BLTR1 Is Decreased in Steroid Resistant Pro-Inflammatory CD28nullCD8+ T Lymphocytes in Patients with COPD-The Spillover Hypothesis Explained?

INTRODUCTION: Pro-inflammatory CD8+ T cells are increased in the lungs and also in the peripheral circulation of both smokers and chronic obstructive pulmonary disease (COPD) patients. The reason for this is unclear but has been described as a spillover from cells in the lungs that may cause the systemic inflammation noted in COPD. We have recently shown an increase in steroid-resistant CD28nullCD8+ senescent lymphocytes in the lungs and peripheral blood in COPD. Leukotreine B4 (LB4) receptor 1 (BLTR1) is involved in recruitment of CD8+ T cells to sites of inflammation, and we hypothesized that it may be involved in the migration of these senescent lymphocytes from the lungs in COPD. METHODS: Via flow cytometry and Western blot BLTR1, IFNγ, and TNFα expression were measured in peripheral blood, BAL, and large proximal and small distal airway CD28±, CD8± T, and NKT-like cells from COPD patients and healthy control subjects (±prednisolone) following in vitro stimulation. Chemotaxis of leucocyte subsets was determined (±LB4 ± prednisolone). RESULTS: There was an increase in BLTR1-CD28nullCD8+ lymphocytes in the lungs and blood in patients with COPD compared with controls. BLTR1-CD28nullCD8+ T and NKT-like cells produce more IFN/TNF than BLTR+ cells and fail to migrate to LTB4. Treatment with 1 µM prednisolone in vitro resulted in upregulation of BLTR1 expression in pro-inflammatory CD28nullCD8+ cells and migration to LB4. CONCLUSIONS: Loss of BLTR1 is associated with an increased inflammatory potential of CD28nullCD8+ T cells and may allow these pro-inflammatory steroid-resistant cells to migrate to peripheral blood. Treatment strategies that upregulate BLTR1 may reduce systemic inflammation and associated co-morbidity in patients with COPD.

Reduction in Rubicon by cigarette smoke is associated with impaired phagocytosis and occurs through lysosomal degradation pathway.

BACKGROUND: A common feature of COPD is a defective lung macrophage phagocytic capacity that can contribute to chronic lung inflammation and infection. The precise mechanisms remain incompletely understood, although cigarette smoke is a known contributor. We previously showed deficiency of the LC3-associated phagocytosis (LAP) regulator, Rubicon, in macrophages from COPD subjects and in response to cigarette smoke. The current study investigated the molecular basis by which cigarette smoke extract (CSE) reduces Rubicon in THP-1, alveolar and blood monocyte-derived macrophages, and the relationship between Rubicon deficiency and CSE-impaired phagocytosis. METHODOLOGY: Phagocytic capacity of CSE-treated macrophages was measured by flow cytometry, Rubicon expression by Western blot and real time polymerase chain reaction, and autophagic-flux by LC3 and p62 levels. The effect of CSE on Rubicon degradation was determined using cycloheximide inhibition and Rubicon protein synthesis and half-life assessment. RESULTS: Phagocytosis was significantly impaired in CSE-exposed macrophages and strongly correlated with Rubicon expression. CSE-impaired autophagy, accelerated Rubicon degradation, and reduced its half-life. Lysosomal protease inhibitors, but not proteasome inhibitors, attenuated this effect. Autophagy induction did not significantly affect Rubicon expression. CONCLUSIONS: CSE decreases Rubicon through the lysosomal degradation pathway. Rubicon degradation and/or LAP impairment may contribute to dysregulated phagocytosis perpetuated by CSE.

Immunolocalization of zinc transporters and metallothioneins reveals links to microvascular morphology and functions.

