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

doi:10.21203/rs.3.rs-2010568/v1

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

https://doi.org/10.21203/rs.3.rs-2010568/v1

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Objective.

Interstitial lung disease (ILD) is a common and highly fatal manifestation of idiopathic inflammatory myopathies (IIMs). T helper (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.

Methods.

11 healthy controls (HC) and 57 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.

Results.

The proportion of Th1 cells decreased and Th2 cells increased in IIMs-ILD compared with IIMs-non-ILD patients (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. Th1 cells proportion decreased in anti-MDA5 antibodies positive patients, while the Th2 cells proportion increased in patients with nonspecific interstitial pneumonia, compared with IIMs-non-ILD patients(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). Th1 proportion decreased, Th2 proportion increased, positivity for ARS or anti-Ro52 antibodies (OR = 0.7122; OR = 1.679; OR = 9.188 and OR = 6.161, respectively) were associated with the occurrence of ILD in IIMs patients.

Conclusion.

A decreased Th1 cell percentage and an elevated Th2 cells percentage in peripheral blood may be the pathogenesis of ILD in IIMs patients and have different effects on different serological and imaging subtypes.

Interstitial lung disease (ILD)

idiopathic inflammatory myopathies (IIMs)

Th 1 cells

Th2 cells

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 interaction 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 the lymphocyte, 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 that how T cells regulate fibrosis are not completely understood, and the factors related to ILD in patients with IIMs are unknown. And 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 IIMs 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 IIMs exclusion criteria and were age- and gender-matched to the IIMs group. We enrolled 53 patients who were diagnosed with IIMs, 30 and 23 were complicated with and without ILD, respectively, based on high-resolution computed tomography (HRCT).

Clinical assessment of patients

The clinical data were obtained from medical records at the time the serum samples were obtained. It contained demographic data and laboratory results. Myositis specific antibodies refer to aminoacyl tRNA synthetase (ARS, anti-PL-7，anti-PL-12，anti-Jo1，anti-EJ，anti-OJ), anti-NXP2, anti-MDA5, anti-SAE, anti-SRP, anti-HMGCR, anti-TIF1γ.

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].

Evaluation of ILD

We assessed the physical functions related to daily living activities of the subjects by the patient Health Assessment Questionnaire (HAQ), which was downloaded from the IMACS official website and evaluated according to its scoring principle[11]. The 6-minute walking test (6MWT) was performed according to the experimental guidelines reported in the literature[12].

Evaluation of muscle strength

The MMT8 rating scale and detailed instructions of the muscle strength test were downloaded from the IMACS website, and the muscle strength test was performed according to its training video.

Classification of HRCT

The HRCT images were divided into usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP), and organizing pneumonia (OP) according to the guidelines for idiopathic interstitial lung disease[13]. Those that did not meet the above classification criteria were not considered classified by CT (Unclassified). HRCT was blindly evaluated by two experts.

Flow cytometry

8 ml 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 FACS Calibur (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 (Q₂₅, Q₇₅). 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.

Baseline demographic and clinical characteristics

Baseline demographic and clinical characteristics are presented in Table 1. A total of 53 patients and sex-matched healthy controls (HC) (n = 11) were enrolled in the study. The 53 patients were further divided into the IIMs-ILD group (n = 30) and IIMs-non-ILD group (n = 23). The average ages in the HC, IIMs-non-ILD, and IIMs-ILD groups were 50.27 ± 8.15, 46.74 ± 12.33 and 52.77 ± 13.91 years respectively. Compared with the IIMs-non-ILD group, patients in the IIMs-ILD group had higher MYOACT scores (0.33 ± 0.13 vs. 0.23 ± 0.12, P = 0.007) and more respiratory symptoms, such as cough (p < 0.001), expectoration (p = 0.007), dyspnea (p = 0.087) and Velcro rales.

