Myeloid Zfhx3 deficiency protects against hypercapnia-induced suppression of host defense against influenza A virus.

Hypercapnia, elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions and that elevated CO2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3) - a mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila - is expressed in mouse and human macrophages. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury, and mortality in hypercapnic mice infected with influenza A virus. To our knowledge, our results establish Zfhx3 as the first known mammalian mediator of CO2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung disease.

PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Myosin Light Chain Dephosphorylation by PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls ( P =2.0×10 -2 , n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation ( P =1.4×10 -6 , n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF ( P =2.9×10 -2 and 6.7×10 -3 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls ( P =5.0×10 -6 , n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls ( P =1.8×10 -2 and 4.1×10 -2 respectively, n= 6,6,5). Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Myeloid Zfhx3 Deficiency Protects Against Hypercapnia-induced Suppression of Host Defense Against Influenza A Virus.

Hypercapnia, elevation of the partial pressure of CO 2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions, and that elevated CO 2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3), mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila , is expressed in mouse and human MØs. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury and mortality in hypercapnic mice infected with influenza A virus. Our results establish Zfhx3 as the first known mammalian mediator of CO 2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung diseases.

Left atrial echocardiographic parameters predict the onset of atrial fibrillation: the SMASH2 scoring system.

BACKGROUND: As AF-associated morbidity and mortality are increasing, there is an acute need for improved surveillance and prevention strategies to reduce the impact of AF and related strokes. Specific echocardiographic parameters that can best predict future onset of AF within 3 months are lacking. METHODS: Twenty patients with AF, as identified by presence of ICD-9 diagnosis code, were compared with a control group of twenty age- and sex-matched patients selected from the same clinic population but without a diagnosis of AF. Transthoracic echocardiograms (TTE) obtained within 90 days prior to first documented AF episode (study group) or obtained closest to first clinic visit (control) were selected for review. RESULTS: Baseline characteristics, including age, BMI, presence of hypertension, hyperlipidemia, diabetes, and heart failure were comparable. Increased left atrial (LA) size (end systolic major axis in 2-chamber view: AF 4.62±0.03 vs control 3.79±0.21, P =0.03), increased mitral inflow (E/A ratio: AF 1.35±0.15 vs control 1.06±0.07, P =0.04), and reduced LA global longitudinal strain (AF -2.69±0.26 vs control - 3.59±0.31, P =0.04) were most closely associated with AF compared with the control group. Multivariate logistic regression was used to develop predictive models for AF onset. A combination of imaging and traditional clinical risk factors was the best AF prediction model with AUC of 0.94, which greatly exceeds the current best predictors published. From these parameters, we developed the SMASH2 scoring system for 90- day AF risk estimation. CONCLUSIONS: Risk factors for AF and early features of atrial cardiomyopathy including male sex, hypertension, LA enlargement, reduced mitral inflow, and reduced LA strain are powerful predictors of AF onset within 90 days, and may be used to prognosticate future AF risk.

Virchow's Triad and the Role of Thrombosis in COVID-Related Stroke.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as a virally transmitted disease. Three months later, SARS-CoV-2 became one of the largest pandemics in recent times, causing more than 235 million cases globally, and accounting for at least 4.8 million deaths to date. SARS-COV-2 infection was initially classified as a respiratory tract infection, but later was recognized as a multisystemic disease compromising gastrointestinal, hematological, cardiac, and neurological systems. With this Review, we aim to describe the epidemiology, risk factors, mechanisms, and management of cerebrovascular events in patients infected with COVID-19. Neurological manifestations related to thromboembolic cerebrovascular events in patients infected with COVID-19 have been frequent and associated with poor prognosis in the majority of cases. A better understanding of the mechanisms of thrombosis and etiologies of this new disease process are necessary to determine how to prevent and treat patients to reduce their length of stay, morbidity, and mortality.

Epithelial cell-specific loss of function of Miz1 causes a spontaneous COPD-like phenotype and up-regulates Ace2 expression in mice.

Cigarette smoking, the leading cause of chronic obstructive pulmonary disease (COPD), has been implicated as a risk factor for severe disease in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we show that mice with lung epithelial cell-specific loss of function of Miz1, which we identified as a negative regulator of nuclear factor κB (NF-κB) signaling, spontaneously develop progressive age-related changes resembling COPD. Furthermore, loss of Miz1 up-regulates the expression of Ace2, the receptor for SARS-CoV-2. Concomitant partial loss of NF-κB/RelA prevented the development of COPD-like phenotype in Miz1-deficient mice. Miz1 protein levels are reduced in the lungs from patients with COPD, and in the lungs of mice exposed to chronic cigarette smoke. Our data suggest that Miz1 down-regulation-induced sustained activation of NF-κB-dependent inflammation in the lung epithelium is sufficient to induce progressive lung and airway destruction that recapitulates features of COPD, with implications for COVID-19.

Hypercapnia Suppresses Macrophage Antiviral Activity and Increases Mortality of Influenza A Infection via Akt1.

