Protease-armed, Pathogenic Extracellular Vesicles Link Smoking and Chronic Obstructive Pulmonary Disease.

Rationale: Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as chronic obstructive pulmonary disease (COPD). Although cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. Objective: To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. Methods: Protease expression and enzymatic activity were measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring after EV transfer into naive recipient mice. Measurements and Main Results: The analyses revealed a unique EV profile defined by neutrophil- and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase 12 (MMP12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naive recipient mice, with an additive effect of NE- and MMP12-expressing EVs. Conclusions: These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naive mice, they provide a novel model to facilitate preclinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway.

Abnormal calcium homeostasis, activation of protease calpain, generation of p25 and hyperactivation of cyclin-dependent kinase 5 (Cdk5) have all been implicated in the pathogenesis of neurogenerative diseases including Alzheimer's disease. We have recently shown that extracellular cold-inducible RNA-binding protein (eCIRP) induces Cdk5 activation via p25. However, the precise molecular mechanism by which eCIRP regulates calcium signaling and calpain remains to be addressed. We hypothesized that eCIRP regulates p25 via Ca2+-dependent calpain activation. eCIRP increased calpain activity and decreased the endogenous calpain inhibitor calpastatin in Neuro 2a (N2a) cells. Calpain inhibition with calpeptin attenuated eCIRP-induced calpain activity and p25. eCIRP specifically upregulated cytosolic calpain 1, and calpain 1 silencing attenuated the eCIRP-induced increase in p25. eCIRP stimulation increased cytosolic free Ca2+, especially in hippocampal neuronal HT22 cells, which was attenuated by the eCIRP inhibitor Compound 23 (C23). Endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP3R) inhibition using 2-aminoethoxy-diphenyl-borate or xestospongin-C (X-C), interleukin-6 receptor alpha (IL-6Rα)-neutralization, and phospholipase C (PLC) inhibition with U73122 attenuated eCIRP-induced Ca2+ increase, while Ca2+ influx across the plasma membrane remained unaffected by eCIRP. Finally, C23, IL-6Rα antibody, U73122 and X-C attenuated eCIRP-induced p25 in HT-22 cells. In conclusion, the current study uncovers eCIRP-triggered Ca2+ release from ER stores in an IL-6Rα/PLC/IP3-dependent manner as a novel molecular mechanism underlying eCIRP's induction of Cdk5 activity and potential involvement in neurodegeneration.

Plasticity towards Rigidity: A Macrophage Conundrum in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, and ultimately fatal diffuse parenchymal lung disease. The molecular mechanisms of fibrosis in IPF patients are not fully understood and there is a lack of effective treatments. For decades, different types of drugs such as immunosuppressants and antioxidants have been tested, usually with unsuccessful results. Although two antifibrotic drugs (Nintedanib and Pirfenidone) are approved and used for the treatment of IPF, side effects are common, and they only slow down disease progression without improving patients' survival. Macrophages are central to lung homeostasis, wound healing, and injury. Depending on the stimulus in the microenvironment, macrophages may contribute to fibrosis, but also, they may play a role in the amelioration of fibrosis. In this review, we explore the role of macrophages in IPF in relation to the fibrotic processes, epithelial-mesenchymal transition (EMT), and their crosstalk with resident and recruited cells and we emphasized the importance of macrophages in finding new treatments.

COVID-19 Patients' Life Events, Emotional Health And Post-Illness Awareness: A Qualitative Study.

PURPOSE: This study aimed to examine the life events, emotional health and post-illness awareness of COVID-19 patients. We interviewed 25 people who experienced COVID-19. DESIGN: The study was executed in qualitative descriptive design. METHODS: The study was conducted with a phenomenological design approach. Data were collected using an in-depth, semi-structured interview method. We interviewed 25 people who experienced COVID-19. FINDINGS: Three themes were identified in the content analysis: "Experiences before COVID-19," "The active experience of COVID-19," "Remnants of COVID-19." We found that when participants first heard they tested positive for COVID-19, the anxiety they experienced turned into panic and fear of death as the duration and severity of the symptoms of the disease increased. That uncertainty, as well as the influence of the media, intensified their fears. However, despite the unfavorable conditions, experiencing the disease led to positive awareness in all the participants. CONCLUSIONS: We observed that the intensity of the symptoms of COVID-19, the uncertainty of the treatment process, and the attitude of the media were important factors in producing fear and anxiety. Experiencing the illness created an awareness that resulted in the participants' questioning the meaning of life. CLINICAL EVIDENCE: Within the scope of preventive health services, there is a need for informative programs of education on COVID-19 prevention, coping, and self-improvement that will be useful in reducing the psychological effects of the disease.

