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. 2021 Jul 27;16(7):e0255365. doi: 10.1371/journal.pone.0255365

The role of interleukin-6 as a prognostic biomarker for predicting acute exacerbation in interstitial lung diseases

Jae Ha Lee 1, Ji Hoon Jang 1, Jin Han Park 1, Hang-Jea Jang 1, Chan Sun Park 2, Sunggun Lee 3, Seong-Ho Kim 3, Ji Yeon Kim 4, Hyun Kuk Kim 1,*
Editor: Minghua Wu5
PMCID: PMC8315549  PMID: 34314462

Abstract

Background

Interstitial lung diseases (ILDs) are chronic, parenchymal lung diseases with a variable clinical course and a poor prognosis. Within various clinical courses, acute exacerbation (AE) is a devastating condition with significant morbidity and high mortality. The aim of this study was to investigate the role of interleukin-6 (IL-6) to predict AE and prognosis in patients with ILD.

Methods

Eighty-three patients who were diagnosed with ILD from 2016 to 2019 at the Haeundae Paik Hospital, Busan, South Korea, were included and their clinical data were retrospectively analyzed.

Results

The median follow-up period was 20 months. The mean age was 68.1 years and 65.1% of the patients were men with 60.2% of patients being ever-smokers. Among ILDs, idiopathic pulmonary fibrosis was the most common disease (68.7%), followed by connective tissue disease-associated ILD (14.5%), cryptogenic organizing pneumonia (9.6%), and nonspecific interstitial pneumonia (6.0%). The serum levels of IL-6 were measured at diagnosis with ILD and sequentially at follow-up visits. During the follow-ups, 15 (18.1%) patients experienced an acute exacerbation (AE) of ILD and among them, four (26.7%) patients died. In the multivariable analysis, high levels of IL-6 (OR 1.014, 95% CI: 1.001–1.027, p = 0.036) along with lower baseline saturations of peripheral oxygen (SpO2) were independent risk factors for AE. In the receiver operating characteristic curve analysis, the area under the curve was 0.815 (p < 0.001) and the optimal cut-off value of serum IL-6 to predict AE was 25.20 pg/mL with a sensitivity of 66.7% and specificity of 80.6%. In the multivariable Cox analysis, a high level of serum IL-6 (HR 1.007, 95% CI: 1.001–1.014, p = 0.018) was only an independent risk factor for mortality in ILD patients.

Conclusions

In our study, a high level of serum IL-6 is a useful biomarker to predict AE and poor prognosis in patients with ILD.

Introduction

Interstitial lung diseases (ILD) are a heterogeneous group of diffuse parenchymal lung disorders with highly variable clinical courses and poor outcomes [1]. Within variable clinical courses, acute exacerbation (AE) is well-known as a life threatening condition with significant morbidity and high mortality [2, 3]. In terms of AE, the incidence in patients with idiopathic pulmonary fibrosis (IPF) is 5–10% per year with a median survival of less than 3 months [4–6].

Previous studies reported that old age, lower lung function including forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLco), and distances or de-saturation during the six minute walk test (6MWT) were risk factors for AE IPF [7, 8]. However, because of limitations of physiologic parameters such as dependency on patient efforts or interobserver variability, predicting AE remains difficult [9].

Serum biomarkers are relatively easy to measure independently of patient effort or observer ability. Within the pathogenesis of AE-IPF, there have been several reports that cytokines may play an important role [10, 11]. Among cytokines, IL-6 is a soluble mediator with pleiotropic effects on inflammation, immune responses, and fibrosis [12, 13]. In a recent study, Shochet et al. reported in experimental research between IPF patients and normal healthy donors that IL-6 trans-signaling components lead to indirect TGF-β, which is well-known as a pro-fibrotic growth factor with an influence on pathway activation and disease progression, suggesting the importance of IL-6 in IPF pathogenesis [14]. Therefore, our aim in this study was to evaluate the role of IL-6 as a biomarker for predicting AE and prognosis in patients with ILD.

Materials and methods

Study subjects

Eighty-three patients who were diagnosed with ILD at the Haeundae-Paik Hospital, Busan, Republic of Korea, from December 2016 to September 2019, were included in this study. Among the patients with ILD, patients who had serum IL-6 levels measured at diagnosis with ILD and at follow-up visits consecutively every 2–3 months were included. All the patients met the diagnostic criteria in the international guidelines set by the American Thoracic Society (ATS) and European Respiratory Society (ERS) [15, 16]. This study was approved by the Institutional Review Board of the Haeundae-Paik Hospital (approval number: 2019-12-036), and the requirement for written informed consent was waived due to the retrospective nature of this study.

Measurement of IL-6

IL-6 samples were taken at the diagnosis with ILD and sequentially at follow-up visits every 2–3 months. Serum IL-6 concentrations (pg/mL) were measured though an electrochemiluminescence immunoassay (ECLIA) using the Cobas e411 analyzer (Hitachi High-Technologies, Japan).

