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. 2021 Jun 4;16(6):e0252594. doi: 10.1371/journal.pone.0252594

IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis

Yu Nakanishi 1, Yasushi Horimasu 1,*, Kakuhiro Yamaguchi 1,#, Shinjiro Sakamoto 1,‡, Takeshi Masuda 1,‡, Taku Nakashima 1,‡, Shintaro Miyamoto 1,#, Hiroshi Iwamoto 1,#, Shinichiro Ohshimo 2,#, Kazunori Fujitaka 1,‡, Hironobu Hamada 3,#, Noboru Hattori 1,‡
Editor: Gernot Zissel4
PMCID: PMC8177514  PMID: 34086758

Abstract

Idiopathic pulmonary fibrosis is a chronic, fibrosing interstitial pneumonia that presents with various clinical courses and progression ranging from rapid to slow. To identify novel biomarkers that can support the diagnosis and/or prognostic prediction of idiopathic pulmonary fibrosis, we performed gene expression analysis, and the mRNA of interleukin-18 binding protein was increasingly expressed in patients with idiopathic pulmonary fibrosis compared with healthy controls. Therefore, we hypothesized that the interleukin-18 binding protein can serve as a diagnostic and/or prognostic biomarker for idiopathic pulmonary fibrosis. We investigated the expression of interleukin-18 binding protein in lung tissue, bronchoalveolar lavage fluid, and serum. Additionally, the correlation between interleukin-18 binding protein expression levels and the extent of fibrosis was investigated using mouse models of lung fibrosis induced by subcutaneous bleomycin injections. Serum interleukin-18 binding protein levels were significantly higher in idiopathic pulmonary fibrosis patients (5.06 ng/mL, interquartile range [IQR]: 4.20–6.35) than in healthy volunteers (3.31 ng/mL, IQR: 2.84–3.99) (p < 0.001). Multivariate logistic regression models revealed that the correlation between serum interleukin-18 binding protein levels and idiopathic pulmonary fibrosis was statistically independent after adjustment for age, sex, and smoking status. Multivariate Cox proportional hazard models revealed that serum interleukin-18 binding protein levels were predictive of idiopathic pulmonary fibrosis disease prognosis independent of other covariate factors (hazard ratio: 1.655, 95% confidence interval: 1.224–2.237, p = 0.001). We also demonstrated a significant positive correlation between lung hydroxyproline expression levels and interleukin-18 binding protein levels in bronchoalveolar lavage fluid from bleomycin-treated mice (Spearman r = 0.509, p = 0.004). These results indicate the utility of interleukin-18 binding protein as a novel prognostic biomarker for idiopathic pulmonary fibrosis.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fibrosing interstitial pneumonia of unknown cause, characterized by persistently progressive fibrosis of the lung interstitium that results in irreversible destruction of the alveolar structure [1]. The median survival of IPF patients has been reported to be approximately 3 years [1]. The clinical course of IPF varies widely from extremely rapid progression to relatively stable [2].

Pirfenidone and nintedanib have been approved as effective antifibrotic agents for IPF, although their efficacy is limited to modification of the extent of pulmonary functional deterioration [3–7]. With no available treatment option that can restore the deteriorated pulmonary function, early diagnosis of IPF as well as accurate prediction of disease progression are quite important for the clinical management of IPF [8, 9].

Biomarkers are commonly defined as objectively measurable indicators of the physiological and/or pathological processes of the organs or the response to therapeutic interventions [10]. For the reasons described above, substantial efforts have been made to identify a biomarker for IPF that can support the diagnosis, prognosis prediction, and the assessment of response to treatment [11]. We have previously performed gene expression analysis and have identified several molecules as potential biomarkers for IPF [12]. Among these, we focused on interleukin-18 binding protein (IL-18BP), which is known to be a decoy receptor for IL-18 [13]. In past reports, IL-18 was upregulated in patients with IPF and the lungs of bleomycin (BLM)-injured mice [14–16], but the role of IL-18BP, which is a natural antagonist of IL-18, is still unknown. Recently, it was reported that administration of IL-18BP to a BLM-injury model improved lung fibrosis [14, 17], but the trend of IL-18BP secreted in vivo is unclear. Therefore, the present study was conducted to clarify whether IL-18BP can serve as a diagnostic and/or prognostic biomarker for IPF.

Methods

Immunohistochemical staining for IL-18BP

Lung tissue sections were obtained from nine patients with IPF who agreed to undergo diagnostic surgical lung biopsies and provided written informed consent and permission to use their samples. The clinical characteristics of these patients were presented in S1 Table. Control lung tissue sections were obtained from healthy areas in the lungs of five patients with lung cancer who had undergone therapeutic surgical resection. The lung tissue sections were stained with ENVISION+ Kit/horseradish peroxidase (HRP) (Dako, Tokyo, Japan) as previously described [12]. Rabbit anti-IL-18BP antibody (ab52914, 1:3000, Lot ID: 161918; Abcam, Cambridge, UK) or rabbit control IgG antibody (ab37415, Lot ID: 3237588–1; Abcam) was added after blocking endogenous peroxidase and proteins. The sections were then incubated with HRP-labelled anti-rabbit IgG antibody followed by the addition of substrate-chromogen and counterstaining with hematoxylin.

In the nine IPF patients and five controls, the positively stained area was quantified using the ImageJ Fiji (National Institutes of Health, USA) software. We randomly selected five fields for each slide, and the average of the percentage of positively stained area in each of the five fields was deemed to be representative of the positively stained area for a patient.

Subjects for Enzyme-Linked Immunosorbent Assay (ELISA) measurements

Between November 2001 and February 2017, 86 consecutive Japanese patients newly diagnosed with stable IPF at Hiroshima University Hospital were recruited, and serum and bronchoalveolar lavage fluid (BALF) samples were obtained. Patients who developed acute exacerbation of IPF within 1 month of diagnosis or had lung cancer at the time of IPF diagnosis were excluded. IPF was diagnosed in accordance with the international diagnostic criteria published in 2018 [18]. Two hundred and fifty healthy subjects who underwent health check-ups were also enrolled as healthy volunteers (HVs) and serum samples were obtained. Each HV underwent pulmonary function tests and chest radiography studies, and those with apparent lung disease, such as interstitial lung disease or chronic obstructive pulmonary disease, were excluded. This study was approved by the Ethics Committees of Hiroshima University Hospital (IRB33) and conducted in accordance with the ethical standards established in the Helsinki Declaration of 1975. All patients and HVs provided informed consent in writing and permission to use their samples.

Serum and BALF measurements

Serum samples, obtained from peripheral blood drawn during the initial assessment of patients with IPF or during the health check-ups for HVs, were stored at -80°C until the time of analysis [19, 20]. Bronchoalveolar lavage was performed as previously described for patients with IPF [21]. Briefly, 50 mL of saline was introduced into the lung, promptly drawn, and this procedure was repeated three times [21]. The BALF was centrifuged promptly, and the supernatant was cryopreserved at -80°C until analysis. BALF was available for 50 IPF patients. IL-18BP and IL-18 concentrations in the serum or BALF were measured using commercially available ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Development of mouse models of lung fibrosis

Wild-type male C57BL/6 mice (Charles River Japan Inc., Yokohama, Japan) were housed in a specific pathogen free environment at an optimal temperature with a 12-h light/dark cycle and were used for experiments at 8–10 weeks of age. On day 0, osmotic minipumps (ALZET 1007D; DURECT, Cupertino, CA) containing either 100 μL of saline vehicle or bleomycin (100 mg/kg) and designed to deliver their contents at 0.5 μL/h for 7 days were implanted under the loose skin on the back of the mice slightly posterior to the scapulae under intraperitoneal anesthesia [22]. Combination anesthesia was prepared with 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol. We monitored the mice in cages until they fully recovered from anesthesia without additional analgesics after osmotic pump implantation because the pump was small enough not to cause any obvious pain to the mice. The osmotic minipumps were removed on day 10 as recommended by the manufacturer. We monitored the body weight of the mice to evaluate health and welfare. Each experiment was performed using 5 or 6 mice based on the estimated mean and standard deviation of BALF IL-18BP in the preliminary experiment. One mouse in the bleomycin group died on day 21. At each time point, mice were euthanized by exsanguination. All mice were handled in accordance with Guidelines for Care and Use of Experimental Animals published by Hiroshima University, and all protocols were approved by the Hiroshima University (Approval #A19-34).