Table 1

Clinical manifestations and laboratory data of IIMs-non-ILD, IIMs- ILD and HCs.
Characteristics	HC(n = 11)	IIMs-non-ILD(n = 23)	IIMs-ILD(n = 30)	p
Demographic data
Age(years), mean ± SD	50.27 ± 8.15	46.74 ± 12.33	52.77 ± 13.91	0.231
Female sex, no./total no(%)	9/11(81.82)	14/23(60.87)	22/30(73.33)	0.404
BMI(kg/m²), mean ± SD		22.31 ± 2.96	22.50 ± 2.86(n = 27)	0.811
Smoking, no./total no(%)
Nonsmoker		17/23(73.91)	26/30(86.67)	0.437
Ex-smoker		4/23(17.39)	2/30(6.67)
Current smoker		2/23(8.70)	2/30(6.67)
Disease duration(months), median(range)				/
IIMs		9(3, 36)	7.5(3.75, 24)	0.76
ILD		/	4(1, 15)	/
Disease evaluation
HAQ(score), median(range)		0.60(0.20, 1.20)	0.70(0.31, 1.44)^*	0.556
6MWD pred(%), mean ± SD		0.64 ± 0.19^§	0.69 ± 0.24 ^ʄ	0.738
MMT(score), median(range)		0.80(0.61, 0.89)	0.85(0.70, 0.98)^**	0.147
MYOACT (score), mean ± SD		0.23 ± 0.12	0.33 ± 0.13	0.007
MITAX (score), mean ± SD		0.24 ± 0.12	0.28 ± 0.17	0.267
Clinical manifestation, no./total no(%)
Fever		0/23	6/30(25.00)	/
Weak		23/23(1.00)	20/30(66.67)	/
Muscle weakness		16/23(69.57)	19/30(63.33)	0.855
Loss of weight		8/23(34.78)	9/30(30.00)	0.942
Cough		2/23(8.70)	16/30(53.33)	༜0.001
Expectoration		1/23(4.35)	11/30(36.67)	0.007
Dyspnea		6/23(26.09)	16/30(53.33)	0.087
Velcro rale		0/23	14/30(46.67)	/
Arthritis		5/23(21.74)	6/30(20.00)	1.00
Dysphagia		6/23(26.09)	5/30(16.67)	0.501
Rash		8/23(34.78)	19/30(63.33)	0.075
Laboratory data
CRP (mg/L), median(range)		4.29(2.35, 6.61)^§§	3.82(2.27, 10.4)^ʄʄ	0.94
Triglyceride(mmol/L), median(range)		1.97(1.67, 2.65)	1.83(1.32, 2.68)	0.243
Cholesterol(mmol/L), median(range)		4.94 ± 1.21	4.92 ± 1.32	0.953
Hb(g), median(range)		133.00(129.00, 135.00)	125.00(118.75, 137.75)	0.184
PLT(×10⁹/L), median(range)		177.00(120.00, 249.00)	199.50(140.00, 225.25)	0.584
WBC(×10⁹/L), median(range)		6.51(5.18, 10.86)	7.08(4.85, 9.58)	0.795
Lymphocyte(×10⁹/L), median(range)		1.59(1.13, 2.33)	1.14(0.82, 1.60)	0.028
CD3 (mm³), median(range)^***		1018.00(635.50, 1624.50)	780.00(501.75, 997.00)	0.105
CD4(mm³), mean ± SD^***		649.54 ± 375.41	431.5 ± 229.56	0.0398
CD8 (mm³), median(range)^***		346.00(209.50, 474.00)	249.00(167.75, 341.25)	0.133
CK (IU/L), median(range)		1311.00(135.00, 3792.00)	108.00(45.00, 704.75)	0.006
LDH (IU/L), median(range)		358.00(239.00, 709.00)	297.50(223.75, 394.75)	0.092
HBDH (IU/L), median(range)		289.00(188.00, 550.00)	221.00(176.50, 322.25)	0.118
IgG (g/L), median(range)		11.00(8.72, 13.80)	14.90(10.33, 17.45)	0.042
IgA (g/L), median(range)		1.85(1.28, 3.35)	2.47(1.61, 3.05)	0.467
IgM (g/L), median(range)		1.06(8.47, 1.52)	1.53(9.80, 2.31)	0.136
C3 (g/L), mean ± SD		0.87 ± 0.17	0.84 ± 0.16 ^§§§	0.521
C4 (g/L), median(range)		0.23(0.16, 0.27)	0.20(0.17, 0.23)^§§§	0.09
ANA(+), no./total no༈%༉		16/23(69.57)	19/30(63.33)	0.855
Anti-Ro-52(+), (no./total no༈%༉)		8/23(34.78)	23/30(76.68)	0.0005
MSA(-), (no./total no༈%༉)		8/21(38.10)	6/30(20.00)	0.705
MDA5(+), (no./total no༈%༉)		2/21(9.52)	9/30(30.00)	0.098
HMGCR/SRP(+), (no./total no༈%༉)		7/21(33.33)	1/30(3.33)	/
ARS(+), (no./total no༈%༉)		2/21(9.52)	14/30(46.68)	0.006
NXP2(+), (no./total no༈%༉)		1/21(4.76)	0/30	/
Mi2(+), (no./total no༈%༉)		4/21(19.05)	2/30(6.67)	0.214
TIF1γ(+), (no./total no༈%༉)		1/21(4.76)	1/30(3.33)	/
Pulmonary function^ʄʄʄ
DLCO pred (%), mean ± SD		107.33 ± 17.50	62.45 ± 19.12	< 0.001
FVC pred(%), median(range)		102.05(97.90, 106.95)	80.75(64.45, 98.15)	0.026
VC pred(%), median(range)		101.25(96.20, 108.63)	77.80(64.20, 96.68)	0.029

