Acoustofluidic Virus Isolation via Bessel Beam Excitation Separation Technology.

The isolation of viruses from complex biological samples is essential for creating sensitive bioassays that assess the efficacy and safety of viral therapeutics and vaccines, which have played a critical role during the COVID-19 pandemic. However, existing methods of viral isolation are time-consuming and labor-intensive due to the multiple processing steps required, resulting in low yields. Here, we introduce the rapid, efficient, and high-resolution acoustofluidic isolation of viruses from complex biological samples via Bessel beam excitation separation technology (BEST). BEST isolates viruses by utilizing the nondiffractive and self-healing properties of 2D, in-plane acoustic Bessel beams to continuously separate cell-free viruses from biofluids, with high throughput and high viral RNA yield. By tuning the acoustic parameters, the cutoff size of isolated viruses can be easily adjusted to perform dynamic, size-selective virus isolation while simultaneously trapping larger particles and separating smaller particles and contaminants from the sample, achieving high-precision isolation of the target virus. BEST was used to isolate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from human saliva samples and Moloney Murine Leukemia Virus from cell culture media, demonstrating its potential use in both practical diagnostic applications and fundamental virology research. With high separation resolution, high yield, and high purity, BEST is a powerful tool for rapidly and efficiently isolating viruses. It has the potential to play an important role in the development of next-generation viral diagnostics, therapeutics, and vaccines.

Characterization of pulmonary vascular remodeling and MicroRNA-126-targets in COPD-pulmonary hypertension.

BACKGROUND: Despite causing increased morbidity and mortality, pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) patients (COPD-PH) lacks treatment, due to incomplete understanding of its pathogenesis. Hypertrophy of pulmonary arterial walls and pruning of the microvasculature with loss of capillary beds are known features of pulmonary vascular remodeling in COPD. The remodeling features of pulmonary medium- and smaller vessels in COPD-PH lungs are less well described and may be linked to maladaptation of endothelial cells to chronic cigarette smoking (CS). MicroRNA-126 (miR126), a master regulator of endothelial cell fate, has divergent functions that are vessel-size specific, supporting the survival of large vessel endothelial cells and inhibiting the proliferation of microvascular endothelial cells. Since CS decreases miR126 in microvascular lung endothelial cells, we set out to characterize the remodeling by pulmonary vascular size in COPD-PH and its relationship with miR126 in COPD and COPD-PH lungs. METHODS: Deidentified lung tissue was obtained from individuals with COPD with and without PH and from non-diseased non-smokers and smokers. Pulmonary artery remodeling was assessed by ⍺-smooth muscle actin (SMA) abundance via immunohistochemistry and analyzed by pulmonary artery size. miR126 and miR126-target abundance were quantified by qPCR. The expression levels of ceramide, ADAM9, and endothelial cell marker CD31 were assessed by immunofluorescence. RESULTS: Pulmonary arteries from COPD and COPD-PH lungs had significantly increased SMA abundance compared to non-COPD lungs, especially in small pulmonary arteries and the lung microvasculature. This was accompanied by significantly fewer endothelial cell markers and increased pro-apoptotic ceramide abundance. miR126 expression was significantly decreased in lungs of COPD individuals. Of the targets tested (SPRED1, VEGF, LAT1, ADAM9), lung miR126 most significantly inversely correlated with ADAM9 expression. Compared to controls, ADAM9 was significantly increased in COPD and COPD-PH lungs, predominantly in small pulmonary arteries and lung microvasculature. CONCLUSION: Both COPD and COPD-PH lungs exhibited significant remodeling of the pulmonary vascular bed of small and microvascular size, suggesting these changes may occur before or independent of the clinical development of PH. Decreased miR126 expression with reciprocal increase in ADAM9 may regulate endothelial cell survival and vascular remodeling in small pulmonary arteries and lung microvasculature in COPD and COPD-PH.

Multi-apical polarity of alveolar stem cells and their dynamics during lung development and regeneration.

