Maor Sauler

Lung endothelial HO-1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury

The lung endothelium is a major target for inflammatory and oxidative stress. Heme oxygenase‐1 (HO‐1) induction is a crucial defense mechanism during oxidant challenges, such as hyperoxia. The role of lung endothelial HO‐1during hyperoxia in vivo is not well defined. We engineered lentiviral vectors with microRNA (miRNA) sequences controlled by vascular endothelium cadherin (VE‐cad) to study the specific role of lung endothelial HO‐1. Wild‐type (WT) murine lung endothelial cells (MLECs) or WT mice were treated with lentivirus and exposed to hyperoxia (95% oxygen). We detected HO‐1 knockdown (~55%) specifically in the lung endothelium. MLECs and lungs showed approximately a 2‐fold increase in apoptosis and ROS generation after HO‐1 silencing. We also demonstrate for the first time that silencing endothelial HO‐1 has the same effect on lung injury and survival as silencing HO‐1 in multiple lung cell types and that HO‐1 regulates caspase 3 activation and autophagy in endothelium during hyperoxia. These studies demonstrate the utility of endothelial‐targeted gene silencing in vivo using lentiviral miRNA constructs to assess gene function and that endothelial HO‐1 is an important determinant of survival during hyperoxia.—Zhang, Y., Jiang, G., Sauler, M., Lee, P. J., Lung endothelial HO‐1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury. FASEB J. 27, 4041–4058 (2013). www.fasebj.org

Endothelial PINK1 Mediates the Protective Effects of NLRP3 Deficiency during Lethal Oxidant Injury

High levels of inspired oxygen, hyperoxia, are frequently used in patients with acute respiratory failure. Hyperoxia can exacerbate acute respiratory failure, which has high mortality and no specific therapies. We identified novel roles for PTEN-induced putative kinase 1 (PINK1), a mitochondrial protein, and the cytosolic innate immune protein NLRP3 in the lung and endothelium. We generated double knockouts (PINK1−/−/NLRP3−/−), as well as cell-targeted PINK1 silencing and lung-targeted overexpression constructs, to specifically show that PINK1 mediates cytoprotection in wild-type and NLRP3−/− mice. The ability to resist hyperoxia is proportional to PINK1 expression. PINK1−/− mice were the most susceptible; wild-type mice, which induced PINK1 after hyperoxia, had intermediate susceptibility; and NLRP3−/− mice, which had high basal and hyperoxia-induced PINK1, were the least susceptible. Genetic deletion of PINK1 or PINK1 silencing in the lung endothelium increased susceptibility to hyperoxia via alterations in autophagy/mitophagy, proteasome activation, apoptosis, and oxidant generation.

Suppression of NLRX1 in chronic obstructive pulmonary disease

Cigarette smoke (CS) and viruses promote the inflammation and remodeling associated with chronic obstructive pulmonary disease (COPD). The MAVS/RIG-I-like helicase (MAVS/RLH) pathway and inflammasome-dependent innate immune pathways are important mediators of these responses. At baseline, the MAVS/RLH pathway is suppressed, and this inhibition must be reversed to engender tissue effects; however, the mechanisms that mediate activation and repression of the pathway have not been defined. In addition, the regulation and contribution of MAVS/RLH signaling in CS-induced inflammation and remodeling responses and in the development of human COPD remain unaddressed. Here, we demonstrate that expression of NLRX1, which inhibits the MAVS/RLH pathway and regulates other innate immune responses, was markedly decreased in 3 independent cohorts of COPD patients. NLRX1 suppression correlated directly with disease severity and inversely with pulmonary function, quality of life, and prognosis. In murine models, CS inhibited NLRX1, and CS-induced inflammation, alveolar destruction, protease induction, structural cell apoptosis, and inflammasome activation were augmented in NLRX1-deficient animals. Conversely, MAVS deficiency abrogated this CS-induced inflammation and remodeling. Restoration of NLRX1 in CS-exposed animals ameliorated alveolar destruction. These data support a model in which CS-dependent NLRX1 inhibition facilitates MAVS/RHL activation and subsequent inflammation, remodeling, protease, cell death, and inflammasome responses.