Measurements of lung hydroxyproline, BALF IL-18BP and BALF IL-18

After tracheostomy, a 19G tube was inserted into the trachea, and the lungs were washed three times with 150 μL of saline vehicle. The recovered fluid was evaluated with a hemocytometer. The remaining BALF was centrifuged, and the supernatants were then collected and stored at -20°C until ELISA was performed. The IL-18BP and IL-18 levels in BALF were measured using custom ELISA kits according to the manufacturer’s instructions (Mouse IL-18 BPc ELISA Kit, RayBiotech, Peachtree Corners, GA, USA and Mouse IL-18 ELISA Kit, MBL, Nagoya, Japan). The ELISA measurements were performed within 2 weeks of sample collection. At the time of sacrifice, the left lungs were removed and homogenized in phosphate buffered saline, followed by acid hydrolysis [23]. The collagen content was estimated by a colorimetric assay for hydroxyproline as described previously [23–25].

Statistical analysis

Continuous variables are expressed as median and interquartile range (IQR). The differences between the two groups were tested using the Mann–Whitney U test or Pearson’s chi-squared test, as appropriate, and those between three or more groups were tested using multiple Mann–Whitney U-tests with Bonferroni correction. Receiver operating characteristic (ROC) analysis was performed to assess the discriminating abilities. The area under the ROC curve (AUC) with 95% confidence interval (CI) was calculated to assess the discrimination power of serum IL-18BP and IL-18 levels. The correlations between two numerical variables were tested using Spearman correlations. Cox proportional hazards analysis was used to identify significant predictors of 3-year survival in IPF patients. Among the patients, those who had been treated with pirfenidone and/or nintedanib for 1 year or longer were classified as having received antifibrotic treatment. Survival was evaluated using the Kaplan–Meier approach, and the prognostic differences between the two groups were tested with the log-rank test. Those who died from non-IPF causes, including cancer, were treated as censored. Statistical analyses were performed using EZR (Saitama Medical Centre, Jichi Medical University, Saitama, Japan) for ROC analysis and JMP ®14 (SAS Institute Inc., Cary, NC, USA) for the other tests. A p value of ≤ 0.05 was considered to be statistically significant for all analyses.

Results

Increased IL-18BP expression was detected in the lung tissue of IPF patients

In order to confirm our previous findings showing increased IL-18BP mRNA expression in IPF lungs [12], immunohistochemical staining for IL-18BP was performed. As shown in Fig 1A–1D, the IL-18BP expression in the lungs of representative three out of nine IPF patients was greater than that in the control lungs. The IPF lungs showed marked expression of IL-18BP in the fibrotic interstitium and bronchiole epithelial cells, as well as in the alveolar macrophages. Furthermore, as shown in S1 Fig, the percentage of the positively stained area was higher in patients with IPF (21.7% [IQR: 20.6–23.1]) than those in control group (10.6% [IQR: 7.5–13.0]) (p = 0.003).

Fig 1. Immunohistochemical expression of IL-18BP in IPF and control lungs.

Fig 1

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(a, c, d) Immunohistochemical staining of IPF lung tissue with anti-IL-18BP antibody presented at x100 magnification. (b) The same field of view as in panel (a) presented at x400 magnification. (e) Immunohistochemical staining of IPF lung tissue with rabbit IgG presented at x100 magnification. (f) Immunohistochemical staining of control lungs with anti-IL-18BP antibody presented at x100 magnification. Scale bar = 100 μm. IL-18BP: interleukin-18 binding protein, IPF: idiopathic pulmonary fibrosis.

Increased serum IL-18BP levels were detected in IPF patients

To further assesses the clinical usefulness of IL-18BP as a biomarker, we also investigated serum IL-18 levels, which had previously been reported to be useful as a clinical biomarker for IPF [16], in both IPF patients and HVs. The characteristics of IPF patients and HVs are shown in Table 1. IPF patients were older and had history of heavier smoking than the HVs. As shown in Fig 2A, both serum IL-18BP and IL-18 levels were significantly higher in IPF patients than in HVs (p < 0.001 and p < 0.001, respectively). Importantly, ROC curve analysis revealed that the AUC value of serum IL-18BP was significantly larger than that of serum IL-18 for discriminating IPF patients from HVs (Fig 2B, p < 0.001).

Table 1. Patient characteristics.

Variables IPF HV p-value
N = 86 N = 250
Age (years) 68.5 (63.0–74.3) 51.0 (43.0–55.3) < 0.001
Sex
 Male / Female 71 / 15 213 / 37 0.604
Smoking history
 Yes / No 70 / 16 147 / 103 < 0.001
 Pack-years 30.8 (16.9–45.8) 12.0 (0.0–32.0) < 0.001
Pulmonary function test
 FVC (% predicted) 70.1 (61.5–81.8) 98.4 (90.3–108.9) < 0.001
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Data are expressed as frequencies or medians (interquartile range).

IPF: idiopathic pulmonary fibrosis, HV: healthy volunteer, FVC: forced vital capacity.

Fig 2. Distribution of serum IL-18BP concentrations and ROC analysis in IPF patients and HVs.

Fig 2

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(a) Box plot graphs showing ranges of serum IL-18BP and IL-18 levels in patients with IPF and HVs. The median IL-18BP levels of serum in patients with IPF and HVs were 5.06 (4.20–6.35) ng/mL and 3.31 (2.84–3.99) ng/mL (p < 0.001). The median IL-18 levels of serum in patients with IPF and HVs were 181.7 (146.6–243.6) pg/mL and 143.9 (108.5–196.8) pg/mL, respectively (p < 0.001). Boxes represent the 25th–75th percentiles; solid lines within the boxes show the median values, whiskers are the 10th and 90th percentiles, and the small dots represent outliers. (b) ROC curve analyses for serum IL-18BP (solid line) and IL-18 (dotted line) levels. The AUC value of serum IL-18BP (0.858, 95% Cl; 0.810–0.906) was significantly larger than that of serum IL-18 (0.661, 95%Cl: 0.595–0.726, p < 0.001). IL-18BP: interleukin-18 binding protein, ROC: receiver operating characteristics, IPF: idiopathic pulmonary fibrosis, HVs: healthy volunteers, IL-18: interleukin-18, AUC: area under the curve, CI: confidence interval.

Serum IL-18BP can independently predict the presence of IPF

Next, we investigated the correlations between serum IL-18BP and serum IL-18 or BALF IL-18BP levels. The correlations between serum IL-18BP and serum IL-18 levels were statistically significant not only in IPF patients (S2 Fig; r = 0.251, p = 0.019) but also in HVs (S2 Fig; r = 0.267, p < 0.001). In IPF patients, there were significant positive correlations between serum IL-18BP and BALF IL-18BP levels (S3 Fig; r = 0.406, p = 0.005).

In order to assess the statistical independence of the associations between the presence of IPF and serum IL-18BP or IL-18 levels, logistic regression analyses were performed. In the multivariate model, a high serum IL-18BP level was revealed to be a statistically independent predictor for the presence of IPF (p = 0.019, Table 2).

Table 2. Logistic regression analysis of variables associated with the discriminating ability of IPF patients or healthy volunteers.

Odds ratio 95% Cl p-value
Univariate analysis
Age, years 1.645 1.428–1.894 < 0.001*
Sex, Male 0.822 0.433–1.626 0.563
Smoking history
 Pack-years 1.029 1.018–1.040 0.001*
FVC (%predicted) 0.910 0.887–0.931 0.001*
Serum IL-18BP (ng/mL)
 Continuous 3.561 2.596–4.885 < 0.001*
Serum IL-18 (pg/mL)
 Continuous 1.007 1.004–1.011 0.001*
Multivariate analysis‡
IL-18BP (ng/mL)
 Continuous 1.791 1.061–3.024 0.019*
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* p < 0.05 (logistic regression analysis).

‡Adjusted for age, sex, smoking history, and FVC (%predicted).

IPF: idiopathic pulmonary fibrosis, 95% Cl: 95% confidence interval, FVC: forced vital capacity, IL-18BP: interleukin-18 binding protein, IL-18: interleukin-18.

High serum IL-18BP was associated with poor prognosis and impaired pulmonary function

As shown in Table 3, in IPF patients, the univariate Cox proportional hazards analysis revealed that increased serum IL-18BP levels, as well as deteriorated lung function was significantly associated with a poor prognosis. Additionally, the multivariate analysis confirmed that the serum IL-18BP level was a statistically independent predictor for the prognosis of IPF patients even when adjusted for age, sex, smoking history, percentage of forced vital capacity (%FVC), percentage of diffused capacity of the lung for carbon monoxide (%DLco), and the use of antifibrotic agents. Based on the ROC analysis shown in S4 Fig, we determined the optimal cut-off serum IL-18BP level that can discriminate those who died from those who did not, during 3 years from diagnosis, as 5.72 ng/mL. As shown in Fig 3A, the log-rank test revealed that participants with high serum IL-18BP levels had significantly poorer prognosis than those with low levels (p = 0.024). Furthermore, as shown in Fig 3B and 3C, serum IL-18BP levels showed a significant inverse correlation with %DLco (r = -0.300, p = 0.016), although it was not significantly correlated with %FVC (r = -0.177, p = 0.156).

