Cigarette Smoke Disturbs the Survival of CD8+ Tc/Tregs Partially through Muscarinic Receptors-Dependent Mechanisms in Chronic Obstructive Pulmonary Disease

Background

CD8⁺ T cells (Cytotoxic T cells, Tc) are known to play a critical role in the pathogenesis of smoking related airway inflammation including chronic obstructive pulmonary disease (COPD). However, how cigarette smoke directly impacts systematic CD8⁺ T cell and regulatory T cell (Treg) subsets, especially by modulating muscarinic acetylcholine receptors (MRs), has yet to be well elucidated.

Methods

Circulating CD8⁺ Tc/Tregs in healthy nonsmokers (n = 15), healthy smokers (n = 15) and COPD patients (n = 18) were evaluated by flow cytometry after incubating with anti-CD3, anti-CD8, anti-CD25, anti-Foxp3 antibodies. Peripheral blood T cells (PBT cells) from healthy nonsmokers were cultured in the presence of cigarette smoke extract (CSE) alone or combined with MRs agonist/antagonist for 5 days. Proliferation and apoptosis were evaluated by flow cytometry using Ki-67/Annexin-V antibodies to measure the effects of CSE on the survival of CD8⁺ Tc/Tregs.

Results

While COPD patients have elevated circulating percentage of CD8⁺ T cells, healthy smokers have higher frequency of CD8⁺ Tregs. Elevated percentages of CD8⁺ T cells correlated inversely with declined FEV1 in COPD. CSE promoted the proliferation and inhibited the apoptosis of CD8⁺ T cells, while facilitated both the proliferation and apoptosis of CD8⁺ Tregs. Notably, the effects of CSE on CD8⁺ Tc/Tregs can be mostly simulated or attenuated by muscarine and atropine, the MR agonist and antagonist, respectively. However, neither muscarine nor atropine influenced the apoptosis of CD8⁺ Tregs.

Conclusion

The results imply that cigarette smoking likely facilitates a proinflammatory state in smokers, which is partially mediated by MR dysfunction. The MR antagonist may be a beneficial drug candidate for cigarette smoke-induced chronic airway inflammation.     

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.     

miR-21a-5p Promotes Inflammation following Traumatic Spinal Cord Injury through Upregulation of Neurotoxic Reactive Astrocyte (A1) Polarization by Inhibiting the CNTF/STAT3/Nkrf Pathway

Reactive astrocytes are implicated in traumatic spinal cord injury (TSCI). Interestingly, naïve astrocytes can easily transform into neurotoxic reactive astrocytes (A1s) with inflammatory stimulation. Previous studies demonstrated that microRNA(miR)-21a-5p was up-regulated in spinal cord tissue after TSCI; however, it is not clear whether this affected reactive astrocyte polarization. Here, we aim to detect the effects of miR-21a-5p on the induction of A1 formation and the underlying mechanisms. Our study found that the expression of miR-21a-5p was significantly increased while that of Cntfr α was decreased, since naïve astrocytes transformed into A1s 3 days post-TSCI; the binding site between miR-21a-5p and Cntfr α was further confirmed in astrocytes. After treatment with CNTF, the levels of A1 markers decreased while that of A2 increased. The expression of A1 markers significantly decreased with the downregulation of miR-21a-5p, while Cntfr α siRNA treatment caused the opposite both in vitro and in vivo. To summarize, miR-21a-5p/Cntfr α promotes A1 induction and might enhance the inflammatory process of TSCI; furthermore, we identified, for the first time, the effect and potential mechanism by which CNTF inhibits naïve astrocytes transformation into A1s. Collectively, our findings demonstrate that targeting miR-21a-5p represents a prospective therapy for promoting the recovery of TSCI.     

Up-Regulation of the Neuronal Form of Nitric Oxide Synthase in Response to Prolonged Muscarinic M1 Receptor Stimulation

Abstract: It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M₁ receptors and nNOS, with 3.3 µ M or 1 m M carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by l -[³H]citrulline and cyclic [³H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [ N-methyl -³H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M₁ receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.     

