Magnetic Resonance Elastography and Computational Modeling Identify Heterogeneous Lung Biomechanical Properties during Cystic Fibrosis.

The lung is a dynamic mechanical organ and several pulmonary disorders are characterized by heterogeneous changes in the lung's local mechanical properties (i.e. stiffness). These alterations lead to abnormal lung tissue deformation (i.e. strain) which have been shown to promote disease progression. Although heterogenous mechanical properties may be important biomarkers of disease, there is currently no non-invasive way to measure these properties for clinical diagnostic purposes. In this study, we use a magnetic resonance elastography technique to measure heterogenous distributions of the lung's shear stiffness in healthy adults and in people with Cystic Fibrosis. Additionally, computational finite element models which directly incorporate the measured heterogenous mechanical properties were developed to assess the effects on lung tissue deformation. Results indicate that consolidated lung regions in people with Cystic Fibrosis exhibited increased shear stiffness and reduced spatial heterogeneity compared to surrounding non-consolidated regions. Accounting for heterogenous lung stiffness in healthy adults did not change the globally averaged strain magnitude obtained in computational models. However, computational models that used heterogenous stiffness measurements predicted significantly more variability in local strain and higher spatial strain gradients. Finally, computational models predicted lower strain variability and spatial strain gradients in consolidated lung regions compared to non-consolidated regions. These results indicate that spatial variability in shear stiffness alters local strain and strain gradient magnitudes in people with Cystic Fibrosis. This imaged-based modeling technique therefore represents a clinically viable way to non-invasively assess lung mechanics during both health and disease.

Building Career Paths for Ph.D., Basic and Translational Scientists in Clinical Departments in the United States: An Official American Thoracic Society Workshop Report.

Rationale: To identify barriers and opportunities for Ph.D., basic and translational scientists to be fully integrated into clinical units. Objectives: In 2022, an ad hoc committee of the American Thoracic Society developed a project proposal and workshop to identify opportunities and barriers for scientists who do not practice medicine to develop successful careers and achieve tenure-track faculty positions in clinical departments and divisions within academic medical centers (AMCs) in the United States. Methods: This document focuses on results from a survey of adult and pediatric pulmonary, critical care, and sleep medicine division chiefs as well as a survey of workshop participants, including faculty in departmental and school leadership roles in both basic science and clinical units within U.S. AMCs. Results: We conclude that full integration of non-clinically practicing basic and translational scientists into the clinical units, in addition to their traditional placements in basic science units, best serves the tripartite mission of AMCs to provide care, perform research, and educate the next generation. Evidence suggests clinical units do employ Ph.D. scientists in large numbers, but these faculty are often hired into non-tenure track positions, which do not provide the salary support, start-up funds, research independence, or space often associated with hiring in basic science units within the same institution. These barriers to success of Ph.D. faculty in clinical units are largely financial. Conclusions: Our recommendation is for AMCs to consider and explore some of our proposed strategies to accomplish the goal of integrating basic and translational scientists into clinical units in a meaningful way.

Macrophage-Targeted Lipid Nanoparticle Delivery of microRNA-146a to Mitigate Hemorrhagic Shock-Induced Acute Respiratory Distress Syndrome.

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro coculture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro. Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose of miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a levels in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have the therapeutic potential to mitigate lung injury during mechanical ventilation.

Macrophage-targeted lipid nanoparticle delivery of microRNA-146a to mitigate hemorrhagic shock-induced acute respiratory distress syndrome.

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury, but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro co-culture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro . Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have therapeutic potential to mitigate lung injury during mechanical ventilation.

Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples.

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and nonresolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFα and IL-1ß, compared with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGFß did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared with direct coculture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity.

Scavenger receptor BI attenuates oxidized phospholipid-induced pulmonary inflammation.