Zinc homeostasis is vital to immune and other organ system functions, yet over a quarter of the world's population is zinc deficient. Abnormal zinc transport or storage protein expression has been linked to diseases, such as cancer and chronic obstructive pulmonary disorder. Although recent studies indicate a role for zinc regulation in vascular functions and diseases, detailed knowledge of the mechanisms involved remains unknown. This study aimed to assess protein expression and localization of zinc transporters of the SLC39A/ZIP family (ZIPs) and metallothioneins (MTs) in human subcutaneous microvessels and to relate them to morphological features and expression of function-related molecules in the microvasculature. Microvessels in paraffin biopsies of subcutaneous adipose tissues from 14 patients undergoing hernia reconstruction surgery were analysed for 9 ZIPs and 3 MT proteins by MQCM (multifluorescence quantitative confocal microscopy). Zinc regulation proteins detected in human microvasculature included ZIP1, ZIP2, ZIP8, ZIP10, ZIP12, ZIP14 and MT1-3, which showed differential localization among endothelial and smooth muscle cells. ZIP1, ZIP2, ZIP12 and MT3 showed significantly (p < 0.05) increased immunoreactivities, in association with increased microvascular muscularization, and upregulated ET-1, α-SMA and the active form of p38 MAPK (Thr180/Tyr182 phosphorylated, p38 MAPK-P). These findings support roles of the zinc regulation system in microvascular physiology and diseases.

COPD is associated with increased pro-inflammatory CD28null CD8 T and NKT-like cells in the small airways.

We previously showed increased steroid-resistant CD28null CD8+ senescent lymphocyte subsets in the peripheral blood from patients with chronic obstructive pulmonary disease (COPD). These cells expressed decreased levels of the glucocorticoid receptor (GCR), suggesting their contribution to the steroid-resistant property of these cells. COPD is a disease of the small airways (SA). We, therefore, hypothesized that there would be a further increase in these steroid-resistant lymphocytes in the lung, particularly in the SA. We further hypothesized that the pro-inflammatory/cytotoxic potential of these cells could be negated using prednisolone with low-dose cyclosporin A. Blood, bronchoalveolar lavage, large proximal, and small distal airway brushings were collected from 11 patients with COPD and 10 healthy aged-matched controls. The cytotoxic mediator granzyme b, pro-inflammatory cytokines IFNγ/TNFα, and GCR were determined in lymphocytes subsets before and after their exposure to 1µM prednisolone and/or 2.5 ng/mL cyclosporin A. Particularly in the SA, COPD subjects showed an increased percentage of CD28null CD8 T-cells and NKT-like cells, with increased expression of granzyme b, IFNγ and TNFα and a loss of GCR, compared with controls. Significant negative correlations between SA GCR expression and IFNγ/TNFα production by T and NKT-like cells (eg, T-cell IFNγ R = -0.834, P = 0.031) and with FEV1 (R = -0.890) were shown. Cyclosporine A and prednisolone synergistically increased GCR expression and inhibited pro-inflammatory cytokine production by CD28null CD8- T and NKT-like cells. COPD is associated with increased pro-inflammatory CD28null CD8+ T and NKT-like cells in the SA. Treatments that increase GCR in these lymphocyte subsets may improve the efficacy of clinical treatment.

Inhibition of LC3-associated phagocytosis in COPD and in response to cigarette smoke.

INTRODUCTION/RATIONALE: In chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD. METHODS: Bronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence. RESULTS: Rubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis. CONCLUSION: LAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

AIM2 nuclear exit and inflammasome activation in chronic obstructive pulmonary disease and response to cigarette smoke.

INTRODUCTION: The role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke. METHODS: Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors; lungs from cigarette smoke-exposed mice; and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1ß. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells. RESULTS: Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1ß, were demonstrated in COPD lungs (n = 9) vs. control (n = 5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 h stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an > 8-fold increase (p < 0.05) of cytoplasmic AIM2 and > 6-fold increase (p < 0.01) of colocalized cleaved IL-1ß speck foci, which were also localized with ASC. CONCLUSION: The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.

LC3-Associated Phagocytosis (LAP): A Potentially Influential Mediator of Efferocytosis-Related Tumor Progression and Aggressiveness.