Expression of miRNA-29 in Pancreatic β Cells Promotes Inflammation and Diabetes via TRAF3

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PGE2 Promotes Apoptosis Induced by Cytokine Deprivation through EP3 Receptor and Induces Bim in Mouse Mast Cells

Increased mast cell numbers are observed at sites of allergic inflammation and restoration of normal mast cell numbers is critical to the resolution of these responses. Early studies showed that cytokines protect mast cells from apoptosis, suggesting a simple model in which diminished cytokine levels during resolution leads to cell death. The report that prostaglandins can contribute both to recruitment and to the resolution of inflammation together with the demonstration that mast cells express all four PGE₂ receptors raises the question of whether a single PGE₂ receptor mediates the ability of PGE₂ to regulate mast cell survival and apoptosis. We report here that PGE₂ through the EP3 receptor promotes cell death of mast cells initiated by cytokine withdrawal. Furthermore, the ability of PGE₂ to limit reconstitution of tissues with cultured mast cells is lost in cell lacking the EP3 receptor. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨ_m), increased caspase-3 activation, chromatin condensation, and low molecular weight DNA cleavage. PGE₂ augmented cell death is dependent on an increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE₂ can contribute to the resolution of inflammation in part by augmenting the removal of inflammatory cells in this case, mast cells.     

Up-Regulation of 5-HT2B Receptor Density and Receptor-Mediated Glycogenolysis in Mouse Astrocytes by Long-Term Fluoxetine Administration

The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca²⁺ concentration ([Ca²⁺]_i]) in these cultures, whereas short-term (one week) treatment with 10 M down-regulates the effects on glycogen and [Ca²⁺]_i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT₂ receptor that is different from the 5-HT_2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT_2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT_2A and 5-HT_2B receptors, but not for the 5-HT_2C receptor, identifying the 5-HT₂ receptor activated by fluoxetine as the 5-HT_2B receptor, the most recently cloned 5-HT₂ receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [³H]mesulergine, a ligand for all three 5-HT₂ receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT_2B-mediated effects are initially down-regulated and subsequently up-regulated.     

Four SNPs in the CHRNA3/5 Alpha-Neuronal Nicotinic Acetylcholine Receptor Subunit Locus Are Associated with COPD Risk Based on Meta-Analyses

Background

Several single nucleotide polymorphisms (SNPs) in an α-neuronal nicotinic acetylcholine receptor subunit (CHRNA3/5) were identified to be associated with chronic obstructive pulmonary disease (COPD) in a study based on a Norwegian population. However, results from subsequent studies have been controversial, particularly in studies recruiting Asians. In the present study, we conducted a comprehensive search and meta-analyses to identify susceptibility SNPs for COPD in the CHRNA3/5 locus.

Methods

A comprehensive literature search was conducted to find studies that have reported an association between SNPs in the CHRNA3/5 locus and COPD risk. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) for each SNP were calculated with the major allele or genotype as the reference group. The influence of individual studies on pooled measures was assessed, in addition to publication bias.

Results

A total of 12 articles with 14 eligible studies were included in this analysis. Association between 4 SNPs in the CHRNA3/5 locus and COPD was evaluated and included rs1051730, rs8034191, rs6495309, and rs16969968. Significant associations between the 4 SNPs and COPD were identified under allele (rs1051730: OR = 1.14, 95%CI = 1.10–1.18; rs8034191: OR = 1.29, 95%CI = 1.18–1.41; rs6495309: OR = 1.26, 95%CI = 1.09–1.45; rs16969968: OR = 1.27, 95%CI = 1.17–1.39) and genotype models. Subgroup analysis conducted for rs1051730 showed a significant association between this SNP and COPD risk in non-Asians (OR = 1.14, 95%CI = 1.10–1.18), but not Asians (OR = 1.23, 95%CI = 0.91–1.67). Rs1051730 and rs6495309 were also significantly associated with COPD after adjusting for multiple variables, including age and smoking status.

Conclusion

Our results indicate that 4 SNPs in the CHRNA3/5 locus are associated with COPD risk. Rs1051730 was particularly associated with COPD in non-Asians, but its role in Asians still needs to be verified. Additional studies will be necessary to assess the effect of rs6495309 on COPD. Although rs1051730 and rs6495309 were shown to be independent risk factors for COPD, validation studies should be performed.     

