Min-Jong Kang

Role of Chitin and Chitinase/Chitinase-Like Proteins in Inflammation, Tissue Remodeling, and Injury

The 18 glycosyl hydrolase family of chitinases is an ancient gene family that is widely expressed from prokaryotes to eukaryotes. In mammals, despite the absence of endogenous chitin, a number of chitinases and chitinase-like proteins (C/CLPs) have been identified. However, their roles have only recently begun to be elucidated. Acidic mammalian chitinase (AMCase) inhibits chitin-induced innate inflammation; augments chitin-free, allergen-induced Th2 inflammation; and mediates effector functions of IL-13. The CLPs BRP-39/YKL-40 (also termed chitinase 3-like 1) inhibit oxidant-induced lung injury, augments adaptive Th2 immunity, regulates apoptosis, stimulates alternative macrophage activation, and contributes to fibrosis and wound healing. In accord with these findings, levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders and often correlate with disease severity. Our understanding of the roles of C/CLPs in inflammation, tissue remodeling, and tissue injury in health and disease is reviewed below.

Cigarette smoke selectively enhances viral PAMP– and virus-induced pulmonary innate immune and remodeling responses in mice

Viral infections have more severe consequences in patients who have been exposed to cigarette smoke (CS) than in those not exposed to CS. For example, in chronic obstructive pulmonary disease (COPD), viruses cause more severe disease exacerbation, heightened inflammation, and accelerated loss of lung function compared with other causes of disease exacerbation. Symptomatology and mortality in influenza-infected smokers is also enhanced. To test the hypothesis that these outcomes are caused by CS-induced alterations in innate immunity, we defined the effects of CS on pathogen-associated molecular pattern-induced (PAMP-induced) pulmonary inflammation and remodeling in mice. CS was found to enhance parenchymal and airway inflammation and apoptosis induced by the viral PAMP poly(I:C). CS and poly(I:C) also induced accelerated emphysema and airway fibrosis. The effects of a combination of CS and poly(I:C) were associated with early induction of type I IFN and IL-18, later induction of IL-12/IL-23 p40 and IFN-gamma, and the activation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF2alpha). Further analysis using mice lacking specific proteins indicated a role for TLR3-dependent and -independent pathways as well as a pathway or pathways that are dependent on mitochondrial antiviral signaling protein (MAVS), IL-18Ralpha, IFN-gamma, and PKR. Importantly, CS enhanced the effects of influenza but not other agonists of innate immunity in a similar fashion. These studies demonstrate that CS selectively augments the airway and alveolar inflammatory and remodeling responses induced in the murine lung by viral PAMPs and viruses.

IL-18 Is Induced and IL-18 Receptor α Plays a Critical Role in the Pathogenesis of Cigarette Smoke-Induced Pulmonary Emphysema and Inflammation

Th1 inflammation and remodeling characterized by local tissue destruction coexist in pulmonary emphysema and other diseases. To test the hypothesis that IL-18 plays an important role in these responses, we characterized the regulation of IL-18 in lungs from cigarette smoke (CS) and room air-exposed mice and characterized the effects of CS in wild-type mice and mice with null mutations of IL-18Rα (IL-18Rα−/−). CS was a potent stimulator and activator of IL-18 and caspases 1 and 11. In addition, although CS caused inflammation and emphysema in wild-type mice, both of these responses were significantly decreased in IL-18Rα−/− animals. CS also induced epithelial apoptosis, activated effector caspases and stimulated proteases and chemokines via IL-18Rα-dependent pathways. Importantly, the levels of IL-18 and its targets, cathepsins S and B, were increased in pulmonary macrophages from smokers and patients with chronic obstructive lung disease. Elevated levels of circulating IL-18 were also seen in patients with chronic obstructive lung disease. These studies demonstrate that IL-18 and the IL-18 pathway are activated in CS-exposed mice and man. They also demonstrate, in a murine modeling system, that IL-18R signaling plays a critical role in the pathogenesis of CS-induced inflammation and emphysema.

Role of CCR5 in IFN-γ–induced and cigarette smoke–induced emphysema

Th1 inflammation and remodeling characterized by tissue destruction frequently coexist in human diseases. To further understand the mechanisms of these responses, we defined the role(s) of CCR5 in the pathogenesis of IFN-gamma-induced inflammation and remodeling in a murine emphysema model. IFN-gamma was a potent stimulator of the CCR5 ligands macrophage inflammatory protein-1alpha/CCL-3 (MIP-1alpha/CCL-3), MIP-1beta/CCL-4, and RANTES/CCL-5, among others. Antibody neutralization or null mutation of CCR5 decreased IFN-gamma-induced inflammation, DNA injury, apoptosis, and alveolar remodeling. These interventions decreased the expression of select chemokines, including CCR5 ligands and MMP-9, and increased levels of secretory leukocyte protease inhibitor. They also decreased the expression and/or activation of Fas, FasL, TNF, caspase-3, -8, and -9, Bid, and Bax. In accordance with these findings, cigarette smoke induced pulmonary inflammation, DNA injury, apoptosis, and emphysema via an IFN-gamma-dependent pathway(s), and a null mutation of CCR5 decreased these responses. These studies demonstrate that IFN-gamma is a potent stimulator of CC and CXC chemokines and highlight the importance of CCR5 in the pathogenesis of IFN-gamma-induced and cigarette smoke-induced inflammation, tissue remodeling, and emphysema. They also demonstrate that CCR5 is required for optimal IFN-gamma stimulation of its own ligands, other chemokines, MMPs, caspases, and cell death regulators and the inhibition of antiproteases.

