IRGM1 links mitochondrial quality control to autoimmunity.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Type I interferon signaling and peroxisomal dysfunction contribute to enhanced inflammatory cytokine production in Irgm1-deficient macrophages.

The human IRGM gene has been linked to inflammatory diseases including sepsis and Crohn's disease. Decreased expression of human IRGM, or of the mouse orthologues Irgm1 and Irgm2, leads to increased production of a number of inflammatory chemokines and cytokines in vivo and/or in cultured macrophages. Prior work has indicated that increased cytokine production is instigated by metabolic alterations and by changes in mitochondrial homeostasis; however, a comprehensive mechanism has not been elucidated. In the studies presented here, RNA deep sequencing and quantitative PCR were used to show that increases in cytokine production, as well as most changes in the transcriptional profile of Irgm1-/- bone marrow-derived macrophages (BMM), are dependent on increased type I IFN production seen in those cells. Metabolic alterations that drive increased cytokines in Irgm1-/- BMM - specifically increases in glycolysis and increased accumulation of acyl-carnitines - were unaffected by quenching type I IFN signaling. Dysregulation of peroxisomal homeostasis was identified as a novel upstream pathway that governs type I IFN production and inflammatory cytokine production. Collectively, these results enhance our understanding of the complex biochemical changes that are triggered by lack of Irgm1 and contribute to inflammatory disease seen with Irgm1-deficiency.

Mechanisms and Effects of Activation of Innate Immunity by Mitochondrial Nucleic Acids.

In recent years, a growing number of roles have been identified for mitochondria in innate immunity. One principal mechanism is that translocation of mitochondrial nucleic acid species from the mitochondrial matrix to the cytosol and endolysosomal lumen in response to an array of microbial and non-microbial environmental stressors has been found to serve as a second messenger event in the cell signaling of the innate immune response. Thus, mitochondrial DNA and RNA have been shown to access the cytosol through several regulated mechanisms involving remodeling of the mitochondrial inner and outer membranes and to access lysosomes via vesicular transport, thereby activating cytosolic (e.g., cyclic GMP-AMP synthase [cGAS]; retinoic acid-inducible gene-I [RIG-I]-like receptors) and endolysosomal (Toll-like Receptor [TLR]7, -9) nucleic acid receptors that induce type I interferons and pro-inflammatory cytokines. In this mini-review, we discuss these molecular mechanisms of mitochondrial nucleic acid mislocalization and their roles in host defense, autoimmunity, and auto-inflammatory disorders. The emergent paradigm is one in which host-derived DNA interestingly serves as a signal amplifier in the innate immune response and also as an alarm signal for disturbances in organellar homeostasis. The apparent vast excess of mitochondria and mitochondrial DNA nucleoids per cell may thus serve to sensitize the cell response to stressors while ensuring an underlying reserve of intact mitochondria to sustain cellular metabolism. An improved understanding of these molecular mechanisms will hopefully afford future opportunities for therapeutic intervention in human disease.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

Production and Release of Proinflammatory Mediators by the Cockroach Allergen Bla g 1 via a Shared Membrane-Destabilization Mechanism.

The cockroach allergen Bla g 1 encloses an exceptionally large hydrophobic cavity, which allows it to bind and deliver unsaturated fatty acid ligands. Bla g 1-mediated delivery of naturally occurring (nMix) ligands has been shown to destabilize lipid membranes, contributing to its digestive/antiviral functions within the source organism. However, the consequences of this activity on Bla g 1 allergenicity following human exposure remain unknown. In this work, we show that Bla g 1-mediated membrane disruption can induce a proinflammatory immune response in mammalian cells via two complementary pathways. At high concentrations, the cytotoxic activity of Bla g 1 induces the release of proinflammatory cytosolic contents including damage-associated molecular patterns (DAMPs) such as heat-shock Protein-70 (HSP70) and the cytokine interleukin-1 (IL-1ß). Sublytic concentrations of Bla g 1 enhanced the ability of phospholipase A2 (PLA2) to extract and hydrolyze phospholipid substrates from cellular membranes, stimulating the production of free polyunsaturated fatty acids (PUFAs) and various downstream inflammatory lipid mediators. Both of these effects are dependent on the presence of Bla g 1's natural fatty-acid (nMix) ligands with CC50 values corresponding to the concentrations required for membrane destabilization reported in previous studies. Taken together, these results suggest that mechanisms through which Bla g 1-mediated lipid delivery and membrane destabilization could directly contribute to cockroach allergic sensitization.