Data are presented as the mean±SD, median and range, n (%) or n/N (%).

*n=24; §n=14; ʄn=14; **n=24; §§n=20; ʄʄn=27; ***IIMs-non-ILD n=13, IIMs-ILD n=22; §§§n=29; ʄʄʄ IIMs-non-ILD n=4, IIMs-ILD n=12.

CRP, C-reactive protein; Hb, Hemoglobin; PLT, Platelets; WBC, White blood cell count; CK, Creatine kinase; LDH, Lactic dehydrogenase; HBDH, Hydroxybutyrate dehydrogenase; ANA, Antinuclear antibodies; MAS, Myositis-specific antibody; ARS, Anti-aminoacyl tRNA synthase; DLCO pred, Percentage of pulmonary carbon monoxide dispersion to estimated value; FVC pred, Percentage of forced lung capacity to estimated value; HAQ, Health assessment questionnaire; 6MWD pred, Percentage of 6-minute walking distance to estimated value; MMT8, Manual muscle strength score of 8 sites; MYOACT, Myositis disease activity assessment visual analog scales; MITAX, myositis intention to treat activity index.

The numbers of lymphocytes (p = 0.028) and CD4 (p = 0.0398) and the levels of CK (p = 0.006) were significantly lower in IIMs-ILD than in IIMs-non-ILD patients. Meanwhile, the frequency of autoantibody positivity, such as anti-Ro52 (23/30(76.68%)vs. 8/23 (34.78%), P = 0.0005) and ARS (14/30 (46.68%) vs. 2/21 (9.52%), P = 0.0005) were significantly higher in patients with ILD than in patients without ILD. Importantly, IIMs-ILD patients had lower pulmonary function (i.e., forced vital capacity (FVC), vital capacity (FVC), and diffusing capacity of the lung for carbon monoxide (DLCO)) than those without ILD (P < 0.05).

CD4+ T lymphocyte cell subsets in peripheral blood

The role of CD4+ lymphocyte cell subsets in the presence of IIMs-ILD patients was studied by analyzing patients with and without ILD and HC. The proportion of Th1 cells was significantly lower in IIMs-ILD patients than in HC and IIMs-non-ILD groups(2.99 (1.59–5.39) vs.7.84 (5.38-8.97) vs. 6.91 (3.48–10.04), p <0.001) (Figure 1a). In contrast, compared with IIMs-non-ILD, the proportion of Th2 cells was significantly increased in IIMs-ILD patients (2.67 (1.79–4.67) vs. 1.62 (0.85–2.66), p =0.006), and there were no significant differences in HC (Figure 1b). There was no significant difference in Tregs or Th17 cells between IIMs-ILD patients and IIMs-non-ILD patients (Figure 1c, 1d).

