Mansoor Syed

Hyperoxia and Interferon-γ–Induced Injury in Developing Lungs Occur via Cyclooxygenase-2 and the Endoplasmic Reticulum Stress–Dependent Pathway

We noted a marked increase in cyclooxygenase-2 (Cox2) and the activation of the endoplasmic reticulum (ER) stress pathway in newborn murine lung on exposure to hyperoxia and IFN-γ. We sought to evaluate Cox2-mediated ER stress pathway activation in hyperoxia-induced and IFN-γ-mediated injury in developing lungs. We applied in vivo genetic gain-of-function and genetic/chemical inhibition, as well as in vitro loss-of-function genetic strategies. Hyperoxia-induced and IFN-γ-mediated impaired alveolarization was rescued by Cox2 inhibition, using celecoxib. The use of small interfering RNA against the ER stress pathway mediator, the C/EBP homologous protein (CHOP; also known as growth arrest and DNA damage-inducible gene 153/GADD153), alleviated cell death in alveolar epithelial cells as well as in hyperoxia-induced and IFN-γ-mediated murine models of bronchopulmonary dysplasia (BPD). In addition, CHOP siRNA also restored alveolarization in the in vivo models. Furthermore, as evidence of clinical relevance, we show increased concentrations of Cox2 and ER stress pathway mediators in human lungs with BPD. Cox2, via CHOP, may significantly contribute to the final common pathway of hyperoxia-induced and IFN-γ-mediated injury in developing lungs and human BPD.

Small molecular modulation of macrophage migration inhibitory factor in the hyperoxia-induced mouse model of bronchopulmonary dysplasia.

BackgroundThe role and mechanism of action of MIF in bronchopulmonary dysplasia (BPD) are not known. We hypothesized that increased MIF signaling would ameliorate the pulmonary phenotype of BPD in the mouse lung.MethodsWe studied newborn wild type (WT), MIF knockout (MIFKO), and lung MIF transgenic (MIFTG) mice in room air and a BPD model, and examined the effects of administering a small molecule MIF agonist and antagonist. Lung morphometry was performed and mRNA and protein expression of vascular mediators were analyzed.ResultsThe pulmonary phenotype of MIFKO and MIFTG mice lungs in room air (RA) and BPD model were comparable to the WT-BPD mice at postnatal (PN) day 14. Vascular endothelial growth factor (VEGF)-A, -R1 and Angiopoietin (Ang)1 mRNA were decreased, and Ang2 increased in the WT-BPD, MIFKO-RA, MIFKO-BPD, MIFTG-RA and MIFTG-BPD mice lungs, compared to appropriate controls. The protein expression of Ang1 in the MIFKO-RA was similar to WT-RA, but decreased in MIFTG-RA, and decreased in all the BPD groups. Ang2 was increased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. Tie2 was increased in WT-BPD compared to WT-RA, but decreased in MIFKO- and MIFTG- RA and BPD groups. VEGFR1 was uniformly decreased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. VEGF-A had a similar expression across all RA and BPD groups. There was partial recovery of the pulmonary phenotype in the WT-BPD model treated with the MIF agonist, and in the MIFTG mice treated with the MIF antagonist.ConclusionsThese data point to the careful regulatory balance exerted by MIF in the developing lung and response to hyperoxia and support the potential therapeutic value of small molecule MIF modulation in BPD.

An Analysis of MIF Structural Features that Control Functional Activation of CD74.

For more than 15 years, the tautomerase active site of macrophage migration inhibitory factor (MIF) and its catalytic residue Pro1 have been being targeted for the development of therapeutics that block activation of its cell surface receptor, CD74. Neither the biological role of the MIF catalytic site nor the mechanistic details of CD74 activation are well understood. The inherently unstable structure of CD74 remains the biggest obstacle in structural studies with MIF for understanding the basis of CD74 activation. Using a novel approach, we elucidate the mechanistic details that control activation of CD74 by MIF surface residues and identify structural parameters of inhibitors that reduce CD74 biological activation. We also find that N-terminal mutants located deep in the catalytic site affect surface residues immediately outside the catalytic site, which are responsible for reduction of CD74 activation.

Increased hyperoxia-induced lung injury in nitric oxide synthase 2 null mice is mediated via angiopoietin 2.