Epithelial cells of diverse tissues are characterized by the presence of a single apical domain. In the lung, electron microscopy studies have suggested that alveolar type-2 epithelial cells (AT2s) en face multiple alveolar sacs. However, apical and basolateral organization of the AT2s and their establishment during development and remodeling after injury repair remain unknown. Thick tissue imaging and electron microscopy revealed that a single AT2 can have multiple apical domains that enface multiple alveoli. AT2s gradually establish multi-apical domains post-natally, and they are maintained throughout life. Lineage tracing, live imaging, and selective cell ablation revealed that AT2s dynamically reorganize multi-apical domains during injury repair. Single-cell transcriptome signatures of residual AT2s revealed changes in cytoskeleton and cell migration. Significantly, cigarette smoke and oncogene activation lead to dysregulation of multi-apical domains. We propose that the multi-apical domains of AT2s enable them to be poised to support the regeneration of a large array of alveolar sacs.

Immunofibrotic drivers of impaired lung function in postacute sequelae of SARS-CoV-2 infection.

BACKGROUNDIndividuals recovering from COVID-19 frequently experience persistent respiratory ailments, which are key elements of postacute sequelae of SARS-CoV-2 infection (PASC); however, little is known about the underlying biological factors that may direct lung recovery and the extent to which these are affected by COVID-19 severity.METHODSWe performed a prospective cohort study of individuals with persistent symptoms after acute COVID-19, collecting clinical data, pulmonary function tests, and plasma samples used for multiplex profiling of inflammatory, metabolic, angiogenic, and fibrotic factors.RESULTSSixty-one participants were enrolled across 2 academic medical centers at a median of 9 weeks (interquartile range, 6-10 weeks) after COVID-19 illness: n = 13 participants (21%) had mild COVID-19 and were not hospitalized, n = 30 participants (49%) were hospitalized but were considered noncritical, and n = 18 participants (30%) were hospitalized and in the intensive care unit (ICU). Fifty-three participants (85%) had lingering symptoms, most commonly dyspnea (69%) and cough (58%). Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and diffusing capacity for carbon monoxide (DLCO) declined as COVID-19 severity increased (P < 0.05) but these values did not correlate with respiratory symptoms. Partial least-squares discriminant analysis of plasma biomarker profiles clustered participants by past COVID-19 severity. Lipocalin-2 (LCN2), MMP-7, and HGF identified by our analysis were significantly higher in the ICU group (P < 0.05), inversely correlated with FVC and DLCO (P < 0.05), and were confirmed in a separate validation cohort (n = 53).CONCLUSIONSubjective respiratory symptoms are common after acute COVID-19 illness but do not correlate with COVID-19 severity or pulmonary function. Host response profiles reflecting neutrophil activation (LCN2), fibrosis signaling (MMP-7), and alveolar repair (HGF) track with lung impairment and may be novel therapeutic or prognostic targets.FundingNational Heart, Lung, and Blood Institute (K08HL130557 and R01HL142818), American Heart Association (Transformational Project Award), the DeLuca Foundation Award, a donation from Jack Levin to the Benign Hematology Program at Yale University, and Duke University.

PINK1 mediates the protective effects of thyroid hormone T3 in hyperoxia-induced lung injury.

