EETs alleviate alveolar epithelial cell senescence by inhibiting endoplasmic reticulum stress through the Trim25/Keap1/Nrf2 axis.

Alveolar epithelial cell (AEC) senescence is a key driver of a variety of chronic lung diseases. It remains a challenge how to alleviate AEC senescence and mitigate disease progression. Our study identified a critical role of epoxyeicosatrienoic acids (EETs), downstream metabolites of arachidonic acid (ARA) by cytochrome p450 (CYP), in alleviating AEC senescence. In vitro, we found that 14,15-EET content was significantly decreased in senescent AECs. Exogenous EETs supplementation, overexpression of CYP2J2, or inhibition of EETs degrading enzyme soluble epoxide hydrolase (sEH) to increase EETs alleviated AECs' senescence. Mechanistically, 14,15-EET promoted the expression of Trim25 to ubiquitinate and degrade Keap1 and promoted Nrf2 to enter the nucleus to exert an anti-oxidant effect, thereby inhibiting endoplasmic reticulum stress (ERS) and alleviating AEC senescence. Furthermore, in D-galactose (D-gal)-induced premature aging mouse model, inhibiting the degradation of EETs by Trifluoromethoxyphenyl propionylpiperidin urea (TPPU, an inhibitor of sEH) significantly inhibited the protein expression of p16, p21, and γH2AX. Meanwhile, TPPU reduced the degree of age-related pulmonary fibrosis in mice. Our study has confirmed that EETs are novel anti-senescence substances for AECs, providing new targets for the treatment of chronic lung diseases.

Human microphysiological models of airway and alveolar epithelia.

Human organ-on-a-chip models are powerful tools for preclinical research that can be used to study the mechanisms of disease and evaluate new targets for therapeutic intervention. Lung-on-a-chip models have been one of the most well-characterized designs in this field and can be altered to evaluate various types of respiratory disease and to assess treatment candidates prior to clinical testing. These systems are capable of overcoming the flaws of conventional two-dimensional (2-D) cell culture and in vivo animal testing due to their ability to accurately recapitulate the in vivo microenvironment of human tissue with tunable material properties, microfluidic integration, delivery of precise mechanical and biochemical cues, and designs with organ-specific architecture. In this review, we first describe an overview of currently available lung-on-a-chip designs. We then present how recent innovations in human stem cell biology, tissue engineering, and microfabrication can be used to create more predictive human lung-on-a-chip models for studying respiratory disease. Finally, we discuss the current challenges and future directions of lung-on-a-chip designs for in vitro disease modeling with a particular focus on immune and multiorgan interactions.

Atractylodin Suppresses TGF-ß-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-ß1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-ß1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.

Vasoactive intestinal peptide attenuates bleomycin-induced murine pulmonary fibrosis by inhibiting epithelial-mesenchymal transition: Restoring autophagy in alveolar epithelial cells.

Vasoactive intestinal peptide (VIP) is an intrapulmonary neuropeptide with multi-function, including anti-fibrosis. However, the exact role of VIP in pulmonary fibrosis has not been documented. Here, we investigated the protective effect of VIP against pulmonary fibrosis in a murine model induced by bleomycin (BLM). We found that the overexpression of VIP mediated by the adenoviral vector significantly attenuated the lung tissue destruction, reduced the deposition of the extracellular matrix, and inhibited the expression of alpha-smooth muscle actin (α-SMA) in the lungs of mice received BLM. Mechanismly, we found that VIP significantly suppressed the transforming growth factor-beta 1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) and inhibited the matrix-producing ability of alveolar epithelial cells in vitro. Furthermore, we found that TGF-ß1 depressed the autophagy and an autophagy inductor partly reversed the TGF-ß1-induced EMT in alveolar epithelial cells. The impaired autophagy was also observed in the lungs of BLM-treated mice, which was restored by VIP treatment. And VIP treatment enhanced autophagy in TGF-ß1-stimulated alveolar epithelial cells, contributing to its anti-EMT effect. In summary, our data, for the first time, show that VIP attenuates BLM-induced pulmonary fibrosis in mice with anti-EMT effect through restoring autophagy in alveolar epithelial cells. This study provides a possibility that inhaled long-acting VIP may be an anti-fibrotic drug in the treatment of pulmonary fibrosis.

Mesenchymal stromal cell aging impairs the self-organizing capacity of lung alveolar epithelial stem cells.

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.

