Peiying Shan

Toll-like receptor 4 deficiency causes pulmonary emphysema

TLRs have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unknown. Given their direct interface with the external environment, TLRs in the lungs are prime candidates to respond to air constituents, namely particulates and oxygen. The mechanism whereby the lung maintains structural integrity in the face of constant ambient exposures is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, usually in the later decades of life and after years of insult, most commonly cigarette smoke. Here we show Tlr4(-/-) mice exhibited emphysema as they aged. Adoptive transfer experiments revealed that TLR4 expression in lung structural cells was required for maintaining normal lung architecture. TLR4 deficiency led to the upregulation of what we believe to be a novel NADPH oxidase (Nox), Nox3, in lungs and endothelial cells, resulting in increased oxidant generation and elastolytic activity. Treatment of Tlr4(-/- )mice or endothelial cells with chemical NADPH inhibitors or Nox3 siRNA reversed the observed phenotype. Our data identify a role for TLR4 in maintaining constitutive lung integrity by modulating oxidant generation and provide insights into the development of emphysema.

Cutting Edge: TLR4 Deficiency Confers Susceptibility to Lethal Oxidant Lung Injury

TLRs have been studied extensively in pathogen-mediated host responses. We use a murine model of lethal oxidant-mediated injury to demonstrate for the first time that mammalian TLR4 is required for survival and lung integrity. Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can lead to respiratory failure and death. TLR4-deficient mice exhibited increased mortality and lung injury during hyperoxia. The enhanced susceptibility of TLR4-deficient mice to hyperoxia was associated with an inability to up-regulate Bcl-2 and phospho-Akt. Restoration of Bcl-2 and phospho-Akt levels by the exogenous transfer of the antioxidant gene heme oxygenase-1 markedly attenuated hyperoxia-induced injury, apoptosis, and mortality in TLR4-deficient mice. Taken together, our results suggest a protective role of TLR4 in oxidant-mediated injury, providing novel mechanistic links among innate immunity, oxidant stress, and apoptosis.

ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo

IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13-induced inflammation and alveolar remodeling. There were associated decreases in IL-13-induced chemokines (MIP-1alpha/CCL-3, MIP-1beta/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of alpha1-antitrypsin. IL-13-induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13-induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13-induced diseases and disorders.

Endothelial STAT3 is essential for the protective effects of HO-1 in oxidant-induced lung injury

Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life‐sustaining measure in critically ill patients. Unfortunately, the oxidant stress generated by prolonged hyperoxia can lead to respiratory failure, multiorgan failure, and death. Although the endothelial cell is known to be a target for hyperoxia‐induced injury, its precise role is unclear. Heme oxygenase‐1 (HO‐1) and “signal transducer and activator of transcription 3” (STAT3) have been found to confer protection against endothelial cell injury. We sought to elucidate the specific roles of HO‐1 and STAT3 in hyperoxic lung and endothelial cell injury. Mice or murine lung endothelial cells (MLEC) administered HO‐1 siRNA exhibited marked injury and death compared with nonspecific siRNA. Overexpression of either HO‐1 or STAT3 confers protection. However, HO‐1 and its reaction product carbon monoxide (CO) lose their protective effects in the presence of STAT3 siRNA in MLEC or in endothelial‐specific, STAT3‐deficient mice. STAT3 overexpression is able to partially rescue HO‐1‐deficient MLEC from hyperoxia‐induced cell death. Our results demonstrate 1) the importance of the endothelium in lethal hyperoxic injury, 2) HO‐1 and CO require endothelial STAT3 for their protective effects, and 3) STAT3 confers endothelial cell protection via both HO‐1‐dependent and independent mechanisms.—Zhang, X., Shan, P., Jiang, G., Zhang, S. S‐M., Otterbein, L. E., Fu, X‐Y., Lee, P. J. Endothelial STAT3 is essential for the protective effects of HO‐1 in oxidant‐induced lung injury. FASEB J. 20, E1528 –E1538 (2006)

VEGF-induced heme oxygenase-1 confers cytoprotection from lethal hyperoxia in vivo

Prolonged exposure to hyperoxia results in hyperoxic acute lung injury (HALI). Vascular endothelial growth factor (VEGF) has been shown to have cytoprotective effects and prolong survival in an in vivo model of HALI. Heme oxygenase‐1 (HO‐1) has protective effects in hyperoxia; therefore, we hypothesized that induction of HO‐1 would be an important contributor to VEGF‐induced cytoprotection. Using inducible, lung‐specific VEGF overexpressing transgenic mice, we demonstrated that VEGF is a potent inducer of HO‐1 mRNA and protein in mouse lung. To evaluate the effect of inhibition of HO‐1 on injury, VEGF transgenic mice were treated with HO‐1 short interfering RNA (HO‐1 siRNA) and exposed to hyperoxia. Total lung lavage protein concentration, TUNEL staining, lipid peroxidation, and wet‐to‐dry ratio were all increased, consistent with increased injury. These findings were corroborated by survival studies in which inhibition of HO‐1 function using tin‐protoporphryin or silencing of HO‐1 with lentiviral HO‐1 short hairpin RNA (ShRNA) significantly decreased survival in hyperoxia. We conclude from these data that VEGF‐induced HO‐1 is an important contributor to cytoprotection in this in vivo model of acute lung injury and that alterations in VEGF function in the lung are likely to be important determinants of the outcome of acute lung injury.—Siner, J. M., Jiang, G., Cohen, Z. I., Shan, P., Zhang, X., Lee, C. G., Elias, J. A., Lee, P. J. VEGF‐induced heme oxygenase‐1 confers cytoprotection from lethal hyper‐oxia in vivo. FASEB J. 21, 1422–1432 (2007)