Hyperoxia can lead to respiratory failure and death. Our previous work demonstrates that oxidant and mitochondrial injury play a critical role in hyperoxia-induced acute lung injury (HALI). Recently, thyroid hormone has been demonstrated to promote mitochondrial survival in other models of lung injury, but its role in hyperoxia is unknown. Adult wild-type (WT) mice were pretreated with either nebulized triiodothyronine (T3, 40 µg/kg) for 1 or 3 days, or with propylthiouracil (PTU, 100 µg/kg), for 3 days. Following pretreatment, WT mice underwent 72 h of hyperoxia exposure. WT and PINK1-/- mice were pretreated with either nebulized T3 (40 µg/kg) for 3 days or no pretreatment before 72 h continuous hyperoxia exposure. Bronchoalveolar lavage (BAL), histological changes in cellular composition, and type I cytokine induction were assessed. Lung lysates for mitochondrial cellular bioenergetics markers were analyzed by Western blot. Hyperoxia caused a significant increase in BAL total cell counts and lung cellular infiltrates. Administration of PTU enhanced HALI, whereas T3 attenuated HALI, inflammation, and oxidants in WT mice. In addition, T3 pretreatment increased mitochondrial biogenesis/fusion/mitophagy and decreased ER stress and apoptosis. PINK1-/- mice were more susceptible to hyperoxia than WT mice. Notably, pretreatment with T3 did not attenuate HALI in PINK1-/- mice. In addition, T3 pretreatment increased mitochondrial anti-ROS potential, improved mitochondrial bioenergetics and mitophagy, and attenuated mitochondria-regulated apoptosis, all in a PINK1-dependent manner. Our results highlight a novel protective role for PINK1 in mediating the cytoprotective effects of thyroid hormone in HALI. Therefore, thyroid hormone may represent a potential therapy for ALI.

Pazopanib ameliorates acute lung injuries via inhibition of MAP3K2 and MAP3K3.

Acute lung injury (ALI) causes high mortality and lacks any pharmacological intervention. Here, we found that pazopanib ameliorated ALI manifestations and reduced mortality in mouse ALI models and reduced edema in human lung transplantation recipients. Pazopanib inhibits mitogen-activated protein kinase kinase kinase 2 (MAP3K2)- and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208 to increase reactive oxygen species (ROS) formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic mutation of p47phox Ser208 to alanine attenuated ALI manifestations and abrogates anti-ALI effects of pazopanib. This myeloid MAP3K2/MAP3K3-p47phox pathway acted via paracrine H2O2 to enhance pulmonary vasculature integrity and promote lung epithelial cell survival and proliferation, leading to increased pulmonary barrier function and resistance to ALI. Thus, pazopanib has the potential to be effective for treating ALI.

MicroRNA miR-24-3p reduces DNA damage responses, apoptosis, and susceptibility to chronic obstructive pulmonary disease.

The pathogenesis of chronic obstructive pulmonary disease (COPD) involves aberrant responses to cellular stress caused by chronic cigarette smoke (CS) exposure. However, not all smokers develop COPD and the critical mechanisms that regulate cellular stress responses to increase COPD susceptibility are not understood. Because microRNAs are well-known regulators of cellular stress responses, we evaluated microRNA expression arrays performed on distal parenchymal lung tissue samples from 172 subjects with and without COPD. We identified miR-24-3p as the microRNA that best correlated with radiographic emphysema and validated this finding in multiple cohorts. In a CS exposure mouse model, inhibition of miR-24-3p increased susceptibility to apoptosis, including alveolar type II epithelial cell apoptosis, and emphysema severity. In lung epithelial cells, miR-24-3p suppressed apoptosis through the BH3-only protein BIM and suppressed homology-directed DNA repair and the DNA repair protein BRCA1. Finally, we found BIM and BRCA1 were increased in COPD lung tissue, and BIM and BRCA1 expression inversely correlated with miR-24-3p. We concluded that miR-24-3p, a regulator of the cellular response to DNA damage, is decreased in COPD, and decreased miR-24-3p increases susceptibility to emphysema through increased BIM and apoptosis.

Endothelial toll-like receptor 4 maintains lung integrity via epigenetic suppression of p16INK4a.

We previously reported that the canonical innate immune receptor toll-like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic-specific, lung- and cell-targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4-/- mice. Lung Ec-silencing of TLR4 in wild-type mice induced emphysema, highlighting the specific and distinct role of Ec-expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16INK4a , a senescence-associated gene. Lung Ec-p16INK4a -silencing prevented TLR4-/- induced emphysema, revealing a new functional role for p16INK4a in lungs. TLR4 suppressed endogenous p16INK4a expression via HDAC2-mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence-related gene expression.