Clinical information

Clinical data were retrospectively obtained from the medical records at January 3, 2020. Pulmonary function testing, a measurement of the diffusing capacity of the lung for carbon monoxide (DLco), forced expiratory volume in one second, and forced vital capacity (FVC) were performed according to the recommendations of the ATS/ERS [17–19]. The results were expressed as percentages of normal predicted values. The 6-minute walk test was performed according to ATS guidelines [20]. Data from the diagnosis to the follow-up clinic visits, which were conducted normally every 2–3 months, and those from hospitalizations were reviewed to determine the development of AE. The diagnostic criteria for AE-ILD, based on Collard et al., were as follows: 1) previous or concurrent IPF diagnosis; 2) acute worsening or development of dyspnea typically within the past month; 3) high-resolution computed tomography (HRCT) with new bilateral ground-glass opacity and/or consolidation superimposed on a background pattern consistent with typical interstitial pneumonia patterns; and 4) deterioration not fully explained by cardiac failure or fluid overload [21].

Statistical analysis

The data presents the frequency and percentage for categorical variables and mean ± standard deviation (SD) for numeric variables. Differences in study participants’ characteristics were compared across subgroups with a chi-square test or Fisher’s exact test for categorical variables and an independent test or Mann-Whitney’s U test for continuous variables as appropriate. To determine if its distribution was normal, we used the Shapiro-Wilk’s test. Univariate and multivariate analyses, using logistic regression, were performed in order to identify prognostic factors which are independently related to AE. The receiver operating characteristic (ROC) curve analysis was performed to assess the sensitivity and specificity of IL-6 for predicting AE. Overall survival (OS) was estimated using the Kaplan-Meier curve. Survival curves were compared between the groups using the log-rank test. Multivariate analyses, using Cox regression with backward and stepwise elimination, were performed in order to identify prognostic factors which are independently related to mortality. All statistical analyses were carried out using SPSS 24.0 (IBM Corp, Armonk, USA), and p values less than 0.05 were considered statistically significant.

Results

Study population

From December 2016 to September 2019, 405 patients with ILD at Haeundae Paik hospital (Busan, Republic of Korea) were screening for this study. Among them, patients who did not perform IL-6 test or follow up at IL-6 test at least 6 months were excluded (Fig 1). A total of 83 patients with ILD were included in this study. The median follow-up period was 20 months. The mean age of the study population was 68.1 years with 65.1% male patients and 60.2% of patients being ever-smokers. Among ILDs, IPF was the most common (68.7%), followed by connective tissue disease–associated (CTD) ILD (14.5%), cryptogenic organizing pneumonia (9.6%), and nonspecific interstitial pneumonia (6.0%) (Table 1).

Fig 1. Flow chart of patients enrollment.

Fig 1

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ILD, interstitial lung disease; IL-6, interleukin-6.

Table 1. Baseline clinical characteristics of the patients.

Characteristics All Patients (n = 83)
Age, years 68.1 ± 10.5
Male, n (%) 54 (65.1)
Ever smokers, n (%) 50 (60.2)
Height, cm 162.3 ± 8.5
Weight, kg 64.7 ± 10.9
BMI, kg/m2 24.5 ± 3.4
Home O2, n (%) 5 (6.0)
Interstitial lung disease
 IPF, n (%) 57 (68.7)
 CTD, n (%) 12 (14.5)
 COP, n (%) 8 (9.6)
 NSIP, n (%) 5 (6.0)
 HP, n (%) 1 (1.2)
Radiologic pattern
 UIP, n (%) 56 (67.4)
 NSIP, n (%) 10 (12.1)
 OP, n (%) 10 (12.1)
 Indeterminate, n (%) 6 (7.2)
Pulmonary function
 FVC, % predicted 73.9 ± 15.6
 DLco, % predicted 59.1 ± 17.0
 TLC, % predicted 72.5 ± 12.4
 FEV1/FVC, % 77.0 ± 8.5
Six-minute walk test
 Distance, m 402.2 ± 99.0
 Baseline SpO2, % 95.9 ± 2.5
 Lowest SpO2, % 90.6 ± 5.9
BAL
 Total WBC count, cell/μL 2,135.7 ± 3,402.5
 Neutrophil, % 28.6 ± 26.3
 Lymphocyte, % 18.6 ± 21.8
PaO2, mmHg 88.0 ± 28.2
BNP, pg/mL 421.1 ± 734.5
Baseline IL-6, pg/mL 14.9 ± 33.8
CRP, mg/dL 2.5 ± 5.5
WBC, cell/μL 8,094.8 ± 3,041.7
LDH, IU/L 264.8 ± 86.3
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Values are presented as mean ± standard deviation or number (%).

