Role of Toll-Like Receptor 2 in Regulation of T-Helper Immune Response in Chronic Obstructive Pulmonary Disease

Objective According to modern views, the differences in the clinical course of chronic obstructive pulmonary disease (COPD) are associated with certain types of T-helper (Th) immune response. Recent data have shown that toll-like receptor 2 (TLR2) is involved in the development of Th immune response. However, TLR2-mediated regulation of Th subpopulation balance in COPD needs to be elucidated. The aim of our work is to determine the mechanisms of TLR2-mediated regulation of Th immune response in COPD of varying severity. Methods The study included 323 smokers/ex-smokers with stable COPD (GOLD I, GOLD II, and GOLD III) and 97 healthy nonsmokers (control group). Serum levels of Th1 (TNF-α and IFN-γ), Th2 (IL-4), Th17 (IL-6 and IL-17A), Treg (IL-10) cytokines, and the percentage of peripheral blood Th cells expressing TLR2 (CD4+CD282+) were assessed by flow cytometry. Serum concentrations of IL-21 (Th17) and TGF-β1 (Treg) were measured using the ELISA method. The predominant Th cytokine profile in serum was determined by calculating the ratios between levels of Th1 and Th17 cytokines. Spearman's correlation test was performed. Results Patients with COPD GOLD II and III with Th1 and Th17 cytokine profiles exhibited an increase in the percentage of CD4+CD282+ cells compared to the control group. In COPD GOLD I–III, positive correlations between CD4+CD282+ cell frequency and Th17 cytokine levels (IL-6, IL-17A, and IL-21) were found. In COPD GOLD I, IL-10 concentration was negatively correlated with the percentages of studied cells; in COPD GOLD II, a positive correlation between these parameters was noted. Conclusions Enhanced TLR2 expression on CD4+ cells shifts cytokine profile toward Th17 phenotype that plays a crucial role in COPD progression. The level of TLR2 expression on peripheral blood CD4+ cells may be considered as a biomarker for diagnosing and predicting the progression of COPD.


Introduction
Nowadays, chronic obstructive pulmonary disease (COPD) is one of the most common chronic inflammatory diseases of the respiratory system [1,2]. According to modern views, there are several pathophysiological COPD phenotypes with different clinical courses of the disease [3][4][5].
Recent research data indicate that the classification of COPD into pathophysiological phenotypes is based on a shift in the balance of subpopulations of T-helper ( or CD4 + ) cells, such as type 1 cells ( 1), 2, 17, and T-regulatory cells (Treg) [6]. Predominant activation of certain subpopulations, which depend on the cytokine microenvironment, determines adaptive immune response involving various mediators and effector cells [7,8]. e 1 immune response is characterized by the enhanced production of pro-inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interferon-c (IFN-c) that trigger the macrophage-mediated inflammatory response. e cytokine IFN-c is also involved in the differentiation of 1 cells through a feedback loop [7,9].
2-lymphocytes mainly produce IL-4 that is essential for their differentiation and promotes the development of immune response involving mast cells and eosinophils [7,8]. e development of 17 immune response is accompanied by an increase in the concentration of IL-17 that is responsible for recruiting neutrophils in the small airways. e essential factors for 17 cell differentiation are IL-6 and IL-21 [10,11]. e main function of Treg is to inhibit the activity of effector cells by secreting anti-inflammatory cytokine IL-10 and transforming growth factor-β (TGF-β) [12,13]. According to the literature, many reactions of cellular immunity, including the cytokine response, are mediated by toll-like receptors (TLRs). e activation of these receptors occurs due to the binding of molecular structures of pathogens (pathogen-associated molecular patterns, PAMPs) and tissue damage products (damage-associated molecular patterns, DAMPs). In humans, 10 TLRs have been identified. ey differ in ligands, signaling pathways, and functions. TLRs may be located on the endosomal membrane (TLR3, TLR7, TLR8, and TLR9) or the cytoplasmic membrane of the cell (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) [14].
A number of publications have reported the participation of endosomal TLRs recognizing viruses, such as TLR3, TLR7, and TLR9, in the pathogenesis of COPD [15][16][17][18][19][20]. However, numerous studies indicate the considerable contribution of membrane TLR2 and TLR4 activated by bacterial PAMPs to the development of the inflammatory process in COPD [21][22][23][24][25]. Altered expression of these TLRs in the tissues of the respiratory tract has been found both in patients with COPD and experimental animals [21,[26][27][28][29]. Genetic studies have shown that polymorphism of the TLR2 and TLR4 genes is associated with the risk of developing and progressing COPD [30][31][32].
As for the role of TLRs in the regulation of immune response, there is currently evidence of the direct involvement of TLR2 in this process in various pathologies [33][34][35][36][37][38], which makes this receptor an interesting object for research. Nevertheless, TLR2-related mechanisms determining the development of the immunoregulatory pathway in COPD are still poorly understood. Taking into account the important role of T-helper immune pathways in COPD progression, the determination of TLR2-mediated mechanisms regulating the balance of subpopulations in COPD offers significant potential to improve the methods for diagnosing COPD, enable new personalized approaches to treatment, and enhance the quality of prediction of disease course. e aim of our work is to determine the mechanisms of TLR2-mediated regulation of immune response in COPD of varying severity. e diagnosis was made in accordance with the Global Strategy for Diagnosis, Management, and Prevention of COPD [2]. Clinical characteristics of the examined groups are presented in Table 1. e exclusion criteria were COPD exacerbations during the last 2 months, acute pathological conditions, exacerbation of chronic diseases, and occupational hazards associated with inhalation of toxic substances. All COPD patients were treated with long-acting m-cholinolytics, and patients with COPD GOLD III were treated with long-acting m-cholinolytics combined with a fixed combination of long-acting β2-agonists and inhaled glucocorticoids [2]. e control group consisted of 97 practically healthy nonsmokers with normal respiratory function (mean age 51.3 ± 2.5 years).

