Type 2 cytokines and scleroderma interstitial lung disease

Interstitial lung disease (ILD) is a life-threatening complication of systemic sclerosis (SSc). Type 2 (Th2) cytokines play a pivotal role in airway disease. Study aim was to evaluate serum level of Th2 interleukin (IL) and chemokine in SSc-ILD. Serum levels of IL-4, IL-5, IL-11, IL-13, IL-21, IL-31 and CXCL-13 were measured by Bio-Plex Multiplex Immunoassays in 60 SSc patients and 20 healthy controls (HC). Pulmonary function tests with diffusion lung capacity for carbon monoxide (DLco) and high resolution computed tomography (HRCT) were performed in SSc patients. ILD is defined as fibrotic changes (ground glass, reticular and honeycombing), assessed by Computer-Aided Lung Informatics for Pathology Evaluation and Ratings (CALIPER) software, affecting at least 10% of the lungs. Serum levels of Th2 cytokines were higher in SSc patients than HC. A linear correlation was observed between ground glass and IL-13 (r = 0.342, p < 0.01), IL-21 (r = 0.345, p < 0.01), IL-31 (r = 0.473, p < 0.001), IL-4 (r = 0.863, p < 0.001), IL-5 (r = 0.249, p < 0.05) and peripheral blood eosinophils (r = 0.463, p < 0.001). We found a negative correlation between DLco and IL-4 (r =  − 0.511, p < 0.001) and peripheral blood eosinophils (r =  − 0.446, p < 0.001). In the logistic regression analysis, IL-4 is associated with DLco ≤ 60% of the predicted [OR 1.039 (CI 95%: 1.015–1.064), p < 0.001], whilst mRSS [OR 1.138 (CI 95%: 1.023–1.266), p < 0.05] and IL-4 [OR 1.017 (CI 95%: 1–1.034), p < 0.05] were associated with ILD. Th2 inflammation could play a key role in early phase of SSc-ILD.


Introduction
Systemic sclerosis (SSc) is an autoimmune disease characterized by immune system dysregulation, endothelial dysfunction and fibrosis of skin and internal organs.Interstitial lung disease (ILD) is common in SSc and about 80% of patients will develop ILD during the disease course [1].Although most patients have a stable or slowly progressive disease, showing a slow decline in lung function or a minimal increase in the extent of pulmonary fibrosis by high resolution computed tomography (HRCT), 25-30% of them will ultimately progress to respiratory failure or death [2].Of the SSc-related deaths, 35% were attributed to ILD [3].
The underlying pathophysiologic mechanisms of SSc-ILD is still not completely understood [2].The pathogenesis of SSc-ILD is initially characterized by an injury to alveolar epithelial and vascular endothelial cells which promotes the activation of the immune system and the recruitment of inflammatory cells and the production of profibrotic mediators, in the attempt to repair the damage, with accumulation of myofibroblasts [4,5].Infiltration by inflammatory T cells is thought to precede the development of lung fibrosis and in this early inflammatory phase, profibrotic type 2 (Th2) cytokines, such as IL-4, IL-5, IL-13, play a key role [6,7].Indeed, an abnormal profibrotic Th2polarized T cell response has been postulated to mediate tissue damage and fibrosis in SSc-ILD as Th2 cytokines lead to the activation of alternative inflammatory pathways and to the transcription of transforming growth factor (TGF)-β, involved in induction and progression of fibrosis [8,9].Moreover, it has been demonstrated that IL-4/IL-13 axis upregulate genes known to be involved in the mechanisms of wound healing and fibrosis in murine models and in vitro, influencing the activation of myofibroblasts [10].
Th2 cytokines also contribute to the differentiation and migration of eosinophils, which are involved in systemic inflammatory process in SSc patients [11][12][13].In the earlier course of ILD, pulmonary function tests (PFTs) show a reduction of the diffusion lung capacity for carbon monoxide (DLco) with preserved forced vital capacity (FVC) and HRCT shows a ground glass pattern with typical subpleural and posterobasal distribution [14].At a later stage, fibroblasts proliferation and activation lead to extracellular matrix deposition and fibrosis of the lung parenchyma.PFTs show a restrictive pattern with a further decline of DLco and a reduction of FVC [14].HRCT shows a reticular pattern with intralobular opacity and thickening of the interlobular septa and, eventually, a honeycombing pattern with traction bronchiectasis [14].Aim of the study was to evaluate the Th2 cytokines serum levels in SSc-ILD.