BMI, body mass index; IPF, idiopathic pulmonary fibrosis; CTD, connective tissue disease-associated interstitial lung disease; COP, cryptogenic organizing pneumonia; NSIP, non-specific interstitial pneumonia; HP, hypersensitivity pneumonitis; UIP, usual interstitial pneumonia; OP, organizing pneumonia; FVC, forced vital capacity; DLco, diffusing capacity of the lungs for carbon monoxide; TLC, total lung capacity; SpO2, saturation of peripheral oxygen; BAL, bronchoalveolar lavage; WBC, white blood cell; PaO2, partial pressure of oxygen; BNP, brain natriuretic peptide; IL-6, interleukin-6; CRP, C-reactive protein; LDH, lactate dehydrogenase.

Baseline characteristics

Most patients exhibited a mild restrictive ventilator defect and reduced DLco. In 6MWT, the mean baseline and minimum SpO2 was 95.9% and 90.6%, respectively. The mean baseline level of serum of IL-6 was 14.9 pg/mL. During the follow-up period, 15 patients (18.1%) suffered AE. The AE group exhibited higher levels of C-reactive proteins (CRP) and white blood cells (WBC) with lower levels of baseline SpO2 compared with the no-AE group (Table 2).

Table 2. Comparison of characteristics between ILD patients with and without AE.

Variable AE (+) No AE(-) P-value
(n = 15) (n = 68)
Number of patients, n (%) 15 (18.1) 68 (81.9)
Age, years 70.3 ± 5.8 67.6 ± 11.2 .477
Male, n (%) 10 (66.7) 44 (64.7) .885
Ever smokers, n (%) 7 (46.7) 43 (64.2) .209
BMI, kg/m2 23.9 ± 4.1 24.6 ± 3.3 .368
Interstitial lung disease
 IPF, n (%) 12 (80.0) 45 (66.2) .296
 CTD, n (%) 3 (20.0) 9 (13.2) .500
Pulmonary function
 FVC, % predicted 69.5 ± 13.1 74.9 ± 15.9 .219
 DLco, % predicted 52.5 ± 16.8 60.5 ± 16.8 .179
 TLC, % predicted 76.0 ± 13.4 71.6 ± 12.4 .536
Six-minute walk test
 Distance, m 355.9 ± 101.9 410.8 ± 96.8 .084
 Baseline SpO2, % 94.3 ± 2.3 96.2 ±2.4 .009
 Lowest SpO2, % 88.8 ± 5.5 90.9 ± 5.9 .153
Baseline IL-6, pg/mL 35.5 ± 68.3 10.5 ± 17.5 .057
Peak IL-6*, pg/mL 133.5 ± 205.9 21.1 ± 35.0 < .001
CRP, mg/dL 7.9 ± 9.9 1.2 ± 2.5 < .001
WBC, cell/μL 9,614.0 ± 4,169.3 7,759.7 ± 2,657.2 .024
LDH, IU/L 317.0 ± 147.7 255.1 ± 67.0 .226
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Values are presented as mean ± standard deviation or number (%).

AE, acute exacerbation; BMI, body mass index; IPF, idiopathic pulmonary fibrosis; CTD, connective tissue disease-associated interstitial lung disease; FVC, forced vital capacity; DLco, diffusing capacity of the lungs for carbon monoxide; TLC, total lung capacity; SpO2, saturation of peripheral oxygen; IL-6, interleukin-6; CRP, C-reactive protein; WBC, white blood cell; LDH, Lactate dehydrogenase.

*Peak IL-6 –highest serum level of IL-6 among sequentially measured IL-6 during follow-up.

Predictive factors for AE

Among the 15 (18.1%) patients with AE (idiopathic AE: 11 patients and triggered AE: 4 patients), IPF patients were most common (80%) while the other patients were classified with CTD (20%). In the univariable logistic regression analysis, high levels of serum IL-6 during the follow-up period, CRP, white blood cell (WBC) count, lactate dehydrogenase (LDH), baseline SpO2, and disease duration were significant predictive factors for AE in patients with ILD (Table 3).

Table 3. Predictive factors for AE in ILD patients assessed using the logistic regression model.