Table 3. Prediction values for 3-year mortality in IPF patients assessed by Cox proportional hazards model (n = 86).

HR 95% Cl p-value
Univariate analysis
Age, years 0.967 0.924–1.013 0.154
Sex, male 1.841 0.643–7.792 0.283
Smoking history
 Pack-years 0.994 0.979–1.007 0.386
FVC (%predicted) 0.958 0.932–0.983 < 0.001*
DLco (%predicted) 0.926 0.888–0.964 < 0.001*
Use of anti-fibrotic agent 2.387 1.082–5.265 0.031*
Serum IL-18BP (ng/mL)
 Continuous 1.185 1.018–1.343 0.031*
BALF IL-18BP (pg/mL)
 Continuous 0.993 0.975–1.008 0.394
Serum IL-18 (pg/mL)
 Continuous 1.000 0.996–1.003 0.897
Multivariate analysis
Serum IL-18BP‡
 Continuous 1.655 1.224–2.237 0.001*
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* p < 0.05 (Cox proportional hazards model).

‡ Adjusted for age, sex, smoking history, FVC (%predicted), DLco (%predicted), and use of anti-fibrotic agent.

IPF: idiopathic pulmonary fibrosis, HR: Hazard ratio, 95% Cl: 95% confidence interval, FVC: forced vital capacity, DLco: diffusion capacity of the lung for carbon monoxide, IL-18BP: interleukin-18 binding protein, BALF: bronchoalveolar lavage fluid, IL-18: interleukin-18.

Fig 3. Correlations between serum IL-18BP levels and pulmonary function or survival outcome of patients with IPF.

Fig 3

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(a) Kaplan–Meier analysis for 3-year survival in patients with IPF, based on serum IL-18BP levels. Patients with higher levels of serum IL-18BP (≥ 5.72 ng/mL) showed significantly poorer survival compared with those with lower serum IL-18BP (< 5.72 ng/mL). (b) Scatter plot graphs showing the correlations between serum IL-18BP levels and %FVC, which showed no significant correlation (Spearman r = -0.177, p = 0.156). (c) Serum IL-18BP levels showed significant negative correlations with %DLco (Spearman r = -0.300, p = 0.016). IL-18BP: interleukin-18 binding protein, IPF: idiopathic pulmonary fibrosis, FVC: forced vital capacity, DLco: diffusion capacity of the lung for carbon monoxide.

BALF IL-18BP levels were associated with the extent of lung fibrosis in the mice

To further investigate the associations between the expression levels of IL-18BP and the extent of lung fibrosis, we established a mouse model of lung fibrosis induced by subcutaneous bleomycin injections and investigated the associations between BALF IL-18BP levels and hydroxyproline expression in the lung (S5 Fig). As shown in Fig 4A, hydroxyproline expression in the lung tissue increased over time in the bleomycin- injected mice. On the other hand, BALF IL-18BP levels showed bimodal elevation with two peaks on day 7 and days 21 to 28 (Fig 4B). In addition, there were significant positive correlations between lung hydroxyproline and BALF IL-18BP levels in the bleomycin- injected mice (Fig 4C, r = 0.509, p = 0.004). Besides, BALF IL-18 levels showed unimodal elevation with a peak on day 7 (Fig 4D).

Fig 4. Chronological change of BALF IL-18BP levels in the bleomycin-injected mice.

Fig 4

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(a) Lung hydroxyproline levels chronologically increased over time in the bleomycin-injected mice. (b) BALF IL-18BP levels showed bimodal elevation with two peaks on day 7 and days 21 to 28 in the bleomycin-injected mice. The significance level was defined α = 0.013 (four comparisons). (c) Lung hydroxyproline levels were significantly correlated with BALF IL-18BP levels (Spearman r = 0.509, p = 0.004). (d) BALF IL-18 levels showed unimodal elevation with a peak on day 7. The significance level was defined α = 0.010 (five comparisons). Horizontal bars represent the median. BALF: bronchoalveolar lavage fluid, IL-18BP: interleukin-18 binding protein, IL-18: interleukin-18. * p < 0.01 (Mann–Whitney U-test with Bonferroni correction).

Discussion

The aim of this study was to clarify whether IL-18BP can serve as a diagnostic and/or prognostic biomarker for IPF. As a result, we demonstrated that IL-18BP expression was increased in the lung tissue and serum of IPF patients and that the serum IL-18BP level was an independent prognostic predictor for IPF patients. We believe that these results are clinically important in that they show the usefulness of IL-18BP as a novel prognostic biomarker for IPF.

The most important finding in the present study was that the serum IL-18BP level could be a prognostic biomarker for IPF. In IPF patients, the expressions of IL-18BP both in the lung tissue and in the serum were increased (Figs 1 and 2), and increased serum IL-18BP levels were significantly associated with decreased %DLco (Fig 3C). Importantly, increased serum IL-18BP levels were significantly associated with poor prognosis of patients with IPF even in the multivariate model (Table 3). Furthermore, BALF IL-18BP levels in the mouse models of subcutaneously injected bleomycin-induced lung fibrosis were positively correlated with lung hydroxyproline expression levels (Fig 4). All of these findings suggest that increased expression of IL-18BP is associated with progressive lung fibrosis, pulmonary dysfunction, and poor prognosis in IPF patients. Additionally, these findings are consistent with the results of our previous gene expression study that demonstrated the increased expression of IL-18BP mRNA in the lung tissue of patients with IPF [12].

Although there are limited reports focusing on the utility of IL-18BP as a biomarker, Ha and colleagues reported that increased levels of serum IL-18BP were associated with increased mortality in total-body gamma irradiation mouse models [26]. We believe that this previous report supports our results. Although the mechanisms underlying the association between the increased expression of IL-18BP and the poor outcome of IPF remain unclear, we considered two possibilities. First, IL-18BP itself is not the cause of the poor outcomes for IPF; although IL-18BP itself has anti-fibrotic and/or anti-inflammatory activity, its increased expression simply reflects increased expression of IL-18, which can promote lung fibrosis. IL-18 is known to have high expression in the fibrotic lung [15, 16], and bleomycin-induced lung fibrosis can be altered by the inhibition of IL-18 expression [15]. Furthermore, IL-18 promotes the proliferation, migration, and collagen production of cardiac fibroblasts [27–31]. It is also known to promote the expression of IL-18BP by the production of interferon-γ [32–35]. IL-18BP is known to be a decoy receptor for IL-18 with substantially higher affinity than IL-18Rα, which is the functional receptor [13]. IL-18BP is known to act as a down-regulator for Th1 responses by reducing the induction of interferon-γ via inhibition of IL-18 activity [36]. Zhang et al. reported that exogenous administration of IL-18BP could attenuate both lipopolysaccharide-induced lung injury and bleomycin-induced lung fibrosis in murine models [14, 37]. In addition, IL-18BP was also reported to attenuate renal and hepatic injury in ischemia–reperfusion murine models through its antioxidant and anti-inflammatory activities [38]. Based on these reports, we can speculate that the increased expression of IL-18BP in IPF patients was not the cause of fibrosis, but a result of the increased expression of IL-18, a profibrotic factor. As shown in Fig 4, the unimodal elevation of BALF IL-18 followed by the bimodal elevation of BALF IL-18BP in our murine models, might support this speculation in part. Further, such different serial changes in expression levels of IL-18BP and IL-18 may result in the different utilities of IL-18BP and IL-18 in predicting the outcome in IPF.

Another possibility is that IL-18BP promotes fibrosis via inhibition of interleukin-37 (IL-37), resulting in poor outcomes for IPF [39]. IL-37 has been reported to have a protective effect in interstitial lung disease through the inhibition of transforming growth factor-β1 signaling and the enhancement of fibroblast autophagy. IL-18BP has been reported to bind to IL-37, although the binding affinity is not high, and to suppress its activation, resulting in the promotion of fibrosis.

