Chemical modification patterns for microRNA therapeutic mimics: a structure-activity relationship (SAR) case-study on miR-200c.

microRNA (miRNA) mimics are an emerging class of oligonucleotide therapeutics, with a few compounds already in clinical stages. Synthetic miRNAs are able to restore downregulated levels of intrinsic miRNAs, allowing for parallel regulation of multiple genes involved in a particular disease. In this work, we examined the influence of chemical modifications patterns in miR-200c mimics, assessing the regulation of a selection of target messenger RNAs (mRNA) and, subsequently, of the whole transcriptome in A549 cells. We have probed 37 mimics and provided an initial set of instructions for designing miRNA mimics with potency and selectivity similar to an unmodified miRNA duplex. Additionally, we have examined the stability of selected mimics in serum. Finally, the selected two modification patterns were translated to two other miRNAs, miR-34a and miR-155. To differing degrees, these designs acted on target mRNAs in a similar manner to the unmodified mimic. Here, for the first time, we describe a structured overview of 'miRNA mimics modification templates' that are chemically stabilised and optimised for use in an in vitro set up and highlight the need of further sequence specific optimization when mimics are to be used beyond in vitro tool experiments.

Spotlight on microRNAs in allergy and asthma.

In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post-transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.

Circulating microRNAs correlate to clinical parameters in individuals with allergic and non-allergic asthma.

BACKGROUND: Asthma is a chronic airway disease affecting millions of people. Better methods to define asthma subgroups using clinical parameters and molecular biomarkers are crucial in the development of personalized medicine. OBJECTIVE: The aim of this study was to determine if circulating microRNAs (miRNAs) may be used to distinguish well-defined asthma groups. METHODS: Blood serum from 116 well-defined subjects, including healthy controls and individuals with allergic or non-allergic asthma, from the West Sweden Asthma Study were included. Serum was analyzed for circulating miRNA expression of miR-126, - 145, -146a, - 155, - 223, and -374a and eosinophil cationic protein (ECP). Correlations between clinical characteristics and circulating miRNA expression as well as potential miRNA gene targets were investigated. RESULTS: A subset of miRNAs were differentially expressed between allergic and non-allergic asthmatic individuals. Alterations in expression of miR-155, -146a, -374a and - 145 were observed in allergic asthmatics in response to inhaled corticosteroid usage. Additionally, miR-223 and miR-374a expression varied in non-allergic asthmatics based on blood eosinophil numbers. Numerous clinical parameters, including lung function measurements, correlated with subsets of miRNAs. Finally, pathway analysis revealed a potential role for inhaled corticosteroid induced miRNAs in leukocyte regulation, IL-6 signaling and glucocorticoid response. CONCLUSION: Circulating miRNA expression was altered in subjects with allergic and non-allergic asthma and correlated to clinical parameters including lung function and potential gene targets involved in immune processes. This combination of clinical and molecular data may be a basis for the further, more precise classification of asthma subgroups. Taken together, these findings would further asthma research and benefit future patients through the discovery of molecular mechanisms as well as identifying asthma subgroups contributing to the development of personalized medicine.

House Dust Mite Induces Bone Marrow IL-33-Responsive ILC2s and TH Cells.

Type 2 innate lymphoid cells (ILC2s) and their adaptive counterpart type 2 T helper (TH2) cells respond to interleukin-33 (IL-33) by producing IL-5, which is a crucial cytokine for eosinophil development in the bone marrow. The aim of this study was to determine if bone marrow ILC2s, TH cells, and eosinophils are locally regulated by IL-33 in terms of number and activation upon exposure to the common aeroallergen house dust mite (HDM). Mice that were sensitized and challenged with HDM by intranasal exposures induced eosinophil development in the bone marrow with an initial increase of IL5Rα+ eosinophil progenitors, following elevated numbers of mature eosinophils and the induction of airway eosinophilia. Bone marrow ILC2s, TH2, and eosinophils all responded to HDM challenge by increased IL-33 receptor (ST2) expression. However, only ILC2s, but not TH cells, revealed increased ST2 expression at the onset of eosinophil development, which significantly correlated with the number of eosinophil progenitors. In summary, our findings suggest that airway allergen challenges with HDM activates IL-33-responsive ILC2s, TH cells, and eosinophils locally in the bone marrow. Targeting the IL-33/ST2 axis in allergic diseases including asthma may be beneficial by decreasing eosinophil production in the bone marrow.

