/dL to 225 mg/dL, while LDL and HDL levels were 94–112 mg/dL and 34–54 mg/dL, respectively [24]. Therefore, it seems that lipid profiles could be considered as excluding criteria for discriminating between atherosclerosis and TAO. Our findings showed neutrophilia and monocytosis in TAO patients as compared with all other study groups. Furthermore, we showed higher NLR and MLR and lower PNR in TAO patients. Previous studies have also linked leukocyte count to the worsening of TAO disease [2, 25]. Fazeli et al. have reported that neutrophilia is an important clinical manifestation in patients with advanced TAO [26, 27]. They have also shown that elevated leukocyte count, known as leukocytosis, may be a risk factor for recurrent TAO-associated thrombosis [28]. A previous study by Ren et al. showed that PNR was significantly lower in patients with TAO compared to healthy individuals and smoker controls. Moreover, they observed that NLR and PNR might be predictive cut-off values for the acute phase of TAO [29]. Thus, our findings highlight that neutrophilia as well as inflammatory markers, including NLR, MLR and PNR may be a valuable prognostic and predictive factors for acute phase of TAO.

In conclusion, higher levels of inflammatory cytokines in TAO patients and their correlation with inflammatory indices such as ESR, CRP, NLR, PLR, and MLR suggest a complex network between cytokines and the induction of inflammation and thrombosis in the context of TAO disease. These data may provide a better understanding of the etiology of TAO and the underlying immunological mechanisms of its development. However, further studies are recommended to determine direct or indirect modulation of inflammatory cytokines pathways through appropriate therapeutic strategies to alleviate potentially detrimental complications in TAO patients.

Funding

No funding was received for this research

Conflict of Interest

The authors declare no conflict of interest, financial or otherwise.

Author Contribution

Reza Darvishvand and Zahra Ghasemi performed the lab work, analyzed the data, and wrote the paper. Athar Faghih collected the samples and data. Hamed Ghoddusi Johari revised the paper. Atefe Ghamar Talepoor conceived, supervised and designed the experiments, wrote and revised the paper.

Ethics Approval

This study was approved by the ethic committee at Shiraz University of Medical Sciences (IR.SUMS.REC.1402.214).

Consent to Participant

Written informed consent was obtained from all research participants.

Consent to Publish

The authors affirm that research participants provided informed consent for publication of manuscript.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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