右美托咪定在老年肺癌合并COPD患者中的麻醉效果及对应激反应和Th1、Th2型细胞因子的影响

摘要 目的：观察右美托咪定在老年肺癌合并慢性阻塞性肺部疾病（COPD）患者中的麻醉效果及对应激反应和辅助T（Th）1、Th2型细胞因子的影响。方法：使用随机数字表法将2019年5月至2022年5月期间在我院行肺叶切除术的100例老年肺癌合并COPD患者分为对照组和治疗组,各为50例。对照组接受常规麻醉,治疗组在对照组基础上接受右美托咪定。对比两组肺动态顺应性、氧合指数、应激反应指标、Th1、Th2型细胞因子,同时记录两组术后并发症发生率。结果：治疗组单肺通气30 min（T2）~术毕（T4）时间点肺动态顺应性、氧合指数高于对照组（P<0.05）。治疗组术后1d皮质醇（Cor）、促肾上腺皮质激素（ACTH）、去甲肾上腺素（NE）、肾上腺素（E）低于对照组（P<0.05）。治疗组术后1 d白细胞介素-2（IL-2）、干扰素-γ ( IFN-γ)、肿瘤坏死因子-α（TNF-α）低于对照组（P<0.05）,IL-4、IL-10高于对照组（P<0.05）。两组术后并发症发生率组间对比未见差异（P>0.05）。结论：右美托咪定对老年肺癌合并COPD患者具有较好的麻醉效果,可改善肺部顺应性,保持氧合指数稳定,减轻机体的应激反应,有效调节Th1、Th2型细胞因子。     

Induction of Murine Macrophage M2 Polarization by Cigarette Smoke Extract via the JAK2/STAT3 Pathway

Cigarette smoking is a major pathogenic factor in lung cancer. Macrophages play an important role in host defense and adaptive immunity. These cells display diverse phenotypes for performing different functions. M2 type macrophages usually exhibit immunosuppressive and tumor-promoting characteristics. Although macrophage polarization toward the M2 phenotype has been observed in the lungs of cigarette smokers, the molecular basis of the process remains unclear. In this study, we evaluated the possible mechanisms for the polarization of mouse macrophages that are induced by cigarette smoking (CS) or cigarette smoke extract (CSE). The results showed that exposure to CSE suppressed the production of reactive oxygen species (ROS) and nitric oxide (NO) and down-regulated the phagocytic ability of Ana-1 cells. The CD163 expressions on the surface of macrophages from different sources were significantly increased in in vivo and in vitro studies. The M1 macrophage cytokines TNF-α, IL-12p40 and enzyme iNOS decreased in the culture supernatant, and their mRNA levels decreased depending on the time and concentration of CSE. In contrast, the M2 phenotype macrophage cytokines IL-10, IL-6, TGF-β1 and TGF-β2 were up-regulated. Moreover, phosphorylation of JAK2 and STAT3 was observed after the Ana-1 cells were treated with CSE. In addition, pretreating the Ana-1 cells with the STAT3 phosphorylation inhibitor WP1066 inhibited the CSE-induced CD163 expression, increased the mRNA level of IL-10 and significantly decreased the mRNA level of IL-12. In conclusion, we demonstrated that the M2 polarization of macrophages induced by CS could be mediated through JAK2/STAT3 pathway activation.     

Epithelial and Stromal Cell Urokinase Plasminogen Activator Receptor Expression Differentially Correlates with Survival in Rectal Cancer Stages B and C Patients

Urokinase plasminogen activator receptor (uPAR) has been proposed as a potential prognostic factor for colorectal cancer (CRC) patient survival. However, CRC uPAR expression remains controversial, especially regarding cell types where uPAR is overexpressed (e.g., epithelium (uPAR^E) or stroma-associated cells (uPAR^S)) and associated prognostic relevance. In this study, two epitope-specific anti-uPAR monoclonal antibodies (MAbs) could discriminate expression of uPAR^E from uPAR^S and were used to examine this association with survival of stages B and C rectal cancer (RC) patients. Using immunohistochemistry, MAbs #3937 and R4 were used to discriminate uPAR^E from uPAR^S respectively in the central and invasive frontal regions of 170 stage B and 179 stage C RC specimens. Kaplan-Meier and Cox regression analyses were used to determine association with survival. uPAR expression occurred in both epithelial and stromal compartments with differential expression observed in many cases, indicating uPAR^E and uPAR^S have different cellular roles. In the central and invasive frontal regions, uPAR^E was adversely associated with overall stage B survival (HR = 1.9; p = 0.014 and HR = 1.5; p = 0.031, respectively) reproducing results from previous studies. uPAR^S at the invasive front was associated with longer stage C survival (HR = 0.6; p = 0.007), reflecting studies demonstrating that macrophage peritumoural accumulation is associated with longer survival. This study demonstrates that different uPAR epitopes should be considered as being expressed on different cell types during tumour progression and at different stages in RC. Understanding how uPAR^E and uPAR^S expression affects survival is anticipated to be a useful clinical prognostic marker of stages B and C RC.     