Sulfatase modifying factor 1 (SUMF1) is associated with Chronic Obstructive Pulmonary Disease.

BACKGROUND: It has been observed that mice lacking the sulfatase modifying factor (Sumf1) developed an emphysema-like phenotype. However, it is unknown if SUMF1 may play a role in Chronic Obstructive Pulmonary Disease (COPD) in humans. The aim was to investigate if the expression and genetic regulation of SUMF1 differs between smokers with and without COPD. METHODS: SUMF1 mRNA was investigated in sputum cells and whole blood from controls and COPD patients (all current or former smokers). Expression quantitative trait loci (eQTL) analysis was used to investigate if single nucleotide polymorphisms (SNPs) in SUMF1 were significantly associated with SUMF1 expression. The association of SUMF1 SNPs with COPD was examined in a population based cohort, Lifelines. SUMF1 mRNA from sputum cells, lung tissue, and lung fibroblasts, as well as lung function parameters, were investigated in relation to genotype. RESULTS: Certain splice variants of SUMF1 showed a relatively high expression in lung tissue compared to many other tissues. SUMF1 Splice variant 2 and 3 showed lower levels in sputum cells from COPD patients as compared to controls. Twelve SNPs were found significant by eQTL analysis and overlapped with the array used for genotyping of Lifelines. We found alterations in mRNA expression in sputum cells and lung fibroblasts associated with SNP rs11915920 (top hit in eQTL), which validated the results of the lung tissue eQTL analysis. Of the twelve SNPs, two SNPs, rs793391 and rs308739, were found to be associated with COPD in Lifelines. The SNP rs793391 was also confirmed to be associated with lung function changes. CONCLUSIONS: We show that SUMF1 expression is affected in COPD patients compared to controls, and that SNPs in SUMF1 are associated with an increased risk of COPD. Certain COPD-associated SNPs have effects on either SUMF1 gene expression or on lung function. Collectively, this study shows that SUMF1 is associated with an increased risk of developing COPD.

The polysome-associated proteins Scp160 and Bfr1 prevent P body formation under normal growth conditions.

Numerous mRNAs are degraded in processing bodies (P bodies) in Saccharomyces cerevisiae. In logarithmically growing cells, only 0-1 P bodies per cell are detectable. However, the number and appearance of P bodies change once the cell encounters stress. Here, we show that the polysome-associated mRNA-binding protein Scp160 interacts with P body components, such as the decapping protein Dcp2 and the scaffold protein Pat1, presumably, on polysomes. Loss of either Scp160 or its interaction partner Bfr1 caused the formation of Dcp2-positive structures. These Dcp2-positive foci contained mRNA, because their formation was inhibited by the presence of cycloheximide. In addition, Scp160 was required for proper P body formation because only a subset of bona fide P body components could assemble into the Dcp2-positive foci in Δscp160 cells. In either Δbfr1 or Δscp160 cells, P body formation was uncoupled from translational attenuation as the polysome profile remained unchanged. Collectively, our data suggest that Bfr1 and Scp160 prevent P body formation under normal growth conditions.

Biomarker-based clustering of patients with chronic obstructive pulmonary disease.