In the present study, there were significant positive correlations between serum IL-18BP and BALF IL-18BP levels in IPF patients. These results would indicate that the elevation of IL-18BP levels in sera of IPF patients reflects the elevation of IL-18BP levels in the alveolar space. Under normal conditions, although relatively weak expression of IL-18BP mRNA was reported in the lung, protein expression has been reported in the appendix, spleen, tonsils, and lymph nodes, but not in the lung [40]. However, marked expression of IL-18BP mRNA has been reported in granulocytes, monocytes, and T-cells even under normal conditions [40]. Therefore, we can speculate that the migrated inflammatory cells can be the main source of IL-18BP expression in the IPF lung and that circulating IL-18BP levels might be also affected by its local expression in the lung. However, we didn’t perform BAL in HVs from the view of medical ethics, so that we couldn’t investigate the correlations between serum IL-18BP and BALF IL-18BP in HVs.

It might be surprising that the use of antifibrotic agents was significantly associated with a poor prognosis (Table 3). However, we consider that is caused by selection bias resulting from the physicians’ and/or patients’ decision, the medical insurance system, and the socio-economic status of the patient.

Although this study showed promising results, it had several limitations. First, the number of subjects included in the immunohistochemical analysis was relatively small. Second, there were significant differences in age and smoking history between HVs and patients with IPF. However, based on the results of the multivariate analyses (Tables 2 and 3), we believe that these differences did not diminish the correlation between IL-18BP and IPF. Third, this study was retrospective, and there was no validation cohort. Thus, multicenter prospective studies are required to confirm the usefulness of IL-18BP as a prognostic biomarker in IPF patients. Fourth, the influence of cryopreservation on the concentration of IL-18BP has not been directly assessed. The human samples have been preserved at -80°C for several years, although the murine samples have been preserved at -20°C for no longer than two weeks. Nevertheless, we believe that the measurement results were substantial because we found that they correlated with lung function and prognosis of patients with IPF. Furthermore, in the murine model, preliminary experiments revealed that serum samples stored at -20°C and those stored at -80°C showed almost comparable levels of IL-18BP. Therefore, we consider that cryopreservation does not significantly affect IL-18BP concentration. Finally, the interactions between IL-18BP and IL-18, or IL-37 were not directly investigated. Therefore, the detailed role of IL-18BP in the pathogenesis of IPF should be clarified in further investigations.

Conclusion

This study showed that IL-18BP was overexpressed in the serum, BALF, and lung tissue of patients with IPF than in those of HVs, and that high serum IL-18BP concentrations were independently associated with poor prognosis in patients with IPF. This study is the first to demonstrate the usefulness of IL-18BP as a prognostic biomarker for IPF.

Supporting information

S1 Fig. Quantification of immunohistochemical staining with IL-18BP.

The percentage of the positively stained area was higher in patients with IPF (21.7% [IQR: 20.6–23.1]) than those in control lung (10.6% [IQR: 7.5–13.0]) (p = 0.003). Horizontal bars represent median. IL-18BP: interleukin-18 binding protein.

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S2 Fig. Correlations between serum IL-18BP levels and IL-18 levels.

(a) Serum IL-18BP and IL-18 levels showed significant positive correlations in HVs (Spearman r = 0.267, p < 0.001). (b) Serum IL-18BP and IL-18 levels showed significant positive correlations in IPF patients (r = 0.251, p = 0.019). IL-18BP: interleukin-18 binding protein, IL-18: interleukin-18, HVs: healthy volunteers, IPF: idiopathic pulmonary fibrosis.

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S3 Fig. Correlation between serum IL-18BP levels and BALF.

Serum IL-18BP and BALF IL-18BP levels showed significant positive correlations in IPF patients (Spearman r = 0.406, p = 0.005). IL-18BP: interleukin-18 binding protein, BALF: bronchoalveolar lavage fluid, IPF: idiopathic pulmonary fibrosis.

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S4 Fig. ROC analysis of serum IL-18BP for 3-years survival in patients with IPF.

ROC analysis of serum IL-18BP levels was performed between those that have died and those that have survived or censored during three years from diagnosis (AUC 0.610, 95% Cl: 0.429–0.732). ROC: receiver operating characteristic, IL-18BP: interleukin-18 binding protein, IPF: idiopathic pulmonary fibrosis, AUC: area under the curve.

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S5 Fig. Summary of experimental model.

Eight-week-old male C57BL/6 mice were subcutaneously implanted with Alzet osmotic minipumps containing either a 200 μL saline vehicle or 100 mg/kg BLM at different doses. Pumps implanted under the back skin of mice slightly caudal to the scapulae were removed on day 10. BLM: bleomycin.

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S1 Table. Characteristics of IPF patients with lung tissue sample available.

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Click here for additional data file. (24.2KB, docx)

Acknowledgments

We are grateful to Dr. Nobuhisa Ishikawa in Hiroshima Prefectural Hospital and Dr. Masaya Taniwaki in Hiroshima Red Cross Hospital & Atomic-bomb Survivors Hospital who played the leading roles in gene expression analysis.

Data Availability

All relevant data is within the manuscript and its Supporting Information files.

Funding Statement

YH has received research funding from Japan Society for the Promotion of Science (https://www.jsps.go.jp/, KAKENHI Grant No. 19K17676). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Gernot Zissel

26 Feb 2021

PONE-D-20-35981

IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript Yu Nakanishi et al, entitled: “IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis” investigated the role of IL-18 in the diagnosis and prognosis of IPF. The paper resulted of interest, however some point need to be improved:

major comments:

Introduction

-The phrase “The prognosis of IPF is known to be poorer than that of certain malignancies” resulted not appropriate in this context, I suggest to delete.

- “although their efficacy is limited to modification of the extent of pulmonary functional deterioration.” Miss references.

- “novel biomarkers that can support an early diagnosis of IPF and/or predict the

progression of the disease are urgently needed.” This sentence resulted vague. Please can the author specify the role of biomarker in IPF? What are the state of art about biomarker in IPF? I also suggest to better define the role of biomarker that the authors intend to emphasize, diagnostic? Prognostic? Response to treatment? Please improve this part of introduction.

- “We previously performed gene expression analysis using surgically resected lung

tissue from seven patients with IPF as well as five patients with non-specific interstitial

pneumonia [10].” Is redundant in the introduction, please move in the discussion section.

“Therefore, we performed the present study to clarify whether IL-18BPcan serve as a clinical biomarker for IPF.” Rephrase this sentence and better explain the purpose of the study

Materials and methods

- Authors excluded patients who develop lung cancer? Because 30% of patients with IPF develop lung within one year. Please clearify this point.

- “Bronchoalveolar lavage was performed as previously described.” Please add the reference

- “cryopreserved at -80℃ until analysis.” Can the concentration of IL-18BP change due to -80°C cryopreserved?

- “The remaining BALF was centrifuged, and the supernatants were then collected and stored at -20℃ until ELISA was performed.” Different method of storage were applied between BAL of Human and Mouse. Please can the authors clearify this discrepancy?

- Results are expressed as mean±SD, however the non parametric test revealed that the variables are not normally distributed. Please can the authors explain why they do not use median and IQR?

Results

- Age and smoking habits differred in the 2 population. What think the authors about this? Can these variable modify the results and the concentrations of IL18-IL18BP?

- “The correlations between serum IL-18BP and serum IL-18 levels were statistically significant not only in IPF patients but also in HVs (S1 Fig).” Please report r and p values.

- “ In IPF patients, there were significant positive correlations between serum IL-18BP and BALF IL-18BP levels (S2 Fig, r = 0.406, p = 0.005).” What think authors about this results?

- “Cox proportional hazards analysis revealed that increased serum IL-18BP levels, as well as low % forced vital capacity (FVC), % diffuse capacity of the lung for carbon monoxide (DLco), and use of antifibrotic agents was significantly associated with a poor prognosis.” You say that the use of antifibrotic agent promote the poor prognosis?

Discussion

- First of all clearify and better explain the aim

- “can serve as a clinical biomarker for IPF” what kind of biomarker? Dagnostic? Prognostic?

- “We believe that these results are clinically important in that they show the usefulness of IL-18BP as a novel predictive biomarker for IPF.” From clinical point of view this result does not help to discriminate IPF than other ILD were normally differential diagnosis occur, but differentiate from Controls, Rarely IPF patients were confused with Controls. Please reformulate.

- “We consider that this previous report supports our results, although the mechanisms underlying how IL-18BP causes a poor outcome in IPF remains unclear.” This is true but can the authors better explain what are the state of art about the knowledge of il-18 in the lung and the implications in ipf pathogenesis?

- “Despite these limitations, we believe that the results of our study are of significance in that we have demonstrated the utility of serum IL-18BP as a prognostic biomarker for IPF for the first time.” The final sentence resulted vague and inappropriate.

