Yusuke Kurita

Autophagy induction by SIRT6 through attenuation of insulin-like growth factor signaling is involved in the regulation of human bronchial epithelial cell senescence.

Cigarette smoke (CS)–induced cellular senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease, and SIRT6, a histone deacetylase, antagonizes this senescence, presumably through the attenuation of insulin-like growth factor (IGF)-Akt signaling. Autophagy controls cellular senescence by eliminating damaged cellular components and is negatively regulated by IGF-Akt signaling through the mammalian target of rapamycin (mTOR). SIRT1, a representative sirtuin family, has been demonstrated to activate autophagy, but a role for SIRT6 in autophagy activation has not been shown. Therefore, we sought to investigate the regulatory role for SIRT6 in autophagy activation during CS-induced cellular senescence. SIRT6 expression levels were modulated by cDNA and small interfering RNA transfection in human bronchial epithelial cells (HBECs). Senescence-associated β-galactosidase staining and Western blotting of p21 were performed to evaluate senescence. We demonstrated that SIRT6 expression levels were decreased in lung homogenates from chronic obstructive pulmonary disease patients, and SIRT6 expression levels correlated significantly with the percentage of forced expiratory volume in 1 s/forced vital capacity. CS extract (CSE) suppressed SIRT6 expression in HBECs. CSE-induced HBEC senescence was inhibited by SIRT6 overexpression, whereas SIRT6 knockdown and mutant SIRT6 (H133Y) without histone deacetylase activity enhanced HBEC senescence. SIRT6 overexpression induced autophagy via attenuation of IGF-Akt-mTOR signaling. Conversely, SIRT6 knockdown and overexpression of a mutant SIRT6 (H133Y) inhibited autophagy. Autophagy inhibition by knockdown of ATG5 and LC3B attenuated the antisenescent effect of SIRT6 overexpression. These results suggest that SIRT6 is involved in CSE-induced HBEC senescence via autophagy regulation, which can be attributed to attenuation of IGF-Akt-mTOR signaling.

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis.

Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by PINK1 and PARK2 knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.

Metformin attenuates lung fibrosis development via NOX4 suppression

BackgroundAccumulation of profibrotic myofibroblasts in fibroblastic foci (FF) is a crucial process for development of fibrosis during idiopathic pulmonary fibrosis (IPF) pathogenesis, and transforming growth factor (TGF)-β plays a key regulatory role in myofibroblast differentiation. Reactive oxygen species (ROS) has been proposed to be involved in the mechanism for TGF-β-induced myofibroblast differentiation. Metformin is a biguanide antidiabetic medication and its pharmacological action is mediated through the activation of AMP-activated protein kinase (AMPK), which regulates not only energy homeostasis but also stress responses, including ROS. Therefore, we sought to investigate the inhibitory role of metformin in lung fibrosis development via modulating TGF-β signaling.MethodsTGF-β-induced myofibroblast differentiation in lung fibroblasts (LF) was used for in vitro models. The anti-fibrotic role of metfromin was examined in a bleomycin (BLM)-induced lung fibrosis model.ResultsWe found that TGF-β-induced myofibroblast differentiation was clearly inhibited by metformin treatment in LF. Metformin-mediated activation of AMPK was responsible for inhibiting TGF-β-induced NOX4 expression. NOX4 knockdown and N-acetylcysteine (NAC) treatment illustrated that NOX4-derived ROS generation was critical for TGF-β-induced SMAD phosphorylation and myofibroblast differentiation. BLM treatment induced development of lung fibrosis with concomitantly enhanced NOX4 expression and SMAD phosphorylation, which was efficiently inhibited by metformin. Increased NOX4 expression levels were also observed in FF of IPF lungs and LF isolated from IPF patients.ConclusionsThese findings suggest that metformin can be a promising anti-fibrotic modality of treatment for IPF affected by TGF-β.

Involvement of PARK2-Mediated Mitophagy in Idiopathic Pulmonary Fibrosis Pathogenesis

Fibroblastic foci, known to be the leading edge of fibrosis development in idiopathic pulmonary fibrosis (IPF), are composed of fibrogenic myofibroblasts. Autophagy has been implicated in the regulation of myofibroblast differentiation. Insufficient mitophagy, the mitochondria-selective autophagy, results in increased reactive oxygen species, which may modulate cell signaling pathways for myofibroblast differentiation. Therefore, we sought to investigate the regulatory role of mitophagy in myofibroblast differentiation as a part of IPF pathogenesis. Lung fibroblasts were used in in vitro experiments. Immunohistochemical evaluation in IPF lung tissues was performed. PARK2 was examined as a target molecule for mitophagy regulation, and a PARK2 knockout mouse was employed in a bleomycin-induced lung fibrosis model. We demonstrated that PARK2 knockdown-mediated mitophagy inhibition was involved in the mechanism for activation of the platelet-derived growth factor receptor (PDGFR)/PI3K/AKT signaling pathway accompanied by enhanced myofibroblast differentiation and proliferation, which were clearly inhibited by treatment with both antioxidants and AG1296, a PDGFR inhibitor. Mitophagy inhibition–mediated activation of PDGFR signaling was responsible for further autophagy suppression, suggesting the existence of a self-amplifying loop of mitophagy inhibition and PDGFR activation. IPF lung demonstrated reduced PARK2 with concomitantly increased PDGFR phosphorylation. Furthermore, bleomycin-induced lung fibrosis was enhanced in PARK2 knockout mice and subsequently inhibited by AG1296. These findings suggest that insufficient mitophagy-mediated PDGFR/PI3K/AKT activation, which is mainly attributed to reduced PARK2 expression, is a potent underlying mechanism for myofibroblast differentiation and proliferation in fibroblastic foci formation during IPF pathogenesis.