Cell Death in the Lung: The Apoptosis-Necroptosis Axis.

Regulated cell death is a major mechanism to eliminate damaged, infected, or superfluous cells. Previously, apoptosis was thought to be the only regulated cell death mechanism; however, new modalities of caspase-independent regulated cell death have been identified, including necroptosis, pyroptosis, and autophagic cell death. As an understanding of the cellular mechanisms that mediate regulated cell death continues to grow, there is increasing evidence that these pathways are implicated in the pathogenesis of many pulmonary disorders. This review summarizes our understanding of regulated cell death as it pertains to the pathogenesis of chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, and pulmonary arterial hypertension.

Effects of bone marrow-derived mesenchymal stromal cells on gene expression in human alveolar type II cells exposed to TNF-α, IL-1ß, and IFN-γ.

The acute respiratory distress syndrome (ARDS) is common in critically ill patients and has a high mortality rate. Mesenchymal stromal cells (MSCs) have demonstrated therapeutic potential in animal models of ARDS, and their benefits occur in part through interactions with alveolar type II (ATII) cells. However, the effects that MSCs have on human ATII cells have not been well studied. Using previously published microarray data, we performed genome-wide differential gene expression analyses of human ATII cells that were (1) unstimulated, (2) exposed to proinflammatory cytokines (CytoMix), or (3) exposed to proinflammatory cytokines plus MSCs. Findings were validated by qPCR. Alveolar type II cells differentially expressed hundreds of genes when exposed either to proinflammatory cytokines or to proinflammatory cytokines plus MSCs. Stimulation with proinflammatory cytokines increased expression of inflammatory genes and downregulated genes related to surfactant function and alveolar fluid clearance. Some of these changes, including expression of some cytokines and genes related to surfactant, were reversed by exposure to MSCs. In addition, MSCs induced upregulation of other potentially beneficial genes, such as those related to extracellular matrix remodeling. We confirmed several of these gene expression changes by qPCR. Thus, ATII cells downregulate genes associated with surfactant and alveolar fluid clearance when exposed to inflammatory cytokines, and mesenchymal stromal cells partially reverse many of these gene expression changes.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

Mitochondrial dysfunction and pulmonary hypertension: cause, effect, or both.

Pulmonary hypertension describes a heterogeneous disease defined by increased pulmonary artery pressures, and progressive increase in pulmonary vascular resistance due to pathologic remodeling of the pulmonary vasculature involving pulmonary endothelial cells, pericytes, and smooth muscle cells. This process occurs under various conditions, and although these populations vary, the clinical manifestations are the same: progressive dyspnea, increases in right ventricular (RV) afterload and dysfunction, RV-pulmonary artery uncoupling, and right-sided heart failure with systemic circulatory collapse. The overall estimated 5-yr survival rate is 72% in highly functioning patients, and as low as 28% for those presenting with advanced symptoms. Metabolic theories have been suggested as underlying the pathogenesis of pulmonary hypertension with growing evidence of the role of mitochondrial dysfunction involving the major proteins of the electron transport chain, redox-related enzymes, regulators of the proton gradient and calcium homeostasis, regulators of apoptosis, and mitophagy. There remain more studies needed to characterize mitochondrial dysfunction leading to impaired vascular relaxation, increase proliferation, and failure of regulatory mechanisms. The effects on endothelial cells and resulting interactions with their microenvironment remain uncharted territory for future discovery. Additionally, on the basis of observations that the "plexigenic lesions" of pulmonary hypertension resemble the unregulated proliferation of tumor cells, similarities between cancer pathobiology and pulmonary hypertension have been drawn, suggesting interactions between mitochondria and angiogenesis. Recently, mitochondria targeting has become feasible, which may yield new therapeutic strategies. We present a state-of-the-art review of the role of mitochondria in both the pathobiology of pulmonary hypertension and potential therapeutic targets in pulmonary vascular processes.

Proteomics data on MAP Kinase Kinase 3 knock out bone marrow derived macrophages exposed to cigarette smoke extract.

