Erythromycin suppresses neutrophil extracellular traps in smoking-related chronic pulmonary inflammation

Neutrophil extracellular traps (NETs) may play a critical role in smoking-related chronic airway inflammation. However, the mechanism by which NETs induced by cigarette smoke initiate the

adaptive immunity in chronic obstructive pulmonary disease (COPD) is not fully understood. In this study, we explored the effects of NETs induced by cigarette smoke on the myeloid dendritic

cells (mDCs) and Th1 and Th17 cells. Additionally, we observed the inhibitory effect of erythromycin on NETs induced by cigarette smoke. We found that elevated NET levels in the sputum of

COPD patients were correlated with the circulating Th1 response, mDC activation and airflow limitation. NETs induced by cigarette smoke extract (CSE) could activate monocyte-derived mDCs and

promote Th1 and Th17 differentiation in vitro. Erythromycin effectively inhibited NET formation induced by CSE. In vivo, erythromycin decreased NETs in the airway and ameliorated emphysema

with Th1 and Th17 cell down-regulation and CD40+ and CD86+ mDCs suppression in mice chronically exposed to cigarette smoke. These findings provide direct evidence that NETs promote the

differentiation of Th1 and Th17 and play a role in the adaptive immunity of smoking-related chronic lung inflammation. Erythromycin is a potential therapeutic strategy for NETs inhibition in

COPD.

Chronic obstructive pulmonary disease (COPD) is a progressive disease associated with abnormal airway and alveolar inflammatory responses to cigarette smoke or other noxious particles and

gases1. Owing to its high mortality and morbidity, COPD has become a serious global health issue2. Cigarette smoking is the major risk factor for COPD and directly promotes airway

neutrophilic inflammation3. Interestingly, cigarette smoke extract (CSE) or nicotine can trigger neutrophil extracellular trap (NET) formation4,5. NETs were initially described as web-like

DNA structures produced by activated neutrophils during the response to pathogens6. More recently, studies have indicated that NETs are involved in many non-infectious disorders and chronic

inflammatory conditions, such as systemic lupus erythematosus7, autoimmune small-vessel vasculitis8, rheumatoid arthritis9, atherosclerosis10, and others11,12,13,14. Previous studies have

revealed that excessive NETs in the sputum are associated with elevated pro-inflammatory cytokine levels and thus, might contribute to lung damage15,16. However, most studies have focused on

the direct pathological effect of NETs17,18, and relatively little is known about the contribution of NETs to adaptive immunity.

The initiation and dysregulation of adaptive immunity are related, in part, to the enhancement and persistence of airway inflammation in COPD. We recently demonstrated that NETs released by

activated polymorphonuclear neutrophils (PMNs) stimulated with CSE promote the activation of plasmacytoid dendritic cells (DCs) in mice and, subsequently initiate pathologic T cell

responses5, suggesting that NETs serve as critical connectors between the innate and adaptive immunity and could serve as a potential target for COPD treatment. However, whether NETs induced

by CSE could activate DCs in human remains to be elucidated.

Persistent neutrophilic infiltration in the airway is a typical feature of smoking-related COPD, which represents an obstacle for conventional therapeutic regimens. The anti-inflammatory and

immuno-modulating effects of macrolides, such as erythromycin, have long been recognised. Emerging evidence has shown that long-term and low-dose erythromycin can inhibit airway

neutrophilic inflammation and reduce acute exacerbations in patients with COPD19. Macrolides are now recommended by a series of clinical guidelines to prevent acute exacerbation of COPD2,20,

but the mechanisms underlying their inhibitory effects on neutrophilic inflammation are still unclear. Several previous studies have reported that erythromycin could attenuate pulmonary

inflammation, protect against the development of emphysema, and alter different types of pulmonary cells in murine models21,22,23,24. However, it is unclear whether erythromycin suppresses

NET formation under stimulation with cigarette smoke and thus, modulates adaptive immunity.

Here, we sought to explore the effects of NETs induced by cigarette smoke on the differentiation of Th1 and Th17. We also observed the effect of erythromycin on NET formation during chronic

inflammation induced by cigarette smoke exposure.

Human blood and sputum samples were collected from 32 patients with COPD and 16 healthy controls. The diagnosis of COPD was based on forced expiratory volume in 1 s (FEV1) and forced vital

capacity (FVC) detected by post-bronchodilator spirometry (FEV1