Meta-Analysis of COVID-19 BAL Single-Cell RNA Sequencing Reveals Alveolar Epithelial Transitions and Unique Alveolar Epithelial Cell Fates.

Single-cell RNA sequencing (scRNA-seq) of BAL cells has provided insights into coronavirus disease (COVID-19). However, reports have been limited by small patient cohorts. We performed a meta-analysis of BAL scRNA-seq data from healthy control subjects (n = 13) and patients with COVID-19 (n = 20), sourced from six independent studies (167,280 high-quality cells in total). Consistent with the source reports, increases in infiltrating leukocyte subtypes were noted, several with type I IFN signatures and unique gene expression signatures associated with transcellular chemokine signaling. Noting dramatic reductions of inferred NKX2-1 and NR4A1 activity in alveolar epithelial type II (AT-II) cells, we modeled pseudotemporal AT-II-to-AT-I progression. This revealed changes in inferred AT-II cell metabolic activity, increased transitional cells, and a previously undescribed AT-I state. This cell state was conspicuously marked by the induction of genes of the epidermal differentiation complex, including the cornified envelope protein SPRR3 (small proline-rich protein 3), upregulation of multiple KRT (keratin) genes, inferred mitochondrial dysfunction, and cell death signatures including apoptosis and ferroptosis. Immunohistochemistry of lungs from patients with COVID-19 confirmed upregulation and colocalization of KRT13 and SPRR3 in the distal airspaces. Forced overexpression of SPRR3 in human alveolar epithelial cells ex vivo did not activate caspase-3 or upregulate KRT13, suggesting that SPRR3 marks an AT-I cornification program in COVID-19 but is not sufficient for phenotypic changes.

Endogenous and Therapeutic 25-Hydroxycholesterols May Worsen Early SARS-CoV-2 Pathogenesis in Mice.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-Hydroxycholesterol (25HC), a product of the activity of cholesterol-25-hydroxylase (CH25H) on cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, 25HC can also amplify inflammation and be converted by CYP7B1 (cytochrome P450 family 7 subfamily B member 1) to 7α,25-dihydroxycholesterol, a lipid with chemoattractant activity, via the G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2)/GPR183 (G protein-coupled receptor 183). Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that although 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 (angiotensin-converting enzyme 2) mouse model in vivo. Treatment with 25HC also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma proinflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points but no change in weight loss. Consistent with these findings, although Ch25h and 25HC were upregulated in the lungs of SARS-CoV-2-infected wild-type mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the ß variant were similar to those in control animals. Taken together, endogenous 25HCs do not significantly regulate early SARS-CoV-2 replication or pathogenesis, and supplemental 25HC may have proinjury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Alveolar Biomarker Profiles in Subphenotypes of the Acute Respiratory Distress Syndrome.