Variable Univariate Analysis Multivariate Analysis
OR (95% CI) P-value OR (95% CI) P-value
Age 1.028 (0.969–1.091) .362
Male 1.091 (0.034–3.561) .885
Ever smokers 0.488 (0.158–1.513) .214
BMI 0.942 (0.791–1.122) .501
Disease duration 1.031 (1.005–1.057) .017
Interstitial lung disease
 IPF 0.489 (0.125–1.908) .303
 CTD 0.610 (0.144–2.592) .503
Radiologic pattern
 UIP 0.115 (0.014–0.930) .430
Pulmonary function
 FVC 0.995 (0.940–1.014) .218
 DLco 0.969 (0.934–1.006) .104
 TLC 1.032 (0.939–1.134) .516
Six-minute walk test
 Distance 0.995 (0.989–1.001) .084
 Baseline SpO2 0.738 (0.573–0.952) .019 0.750 (0.568–0.991) .043
 Lowest SpO2 0.948 (0.862–1.042) .265
PaO2 0.994 (0.969–1.019) .628
Baseline IL-6 1.018 (0.999–1.037) .062
*Peak IL-6 1.017 (1.006–1.028) .002 1.014 (1.001–1.027) .036
CRP 1.223 (1.054–1.442) .009
WBC 1.000 (1.000–1.000) .045
LDH 1.007 (1.000–1.013) .049
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OR: odds ratio; CI: confidence interval; BMI, body mass index; IPF, idiopathic pulmonary fibrosis; CTD, connective tissue disease-associated interstitial lung disease; UIP, usual interstitial pneumonia; FVC, forced vital capacity; DLco, diffusing capacity of the lungs for carbon monoxide; TLC, total lung capacity; SpO2, saturation of peripheral oxygen; PaO2, partial pressure of oxygen; IL-6, interleukin-6; CRP, C-reactive protein; WBC, white blood cell; LDH, lactate dehydrogenase.

*Peak IL-6 –highest serum level of IL-6 among sequentially measured IL-6 during follow-up.

In the multivariable analysis, high levels of serum IL-6 (OR 1.014, 95% CI: 1.001–1.027, p = 0.036) during the follow-up period and lower baseline SpO2 (OR 0.750, 95% CI: 0.568–0.991, p = 0.043) were independent predictive factors for AE. In the receiver operation characteristics curves analysis, high levels of serum IL-6 were useful in predicting for AE (area under the curve = 0.815, 95% CI: 0.704–0.927, p < 0.001) in patients with ILD. The best cut-off level was 25.20 pg/mL with a sensitivity of 66.7% and specificity of 80.6% (Fig 2).

Fig 2. Receiver Operating Characteristic (ROC) curve of IL-6 to predict AE.

Fig 2

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IL-6, interleukin 6.

Survival analysis

During the follow-up period, four patients (4.8%) died. All deaths occurred in the AE groups and all causes of mortality were AE ILD. In the AE groups, 2-year and 3-year survival rates were 92.3% and 65.9%, respectively (S1 Fig). In a comparison of survival between high and low levels of IL-6 groups, the survival rate was significantly high in the low IL-6 group (Log rank test, p = 0.018) (Fig 3).

Fig 3. Comparison of Kaplan–Meier survival curves between high and low levels of IL-6.

Fig 3

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IL-6, interleukin 6.

In a univariable Cox analysis, older age, high levels of IL-6, and shorter distances during 6MWT were significant prognostic factors for mortality. However, in multivariable Cox analysis, only high levels of IL-6 were found to be a significant factor affecting overall survival (HR 1.007, 95% CI: 1.001–1.014, p = 0.018) (Table 4).

Table 4. Prognostic factors for mortality in patients with ILD assessed using cox proportional hazards model.

Variable Univariate Analysis Multivariate Analysis
HR (95% CI) P-value HR (95% CI) P-value
Age 1.211 (1.002–1.465) .048
Male 0.206 (0.021–1.993) .172
Ever smokers 0.016 (0.000–51.983) .317
BMI 0.746 (0.504–1.104) .143
Disease duration 0.810 (0.581–1.130) .215
Interstitial lung disease
 IPF 0.667 (0.068–6.505) .728
 CTD 0.857 (0.089–8.294) .894
Radiologic pattern
 UIP 0.031 (0.001–598.0) .491
Pulmonary function
 FVC 1.031 (0.936–1.135) .538
 DLco 1.011 (0.946–1.081) .742
 TLC 1.177 (0.903–1.535) .229
Six-minute walk test
 Distance 0.988 (0.976–0.999) .037
 Baseline SpO2 0.802 (0.502–1.280) .355
 Lowest SpO2 0.992 (0.809–1.217) .938
PaO2 0.998 (0.929–1.072) .955
Baseline IL-6 0.992 (0.953–1.033) .694
*Peak IL-6 1.007 (1.001–1.014) .018 1.007 (1.001–1.014) .018
CRP 0.914 (0.640–1.305) .620
LDH 0.999 (0.985–1.013) .882
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HR: hazards ratio; CI: confidence interval; BMI, body mass index; IPF, idiopathic pulmonary fibrosis; CTD, connective tissue disease-associated interstitial lung disease; UIP, usual interstitial pneumonia; FVC, forced vital capacity; DLco, diffusing capacity of the lungs for carbon monoxide; TLC, total lung capacity; SpO2, saturation of peripheral oxygen; PaO2, partial pressure of oxygen; IL-6, interleukin-6; CRP, C-reactive protein; WBC, white blood cell; LDH, lactate dehydrogenase.