This data article reports changes in the phosphoproteome and total proteome of cigarette smoke extract (CSE) exposed WT and MAP Kinase Kinase 3 knock out (MKK3-/-) bone marrow derived macrophages (BMDM). The dataset generated is helpful for understanding the mechanism of CSE induced inflammation and the role of MAP kinase signaling pathway. The cellular proteins were labeled with isobaric tags for relative and absolute quantitation (iTRAQ®) reagents and analyzed by LC-MS/MS. The standard workflow module for iTRAQ® quantification within the Proteome Discoverer was utilized for the data analysis. Ingenuity Pathway Analysis (IPA) software and Reactome was used to identify enriched canonical pathways and molecular networks (Mannam et al., 2016) [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED) with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs.

SH2 Domain-Containing Phosphatase-2 Is a Novel Antifibrotic Regulator in Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease with dismal prognosis and no cure. The potential role of the ubiquitously expressed SH2 domain-containing tyrosine phosphatase-2 (SHP2) as a therapeutic target has not been studied in IPF. OBJECTIVES: To determine the expression, mechanistic role, and potential therapeutic usefulness of SHP2 in pulmonary fibrosis. METHODS: The effects of SHP2 overexpression and inhibition on fibroblast response to profibrotic stimuli were analyzed in vitro in primary human and mouse lung fibroblasts. In vivo therapeutic effects were assessed in the bleomycin model of lung fibrosis by SHP2-lentiviral administration and transgenic mice carrying a constitutively active SHP2 mutation. MEASUREMENTS AND MAIN RESULTS: SHP2 was down-regulated in lungs and lung fibroblasts obtained from patients with IPF. Immunolocalization studies revealed that SHP2 was absent within fibroblastic foci. Loss of SHP2 expression or activity was sufficient to induce fibroblast-to-myofibroblast differentiation in primary human lung fibroblasts. Overexpression of constitutively active SHP2 reduced the responsiveness of fibroblasts to profibrotic stimuli, including significant reductions in cell survival and myofibroblast differentiation. SHP2 effects were mediated through deactivation of fibrosis-relevant tyrosine kinase and serine/threonine kinase signaling pathways. Mice carrying the Noonan syndrome-associated gain-of-function SHP2 mutation (SHP2D61G/+) were resistant to bleomycin-induced pulmonary fibrosis. Restoration of SHP2 levels in vivo through lentiviral delivery blunted bleomycin-induced pulmonary fibrosis. CONCLUSIONS: Our data suggest that SHP2 is an important regulator of fibroblast differentiation, and its loss as observed in IPF facilitates profibrotic phenotypic changes. Augmentation of SHP2 activity or expression should be investigated as a novel therapeutic strategy for IPF.

MKK3 influences mitophagy and is involved in cigarette smoke-induced inflammation.

Cigarette smoking is the primary risk factor for COPD which is characterized by excessive inflammation and airflow obstruction of the lung. While inflammation is causally related to initiation and progression of COPD, the mitochondrial mechanisms that underlie the associated inflammatory responses are poorly understood. In this context, we have studied the role played by Mitogen activated protein (MAP) kinase kinase 3 (MKK3), a dual-specificity protein kinase, in cigarette smoke induced-inflammation and mitochondrial dysfunction. Serum pro-inflammatory cytokines were significantly elevated in WT but not in MKK3-/- mice exposed to Cigarette smoke (CS) for 2 months. To study the cellular mechanisms of inflammation, bone marrow derived macrophages (BMDMs), wild type (WT) and MKK3-/-, were exposed to cigarette smoke extract (CSE) and inflammatory cytokine production and mitochondrial function assessed. The levels of IL-1ß, IL-6, and TNFα were increased along with higher reactive oxygen species (ROS) and P-NFκB after CSE treatment in WT but not in MKK3-/- BMDMs. CSE treatment adversely affected basal mitochondrial respiration, ATP production, maximum respiratory capacity, and spare respiratory capacity in WT BMDMs only. Mitophagy, clearance of dysfunctional mitochondria, was up regulated in CS exposed WT mice lung tissue and CSE exposed WT BMDMs, respectively. The proteomic analysis of BMDMs by iTRAQ (isobaric tags for relative and absolute quantitation) showed up regulation of mitochondrial dysfunction associated proteins in WT and higher OXPHOS (Oxidative phosphorylation) and IL-10 signaling proteins in MKK3-/- BMDMs after CSE exposure, confirming the critical role of mitochondrial homeostasis. Interestingly, we found increased levels of p-MKK3 by immunohistochemistry in COPD patient lung tissues that could be responsible for insufficient mitophagy and disease progression. This study identifies MKK3 as a negative regulator of mitochondrial function and inflammatory responses to CS and suggests that MKK3 could be a therapeutic target.

