Decreased Th1 Cells and Increased Th2 Cells in Peripheral Blood Are Associated with Idiopathic Inflammatory Myopathies Patients with Interstitial Lung Disease

Interstitial lung disease (ILD) is a highly fatal manifestation of idiopathic inflammatory myopathies (IIMs). Th cells play important roles in the initiation of ILD. Here, we investigated the clinical significance of peripheral blood Th cells in IIMs-ILD patients. Eleven healthy controls (HC) and 53 patients diagnosed with IIMs were included, including 30 with ILD (IIMs-ILD) and 23 without ILD (IIMs-non-ILD). Circulating Th1, Th2, Th17, and Treg cells were examined by flow cytometry, and their correlation with clinical and laboratory findings was analyzed by Spearman’s correlation and logistic regression. The proportion of Th1 cells decreased and Th2 cells increased in IIMs-ILD compared with IIMs-non-ILD (median (quartile): 2.99 (1.59–5.39) vs. 6.91 (3.48–10.04), p < 0.001; 2.67 (1.79–4.67) vs. 1.62 (0.85–2.66), p = 0.006) and correlated with disease activity. The Th1-cell proportion decreased in anti-MDA5 antibody-positive patients, while the Th2 cell proportion increased in patients with nonspecific interstitial pneumonia compared with IIMs-non-ILD (2.66 (1.06–4.35) vs. 6.91 (3.48–10.04), p = 0.002; 3.09 (2.03–5.72) vs. 1.62 (0.85–2.66), p = 0.016). Univariate analysis showed that a lower Th1 proportion, higher Th2 proportion increased, lower CK level, positivity for ARS, or anti-Ro52 antibodies (OR = 0.7122; OR = 1.679; OR = 0.9993; OR = 9.188; and OR = 6.161, respectively) were associated with the occurrence of ILD in IIMs patients. Decreased Th1 cells and elevated Th2 cells in peripheral blood may be involved in the pathogenesis of ILD in IIMs patients and have different effects on different serological and imaging subtypes.


INTRODUCTION
Idiopathic inflammatory myopathies (IIMs) are a group of heterogeneous systemic autoimmune diseases predominantly affecting extremity proximal muscles but also the lungs, heart, and gastrointestinal tract [1]. The exact etiology of IIMs remains unknown, and the interactions of environmental factors and genetic abnormalities are the main risk factors [2,3].
IIMs most commonly affect the lungs, frequently presenting as interstitial lung disease (ILD) and regularly causing death [4,5]. ILD is characterized by the accumulation of collagen and other extracellular matrix (ECM) components and has a complex and diverse pathogenesis involving various immune cells [6]. It is widely known that the immune response plays an essential role in fibrosis and fibrotic diseases [6]. T cells, which account for the largest number and types of lymphocytes, are the main effector cells mediating the cellular immune response [7]. Several studies have confirmed that the effects of T cells are consistent with fibrosis [6,8]. However, the underlying mechanisms by which T cells regulate fibrosis are not completely understood, and the factors related to ILD in patients with IIMs are unknown. Few studies have recorded the role of peripheral CD4 + T cell subsets in IIMs-ILD. In the present study, we investigated subpopulations of CD4 + T cells using flow cytometry in the peripheral blood of IIM patients with and without ILD, exploring the pathogenesis of ILD in patients with IIMs.

Subjects
This is a single-center cross-sectional study. In this study, all subjects who were admitted to the Department of Rheumatology in West China Hospital of Sichuan University from January 2020 to January 2021 were recruited. All subjects were ≥ 18 years old and met the Bohan and Peter criteria [9]. Patients with malignant disease, severe lung infection, and other known causes of ILD were excluded. The control group was healthy volunteers, all of whom met the above IIM exclusion criteria and were age-and gender-matched to the IIMs group. We enrolled 53 patients who were diagnosed with IIMs, and 30 and 23 were complicated with and without ILD, respectively, based on high-resolution computed tomography (HRCT).