*Peak IL-6 –highest serum level of IL-6 among sequentially measured IL-6 during follow-up.

Discussion

In our study, high levels of serum IL-6 during the follow-up period and lower baseline levels of SpO2 were independent predictive factors for AE in patients with ILD. The best cut-off level of IL-6 to predict AE was 25.20 pg/mL. In addition, high levels of IL-6 during the follow-up period were an independent prognostic factor for mortality in patients with ILD.

Within heterogeneous and diverse clinical courses of ILD, AE is a clinically significant event resulting high mortality. Despite the efforts of many researchers, the actual pathogenesis of AE is not well-known and the mortality of AE remains high. Recently, there have been increasing reports about plasma biomarkers that may be useful for predicting AE in patients with ILD in the pathobiology of AE ILD. Among them, previous studies reported that IL-6 also may be a useful predictor of AE [22, 23]. Firstly, IL-6 is considered a pro-inflammatory cytokine in the acute phase of inflammation, which is produced by various stimuli as well as a promotor specific immune response [24]. In a previous study, the authors suggested that IL-6, as a systemic pro-inflammatory cytokine, played an important role in the innate cells of the pathogenesis of IPF [11]. In addition to the role of IL-6 as a pro-inflammatory cytokine, there were some reports that IL-6 is associated with a fibrotic response and can promote fibrosis by driving chronic inflammation and activating the TGF-β pathway [25]. In a rat model of experimental fibrosis, IL-6 concentrations were shown to be increased in rats and were associated with a proliferative response in fibroblasts [12, 13]. Although the actual role of IL-6 in AE ILD remains elusive, we assumed that IL-6 could be an important plasma biomarker because IL-6 possesses both the nature of pro-inflammatory and pro-fibrotic mediators. Further studies must be performed in order to elucidate the role of IL-6 in patients with AE ILD.

Our study showed that impairment of oxygenation in the resting state was a useful predictor of AE ILD. A previous study also supported our results in this study. In 1,019 patients with ILD (AE group 193, non-AE group 826), Suzuki et al. found that lower levels of partial pressure of oxygen (PaO2) in arterial blood gas in the resting state was an independent risk factor for AE (HR = 0.98, 95% CI: 0.97–0.99, p = 0.013) [26]. In the analysis of INPULSIS trial data to investigate risk factors for AE, Collard et al. reported that among the patients with investigator-reported AE, the use of baseline supplemental oxygen was an independent risk factor for AE (HR = 2.47, 95% CI: 1.37–4.47, p = 0.001) [27]. In 108 patients with IPF, a previous study reported similar results with our study. Okuda et al. showed that the minimum SpO2 level of 88% or less during 6MW was a predictive factor for predicting AE (HR 0.86, 95% CI: 0.80–0.93, p < 0.001) [28]. Therefore, when low baseline levels of SpO2 were observed in addition to the minimal level of SpO2 during 6MWT, we needed to also consider low baseline levels of SpO2 as a risk factor for AE.

In our study, high levels of IL-6 at sequential measurements during the follow-up period was an independent risk factor for AE in patients with ILD. Recently in 41 patients with IPF, Spyros et al. reported that high levels of IL-6 characterized the early diagnosis of AE-IPF (IL-6 between the AE-IPF group vs stable IPF group, 6.2 pg/mL vs 2.1pg/mL, p = 0.002). The authors suggested that the role of pro-inflammatory and pro-fibrotic markers on IL-6 may play an important factor and further studies are necessary to clarify the enigma of AE pathogenesis [22]. Also, in our study, a high serum IL-6 level of 25.20 pg/mL was the most discriminatory optimal cut-off value predicting AE in patients with ILD. So far, the optimal cut-off value of IL-6 to predict AE has not been determined. Therefore, in cases of high levels of IL-6 above 25.20 pg/mL in patients with ILD, it is necessary to suspect AE.

In our study, only high levels of IL-6 were an independent risk factor for mortality. A previous study supported our result. Among the 41 IPF patients, high levels of IL-6 were associated with mortality (OR 1.056, 95% CI 1.008–1.105, p = 0.021) [22]. In addition, in 67 patients with IPF (stable IPF 20 and AE IPF 47), Collard et al. showed that IL-6 was higher in the AE IPF group compared to the stable IPF group (10.1 pg/mL vs 5.3 pg/mL, p = 0.004). However, IL-6 was not associated with high mortality (OR 0.26, 95% CI: 0.06–1.24, p = 0.09). Our study results suggested that high levels of IL-6 are associated with the occurrence of AE resulting in high mortality. However, it is not clear whether high levels of IL-6 is a direct risk factor for mortality or predictor for the severity of AE because all deaths occurred in AE groups and groups with high levels of IL-6. Further studies are necessary to elucidate the relationship between degrees of IL-6 and the severity of AE suggesting high mortality.