One aim of cancer therapies is to induce apoptosis of tumor cells. Efficient removal of the apoptotic cells requires coordinated efforts between the processes of efferocytosis and LC3-associated phagocytosis (LAP). However, this activity has also been shown to produce anti-inflammatory and immunosuppressive signals that can be utilized by live tumor cells to evade immune defense mechanisms, resulting in tumor progression and aggressiveness. In the absence of LAP, mice exhibit suppressed tumor growth during efferocytosis, while LAP-sufficient mice show enhanced tumor progression. Little is known about how LAP or its regulators directly affect efferocytosis, tumor growth and treatment responses, and identifying the mechanisms involved has the potential to lead to the discovery of novel approaches to target cancer cells. Also incompletely understood is the direct effect of apoptotic cancer cells on LAP. This is particularly important as induction of apoptosis by current cytotoxic cancer therapies can potentially stimulate LAP following efferocytosis. Herein, we highlight the current understanding of the role of LAP and its relationship with efferocytosis in the tumor microenvironment with a view to presenting novel therapeutic strategies.

Amelioration of cardiac dysfunction and ventricular remodeling after myocardial infarction by danhong injection are critically contributed by anti-TGF-ß-mediated fibrosis and angiogenesis mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI) is a standardized product extracted from Radix et Rhizoma Salviae Miltiorrhizae and Flos Carthami , which has been long applied mainly used to treat ischemic encephalopathy and cardiac diseases including myocardial infarction and angina in clinical practice. AIM OF THE STUDY: Aim of this study was to investigate the salutary effects of DHI by slowing ventricular remodeling and improving cardiac function after myocardial infarction (MI) in rats. MATERIALS AND METHODS: In this study, Male Sprague-Dawley rats were subjected to ligation on left anterior descending coronary artery to establish MI models and valsartan was selected as positive control. Cardiac function examination was conducted at the 1st, 3rd, 7th, 14th and the 28th days after LAD. Haematoxylin and Eosin (HE) staining and Masson staining were conducted to observe cardiac pathology and morphological changes levels of VEGF, TGF-ß, MMP-2, and MMP-9 in the myocardial tissue were determined in gene and protein expressions. RESULTS: After 3 days post-treatment and thereafter, EF and FS in DHI group were greater than that of model group (p<0.05). Compared with the MI group, ratio of infarct was markedly decreased in treated-DHI group(p<0.05). TGF-ß1 protein and fibrosis-related proteins MMP-2 and MMP-9 were up-regulated after MI, and they were significantly suppressed by the administration of DHI(p<0.05 and p<0.01, respectively). Moreover, DHI improved the mRNA expression of VEGF and increased the blood vessel density of myocardial infarct border zone. DHI decreased the expression of cell apoptosis protein of caspase-3 and increased the anti-apoptotic protein, bcl-2. CONCLUSIONS: We provided direct evidences that DHI improves cardiac remodeling and preserves ventricular function post-MI in rats. DHI conferred cardio-protection in rats with MI via anti-myocardial apoptosis, angiogenesis, reduction of myocardial fibrosis and many other aspects of joint actions.

Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling.

The traditional Chinese medicine Danshensu (DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre-treatment of DSS significantly improved cell survival and protected against I/R-induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin (mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy- and apoptosis-related proteins (including p62, LC3-II, Beclin-1, Bax, and Caspase-3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre-treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation.

Danzhi Qing'e (DZQE) activates AMPK pathway and regulates lipid metabolism in a rat model of perimenopausal hyperlipidaemia.