Inducible Activation of TLR4 Confers Resistance to Hyperoxia-Induced Pulmonary Apoptosis

TLRs are essential mediators of host defense against infection via recognition of unique microbial structures. Recent observations indicate that TLR4, the principal receptor for bacterial LPS, may also be activated by noninfectious stimuli including host-derived molecules and environmental oxidant stress. In mice, susceptibility to ozone-induced lung permeability has been linked to the wild-type allele of TLR4, whereas deficiency of TLR4 predisposes to lethal lung injury in hyperoxia. To precisely characterize the role of lung epithelial TLR4 expression in the host response to oxidant stress, we have created an inducible transgenic mouse model that targets the human TLR4 signaling domain to the airways. Exposure of induced transgenic mice to hyperoxia revealed a significant reduction in pulmonary apoptosis compared with controls. This phenotype was associated with sustained up-regulation of antiapoptotic molecules such as heme oxygenase-1 and Bcl-2, yet only transient activation of the transcription factor NF-κB. Specific in vivo knockdown of pulmonary heme oxygenase-1 or Bcl-2 expression by intranasal administration of short interfering RNA blocked the effect of TLR4 signaling on hyperoxia-induced lung apoptosis. These results define a novel role for lung epithelial TLR4 as a modulator of cellular apoptosis in response to oxidant stress.

Aging Enhances the Basal Production of IL-6 and CCL2 in Vascular Smooth Muscle Cells

Objective— Increased circulating cytokine levels are a prominent feature of aging that may contribute to atherosclerosis. However, the role vascular cells play in chronic inflammation induced by aging is not clear. Here, we examined the role of aging on inflammatory responses of vascular cells. Methods and Results— In an ex vivo culture system, we examined the inflammatory response of aortas from young (2–4 months) and aged (16–18 months) mice under nonstimulatory conditions. We found that basal levels of interleukin-6 were increased in aged aortas. Aged aortic vascular smooth muscle cells (VSMC) exhibited a higher basal secretion of interleukin-6 than young VSMC. Gene and protein expression analysis revealed that aged VSMC exhibited upregulation of chemokines (eg, CCL2), adhesion molecules (eg, intracellular adhesion molecule 1), and innate immune receptors (eg, Toll-like receptor [TLR] 4), which all contribute to atherosclerosis. Using VSMC from aged TL4−/− and Myd88−/− mice, we demonstrate that signaling via TLR4 and its signal adaptor, MyD88, are in part responsible for the age-elevated basal interleukin-6 response. Conclusion— Aging induces a proinflammatory phenotype in VSMC due in part to increased signaling of TLR4 and MyD88. Our results provide a potential explanation as to why aging leads to chronic inflammation and enhanced atherosclerosis.

Endothelial MKK3 Is a Critical Mediator of Lethal Murine Endotoxemia and Acute Lung Injury

Sepsis is a leading cause of intensive care unit admissions, with high mortality and morbidity. Although outcomes have improved with better supportive care, specific therapies are limited. Endothelial activation and oxidant injury are key events in the pathogenesis of sepsis-induced lung injury. The signaling pathways leading to these events remain poorly defined. We sought to determine the role of MAPK kinase 3 (MKK3), a kinase of the p38 group, in the pathogenesis of sepsis. We used a murine i.p. LPS model of systemic inflammation to mimic sepsis. Lung injury parameters were assessed in lung tissue and bronchoalveolar lavage specimens. Primary lung endothelial cells were cultured and assessed for mediators of inflammation and injury, such as ICAM-1, AP-1, NF-κB, and mitochondrial reactive oxygen species. Our studies demonstrate that MKK3 deficiency confers virtually complete protection against organ injury after i.p. LPS. Specifically, MKK3−/− mice were protected against acute lung injury, as assessed by reduced inflammation, mitochondrial reactive oxygen species generation, endothelial injury, and ICAM-1 expression after LPS administration. Our results show that endothelial MKK3 is required for inflammatory cell recruitment to the lungs, mitochondrial oxidant-mediated AP-1, NF-κB activation, and ICAM-1 expression during LPS challenge. Collectively, these studies identify a novel role for MKK3 in lethal LPS responses and provide new therapeutic targets against sepsis and acute lung injury.

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.

A Protective Hsp70–TLR4 Pathway in Lethal Oxidant Lung Injury

Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can exacerbate respiratory failure. Our previous study showed that TLR4 confers protection against hyperoxia-induced lung injury and mortality. Hsp70 has potent cytoprotective properties and has been described as a TLR4 ligand in cell lines. We sought to elucidate the relationship between TLR4 and Hsp70 in hyperoxia-induced lung injury in vitro and in vivo and to define the signaling mechanisms involved. Wild-type, TLR4−/−, and Trif−/− (a TLR4 adapter protein) murine lung endothelial cells (MLECs) were exposed to hyperoxia. We found markedly elevated levels of intracellular and secreted Hsp70 from wild-type mice lungs and MLECs after hyperoxia. We confirmed that Hsp70 and TLR4 coimmunoprecipitate in lung tissue and MLECs. Hsp70-mediated NF-κB activation appears to depend upon TLR4. In the absence of TLR4, Hsp70 loses its protective effects in endothelial cells. Furthermore, these protective properties of Hsp70 are TLR4 adapter Trif dependent and MyD88 independent. Hsp70-deficient mice have increased mortality during hyperoxia, and lung-targeted adenoviral delivery of Hsp70 effectively rescues both Hsp70-deficient and wild-type mice. To our knowledge, our studies are the first to define an Hsp70–TLR4–Trif cytoprotective axis in the lung and endothelial cells. This pathway is a potential therapeutic target against a range of oxidant-induced lung injuries.