Determination of TLR2 Expression on Peripheral Blood
Cells.

Statistical Analysis.
Statistical data processing was performed using the Statistica 6.0 software. To check the normality of the distribution for the quantitative parameters, the Kolmogorov-Smirnov test was used. e results of statistical analysis of data with nonnormal distribution were presented as median, upper, and lower quartiles. Statistical significance of the differences was calculated using Mann-Whitney U test. e correlation analysis was done using Spearman's correlation coefficient (r). e p-value of less than 0.05 was considered to be significant. e receiver operating characteristic (ROC) curve was calculated to measure the sensitivity and specificity of the level of TLR2 expression on circulating CD4 + cells as a diagnostic and prognostic biomarker for COPD. An area under the ROC curve (AUC) with a 95% confidence interval (CI) was evaluated. A probability of p < 0.05 was considered as the threshold of significance.

TLR2 Expression on Circulating CD4 + Cells in COPD of Varying Severity in relation to the Predominant Serum
Cytokine Profile. In this study, the groups of patients with predominant 1 and 17 cytokine profiles have been identified. Due to the fact that the serum levels of the key 2 cytokine IL-4 in patients with COPD of varying severity were lower or only slightly different from the values of the control group, patients with a 2 cytokine profile have not been distinguished.
Differences in the changes of TLR2 expression on circulating CD4 + cells were found between COPD patients with 1 and 17 cytokine profiles during disease progression ( Table 2).
In patients with COPD GOLD II and III with a predominance of 1 immunoregulatory pathway, the relative number of peripheral blood CD4 + CD282 + cells was statistically significantly elevated by 68% (p < 0.05) and 92% (p < 0.05), respectively, compared to the control group. In patients with COPD GOLD II and III with 17 cytokine profile, the percentage of the studied cells was statistically significantly increased by 141% (p < 0.001) and 258% (p < 0.001), respectively, relative to the control values, as well as by 40% (p < 0.01) and 87% (p < 0.001), respectively, compared to COPD patients with a 1 cytokine profile.
us, the greatest enhancement of TLR2 expression on peripheral blood cells during COPD progression was found in patients with a 17 cytokine profile.