Many tissues in the body are capable of regenerating by replacing defective or worn-out cells with new ones. This process relies heavily on stem cells, which are precursor cells that lack a set role in the body and can develop into different types of cells under the right conditions. Tissues often have their own pool of stem cells that they use to replenish damaged cells. But as we age, this regeneration process becomes less effective. Many of our organs, such as the lungs, are lined with epithelial cells. These cells form a protective barrier, controlling what substances get in and out of the tissue. Alveoli are parts of the lungs that allow oxygen and carbon dioxide to move between the blood and the air in the lungs. And alveoli rely on an effective epithelial cell lining to work properly. To replenish these epithelial cells, alveoli have pockets, in which a type of epithelial cell, known as AEC2, lives. These cells can serve as stem cells, developing into a different type of cell under the right conditions. To work properly, AEC2 cells require close interactions with another type of cell called L-MSC, which supports the maintenance of other cells and also has the ability to differentiate into several other cell types. Both cell types can be found close together in these stem cell pockets. So far, it has been unclear how aging affects how these cells work together to replenish the epithelial lining of the alveoli. To investigate, Chanda et al. probed AEC2s and L-MSCs in the alveoli of young and old mice. The researchers collected both cell types from young (2-3 months) and aged (22-24 months) mice. Various combinations of these cells were grown to form 3D structures, mimicking how the cells grow in the lungs. Young L-MSCs formed normal 3D structures with both young and aged AEC2 cells. But aged L-MSCs developed abnormal, loose structures with AEC2 cells (both young and old cells). Aged L-MSCs were found to have higher levels of an enzyme (called Nox4) that produces oxidants and other 'pro-aging' factors, compared to young L-MSCs. However, reducing Nox4 levels in aged L-MSCs allowed these cells to form normal 3D structures with young AEC2 cells, but not aged AEC2 cells. These findings highlight the varying effects specific stem cells have, and how their behaviour is affected by pro-aging factors. Moreover, the pro-aging enzyme Nox4 shows potential as a therapeutic target ­ downregulating its activity may reverse critical effects of aging in cells.

Co-culture of type I and type II pneumocytes as a model of alveolar epithelium.

The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them. We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines which bear features of type I (hAELVi) and type II (NCI-H441) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation. The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.

Stretch increases alveolar type 1 cell number in fetal lungs through ROCK-Yap/Taz pathway.

Accurate fluid pressure in the fetal lung is critical for its development, especially at the beginning of the saccular stage when alveolar epithelial type 1 (AT1) and type 2 (AT2) cells differentiate from the epithelial progenitors. Despite our growing understanding of the role of physical forces in lung development, the molecular mechanisms that regulate the transduction of mechanical stretch to alveolar differentiation remain elusive. To simulate lung distension, we optimized both an ex vivo model with precision cut lung slices and an in vivo model of fetal tracheal occlusion. Increased mechanical tension showed to improve alveolar maturation and differentiation toward AT1. By manipulating ROCK pathway, we demonstrate that stretch-induced Yap/Taz activation promotes alveolar differentiation toward AT1 phenotype via ROCK activity. Our findings show that balanced ROCK-Yap/Taz signaling is essential to regulate AT1 differentiation in response to mechanical stretching of the fetal lung, which might be helpful in improving lung development and regeneration.

Comprehensive Transcriptomic Analysis Identifies Novel Antiviral Factors Against Influenza A Virus Infection.

Influenza A virus (IAV) has a higher genetic variation, leading to the poor efficiency of traditional vaccine and antiviral strategies targeting viral proteins. Therefore, developing broad-spectrum antiviral treatments is particularly important. Host responses to IAV infection provide a promising approach to identify antiviral factors involved in virus infection as potential molecular drug targets. In this study, in order to better illustrate the molecular mechanism of host responses to IAV and develop broad-spectrum antiviral drugs, we systematically analyzed mRNA expression profiles of host genes in a variety of human cells, including transformed and primary epithelial cells infected with different subtypes of IAV by mining 35 microarray datasets from the GEO database. The transcriptomic results showed that IAV infection resulted in the difference in expression of amounts of host genes in all cell types, especially those genes participating in immune defense and antiviral response. In addition, following the criteria of P<0.05 and |logFC|≥1.5, we found that some difference expression genes were overlapped in different cell types under IAV infection via integrative gene network analysis. IFI6, IFIT2, ISG15, HERC5, RSAD2, GBP1, IFIT3, IFITM1, LAMP3, USP18, and CXCL10 might act as key antiviral factors in alveolar basal epithelial cells against IAV infection, while BATF2, CXCL10, IFI44L, IL6, and OAS2 played important roles in airway epithelial cells in response to different subtypes of IAV infection. Additionally, we also revealed that some overlaps (BATF2, IFI44L, IFI44, HERC5, CXCL10, OAS2, IFIT3, USP18, OAS1, IFIT2) were commonly upregulated in human primary epithelial cells infected with high or low pathogenicity IAV. Moreover, there were similar defense responses activated by IAV infection, including the interferon-regulated signaling pathway in different phagocyte types, although the differentially expressed genes in different phagocyte types showed a great difference. Taken together, our findings will help better understand the fundamental patterns of molecular responses induced by highly or lowly pathogenic IAV, and the overlapped genes upregulated by IAV in different cell types may act as early detection markers or broad-spectrum antiviral targets.