Damage associated molecular patterns (DAMPs) are molecules released from dead/dying cells following toxicant and/or environmental exposures that activate the immune response through binding of pattern recognition receptors (PRRs). Excessive production of DAMPs or failed clearance leads to chronic inflammation and delayed inflammation resolution. One category of DAMPs are oxidized phospholipids (oxPLs) produced upon exposure to high levels of oxidative stress, such as following ozone (O3) induced inflammation. OxPLs are bound by multiple classes of PRRs that include scavenger receptors (SRs) such as SR class B-1 (SR-BI) and toll-like receptors (TLRs). Interactions between oxPLs and PRRs appear to regulate inflammation; however, the role of SR-BI in oxPL-induced lung inflammation has not been defined. Therefore, we hypothesize that SR-BI is critical in protecting the lung from oxPL-induced pulmonary inflammation/injury. To test this hypothesis, C57BL/6J (WT) female mice were dosed with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (oxPAPC) by oropharyngeal aspiration which increased pulmonary SR-BI expression. Following oxPAPC exposure, SR-BI deficient (SR-BI-/-) mice exhibited increased lung pathology and inflammatory cytokine/chemokine production. Lipidomic analysis revealed that SR-BI-/- mice had an altered pulmonary lipidome prior to and following oxPAPC exposure, which correlated with increased oxidized phosphatidylcholines (PCs). Finally, we characterized TLR4-mediated activation of NF-κB following oxPAPC exposure and discovered that SR-BI-/- mice had increased TLR4 mRNA expression in lung tissue and macrophages, increased nuclear p65, and decreased cytoplasmic IκBα. Overall, we conclude that SR-BI is required for limiting oxPAPC-induced lung pathology by maintaining lipid homeostasis, reducing oxidized PCs, and attenuating TLR4-NF-κB activation, thereby preventing excessive and persistent inflammation.

Ozone impairs endogenous compensatory responses in allergic asthma.

Asthma is a chronic inflammatory airway disease characterized by acute exacerbations triggered by inhaled allergens, respiratory infections, or air pollution. Ozone (O3), a major component of air pollution, can damage the lung epithelium in healthy individuals. Despite this association, little is known about the effects of O3 and its impact on chronic lung disease. Epidemiological data have demonstrated that elevations in ambient O3 are associated with increased asthma exacerbations. To identify mechanisms by which O3 exposure leads to asthma exacerbations, we developed a two-hit mouse model where mice were sensitized and challenged with three common allergens (dust mite, ragweed and Aspergillus fumigates, DRA) to induce allergic inflammation prior to exposure to O3 (DRAO3). Changes in lung physiology, inflammatory cells, and inflammation were measured. Exposure to O3 following DRA significantly increased airway hyperreactivity (AHR), which was independent of TLR4. DRA exposure resulted in increased BAL eosinophilia while O3 exposure resulted in neutrophilia. Additionally, O3 exposure following DRA blunted anti-inflammatory and antioxidant responses. Finally, there were significantly less monocytes and innate lymphoid type 2 cells (ILC2s) in the dual challenged DRA-O3 group suggesting that the lack of these immune cells may influence O3-induced AHR in the setting of allergic inflammation. In summary, we developed a mouse model that mirrors some aspects of the clinical course of asthma exacerbations due to air pollution and identified that O3 exposure in the asthmatic lung leads to impaired endogenous anti-inflammatory and antioxidant responses and alterations inflammatory cell populations.

Biomechanical Force and Cellular Stiffness in Lung Fibrosis.

Lung fibrosis is characterized by the continuous accumulation of extracellular matrix (ECM) proteins produced by apoptosis-resistant (myo)fibroblasts. Lung epithelial injury promotes the recruitment and activation of fibroblasts, which are necessary for tissue repair and restoration of homeostasis. However, under pathologic conditions, a vicious cycle generated by profibrotic growth factors/cytokines, multicellular interactions, and matrix-associated signaling propagates the wound repair response and promotes lung fibrosis characterized not only by increased quantities of ECM proteins but also by changes in the biomechanical properties of the matrix. Importantly, changes in the biochemical and biomechanical properties of the matrix itself can serve to perpetuate fibroblast activity and propagate fibrosis, even in the absence of the initial stimulus of injury. The development of novel experimental models and methods increasingly facilitates our ability to interrogate fibrotic processes at the cellular and molecular levels. The goal of this review is to discuss the impact of ECM conditions in the development of lung fibrosis and to introduce new approaches to more accurately model the in vivo fibrotic microenvironment. This article highlights the pathologic roles of ECM in terms of mechanical force and the cellular interactions while reviewing in vitro and ex vivo models of lung fibrosis. The improved understanding of the fundamental mechanisms that contribute to lung fibrosis holds promise for identification of new therapeutic targets and improved outcomes.