What is the central question of this study? Does Danzhi Qing'e (DZQE) regulate lipid metabolism and improve ovarian function in a rat model of perimenopausal hyperlipidaemia, and could this effect be mediated through the AMPK pathway? What is the main finding and its importance? We revealed that DZQE is a pharmacotherapy that could activate the AMPK pathway to improve ovarian function and lipid metabolism during perimenopause complicated with hyperlipidaemia syndrome in an animal model. Thus, this study provides a novel therapeutic option for treating perimenopausal syndrome and highlights the therapeutic potential of DZQE in perimenopausal rats. Menopause is an important event in a woman's life. During perimenopause, accompanied by development of osteoporosis and dyslipidaemia, ovarian function gradually declines. Dyslipidaemia is a risk factor for cardiovascular disorders, cerebrovascular disease and breast cancer in postmenopausal women. All of these contribute to impairment of liver function, particularly fatty liver disease, because liver dysfunction is associated with ovarian dysfunction and hyperlipidaemia. The aim of this study was to define a therapeutic approach to improve ovarian function and attenuate lipid accumulation in order to prevent perimenopause-induced ovarian dysfunction and hyperlipidaemia. Four-week-old female Wistar rats were injected i.p. with 4-vinylcyclohexene diepoxide (4-VCD) and fed with a high-fat diet (HFD) to serve as a model of perimenopause complicated with hyperlipidaemia. The 4-VCD induces perimenopause, while the HFD causes hyperlipidaemia. Five days after administration of 4-VCD, the 4-VCD + HFD-treated rats were assessed daily for oestrous cycle stage by vaginal cytology. Rats were then assigned into groups, in which 2.5, 5.0 or 10.0 g kg-1 Danzhi Qing'e (DZQE) or estradiol valerate was administered intragastrically for 8 weeks. Expression levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), oestrogen and testosterone measured by enzyme-linked immunosorbent assay served as biomarkers for perimenopause and ovarian dysfunction. The expression levels of AMPK and acetyl-CoA carboxylase in the liver were determined with Western blotting, and aspartate aminotransferase and alanine aminotransferase were analysed using an automated biochemical analyser to examine liver function. The DZQE improved ovarian function by upregulating oestrogen and testosterone concentrations in serum and downregulating FSH and LH serum concentrations. Moreover, DZQE reduced serum concentrations of triglyceride, total cholesterol and low-density lipoprotein in a dose-dependent manner to regulate lipid levels during perimenopause. Furthermore, DZQE increased AMPK at both the transcriptional and translational levels and decreased the expression of SREBP-1c gene as well as its downstream target gene, fatty acid synthase. Danzhi Qing'e improved dyslipidaemia during menopause and also had an effect on liver function. Danzhi Qing'e is an effective Chinese herbal compound, which improves ovarian function and lipid metabolism in perimenopause complicated with hyperlipidaemia at least in part through the AMPK pathway.

Gastrodin Reduces Blood Pressure by Intervening with RAAS and PPARγ in SHRs.

Gastrodin is a bioactive compound extracted from traditional Chinese medicine, Gastrodia elata Bl. It has a definite effect on reducing blood pressure in hypertensive patients. However, the mechanisms of gastrodin in lowering blood pressure still remain unclear. In this study, 4 weeks of administration of gastrodin (100 mg/kg/d intraperitoneally injected) decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) (190.2 ± 8.9 versus 169.8 ± 6.4, P < 0.01). Among SHRs receiving gastrodin treatment, angiotensin II (Ang II) and aldosterone (ALD) in serum were significantly decreased (2022.1 ± 53.0 versus 1528.7 ± 93.9, 213.33 ± 35.17 versus 179.65 ± 20.31, and P < 0.01, P < 0.05, resp.) and dramatically downregulated expression of angiotensin type 1 receptor (AT1R) (4.9 ± 0.9 versus 2.6 ± 0.9, P < 0.05) in myocardium in both mRNA and protein levels compared with their corresponding groups without gastrodin treatment. Additionally, gastrodin increased the mRNA expression (0.18 ± 0.07 versus 0.82 ± 0.10, P < 0.01) and protein synthesis (0.40 ± 0.10 versus 0.34 ± 0.10, P < 0.01) of peroxisome proliferator-activated receptor γ (PPARγ) in myocardium tissues. Overall, our data demonstrated that gastrodin was able to decrease the SBP in SHR. Furthermore, this study showed that gastrodin intervened with the renin-angiotensin-aldosterone system (RAAS) and PPARγ effectively, which indicates its antihypertensive mechanism.