J Korean Med Sci.  2024 Jan;39(1):e13. 10.3346/jkms.2024.39.e13.

S100 Calcium-Binding Protein A9, a Potential Novel Diagnostic Biomarker for Idiopathic Pulmonary Fibrosis

• Lee JU
• Kim MK
• Kim MS
• Lee SJ
• Park Sl
• Chang HS
• Park JS
• Park CS

• KMID: 2550787
• DOI: http://doi.org/10.3346/jkms.2024.39.e13

Abstract

Background
Neutrophilic inflammation is a characteristic feature of idiopathic pulmonary fibrosis (IPF). S100 calcium-binding protein A9 (S100A9) is a neutrophil-derived protein involved in the development of neutrophil-related chronic inflammatory disorders. However, the role of S100A9 in IPF remains unclear.
Methods
We used enzyme-linked immunosorbent assays to measure S100A9 levels in bronchoalveolar lavage fluid (BALF) and serum obtained from healthy controls (HCs) and patients with IPF, non-specific interstitial pneumonia, hypersensitivity pneumonitis, and sarcoidosis.
Results
Compared with HCs, BALF S100A9 levels were significantly higher in IPF patients (P < 0.001), patients with hypersensitivity pneumonitis (P = 0.043), and patients with nonspecific interstitial pneumonia (P < 0.001). The S100A9 level in BALF of 0.093 ng/mL could distinguish IPF patients from HCs, with a specificity of 78.8% and a sensitivity of 81.6%. Similarly, the S100A9 level in BALF of 0.239 ng/mL had a specificity of 64.7% and a sensitivity of 66.7% for distinguishing IPF patients from patients with other interstitial lung diseases. Additionally, BALF S100A9 levels were significantly correlated with neutrophil counts (r = 0.356, P < 0.001) in BALF. IPF patients with S100A9 levels in BALF > 0.533 ng/ mL had lower survival rates, compared with patients who had levels ≤ 0.553 ng/mL (n = 49; hazard ratio [HR], 3.62; P = 0.021). Combination analysis revealed that IPF patients with S100A9 levels in BALF> 0.553 ng/mL or neutrophil percentages > 49.1% (n = 43) had significantly lower survival rates than patients with S100A9 levels in BALF ≤ 0.553 ng/mL and neutrophil percentages ≤ 49.1% (n = 41) (HR, 3.91; P = 0.014). Additionally, patients with serum S100A9 levels > 0.077 ng/mL (n = 29) had significantly lower survival rates than patients with levels ≤ 0.077 ng/mL (n = 53, HR, 2.52; P = 0.013). S100A9 was expressed on neutrophils and macrophages in BALF from IPF patients as well as α-smooth muscle actin positive cells in the lung tissues.
Conclusion
S100A9 is involved in the development and progression of IPF. Moreover, S100A9 levels in BALF and serum may be surrogate markers for IPF diagnosis and survival prediction, particularly when analyzed in combination with neutrophil percentages.

Keyword

IPF; S100A9; BAL Fluid; Serum; Diagnosis; Prognosis

Figure

• 

  Fig. 1 S100A9 levels, correlation with neutrophil percentage in BALF, and ROC analysis. (A) S100A9 levels were detected in 12 of 33 HCs, 76 of 87 patients with IPF, 17 of 22 patients with NSIP, 15 of 19 patients with HP, and 6 of 10 patients with sarcoidosis. Data are presented as medians with interquartile ranges. (B) Correlation between S100A9 levels and neutrophil percentages in BALF (n = 87, r = 0.356, P = 0.007). (C) ROC curves for S100A9 levels in IPF patients and HCs. A cutoff value of 0.093 ng/mL had an AUC of 0.833, specificity of 78.8%, and sensitivity of 81.6% for distinguishing IPF patients from HCs. (D) A cutoff level of 0.239 ng/mL for S100A9 exhibited a specificity of 64.7% and a sensitivity of 66.7% for distinguishing between IPF patients and patients with other ILDs (AUC = 0.661).BALF = bronchoalveolar lavage fluid, S100A9 = S100 calcium-binding protein A9, HC = healthy control, IPF = idiopathic pulmonary fibrosis, NSIP = non-specific interstitial fibrosis, HP = hypersensitivity pneumonitis, AUC = area under the ROC curve, ILD = interstitial lung disease, ROC = receiver operating characteristic.*P < 0.05; **P < 0.001.

• 

  Fig. 2 Survival rates in relation to S100A9 levels and neutrophil percentages in bronchoalveolar lavage fluid from IPF patients. Kaplan–Meier analysis was used to analyze 84 IPF patients who were followed up for 1–10 years. (A) Comparison of survival rates between patients with S100A9 levels > 0.553 ng/mL (n = 35, dotted line) and patients with S100A9 levels ≤ 0.553 ng/mL (n = 49, solid line) (HR, 3.62; 95.0% CI, 1.13–11.63; P = 0.021). (B) Comparison of survival rates between patients with neutrophil percentages > 49.1% (n = 17, dotted line) and patients with neutrophil percentages ≤ 49.1% (n = 67, solid line) (HR, 5.38; 95.0% CI, 2.07–13.98; P = 0.003). (C) Comparison of survival rates between patients with S100A9 levels > 0.553 ng/mL or neutrophil percentages > 49.1% (n = 43, dotted line) and patients with S100A9 levels ≤ 0.553 ng/mL and neutrophil percentages ≤ 49.1% (n = 41, solid line) (HR, 3.91; 95.0% CI, 1.62–9.39; P = 0.014).S100A9 = S100 calcium-binding protein A9, Neu = neutrophil, IPF = idiopathic pulmonary fibrosis, HR = hazard ratio, CI = confidence interval.

• 

  Fig. 3 S100A9 levels in serum from IPF patients and relationship with survival rates. (A) Serum S100A9 levels were detected in 19 pf 40 HCs and in 53 of 90 IPF patients. Data are expressed as medians with 25.0% and 75.0% quartiles, with statistical significance denoted as *P < 0.05 vs. HCs. (B) Correlation between S100A9 levels in serum and BALF (r = 0.320, P = 0.023). (C) Kaplan–Meier survival curves comparing survival rates between IPF patients with S100A9 levels > 0.077 ng/mL (n = 29, dotted line) and those with S100A9 levels ≤ 0.077 (n = 53, solid line) ng/mL (hazard ratio, 2.52; 95.0% confidence interval, 1.15–5.51; P = 0.013).S100A9 = S100 calcium-binding protein A9, IPF = idiopathic pulmonary fibrosis, HC = healthy control, BALF = bronchoalveolar lavage fluid.

• 

  Fig. 4 Immunofluorescence staining of S100A9 in lung tissues and BALF cells of IPF patients. (A) S100A9 (green) and α-SMA (red) were co-stained with PE-(red) or FITC-conjugated antibodies (green), respectively (200× magnification). (B) Images show colocalization of pan-macrophage marker CD163 (Alexa 488) (green) and S100A9 (Alexa 594) (red), along with DAPI-stained nuclei (blue) (400× magnification). Control: lung tissue obtained from normal lungs of the patients who underwent surgery for stage I or II lung cancer.BALF = bronchoalveolar lavage fluid, IPF = idiopathic pulmonary fibrosis, S100A9 = S100 calcium-binding protein A9, SMA = smooth muscle actin, PE = phycoerythrin, FITC = fluorescein isothiocyanate, DAPI = 4′,6-diamidino-2-phenylindole, H&E = hematoxylin & eosin.

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