Epidermal growth factor strongly affects epithelial Na+ transport and barrier function in fetal alveolar cells, with minor sex-specific effects.

Male sex remains an independent risk factor for respiratory distress syndrome (RDS) in preterm infants. Insufficient Na+ transport-mediated alveolar fluid clearance contributes to RDS development and we previously demonstrated sex-specific differences in Na+ transport. The epidermal growth factor (EGF) is important during fetal lung development with possible influence on Na+ transport. Sex-specific effects of EGF during surfactant synthesis were shown. We thus determined whether EGF exerts sex-specific effects on Na+ transport in fetal alveolar cells. We analyzed sex-specific fetal distal lung epithelial (FDLE) cells exposed to EGF and related ligands with Ussing chambers, RT-qPCR and Western blots. EGF strongly reduced the epithelial Na+ channel (ENaC) mRNA levels in both male and female FDLE cells. This was corroborated by a markedly reduced ENaC activity, while amiloride-insensitive pathways as well as barrier function were raised by EGF. In contrast to chronic effects, acute effects of EGF were sex-specific, because Na+ transport was reduced only in males. AKT phosphorylation was elevated only in female cells, while pERK1/2 was increased in both male and female cells. EGF showed certain sex- and time-dependent effects in FDLE cells. Nevertheless, the results suggest that EGF is an unlikely cause for the sex-specific differences in Na+ transport.

Linking Fibrotic Remodeling and Ultrastructural Alterations of Alveolar Epithelial Cells after Deletion of Nedd4-2.

Our previous study showed that in adult mice, conditional Nedd4-2-deficiency in club and alveolar epithelial type II (AE2) cells results in impaired mucociliary clearance, accumulation of Muc5b and progressive, terminal pulmonary fibrosis within 16 weeks. In the present study, we investigated ultrastructural alterations of the alveolar epithelium in relation to interstitial remodeling in alveolar septa as a function of disease progression. Two, eight and twelve weeks after induction of Nedd4-2 knockout, lungs were fixed and subjected to design-based stereological investigation at the light and electron microscopic level. Quantitative data did not show any abnormalities until 8 weeks compared to controls. At 12 weeks, however, volume of septal wall tissue increased while volume of acinar airspace and alveolar surface area significantly decreased. Volume and surface area of alveolar epithelial type I cells were reduced, which could not be compensated by a corresponding increase of AE2 cells. The volume of collagen fibrils in septal walls increased and was linked with an increase in blood-gas barrier thickness. A high correlation between parameters reflecting interstitial remodeling and abnormal AE2 cell ultrastructure could be established. Taken together, abnormal regeneration of the alveolar epithelium is correlated with interstitial septal wall remodeling.

Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis.

Recent studies have identified impaired type 2 alveolar epithelial cell (ATII) renewal in idiopathic pulmonary fibrosis (IPF) human organoids and severe fibrosis when ATII is defective in mice. ATIIs function as progenitor cells and require supportive signals from the surrounding mesenchymal cells. The mechanisms by which mesenchymal cells promote ATII progenitor functions in lung fibrosis are incompletely understood. We identified growth hormone receptor (GHR) is mainly expressed in mesenchymal cells, and its expression is substantially decreased in IPF lungs. Higher levels of GHR expression correlated with better lung function in patients with IPF. Profibrotic mesenchymal cells retarded ATII growth and were associated with suppressed vesicular GHR expression. Vesicles enriched with Ghr promote ATII proliferation and diminished pulmonary fibrosis in mesenchymal Ghr-deficient mice. Our findings demonstrate a previously unidentified mesenchymal paracrine signaling coordinated by GHR that is capable of supporting ATII progenitor cell renewal and limiting the severity of lung fibrosis.