The role of eCIRP in bleomycin-induced pulmonary fibrosis in mice.

OBJECTIVE AND DESIGN: We examined the role of eCIRP in the pathogenesis of bleomycin-induced pulmonary fibrosis (PF). MATERIAL AND METHODS: Publicly available gene expression omnibus datasets were analyzed for the expression of CIRP in lung samples from patients with PF. Wild type (WT) or CIRP-/- mice received daily injections of 10 µg/g bleomycin for 10 days. A subset of bleomycin-injected WT mice was treated with the eCIRP antagonist C23 (8 µg/g/day) from day 10 to day 19. At three weeks, transthoracic echocardiography was performed to measure the degree of pulmonary hypertension, and lung tissues were collected and analyzed for markers of fibrosis. RESULTS: Analysis of the mRNA data of human lung samples showed a significant positive correlation between CIRP and α-smooth muscle actin (α-SMA), an important marker of fibrosis. Moreover, the expression of CIRP was higher in patients with acute exacerbation of PF than in patients with stable PF. CIRP-/- mice showed attenuated induction of α-SMA and collagens (Col1a1, Col3a1), reduced hydroxyproline content, decreased histological fibrosis scores, and improved pulmonary hypertension as compared to WT mice. WT mice treated with C23 also had significant attenuation of the above endpoint measure. CONCLUSIONS: Our study demonstrates that eCIRP plays a key role in promoting the development of PF, and blocking eCIRP with C23 can significantly attenuate this process.

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4.

Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern (DAMP), has been recently shown to play a critical role in promoting the development of bleomycin-induced pulmonary fibrosis. Although fibroblast activation is a critical component of the fibrotic process, the direct effects of eCIRP on fibroblasts have never been examined. We studied eCIRP's role in the induction of inflammatory phenotype in pulmonary fibroblasts and its connection to bleomycin-induced pulmonary fibrosis in mice. We found that eCIRP causes the induction of proinflammatory cytokines and differentially expression-related pathways in a TLR4-dependent manner in pulmonary fibroblasts. Our analysis further showed that the accessory pathways MD2 and Myd88 are involved in the induction of inflammatory phenotype. In order to study the connection of the enrichment of these pathways in priming the microenvironment for pulmonary fibrosis, we investigated the gene expression profile of lung tissues from mice subjected to bleomycin-induced pulmonary fibrosis collected at various time points. We found that at day 14, which corresponds to the inflammatory-to-fibrotic transition phase after bleomycin injection, TLR4, MD2, and Myd88 were induced, and the transcriptome was differentially enriched for genes in those pathways. Furthermore, we also found that inflammatory cytokines gene expressions were induced, and the cellular responses to these inflammatory cytokines were differentially enriched on day 14. Overall, our results show that eCIRP induces inflammatory phenotype in pulmonary fibroblasts in a TLR4 dependent manner. This study sheds light on the mechanism by which eCIRP induced inflammatory fibroblasts, contributing to pulmonary fibrosis.

Vitamin D modulates E-cadherin turnover by regulating TGF-ß and Wnt signalings during EMT-mediated myofibroblast differentiation in A459 cells.

Vitamin D (VitD) has an anti-fibrotic effect on fibrotic lungs. It reduces epithelial-mesenchymal transition (EMT) on tumors. We aimed to investigate target proteins of VitD for the regression of EMT-mediated myofibroblast differentiation. A group of A549 cells were treated with 5 % cigarette smoke extract (CSE) and 5 %CSE + TGF-ß (5 ng/ml) to induce EMT. The others were treated with 50 nM VitD 30 min before %5CSE and TGF-ß treatments. All cells were collected at 24, 48 and 72 h following 5 %CSE and TGF-ß administrations. The expression of p120ctn and NEDD9 proteins acted on E-cadherin turnover in addition to activations of TGF-ß and Wnt pathways were examined in these cells and fibrotic human lungs. CSE and TGF-ß induced EMT by reducing E-cadherin, p-VDR, SMAD7 and DKK1, increasing α-SMA, p120ctn, Kaiso, NEDD9 and stimulating TGF-ß and Wnt/ß-catenin signalings in A549 cells. VitD administration reversed these alterations and regressed EMT. Co-immunoprecipitation analysis revealed p-VDR interaction with ß-catenin and Kaiso in fibrotic and non-fibrotic human lungs. VitD pre-treatments reduced TGF-ß and Wnt/ß-catenin signalings by increasing p-VDR, protected from E-cadherin degradation and led to the regression of EMT in A549 cells treated with CSE and TGF-ß. Finally, VitD supplementation combined with anti-fibrotic therapeutics can be suggested for treatment of pulmonary fibrosis, which may be developed by smoking, in cases of VitD deficiency.