Conclusion

- The conclusions are inappropriate. Please better specify the role of our research avoiding vague sentences.

Reviewer #2: The manuscript “IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis” by Nakanishi et al. explores the novel biomarker of IL-18BP as a prognostic indicator in IPF. It is proposed that this protein may play a role in the pathogenesis of the disease and can indicate the extent of fibrosis. In this paper IL-18BP is studied through analysis of human samples, including blood, BALF, and lung, and through murine bleomycin-induced models of pulmonary fibrosis. This is an important topic in the field as there is currently a lack of clinically relevant prognostic biomarkers in IPF, and the authors should be applauded for their work in addressing this matter. However, the manuscript would be improved with the consideration of the following comments:

Introduction

“Recently, it was reported that administration of IL- 18BP to a BLM-injury model improved lung fibrosis, but the trend of IL-18BP secreted in vivo is unclear.” Could the authors please provide a citation for the study that this statement applies to?

Methods

Immunohistochemical staining:

Could the authors please provide further information on the surgical lung biopsies? Were these tissues reviewed by a pathologist? What were the inclusion/exclusion criteria? How many tissues were collected? What dates were these tissues collected from? Are demographic/clinical characteristics available for these subjects?

Subjects for ELISA measurements:

Could the authors please clarify what samples were collected from these subjects?

Results

Figure 1:

How many tissues were stained? It would be beneficial to include images from other IPF and control subjects as well, to confirm findings across multiple samples.

Please include a quantitative measure of staining to strengthen the findings.

Figure 3:

In panel c the authors state there is a positive correlation, but the plot demonstrates a negative correlation. Please adjust.

Discussion

The findings of this paper convey the significance of IL-18BP in both the circulatory and lung tissue compartments in IPF. Although the authors state it is expressed in bronchiole epithelial cells and alveolar macrophages in the IHC results, is there any existing literature on the cell types that IL-18BP is typically expressed in, in both the blood and lung tissue? It would be beneficial for the authors to include this, as well as how this may relate to the results of this study.

Conclusion

The authors state that IL-18BP “might be a potential therapeutic target of IPF.” It would be useful for the authors to further explain this in the discussion section, given what is known about its mechanism in conjunction with the findings of this manuscript.

General

The manuscript should be diligently edited for grammar and syntax errors.

Reviewer #3: This paper may be of interest to those researching IPF; a disease which requires better predictive biomarkers of disease prognosis.

In the introduction the authors explain and highlight the need for early diagnosis of IPF and the requirement for predictive biomarkers of prognosis for clinical management. They explain how they arrived at their hypothesis (from a previous publication by this group) that IL-18BP may serve as a clinical biomarker for IPF. The methods are sufficiently detailed.

The authors suggest that IL-18BP is elevated in IPF lung tissue compared to non-cancerous lung tissue from lung cancer patients. They address the limitation of this analysis due to the number of subjects being relatively small, however n numbers are not provided. N numbers should be provided in the methods, results or figure legend and it should be made clear that the images shown in figure 1 are representative of a larger cohort.

The authors show that both IL-18 and IL-18BP levels are increased in IPF patient serum compared to healthy controls and that serum IL-18BP may be more effective than IL-18 at discriminating IPF patients from healthy controls. The authors go on to show that serum IL-18 and IL-18BP levels correlate with each other in both IPF and healthy patients. The authors refer to ‘S1 fig’ to demonstrate this finding, however these graphs need titles to explain which refers to healthy and which refers to IPF data.

The authors show that IPF serum IL-18BP and IPF BALF IL-18BP levels also correlate with each other, however due to the lack of healthy BALF the same comparison could not made for healthy patients and this should be considered as a limitation when making any conclusions from this. However, this does not particularly affect the overall findings of this manuscript.

The authors perform univariate analysis to demonstrate that IL-18BP (as well as other factors) are statistically correlated with a poor prognosis defined as survival of less than 3 years from diagnosis. Indeed, further multivariate analysis suggests IL-18BP may be an independent predictor of prognosis independent of other listed factors. I am not an expert in these types of statistical analyses and so cannot comment on their robustness.

The authors describe in fig 3 that IL-18BP is inversely correlated with %DLco and %FVC which further supports IL-18BP as a biomarker of prognosis in IPF. In figure 3 they also use a Kaplan-Meier plot to illustrate that IPF patients with higher serum levels of IL-18BP (>5.72 ng/ml) showed poorer survival compared to those with lower IL-18BP levels. The authors refer to ‘S3 Fig’ to explain how they determined 5.72ng/ml as the optimal cut-off for predicting the 3 year survival of patients, however I do not understand how this figure explains this and further explanation is required.

Finally, the authors use a mouse model of lung fibrosis to corroborate their findings in humans, illustrating that increases in IL-18 and IL-18BP are associated with increased hydroxyproline expression in lung tissue. I question why they felt that they needed to use a mouse model to confirm what they had already demonstrated in humans. The advantage of using a mouse model here would be to try and understand a pathogenic role for IL-18BP in lung fibrosis however this was not done. For example, in the discussion they speculate two potential roles for IL-18BP in fibrosis; a pathogenic role via inhibition of IL-37 and a non-pathogenic role/protective role simply as a consequence of increased IL-18 expression. Both of these are valid hypotheses that could quite easily have been investigated in the mouse model used. Did the authors look for increased expression of IL-37 in this model to support this?

The authors conclude that IL-18BP is elevated in IPF and may be a predictive biomarker of prognosis. They go on to suggest that IL-18BP may be a key molecule in the development and progression of IPF and might be a potential therapeutic target. I don’t see any evidence in this paper that supports the claim that IL-18BP has a pathogenic role in IPF or that by inhibiting it would impact the course of disease (again where the mouse model could have been used to explore this mechanism). We know from experience that simply because something is elevated in disease it does not mean it is pathogenic or make it a therapeutic target. The known actions of IL-18BP could equally support an attempt by the body to protect against disease progression in patients with rapidly progressing disease.

Overall, the paper is sound with these minor revisions:

1) Include n numbers for figure 1 and explain images shown are representative.

2) Add graph titles to ‘S1 fig’ to discriminate between IPF and healthy controls.

3) Include lack of healthy BALF as a limitation to this study when discussing correlations between IL-18BP in serum an BALF.

4) Further explanation of how they determined 5.72ng/ml IL-18BP as the optimal cut-off for predicting the 3 year survival of patients, as it is not clear from ‘S3 fig’.

5) It is a shame that the mouse model wasn’t used to investigate the role of IL-18BP in fibrosis. Did the authors consider looking at IL-37 in this model to support their hypothesis in the discussion?

6) If the authors have any evidence to support a pathogenic role of IL-18BP in IPF it should be included to support the claim that “IL-18BP may be a key molecule in the development and progression of IPF and might be a potential therapeutic target of IPF” rather than it is simply elevated in disease and a potential clinical biomarker which is otherwise what they have demonstrated. Alternatively, make it clear that this is a hypothesis of this group and further experiments are required. Literature suggests that IL-18BP is potentially protective in fibrosis and its inhibition could actually exacerbate disease.

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PLoS One. 2021 Jun 4;16(6):e0252594. doi: 10.1371/journal.pone.0252594.r002

Author response to Decision Letter 0

31 Mar 2021

March, 29, 2021

PLOS ONE

COMMENTS FOR THE AUTHOR:

PONE-D-20-35981

IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis

Joerg Heber

Editors-in-Chief

PLOS ONE

Dear Editor:

We wish to re-submit the manuscript titled “IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis.” The manuscript ID is PONE-D-20-35981.

We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. I look forward to working with you and the reviewers to move this manuscript closer to publication in PLOS ONE.

The responses to all comments have been prepared and attached word file 'Revised Manuscript with Track Changes' and 'Manuscript'.

Thank you for your consideration. I look forward to hearing from you.

Sincerely,

Yasushi Horimasu

Department of Respiratory Internal Medicine, Hiroshima University Hospital

1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.

Tel: 082-254-8551

E-mail: yasushi17@hiroshima-u.ac.jp

First, we revised our manuscript in accordance with the following Journal requirements:

2. Please address the following items with respect to animal research procedures and welfare: (1) clearly describe the post-operative analgesics and supportive care that you had provided to the animals following the osmotic pump implantation procedure. (2) Provide an explanation for how you arrived at 6 animals per group. Did you conduct a power analysis or any other type of statistical test; (3) state the rate of mortality during the experiment (if any); (4) discuss humane endpoints, that is: did you have a plan in place to euthanize animals who became severely ill during the study (prior to the experimental endpoints); (4) state the monitoring parameters for the animals (the clinical and behavioral signs used to evaluate the health and welfare). (5) State the method of euthanasia.