Macrophage migration inhibitory factor deficiency in chronic obstructive pulmonary disease

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains poorly understood. Cellular senescence and apoptosis contribute to the development of COPD; however, crucial regulators of these underlying mechanisms remain unknown. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that antagonizes both apoptosis and premature senescence and may be important in the pathogenesis of COPD. This study examines the role of MIF in the pathogenesis of COPD. Mice deficient in MIF (Mif(-/-)) or the MIF receptor CD74 (Cd74(-/-)) and wild-type (WT) controls were aged for 6 mo. Both Mif(-/-) and Cd74(-/-) mice developed spontaneous emphysema by 6 mo of age compared with WT mice as measured by lung volume and chord length. This was associated with activation of the senescent pathway markers p53/21 and p16. Following exposure to cigarette smoke, Mif(-/-) mice were more susceptible to the development of COPD and apoptosis compared with WT mice. MIF plasma concentrations were measured in a cohort of 224 human participants. Within a subgroup of older current and former smokers (n = 72), MIF concentrations were significantly lower in those with COPD [8.8, 95%CI (6.7-11.0)] compared with those who did not exhibit COPD [12.7 ng/ml, 95%CI (10.6-14.8)]. Our results suggest that both MIF and the MIF receptor CD74 are required for maintenance of normal alveolar structure in mice and that decreases in MIF are associated with COPD in human subjects.

Transcription factor ICBP90 regulates the MIF promoter and immune susceptibility locus

The immunoregulatory cytokine macrophage migration inhibitory factor (MIF) is encoded in a functionally polymorphic locus that is linked to the susceptibility of autoimmune and infectious diseases. The MIF promoter contains a 4-nucleotide microsatellite polymorphism (-794 CATT) that repeats 5 to 8 times in the locus, with greater numbers of repeats associated with higher mRNA levels. Because there is no information about the transcriptional regulation of these common alleles, we used oligonucleotide affinity chromatography and liquid chromatography-mass spectrometry to identify nuclear proteins that interact with the -794 CATT5-8 site. An analysis of monocyte nuclear lysates revealed that the transcription factor ICBP90 (also known as UHRF1) is the major protein interacting with the MIF microsatellite. We found that ICBP90 is essential for MIF transcription from monocytes/macrophages, B and T lymphocytes, and synovial fibroblasts, and TLR-induced MIF transcription is regulated in an ICBP90- and -794 CATT5-8 length-dependent manner. Whole-genome transcription analysis of ICBP90 shRNA-treated rheumatoid synoviocytes uncovered a subset of proinflammatory and immune response genes that overlapped with those regulated by MIF shRNA. In addition, the expression levels of ICBP90 and MIF were correlated in joint synovia from patients with rheumatoid arthritis. These findings identify ICBP90 as a key regulator of MIF transcription and provide functional insight into the regulation of the polymorphic MIF locus.

Reporting of Multivariable Methods in the Medical Literature

Background Multivariable models are frequently used in the medical literature, but many clinicians have limited training in these analytic methods. Our objective was to assess the prevalence of multivariable methods in medical literature, quantify reporting of methodological criteria applicable to most methods, and determine if assumptions specific to logistic regression or proportional hazards analysis were evaluated. Methods We examined all original articles in Annals of Internal Medicine, British Medical Journal, Journal of the American Medical Association, Lancet, and New England Journal of Medicine, from January through June 2006. Articles reporting multivariable methods underwent a comprehensive review; reporting of methodological criteria was based on each articles primary analysis. Results Among 452 articles, 272 (60%) used multivariable analysis; logistic regression (89 [33%] of 272) and proportional hazards (76 [28%] of 272) were most prominent. Reporting of methodological criteria, when applicable, ranged from 5% (12/265) for assessing influential observations to 84% (222/265) for description of variable coding. Discussion of interpreting odds ratios occurred in 13% (12/89) of articles reporting logistic regression as the primary method and discussion of the proportional hazards assumption occurred in 21% (16/76) of articles using Cox proportional hazards as the primary method. Conclusions More complete reporting of multivariable analysis in the medical literature can improve understanding, interpretation, and perhaps application of these methods.

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.

Standard Nonspecific Therapies in the Management of Pulmonary Arterial Hypertension

Recent advances in pulmonary arterial hypertension (PAH) research have created a new era of PAH-specific therapies. Although these therapeutics have revolutionized PAH therapy, their innovation was predated by supportive but nonspecific medical therapies adapted from their use in more common cardiopulmonary diseases. These therapies include oxygen therapy, diuretics, digoxin, anticoagulation, and high-dose calcium channel blockers. Expert opinion continues to support the use of adjunct therapies based on current pathologic understandings of PAH combined with some evidence extrapolated from small studies. This article discusses why these therapies continue to play an important role in the treatment of patients with PAH.