All-Inkjet-Printed 3D Alveolar Barrier Model with Physiologically Relevant Microarchitecture.

With the outbreak of new respiratory viruses and high mortality rates of pulmonary diseases, physiologically relevant models of human respiratory system are urgently needed to study disease pathogenesis, drug efficacy, and pharmaceutics. In this paper, a 3D alveolar barrier model fabricated by printing four human alveolar cell lines, namely, type I and II alveolar cells (NCI-H1703 and NCI-H441), lung fibroblasts (MRC5), and lung microvascular endothelial cells (HULEC-5a) is presented. Automated high-resolution deposition of alveolar cells by drop-on-demand inkjet printing enables to fabricate a three-layered alveolar barrier model with an unprecedented thickness of ≈10 µm. The results show that the 3D structured model better recapitulate the structure, morphologies, and functions of the lung tissue, compared not only to a conventional 2D cell culture model, as expected, but also a 3D non-structured model of a homogeneous mixture of the alveolar cells and collagen. Finally, it is demonstrated that this thin multilayered model reproduce practical tissue-level responses to influenza infection. Drop-on-demand inkjet-printing is an enabling technology for customization, scalable manufacturing, and standardization of their size and growth, and it is believed that this 3D alveolar barrier model can be used as an alternative to traditional test models for pathological and pharmaceutical applications.

The History and Mystery of Alveolar Epithelial Type II Cells: Focus on Their Physiologic and Pathologic Role in Lung.

Alveolar type II (ATII) cells are a key structure of the distal lung epithelium, where they exert their innate immune response and serve as progenitors of alveolar type I (ATI) cells, contributing to alveolar epithelial repair and regeneration. In the healthy lung, ATII cells coordinate the host defense mechanisms, not only generating a restrictive alveolar epithelial barrier, but also orchestrating host defense mechanisms and secreting surfactant proteins, which are important in lung protection against pathogen exposure. Moreover, surfactant proteins help to maintain homeostasis in the distal lung and reduce surface tension at the pulmonary air-liquid interface, thereby preventing atelectasis and reducing the work of breathing. ATII cells may also contribute to the fibroproliferative reaction by secreting growth factors and proinflammatory molecules after damage. Indeed, various acute and chronic diseases are associated with intensive inflammation. These include oedema, acute respiratory distress syndrome, fibrosis and numerous interstitial lung diseases, and are characterized by hyperplastic ATII cells which are considered an essential part of the epithelialization process and, consequently, wound healing. The aim of this review is that of revising the physiologic and pathologic role ATII cells play in pulmonary diseases, as, despite what has been learnt in the last few decades of research, the origin, phenotypic regulation and crosstalk of these cells still remain, in part, a mystery.

Delta-like 4 is required for pulmonary vascular arborization and alveolarization in the developing lung.

The molecular mechanisms by which endothelial cells (ECs) regulate pulmonary vascularization and contribute to alveolar epithelial cell development during lung morphogenesis remain unknown. We tested the hypothesis that delta-like 4 (DLL4), an EC Notch ligand, is critical for alveolarization by combining lung mapping and functional studies in human tissue and DLL4-haploinsufficient mice (Dll4+/lacz). DLL4 expressed in a PECAM-restricted manner in capillaries, arteries, and the alveolar septum from the canalicular to alveolar stage in mice and humans. Dll4 haploinsufficiency resulted in exuberant, nondirectional vascular patterning at E17.5 and P6, followed by smaller capillaries and fewer intermediate blood vessels at P14. Vascular defects coincided with polarization of lung EC expression toward JAG1-NICD-HES1 signature and decreased tip cell-like (Car4) markers. Dll4+/lacZ mice had impaired terminal bronchiole development at the canalicular stage and impaired alveolarization upon lung maturity. We discovered that alveolar type I cell (Aqp5) markers progressively decreased in Dll4+/lacZ mice after birth. Moreover, in human lung EC, DLL4 deficiency programmed a hypersprouting angiogenic phenotype cell autonomously. In conclusion, DLL4 is expressed from the canalicular to alveolar stage in mice and humans, and Dll4 haploinsufficiency programs dysmorphic microvascularization, impairing alveolarization. Our study reveals an obligate role for DLL4-regulated angiogenesis in distal lung morphogenesis.

Epithelium- and endothelium-derived exosomes regulate the alveolar macrophages by targeting RGS1 mediated calcium signaling-dependent immune response.