Subjects
Sixty SSc patients, fulfilling the American College of Rheumatology/European League Against Rheumatism Collaborative Criteria (2013 ACR/EULAR) for SSc [15] and twenty healthy controls (HC), matched for sex and age and recruited among healthcare workers, were enrolled in this study.
Cardiopulmonary diseases not related to SSc, pulmonary arterial hypertension (PAH), heart and hepatic failure, end stage renal diseases, malignancies, allergic diseases were exclusion criteria.Smokers, pregnant or breastfeeding women and patients treated in the last 6 months with immunosuppressive agents and corticosteroids at an equivalent dose of prednisone ≥ 10 mg/day were also excluded.
The study was conducted according to the Declaration of Helsinki and all participants provided written informed consent.The study was approved by the ethics committee of Sapienza University (IRB 0304).

Clinical assessment
The modified Rodnan skin score (mRSS) was used to assess skin involvement and the disease subset was assessed according to LeRoy et al. [16] Disease activity and disease severity were measured by the disease activity index (DAI) [17] and disease severity scale (DSS) [18], respectively.

Nailfold videocapillaroscopy
Nailfold videocapillaroscopy (NVC) was performed at the level of the distal phalanx of the second, third and fourth fingers of both hands using a videocapillaroscope equipped with a 500 × magnification lens (Pinnacle Studio Version 8 software, Corel, Ottawa, Canada).According to Cutolo et al. [19], the capillaroscopic images were classified in early, active, and late pattern.The NVC was performed by the same experienced operator, blinded for laboratory assessment and for other clinical characteristics of SSc patients.

Pulmonary function tests
PFTs parameters [forced expiratory volume in the 1st second (FEV1), FVC] and single-breath DLco were recorded by the same blinded experienced operator with a Quark PFT 2 spirometer (Cosmed, Rome, Italy) and expressed according to the standards recommended by the American/ European Respiratory Society [20].PFTs were performed at enrollment in this study.

Chest high-resolution computed tomography
The HRCT performed during the study or in the previous 6 months were evaluated by the same expert radiologist.ILD was defined as fibrotic changes affecting at least 10% of the lung parenchyma according to previous study [21].Radiological patterns (normal, ground glass, reticular, and honeycombing) were measured and expressed as percentage of total lung parenchyma through Computer-Aided Lung Informatics for Pathology Evaluation and Ratings (CALIPER) software by the same blinded radiologist [22].

Laboratory assessment
Peripheral venous blood samples were collected in tubes and centrifuged at 3000 × g for 15 min at 19 °C.Serum samples were aliquoted into 1.5 mL Eppendorf tubes and stored at − 80 °C until the time of assay.The assessment of serum levels of IL-4, IL-5, IL-11, IL-13, IL-21, IL-31, CXCL-13 cytokines was carried out using a Bio-Plex Multiplex Immunoassays allow to simultaneously measure analytes available as ready-to-use premixed multiplex panels (BIO-RAD, Hercules, CA, USA).All blood tests were performed in a single analytical session, following the instructions provided by the manufacturers.Each sample was tested twice to minimize eventual discrepancies, and all tests were performed in the same laboratory with the same instrument by the same experienced operator, blinded of the clinical information of the handled sample.

Statystical analysis
SPSS version 26.0 software (Bioz, Los Altos, CA) was used for statistical analysis.After an evaluation of normality by using the Shapiro-Wilk test, continuous variables were expressed as median and interquartile range (IQR).Student's or Mann-Whitney's t-test were performed to evaluate differences between groups.Differences between categorical variables were evaluated by the chi-square or Fisher's exact test, as appropriate.The Pearson or Spearman correlation test was used for bivariate correlations.