Correlations between TLR2 Expression on Circulating CD4 + Cells, Serum
Cytokine Profile, and Clinical Parameters in COPD of Varying Severity.
e results of correlation analysis between the percentage of circulating CD4 + cells expressing TLR2, serum levels of cytokines, and clinical parameters in COPD of varying severity are presented in Table 3.
In COPD GOLD I, strong positive correlations were revealed between the relative number of circulating cells expressing TLR2 and serum levels of IL-6 (r � 0.71 and p < 0.05), IL-17A (r � 0.77 and p < 0.05), and IL-21 (r � 0.79 and p < 0.05). e percentage of peripheral blood CD4 + CD282 + cells was negatively correlated with serum concentration of IL-10 (r � −0.63 and p < 0.05) in this group of patients. In COPD GOLD II, there were positive correlations between the relative number of studied cells and serum levels of IL-21 (r � 0.85 and p < 0.05), IL-6 (r � 0.69 and p < 0.05), IL-17A (r � 0.67 and p < 0.05), and IL-10 (r � 0.53 and p < 0.05). In COPD GOLD III, strong direct correlations were found between the percentage of peripheral blood CD4 + CD282 + cells and serum content of IL-17A (r � 0.96 and p < 0.05), IL-21 (r � 0.94 and p < 0.05), and IL-6 (r � 0.87 and p < 0.05). Increased number of circulating CD4 + CD282 + cells was negatively correlated with the clinical parameters, such as FEV1/FVC (r � −0.87 and p < 0.05) and FEV1% predicted (r � −0.68 andp < 0.05). erefore, positive correlations were found between the percentage of circulating CD4 + CD282 + cells and serum levels of 17 cytokines (IL-6, IL-17A, and IL-21) at all stages of COPD. e serum concentration of IL-10 (Treg cytokine) was negatively correlated with the percentage of studied cells in COPD GOLD I and positively correlated with this parameter in COPD GOLD II. Negative correlations were found between TLR2 expression on cells and clinical parameters characterizing bronchial obstruction (FEV1/FVC and FEV1% predicted).

Receiver Operating Characteristic Curve
Analysis. e AUC value for the level of TLR2 expression on circulating CD4 + cells (percentage of CD4 + CD282 + cells in the peripheral blood) as a diagnostic marker for COPD was 0.952 (95% CI: 0.887-1.017 and p < 0.05) with a cut-off of 1.4% (discriminating between COPD patients and healthy subjects) of the cells having a sensitivity of 86.7% and a Canadian Respiratory Journal 3 specificity of 100%. e parameter was also found to be a promising prognostic biomarker of COPD progression with an AUC of 0.984 (95% CI: 0.943-1.025 and p < 0.05). e optimal cut-off biomarker value to distinguish patients with COPD GOLD II and GOLD III from patients with COPD GOLD I was 1.7% of the cells with a sensitivity of 95.2% and a specificity of 88.9%. erefore, the biomarker has high diagnostic and prognostic value in COPD.