This study has some limitations. First, it was a retrospective study conducted in a single center with a limited number of ILD patients. Also, the number of AE cases and deaths was too small to evaluate the risk factors for AE and deaths. However, the baseline characteristics of our subjects with the incidence and mortality rates were similar to those of patients in previous reports. Second, the estimation of baseline oxygenation was performed by SpO2 despite differences in arterial oxygen pressure or alveolar-arterial oxygen. Even though the level of SpO2 was a statistically significant factor for predicting AE, PaO2 was not included in our study. This is because SpO2 was measured in most patients, but the data for oxygenation by arterial blood gas sampling was obtained only in a small number of patients. In addition, SpO2 was well-validated for its correlation with PaO2 on arterial blood gas analysis in a previous study [29]. Third, in this study, the sequential measurement of IL-6 may also be useful for predicting AE in the CTD-ILD group. However, the number of CTD-ILD patients was too small and the incidence of AE in patients with CTD-ILD was relatively high compared to previous studies [30]. Therefore, selection bias may exist in the CTD-group for the role of IL-6 in predicting AE. In the near future, studies with more CTD-ILD patients may be needed to evaluate the role of IL-6 as a prognostic factor in CTD-ILD patients.

Conclusion

High serum levels of IL-6 during the follow-up period and lower baseline oxygenation were useful biomarkers for predicting AE. The optimal cut-off value of IL-6 was 25.20 pg/mL. In addition, high levels of serum IL-6 were a useful maker for predicting mortality.

Supporting information

S1 Fig. Comparison of survival curves with or without acute exacerbation groups.

(TIF)

Click here for additional data file. (492.1KB, tif)

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by 2019 Inje university research grant (20190041). The grant did not have any influence on the research results and was a support to promote the research activities.

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Decision Letter 0

Minghua Wu

9 Apr 2021

PONE-D-21-04098

The Role of Interleukin-6 as a Prognostic Biomarker for Predicting Acute Exacerbation in Interstitial Lung Diseases

PLOS ONE

Dear Dr. Kim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Comments:

  1. Table 1 & 2: Add % to the patient characteristic population ratio even explained at the bottom of the table, for example,  IPF, n (%)--- 57 (68.7).

  2. Figure 2: survival analysis, please correct mislabeling (IL6 level).

  3. As a predictive factor for AE in the CTD-group, n=3 is too small for biomarker study. I agree to the reviewer’s comments to remove CTD group from study.

     I hope reviewer's comments is helpful for improving manuscript.

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Kind regards,

Minghua Wu, M.D., Ph.D.

Academic Editor

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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**********

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Reviewer #1: The manuscript by Dr. Lee et al demonstrated a Role of Interleukin-6 as a Prognostic Biomarker for Predicting Acute Exacerbation in Interstitial Lung Diseases. The authors followed up 83 patients who were diagnosed with ILD from 2016 to 2019 at the Haeundae Paik Hospital, Busan, South Korea. They measured the lung functional parameters and the serum levels of IL-6 at diagnosis with ILD and sequentially at follow-up visits. From the studies, the authors demonstrated that high levels of IL6 (OR 1.014, 95% CI: 1.001–1.027, p = 0.036) along with lower baseline saturations of peripheral oxygen (SpO2) were independent risk factors for AE. They also performed the receiver operating characteristic curve analysis, and found that the serum IL-6 to predict AE was 25.20 pg/mL with a sensitivity of 66.7% and specificity of 80.6%. Finally, the authors concluded that a high level of serum IL-6 is a useful biomarker to predict AE and poor prognosis in patients with ILD.

This manuscript demonstrated an important role of IL-6 as a biomarker for IPF-AE. Since the increased serum IL-6 and IL-8 level has already been reported (Papiris SA et al, Cytokine. 2018 Feb;102:168-172), the manuscript is somehow lack of novelty. However, the authors performed a longitudinal study to check IL-6 levels overtime. If the authors have already have the data, it will be interesting to show IL-6 levels overtime to see the dynamic changes of IL6 before the onset of the AE.

Minor comments.

Figure 2. Is the labels for “Peak IL-6 level>25.20” and “Peak IL-6 level<25.20” flipped?

Reviewer #2: Review of the manuscript: “The Role of Interleukin-6 as a Prognostic Biomarker for Predicting Acute Exacerbation in Interstitial Lung Diseases”

This retrospective study aimed to assess the merit of IL-6 to predict acute exacerbation (AE) of intestinal lung disease (ILD).