OBJECTIVES: We sought to determine whether hyperinflammatory acute respiratory distress syndrome (ARDS) and hypoinflammatory ARDS, which have been associated with differences in plasma biomarkers and mortality risk, also display differences in bronchoalveolar lavage (BALF) biomarker profiles. We then described the relationship between hyperinflammatory ARDS and hypoinflammatory ARDS to novel subphenotypes derived using BALF biomarkers. DESIGN: Secondary analysis of a randomized control trial testing omega-3 fatty acids for the treatment of ARDS. SETTING: Five North American intensive care units. PATIENTS: Adults (n = 88) on invasive mechanical ventilation within 48 hours of ARDS onset. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We classified 57 patients as hypoinflammatory and 31 patients as hyperinflammatory using a previously validated logistic regression model. Of 14 BALF biomarkers analyzed, interleukin-6 and granulocyte colony stimulating factor were higher among patients with hyperinflammatory ARDS compared with hypoinflammatory ARDS, though the differences were not robust to multiple hypothesis testing. We then performed a de novo latent class analysis of the 14 BALF biomarkers to identify two classes well separated by alveolar profiles. Class 2 (n = 63) displayed significantly higher interleukin-6, von Willebrand factor, soluble programmed cell death receptor-1, % neutrophils, and other biomarkers of inflammation compared with class 1 (n = 25). These BALF-derived classes had minimal overlap with the plasma-derived hyperinflammatory and hypoinflammatory classes, and the majority of both plasma-derived classes were in BALF-derived class 2 and characterized by high BALF biomarkers. Additionally, the BALF-derived classes were associated with clinical severity of pulmonary disease, with class 2 exhibiting lower Pao2 to Fio2 and distinct ventilatory parameters, unlike the plasma-derived classes, which were only related to nonpulmonary organ dysfunction. CONCLUSIONS: Hyperinflammatory and hypoinflammatory ARDS subphenotypes did not display significant differences in alveolar biologic profiles. Identifying ARDS subgroups using BALF measurements is a unique approach that complements information obtained from plasma, with potential to inform enrichment strategies in trials of lung-targeted therapies.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Serum antioxidant vitamins and respiratory morbidity and mortality: a pooled analysis.

BACKGROUND: Oxidative stress plays a key role in the pathogenesis of respiratory diseases; however, studies on antioxidant vitamins and respiratory outcomes have been conflicting. We evaluated whether lower serum levels of vitamins A, C, D, and E are associated with respiratory morbidity and mortality in the U.S. adult population. METHODS: We conducted a pooled analysis of data from the 1988-1994 and 1999-2006 National Health and Nutrition Examination Survey (participants aged ≥ 20 years). We estimated covariate-adjusted odds ratios (aOR) per interquartile decrease in each serum vitamin level to quantify associations with respiratory morbidity, and covariate-adjusted hazard ratios (aHR) to quantify associations with respiratory mortality assessed prospectively through 2015. Vitamin supplementation and smoking were evaluated as potential effect modifiers. RESULTS: Lower serum vitamin C increased the odds of wheeze among all participants (overall aOR: 1.08, 95% CI: 1.01-1.16). Among smokers, lower serum α-tocopherol vitamin E increased the odds of wheeze (aOR: 1.11, 95% CI: 1.04-1.19) and chronic bronchitis/emphysema (aOR: 1.13, 95% CI: 1.03-1.24). Conversely, lower serum γ-tocopherol vitamin E was associated with lower odds of wheeze and chronic bronchitis/emphysema (overall aORs: 0.85, 95% CI: 0.79-0.92 and 0.85, 95% CI: 0.76-0.95, respectively). Lower serum vitamin C was associated with increased chronic lower respiratory disease (CLRD) mortality in all participants (overall aHR: 1.27, 95% CI: 1.07-1.51), whereas lower serum 25-hydroxyvitamin D (25-OHD) tended to increase mortality from CLRD and influenza/pneumonia among smokers (aHR range: 1.33-1.75). Mortality from influenza/ pneumonia increased with decreasing serum vitamin A levels in all participants (overall aHR: 1.21, 95% CI: 0.99-1.48). In pooled analysis, vitamin C deficiency and 25-OHD insufficiency were associated with mortality from influenza/pneumonia, increasing mortality risk up to twofold. CONCLUSIONS: Our analysis of nationally representative data on over 34,000 participants showed that lower serum levels of vitamins A, C, D, and α-tocopherol vitamin E are associated with increased respiratory morbidity and/or mortality in U.S. adults. The results underscore the importance of antioxidant vitamins in respiratory health.

Regulatory mechanisms of neutrophil migration from the circulation to the airspace.