Discussion
Currently, it is generally accepted that COPD is a heterogeneous disease that includes several phenotypes characterized by different clinical manifestations and pathophysiological reactions [3][4][5]39]. According to modern concepts, COPD phenotypes can be differentiated based on cytokine profiles associated with the development of a certain type of immune response [6][7][8]. Our previous studies have shown that 1 and 17 types of immune response determine the clinical course of COPD. It has been found that inflammation developing according to the 17 type of immune response is characterized by more pronounced airway obstruction and a higher risk of infectious complications compared to 1-dependent inflammation [39]. Solleiro-Villavicencio et al. showed that biomass smoke-induced COPD is associated with the 2 cytokine profile [40]. Additionally, there is evidence that patients with asthma-COPD overlap syndrome also have 2 immune response [41]. However, our study included smokers and exsmokers with COPD, and no patient with the predominant 2 cytokine profile was identified. Accumulating evidence demonstrated that COPD progression is associated with the development of the 17 immunoregulatory pathway and imbalance between 17 and Treg cell subpopulations [11-13, 42, 43].
Toll-like receptors are known to be implicated in the regulation of cytokine response in adaptive and innate immune cells. According to the results of numerous studies, TLR2 is one of the most important contributors to the pathogenesis of COPD [22][23][24][25]. In COPD, ligands for TLR2 can be PAMPs (lipopeptides, glycolipids, lipoteichoic acid (LTA), peptidoglycan (PGN), etc.) of bacteria colonizing the lower airways (Streptococcus pneumoniae, nontypeable Haemophilus influenzae (NTHi), Moraxella catarrhalis, and Pseudomonas aeruginosa) [44], as well as DAMPs releasing as a result of lung tissue exposure to negative environmental factors, such as smoking [29] and air pollution (high-mobility group protein B1 (HMGPB1), heat shock proteins (HSP60, HSP70), β-defensin, etc.) [45]. Upon binding to a ligand, TLR2 triggers the myeloid differentiation primary response gene 88 (MyD88) dependent signaling pathway culminating in the activation of nuclear transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) or activating protein-1 (AP-1). e activation of TLR2 signaling in target cells leads to the synthesis of pro-inflammatory mediators, such as TNF-α, IL-6, IL-8, chemokine C-C motif receptor 5/regulated on activation, normal T cell expressed and secreted (CCL5/RANTES), and others [14].
Being accumulated in the lungs during the development of the inflammatory response in COPD, TLR2 ligands can also enter the bloodstream and initiate the TLR2 signaling pathway in circulating immune cells. However, there are few studies regarding TLR2 expression on immune blood cells in COPD. Some research results indicate the inhibition of TLR2-dependent cytokine secretion by leukocytes in the last stages of COPD [46]. ere is evidence of a decreased  Notes. Data are presented as median, upper, and lower quartiles. Statistical significance of the differences between the groups of COPD patients and the control group (Mann-Whitney U test): * p < 0.05 and * * * p < 0.001. Statistical significance of the differences between the groups of patients with COPD of the same severity but with different cytokine profiles (Mann-Whitney U test): ## p < 0.01 and ### p < 0.001.
number of circulating monocytes expressing TLR2 and an impaired monocyte-dependent immune response as COPD progresses [46][47][48]. A number of researchers reported an increase in TLR2-mediated cytokine synthesis by blood neutrophils in COPD patients [21,49]. ere are almost no studies on TLR2 expression on circulating cells in COPD. Current research data suggest that TLR2 significantly contributes to the regulation of differentiation and functioning of cells [50,51]. Experimental studies have shown that the initiation of the TLR2 signaling pathway in T-cell receptor (TCR) activated CD4 + cells enhances their proliferation, survival, and chemotaxis and modulates cytokine synthesis and other effector functions [50][51][52]. However, the role of the TLR2 signaling pathway in the development of immune response in COPD needs to be elucidated.
We have analyzed the changes in TLR2 expression on circulating CD4 + cells in COPD of varying severity in relation to the predominant serum cytokine profile. As a result, a statistically significant increase in the number of peripheral blood CD4 + CD282 + cells has been found in patients with COPD GOLD II and III with both 1 and 17 cytokine profiles compared to the control group. At the same time, TLR2 expression was significantly higher in patients with COPD GOLD II and III with the 17 cytokine profile than in COPD patients with the 1 cytokine profile. It should be noted that patients with 17 immune response predominated in the groups of patients with COPD GOLD II and III.
Despite the elevated TLR2 expression on T-helpers in COPD patients with the 1 cytokine profile, no statistically significant correlations between the relative number of circulating CD4 + cells expressing TLR2 and the levels of 1-associated cytokines have been found. e increased expression of TLR2 on T-helpers in patients with 1 cytokine profile may be related to the mechanisms of regulation of common T-cell functions (proliferation and survival) during the development of inflammation [50,51].
Our study has shown strong correlations between the relative content of circulating CD4 + CD282 + cells and serum levels of 17 cytokines (IL-6, IL-17A, and IL-21) in patients with COPD GOLD I. It has been reported that the stimulation of CD4 + cells by TLR2 ligands leads to the activation of key transcription factors of 17 cells (retinoic-acid-receptor-related orphan nuclear receptor ct (RORct, RORα, RORC) and the production of effector 17 cytokines (IL-6, IL-17A, IL-17F, IL-22, and IL-21) [33][34][35][36]53]. Consequently, the TLR2/MyD88 signaling pathway is already involved in the development of the 17 immunoregulatory pathway in the early stage of COPD. In the group of patients with COPD GOLD II, a strong correlation has been found between the percentage of cells expressing TLR2 and the concentration of IL-21 promoting 17 cell differentiation that indicates the involvement of TLR2 in maintaining 17 immune response during COPD progression. In patients with COPD GOLD III, the relative number of cells expressing TLR2 was strongly correlated with levels of IL-6, IL-21, and IL-17A. us, our findings suggest an important contribution of TLR2 to the progression of COPD associated with 17 immune response. Our data are consistent with accumulated evidence from in vitro studies confirming that the activation of TLR2 signaling pathway on CD4 + cells promotes the development of 17 immune response in a number of pathologies, including acute disseminated encephalomyelitis [33], multiple sclerosis [37], systemic lupus erythematosus [36], hypersensitivity pneumonitis [34], and hepatitis B [35,38].