(1) We monitored the mice in cages until they reached full recovery from anesthesia without additional analgesics after osmotic pump implantation, because the pomp was small enough not to cause any obvious pain to the mice. This point was clearly stated in the revised manuscript (Line 135).

(2) We decided the number of animals based on the estimated mean and standard deviation of BALF IL-18BP in the preliminary experiment. This point was added in the revised manuscript (Line 139).

(3) One mouse in the bleomycin group died on day21. This information was also included in the revised manuscript (Line 141).

(4) We didn’t have a plan in place to euthanize animals who became severely ill because no mouse died or got severely ill in the preliminary experiment. Additionally, this study was conducted in compliance with Guidelines for Care and Use of Experimental Animals published by Hiroshima University. Therefore, the description was added (Line 142).

(4) We monitored the body weight of the mice to evaluate the health and welfare. Therefore, the description was added (Line 138).

(5) The method of euthanasia was exsanguination after anesthesia. This statement was included in the revised manuscript (Line 142).

Next, responses for each reviewer's comment are as follows.

Reviewer #1:

major comments:

Introduction

COMMENT #1.

-The phrase “The prognosis of IPF is known to be poorer than that of certain malignancies” resulted not appropriate in this context, I suggest to delete.

RESPONSE #1.

We deleted this phrase in accordance with the reviewer’s comment (Lines 55).

COMMENT #2.

- “although their efficacy is limited to modification of the extent of pulmonary functional deterioration.” Miss references.

RESPONSE #2.

Thank you very much for your suggestion. We changed the insertion point of the references from the middle of the sentence to the end (Lines 60).

COMMENT #3.

- “novel biomarkers that can support an early diagnosis of IPF and/or predict the progression of the disease are urgently needed.” This sentence resulted vague. Please can the author specify the role of biomarker in IPF? What are the state of art about biomarker in IPF? I also suggest to better define the role of biomarker that the authors intend to emphasize, diagnostic? Prognostic? Response to treatment? Please improve this part of introduction.

RESPONSE #3.

We admit that the reviewer’s comment is critically important. In order to clearly state the role of biomarker in IPF and also to define the role of biomarker that we intend to emphasize, we altered the final paragraph of the Introduction section in the revised manuscript (Line 63 to 67).

COMMENT #4.

- “We previously performed gene expression analysis using surgically resected lung tissue from seven patients with IPF as well as five patients with non-specific interstitial pneumonia [10].” Is redundant in the introduction, please move in the discussion section.

RESPONSE #4.

In accordance with the reviewer’s recommendation, we simplified the sentence (Line 67) and briefly restated in discussion section (Line 333).

COMMENT #5.

- “Therefore, we performed the present study to clarify whether IL-18BP can serve as a clinical biomarker for IPF.” Rephrase this sentence and better explain the purpose of the study.

RESPONSE #5.

We agree with the reviewer's comment. With COMMENT #3 also in mind, we altered the last sentence in the Introduction section (Line 74).

Materials and methods

COMMENT #6.

- Authors excluded patients who develop lung cancer? Because 30% of patients with IPF develop lung within one year. Please clearify this point.

RESPONSE #6.

Based on the reviewer’s comment, we clarified that those with lung cancer at the time of IPF diagnosis were excluded in this study in “Subjects for enzyme-linked immunosorbent assay (ELISA) measurements” subsection (Line 102). Further, in the survival analysis, those who died from non-IPF causes, including cancer, were treated as censored. This statement was added in “Statistical analysis” subsection (Line 175).

COMMENT #7.

- “Bronchoalveolar lavage was performed as previously described.” Please add the reference

RESPONSE #7.

We added the reference number #21 in accordance with the reviewer’s comment (Line 118).

COMMENT #8.

- “cryopreserved at -80℃ until analysis.” Can the concentration of IL-18BP change due to -80°C cryopreserved?

COMMENT #9.

- “The remaining BALF was centrifuged, and the supernatants were then collected and stored at -20℃ until ELISA was performed.” Different method of storage were applied between BAL of Human and Mouse. Please can the authors clearify this discrepancy?

RESPONSE #8 and #9.

We preserved the human samples at -80℃ because they need to be preserved for several years. There are some reports demonstrating the successful measurement of IL-18BP with cryopreserved samples [1-2]. Although the influence of cryopreservation to the concentration of IL-18BP has not been directly assessed, we believe that the measurement results were substantial because we found they correlated with lung function and prognosis of patients with IPF. On the other hand, the storage period of murine samples was shorter than two weeks. Therefore, we planned to preserve murine samples at -20℃. We added these descriptions in Method section (Line 155). Furthermore, in the murine model, serum samples stored at -20℃ and those stored at -80℃ showed almost comparable levels of IL-18BP. Therefore, we consider that the cryopreservation does not significantly affect IL-18BP concentration. These points were included as the limitation in the revised manuscript (Line 393 to 401).

[1] Mediators Inflamm. 2014; 2014: 165742.

[2] Int J Cancer. 2011; 129: 424–432.

COMMENT #10.

- Results are expressed as mean±SD, however the non parametric test revealed that the variables are not normally distributed. Please can the authors explain why they do not use median and IQR?

RESPONSE #10.

We admit that the reviewer's concern is reasonable. We changed the presentation of the numerical variable to median and IQR.

(Line 37, 162, 189, 219, S1 Fig, and Table1).

Results

COMMENT #11.

- Age and smoking habits differred in the 2 population. What think the authors about this? Can these variable modify the results and the concentrations of IL18-IL18BP?

RESPONSE #11.

As the reviewer pointed out, age and smoking habits were different between HVs and patients with IPF. This is the case because HVs were mostly consisted of young office workers. To avoid or minimize the influence of these differences to our result, we had included age and smoking history in the multivariate model both in the logistic regression analysis (Table 2) and in the Cox analysis (Table 3). Furthermore, we also included these points in the limitation of the revised manuscript (Line 387 to 392).

COMMENT #12.

- “The correlations between serum IL-18BP and serum IL-18 levels were statistically significant not only in IPF patients but also in HVs (S1 Fig).” Please report r and p values.

RESPONSE #12.

Thank you for your suggestion. We reported r and p values (Line 234).

COMMENT #13.

- “ In IPF patients, there were significant positive correlations between serum IL-18BP and BALF IL-18BP levels (S2 Fig, r = 0.406, p = 0.005).” What think authors about this results?

RESPONSE #13.

We admit that the reviewer’s comment is critically important. We consider that this result would indicate that the elevation of IL-18BP levels in sera of IPF patients reflects the elevation of IL-18BP levels in the alveolar space. In the normal condition, the expression of IL-18BP was reported to be ubiquitous in the lymphoid tissue, although in those with IPF, circulating IL-18BP levels were mainly affected by its local expression in the lung. These points were addressed in the new paragraph in Discussion section (Line 370 to 382).

COMMENT #14.

- “Cox proportional hazards analysis revealed that increased serum IL-18BP levels, as well as low % forced vital capacity (FVC), % diffuse capacity of the lung for carbon monoxide (DLco), and use of antifibrotic agents was significantly associated with a poor prognosis.” You say that the use of antifibrotic agent promote the poor prognosis?

RESPONSE #14.

We agree that this sentence is quite miss-leading. we consider that this “ostensible” association between use of antifibrotic agents and poor prognosis was caused by the selection bias resulting from physicians’ and/or patients' decision, medical insurance system and economical status of the patients. Therefore, we altered the description in Result section (Line 254), and inserted the interpretation of this “ostensible” association in Discussion section (Line 383 to 386).

Discussion

COMMENT #15.

- First of all clearify and better explain the aim

RESPONSE #15.

We admit that the beginning sentence of Discussion section was roundabout. In accordance with the reviewer’s comment, we modified the description of the aim of the study in the beginning of discussion (Line 317).

COMMENT #16.

- “can serve as a clinical biomarker for IPF” what kind of biomarker? Dagnostic? Prognostic?

RESPONSE #16.

Our initial hypothesis was that IL-18BP can serve as both diagnostic and prognostic biomarker. Therefore, the aim of this study was to clarify whether IL-18BP can serve as a diagnostic and/or prognostic biomarker for IPF. We have clearly describe this aim in the revised manuscript (Line 320).

COMMENT #17.

- “We believe that these results are clinically important in that they show the usefulness of IL-18BP as a novel predictive biomarker for IPF.” From clinical point of view this result does not help to discriminate IPF than other ILD were normally differential diagnosis occur, but differentiate from Controls, Rarely IPF patients were confused with Controls. Please reformulate.