These data showed that Th1 and Th2 cells may play a major role in the progress of ILD in IIMs patients. Our analysis further investigated whether there was any difference in the ratio of Th1 to Th2 cells, Th1 to Treg, Th2 to Treg cells, and Th17 to Treg cells among the three groups. We found that the Th1/Th2 ratio was significantly lower in patients with ILD than in patients without ILD (0.99 (0.51–2.03) vs. 4.00 (2.20–6.56), p<0.001) (Figure 1e). Meanwhile, the ratio in patients with ILD was also lower than that in HC (0.99 (0.51–2.03) vs.2.73(2.37-3.56), p=0.0645) (Figure 1e). The Th1/Treg ratio was decreased in IIMs-ILD patients compared with IIMs-non-ILD patients, while the difference between IIMs-ILD patients and HC was not statistically significant (Figure 1f). The Th2 to Treg ratios did not differ significantly among the three groups (Figure 1g). The Th17 to Treg ratio was higher in the IIMs-non-ILD group than in HC and IIMs-ILD groups (1.42(0.82–2.30)vs.0.38(0.19-0.59),vs.0.48(0.28-1.28), p=0.003) (Figure 1h).

Since autoantibodies are associated with IIMs, such as anti-MDA5, ARS affects the prognosis, patient survival, and treatment response in patients with IIMs-ILD [1]. We analyzed whether there was any difference in the proportion of Th1 and Th2 cells or the ratio of Th1 to Th2 cells between patients who tested positive for these antibodies in the three groups. As shown in Figure 2a, the percentage of Th1 cells was significantly decreased in anti-MDA5-positive IIMs-ILD patients compared with IIMs-non-ILD patients and HC (2.66(1.06–4.35) vs. 6.91 (3.48–10.04), p<0.05). There were no significant differences in the proportion of Th2 cells among the three groups (Figure 2b, P > 0.05).The ratio of Th1 to Th2 cells in anti-MDA5-positive IIMs-ILD patients and ARS-positive IIMs-ILD patients was lower than that in IIMs-non-ILD patients (1.00(0.51–2.08) vs. 4.00 (2.20–6.56), p<0.05; 1.24(0.83–2.69) vs. 4.00 (2.20–6.56), p<0.05) (Figure 2c).

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]. 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 (Figure 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) (Figure 2e). Meanwhile, the ratio of Th1 to Th2 cells was significantly lower in patients with the NSIP pattern than in IIMs-non-ILD patients (1.00(0.45–2.71) vs. 4.00 (2.20–6.56), p<0.05)( Figure 2f).

Correlation between Th1, Th2, Th1/Treg ratio and Clinical Characteristics

As shown in Figure 3, the percentage of Th1 cells was positively correlated with the percentage of actual 6-minute walking distance to the estimated value (6MWD pred %) (r =0.380, p = 0.035). Moreover, the percentage of Th1 cells was negatively correlated with MITAX pulmonary disease activity (r = - 0.278, p = 0.047), MYOCAT pulmonary disease activity (r =-0.447, p <0.001), MYOCAT global disease activity (r =-0.314, p =0.024), HAQ (r =-0.350, p =0.013), and the levels of KL-6 (r =-0.575, p =0.031), IL-6 (r =-0.650, p =0.016), and CRP (r =-0.327, p =0.030) in all IIMs patients. In contrast, the percentage of Th2 cells was positively correlated with the level of CRP (r =0.154, p =0.024), MITAX pulmonary disease activity (r =0.307, p = 0.030) and MYOCAT pulmonary disease activity (r =0.282, p =0.041) in all IIMs patients. In addition, the ratio of Th17 to Treg cells was positively correlated with lung function FEV1/FVC (r =0.656, p =0.003) in all IIMs patients. The ratio of Th1 to Th2 cells, Th1 to Treg cells, and Th17 to Treg cells was not correlated with disease activity or inflammatory marker levels in all IIMs patients.