The neutrophil, a short-lived effector leukocyte of the innate immune system best known for its proteases and other degradative cargo, has unique, reciprocal physiological interactions with the lung. During health, large numbers of 'marginated' neutrophils reside within the pulmonary vasculature, where they patrol the endothelial surface for pathogens and complete their life cycle. Upon respiratory infection, rapid and sustained recruitment of neutrophils through the endothelial barrier, across the extravascular pulmonary interstitium, and again through the respiratory epithelium into the airspace lumen, is required for pathogen killing. Overexuberant neutrophil trafficking to the lung, however, causes bystander tissue injury and underlies several acute and chronic lung diseases. Due in part to the unique architecture of the lung's capillary network, the neutrophil follows a microanatomic passage into the distal airspace unlike that observed in other end-organs that it infiltrates. Several of the regulatory mechanisms underlying the stepwise recruitment of circulating neutrophils to the infected lung have been defined over the past few decades; however, fundamental questions remain. In this article, we provide an updated review and perspective on emerging roles for the neutrophil in lung biology, on the molecular mechanisms that control the trafficking of neutrophils to the lung, and on past and ongoing efforts to design therapeutics to intervene upon pulmonary neutrophilia in lung disease.

Scavenger Receptor BI Attenuates IL-17A-Dependent Neutrophilic Inflammation in Asthma.

Asthma is a common respiratory disease currently affecting more than 300 million worldwide and is characterized by airway inflammation, hyperreactivity, and remodeling. It is a heterogeneous disease consisting of corticosteroid-sensitive T-helper cell type 2-driven eosinophilic and corticosteroid-resistant, T-helper cell type 17-driven neutrophilic phenotypes. One pathway recently described to regulate asthma pathogenesis is cholesterol trafficking. Scavenger receptors, in particular SR-BI (scavenger receptor class B type I), are known to direct cellular cholesterol uptake and efflux. We recently defined SR-BI functions in pulmonary host defense; however, the function of SR-BI in asthma pathogenesis is unknown. To elucidate the role of SR-BI in allergic asthma, SR-BI-sufficient (SR-BI+/+) and SR-BI-deficient (SR-BI-/-) mice were sensitized (Days 0 and 7) and then challenged (Days 14, 15, and 16) with a house dust mite (HDM) preparation administered through oropharyngeal aspiration. Airway inflammation and cytokine production were quantified on Day 17. When compared with SR-BI+/+ mice, the HDM-challenged SR-BI-/- mice had increased neutrophils and pulmonary IL-17A production in BAL fluid. This augmented IL-17A production in SR-BI-/- mice originated from a non-T-cell source that included neutrophils and alveolar macrophages. Given that SR-BI regulates adrenal steroid hormone production, we tested whether the changes in SR-BI-/- mice were glucocorticoid dependent. Indeed, SR-BI-/- mice were adrenally insufficient during the HDM challenge, and corticosterone replacement decreased pulmonary neutrophilia and IL-17A production in SR-BI-/- mice. Taken together, these data indicate that SR-BI dampens pulmonary neutrophilic inflammation and IL-17A production in allergic asthma at least in part by maintaining adrenal function.

Efficient CD4Cre-Mediated Conditional KRas Expression in Alveolar Macrophages and Alveolar Epithelial Cells Causes Fatal Hyperproliferative Pneumonitis.

The CD4Cre transgenic model has been widely used for T cell-specific gene manipulation. We report unexpected highly efficient Cre-mediated recombination in alveolar macrophages (AMFs), bronchial epithelial cells (BECs), and alveolar epithelial cells (AECs) in this strain of mice. Different from CD4 T cells, AMFs, AECs, and BECs do not express detectable Cre protein, suggesting that Cre protein is either very transiently expressed in these cells or only expressed in their precursors. Mice carrying a conditional constitutively active KRas (caKRas) allele and the CD4Cre transgene contain not only hyperactivated T cells but also develop severe AMF accumulation, AEC and BEC hyperplasia, and adenomas in the lung, leading to early lethality correlated with caKRas expression in these cells. We propose that caKRas-CD4Cre mice represent, to our knowledge, a novel model of proliferative pneumonitis involving macrophages and epithelial cells and that the CD4Cre model may offer unique usefulness for studying gene functions simultaneously in multilineages in the lung. Our observations, additionally, suggest that caution in data interpretation is warranted when using the CD4Cre transgenic model for T cell-specific gene manipulation, particularly when lung pathophysiological status is being examined.