RESPONSE #17.

I think the reviewer's concerns are quite important. In the present study, IL-18BP was shown to be useful in prognosis prediction of IPF, although its usefulness in differential diagnosis of ILDs was not demonstrated. Therefore, we changed the words “predictive biomarker” to “prognostic biomarker” (Line 320).

COMMENT #18.

- “We consider that this previous report supports our results, although the mechanisms underlying how IL-18BP causes a poor outcome in IPF remains unclear.” This is true but can the authors better explain what are the state of art about the knowledge of il-18 in the lung and the implications in ipf pathogenesis?

RESPONSE #18.

Thank you for your suggestion. We have speculated on two possible links between IL-18BP and poor outcome of IPF and these explanations were described in the next paragraph of the original manuscript. We have reformulated the paragraph structure for better understanding (Line 339 to 369).

COMMENT #19.

- “Despite these limitations, we believe that the results of our study are of significance in that we have demonstrated the utility of serum IL-18BP as a prognostic biomarker for IPF for the first time.” The final sentence resulted vague and inappropriate.

RESPONSE #19.

Thank you for your suggestion. As the reviewer pointed out, this sentence is vague and it overlapped with the descriptions in Conclusion section. Therefore, we deleted this sentence (Line 404).

Conclusion

COMMENT #20.

- The conclusions are inappropriate. Please better specify the role of our research avoiding vague sentences.

RESPONSE #20.

Thank you for your suggestion. We changed to a clearer and less ambiguous sentence (Line 408).

Reviewer #2:

Introduction

COMMENT #1.

“Recently, it was reported that administration of IL- 18BP to a BLM-injury model improved lung fibrosis, but the trend of IL-18BP secreted in vivo is unclear.” Could the authors please provide a citation for the study that this statement applies to?

RESPONSE #1.

Thank you for your suggestion. We added the appropriate citation (Line 74).

Methods

Immunohistochemical staining:

COMMENT #2.

Could the authors please provide further information on the surgical lung biopsies? Were these tissues reviewed by a pathologist? What were the inclusion/exclusion criteria? How many tissues were collected? What dates were these tissues collected from? Are demographic/clinical characteristics available for these subjects?

RESPONSE #2.

We agree that the reviewer’s comments are important. First, all tissue samples were reviewed by pathologists to confirm the pathological diagnosis of UIP. Next, in principle, we tried to perform surgical lung biopsy for all patients without typical UIP pattern in HRCT. However, it was impossible frequently because of poor lung function or patients’ rejection. Actually, lung tissue was available in 9 patients. These explanations were added in “Immunohistochemical staining for IL-18BP” subsection (Line 80). Finally, we included biopsy date (days from the time of diagnosis) as well as demographic/clinical characteristics of these patients in S1 table.

Subjects for ELISA measurements:

COMMENT #3.

Could the authors please clarify what samples were collected from these subjects?

RESPONSE #3.

In accordance with the reviewers’ comment, we altered the description in this subsection to clarify what kind of samples were obtained (Line 101 and 107).

Results

Figure 1:

COMMENT #4.

How many tissues were stained? It would be beneficial to include images from other IPF and control subjects as well, to confirm findings across multiple samples.

COMMENT #5.

Please include a quantitative measure of staining to strengthen the findings.

RESPONSE #4 and 5.

As we mentioned in response to your Comment #2, lung tissue was available in nine of 86 IPF patients. Additionally, five samples were collected as control. We included two additional images of IPF patients in the revised Fig 1. As the reviewer pointed out in COMMENT#5, we performed quantitative measurement of IL-18BP expression using ImageJ Fiji software. These process were included in the Methods section and the results were included in the revised manuscript (Line 80 to 85, Line 92 to 96, Line 186, Line 189 to 192 and S1 Fig).

Figure 3:

COMMENT #6.

In panel c the authors state there is a positive correlation, but the plot demonstrates a negative correlation. Please adjust.

RESPONSE #6.

We agree that the description was mistaken. We revised it as the reviewer pointed out (Line 286).

Discussion

COMMENT #7.

The findings of this paper convey the significance of IL-18BP in both the circulatory and lung tissue compartments in IPF. Although the authors state it is expressed in bronchiole epithelial cells and alveolar macrophages in the IHC results, is there any existing literature on the cell types that IL-18BP is typically expressed in, in both the blood and lung tissue? It would be beneficial for the authors to include this, as well as how this may relate to the results of this study.

RESPONSE #7.

RESPONSE #7.

We agree that the reviewer’s comment is of critical importance. Unfortunately, we couldn’t find such literature, but in the Human Protein Atlas, an online bioinformatics database, marked expression of IL-18BP mRNA in granulocytes, monocytes and T-cells is reported. Therefore, we can speculate that the migrated inflammatory cells can be the main source of IL-18BP expression in the IPF lung. These points were included in Discussion section of the revised manuscript (Line 373 to 380).

Conclusion

COMMENT #8.

The authors state that IL-18BP “might be a potential therapeutic target of IPF.” It would be useful for the authors to further explain this in the discussion section, given what is known about its mechanism in conjunction with the findings of this manuscript.

RESPONSE #8.

We agree that the reviewer’s comment is reasonable, however, we have decided to delete this sentence in accordance with Comment #20 of Reviewer 1. We stated that IL-18BP “might be a potential therapeutic target of IPF” based on the works reported by Zhang and coworkers revealing that exogenous IL-18BP administration could attenuate bleomycin-induced lung fibrosis in murine models [14]. However, because this statement is not derived directly from our presenting study, we considered this statement as inappropriate as the conclusion of our manuscript (Line 408).

General

COMMENT #9.

The manuscript should be diligently edited for grammar and syntax errors.

RESPONSE #9.

In accordance with the reviewer’s comment, we would get English proofreading before resubmission.

Reviewer #3: This paper may be of interest to those researching IPF; a disease which requires better predictive biomarkers of disease prognosis.

COMMENT #1.

In the introduction the authors explain and highlight the need for early diagnosis of IPF and the requirement for predictive biomarkers of prognosis for clinical management. They explain how they arrived at their hypothesis (from a previous publication by this group) that IL-18BP may serve as a clinical biomarker for IPF. The methods are sufficiently detailed.

RESPONSE #1.

We acknowledge your kind feedback.

COMMENT #2.

The authors suggest that IL-18BP is elevated in IPF lung tissue compared to non-cancerous lung tissue from lung cancer patients. They address the limitation of this analysis due to the number of subjects being relatively small, however n numbers are not provided. N numbers should be provided in the methods, results or figure legend and it should be made clear that the images shown in figure 1 are representative of a larger cohort.

RESPONSE #2.

We admit that the reviewer’s comment is reasonable. Actually, lung tissue was available in 9 patients with IPF. We included this information in Methods section and the background characteristics of these patients were presented in supplementary Table 1 in the revised manuscript (Line 80 to 83 and S1 Table). Further, we added some other representative pictures of lung tissue in Fig 1 (Line 186 and Fig 1).

COMMENT #3.

The authors show that both IL-18 and IL-18BP levels are increased in IPF patient serum compared to healthy controls and that serum IL-18BP may be more effective than IL-18 at discriminating IPF patients from healthy controls. The authors go on to show that serum IL-18 and IL-18BP levels correlate with each other in both IPF and healthy patients. The authors refer to ‘S1 fig’ to demonstrate this finding, however these graphs need titles to explain which refers to healthy and which refers to IPF data.

RESPONSE #3.

In accordance with the reviewer’s comment, we added titles at the top of the graphs (S2 Fig)

COMMENT #4.

The authors show that IPF serum IL-18BP and IPF BALF IL-18BP levels also correlate with each other, however due to the lack of healthy BALF the same comparison could not made for healthy patients and this should be considered as a limitation when making any conclusions from this. However, this does not particularly affect the overall findings of this manuscript.

RESPONSE #4.

We agree with the reviewer’s comment. From the view of medical ethics, we didn’t perform BAL in healthy subjects (Line 380).

COMMENT #5.

The authors describe in fig 3 that IL-18BP is inversely correlated with %DLco and %FVC which further supports IL-18BP as a biomarker of prognosis in IPF. In figure 3 they also use a Kaplan-Meier plot to illustrate that IPF patients with higher serum levels of IL-18BP (>5.72 ng/ml) showed poorer survival compared to those with lower IL-18BP levels. The authors refer to ‘S3 Fig’ to explain how they determined 5.72ng/ml as the optimal cut-off for predicting the 3 years survival of patients, however I do not understand how this figure explains this and further explanation is required.