Rationale: COPD has been associated repeatedly with single biomarkers of systemic inflammation, ignoring the complexity of inflammatory pathways. This study aimed to cluster patients with COPD based on systemic markers of inflammatory processes and to evaluate differences in their clinical characterisation and examine how these differences may relate to altered biological pathways. Methods: 213 patients with moderate-to-severe COPD in a clinically stable state were recruited and clinically characterised, which included a venous blood sample for analysis of serum biomarkers. Patients were clustered based on the overall similarity in systemic levels of 57 different biomarkers. To determine interactions among the regulated biomarkers, protein networks and biological pathways were examined for each patient cluster. Results: Four clusters were identified: two clusters with lower biomarker levels (I and II) and two clusters with higher biomarker levels (III and IV), with only a small number of biomarkers with similar trends in expression. Pathway analysis indicated that three of the four clusters were enriched in Rage (receptor for advanced glycation end-products) and Oncostatin M pathway components. Although the degree of airflow limitation was similar, the clinical characterisation of clusters ranged from 1) better functional capacity and health status and fewer comorbidities; 2) more underweight, osteoporosis and static hyperinflation; 3) more metabolically deranged; and 4) older subjects with worse functional capacity and higher comorbidity load. Conclusions: These new insights may help to understand the functionally relevant inflammatory interactions in the pathophysiology of COPD as a heterogeneous disease.

Gymnotic uptake of AntimiRs alter microRNA-34a levels in 2D and 3D epithelial cell culture.

MicroRNAs are attractive therapeutic targets in many diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Among microRNA inhibitors antimiRs have been proven successful in lowering aberrant microRNA levels in the clinic. We present a set of antimiRs targeting miR-34a, which has been shown to be dysregulated in chronic lung diseases. The tool compounds were taken up by a bronchial epithelial cell line and primary human bronchial epithelial cells, followed by efficient knockdown of miR-34a. Similar results were observed in 3D differentiated primary human bronchial epithelial cells cultured at the air-liquid interface. Varying chemical properties of antimiRs had significant impact on cellular uptake and potency, resulting in effective tool compounds for use in lung-relevant cellular systems. This report demonstrates gymnotic antimiR uptake and activity in 3D epithelial cell culture after apical administration, mimicking inhalation conditions.

Physiological estrogen levels are dispensable for the sex difference in immune responses during allergen-induced airway inflammation.

Women show an increased prevalence of adult-onset asthma compared to men and previous studies have shown that testosterone inhibits while estrogen worsens allergen-induced airway inflammation. However, detailed knowledge about the aggravating effects of estrogen on immune responses remain unclear. Defining the effects of physiological levels of estrogen on immune responses in asthma would aid in the development of improved treatment strategies. In this study, the importance of estrogen for the sex difference in asthma was determined using a murine model of house dust mite (HDM)-induced airway inflammation on intact female and male mice, as well as on ovariectomized (OVX) female mice treated with a physiological dose of 17ß-estradiol (E2). Innate and adaptive immune responses were defined in bronchoalveolar lavage fluid, mediastinal lymph node (mLN) and lung tissue. The results reveal increased numbers of lung eosinophils, macrophages, and dendritic cells in female but not in male mice after HDM challenge. Females also exhibit higher numbers of Th17 cells in both mLN and lung in response to HDM. However, treatment of OVX mice with physiological levels of E2 does not influence any of the analyzed cell populations. Together, this study confirms the previously reported sex difference in allergen-induced airway inflammation and show that female mice mount stronger innate and adaptive immune responses to HDM challenge, but these effects are not mediated by physiological levels of E2.

Single-nucleotide polymorphisms in the sulfatase-modifying factor 1 gene are associated with lung function and COPD.