Cross-linking Proteomics Indicates Effects of Simvastatin on the TLR2 Interactome and Reveals ACTR1A as a Novel Regulator of the TLR2 Signal Cascade.

Toll-like receptor 2 (TLR2) is a pattern recognition receptor that, upon ligation by microbial molecules, interacts with other proteins to initiate pro-inflammatory responses by the cell. Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors), drugs widely prescribed to reduce hypercholesterolemia, are reported to have both pro- and anti-inflammatory effects upon cells. Some of these responses are presumed to be driven by effects on signaling proteins at the plasma membrane, but the underlying mechanisms remain obscure. We reasoned that profiling the effect of statins on the repertoire of TLR2-interacting proteins might provide novel insights into the mechanisms by which statins impact inflammation. In order to study the TLR2 interactome, we designed a coimmunoprecipitation (IP)-based cross-linking proteomics study. A hemagglutinin (HA)-tagged-TLR2 transfected HEK293 cell line was used to precipitate the TLR2 interactome upon cell exposure to the TLR2 agonist Pam3CSK4 and simvastatin, singly and in combination. To stabilize protein interactors, we used two different chemical cross-linkers with different spacer chain lengths. Proteomic analysis revealed important combinatorial effects of simvastatin and Pam3CSK4 on the TLR2 interactome. After stringent data filtering, we identified alpha-centractin (ACTR1A), an actin-related protein and subunit of the dynactin complex, as a potential interactor of TLR2. The interaction was validated using biochemical methods. RNA interference studies revealed an important role for ACTR1A in induction of pro-inflammatory cytokines. Taken together, we report that statins remodel the TLR2 interactome, and we identify ACTR1A, a part of the dynactin complex, as a novel regulator of TLR2-mediated immune signaling pathways.

Leucine-rich repeats and calponin homology containing 4 (Lrch4) regulates the innate immune response.

Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease.

Validation of Questionnaire-based Case Definitions for Chronic Obstructive Pulmonary Disease.

BACKGROUND: Various questionnaire-based definitions of chronic obstructive pulmonary disease (COPD) have been applied using the US representative National Health and Nutrition Examination Survey (NHANES), but few have been validated against objective lung function data. We validated two prior definitions that incorporated self-reported physician diagnosis, respiratory symptoms, and/or smoking. We also validated a new definition that we developed empirically using gradient boosting, an ensemble machine learning method. METHODS: Data came from 7,996 individuals 40-79 years who participated in NHANES 2007-2012 and underwent spirometry. We considered participants "true" COPD cases if their ratio of postbronchodilator forced expiratory volume in 1 second to forced vital capacity was below 0.7 or the lower limit of normal. We stratified all analyses by smoking history. We developed a gradient boosting model for smokers only; predictors assessed (25 total) included sociodemographics, inhalant exposures, clinical variables, and respiratory symptoms. RESULTS: The spirometry-based COPD prevalence was 26% for smokers and 8% for never smokers. Among smokers, using questionnaire-based definitions resulted in a COPD prevalence ranging from 11% to 16%, sensitivity ranging from 18% to 35%, and specificity ranging from 88% to 92%. The new definition classified participants based on age, bronchodilator use, body mass index (BMI), smoking pack-years, and occupational organic dust exposure, and resulted in the highest sensitivity (35%) and specificity (92%) among smokers. Among never smokers, the COPD prevalence ranged from 4% to 5%, and we attained good specificity (96%) at the expense of sensitivity (9-10%). CONCLUSION: Our results can be used to parametrize misclassification assumptions for quantitative bias analysis when pulmonary function data are unavailable.

Association of urinary levels of bisphenols F and S used as bisphenol A substitutes with asthma and hay fever outcomes.