Discussion
In this study, we demonstrated increased Th2 cytokines serum levels in SSc patients compared to HC.Moreover, we found a positive linear correlation between serum Th2 cytokines and the extent of ground glass pattern assessed by CALIPER software in SSc patients.Finally, IL-4 was associated with DLco ≤ 60% of the predicted and with fibrotic involvement of ≥ 10% of lung parenchyma in SSc patients.
In our study, we demonstrated that serum level of Th2 interleukin and chemokine are increased in SSc patients than HC.In SSc pathogenesis, the role of Th2 inflammation is misunderstood.The role of inflammation, especially Th2 inflammation, in the SSc ILD has long been debated [23,24].Th2 cytokines, released in early ILD inflammatory phase, lead to the activation of alternative inflammatory pathways and to the transcription of TGF-β, involved in induction and progression of fibrosis [9].According to previous studies [11,25], we demonstrated increased Th2 cytokines serum levels in SSc patients compared to HC.Moreover, and more importantly, we found a positive linear correlation between the percentage of ground glass on HRCT and Th2 cytokines serum levels (IL-4, IL-31, IL-21, IL-13 and IL-5) in SSc patients.IL-21 seems to stimulate a Th2 dependent fibrotic response by acting as a trigger on IL-4 and IL-13, indeed it has been shown that there is an association between IL-4 and IL-21/IL-21R mRNAs, suggesting that IL-21 can promote fibrosis by facilitating the development of a Th2 response [26].It is well known that IL-4/IL-13 axis upregulate genes known to be involved in the mechanisms of wound healing and fibrosis in murine models and in vitro [10].IL-13 is required for an effective Th2 immune response and appears to have a direct profibrotic function [24].IL-13 promotes epithelial metaplasia and remodeling of the airways by acting on the same receptor as IL-4, acting directly on the production of TGF-β or stimulating its signaling mediated by Smad [10].Several studies suggested that Th2 cytokines contributed to the SSc pathogenesis and it is well known that some of these cytokines play a key role in the differentiation and migration of eosinophils, which are involved in inflammatory process in SSc patients [13].IL-4 and IL-13 promote the secretion of IL-5 to activate eosinophils inducing fibroblast proliferation and promoting the expression of TGF-β and the epithelial-mesenchymal transition [27].There is evidence that Th2 cells play important roles during the inflammatory/maintenance phase of pulmonary fibrosis.Th2 cytokines IL-4, IL-5, and IL-13 have been causally linked to the development of fibrosis in several chronic inflammatory diseases.Overexpression of IL-4 or IL-13 in the lung of murine models showed a profibrotic role of both cytokines influencing the activation of myofibroblasts [10].IL-5 can also promote fibrosis in the lung by recruiting eosinophils that produce TGF-β, platelet derived growth factor (PDGF), and IL-13.
It is well known the correlation between early ILD inflammatory phase and ground glass pattern on HRCT [28].In this study, for the first time, we demonstrated a correlation between increased Th2 cytokine serum levels and the extent of ground glass pattern on HRCT.We may suppose that Th2 cytokine are involved in pulmonary  inflammation in SSc patients and that have a pivotal role in damage repair mechanisms.Afterwards, Th2 cytokines are involved in induction and maintenance of fibrotic pathways (i.e.Smad) that lead to the activation of TGF-β, PDGF and IL-6 [28].Increased expression of genes regulated by TGF-β has been confirmed in patients with progressive lung fibrosis.In our study, reticular and honeycombing pattern showed no correlation or showed a negative linear correlation with serum IL-4.Therefore, we may suppose that in the advanced stages of ILD profibrotic cytokines prevail, such as IL-6 and TGF-β, and Th2 cytokines are less secreted.Ando et al. [29] demonstrated higher circulating eosinophils in SSc patients compared to patients with other systemic autoimmune diseases and they found an association between eosinophils count and severity of radiological ILD only in SSc patients.In our study we demonstrated a positive linear correlation between circulating eosinophil count and ground glass pattern measured by CALIPER software.
We also found a negative linear correlation between DLco and IL-4, IL-5, IL-31 and eosinophils.Moreover, in logistic regression analysis, IL-4 was the only variable associated to DLco ≤ 60% of the predicted.A previous study found a negative correlation between elevated IL-31 serum levels and DLco [25].Previous studies demonstrated a negative linear correlation between eosinophils count in bronchoalveolar lavage fluid (BAL) and DLco [30,31].These findings support the role of Th2 inflammation in early stages of ILD characterized by a reduction of DLco with preserved FVC.
This study has some limitations: the monocentric design, the lack of data about Th2 cytokines and eosinophils in BAL and absence of fractional exhaled nitric oxide (FeNO) test.

Conclusions
We can conclude that serum level of Th2 cytokines were higher in SSc patients than HC.Th2 cytokines might have a key role in early phase of ILD in SSc patients and IL-4 was associated with DLco ≤ 60% of the predicted and with fibrotic involvement of ≥ 10% of lung parenchyma in SSc patients.Future large studies are needed to confirm these findings.

Fig. 2
Fig. 2 Bivariate correlations between Th2 cytokines serum levels and diffusing capacity of lung for carbon monoxide (DLco) in systemic sclerosis (SSc) patients.A: Negative linear correlation between IL-4

Fig. 3
Fig. 3 Peripheral blood eosinophil count in systemic sclerosis (SSc) patients and its association with variables of interstitial lung disease (ILD).A: Positive linear correlation between peripheral blood eosinophil count and ground glass CALIPER pattern (r = 0.463, p < 0.001); B: N egative linear correlation between peripheral blood eosinophil

Table 1
Demographic and clinical features of systemic sclerosis (SSc) patients SSc Systemic sclerosis; HC Healthy controls; IQR Interquartile range

Table 3
Logistic regression analysis showing the association between interstitial lung disease (ILD) or Diffusing Capacity of Lung for Carbon Monoxide (DLco) ≤ 60% of the predicted and independent variables mRSS Modified Rodnan skin score; DLco diffusing capacity of lung for carbon monoxide; ILD interstitial lung disease; OR odds ratio; CI confidence interval