Comments

- Limitation: an important limitation of the paper is the low number of patients, with probable insufficient power (the power of the study sample needs to be determined) and the heterogeneity of included diseases Moreover, the OR and HR of IL6 to predict the occurrence of AE were low, close to 1, the question is their relevance in clinical pertinence

- Please precise the number of screened patients, those who fulfilled inclusion and exclusion criteria leading to the final number of recruited patients

- The ILD pattern on HRCT scan should me precised: UIP, NSIP…

- Please provide the detailed causes of AE. Please confirm that infections have been formally ruled out. It is important since infections may increase CRP and thus, IL-6 levels.

- Logistic regression analysis: please explain how were entered the different covariates in the model (stepwise, backward…). In this model, IL-6 was entered as a continuous variable; it would be interesting to consider IL-6 as a dichotomous variable (Lower/higher)

- Multivariate analyses: gender, disease duration, and HRCT scan pattern should be included in the models

- The pertinence to include in the models both CRP and IL-6 is questionable given the strong relationship between these 2 variables

- Kaplan-Meier survival curve: a table with the number of patients should be added below the figure

- Given the very low number of patients with CTD-ILD, and the low frequency of AE, the reviewer suggests the deletion of the sub analysis of the CTD-group

- How were Handled missing data?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Jul 27;16(7):e0255365. doi: 10.1371/journal.pone.0255365.r002

Author response to Decision Letter 0

28 May 2021

Dear Editor,

We would like to thank you for the opportunity to resubmit a revised copy of our manuscript. We would also like to take this opportunity to express our thanks to the reviewers for the positive feedback and helpful comments for correction or modification.

We have made substantial changes to the manuscript according to the reviewers’ comments. The changes made are marked in red in the revised manuscript. Our point-by-point responses to the reviewers' comments are provided below. We hope the revised manuscript is suitable for publication in PLOS ONE.

[Response to Academic Editor's comments]

Q1. Table 1 & 2: Add % to the patient characteristic population ratio even explained at the bottom of the table, for example, IPF, n (%)--- 57 (68.7).

R1. Thank you for pointing this out. Based on this comment, we add % in the Table 1 & 2.

Q2. Figure 2: survival analysis, please correct mislabeling (IL6 level).

R1. Thank you for your comment. Sorry for our mistake. This is a typing error, and was corrected.

Q3. As a predictive factor for AE in the CTD-group, n=3 is too small for biomarker study. I agree to the reviewer’s comments to remove CTD group from study.

R1. Thank you for pointing this out. We agree to your comment. The paragraph “Predictive factor for AE in the CTD-group” removed from the manuscript.

[Journal Requirements]

Q1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

R1. Thank you for pointing this out. Based on this comment, we ensured that our manuscript meets PLOS ONE's style requirements.

Q2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

R2. Thank you for pointing this out. We updated an ORCID iD for the corresponding author in Editorial Manager.

Q3. Thank you for stating in the text of your manuscript "the requirement for written informed consent was waived due to the retrospective nature of this study". Please also add this information to your ethics statement in the online submission form."

R3. Thank you for pointing this out. We add the statement "the requirement for written informed consent was waived due to the retrospective nature of this study" to the ethic statement in the online submission form.

Q4. Thank you for providing the date(s) when patient medical information was initially recorded. Please also include the date(s) on which your research team accessed the databases/records to obtain the retrospective data used in your study.

R4. Thank you for pointing this out. We added the date in medical records in Clinical information of MATERIALS AND METHODS as below.

[MATERIALS AND METHODS, Clinical information]

Clinical data were retrospectively obtained from the medical records at January 3, 2020.

Q5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

This work was supported by the 2019 Inje University Research Grant (20190041). We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

This study was supported by Inje university research grant. The grant has nothing to do with the result of this research and is only to encourage the research.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

R5. Thank you for pointing this out. We deleted this sentence in the Acknowledgments and also inserted the sentence at the end of the cover letter.

[Response to Reviewer 1's comments]

Q1. Figure 2. Is the labels for “Peak IL-6 level>25.20” and “Peak IL-6 level<25.20” flipped?

R1. Thank you for your comment. Sorry for our mistake. This is a typing error, and was corrected.

[Response to Reviewer 2's comments]

Q1. Limitation: an important limitation of the paper is the low number of patients, with probable insufficient power (the power of the study sample needs to be determined) and the heterogeneity of included diseases Moreover, the OR and HR of IL6 to predict the occurrence of AE were low, close to 1, the question is their relevance in clinical pertinence

R1. Thank you for pointing this out. I agree with your opinion. In this study, the value of OR of IL-6 to predict to acute exacerbation and mortality was 1.014 (95% CI: 1.001 – 1.027) and 1.007 (95%CI: 1.001 – 1.014), respectively. However, the P–value was statistically significant. The diagnosis of acute exacerbation is still difficult and very important for good prognosis. Although revised diagnostic criteria of AE IPF, early and accurate diagnosis of AE ILD is still big challenging. In our county, other biomarker including KL-6 and SP-D had not been available. Therefore, we evaluated the role of IL-6 to predict AE. As you know, there cannot be a perfect biomarker. We expect that IL-6 might be helpful to predict to AE in addition to other method. Also IL-6 can be more useful as an acute reactant than other biomarker.