Regulation of Nasal Airway Homeostasis and Inflammation in Mice by SHP-1 and Th2/Th1 Signaling Pathways

Allergic rhinitis is a chronic inflammatory disease orchestrated by Th2 lymphocytes. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 is known to be a negative regulator in the IL-4α/STAT-6 signaling pathway of the lung. However, the role of SHP-1 enzyme and its functional relationship with Th2 and Th1 cytokines are not known in the nasal airway. In this study, we aimed to study the nasal inflammation as a result of SHP-1 deficiency in viable motheaten (mev) mice and to investigate the molecular mechanisms involved. Cytology, histology, and expression of cytokines and chemokines were analyzed to define the nature of the nasal inflammation. Targeted gene depletion of Th1 (IFN-γ) and Th2 (IL-4 and IL-13) cytokines was used to identify the critical pathways involved. Matrix metalloproteinases (MMPs) were studied to demonstrate the clearance mechanism of recruited inflammatory cells into the nasal airway. We showed here that mev mice had a spontaneous allergic rhinitis-like inflammation with eosinophilia, mucus metaplasia, up-regulation of Th2 cytokines (IL-4 and IL-13), chemokines (eotaxin), and MMPs. All of these inflammatory mediators were clearly counter-regulated by Th2 and Th1 cytokines. Deletion of IFN-γ gene induced a strong Th2-skewed inflammation with transepithelial migration of the inflammatory cells. These findings suggest that SHP-1 enzyme and Th2/Th1 paradigm may play a critical role in the maintenance of nasal immune homeostasis and in the regulation of allergic rhinitis.     

Budesonide/Formoterol Enhances the Expression of Pro Surfactant Protein-B in Lungs of COPD Patients

Rationale & Aim

Pulmonary surfactants are essential components of lung homeostasis. In chronic obstructive pulmonary disease (COPD), surfactant expression decreases in lungs whereas, there is a paradoxical increase in protein expression in plasma. The latter has been associated with poor health outcomes in COPD. The purpose of this study was to determine the relationship of surfactants and other pneumoproteins in bronchoalveolar lavage (BAL) fluid and plasma to airflow limitation and the effects of budesonide/formoterol on this relationship.

Methods

We recruited (clinical trials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT00569712","term_id":"NCT00569712"}}NCT00569712) 7 smokers without COPD and 30 ex and current smokers with COPD who were free of exacerbations for at least 4 weeks. All subjects were treated with budesonide/formoterol 400/12 µg twice a day for 4 weeks. BAL fluid and plasma samples were obtained at baseline and the end of the 4 weeks. We measured lung-predominant pneumoproteins: pro-Surfactant Protein-B (pro-SFTPB), Surfactant Protein-D (SP-D), Club Cell Secretory Protein-16 (CCSP-16) and Pulmonary and Activation-Regulated Chemokine (PARC/CCL-18) in BAL fluid and plasma.

Results

BAL Pro-SFTPB concentrations had the strongest relationship with airflow limitation as measured by FEV₁/FVC (Spearman rho = 0.509; p = 0.001) and FEV₁% of predicted (Spearman rho =  0.362; p = 0.028). Plasma CCSP-16 concentrations were also significantly related to airflow limitation (Spearman rho = 0.362; p = 0.028 for FEV₁% of predicted). The other biomarkers in BAL fluid or plasma were not significantly associated with airflow limitation. In COPD subjects, budesonide/formoterol significantly increased the BAL concentrations of pro-SFTPB by a median of 62.46 ng/ml (p = 0.022) or 48.7% from baseline median value.

Conclusion

Increased severity of COPD is associated with reduced Pro-SFTPB levels in BAL fluid. Short-term treatment with budesonide/formoterol increases these levels in BAL fluid. Long term studies will be needed to determine the clinical relevance of this observation.