Evaluation of Disease Activity in IIMs
The myositis disease activity core set, which was established by the International Myositis Assessment and Clinical Studies (IMACS) group, was used to evaluate disease activity for IIMs, including myositis activity assessment visual analog scales (MYOACT) and the myositis intention-to-treat activity index (MITAX) [10]. Both assess seven individual organ systems (constitutional, cutaneous, skeletal, gastrointestinal, pulmonary, cardiovascular, muscle) [10].
The Patient Health Assessment Questionnaire (HAQ) was used to assess the subject's physical function related to activities of daily living, and the MMT8 rating scale was used to conduct the muscle strength test through the training videos according to the instructions. Both evaluation criteria were downloaded from The IMACS official website [11].

Flow Cytometry
Eight milliliters of peripheral venous blood was extracted from the EDTA anticoagulation tubes and separated into peripheral mononuclear cells (PBMCs) by the Ficoll-Hypaque gradient method in all patients and controls. CD4 + T cells were isolated from PBMCs using CD4 + T cell-specific microbeads according to the manufacturer's instructions. For intracellular cytokine staining, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) and ionomycin (1 µg/ml) in the presence of GolgiStop (1 µL/ml) for 4-6 h. The cells were harvested and stained with anti-CD4-FITC antibodies to facilitate the measurement of Th1, Th2, Th17, and Treg cells. The labeled cells were analyzed by two-or three-color flow cytometry using FACSCalibur (Becton Dickinson).

Statistical Analysis
SigmaPlot 12.5 and/or GraphPad Prism 8.0.2 software were used for data analysis. Data are presented as the mean ± standard deviation (M ± SD) or median (Q 25 , Q 75 ). One-way ANOVA and Mann-Whitney U tests were conducted for multiple or two group comparisons, respectively. The correlations were analyzed with the Spearman correlation coefficient. Logistic regression analysis was conducted to determine which cell type was an independent risk factor for ILD in IIMs. p values < 0.05 were considered statistically significant.
In addition, different types of lung imaging in ILD patients, including nonspecific interstitial pneumonitis (NSIP), usual interstitial pneumonia (UIP), organizing pneumonia (OP), and unclassifiable patterns, affect the response and treatment-related prognosis [14], and there are few reports on the relationship between T cell subsets and imaging subtypes. To understand their relationship, we evaluated the proportions of Th1 and Th2 cells and the ratio of Th1 to Th2 cells in the three groups. There were no significant differences in the proportion of Th1 cells among the three groups (Fig. 2d, p > 0.05). The percentage of Th2 cells was higher in the NSIP pattern IIMs-ILD patients than in the IIMs-non-ILD patients (3.09 (2.03-5.72) vs. 1.62 (0.85-2.66), p < 0.05) (Fig. 2e). Meanwhile, the ratio of Th1 to Th2 cells was significantly lower in patients with the NSIP pattern than in IIMsnon-ILD patients (1.00 (0.45-2.71) vs. 4.00 (2.20-6.56), p < 0.05) (Fig. 2f).