Q2. Please precise the number of screened patients, those who fulfilled inclusion and exclusion criteria leading to the final number of recruited patients

R2. Thank you for pointing this out. According to reviewer’s comment, we added flow-chart for enrollment (Fig 1) in Study population of RESUTLS as below

[Study population in RESULTS]

From December 2016 to September 2019, 405 patients with ILD at Haeundae Paik hospital (Busan, Republic of Korea) were screening for this study. Among them, patients who did not perform IL-6 test or follow up at IL-6 test at least 6 months were excluded (Fig 1).

Q3. The ILD pattern on HRCT scan should me precised: UIP, NSIP…

R3. Thank you for pointing this out. We added Radiologic pattern on HRCT to the table 1.

Q4. Please provide the detailed causes of AE. Please confirm that infections have been formally ruled out. It is important since infections may increase CRP and thus, IL-6 levels.

R4. Thank you for pointing this out. As a result of re-analysis, 11 patients and 4 patients were reclassified as idiopathic and triggered AE, respectively. Triggered AE was defined as proven pathogen or increased level of procalcitonin. We added detail data in Predictive factors in RESULTS as below.

[RESULTS, Predictive factors]

Among the 15 (18.1%) patients with AE (idiopathic AE: 11 patients and triggered AE: 4 patients), IPF patients were most common (80%) while the other patients were classified with CTD (20%).

Q5. Logistic regression analysis: please explain how were entered the different covariates in the model (stepwise, backward…). In this model, IL-6 was entered as a continuous variable; it would be interesting to consider IL-6 as a dichotomous variable (Lower/higher)

R5. Thank your comment. We modified the sentence in Statistical analysis as below.

[MATERIALS AND METHODS, Statistical analysis]

Multivariate analyses, using Cox regression with backward and stepwise elimination, were performed in order to identify prognostic factors which are independently related to mortality.

Q6. Multivariate analyses: gender, disease duration, and HRCT scan pattern should be included in the models

R6. Thank you for comment. According to comment, we added the variables including gender, disease duration and radiologic pattern to the table 3 and 4. However, NSIP, OP and indeterminate pattern were excluded from multivariate analysis because they caused statistical error.

Q7. The pertinence to include in the models both CRP and IL-6 is questionable given the strong relationship between these 2 variables

R7. Thank you for your comment. CRP is produced by hepatocyte in the liver in response to stimulation of pro-inflammatory cytokines. Therefore, CRP has been used as biomarker for inflammation. In several study, elevated level of CRP was reported as useful biomarker to predict AE ILD. In this study, baseline level of CRP was significantly different between AE group and non-AE group. However, high level of CRP was not useful to predict AE and mortality, although IL-6 was useful to predict both. In this study, the role of CRP and IL-6 was different to predict AE and mortality. In the field of ILD, we thought the role of CRP and IL-6 are different and independent.

Q8. Kaplan-Meier survival curve: a table with the number of patients should be added below the figure

R8. Thank you for your comment. We added a table with the number of patients in Figure 3.

Q9. Given the very low number of patients with CTD-ILD, and the low frequency of AE, the reviewer suggests the deletion of the sub analysis of the CTD-group

R9. Thank you for your comment. We agree your opinion. The paragraph “Predictive factor for AE in the CTD-group” was removed from the manuscript.

Q10. How were Handled missing data?

R10. Thank you for comment. There was some missing data. Therefore, the missing data was null out and the remaining data was analyzed. Variables with many missing data such as RVSP, were excluded from the statistical analysis because they caused statistical error.

Sincerely,

Hyun Kuk Kim, M.D., Ph.D.

Department of Pulmonary and Critical Care Medicine, Inje University Haeundae Paik Hospital, Inje University College of Medicine, Haeundae-ro 875, Haeundae-gu, 48108, Busan, Republic of Korea.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (392.6KB, docx)

Decision Letter 1

Minghua Wu

15 Jul 2021

The Role of Interleukin-6 as a Prognostic Biomarker for Predicting Acute Exacerbation in Interstitial Lung Diseases

PONE-D-21-04098R1

Dear Dr. Kim,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Minghua Wu, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

Acceptance letter

Minghua Wu

19 Jul 2021

PONE-D-21-04098R1

The Role of Interleukin-6 as a Prognostic Biomarker for Predicting Acute Exacerbation in Interstitial Lung Diseases

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on behalf of

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Academic Editor

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    Supplementary Materials

    S1 Fig. Comparison of survival curves with or without acute exacerbation groups.

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