In our study, correlations between the levels of TLR2 expression on circulating cells and the serum content of the anti-inflammatory cytokine IL-10 have been identified.
is cytokine is responsible for the limitation of tissue damage during the inflammatory process by inhibiting the macrophage activity and production of TNF-α, IL-6, and other pro-inflammatory cytokines [54]. Available data on the systemic levels of IL-10 in COPD are rather contradictory. A number of studies have demonstrated high serum IL-10 levels in patients with COPD [55,56]. At the same time, there are publications indicating a low concentration of IL-10 in the blood in severe COPD [54]. Additionally, there is evidence of the lack of changes in IL-10 levels with the pathology progression [57]. ese findings indicate the complex mechanisms of the participation of this cytokine in the pathogenesis of COPD.
Our analysis has shown that the percentage of CD4 + CD282 + cells was inversely correlated with IL-10 levels in the peripheral blood in COPD GOLD I. e serum concentration of this cytokine was positively correlated with the percentage of CD4 + CD282 + cells in COPD GOLD II. It can be assumed that an increase in IL-10 levels serves as a compensatory mechanism aimed at maintaining the balance between pro-and anti-inflammatory mediators during COPD progression. In the group of patients with COPD GOLD III, there was no correlation between the number of CD4 + CD282 + cells and IL-10 levels. It may be due to glucocorticoids used in the therapy of COPD GOLD III. ese drugs are known to have potent immunoregulatory effects, so they could influence IL-10 levels in these patients [58].
IL-10 is one of the important cytokines produced by Tregs that play a unique role in controlling the inflammatory response. Currently, there is increasing evidence that TLR2 ligands are involved in the direct modulation of Treg cell functions. However, the data presented in the literature are in marked contrast. In particular, TLR2 stimulation has been shown to reduce the suppressive activity of activated Tregs through mechanisms that are not fully understood yet [59][60][61]. Nyirenda et al. have shown that the stimulation of TLR2 on Tregs leads to the polarization of these cells into 17-like phenotype through an increase in the production of IL-6 that exerts an autocrine effect on these cells and promotes switching of the Jak/STAT5/FoxP3 signaling pathway to the Jak/STAT3/RORC pathway [37,53]. us, according to modern concepts, TLR2 may be responsible for a decrease in the suppressive function of Tregs, regulating the Treg/ 17 balance [37,53,62]. At the same time, Zanin-Zhorov et al. have shown the opposite effect of Treg activation by endogenous TLR2 ligands that led to an increase in its suppressive action mediated by IL-10 production [52]. In addition, there is evidence that activation of the TLR2 signaling pathway can enhance Treg proliferation and IL-10 Canadian Respiratory Journal synthesis by these cells [63]. e positive and negative effects of TLR2 ligands on Tregs are intriguing and may be associated with using different ligands (synthetic and endogenous) and experimental conditions. Still, further research is needed to understand fully the role of the TLR2 signaling pathway in Treg functioning. It should be noted that in addition to Tregs, other cell subpopulations can make a significant contribution to the production and secretion of IL-10. erefore, TLR2 signaling appears to be involved in the control of 17/Treg balance in COPD, but the exact mechanism needs to be further clarified.
Negative correlations were found between TLR2 expression on peripheral blood cells and clinical parameters characterizing bronchial obstruction (FEV1/FVC and FEV1% predicted) in patients with COPD. ese data indicate an important role of the TLR2 signaling pathway in the development of local inflammation in COPD and confirm our assumption about its participation in the progression of this disease. e results of ROC curve analysis suggest the possibility of using the level of TLR2 expression on peripheral blood T-helpers for diagnosis and predicting the prognosis of COPD. e strength of this study is a new viewpoint on the role of innate immunity receptors in the development of T-helper immune response in COPD, which will expand the understanding of the pathogenesis of the disease. e limitation is that we have studied the mechanisms of TLR2mediated regulation of immune response in COPD in the peripheral blood, without regard to local inflammation. Another limitation is the inclusion of only smokers and exsmokers with COPD in the study and the absence of a cohort of nonsmokers with COPD. An additional limitation is the lack of data on the intracellular expression of cytokines in CD4 + cells. Further studies devoted to this issue will make it possible to determine more precise mechanisms of the regulation of immune response in COPD.

Conclusion
It can be concluded that increased TLR2 expression on CD4 + cells contributes to the polarization of the immune response toward the 17 pathway in COPD, which ultimately leads to the deterioration of respiratory function and the progression of this pathology. However, the exact mechanisms of this process require further research. e level of TLR2 expression on circulating T-helper cells can be used as a diagnostic and prognostic marker in COPD. More indepth studies will help answer the question of whether TLR2 is a promising therapeutic target for the treatment of this disease.

Data Availability
Access to data is restricted due to patient privacy.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.