Manipulation Of The Mononuclear Phagocyte Phenotype Switching By Mineralocorticoid Receptor Based Regulation Strategy In Experimental Lung Fibrosis

Objective: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive,fibrosing interstitial pneumonia of unknown etiology, and is largely unaffectedby currently available medical therapies. The precise mechanisms of IPFremain incompletely understood, several areas which have been exploredincluding inflammation and immune mechanisms, epithelial injury andimpaired wound repair, oxidative stress and oxidative signaling andprocoagulant mechanisms, whereas the abnormal regulation of inflammationstill play an important role in the development of pulmonary fibrosis. Theheterogeneity of mononuclear phagocytes, one of the hotspots in immune-related diseases, seem to reflect developmental stages with distinctphysiological roles. Recent evidence demonstrated that there is a closerelationship between the phenotype of mononuclear phagocytes and theprogression of pulmonary fibrosis. In addition to the cardiovascular systemand kidney cells, mineralocorticoid receptor (MR) also expressed in themonocytes and macrophages. It has been demonstrated that MR is involved inthe regulation of monocyte-macrophage phenotype. Liposomes naturallytarget cells of the mononuclear phagocytic system (MPS), especiallymacrophages. Therefore, drug-loaded liposomes act as an efficient meanstargeting to MPS cells.The present study was designed to address the dynamic changes ofmononuclear phagocytes in the circulating, lung alveolar and interstitialcompartments in bleomycin-induced pulmonary injury model, and explore therelationship between polarization of mononuclear phenotype with lunginflammation and fibrosis. Further more, we investigated the effect ofspironolactone liposome, targeting mineralocorticoid receptor, on the regula- tion of mononuclear phenotype as well as the therapeutic potential inbleomycin-induced acute lung injury and fibrosis.Method:100C57BL/6J mice were randomly divided into normal salinegroup (NS), bleomycin group (BLM), mice were lightly anesthetized withether and followed by oropharyngeal installation of40μL of sterile saline orthe same volume of bleomycine A5(2.5mg/kg). At1,3,7,14or21days,animals were sacrificed by exsanguinations under anesthesia. Blood, BALFand lung tissues were collected for the following assays.The left lobe from non-lavaged lung was used for pathologicalexaminations, inflammation score (IS), collagen volume fraction (CVF) andα-SMA positive area represent the extent of pulmonary inflammation andfibrosis. The bronchoalveolar lavage fluid (BALF) was collected for total cellcounts, differential cell counts and flow cytometry analysis. Cytokines in theBALF were measured by commercially available ELISA kits. Real-time PCRwas performed for detecting the mRNA expression levels of cytokines in lungtissue. Hydroxyproline (HYP) was measured by Chloramines T method. Thedynamic changes of different subsets of circulating monocyte, alveolarmacrophage and interstitial macrophage were analysed by flow cytometry.To detect the role of MR antagonist on bleomycin-induced experimentalpulmonary fibrosis, male C57BL/6J mice were divided into normal salinegoup (NS), bleomycin group (BLM), bleomycin plus liposome control group(BLM+LC), bleomycin plus spironolactone liposome group (BLM+LS) andbleomycin plus free spironolactone (BLM+SP). From the day of thebleomycine administration, BLM+LC, BLM+LS and BLM+SP groups weredelivered by oral gavage of blank liposome, spironolactone liposome and freespironolactone, respectively. The treatments continued for21days and alldetections were as same as before.Result: HE-stained lung sections showed significant peribronchial andinterstitial infiltrates of inflammatory cells and thickening of the alveolar walls3days after bleomycin challenge. The magnitude of inflammation (presentedas IS) of the BLM groups was significantly higher than the NS group from day 1to day21, which was gradually increased to a peak on day7and showed adownward trend in the BLM group. The degree of inflammation significantlydecreased in BLM+LS and BLM+SP groups compared with their respectivecontrol group on day7(P <0.05), we found no difference between thedifferent dosage forms of spironolactone. BLM