The Toll-Like Receptor 5 agonist flagellin prevents Non-typeable Haemophilus influenzae-induced exacerbations in cigarette smoke-exposed mice

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. The major bacterial cause of COPD exacerbations is non-typeable Haemophilus influenzae (NTHi). This susceptibility to infection involves a defective production of interleukin (IL)-22 which plays an important role in mucosal defense. Prophylactic administration of flagellin, a Toll-like receptor 5 (TLR5) agonist, protects healthy mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might prevent COPD exacerbations due to NTHi. Mice were chronically exposed to cigarette smoke and then infected with NTHi. According our preventive or therapeutic protocol, flagellin was administered intraperitoneally. Cigarette smoke-exposed mice treated with flagellin showed a lower bacterial load in the airways, the lungs and the blood. This protection was associated with an early neutrophilia, a lower production of pro-inflammatory cytokines and an increased IL-22 production. Flagellin treatment decreased the recruitment of inflammatory cells and the lung damages related to exacerbation. Protective effect of flagellin against NTHi was altered by treatment with anti-IL-22 blocking antibodies in cigarette smoke-exposed mice and in Il22−/− mice. Flagellin treatment also amplified the production of the β-defensin2 anti-bacterial peptides. This study shows that stimulation of innate immunity by a TLR5 ligand is a potent antibacterial treatment in cigarette smoke exposed mice, suggesting innovative therapeutic strategies against acute exacerbation in COPD.


Introduction
Chronic obstructive pulmonary disease (COPD) is characterized by a progressive and irreversible decline in lung function 1 . Being the third leading cause of death worldwide, it is mainly caused by chronic exposure to cigarette smoke (CS) or pollutants 2 . Inhalation of CS essentially leads to activation of epithelial cells and macrophages responsible for the mobilization of effector and immuno-modulatory cells including neutrophils and natural killer T (NKT) cells 3,4 . The chronic inflammatory response progressively leads to airway remodeling, impaired mucociliary clearance and parenchymal destruction in the lungs, further culminating in irreversible airflow limitation 5 . These components are involved in the increased susceptibility of COPD patients to bacterial and viral airway infections.
Airway colonization with bacteria such as Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis contributes to the pathogenesis and clinical course of the disease 6 . This colonization is responsible for lung infection leading to exacerbations of the disease, which have a strong impact on health status, exercise capacity, lung function, and mortality. Non-typeable Haemophilus influenzae (NTHi), a Gram-negative coccobacillus that lacks a polysaccharide capsule, is an important cause of COPD exacerbations and comorbidity 7,8 . Acute exacerbations invariably scarred the chronic course of COPD 9 . Bacterial infections are first controlled by the innate immune system, which implicated pathogen-associated molecular pattern (PAMP) recognition by Toll-like receptors (TLR) such as those recognizing flagellin (TLR5) responsible for the mobilization of effector cells 10 . During COPD, bacterial infection is characterized by an increased influx of immune cells, including neutrophils, macrophages, dendritic cells (DC) and T lymphocytes 3, 11, 12 . However, this response is not effective enough to clear the pathogens. In this context, we recently reported a defective production of IL-22 in response to bacteria both in COPD patients and mice chronically Created with an evaluation copy of Aspose.Words. To discover the full versions of our APIs please visit: https://products.aspose.com/words/ 4 exposed to CS, whereas IL-17 production is only altered after infection by S. pneumoniae 13,14 . Interestingly, the Th17 cytokines IL-17 and IL In this study, we hypothesized that systemic administration of flagellin could limit the development of NTHi-induced COPD through eliciting an appropriate protective IL-22 response. We reported here that systemic stimulation of the innate immunity by flagellin from Salmonella enterica serovar Typhimurium (FliC) could prevent COPD exacerbation induced by NTHi. We also showed that the protective effect of flagellin against NTHi was dependent of IL-22 and associated with the upregulation of anti-microbial peptides.
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WT mice were daily exposed to cigarette smoke (CS) during 12 weeks (5 cigarettes/day, 5 days / week) to mimic COPD pathogenesis 4 . 3R4F research cigarettes were obtained from the University of Kentucky Tobacco and Health Research Institute (Lexington, KY, USA).
The control group was exposed to ambient air. After 12 weeks of CS or air exposure, mice were either treated intranasally with phosphate buffered saline (

Mice infection and flagellin treatment
NTHi 3224A strain was grown to log-phase in brain-heart infusion (BHI) broth (AES Laboratory) supplemented with 10µg/ml haematin and 10µg/ml nicotinamide adenine