Supplemental oxygen is frequently prescribed. However, prolonged exposure to high concentrations of oxygen causes hyperoxic acute lung injury (HALI), which manifests as acute respiratory distress syndrome in adults and leads to bronchopulmonary dysplasia in newborns (NBs). Nitric oxide (NO), NO synthases (NOSs), and angiopoietin (Ang) 2 have been implicated in the pathogenesis of HALI. However, the mechanisms of the contributions of NOS/NO and the relationship(s) between NOS/NO and Ang2 have not been addressed. In addition, the relevance of these moieties in adults and NBs has not been evaluated. To address these issues, we compared the responses in hyperoxia of wild-type (NOS [+/+]) and NOS null (-/-) young adult and NB mice. When compared with NOS2(+/+) adult controls, NOS2(-/-) animals manifest exaggerated alveolar-capillary protein leak and premature death. These responses were associated with enhanced levels of structural cell death, enhanced expression of proapoptotic regulatory proteins, and Ang2. Importantly, silencing RNA knockdown of Ang2 decreased the levels of cell death and the expression of proapoptotic mediators. These effects were at least partially NOS2 specific, and were development dependent, because survival was similar in adult NOS3(+/+) and NOS3(-/-) mice and NB NOS2(+/+) and NOS2(-/-) mice, respectively. These studies demonstrate that NOS2 plays an important protective role in HALI in adult animals. They also demonstrate that this response is mediated, at least in part, by the ability of NOS2 to inhibit hyperoxia-induced Ang2 production and thereby decrease Ang2-induced tissue injury.

Conditional overexpression of TGFβ1 promotes pulmonary inflammation, apoptosis and mortality via TGFβR2 in the developing mouse lung.

BackgroundEarlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.MethodsWe utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.ResultsOur data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.ConclusionsOur study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.

Targeting distinct tautomerase sites of D-DT and MIF with a single molecule for inhibition of neutrophil lung recruitment

We report a new inflammatory activity for extracellular D‐dopachrome tautomerase (D‐DT), the recruitment of neutrophils to the lung on D‐DT intratracheal installation of C57BL/6J mice with an EC50 of 5.6 μg. We also find that D‐DT and macrophage migration inhibitory factor (MIF) have additive effects in neutrophil recruitment. Although the tautomerase site of D‐DT and its homologue MIF are biophysically very different, 4‐iodo‐6‐phenylpyrimidine (4‐IPP) forms a co‐valent bond with Pro‐1 of both proteins, resulting in a 6‐phenylpyrimidine (6‐PP) adduct. Recruitment of neutrophils to the lung for the 6‐PP adducts of D‐DT and MIF are reduced by ~50% relative to the apo proteins, demonstrating that an unmodified Pro‐1 is important for this activity, but there is no cooperativity in inhibition of the proteins together. The differences in the binding mode of the 6‐PP adduct for D‐DT was determined by crystallographic studies at 1.13 Å resolution and compared to the structure of the MIF‐6‐PP complex. There are major differences in the location of the 6‐PP adduct to the D‐DT and MIF active sites that provide insight into the lack of cooperativity by 4‐IPP and into tuning the properties of the covalent inhibitors of D‐DT and MIF that are necessary for the development of therapeutic small molecules against neutrophil damage from lung infections such as Pseudomonas aeruginosa in cystic fibrosis and immunocompromised patients.—Rajasekaran, D., Zierow, S., Syed, M., Bucala, R., Bhandari, V., Lolis, E. J., Targeting distinct tautomerase sites of D‐DT and MIF with a single molecule for inhibition of neutrophil lung recruitment. FASEB J. 28, 4961–4971 (2014). www.fasebj.org

Hyperoxia exacerbates postnatal inflammation-induced lung injury in neonatal BRP-39 null mutant mice promoting the M1 macrophage phenotype.

Rationale. Hyperoxia exposure to developing lungs—critical in the pathogenesis of bronchopulmonary dysplasia—may augment lung inflammation by inhibiting anti-inflammatory mediators in alveolar macrophages. Objective. We sought to determine the O2-induced effects on the polarization of macrophages and the role of anti-inflammatory BRP-39 in macrophage phenotype and neonatal lung injury. Methods. We used RAW264.7, peritoneal, and bone marrow derived macrophages for polarization (M1/M2) studies. For in vivo studies, wild-type (WT) and BRP-39−/− mice received continuous exposure to 21% O2 (control mice) or 100% O2 from postnatal (PN) 1 to PN7 days, along with intranasal lipopolysaccharide (LPS) administered on alternate days (PN2, -4, and -6). Lung histology, bronchoalveolar lavage (BAL) cell counts, BAL protein, and cytokines measurements were performed. Measurements and Main Results. Hyperoxia differentially contributed to macrophage polarization by enhancing LPS induced M1 and inhibiting interleukin-4 induced M2 phenotype. BRP-39 absence led to further enhancement of the hyperoxia and LPS induced M1 phenotype. In addition, BRP-39−/− mice were significantly more sensitive to LPS plus hyperoxia induced lung injury and mortality compared to WT mice. Conclusions. These findings collectively indicate that BRP-39 is involved in repressing the M1 proinflammatory phenotype in hyperoxia, thereby deactivating inflammatory responses in macrophages and preventing neonatal lung injury.

MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic concentrations

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. In addition to its known receptor-mediated biological activities, MIF possesses a catalytic site of unknown function between subunits of a homotrimer. Each subunit contributes three β-strands to adjacent subunits to form a core seven-stranded β-sheet for each monomer. MIF monomers, dimers, or trimers have been reported, but the active form that binds and activates the MIF receptor (CD74) is still a matter of debate. A cysteine mutant (N110C) that covalently locks MIF into a trimer by forming a disulfide with Cys-80 of an adjacent subunit is used to study this issue. Partial catalytic activity and receptor binding to CD74 are retained by N110C (locked trimer), but there is no cellular signaling. Wild-type MIF-induced cellular signaling, in vivo lung neutrophil accumulation, and alveolar permeability are inhibited with a fivefold excess of N110C. NMR and size-exclusion chromatography with light scattering reveal that N110C can form a higher-order oligomer in equilibrium with a single locked trimer. The X-ray structure confirms a local conformational change that disrupts the subunit interface and results in global changes responsible for the oligomeric form. The structure also confirms these changes are consistent for the partial catalytic and receptor binding activities. The absence of any potential monomer and the retention of partial catalytic and receptor binding activities despite changes in conformation (and dynamics) in the mutant support an endogenous MIF trimer that binds and activates CD74 at nanomolar concentrations. This conclusion has implications for therapeutic development.

TREM-1-accentuated lung injury via miR-155 is inhibited by LP17 nanomedicine

Triggering receptors expressed on myeloid cell-1 (TREM-1) is a superimmunoglobulin receptor expressed on myeloid cells. Synergy between TREM-1 and Toll-like receptor amplifies the inflammatory response; however, the mechanisms by which TREM-1 accentuates inflammation are not fully understood. In this study, we investigated the role of TREM-1 in a model of LPS-induced lung injury and neutrophilic inflammation. We show that TREM-1 is induced in lungs of mice with LPS-induced acute neutrophilic inflammation. TREM-1 knockout mice showed an improved survival after lethal doses of LPS with an attenuated inflammatory response in the lungs. Deletion of TREM-1 gene resulted in significantly reduced neutrophils and proinflammatory cytokines and chemokines, particularly IL-1β, TNF-α, and IL-6. Physiologically deletion of TREM-1 conferred an immunometabolic advantage with low oxygen consumption rate (OCR) sparing the respiratory capacity of macrophages challenged with LPS. Furthermore, we show that TREM-1 deletion results in significant attenuation of expression of miR-155 in macrophages and lungs of mice treated with LPS. Experiments with antagomir-155 confirmed that TREM-1-mediated changes were indeed dependent on miR-155 and are mediated by downregulation of suppressor of cytokine signaling-1 (SOCS-1) a key miR-155 target. These data for the first time show that TREM-1 accentuates inflammatory response by inducing the expression of miR-155 in macrophages and suggest a novel mechanism by which TREM-1 signaling contributes to lung injury. Inhibition of TREM-1 using a nanomicellar approach resulted in ablation of neutrophilic inflammation suggesting that TREM-1 inhibition is a potential therapeutic target for neutrophilic lung inflammation and acute respiratory distress syndrome (ARDS).

Hyperoxia causes miR-34a-mediated injury via angiopoietin-1 in neonatal lungs

Hyperoxia-induced acute lung injury (HALI) is a key contributor to the pathogenesis of bronchopulmonary dysplasia (BPD) in neonates, for which no specific preventive or therapeutic agent is available. Here we show that lung micro-RNA (miR)-34a levels are significantly increased in lungs of neonatal mice exposed to hyperoxia. Deletion or inhibition of miR-34a improves the pulmonary phenotype and BPD-associated pulmonary arterial hypertension (PAH) in BPD mouse models, which, conversely, is worsened by miR-34a overexpression. Administration of angiopoietin-1, which is one of the downstream targets of miR34a, is able to ameliorate the BPD pulmonary and PAH phenotypes. Using three independent cohorts of human samples, we show that miR-34a expression is increased in type 2 alveolar epithelial cells in neonates with respiratory distress syndrome and BPD. Our data suggest that pharmacologic miR-34a inhibition may be a therapeutic option to prevent or ameliorate HALI/BPD in neonates.Hyperoxia contributes to lung injury in bronchopulmonary dysplasia. The authors show that hyperoxia increases miR-34a expression in human neonates and in mouse models, and that pathology is ameliorated by miR-34a inhibition or by administration of its target angiopoietin-1