Amphiregulin, an Epidermal Growth Factor Receptor Ligand, Plays an Essential Role in the Pathogenesis of Transforming Growth Factor-β-induced Pulmonary Fibrosis

Background: The interaction between TGF-β and EGFR signaling in the pathogenesis of pulmonary fibrosis has not been defined. Results: Amphiregulin (AR), a EGFR ligand, is induced by TGF-β stimulation and regulates TGF-β-induced fibroblast proliferation and pulmonary fibrosis. Conclusion: AR mediates TGF-β-stimulated pulmonary fibrosis through activation of EGFR signaling pathway. Significance: AR or AR-activated EGFR signaling is crucial in the pathogenesis of TGF-β-induced pulmonary fibrosis. Dysregulated amphiregulin (AR) expression and EGR receptor (EGFR) activation have been described in animal models of pulmonary fibrosis and in patients with idiopathic pulmonary fibrosis. However, the exact role of AR in the pathogenesis of pulmonary fibrosis has not been clearly defined. Here, we show that a potent profibrogenic cytokine TGF-β1 significantly induced the expression of AR in lung fibroblasts in vitro and in murine lungs in vivo. AR stimulated NIH3T3 fibroblast cell proliferation in a dose-dependent manner. Silencing of AR expression by siRNA or chemical inhibition of EGFR signaling, utilizing AG1478 and gefitinib, significantly reduced the ability of TGF-β1 to stimulate fibroblast proliferation and expression of α-smooth muscle actin, collagen, and other extracellular matrix-associated genes. TGF-β1-stimulated activation of Akt, ERK, and Smad signaling was also significantly inhibited by these interventions. Consistent with these in vitro findings, AR expression was impressively increased in the lungs of TGF-β1 transgenic mice, and either siRNA silencing of AR or chemical inhibition of EGFR signaling significantly reduced TGF-β1-stimulated collagen accumulation in the lung. These studies showed a novel regulatory role for AR in the pathogenesis of TGF-β1-induced pulmonary fibrosis. In addition, these studies suggest that AR, or AR-activated EGFR signaling, is a potential therapeutic target for idiopathic pulmonary fibrosis associated with TGF-β1 activation.

The chitinase-like proteins breast regression protein-39 and YKL-40 regulate hyperoxia-induced acute lung injury.

RATIONALE Prolonged exposure to 100% O(2) causes hyperoxic acute lung injury (HALI), characterized by alveolar epithelial cell injury and death. We previously demonstrated that the murine chitinase-like protein, breast regression protein (BRP)-39 and its human homolog, YKL-40, inhibit cellular apoptosis. However, the regulation and roles of these molecules in hyperoxia have not been addressed. OBJECTIVES We hypothesized that BRP-39 and YKL-40 (also called chitinase-3-like 1) play important roles in the pathogenesis of HALI. METHODS We characterized the regulation of BRP-39 during HALI and the responses induced by hyperoxia in wild-type mice, BRP-39-null (-/-) mice, and BRP-39(-/-) mice in which YKL-40 was overexpressed in respiratory epithelium. We also compared the levels of tracheal aspirate YKL-40 in premature newborns with respiratory failure. MEASUREMENTS AND MAIN RESULTS These studies demonstrate that hyperoxia inhibits BRP-39 in vivo in the murine lung and in vitro in epithelial cells. They also demonstrate that BRP-39(-/-) mice have exaggerated permeability, protein leak, oxidation, inflammatory, chemokine, and epithelial apoptosis responses, and experience premature death in 100% O(2). Lastly, they demonstrate that YKL-40 ameliorates HALI, prolongs survival in 100% O(2), and rescues the exaggerated injury response in BRP-39(-/-) animals. In accord with these findings, the levels of tracheal aspirate YKL-40 were lower in premature infants treated with hyperoxia for respiratory failure who subsequently experienced bronchopulmonary dysplasia or death compared with those that did not experience these complications. CONCLUSIONS These studies demonstrate that hyperoxia inhibits BRP-39/YKL-40, and that BRP-39 and YKL-40 are critical regulators of oxidant injury, inflammation, and epithelial apoptosis in the murine and human lung.

