Upregulation of leukocyte immunoglobulin-like receptor B4 on interstitial macrophages in COPD; their possible protective role against emphysema formation.

BACKGROUND: Leukocyte immunoglobulin-like receptor B4 (LILRB4) is one of the inhibitory receptors in various types of immune cells including macrophages. Previous reports suggested that LILRB4 could be involved in a negative feedback system to prevent excessive inflammatory responses. However, its role has been unclear in chronic obstructive pulmonary disease (COPD), in which macrophages play a crucial role in the pathogenesis. In this study, we aimed to examine the changes of LILRB4 on macrophages both in the lung specimens of COPD patients and the lungs of a mouse emphysema model. We then tried to compare the differences in both inflammation and emphysematous changes of the model between wild-type and LILRB4-deficient mice in order to elucidate the role of LILRB4 in the pathogenesis of COPD. METHODS: We prepared single-cell suspensions of resected lung specimens of never-smokers (n = 21), non-COPD smokers (n = 16), and COPD patients (n = 14). The identification of LILRB4-expressing cells and the level of LILRB4 expression were evaluated by flow cytometry. We analyzed the relationships between the LILRB4 expression and clinical characteristics including respiratory function. In the experiments using an elastase-induced mouse model of emphysema, we also analyzed the LILRB4 expression on lung macrophages. We compared inflammatory cell accumulation and emphysematous changes induced by elastase instillation between wild-type and LILRB4-deficient mice. RESULTS: The levels of surface expression of LILRB4 are relatively high on monocyte linage cells including macrophages in the human lungs. The percentage of LILRB4+ cells in lung interstitial macrophages was increased in COPD patients compared to non-COPD smokers (p = 0.018) and correlated with the severity of emphysematous lesions detected by CT scan (rs = 0.559, p < 0.001), whereas the amount of smoking showed no correlation with LILRB4 expression. Increased LILRB4 on interstitial macrophages was also observed in elastase-treated mice (p = 0.008). LILRB4-deficient mice showed severer emphysematous lesions with increased MMP-12 expression in the model. CONCLUSIONS: LILRB4 on interstitial macrophages was upregulated both in human COPD lungs and in a mouse model of emphysema. This upregulated LILRB4 may have a protective effect against emphysema formation, possibly through decreasing MMP-12 expression in the lungs.

Administration route governs the therapeutic efficacy, biodistribution and macrophage targeting of anti-inflammatory nanoparticles in the lung.

BACKGROUND: Uncontrolled inflammation is a central problem for many respiratory diseases. The development of potent, targeted anti-inflammatory therapies to reduce lung inflammation and re-establish the homeostasis in the respiratory tract is still a challenge. Previously, we developed a unique anti-inflammatory nanodrug, P12 (made of hexapeptides and gold nanoparticles), which can attenuate Toll-like receptor-mediated inflammatory responses in macrophages. However, the effect of the administration route on its therapeutic efficacy and tissue distribution remained to be defined. RESULTS: In this study, we systematically compared the effects of three different administration routes [the intratracheal (i.t.), intravenous (i.v.) and intraperitoneal (i.p.)] on the therapeutic activity, biodistribution and pulmonary cell targeting features of P12. Using the LPS-induced ALI mouse model, we found that the local administration route via i.t. instillation was superior in reducing lung inflammation than the other two routes even treated with a lower concentration of P12. Further studies on nanoparticle biodistribution showed that the i.t. administration led to more accumulation of P12 in the lungs but less in the liver and other organs; however, the i.v. and i.p. administration resulted in more nanoparticle accumulation in the liver and lymph nodes, respectively, but less in the lungs. Such a lung favorable distribution was also determined by the unique surface chemistry of P12. Furthermore, the inflammatory condition in the lung could decrease the accumulation of nanoparticles in the lung and liver, while increasing their distribution in the spleen and heart. Interestingly, the i.t. administration route helped the nanoparticles specifically target the lung macrophages, whereas the other two administration routes did not. CONCLUSION: The i.t. administration is better for treating ALI using nanodevices as it enhances the bioavailability and efficacy of the nanodrugs in the target cells of the lung and reduces the potential systematic side effects.

Silica particles disorganize the polarization of pulmonary macrophages in mice.

Silicosis is a fatal fibrotic lung disease caused by long-term silica particle exposure, in which pulmonary macrophages play an important role. However, the relationship between macrophage polarization and silicosis remains unclear. We established an experimental silicosis mouse model to investigate macrophage polarization during silicosis development. C57BL/c mice were exposed to silica by intra-tracheal instillation and sacrificed at different time points. Lung tissues and bronchoalveolar lavage fluid were collected for flow cytometry, quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assays, western blotting, and histology examinations. The polarization of pulmonary macrophages was dysregulated during silicosis development. In the early stage of silicosis, M1 macrophages were induced and played a leading role in eliciting inflammatory; in the late stage, M2 macrophages were induced to promote tissue repair. Levels of several cytokines in lung tissue microenvironment changed with macrophage polarization. Inflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-1ß and IL-6 were upregulated in the inflammation stage, while the anti-inflammatory cytokine IL-10 was upregulated in the fibrosis stage. Furthermore, we found that STAT (signal transducer and activator of transcription) and IRF (interferon regulatory factor) signaling pathway were involved in the regulation of macrophage polarization in silicosis. In summary, macrophage polarization is closely related to the occurrence and development of silicosis and may be a key point for further elucidating silicosis pathogenesis.

Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide.

Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs -1, -4, and -6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam3CSK4. In addition, the presence of EVs reduced inflammatory responses in Pam3CSK4-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression.