RESPONSE 5.

In accordance with the reviewer’s comment, we altered the legend for S4 Fig as well as the description in “High serum IL-18BP was associated with poor prognosis and impaired pulmonary function” subsection of Result section (Line 261 and 580).

COMMENT #6.

Finally, the authors use a mouse model of lung fibrosis to corroborate their findings in humans, illustrating that increases in IL-18 and IL-18BP are associated with increased hydroxyproline expression in lung tissue. I question why they felt that they needed to use a mouse model to confirm what they had already demonstrated in humans. The advantage of using a mouse model here would be to try and understand a pathogenic role for IL-18BP in lung fibrosis however this was not done. For example, in the discussion they speculate two potential roles for IL-18BP in fibrosis; a pathogenic role via inhibition of IL-37 and a non-pathogenic role/protective role simply as a consequence of increased IL-18 expression. Both of these are valid hypotheses that could quite easily have been investigated in the mouse model used. Did the authors look for increased expression of IL-37 in this model to support this?

RESPONSE #6.

We admit that our mouse model experiments were insufficient to fully understand a pathogenic role for IL-18BP in lung fibrosis. Our primal hypothesis was that the increased expression of IL-18BP in IPF patients was not the cause of fibrosis, but the result of the increased expression of IL-18, a profibrotic factor. We considered that the unimodal elevation of BALF IL-18 followed by the bimodal elevation of BALF IL-18BP in our murine models might support this hypothesis in part. To explain these points more clearly, we reconstructed the 3rd and 4th paragraphs in Discussion section (Line 339 to 369). Of course, as the reviewer pointed out, we should have checked the expression of IL-37, but it was impossible mainly due to our financial limitation. We also included these weakness of this study in the limitation of the study (Line 403).

COMMENT #7.

The authors conclude that IL-18BP is elevated in IPF and may be a predictive biomarker of prognosis. They go on to suggest that IL-18BP may be a key molecule in the development and progression of IPF and might be a potential therapeutic target. I don’t see any evidence in this paper that supports the claim that IL-18BP has a pathogenic role in IPF or that by inhibiting it would impact the course of disease (again where the mouse model could have been used to explore this mechanism). We know from experience that simply because something is elevated in disease it does not mean it is pathogenic or make it a therapeutic target. The known actions of IL-18BP could equally support an attempt by the body to protect against disease progression in patients with rapidly progressing disease.

RESPONSE #7.

As the reviewer pointed out, we agree that the statement in Conclusion section was inappropriate and misleading. What we have demonstrated in this study was simply that IL-18BP was elevated in disease and it can be a prognostic biomarker for IPF. Therefore, we changed the statement in Conclusion section (Line 407).

Overall, the paper is sound with these minor revisions:

COMMENT #8.

1) Include n numbers for figure 1 and explain images shown are representative.

RESPONSE #8.

We investigated the lung tissue derived from nine patients with IPF. In accordance with the comment of reviewer 2, we included three representative images from these nine patients in the revised version (Line 80 to 83, Line 186 and Fig 1).

COMMENT #9.

2) Add graph titles to ‘S1 fig’ to discriminate between IPF and healthy controls.

RESPONSE #9.

In accordance with the reviewer’s comment, we added titles at the top of the graphs (S2 Fig)

COMMENT #10.

3) Include lack of healthy BALF as a limitation to this study when discussing correlations between IL-18BP in serum an BALF.

RESPONSE #10.

We agree with the reviewer’s comment. From the view of medical ethics, we didn’t perform BAL in healthy subjects (Line 380).

COMMENT #11.

4) Further explanation of how they determined 5.72ng/ml IL-18BP as the optimal cut-off for predicting the 3 year survival of patients, as it is not clear from ‘S3 fig’.

RESPONSE #11.

In accordance with the reviewer’s comment, we altered the legend for S3 Fig as well as the description in “High serum IL-18BP was associated with poor prognosis and impaired pulmonary function” subsection of Result section (Line 261 and 580).

COMMENT #12.

5) It is a shame that the mouse model wasn’t used to investigate the role of IL-18BP in fibrosis. Did the authors consider looking at IL-37 in this model to support their hypothesis in the discussion?

RESPONSE #12.

We admit that our mouse model experiments were insufficient to fully understand a pathogenic role for IL-18BP in lung fibrosis. Our primal hypothesis was that the increased expression of IL-18BP in IPF patients was not the cause of fibrosis, but the result of the increased expression of IL-18, a profibrotic factor. We considered that the unimodal elevation of BALF IL-18 followed by the bimodal elevation of BALF IL-18BP in our murine models might support this hypothesis in part. To explain these points more clearly, we reconstructed the 3rd and 4th paragraphs in Discussion section (Line 339 to 369). Of course, as the reviewer pointed out, we should have checked the expression of IL-37, but it was impossible mainly due to our financial limitation (Line 403).

COMMENT #13.

6) If the authors have any evidence to support a pathogenic role of IL-18BP in IPF it should be included to support the claim that “IL-18BP may be a key molecule in the development and progression of IPF and might be a potential therapeutic target of IPF” rather than it is simply elevated in disease and a potential clinical biomarker which is otherwise what they have demonstrated. Alternatively, make it clear that this is a hypothesis of this group and further experiments are required. Literature suggests that IL-18BP is potentially protective in fibrosis and its inhibition could actually exacerbate disease.

RESPONSE #13.

As the reviewer pointed out, we agree that the statement in Conclusion section was inappropriate and misleading. What we have demonstrated in this study was simply that IL-18BP was elevated in disease and it can be a prognostic biomarker for IPF. Therefore, we changed the statement in Conclusion section (Line 407).

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Decision Letter 1

Gernot Zissel

19 May 2021

IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis

PONE-D-20-35981R1

Dear Dr. Horimasu,

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,

Gernot Zissel, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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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 #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: I Don't Know

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: The authors have addressed all the previous concerns and this reviewer finds the manuscript much improved.

Reviewer #3: An interesting piece of work that presents evidence to support a role of IL-18BP as a prognostic marker in IPF. Further investigations into understanding the potential pathogenic mechanism of the IL-18 pathway in IPF will be of interest. All comments have been responded to sufficiently.

**********

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Reviewer #2: No

Reviewer #3: No

Acceptance letter

Gernot Zissel

27 May 2021

PONE-D-20-35981R1

IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis

Dear Dr. Horimasu:

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    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Quantification of immunohistochemical staining with IL-18BP.

    The percentage of the positively stained area was higher in patients with IPF (21.7% [IQR: 20.6–23.1]) than those in control lung (10.6% [IQR: 7.5–13.0]) (p = 0.003). Horizontal bars represent median. IL-18BP: interleukin-18 binding protein.

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    S2 Fig. Correlations between serum IL-18BP levels and IL-18 levels.

    (a) Serum IL-18BP and IL-18 levels showed significant positive correlations in HVs (Spearman r = 0.267, p < 0.001). (b) Serum IL-18BP and IL-18 levels showed significant positive correlations in IPF patients (r = 0.251, p = 0.019). IL-18BP: interleukin-18 binding protein, IL-18: interleukin-18, HVs: healthy volunteers, IPF: idiopathic pulmonary fibrosis.

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    S3 Fig. Correlation between serum IL-18BP levels and BALF.

    Serum IL-18BP and BALF IL-18BP levels showed significant positive correlations in IPF patients (Spearman r = 0.406, p = 0.005). IL-18BP: interleukin-18 binding protein, BALF: bronchoalveolar lavage fluid, IPF: idiopathic pulmonary fibrosis.

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    S4 Fig. ROC analysis of serum IL-18BP for 3-years survival in patients with IPF.

    ROC analysis of serum IL-18BP levels was performed between those that have died and those that have survived or censored during three years from diagnosis (AUC 0.610, 95% Cl: 0.429–0.732). ROC: receiver operating characteristic, IL-18BP: interleukin-18 binding protein, IPF: idiopathic pulmonary fibrosis, AUC: area under the curve.

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    S5 Fig. Summary of experimental model.

    Eight-week-old male C57BL/6 mice were subcutaneously implanted with Alzet osmotic minipumps containing either a 200 μL saline vehicle or 100 mg/kg BLM at different doses. Pumps implanted under the back skin of mice slightly caudal to the scapulae were removed on day 10. BLM: bleomycin.

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    S1 Table. Characteristics of IPF patients with lung tissue sample available.

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    Attachment

    Submitted filename: Response to Reviewers.docx

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    Data Availability Statement

    All relevant data is within the manuscript and its Supporting Information files.

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