DISCUSSION
Interstitial lung disease (ILD) is the most prominent complication of idiopathic inflammatory myopathies (IIMs), which seriously reduce life expectancy, and there are very few effective treatments [1,4]. A variety of cells, including T cells, are involved in the development of ILD [8], and the underlying mechanisms are not completely understood. In this study, we detected CD4 + T cells in the peripheral blood of patients with IIMs by flow cytometry.
As the first step of fibrosis, the inflammatory response plays an important role in initiating and amplifying fibrosis [6]. T helper cells are important cells mediating inflammation, but the detailed mechanism of the fibrosis process is not clear.
Th1 cells orientate cell-mediated immunity and phagocyte-dependent inflammation by secreting IL-2 and IFN-γ [15]. IFN-γ has been identified as an antifibrotic cytokine that is able to inhibit fibrosis by inhibiting collagen fibers produced by fibroblasts, overexpressing MMPs and accelerating the decomposition of the extracellular matrix [6,16,17]. Several studies have been used to document the potent antifibrotic activity of IFN-γ [16]. For example, Keane MP et al. reported that IL-12 attenuates bleomycin-induced pulmonary fibrosis via modulation of IFN-γ production [17]. However, a study involving 9 patients with rapidly progressive interstitial lung disease (RPILD) associated with DM suggested that there was a dramatic increase in IFN-γ in the peripheral blood of patients with RPILD, which was positively correlated with scores of pulmonary fibrosis, and abundant IFN-γ-positive cells existed in lung tissue immunohistostaining [18]. IL-2 is a T cell growth factor that has two different functions, maintaining Treg cells to control immune responses (low dose) and stimulating conventional T cells to promote immune responses (high dose) according to the dose [19]. Animal studies suggested that low-dose IL-2 therapy improved the phenotype of lung fibrosis mice, with a significant reduction in pulmonary parenchymal fibrosis and lung vascular remodeling [20]. The occurrence of DM-ILD patients is correlated with decreased Treg cells in peripheral blood [21], and treatment with IL-2 at a low-dose results in the elevation of Treg cells [21]. It is worth noting that Zou J et al. reported that pulmonary function was improved by IL-2 receptor antagonists in four patients with IIMs with RPILD [22]. This may be due to IL-2 receptor antagonists inhibiting the proinflammatory effects of T cells and thus inhibiting fibrosis. This shows that complexity remains between Th1 cells and the mechanism of fibrosis, and Th1 function differs in various tissues and organs. In our research, patients diagnosed with IIMs-ILD had a profound decrease in Th1 cells, which was negatively correlated with scores of pulmonary disease activity and systematic disease activity.
Th2 cytokine production, including IL-4, IL-5, and IL-13, evokes strong antibody responses and eosinophil accumulation, which are involved in the pathological process of type 2 immune-induced fibrosis [13,15]. In fact, these cytokines have distinct roles in the regulation of fibrosis. The levels of IL-4 were increased in the serum and bronchoalveolar lavage fluid (BALF) of rheumatoid arthritis (RA) patients with ILD [24]. Some researchers have reported that IL-4 is almost twice as powerful as transforming growth factor-β (TGF-β) in mediating fibrosis [23]. Interestingly, IL-13 has greater fibrogenic activity than IL-4, and both cytokines use the same signal transducer and signaling pathway [23]. IL-13 deficiency or defective IL-13 signaling reduced mouse models of lung fibrosis, but overexpression of IL-13 increased lung fibrosis [8,23]. Increased IL-13 was observed in the alveolar lavage fluid and lung tissue in patients with IPF [23]. However, it was disappointing that an IL-13 receptor antagonist (lebrikizumab) did not improve pulmonary function in patients with IPF in phase II clinical trials [25]. In addition, basophils, mast cells, eosinophils, and ILC2s interact with Th2 cells to accelerate the process of fibrosis [23,26]. In our research, IIMs-ILD patients had a considerable increase in Th2 cells and a decrease in the ratio of Th1 to Th2 cells, suggesting that Th2 cells are critical in the pathogenesis of IIMs-ILD, hence making targeting Th2 cell therapy possible for new treatment in patients diagnosed with IIMs-ILD. For further exploration between Th1/Th2 and fibrosis, patients with IIMs-ILD were analyzed in subgroups according to HRCT. The NSIP pattern is commonly found in interstitial lung disease due to IIMs and is associated with a better response to immunosuppressive therapies and a more favorable prognosis [27]. Several studies have reported that, compared with UIP, NSIP is characterized by an elevated Th1 type, both on histology and bronchoalveolar lavage [27]. However, our study found that, compared with HC, increased Th2 cells and an extremely decreased ratio of Th1 to Th2 cells seemed in the peripheral blood of patients with NSIP; nevertheless, there was no significant difference in comparison with patients with UIP. On the one hand, discrepancies in the expression of peripheral blood and tissue cells may contribute to this; on the other hand, the lack of patients in the UIP group may affect the result. We also analyzed the discrepancy in the expression of peripheral blood in IIMs-ILD patients with different serological myositis-specific antibodies. We found that there was a decreased ratio of Th1 to Th2 in patients with positive results for anti-MDA5 antibodies and ARS. Decreased Th1 cells are a striking feature in patients with positive results for anti-MDA5 antibodies. Anti-MDA5 antibody, which is specific to patients with DM and is correlated with ILD and RPILD, is an independent risk factor for death [5]. The rapid progression and poor prognosis of DM patients with this subtype are difficulties in clinical treatment [5]. Consistent with our study, a study reported that ILD patients with poor prognosis and anti-MDA5 antibody-positive patients, indicating higher disease activity, frequently had a low ratio of IL-4 to IFN-γ [18].
The effects of Treg cells remain controversial in fibrosis [8]. Treg cells induce collagen fiber deposition by promoting the growth and repair of TGF-β, while they reverse fibrosis by producing IL-10, modulating the inflammatory response [8]. A study involving DM/PM reported that the decline in Treg cells was strongly correlated with the presence of ILD [21]. Th17 cells promote fibrosis by secreting IL-17A [8]. In addition, plateletderived growth factors produced by Th17 cells participate in angiogenesis, accordingly promoting fibrosis [8]. However, there were no significant differences in Treg cells or Th17 cells in IIMs-ILD patients. This may be because IIMs are a group of heterogeneous diseases, the different clinical subtypes of the disease may vary, and our research contains more clinical subtypes.
The effects of T cells in the process of fibrosis are complicated. T cells interact with lung epithelial cells and fibroblasts, which play different roles in different stages of fibrosis. In patients with ILD due to different connective tissue diseases, the expression of T cells varies. A study including 142 patients with Sjögren's syndrome indicated that decreased Th1 cells and an increased ratio of Th1 to Th2 cells were risk factors that could lead to ILD in patients with Sjögren's syndrome [28]. Th2 cytokines-IL4-are critical in ILD patients with RA (RA-ILD) and systemic sclerosis (SSc-ILD) [24,29]. Our research supported the view that Th1 cells suppress fibrosis and Th2 cells promote fibrosis, highlighting the potential effects of Th1 cells in the pathophysiological process of IIM patients with anti-MDA5 antibody positivity. Apart from this, the treatment of Th2 cell regulation may be a novel direction for IIMs-ILD patients with NSIP.
Our study suggests that CK levels are negatively correlated with ILD in patients with IIMs, which is consistent with the results of previous reports [30,31]. These reports indicate that IIM patients with higher CK levels (severe muscle involvement) have less involvement of extramural organs, such as immune-mediated necrotizing myopathy; patients with IIMs with lower CK levels (slight muscle involvement) have more severe skin and lung involvement, such as amyopathic dermatomyositis. The above evidence suggests that CK may be a regulator of organ involvement in IIMs. In addition, some studies have confirmed that ARS or anti-Ro52 positivity is associated with IIMs-ILD [30,32]. However, the anti-Ro52 antibody often co-occurs with ARS, so confounding bias due to ARS needs to be excluded. Therefore, more studies and a larger sample size are needed to verify this result.
There are some limitations in our research. First, this was a single-center cross-sectional study. Second, immunosuppressive therapy may alter the levels of circulating cells, which requires us to initiate studies in untreated patients or animals. Finally, the sample size was insufficient, which may be the reason why most of the correlations were weak or moderate. There were many weak or moderate associations in this study, which may require a larger sample size to better understand the association between indicators. Therefore, the above results warrant confirmation in a larger cohort study.