IL-18 induces emphysema and airway and vascular remodeling via IFN-γ, IL-17A, and IL-13.

RATIONALE Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation, alveolar destruction, and airway and vascular remodeling. However, the mechanisms that lead to these diverse alterations have not been defined. OBJECTIVES We hypothesized that IL-18 plays a central role in the pathogenesis of these lesions. METHODS We generated and characterized lung-specific, inducible IL-18 transgenic mice. MEASUREMENTS AND MAIN RESULTS Here we demonstrate that the expression of IL-18 in the mature murine lung induces inflammation that is associated with the accumulation of CD4(+), CD8(+), CD19(+), and NK1.1(+) cells; emphysema; mucus metaplasia; airway fibrosis; vascular remodeling; and right ventricle cardiac hypertrophy. We also demonstrate that IL-18 induces type 1, type 2, and type 17 cytokines with IFN-γ-inhibiting macrophage, lymphocyte, and eosinophil accumulation while stimulating alveolar destruction and genes associated with cell cytotoxicity and IL-13 and IL-17A inducing mucus metaplasia, airway fibrosis, and vascular remodeling. We also highlight interactions between these responses with IL-18 inducing IL-13 via an IL-17A-dependent mechanism and the type 1 and type17/type 2 responses counterregulating each another. CONCLUSIONS These studies define the spectrum of inflammatory, parenchymal, airway, and vascular alterations that are induced by pulmonary IL-18; highlight the similarities between these responses and the lesions in COPD; and define the selective roles that type 1, type 2, and type 17 responses play in the generation of IL-18-induced pathologies.

Role of Breast Regression Protein–39 in the Pathogenesis of Cigarette Smoke–Induced Inflammation and Emphysema

The exaggerated expression of chitinase-like protein YKL-40, the human homologue of breast regression protein-39 (BRP-39), was reported in a number of diseases, including chronic obstructive pulmonary disease (COPD). However, the in vivo roles of YKL-40 in normal physiology or in the pathogenesis of specific diseases such as COPD remain poorly understood. We hypothesized that BRP-39/YKL-40 plays an important role in the pathogenesis of cigarette smoke (CS)-induced emphysema. To test this hypothesis, 10-week-old wild-type and BRP-39 null mutant mice (BRP-39(-/-)) were exposed to room air (RA) and CS for up to 10 months. The expression of BRP-39 was significantly induced in macrophages, airway epithelial cells, and alveolar Type II cells in the lungs of CS-exposed mice compared with RA-exposed mice, at least in part via an IL-18 signaling-dependent pathway. The null mutation of BRP-39 significantly reduced CS-induced bronchoalveolar lavage and tissue inflammation. However, CS-induced epithelial cell apoptosis and alveolar destruction were further enhanced in the absence of BRP-39. Consistent with these findings in mice, the tissue expression of YKL-40 was significantly increased in the lungs of current smokers compared with the lungs of ex-smokers or nonsmokers. In addition, serum concentrations of YKL-40 were significantly higher in smokers with COPD than in nonsmokers or smokers without COPD. These studies demonstrate a novel regulatory role of BRP-39/YKL-40 in CS-induced inflammation and emphysematous destruction. These studies also underscore that maintaining physiologic concentrations of YKL-40 in the lung is therapeutically important in preventing excessive inflammatory responses or emphysematous alveolar destruction.

Crucial role of calbindin-D28k in the pathogenesis of Alzheimer's disease mouse model.

Calbindin-D28k (CB), one of the major calcium-binding and buffering proteins, has a critical role in preventing a neuronal death as well as maintaining calcium homeostasis. Although marked reductions of CB expression have been observed in the brains of mice and humans with Alzheimer disease (AD), it is unknown whether these changes contribute to AD-related dysfunction. To determine the pathogenic importance of CB depletions in AD models, we crossed 5 familial AD mutations (5XFAD; Tg) mice with CB knock-out (CBKO) mice and generated a novel line CBKO·5XFAD (CBKOTg) mice. We first identified the change of signaling pathways and differentially expressed proteins globally by removing CB in Tg mice using mass spectrometry and antibody microarray. Immunohistochemistry showed that CBKOTg mice had significant neuronal loss in the subiculum area without changing the magnitude (number) of amyloid β-peptide (Aβ) plaques deposition and elicited significant apoptotic features and mitochondrial dysfunction compared with Tg mice. Moreover, CBKOTg mice reduced levels of phosphorylated mitogen-activated protein kinase (extracellular signal-regulated kinase) 1/2 and cAMP response element-binding protein at Ser-133 and synaptic molecules such as N-methyl-D-aspartate receptor 1 (NMDA receptor 1), NMDA receptor 2A, PSD-95 and synaptophysin in the subiculum compared with Tg mice. Importantly, this is the first experimental evidence that removal of CB from amyloid precursor protein/presenilin transgenic mice aggravates AD pathogenesis, suggesting that CB has a critical role in AD pathogenesis.

MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.