Mechanobiology of Pulmonary Diseases: A Review of Engineering Tools to Understand Lung Mechanotransduction.

Cells within the lung micro-environment are continuously subjected to dynamic mechanical stimuli which are converted into biochemical signaling events in a process known as mechanotransduction. In pulmonary diseases, the abrogated mechanical conditions modify the homeostatic signaling which influences cellular phenotype and disease progression. The use of in vitro models has significantly expanded our understanding of lung mechanotransduction mechanisms. However, our ability to match complex facets of the lung including three-dimensionality, multicellular interactions, and multiple simultaneous forces is limited and it has proven difficult to replicate and control these factors in vitro. The goal of this review is to (a) outline the anatomy of the pulmonary system and the mechanical stimuli that reside therein, (b) describe how disease impacts the mechanical micro-environment of the lung, and (c) summarize how existing in vitro models have contributed to our current understanding of pulmonary mechanotransduction. We also highlight critical needs in the pulmonary mechanotransduction field with an emphasis on next-generation devices that can simulate the complex mechanical and cellular environment of the lung. This review provides a comprehensive basis for understanding the current state of knowledge in pulmonary mechanotransduction and identifying the areas for future research.

Nanoparticle delivery of microRNA-146a regulates mechanotransduction in lung macrophages and mitigates injury during mechanical ventilation.

Mechanical ventilation generates injurious forces that exacerbate lung injury. These forces disrupt lung barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides including microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We use humanized in-vitro systems, mouse models, and biospecimens from patients to elucidate the expression dynamics of miR-146a needed to decrease lung injury during mechanical ventilation. We find that the endogenous increase in miR-146a following injurious ventilation is not sufficient to prevent lung injury. However, when miR-146a is highly overexpressed using a nanoparticle delivery platform it is sufficient to prevent injury. These data indicate that the endogenous increase in microRNA-146a during mechanical ventilation is a compensatory response that partially limits injury and that nanoparticle delivery of miR-146a is an effective strategy for mitigating lung injury during mechanical ventilation.

PolyADP-Ribosylation of NFATc3 and NF-κB Transcription Factors Modulate Macrophage Inflammatory Gene Expression in LPS-Induced Acute Lung Injury.

Pulmonary macrophages play a critical role in the recognition of pathogens, initiation of host defense via inflammation, clearance of pathogens from the airways, and resolution of inflammation. Recently, we have shown a pivotal role for the nuclear factor of activated T-cell cytoplasmic member 3 (NFATc3) transcription factor in modulating pulmonary macrophage function in LPS-induced acute lung injury (ALI) pathogenesis. Although the NFATc proteins are activated primarily by calcineurin-dependent dephosphorylation, here we show that LPS induces posttranslational modification of NFATc3 by polyADP-ribose polymerase 1 (PARP-1)-mediated polyADP-ribosylation. ADP-ribosylated NFATc3 showed increased binding to iNOS and TNFα promoter DNA, thereby increasing downstream gene expression. Inhibitors of PARP-1 decreased LPS-induced NFATc3 ribosylation, target gene promoter binding, and gene expression. LPS increased NFAT luciferase reporter activity in lung macrophages and lung tissue that was inhibited by pretreatment with PARP-1 inhibitors. More importantly, pretreatment of mice with the PARP-1 inhibitor olaparib markedly decreased LPS-induced cytokines, protein extravasation in bronchoalveolar fluid, lung wet-to-dry ratios, and myeloperoxidase activity. Furthermore, PARP-1 inhibitors decreased NF-кB luciferase reporter activity and LPS-induced ALI in NF-кB reporter mice. Thus, our study demonstrates that inhibiting NFATc3 and NF-кB polyADP-ribosylation with PARP-1 inhibitors prevented LPS-induced ALI pathogenesis.

