Clarithromycin attenuates IL-13–induced periostin production in human lung fibroblasts

The immunomodulatory effects of macrolides were first described in patients with diffuse panbronchiolitis in 1998 [1]. Macrolide immunomodulation was found to be independent of antibiotic properties [2]. Their effects include modulation (both increasing and decreasing) of inflammatory cytokine production, decreasing airway mucus hypersecretion, and blocking bacterial biofilm formation and virulence factor production [2‐5]. Macrolide therapy has been recommended for chronic obstructive pulmonary disease, cystic fibrosis, non-cystic fibrosis bronchiectasis, and severe asthma [6‐10]. In patients with asthma, long-term macrolide therapy was reported to improve airflow, quality of life, and airway hypersensitiveness [11].

Periostin is an extracellular matrix protein that is associated with eosinophilic airway inflammation and the severity of asthma. Periostin may enhance type 2 inflammation and mucus hypersecretion [12‐15]. Periostin is reported to be the most robust biomarker predicting the effectiveness of lebrikizumab, an anti-IL-13 antibody, for treating asthma [16‐18]. As macrolides also affect type 2-dominated inflammation in asthma, we hypothesized that macrolide therapy may attenuate IL-13 stimulated periostin production and inflammatory gene expression in human lung fibroblasts.

It has been consistently reported that macrolides with 14- and 15-member rings have much greater immunomodulatory effects than the 16-member ring macrolides [2]. In this study, we showed that clarithromycin, a 14-member–ring macrolide, showed the strongest inhibitory effects on periostin expression induced by IL-13 among the examined macrolides. Erythromycin, another 14-member–ring macrolide, showed fewer inhibitory effects while josamycin, a 16-member–ring macrolide, had no such effects. The details of what causes these differences in the inhibitory effects are thus far unclear; however, our results are consistent with these reports in that clarithromycin had the greatest suppressive effect on IL-13–induced periostin expression [2].

The Janus kinase (JAK)-STAT6 pathway is key to IL-13 signaling [24]. We confirmed that clarithromycin suppressed the phosphorylation of STAT6, but the ability of clarithromycin to attenuate periostin production may not entirely be explained by inhibiting STAT6 phosphorylation. There are several reports that STAT6 inhibition stops most periostin expression in lung fibroblasts [25]. These data indicate that STAT6 is the exclusive regulator of periostin expression in lung fibroblasts. On the other hand, we have recently demonstrated that the periostin level is decreased in bronchial epithelial cells by inhibitors against extracellular signal-regulated kinase (ERK) and nuclear factor-kappa B (NF-κB) in addition to STAT6, suggesting that in other cell types, the ERK and NF-κB pathways are involved in periostin production [26]. Additionally, it has been reported that the ERK signaling pathway positively regulates JAK1/STAT6 activity in T cells [27]. On the other hand, macrolides are known to decrease ERK and NF-κB signaling pathways [28]. Tanabe et al. reported that clarithromycin attenuates these pathways and the JAK-STAT6 pathway [20]. Taken together, these results suggest that macrolides may attenuate periostin production via the ERK or NF-κB signaling pathways in addition to the JAK/STAT6 pathway. Clarithromycin may affect STAT6 signaling by downregulating IL-13Rα1/IL-4Rα. We performed flow cytometry to investigate the surface expression of the cytokine receptors on MRC5 cells upon treatment with clarithromycin (Additional file 2: Figure S1A). The expression of IL-13Rα1 was not affected by clarithromycin. Although a statistically significant decrease of IL-4Rα expression was observed, it seemed too slight to explain the considerable attenuation of the periostin production by clarithromycin. To confirm the expression of these receptors at the transcriptional level, we also performed quantitative PCR, finding no suppressive effect by clarithromycin (Additional file 2: Figure S1B). Consequently, we conclude that the inhibition of the STAT6 signaling by clarithromycin is not mainly due to downregulation of the IL-13Rα1/IL-4Rα expression.

We found that clarithromycin showed significantly suppressive effects on IL-13–inducble genes (Fig. 4). These specific genes, whose expression was attenuated by clarithromycin after IL-13 exposure, were dominantly categorized as “extracellular region,” “plasma membrane,” and “defense response” genes, among which asthma-related CD40, NOS2, and CXCL1 (Table 1A) were included. The improvement of asthma symptoms by clarithromycin may be attributed to the downregulation of these genes in addition to periostin. In contrast to suppression of genes activated by IL-13, constitutive expression of these genes was less affected by clarithromycin. Macrolides are classified as ‘immunomodulators’ and decrease hyperinflammation without impairing the normal immune system against infection, as differentiated from immunosuppressive agents such as glucocorticosteroids [2]. The detailed mechanism of how macrolides select for suppressive genes still remains unclear; however, our present finding that clarithromycin selectively suppresses IL-13–inducible genes including periostin may shed light on this mechanism. Extracellular matrix proteins constitute a positive feedback loop in lung fibrosis [23, 29]. Masuoka et al. showed that type 2 cytokines stimulated fibroblasts to produce periostin, interacting with αv integrin, a functional periostin receptor, on keratinocytes [23]. Inhibition of periostin or αv integrin prevented the development or progression of allergen-induced skin inflammations, including fibrosis. Macrolides are reported to have anti-fibrotic effects [30], implying that they may attenuate fibrosis by modulating extracellular matrix proteins. Serum periostin levels are significantly increased in asthmatic patients [12]. The role of periostin on fibrogenesis has been explored, showing that epithelial cell-derived periostin increased secretion of type 1 collagen from airway fibroblasts [14]. Attenuation of periostin production by macrolides may decrease both asthmatic airway inflammation and fibrosis.

This study has a certain limitation. We selected the concentration of clarithromycin (5.0 × 10⁻⁵ M) based on a previous report showing the clarithromycin concentration in epithelial lining fluid after taking clarithromycin [19]. Our study and most of the studies assessing the effects of macrolides used the unified concentrations for each drug when comparing the immunomodulatory effects among macrolides with different types of rings [4, 5]. We did not evaluate whether these drugs at the same concentrations were equally efficacious with other assay such as bactericidal activity. Thus, the results do not necessarily prove actual intrinsic differences in the inhibitory efficacy of these drugs.

Clarithromycin suppressed IL-13–induced periostin production in human lung fibroblasts, in part through inhibition of STAT6 phosphorylation. This suggests a novel mechanism of the immunomodulatory effect of clarithromycin in asthmatic airway inflammation and fibrosis.