Widespread activation of immunity and pro-inflammatory programs in peripheral blood leukocytes of HIV-infected patients with impaired lung gas exchange.

HIV infection is associated with impaired lung gas transfer as indicated by a low diffusing capacity (DLCO), but the mechanisms are not well understood. We hypothesized that HIV-associated gas exchange impairment is indicative of system-wide perturbations that could be reflected by alterations in peripheral blood leukocyte (PBL) gene expression. Forty HIV-infected (HIV(+)) and uninfected (HIV(-)) men with preserved versus low DLCO were enrolled. All subjects were current smokers and those with acute illness, lung diseases other than COPD or asthma were excluded. Total RNA was extracted from PBLs and hybridized to whole-genome microarrays. Gene set enrichment analysis (GSEA) was performed between HIV(+) versus HIV(-) subjects with preserved DLCO and those with low DLCO to identify differentially activated pathways. Using pathway-based analyses, we found that in subjects with preserved DLCO, HIV infection is associated with activation of processes involved in immunity, cell cycle, and apoptosis. Applying a similar analysis to subjects with low DLCO, we identified a much broader repertoire of pro-inflammatory and immune-related pathways in HIV(+) patients relative to HIV(-) subjects, with up-regulation of multiple interleukin pathways, interferon signaling, and toll-like receptor signaling. We confirmed elevated circulating levels of IL-6 in HIV(+) patients with low DLCO relative to the other groups. Our findings reveal that PBLs of subjects with HIV infection and low DLCO are distinguished by widespread enrichment of immuno-inflammatory programs. Activation of these pathways may alter the biology of circulating leukocytes and play a role in the pathogenesis of HIV-associated gas exchange impairment.

An endothelial TLR4-VEGFR2 pathway mediates lung protection against oxidant-induced injury.

TLR4 deficiency causes hypersusceptibility to oxidant-induced injury. We investigated the role of TLR4 in lung protection, using used bone marrow chimeras; cell-specific transgenic modeling; and lentiviral delivery in vivo to knock down or express TLR4 in various lung compartments; and lung-specific VEGF transgenic mice to investigate the effect of TLR4 on VEGF-mediated protection. C57/BL6 mice were exposed to 100% oxygen in an enclosed chamber and assessed for survival and lung injury. Primary endothelial cells were stimulated with recombinant VEGF and exposed to hyperoxia or hydrogen peroxide. Endothelium-specific expression of human TLR4 (as opposed to its expression in epithelium or immune cells) increased the survival of TLR4-deficent mice in hyperoxia by 24 h and decreased LDH release and lung cell apoptosis after 72 h of exposure by 30%. TLR4 expression was necessary and sufficient for the protective effect of VEGF in the lungs and in primary endothelial cells in culture. TLR4 knockdown inhibited VEGF signaling through VEGF receptor 2 (VEGFR2), Akt, and ERK pathways in lungs and primary endothelial cells and decreased the availability of VEGFR2 at the cell surface. These findings demonstrate a novel mechanism through which TLR4, an innate pattern receptor, interacts with an endothelial survival pathway.

MKK3 deletion improves mitochondrial quality.