IRAK-M Regulates Monocyte Trafficking to the Lungs in Response to Bleomycin Challenge.

Idiopathic pulmonary fibrosis is a deadly disease characterized by excessive extracellular matrix deposition in the lungs, resulting in decreased pulmonary function. Although epithelial cells and fibroblasts have long been the focus of idiopathic pulmonary fibrosis research, the role of various subpopulations of macrophages in promoting a fibrotic response is an emerging target. Healthy lungs are composed of two macrophage populations, tissue-resident alveolar macrophages and interstitial macrophages, which help to maintain homeostasis. After injury, tissue-resident alveolar macrophages are depleted, and monocytes from the bone marrow (BM) traffic to the lungs along a CCL2/CCR2 axis and differentiate into monocyte-derived alveolar macrophages (Mo-AMs), which is a cell population implicated in murine models of pulmonary fibrosis. In this study, we sought to determine how IL-1R-associated kinase-M (IRAK-M), a negative regulator of TLR signaling, modulates monocyte trafficking into the lungs in response to bleomycin. Our data indicate that after bleomycin challenge, mice lacking IRAK-M have decreased monocyte trafficking and reduced Mo-AMs in their lungs. Although IRAK-M expression did not regulate differences in chemokines, cytokines, or adhesion molecules associated with monocyte recruitment, IRAK-M was necessary for CCR2 upregulation following bleomycin challenge. This finding prompted us to develop a competitive BM chimera model, which demonstrated that expression of BM-derived IRAK-M was necessary for monocyte trafficking into the lung and for subsequent enhanced collagen deposition. These data indicate that IRAK-M regulates monocyte trafficking by increasing the expression of CCR2, resulting in enhanced monocyte translocation into the lung, Mo-AM differentiation, and development of pulmonary fibrosis.

Macrophage HIF-1α mediates obesity-related adipose tissue dysfunction via interleukin-1 receptor-associated kinase M.

Hypoxia leading to stabilization of hypoxia-inducible factor 1α (HIF-1α) serves as an early upstream initiator for adipose tissue (AT) dysfunction. Monocyte-derived macrophage infiltration in AT contributes to inflammation, fibrosis and obesity-related metabolic dysfunction. It was previously reported that myeloid cell-specific deletion of Hif-1α protected against high-fat diet (HFD)-induced AT dysfunction. Prolyl hydroxylases (PHDs) are key regulators of HIF-1α. We examined the effects of myeloid cell-specific upregulation and stabilization of Hif-1α via deletion of prolyl-hydroxylase 2 (Phd2) and whether interleukin-1 receptor associated kinase-M (Irak-M), a known downstream target of Hif-1α, contributes to Hif-1α-induced AT dysfunction. Our data show that with HFD, Hif-1α and Irak-M expressions were increased in the AT macrophages of Phd2flox/flox/LysMcre mice compared with LysMcre mice. With HFD, Phd2flox/flox/LysMcre mice exhibited increased AT inflammation, fibrosis, and systemic insulin resistance compared with control mice. Furthermore, Phd2flox/flox/LysMcre mice bone marrow-derived macrophages exposed to hypoxia in vitro also had increased expressions of both Hif-1α and Irak-M. In wild-type mice, HFD induced upregulation of both HIF-1a and Irak-M in adipose tissue. Despite equivalent expression of Hif-1α compared with wild-type mice, globally-deficient Irak-M mice fed a HFD exhibited less macrophage infiltration, decreased inflammation and fibrosis and improved glucose tolerance. Global Irak-M deficiency was associated with an alternatively-activated macrophage phenotype in the AT after HFD. Together, these data show for the first time that an Irak-M-dependent mechanism likely mediates obesity-related AT dysfunction in conjunction with Hif-1α upregulation.

Depletion of microRNA-451 in response to allergen exposure accentuates asthmatic inflammation by regulating Sirtuin2.