Single nucleotide polymorphisms (SNPs) in various genes have been shown to associate with COPD, suggesting a role in disease pathogenesis. Sulfatase modifying factor (SUMF1) is a key modifier in connective tissue remodelling, and we have shown previously that several SNPs in SUMF1 are associated with COPD. The aim of this study was to investigate the association between SUMF1 SNPs and advanced lung function characteristics. Never-, former and current smokers with (n=154) or without (n=405) COPD were genotyped for 21 SNPs in SUMF1 and underwent spirometry, body plethysmography, diffusing capacity of the lung for carbon monoxide (D LCO) measurement and impulse oscillometry. Four SNPs (rs793391, rs12634248, rs2819590 and rs304092) showed a significantly decreased odds ratio of having COPD when heterozygous for the variance allele, together with a lower forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) ratio and an impaired peripheral resistance and reactance. Moreover, individuals homozygous for the variance allele of rs3864051 exhibited a strong association to COPD, a lower FEV1/FVC, FEV1 and D LCO, and an impaired peripheral resistance and reactance. Other SNPs (rs4685744, rs2819562, rs2819561 and rs11915920) were instead associated with impaired lung volumes and exhibited a lower FVC, total lung capacity and alveolar volume, in individuals having the variance allele. Several SNPs in the SUMF1 gene are shown to be associated with COPD and impaired lung function. These genetic variants of SUMF1 may cause a deficient sulfation balance in the extracellular matrix of the lung tissue, thereby contributing to the development of COPD.

Rapamycin Dampens Inflammatory Properties of Bone Marrow ILC2s in IL-33-Induced Eosinophilic Airway Inflammation.

The alarmin cytokine interleukin (IL)-33 plays an important proinflammatory role in type 2 immunity and can act on type 2 innate lymphoid cells (ILC2s) and type 2 T helper (TH2) cells in eosinophilic inflammation and asthma. The mechanistic target of rapamycin (mTOR) signaling pathway drives immune responses in several inflammatory diseases, but its role in regulating bone marrow responses to IL-33 is unclear. The aim of this study was to determine the role of the mTORC1 signaling pathway in IL-33-induced bone marrow ILC2 responses and its impact on IL-33-induced eosinophilia. Wild-type mice were intranasally exposed to IL-33 only or in combination with the mTORC1 inhibitor, rapamycin, intraperitoneally. Four groups were included in the study: saline-treated (PBS)+PBS, rapamycin+PBS, PBS+IL-33 and rapamycin+IL-33. Bronchoalveolar lavage fluid (BALF), serum and bone marrow cells were collected and analyzed by differential cell count, enzyme-linked immunosorbent assay and flow cytometry. IL-33 induced phosphorylation of the mTORC1 protein rpS6 in bone marrow ILC2s both ex vivo and in vivo. The observed mTOR signal was reduced by rapamycin treatment, indicating the sensitivity of bone marrow ILC2s to mTORC1 inhibition. IL-5 production by ILC2s was reduced in cultures treated with rapamycin before stimulation with IL-33 compared to IL-33 only. Bone marrow and airway eosinophils were reduced in mice given rapamycin before IL-33-exposure compared to mice given IL-33 only. Bone marrow ILC2s responded to IL-33 in vivo with increased mTORC1 activity and rapamycin treatment successfully decreased IL-33-induced eosinophilic inflammation, possibly by inhibition of IL-5-producing bone marrow ILC2s. These findings highlight the importance of investigating specific cells and proinflammatory pathways as potential drivers of inflammatory diseases, including asthma.

The Serum/Glucocorticoid-Regulated Kinase 1 Is Targeted by miR-19a in CD4+ T Cells.

The polarization of CD4+ T cells into different T helper subsets is an important process in many diseases, including asthma. Part of the adaptive immune system, T cells are responsible for propagating signals to alert and prime the immune system. MicroRNAs (miRNAs) are small non-coding RNAs that act on numerous targets in the cell to regulate a variety of cellular processes, including roles in T cell polarization. In this study, we aimed to identify genes dysregulated in peripheral blood mononuclear cells from individuals with asthma. Moreover, we sought to examine miRNAs that may regulate the candidate genes and explore their functional relationship. Utilizing a focused gene array, we identified the serum/glucocorticoid-regulated kinase 1 (SGK1) gene to be upregulated in circulating peripheral blood mononuclear cells, which included T cells, from individuals with asthma. Several miRNAs were bioinformatically identified to target SGK1, but miR-19a was the only screened candidate that negatively correlated to SGK1 expression. Further analysis of the miR-19a-SGK1 relationship showed a negative correlation in CD4+ T cells in situ and direct binding in vitro during T cell activation. Moreover, we observed a negative correlation of miR-19a and SGK1 during early type 2 polarization of CD4+ naïve human T cells. Thus, we suggest that miR-19a has a role in binding and regulating SGK1 transcript levels during T cell development.