Hypercapnia (HC), elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that HC inhibits multiple macrophage and neutrophil antimicrobial functions and increases the mortality of bacterial pneumonia in mice. In this study, we show that normoxic HC increases viral replication, lung injury, and mortality in mice infected with influenza A virus (IAV). Elevated CO2 increased IAV replication and inhibited antiviral gene and protein expression in macrophages in vivo and in vitro. HC potentiated IAV-induced activation of Akt, whereas specific pharmacologic inhibition or short hairpin RNA knockdown of Akt1 in alveolar macrophages blocked HC's effects on IAV growth and the macrophage antiviral response. Our findings suggest that targeting Akt1 or the downstream pathways through which elevated CO2 signals could enhance macrophage antiviral host defense and improve clinical outcomes in hypercapnic patients with advanced lung disease.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Metformin Targets Mitochondrial Electron Transport to Reduce Air-Pollution-Induced Thrombosis.

Urban particulate matter air pollution induces the release of pro-inflammatory cytokines including interleukin-6 (IL-6) from alveolar macrophages, resulting in an increase in thrombosis. Here, we report that metformin provides protection in this murine model. Treatment of mice with metformin or exposure of murine or human alveolar macrophages to metformin prevented the particulate matter-induced generation of complex III mitochondrial reactive oxygen species, which were necessary for the opening of calcium release-activated channels (CRAC) and release of IL-6. Targeted genetic deletion of electron transport or CRAC channels in alveolar macrophages in mice prevented particulate matter-induced acceleration of arterial thrombosis. These findings suggest metformin as a potential therapy to prevent some of the premature deaths attributable to air pollution exposure worldwide.

Multidimensional Assessment of the Host Response in Mechanically Ventilated Patients with Suspected Pneumonia.

Rationale: The identification of informative elements of the host response to infection may improve the diagnosis and management of bacterial pneumonia. Objectives: To determine whether the absence of alveolar neutrophilia can exclude bacterial pneumonia in critically ill patients with suspected infection and to test whether signatures of bacterial pneumonia can be identified in the alveolar macrophage transcriptome. Methods: We determined the test characteristics of alveolar neutrophilia for the diagnosis of bacterial pneumonia in three cohorts of mechanically ventilated patients. In one cohort, we also isolated macrophages from alveolar lavage fluid and used the transcriptome to identify signatures of bacterial pneumonia. Finally, we developed a humanized mouse model of Pseudomonas aeruginosa pneumonia to determine if pathogen-specific signatures can be identified in human alveolar macrophages. Measurements and Main Results: An alveolar neutrophil percentage less than 50% had a negative predictive value of greater than 90% for bacterial pneumonia in both the retrospective (n = 851) and validation cohorts (n = 76 and n = 79). A transcriptional signature of bacterial pneumonia was present in both resident and recruited macrophages. Gene signatures from both cell types identified patients with bacterial pneumonia with test characteristics similar to alveolar neutrophilia. Conclusions: The absence of alveolar neutrophilia has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection. Macrophages can be isolated from alveolar lavage fluid obtained during routine care and used for RNA-Seq analysis. This novel approach may facilitate a longitudinal and multidimensional assessment of the host response to bacterial pneumonia.

Hypercapnia increases airway smooth muscle contractility via caspase-7-mediated miR-133a-RhoA signaling.

The elevation of carbon dioxide (CO2) in tissues and the bloodstream (hypercapnia) occurs in patients with severe lung diseases, including chronic obstructive pulmonary disease (COPD). Whereas hypercapnia has been recognized as a marker of COPD severity, a role for hypercapnia in disease pathogenesis remains unclear. We provide evidence that CO2 acts as a signaling molecule in mouse and human airway smooth muscle cells. High CO2 activated calcium-calpain signaling and consequent smooth muscle cell contraction in mouse airway smooth muscle cells. The signaling was mediated by caspase-7-induced down-regulation of the microRNA-133a (miR-133a) and consequent up-regulation of Ras homolog family member A and myosin light-chain phosphorylation. Exposure of wild-type, but not caspase-7-null, mice to hypercapnia increased airway contraction and resistance. Deletion of the Caspase-7 gene prevented hypercapnia-induced airway contractility, which was restored by lentiviral transfection of a miR-133a antagonist. In a cohort of patients with severe COPD, hypercapnic patients had higher airway resistance, which improved after correction of hypercapnia. Our data suggest a specific molecular mechanism by which the development of hypercapnia may drive COPD pathogenesis and progression.

Reactive oxygen species as signaling molecules in the development of lung fibrosis.

Pulmonary fibrosis is a relatively rare but devastating disease characterized by the excessive deposition of extracellular matrix. The increased matrix results in reduced lung compliance and increased work of breathing, while the obliteration of alveolar-capillary structures can result in hypoxemia and pulmonary hypertension, which manifests clinically as worsening shortness of breath, respiratory failure, and death. Unbiased genome-wide association studies combined with animal models suggest that damage to the alveolar epithelium is the initiating factor in pulmonary fibrosis. This epithelial injury leads to the activation and proliferation of myofibroblasts that secrete extracellular matrix proteins characteristic of fibrosis. The best described molecular link between alveolar epithelial dysfunction and myofibroblast activation and proliferation is the profibrotic cytokine transforming growth factor-ß (TGF-ß). We and others have found that mitochondrial and NAD(P)H oxidase-generated reactive oxygen species (ROS) play a signaling role to enhance TGF-ß signaling and promote fibrosis. The purpose of this article is to review how ROS signaling leads to the activation of TGF-ß. We suggest that an improved understanding of these pathways might explain the failure of nonselective antioxidants to improve outcomes in patients with pulmonary fibrosis and might identify novel targets for therapy.

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.