Research Progress in the Influencing Factors in Particles Uptaking of Alveolar Macrophages / 中国药师

Pulmonary drug delivery has become a research focus in recent years, while the action between particles and pulmonary macrophages after particles transportation to lung tissue was paid little attention. Therapeutic efficacy can be enhanced by regulating the particles uptake action of pulmonary macrophages according to different diseases. Referring to a lot of articles, the relationship between common diseases and macrophages was reviewed and the influencing factors in the particles uptake of alveolar macrophages were sum-marized. The review laid foundation for the development of preparations and clinical application of pulmonary drug delivery systems.

Effects of CORM-2 on mitochondrial fission protein Fis1 in the LPS-activated lung macrophages of rats through p38MAPK signaling pathway / 中华急诊医学杂志

Objective To investigate the effects of CORM-2 via p38 mitogeu-activated protein kinase (p38MAPK) signaling pathway on the expression of the mitochondrial fission protein 1 (Fisl) in lipopolysaccharide (LPS)-induced mouse pulmonary macrophages.Methods The rat subculture alveolar macrophages were seeded on 96 well plates with 2 × 105/ml densities.After 24 hours of culture,it was divided into 4 groups by random number table method:normal control group (group C),group LPS (group L),CO releasing agent CORM-2 + LPS group (group LC),p38MAPK inhibitor SB203580 + CORM-2 + LPS group (group LCS).When the cells were incubated for 24 hours,the mitochondrial MDA content and SOD activity were determined by ELISA kit,the levels of HO-1、mitochondrial fission protein Fis1 and p38 were determined by Western blot,the expressions of HO-1 and mitochondrial fission protein Fis1 were detected by RT-PCR.Results Compared with the C group,the levels of MDA [(2.43 ±0.12) vs.(3.59 ±0.07)],HO-1 [(1.31±0.27) vs.(1.65±0.41)],Fis1 [(1.27±0.23) vs.(1.65±0.41)] andp38 [(1.01 ±0.24) vs.(1.36 ±0.17)] in group L were increased,and the activity of SOD [(81.7 ± 1.62) vs.(54.7 ± 1.62)] was decreased (P ＜ 0.05);Compared with the group L,the MDA content [(3.59 ± 0.07) vs.(3.08 ±0.52)] and the level of Fis1 [(2.01 ±0.35) vs.(1.48 ±0.39)] in group LC were down-regulated,and the levels of SOD [(54.7 ± 1.62) vs.(67.4 ± 1.32)]、and the expressions of HO-1 [(1.65±0.41)vs.(2.25±0.18)] andp38 [(1.36±0.17) vs.(1.78±0.23)] wereup-regulated (P ＜0.05).Compared with the group LC,the MDA content [(3.08 ±0.52) vs.(4.16 ±0.19)] and the expression of Fis1 [(1.48 ±0.39) vs.(1.96 ±0.31)] in group LCS were increased,and the level of SOD [(67.4±1.32)vs.(45.9±1.52)]、and the expressions of HO-1 [(2.25±0.18)vs.(1.78± 0.19)] and p38 [(1.78 ±0.23) vs.(1.12 ±0.29)] were decreased (P ＜0.05).Conclusions HO-1/CO system inhibits the expression of Fis1 in LPS-induced lung macrophages,which may be regulated by p38MAPK signaling pathway.

Particulate matter exposure induces the autophagy of macrophages via oxidative stress-mediated PI3K/AKT/mTOR pathway.

Many epidemiological investigations have consistently demonstrated the immunotoxicity of fine particulate matter (PM2.5), but the underlying molecular mechanism remains unclear and needs to be elucidated. In this work, the immune cells, including pulmonary macrophages of SD rats and Raw264.7 cells, were applied to further investigate the effect of PM2.5 on cell autophagy of macrophages, thus clarified the possible molecular mechanism of immunotoxicity caused by PM2.5. SD rats were exposed to summer (0.2, 0.6, 1.5 mg kg-1 b.w.) and winter (0.3, 1.5, 2.7 mg kg-1 b.w.) PM2.5 adopting the intratracheal instillation method. The exposure was performed one time every 3 days and continued for 2 months. The data showed that PM2.5 exposure decreased numbers of immune cells in pulmonary macrophages of SD rats. In addition, PM2.5 could induce the cell autophagy through the increased LC3 and decreased p62 mRNA and protein levels of pulmonary macrophages in SD rats and Raw264.7 cells in a concentration-dependent manner. Strikingly, PM2.5-induced oxidative stress was observed. However, NAC supplement (the ROS inhibitor) significantly reversed PM2.5-caused effects. Additionally, the PI3K/AKT/mTOR pathway was activated in PM2.5-treated cells and NAC had an important inhibitory effect. These results demonstrated that PM2.5 exposures induced autophagy of pulmonary macrophages via the oxidative stress-mediated PI3K/AKT/mTOR pathway, which may contribute to explain the molecular mechanism of immunotoxicity caused by PM2.5 and provide the theoretical foundation for environment toxicology of PM2.5.

Functional and morphological differences between rat alveolar and interstitial macrophages / 第三军医大学学报

Objective To observe the difference of morphology and phagocytosis between alveolar macrophages (AMs) and pulmonary interstitial macrophages (IMs). Methods AMs were collected by lung lavage and IMs by treatment of the lung tissue with DNAse and collagenase. The two cell populations were analyzed with respect to morphology by transmission electron microscopy, and the variation of these macrophages of phagocytosis were tested by malachite green colorimetry. Results There were great differences in morphology between AMs and IMs. The phagocytosis of AMs was much stronger than that of IMs. Conclusion There is functional and morphological heterogeneity between AMs and IMs. IMs should not be regarded as the precursors to AMs.