The non-antibiotic macrolide EM900 attenuates HDM and poly(I:C)-induced airway inflammation with inhibition of macrophages in a mouse model
非抗生素大环内酯类 EM900 在小鼠模型中通过抑制巨噬细胞减轻 HDM 和 poly (I: C) 诱导的气道炎症
- 作者列表："Sadamatsu, Hironori","Takahashi, Koichiro","Tashiro, Hiroki","Kato, Go","Noguchi, Yoshihiko","Kurata, Keigo","Ōmura, Satoshi","Kimura, Shinya","Sunazuka, Toshiaki","Sueoka-Aragane, Naoko
Objective Macrolides have been reported to reduce the exacerbation of severe asthma. The aim of this study was to clarify the effects and mechanisms of EM900, a non-antibiotic macrolide, on allergic airway inflammation. Methods Mice were sensitized and challenged by house dust mite (HDM), then exposed to polyinosinic-polycytidylic acid (poly(I:C)) as a model of asthma complicated with viral infection. Mice were administered with EM900. Airway inflammation was assessed from inflammatory cells in bronchoalveolar lavage fluid (BALF) and cytokines in lung tissues. Lung interstitial macrophages were counted by flow cytometry. Cytokine production, phosphorylation of NF-κB, and p38 in macrophages were examined by ELISA and western blotting. Results Counts of cells in BALF and concentrations of IL-13, IL-5, RANTES, IL-17A, and MIP-2 were significantly decreased by EM900 compared to those without EM900. Percentages of lung interstitial macrophages were significantly decreased with EM900. Concentrations of IL-6, RANTES, and MIP-2 induced by HDM and poly(I:C) were significantly suppressed by EM900 through the suppression of NF-κB and p38 phosphorylation in macrophages. Conclusions HDM and poly(I:C)-induced airway inflammation is attenuated by EM900 with the inhibition of lung interstitial macrophages. Clinical use of EM900 is expected, because EM900 has inhibitory effects against airway inflammation without inducing bacterial drug resistance.
目的已报道大环内酯类药物可减少严重哮喘的恶化。本研究的目的是阐明非抗生素大环内酯类 EM900 对过敏性气道炎症的作用和机制。方法用屋尘螨 (HDM) 致敏小鼠，然后用聚肌胞-聚胞苷酸 (poly (I: C)) 作为哮喘合并病毒感染的模型。小鼠给予 em900。从支气管肺泡灌洗液 (BALF) 中的炎症细胞和肺组织中的细胞因子评估气道炎症。流式细胞术计数肺间质巨噬细胞。通过 ELISA 和 western blotting 检测巨噬细胞中细胞因子的产生、 NF-κ b 的磷酸化和 p38。结果 EM900 组 BALF 中细胞计数及 IL-13 、 IL-5 、 RANTES 、 IL-17A 、 MIP-2 浓度均显著低于对照组。Em900 使肺间质巨噬细胞百分比显著降低。EM900 通过抑制巨噬细胞中 NF-κ b 和 p38 磷酸化，显著抑制 HDM 和 poly (I: C) 诱导的 IL-6 、 RANTES 和 MIP-2 的浓度。结论 EM900 可通过抑制肺间质巨噬细胞减轻 HDM 和 poly (I: C) 诱导的气道炎症。预计临床使用 EM900，因为 EM900 对气道炎症有抑制作用，而不会诱导细菌耐药。
METHODS:BACKGROUND AND PURPOSE:A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD). EXPERIMENTAL APPROACH:C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed. KEY RESULTS:Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K2 , and S1P receptors (S1P2 and S1P3 ) has been detected in the lung of smoking mice. Sphingosine kinases inhibition reversed the increased cholinergic response in airways of smoking mice. CONCLUSIONS AND IMPLICATIONS:S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.
METHODS::The interim results from this 90-day multi-dose, inhalation toxicology study with life-time post-exposure observation has shown an important fundamental difference in persistence and pathological response in the lung between brake dust derived from brake-pads manufactured with chrysotile, TiO2 or chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos. In the brake dust exposure groups no significant pathological response was observed at any time. Slight macrophage accumulation of particles was noted. Wagner-scores, were from 1 to 2 (1 = air-control group) and were similar to the TiO2 group. Chrysotile being biodegradable, shows a weakening of its matrix and breaking into short fibers & particles that can be cleared by alveolar macrophages and continued dissolution. In the chrysotile exposure groups, particle laden macrophage accumulation was noted leading to a slight interstitial inflammatory response (Wagner-score 1-3). There was no peribronchiolar inflammation and occasional very slight interstitial fibrosis. The histopathology and the confocal analyses clearly differentiate the pathological response from amphibole asbestos, crocidolite and amosite, compared to that from the brake dust and chrysotile. Both crocidolite and amosite induced persistent inflammation, microgranulomas, and fibrosis (Wagner-scores 4), which persisted through the post exposure period. The confocal microscopy of the lung and snap-frozen chestwalls quantified the extensive inflammatory response and collagen development in the lung and on the visceral and parietal surfaces. The interim results reported here, provide a clear basis for differentiating the effects from brake dust exposure from those following amphibole asbestos exposure. The subsequent results through life-time post-exposure will follow.
METHODS::The respiratory tract is lined by a pseudo-stratified epithelium from the nose to terminal bronchioles. This first line of defense of the lung against external stress includes five main cell types: basal, suprabasal, club, goblet and multiciliated cells, as well as rare cells such as ionocytes, neuroendocrine and tuft/brush cells. At homeostasis, this epithelium self-renews at low rate but is able of fast regeneration upon damage. Airway epithelial cell lineages during regeneration have been investigated in the mouse by genetic labeling, mainly after injuring the epithelium with noxious agents. From these approaches, basal cells have been identified as progenitors of club, goblet and multiciliated cells, but also of ionocytes and neuroendocrine cells. Single-cell RNA sequencing, coupled to lineage inference algorithms, has independently allowed the establishment of comprehensive pictures of cell lineage relationships in both mouse and human. In line with genetic tracing experiments in mouse trachea, studies using single-cell RNA sequencing (RNAseq) have shown that basal cells first differentiate into club cells, which in turn mature into goblet cells or differentiate into multiciliated cells. In the human airway epithelium, single-cell RNAseq has identified novel intermediate populations such as deuterosomal cells, 'hybrid' mucous-multiciliated cells and progenitors of rare cells. Novel differentiation dynamics, such as a transition from goblet to multiciliated cells have also been discovered. The future of cell lineage relationships in the respiratory tract now resides in the combination of genetic labeling approaches with single-cell RNAseq to establish, in a definitive manner, the hallmarks of cellular lineages in normal and pathological situations.