Factors affecting ILD in patients with IIMs

Furthermore, we used logistic regression models to explore how the factors correlated with ILD in IIM patients. As Figure 4 illustrates, univariate logistic regression analyses suggested that the levels of CK (OR=0.9995, 95% CI= (0.9990-0.9999), P=0.0059), the number of lymphocytes (OR= 0.3819, 95% CI= (0.1355-0.8972), P= 0.0479), positivity for anti-Ro52 (OR= 6.161, 95% CI= (1.923-21.84), P= 0.0031) and ARS (OR=9.188, 95% CI= (2.164-64.05), P=0.0072), the percentage of Th1 cells (OR=0.7122, 95% CI= (0.5551-0.8701), P=0.0028), and Th2 cells (OR=1.679, 95% CI= (1.164-2.648), P=0.012), and the ratio of Th1 to Th2 cells (OR=0.7155, 95% CI= (0.5246-0.8927), P=0.0142) were associated with ILD in IIMs.In addition, the levels of CK (OR=0.9993, 95% CI= (0.9986 to 0.9998), P=0.0257) that were found on multivariable analysis were associated with decreased ILD in IIMs patients. Concurrently, positivity for ARS (OR=13.55, 95% CI= (1.260 to 384.4), P=0.0568), the percentage of Th1 cells (OR=0.7198, 95% CI= (0.4792 to 0.9670), P=0.0614) and Th2 cells (OR=2.154, 95% CI= (1.064 to 5.893), P=0.0700) were related to ILD in IIMs patients and achieved borderline statistical significance.

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 DM-RPILD 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. It 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[15, 13]. 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 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, 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 is 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 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, platelet-derived growth factors produced by Th17 cells participate in angiogenesis, accordingly promoting fibrosis[8]. However, there were no significant differences in Treg cells, Th17 cells in IIMs-ILD patients. This may be due to the fact that IIMs is a group of heterogeneous diseases, and 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 IIMs 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.

There are some limitations in our research. Firstly, this is a single-center cross-sectional study. Secondly, immunosuppressive therapy may alter the levels of circulating cells, which requires us to initiate studies in untreated patients or animals. Finally, the sample size is insufficient. Therefore, the above results warrant confirmation in a larger cohort study.

Our study confirmed that positivity for anti-Ro52 antibody, anti-synthetase antibodies, and increased Th2 cells in peripheral blood are independent risk factors in patients with IIMs-ILD. Moreover, Th1 and Th2 cell polarization was different in IIMs-ILD patients with different serological and imaging, providing potential therapeutic targets for precision therapy.

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, that no ethical approval is required.

Consent for publication

All patients and controls provided written informed consent.

Availability of data

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

Competing interests

All authors declare that they have no conflict of interest.

Funding

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

Authors' information

Lu Cheng, MD^{1, 2,3†}, Yanhong Li, PhD^{1, 2,3†}, Yubin Luo, PhD^{1, 2,3},Yu Zhou, MD⁴, Ji Wen, PhD^{1, 2,3}, Yinlan Wu, MD^{1, 2,3}, Xiuping Liang, PhD^{1, 2,3}, Tong Wu, PhD^{1, 2,3}, Chunyu Tan, PhD^{1, 2,3*}, Yi Liu, PhD^{1, 2,3*}

¹Department of Rheumatology and Immunology, West China Hospital, Sichuan University, Chengdu, China.²Rare Diseases Center, West China Hospital, Sichuan University, Chengdu, China.³Institute of Immunology and Inflammation, Frontiers Science Center for Disease-related Molecular Network, West China Hospital.⁴ Department of Respiratory and Critical Care Medicine, Chengdu First People’s Hospital, Chengdu, China

^†These authors contributed equally.

^* Corresponding authors: Chunyu Tan, PhD, and Yi Liu, PhD, Department of Rheumatology and Immunology, West China Hospital, Sichuan University, No.37 Guoxue Alley; Chengdu 610041, China. Email: [email protected] (Chunyu Tan) and [email protected](Yi Liu)

Authors' contributions

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.

Acknowledgements

Not applicable

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

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