Interplay Between the IL-33/ST2 Axis and Bone Marrow ILC2s in Protease Allergen-Induced IL-5-Dependent Eosinophilia.

Background: Eosinophils develop from CD34+ progenitor cells in the bone marrow under the influence of interleukin (IL)-5. Several cell types produce IL-5, including type 2 innate lymphoid cells (ILC2s). The alarmin cytokine IL-33 is known to activate ILC2s in mucosal tissues, but little is known about IL-33-responsive ILC2s in the bone marrow in allergen-induced airway inflammation. Methods: Wild type (WT) and Rag1 deficient (Rag1-/-) mice, which lack mature T and B cells, received intranasal doses of papain to induce acute allergic inflammation. In some experiments, mice were pre-treated with anti-IL-5 prior to the papain challenge. Furthermore, recombinant IL-33 was administered to WT mice, Rag1-/- mice, lymphocyte deficient mice (Rag2-/-Il2rg-/-) and to ex vivo whole bone marrow cultures. Bone marrow eosinophils and ILC2s were analyzed by flow cytometry. Eosinophil count was assessed by differential cell count and secreted IL-5 from bone marrow cells by ELISA. Results: Intranasal administration of papain or IL-33 increased the number of mature eosinophils in the bone marrow despite the absence of adaptive immune cells in Rag1-/- mice. In parallel, an increased number of eosinophils was observed in the airways together with elevated levels of Eotaxin-2/CCL24. Bone marrow ILC2s were increased after papain or IL-33 administration, whereas ILC2s was found to be increased at baseline in Rag1-/- mice compared to WT mice. An upregulation of the IL-33 receptor (ST2) expression on bone marrow ILC2s was observed after papain challenge in both Rag1-/- and WT mice which correlated to increased number of bone marrow eosinophilia. Furthermore, an increased number of ST2+ mature eosinophils in the bone marrow was observed after papain challenge, which was further dependent on IL-5. In addition, bone marrow-derived ILC2s from both mouse strains produced large amounts of IL-5 ex vivo after IL-33 stimulation of whole bone marrow cultures. In contrast, IL-33-induced bone marrow and airway eosinophilia were abolished in the absence of ILC2s in Rag2-/-Il2rg-/- mice and no production of IL-5 was detected in IL-33-stimulated bone marrow cultures. Conclusion: These findings establish bone marrow ILC2s and the IL-33/ST2 axis as promising targets for modulation of uncontrolled IL-5-dependent eosinophilic diseases including asthma.

Expression, activity and localization of lysosomal sulfatases in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death world-wide. Recently, we showed that COPD is associated with gene polymorphisms in SUMF1, a master regulator of sulfatases. Sulfatases are involved in extracellular matrix remodeling and activated by SUMF1, but their role in the lung is poorly described. We aimed to examine how sulfatases are affected in the airways of patients with COPD compared to ever smokers and never smokers. We observed that mRNA expression of the sulfatases GALNS, GNS and IDS was increased, while protein expression of many sulfatases was decreased in COPD fibroblasts. Several sulfatases, including GALNS, IDS, and SGSH, showed increased activity in COPD fibroblasts. Examination of different sulfatases by immunofluorescence showed that IDS, ARSB, GNS and SGSH in fibroblasts were localized to sites other than their reported destination. Using a master panel from different organs, RNA expression of all sulfatases could be observed in lung tissue. Additionally, immunohistochemistry on lung biopsies indicated differing expression of sulfatases in COPD patients. In conclusion, mRNA, protein expression, sulfatase activity levels, and localization of sulfatases are altered in lung fibroblasts and lung tissue from COPD patients and may be mechanistically important in COPD pathogenesis. This could contribute to the understanding of the disease mechanism in COPD and in the long run, to lead to more individualized therapies.

Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae.

Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5' cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, the RNAs are subjected to adapter ligation and subsequently proceeded to Next-generation sequencing (NGS). Due to the fast and efficient cross-linking and quenching steps, this protocol is also suitable for transiently induced mRNP granules. Examples include processing bodies (PBs) or stress granules (SGs) triggered by extrinsic stressors. Its reproducibility and broad applications make this protocol a useful and powerful tool to study the RNA compositions of specific RNPs.

MicroRNAs in type 2 immunity.

Type 2 immunity drives the pathology of allergic diseases and is necessary for expulsion of parasitic worms as well as having important implications in tumor progression. Over the last decade, a new research field has emerged describing a significant link between type 2 immunity and cancer development, called AllergoOncology. Thus, type 2 immune responses must be carefully regulated to mediate effective protection against damaging environmental factors, yet avoid excessive activation and immunopathology. Regulation of gene expression by microRNAs is required for normal behavior of most mammalian cells and has been studied extensively in the context of cancer. Although microRNA regulation of the immune system in cancer is well established and includes type 2 immune reactions in the tumor microenvironment, the involvement of microRNAs in these responses initiated by allergens, parasites or other environmental factors is just emerging. In this review, we focus on recent advances which increase the understanding of microRNA-mediated regulation of key mechanisms of type 2 immunity.

Context-dependent deposition and regulation of mRNAs in P-bodies.

Cells respond to stress by remodeling their transcriptome through transcription and degradation. Xrn1p-dependent degradation in P-bodies is the most prevalent decay pathway, yet, P-bodies may facilitate not only decay, but also act as a storage compartment. However, which and how mRNAs are selected into different degradation pathways and what determines the fate of any given mRNA in P-bodies remain largely unknown. We devised a new method to identify both common and stress-specific mRNA subsets associated with P-bodies. mRNAs targeted for degradation to P-bodies, decayed with different kinetics. Moreover, the localization of a specific set of mRNAs to P-bodies under glucose deprivation was obligatory to prevent decay. Depending on its client mRNA, the RNA-binding protein Puf5p either promoted or inhibited decay. Furthermore, the Puf5p-dependent storage of a subset of mRNAs in P-bodies under glucose starvation may be beneficial with respect to chronological lifespan.

Endoplasmic reticulum, Golgi, and lysosomes are disorganized in lung fibroblasts from chronic obstructive pulmonary disease patients.

Chronic Obstructive Pulmonary Disease (COPD) is often caused by smoking and other stressors. This causes oxidative stress, which induces numerous changes on both the transcriptome and proteome of the cell. We aimed to examine if the endomembrane pathway, including the endoplasmic reticulum (ER), Golgi, and lysosomes, was disrupted in fibroblasts from COPD patients as opposed to healthy ever-smokers or never-smokers, and if the response to stress differed. Different cellular compartments involved in the endomembrane pathway, as well as mRNA expression and apoptosis, were examined before and after the addition of stress in lung fibroblasts from never-smokers, ever-smokers, and patients with COPD. We found that the ER, Golgi, and lysosomes were disorganized in fibroblasts from COPD patients under baseline conditions. After a time course with ER stress inducing chemicals, changes to the phenotypes of cellular compartments in COPD patient fibroblasts were observed, and the expression of the ER stress-induced gene ERP72 was upregulated more in the COPD patient's cells compared to ever-smokers or never-smokers. Lastly, a tendency of increased active Caspase-3 was observed in COPD fibroblasts. Our results show that COPD patients have phenotypic changes in the lung fibroblasts endomembrane pathway, and respond differently to stress. Furthermore, these fibroblasts were cultured for several weeks outside the body, but they were not able to regain proper ER structure, indicating that the internal changes to the endomembrane system are permanent in smokers. This vulnerability to cellular stress might be a cause as to why some smokers develop COPD.