CONCLUSIONS
In this study, we found that a decrease in CK and Th1 cells, an increase in Th2 cells, and positivity for ARS or anti-Ro52 in peripheral blood were associated with the development of ILD in patients with IIMs. Moreover, Th1 and Th2 cell polarization was different in IIMs-ILD patients with different serological and imaging results, providing potential therapeutic targets for precision therapy.

AUTHOR CONTRIBUTION
LY, TCY, and LYB conceived and designed the study. LY and TCY guided the study. CL, LYH, ZY, WJ, WYL, LXP, and WT collected the clinical samples and assessed the disease activity. CL, LYH, LYB, ZY, WYL, WJ, and LXP performed flow cytometry and analyzed the data. All authors drafted and revised the manuscript. All authors drafted and revised the manuscript. Lu Cheng and Yanhong Li contributed equally to this work.

FUNDING
This work was supported by the National Natural Science Foundation of China (82001728,81973540), the 1·3·5 project for Outstanding interdisciplinary project of West China Hospital, Sichuan University (ZYGD18015, ZYJC18003, ZYJC18024), the Sichuan Science and Technology Program (20YYJC3358), and the zero to one Innovative Research Program of Sichuan University (2022SCUH0020).

DATA AVAILABILITY
The data supporting the conclusions of this article are included within the article and its additional file.

DECLARATIONS Ethics Approval and Consent to Participate
The study was approved by the ethical committee of West China Hospital of Sichuan University (no. 695 in 2020) and complied with the Declaration of Helsinki. The study did not involve animal studies, and no ethical approval was needed.

Consent for Publication
All patients and controls provided written informed consent.