Endothelial CD74 mediates macrophage migration inhibitory factor protection in hyperoxic lung injury

Exposure to hyperoxia results in acute lung injury. A pathogenic consequence of hyperoxia is endothelial injury. Macrophage migration inhibitory factor (MIF) has a cytoprotective effect on lung endothelial cells; however, the mechanism is uncertain. We postulate that the MIF receptor CD74 mediates this protective effect. Using adult wild‐type (WT), MIF‐deficient (Mif‐/‐), CD74‐deficient (Cd74‐/‐) mice and MIF receptor inhibitor treated mice, we report that MIF deficiency or inhibition of MIF receptor binding results in increased sensitivity to hyperoxia. Mif‐/‐ and Cd74‐/‐ mice demonstrated decreased median survival following hyperoxia compared to WT mice. Mif‐/‐ mice demonstrated an increase in bronchoalveolar protein (48%) and lactate dehydrogenase (LDH) (68%) following 72 hours of hyperoxia. Similarly, treatment with MIF receptor antagonist resulted in a 59% and 91% increase in bronchoalveolar lavage protein and LDH, respectively. Inhibition of CD74 in primary murine lung endothelial cells (MLECs) abrogated the protective effect of MIF, including decreased hyperoxia‐mediated AKT phosphorylation and a 20% reduction in the anti‐apoptotic effect of exogenous MIF. Treatment with MIF decreased hyperoxia‐mediated H2AX phosphorylation in a CD74‐dependent manner. These data suggest that therapeutic manipulation of the MIF‐CD74 axis in lung endothelial cells may be a novel approach to protect against acute oxidative stress.—Sauler, M., Zhang, Y., Min, J.‐N., Leng, L., Shan, P., Roberts, S., Jorgensen, W. L., Bucala, R., Lee, P. J. Endothelial CD74 mediates macrophage migration inhibitory factor protection in hyperoxic lung injury. FASEB J. 29, 1940‐1949 (2015). www.fasebj.org

MIF allele-dependent regulation of the MIF coreceptor CD44 and role in rheumatoid arthritis

Significance High-expression alleles of the cytokine macrophage migration inhibitory factor (MIF) are associated with severe joint destruction in autoimmune arthritis, but the mechanism for this effect is unknown. High-genotypic MIF-expressing joint fibroblasts produce high levels of MIF under inflammatory stimulation to up-regulate the surface expression of the MIF signaling coreceptor CD44 and promote its alternative splicing into invasive, tumor-associated isoforms, which contribute to the invasive and tissue-destructive character of the rheumatoid joint synovium. These findings support a precision medicine approach to the treatment of rheumatoid arthritis by pharmacologically targeting the MIF pathway in high-genotypic MIF-expressing patients. Fibroblast-like synoviocytes mediate joint destruction in rheumatoid arthritis and exhibit sustained proinflammatory and invasive properties. CD44 is a polymorphic transmembrane protein with defined roles in matrix interaction and tumor invasion that is also a signaling coreceptor for macrophage migration inhibitory factor (MIF), which engages cell surface CD74. High-expression MIF alleles (rs5844572) are associated with rheumatoid joint erosion, but whether MIF signaling through the CD74/CD44 receptor complex promotes upstream autoimmune responses or contributes directly to synovial joint destruction is unknown. We report here the functional regulation of CD44 by an autocrine pathway in synovial fibroblasts that is driven by high-expression MIF alleles to up-regulate an inflammatory and invasive phenotype. MIF increases CD44 expression, promotes its recruitment into a functional signal transduction complex, and stimulates alternative exon splicing, leading to expression of the CD44v3–v6 isoforms associated with oncogenic invasion. CD44 recruitment into the MIF receptor complex, downstream MAPK and RhoA signaling, and invasive phenotype require MIF and CD74 and are reduced by MIF pathway antagonists. These data support a functional role for high-MIF expression alleles and the two-component CD74/CD44 MIF receptor in rheumatoid arthritis and suggest that pharmacologic inhibition of this pathway may offer a specific means to interfere with progressive joint destruction.