The incidence of asthma has increased from 5.5% to near 8% of the population, which is a major health concern. The hallmarks of asthma include eosinophilic airway inflammation that is associated with chronic airway remodeling. Allergic airway inflammation is characterized by a complex interplay of resident and inflammatory cells. MicroRNAs (miRNAs) are small noncoding RNAs that function as posttranscriptional modulators of gene expression. However, the role of miRNAs, specifically miR-451, in the regulation of allergic airway inflammation is unexplored. Our previous findings showed that oxidant stress regulates miR-451 gene expression in macrophages during an inflammatory process. In this paper, we examined the role of miR-451 in regulating macrophage phenotype using an experimental poly-allergenic murine model of allergic airway inflammation. We found that miR-451 contributes to the allergic induction of CCL17 in the lung and plays a key role in proasthmatic macrophage activation. Remarkably, administration of a Sirtuin 2 (Sirt2) inhibitor diminished alternate macrophage activation and markedly abrogated triple-allergen [dust mite, ragweed, Aspergillus fumigatus (DRA)]-induced lung inflammation. These data demonstrate a role for miR-451 in modulating allergic inflammation by influencing allergen-mediated macrophages phenotype.

Sirtuin 2 enhances allergic asthmatic inflammation.

Allergic eosinophilic asthma is a chronic condition causing airway remodeling resulting in lung dysfunction. We observed that expression of sirtuin 2 (Sirt2), a histone deacetylase, regulates the recruitment of eosinophils after sensitization and challenge with a triple antigen: dust mite, ragweed, and Aspergillus fumigatus (DRA). Our data demonstrate that IL-4 regulates the expression of Sirt2 isoform 3/5. Pharmacological inhibition of Sirt2 by AGK2 resulted in diminished cellular recruitment, decreased CCL17/TARC, and reduced goblet cell hyperplasia. YM1 and Fizz1 expression was reduced in AGK2-treated, IL-4-stimulated lung macrophages in vitro as well as in lung macrophages from AGK2-DRA-challenged mice. Conversely, overexpression of Sirt2 resulted in increased cellular recruitment, CCL17 production, and goblet cell hyperplasia following DRA challenge. Sirt2 isoform 3/5 was upregulated in primary human alveolar macrophages following IL-4 and AGK2 treatment, which resulted in reduced CCL17 and markers of alternative activation. These gain-of-function and loss-of-function studies indicate that Sirt2 could be developed as a treatment for eosinophilic asthma.

FoxO1 is a critical regulator of M2-like macrophage activation in allergic asthma.

BACKGROUND: The pathogenesis of asthma and airway obstruction is the result of an abnormal response to different environmental exposures. The scientific premise of our study was based on the finding that FoxO1 expression is increased in lung macrophages of mice after allergen exposure and human asthmatic patients. Macrophages are capable of switching from one functional phenotype to another, and it is important to understand the mechanisms involved in the transformation of macrophages and how their cellular function affects the peribronchial stromal microenvironment. METHODS: We employed a murine asthma model, in which mice were treated by intranasal insufflation with allergens for 2-8 weeks. We used both a pharmacologic approach using a highly specific FoxO1 inhibitor and genetic approaches using FoxO1 knockout mice (FoxO1fl/fl LysMcre). Cytokine level in biological fluids was measured by ELISA and the expression of encoding molecules by NanoString assay and qRT-PCR. RESULTS: We show that the levels of FoxO1 gene are significantly elevated in the airway macrophages of patients with mild asthma in response to subsegmental bronchial allergen challenge. Transcription factor FoxO1 regulates a pro-asthmatic phenotype of lung macrophages that is involved in the development and progression of chronic allergic airway disease. We have shown that inhibition of FoxO1 induced phenotypic conversion of lung macrophages and downregulates pro-asthmatic and pro-fibrotic gene expression by macrophages, which contribute to airway inflammation and airway remodeling in allergic asthma. CONCLUSION: Targeting FoxO1 with its downstream regulator IRF4 is a novel therapeutic target for controlling allergic inflammation and potentially reversing fibrotic airway remodeling.