Alveolar macrophages (AM) maintain airway immune balance; however, the regulation of heterogeneity of AMs is incompletely understood. We demonstrate that RGS1 coregulates the immunophenotype of AM subpopulations, including pro- and anti-inflammatory, injury- and repair-associated, and pro- and antifibrotic phenotypes, through the PLC-IP3R signal-dependent intracellular Ca2+ response. Flt3+ AMs and Tie2+ AMs had different immune properties, and RGS1 expression in the cells was targeted by exosomes (EXOs) containing miR-223 and miR-27b-3p that were derived from vascular endothelial cells (EnCs) and type II alveolar epithelial cells (EpCs-II), respectively. Imbalance of AMs was correlated with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis (PF) caused a lack of secretion of CD31+ and CD74+ EXOs derived from EnCs and EpCs-II. Timely treatment with EXOs significantly improved endotoxin-induced ALI/ARDS and bleomycin-induced PF in mice. Thus, EnC- and EpC-II-derived EXOs regulate the immune balance of AMs and can be used as potential therapeutic drugs.

Second-generation lung-on-a-chip with an array of stretchable alveoli made with a biological membrane.

The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.

IGF-1 Receptor Signaling Regulates Type II Pneumocyte Senescence and Resulting Macrophage Polarization in Lung Fibrosis.

PURPOSE: Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging. METHODS AND MATERIALS: Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63). RESULTS: IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 µg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis. CONCLUSIONS: This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.

Preventing the development of severe COVID-19 by modifying immunothrombosis.

BACKGROUND: COVID-19-associated acute respiratory distress syndrome (ARDS) is associated with significant morbidity and high levels of mortality. This paper describes the processes involved in the pathophysiology of COVID-19 from the initial infection and subsequent destruction of type II alveolar epithelial cells by SARS-CoV-2 and culminating in the development of ARDS. MAIN BODY: The activation of alveolar cells and alveolar macrophages leads to the release of large quantities of proinflammatory cytokines and chemokines and their translocation into the pulmonary vasculature. The presence of these inflammatory mediators in the vascular compartment leads to the activation of vascular endothelial cells platelets and neutrophils and the subsequent formation of platelet neutrophil complexes. These complexes in concert with activated endothelial cells interact to create a state of immunothrombosis. The consequence of immunothrombosis include hypercoagulation, accelerating inflammation, fibrin deposition, migration of neutrophil extracellular traps (NETs) producing neutrophils into the alveolar apace, activation of the NLRP3 inflammazome, increased alveolar macrophage destruction and massive tissue damage by pyroptosis and necroptosis Therapeutic combinations aimed at ameliorating immunothrombosis and preventing the development of severe COVID-19 are discussed in detail.

Curcumin Targets p53-Fibrinolytic System in TGF-ß1 Mediated Alveolar Epithelial Mesenchymal Transition in Alveolar Epithelial Cells.

AIMS: We aim to investigate curcumin interaction with p53-fibrinolytic system, smad dependent and independent pathways underlying their prime role during lung injury and fibrosis. BACKGROUND: Curcumin, an active component of Curcuma longa plant, substantially modulates respiratory conditions. TGF-ß1 plays a central role in lung remodeling by balancing extracellular matrix (ECM) production and degradation, which is a hallmark for alveolar EMT. However, the crosstalk of curcumin is not known yet with TGF- ß1 mediated p53-Fibrinolytic system regulating alveolar EMT leading to IPF. In the present study, the potential molecular mechanism of curcumin in TGF-ß1 mediated p53-fibrinolytic system in basal alveolar epithelial cells was explored. OBJECTIVES: To understand the potential molecular mechanism of curcumin in TGF-ß1 mediated p53-fibrinolytic system in basal alveolar epithelial cells. METHODS: Basal alveolar epithelial cells were treated with TGF- ß1 to induce alveolar EMT and after 24 hrs curcumin was administered to study its anti-fibrotic effects. Molecular techniques like immunoblot, RT-PCR and immunofluorescence were performed to assess the anti-fibrotic role of curcumin on EMT markers, IL-17A, p53-smad interaction to investigate the anti-fibrotic role of curcumin. RESULTS: The results indicated that TGF-ß1-induced EMT in A549 cells exhibited altered expression of the IL-17A, p53-fibrinolytic markers and EMT markers at the mRNA and protein level. Intervention with curcumin attenuated alveolar EMT and inactivated TGF-ß1 induced Smad/non Smad signaling pathways via blocking p53-fibrinolytic system. CONCLUSION: This study provides the first evidence of the dynamic response of curcumin on TGF- ß1 mediated p53-fibrinolytic system during alveolar injury in vitro.