Sepsis, a severe response to infection, leads to excessive inflammation and is the major cause of mortality in intensive care units. Mitochondria have been shown to influence the outcome of septic injury. We have previously shown that MAP kinase kinase 3 (MKK3)(-/-) mice are resistant to septic injury and MKK3(-/-) macrophages have improved mitochondrial function. In this study we examined processes that lead to improved mitochondrial quality in MKK3(-/-) mouse embryonic fibroblasts (MEFs) and specifically the role of mitophagy in mitochondrial health. MKK3(-/-) MEFs had lower inflammatory cytokine release and oxidant production after lipopolysaccharide (LPS) stimulation, confirming our earlier observations. MKK3(-/-) MEFs had better mitochondrial function as measured by mitochondrial membrane potential (MMP) and ATP, even after LPS treatment. We observed higher mitophagy in MKK3(-/-) MEFs compared to wild type (WT). Transmission electron microscopy studies showed longer and larger mitochondria in MKK3(-/-) MEFs, indicative of healthier mitochondria. We performed a SILAC (stable isotope labeling by/with amino acids in cell culture) study to assess differences in mitochondrial proteome between WT and MKK3(-/-) MEFs and observed increased expression of tricarboxylic acid (TCA) cycle enzymes and respiratory complex subunits. Further, inhibition of mitophagy by Mdivi1 led to loss in MMP and increased cytokine secretion after LPS treatment in MKK3(-/-) MEFs. In conclusion, this study demonstrates that MKK3 influences mitochondrial quality by affecting the expression of mitochondrial proteins, including TCA cycle enzymes, and mitophagy, which consequently regulates the inflammatory response. Based on our results, MKK3 could be a potential therapeutic target for inflammatory diseases like sepsis.

Role of macrophage migration inhibitory factor in age-related lung disease.

The prevalence of many common respiratory disorders, including pneumonia, chronic obstructive lung disease, pulmonary fibrosis, and lung cancer, increases with age. Little is known of the host factors that may predispose individuals to such diseases. Macrophage migration inhibitory factor (MIF) is a potent upstream regulator of the immune system. MIF is encoded by variant alleles that occur commonly in the population. In addition to its role as a proinflammatory cytokine, a growing body of literature demonstrates that MIF influences diverse molecular processes important for the maintenance of cellular homeostasis and may influence the incidence or clinical manifestations of a variety of chronic lung diseases. This review highlights the biological properties of MIF and its implication in age-related lung disease.

MKK3 mediates inflammatory response through modulation of mitochondrial function.

Mitochondria are increasingly recognized as drivers of inflammatory responses. MAP kinase kinase 3 (MKK3), a dual-specificity protein kinase, is activated in inflammation and in turn activates p38 MAP kinase signaling. Here we show that MKK3 influences mitochondrial function and acts as a critical mediator of inflammation. MKK3-deficient (MKK3(-/-)) mice and bone marrow-derived macrophages (BMDMs) secreted smaller amounts of cytokines than wild type (WT) after lipopolysaccharide (LPS) exposure. There was improved mitochondrial function, as measured by basal oxygen consumption rate, mitochondrial membrane potential, and ATP production, in MKK3(-/-) BMDMs. After LPS exposure, MKK3(-/-) BMDMs did not show a significant increase in cellular reactive oxygen species production or in mitochondrial superoxide compared to WT. Activation of two important inflammatory mediators, i.e., the nuclear translocation of NF-κB and caspase-1 activity (a key component of the inflammasome), was lower in MKK3(-/-) BMDMs. p38 and JNK activation was lower in MKK3(-/-) BMDMs compared to WT after exposure to LPS. Knockdown of MKK3 by siRNA in wild-type BMDMs improved mitochondrial membrane potential, reduced LPS-induced caspase-1 activation, and attenuated cytokine secretion. Our studies establish MKK3 as a regulator of mitochondrial function and inflammatory responses to LPS and suggest that MKK3 may be a therapeutic target in inflammatory disorders such as sepsis.