administration inducedexcessive collagen deposition in the lung and the CVF of the BLM groupsgradually increased from day7to day21. Spironolactone treatment couldsignificantly reduced the collagen deposition as well as α-SMA positive areaon day21(all P <0.05). The average values of fibrosis index were lower inspironolactone lipsome group compared with free spironolactone group. Thedynamic changes of hydroxyproline content were consistent with pathologicalexamination.Compared with the NS group, total cell counts increased in the BLMgroup from day3to day21（all P <0.01）, which reached the maximum onday7. The absolute counts of Mφ increased significantly on day7, which wasconsistent with the change of total cell counts. However, a significant increaseof neutrophils was observed from day1to day7after BLM administration (allP <0.01) and then showed a downward trend. The number of lymphocytes inBALF increased dramatically since day3to day21, which increased to a peakon day14. The total and differential counts of inflammatory cells werestatistically decreased in BLM+LS and BLM+SP groups compared with theirrespective control group on day7.Bleomycin treatment induced increased mRNA expression levels ofIL-1β, CCL2and TNF-α during acute inflammation phase, Col I and Col IIIexpression levels were increased during fibrosis stage, all had statisticalsignificance. Compared with their respective control groups, spironolactoneliposome treatment was associated with downregulated CCL2, TNF-α andIL-1β both at the mRNA and the protein levels，while we failed to observedown-regulation of CCL2mRNA expression and TNF-α protein from day7inBLM+SP group. In addition, markers for M2polarization, such as arginase-1mRNA level in lung tissue and IL-4protein content in BALF were downregulated by spironolactone. The mean level of IL-4was lower in LSgroup than that was in SP group, although there was no statistical difference.Circulating monocytes showed a dynamic expression of Ly6C afterbleomycin administration compared with the NS group. The percentage ofLy6Chimonocytes immediately increased on day1after bleomycin challenge(P <0.01), reached the maximal level on day3and gradually decreased untilday21. The percentage of Ly6Clomonocyte subsets showed reciprocalchanges. In the NS group, from day1until day21, M1-like AMφ wasconsistently the predominant cell type of AMφ in BALF. However, there wasan increasing trend of M2-like AMφ in the BLM-treated mice from day3,which reached the plateau level on day14, followed by a decreasing trend. Asfor IMφ from enzymatically digested lung tissue, there was no obvious changeof M1to M2ratio from day1to day14after BLM challenge, however, anincrease of M2-like IMφ was observed on day21(P <0.01). Linear correlationanalysis revealed that the percent of Ly6Chimonocytes was positivelycorrelated with lung inflammatory response and M2-like AMφ was positivelycorrelated with collagen deposition. Accordingly, Ly6Clomonocytes andM1-like AMφ were both negatively correlated with the above histologicalchanges, respectively. Of note, there were no statistical associations betweenIMφ and lung pathology. Spironolactone lipsome treatment could significantlyreduce bleomycin-induced the Ly6Chimonocyte pool expansion on day3(P <0.01).The increased tendency of M2-like AMφ induced by BLM wassignificantly normalized by MR antagonism on day14(P <0.01).Spironolactone lipsome and free spironolactone have similar effect onregulating the phenotype of mononuclear phagocytes, and there was nostatistically significant difference.Conclusion: the present study demonstrates the detailed dynamicchanges of circulating monocytes, AMφ and IMφ in a C57BL/6J model ofBLM-induced lung injury and fibrosis. Specifically, a rapid increase ofcirculating Ly6Chimonocytes after BLM challenge is followed by subsequentexpansion of M2-like AMφ, which are closely associated with lung inflammatory response and fibrosis, respectively. there was no obviousphenotypic change of IMφ from enzymatically digested lung tissue. MRantagonism by spironolactone lipsome could attenuate bleomycin-inducedacute pulmonary injury and fibrosis, partially by reducing circulatinginflammatory Ly6Chimonocyte expansion and inhibiting alternativelyactivation of mononuclear phagocyte in alveolar compartment.