Background
Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of disorders clinically characterized by chronic muscle weakness, low muscle endurance, and the presence of inflammatory cell infiltrates in muscle tissue [
1]. Polymyositis (PM), dermatomyositis (DM), and clinically amyopathic dermatomyositis (CADM) are subsets of IIMs that frequently affect the lungs. Interstitial lung disease (ILD) is a common pulmonary manifestation considered a common cause of morbidity and mortality in myositis [
2,
3]. Risk factors for ILD in patients with myositis include genetic predisposition and myositis-specific autoantibodies [
4,
5]; however, little is known about the clinical course and pathogenesis of myositis-associated ILD. Cellular profiles in bronchoalveolar lavage fluid (BALF) can be used to help diagnose ILD, but only serve to rule out infection in the current clinical differential diagnosis in patients with myositis [
6]. However, some reports suggest that the presence of neutrophils in BALF correlates with poor clinical course [
2,
7,
8].
Defensins are small, arginine-rich, cationic peptides that exhibit antimicrobial activity [
9]. Human cells express α- and β-defensins, and among the six known α-defensins, human neutrophil peptides (HNPs) 1 to 4 are mainly found in neutrophils, whereas human defensins 5 and 6 are primarily expressed in intestinal Paneth cells and the respiratory and female reproductive tracts [
10]. In addition to their antimicrobial functions, defensins might also regulate inflammatory responses [
11]. We previously identified elevated plasma and BALF HNP levels in patients with various inflammatory lung diseases, including systemic sclerosis-associated ILD, with these levels correlated with neutrophils in BALF [
12‐
19]. These results indicated that plasma and BALF HNP levels play a pivotal role and might serve as biomarkers of other connective-tissue-disease-associated ILD. Here, we evaluated HNP concentrations in BALF and plasma samples from patients with myositis-associated ILD to determine whether HNPs could be used as markers of myositis-associated ILD.
Discussion
In this study, we found elevated HNP concentrations in the plasma and BALF from patients with myositis-associated ILD as compared with that observed in those from healthy controls. Notably, plasma HNP levels were associated with total cell counts in the BALF, whereas those in BALF samples were associated with the interstitial pneumonia marker SP-A and the percentage of neutrophils in BALF. Furthermore, BALF HNP levels in patients with anti-ARS-antibody positive myositis-associated ILD were significantly correlated with the extent of reticular opacities and negatively correlated with consolidation in HRCT findings.
Previous studies demonstrated that BALF HNP levels are associated with the prevalence of neutrophils in BALF samples and disease activity in patients with various lung diseases, including connective-tissue-disease-associated ILD [
14‐
19]. Moreover, several reports suggest that neutrophils in the BALF correlate with poor clinical course in patients with PM/DM [
2,
7,
8]. Consistent with these findings, the present study showed that BALF HNP levels correlated with the amount of neutrophils in the BALF of patients with myositis-associated ILD. Neutrophils release granular and nuclear contents called neutrophil extracellular traps (NETs), including HNPs, in response to different classes of microorganisms, soluble factors, and host molecules [
28]. Zang et al. [
29] demonstrated that patients with PM/DM have the capacity to form NETs that could not be completely degraded, particularly in patients with PM/DM-ILD. Moreover, they reported that abnormal NET regulation might be involved in PM/DM pathogenesis and could be a factor that initiates and/or aggravates ILD [
29]. The authors also reported a higher percentage of low-density granulocytes (LDGs) along with enhanced NET-formation capabilities in patients with DM as compared with healthy controls, and that this percentage was also higher in DM patients with ILD than in those without. Additionally, LDG percentage was positively correlated with lung disease activity scores [
30]. In line with these reports, the present results showed that increased HNP levels in the plasma and BALF from patients with myositis-associated ILD suggested that neutrophils are likely to release NETs, including HNPs, which are difficult to degrade in patients with myositis-associated ILD.
Our results also showed that BALF HNP levels correlated with SP-A levels, suggesting the existence of interstitial lung injury [
31]; however, this was not observed in HRCT findings for all patients. Additionally, these levels in patients with anti-ARS-antibody positive myositis-associated ILD were significantly correlated with the extent of reticular opacities according to HRCT findings, which we previously reported in patients with systemic sclerosis-associated ILD [
19]. Reticular opacities are common HRCT findings in anti-ARS-antibody positive associated ILD with or without myositis [
32‐
34] and reflect fibrosis in anti-ARS-antibody positive ILD [
33]. These findings indicate that HNP levels in BALF reflect the fibrotic change in anti-ARS-antibody positive associated ILD. Furthermore, we previously reported that HNPs induce the production of cytokines and growth factors, which act on lung fibroblasts and epithelial cells to induce pulmonary fibrosis and collagen production in vitro [
9,
35,
36]. This might suggest that HNPs in the lung both indicate and induce fibrotic change. Further studies are needed to determine whether neutrophil-derived HNPs play a role in the pathogenesis of myositis-associated ILD.
We also observed that plasma HNPs were associated with total cell counts in BALF, which might reflect the lung inflammation observed in patients with myositis-associated ILD. Although the precise mechanism of plasma HNP production is not well understood, these factors are likely derived from neutrophil-precursor cells in the bone marrow following stimulation by inflammatory mediators [
18,
37]. Therefore, these results might suggest that lung inflammation elicits increased plasma HNP levels. Nevertheless, further studies are needed to clarify the functional significance of plasma HNPs in patients with myositis-associated ILD.
Although we found that HNP levels were associated with several clinical parameters and suggested to play a role in myositis-associated ILD, it remains unclear whether HNPs can be used as disease markers in myositis-associated ILD. This might be because our definition of myositis-associated ILD included different disease types (PM, DM, or CADM) or the use of myositis-specific antibodies (anti-ARS and anti-MDA5).
The present study has several limitations. First, because the patient population was strictly seen by physicians in the respiratory department, our study only examined HNP levels in patients with myositis-associated ILD, but not those without the ILD component. Therefore, we were unable to confirm that lung pathology was directly responsible for the observed differences in HNP levels. Second, we did not show values for plasma or BALF HNPs, which can discriminate between various types of ILD or infectious processes. We previously reported elevated HNP levels in several types of ILD, as well as according to infectious status [
14‐
19], indicating that increased levels of HNPs were nonspecific in myositis-associated ILD. Additionally, the small patient cohort limited the clinical application of these findings; therefore, a larger patient population should be examined using a prospective study model in future investigations.
Acknowledgments
We thank Mr. Atsushi Yokoyama and Mrs. Yoshiko Akiyama (Nagasaki University Hospital) for their invaluable technical assistance with the ELISA assays for HNP quantification.