BACKGROUND: Bisphenols F (BPF) and S (BPS) are bisphenol A (BPA) analogs used as substitutes in consumer products. Despite previous reports of BPA's association with asthma, no studies have examined its structural analogs in relation to asthma and allergy outcomes. OBJECTIVE: To examine the association of urinary BPF, BPS, and BPA with asthma and hay fever in a US representative sample. METHODS: We analyzed data from 3,538 participants aged 12 years or older in the 2013-2016 National Health and Nutrition Examination Survey (NHANES). Children aged 6-11 years (N = 738), who did not have all covariate data available, were analyzed separately. Covariate-adjusted logistic regression was used to assess the association of the exposures with the outcomes. RESULTS: BPF, BPS, and BPA were detected in 57.1%, 88.4%, and 94.8% of the urine samples, respectively. Urinary BPF detection was positively associated with current asthma (odds ratio [OR]: 1.54, 95% confidence interval [CI]: 1.16-2.04) and hay fever (OR: 1.66, 95% CI: 1.12-2.46). Urinary BPS was associated with increased odds of current asthma in men (OR: 1.64, 95% CI: 1.13-2.40) and urinary BPA was associated with increased odds of asthma without hay fever in children aged 6-11 years (OR: 2.65, 95% CI: 1.05-6.68). CONCLUSION: Our nationally-representative findings document that BPF and BPS exposure is common in the US and that exposure to these BPA analogs is associated with asthma and/or hay fever. Our results suggest that BPF and BPS may not be safe alternatives to BPA; however, prospective studies should be conducted to confirm these results.

Macrophage polarization in innate immune responses contributing to pathogenesis of chronic kidney disease.

Chronic kidney disease (CKD) is characterized by inflammation, injury and fibrosis. Dysregulated innate immune responses mediated by macrophages play critical roles in progressive renal injury. The differentiation and polarization of macrophages into pro-inflammatory 'M1' and anti-inflammatory 'M2' states represent the two extreme maturation programs of macrophages during tissue injury. However, the effects of macrophage polarization on the pathogenesis of CKD are not fully understood. In this review, we discuss the innate immune mechanisms underlying macrophage polarization and the role of macrophage polarization in the initiation, progression, resolution and recurrence of CKD. Macrophage activation and polarization are initiated through recognition of conserved endogenous and exogenous molecular motifs by pattern recognition receptors, chiefly, Toll-like receptors (TLRs), which are located on the cell surface and in endosomes, and NLR inflammasomes, which are positioned in the cytosol. Recent data suggest that genetic variants of the innate immune molecule apolipoprotein L1 (APOL1) that are associated with increased CKD prevalence in people of African descent, mediate an atypical M1 macrophage polarization. Manipulation of macrophage polarization may offer novel strategies to address dysregulated immunometabolism and may provide a complementary approach along with current podocentric treatment for glomerular diseases.

Alveolar Macrophage ABCG1 Deficiency Promotes Pulmonary Granulomatous Inflammation.

Pulmonary granuloma formation is a complex and poorly understood response to inhaled pathogens and particulate matter. To explore the mechanisms of pulmonary granuloma formation and maintenance, our laboratory has developed a multiwall carbon nanotube (MWCNT)-induced murine model of chronic granulomatous inflammation. We have demonstrated that the MWCNT model closely mimics pulmonary sarcoidosis pathophysiology, including the deficiency of alveolar macrophage ATP-binding cassette (ABC) lipid transporters ABCA1 and ABCG1. We hypothesized that deficiency of alveolar macrophage ABCA1 and ABCG1 would promote pulmonary granuloma formation and inflammation. To test this hypothesis, the effects of MWCNT instillation were evaluated in ABCA1, ABCG1, and ABCA1/ABCG1 myeloid-specific knockout (KO) mice. Histological examination revealed significantly larger pulmonary granulomas in ABCG1-KO and ABCA1/ABCG1 double-KO animals when compared with wild-type animals. Evaluation of BAL cells indicated increased expression of CCL2 and osteopontin, genes shown to be involved in the formation and maintenance of pulmonary granulomas. Single deficiency of alveolar macrophage ABCA1 did not affect MWCNT-induced granuloma formation or proinflammatory gene expression. These observations indicate that the deficiency of alveolar macrophage ABCG1 promotes pulmonary granulomatous inflammation and that this is augmented by additional deletion of ABCA1.