Pirfenidone inhibits myofibroblast differentiation and lung fibrosis development during insufficient mitophagy.

BackgroundPirfenidone (PFD) is an anti-fibrotic agent used to treat idiopathic pulmonary fibrosis (IPF), but its precise mechanism of action remains elusive. Accumulation of profibrotic myofibroblasts is a crucial process for fibrotic remodeling in IPF. Recent findings show participation of autophagy/mitophagy, part of the lysosomal degradation machinery, in IPF pathogenesis. Mitophagy has been implicated in myofibroblast differentiation through regulating mitochondrial reactive oxygen species (ROS)-mediated platelet-derived growth factor receptor (PDGFR) activation. In this study, the effect of PFD on autophagy/mitophagy activation in lung fibroblasts (LF) was evaluated, specifically the anti-fibrotic property of PFD for modulation of myofibroblast differentiation during insufficient mitophagy.MethodsTransforming growth factor-β (TGF-β)-induced or ATG5, ATG7, and PARK2 knockdown-mediated myofibroblast differentiation in LF were used for in vitro models. The anti-fibrotic role of PFD was examined in a bleomycin (BLM)-induced lung fibrosis model using PARK2 knockout (KO) mice.ResultsWe found that PFD induced autophagy/mitophagy activation via enhanced PARK2 expression, which was partly involved in the inhibition of myofibroblast differentiation in the presence of TGF-β. PFD inhibited the myofibroblast differentiation induced by PARK2 knockdown by reducing mitochondrial ROS and PDGFR-PI3K-Akt activation. BLM-treated PARK2 KO mice demonstrated augmentation of lung fibrosis and oxidative modifications compared to those of BLM-treated wild type mice, which were efficiently attenuated by PFD.ConclusionsThese results suggest that PFD induces PARK2-mediated mitophagy and also inhibits lung fibrosis development in the setting of insufficient mitophagy, which may at least partly explain the anti-fibrotic mechanisms of PFD for IPF treatment.

Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

ABSTRACT Accumulation of profibrotic myofibroblasts is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF) pathogenesis. TGFB (transforming growth factor β) is one of the major profibrotic cytokines for myofibroblast differentiation and NOX4 (NADPH oxidase 4) has an essential role in TGFB-mediated cell signaling. Azithromycin (AZM), a second-generation antibacterial macrolide, has a pleiotropic effect on cellular processes including proteostasis. Hence, we hypothesized that AZM may regulate NOX4 levels by modulating proteostasis machineries, resulting in inhibition of TGFB-associated lung fibrosis development. Human lung fibroblasts (LF) were used to evaluate TGFB-induced myofibroblast differentiation. With respect to NOX4 regulation via proteostasis, assays for macroautophagy/autophagy, the unfolded protein response (UPR), and proteasome activity were performed. The potential anti-fibrotic property of AZM was examined by using bleomycin (BLM)-induced lung fibrosis mouse models. TGFB-induced NOX4 and myofibroblast differentiation were clearly inhibited by AZM treatment in LF. AZM-mediated NOX4 reduction was restored by treatment with MG132, a proteasome inhibitor. AZM inhibited autophagy and enhanced the UPR. Autophagy inhibition by AZM was linked to ubiquitination of NOX4 via increased protein levels of STUB1 (STIP1 homology and U-box containing protein 1), an E3 ubiquitin ligase. An increased UPR by AZM was associated with enhanced proteasome activity. AZM suppressed lung fibrosis development induced by BLM with concomitantly reduced NOX4 protein levels and enhanced proteasome activation. These results suggest that AZM suppresses NOX4 by promoting proteasomal degradation, resulting in inhibition of TGFB-induced myofibroblast differentiation and lung fibrosis development. AZM may be a candidate for the treatment of the fibrotic lung disease IPF.

A case of segmental zoster paresis with enhanced anterior and posterior spinal roots on MRI

Herpes zoster is an infectious disease caused by reactivation of the varicella zoster virus (VZV) in the dorsal root ganglia [2]. Neurological complications other than sensory abnormalities such as encephalitis, myelitis, and various lower motor neuron diseases such as polyradiculoneuritis as segmental radiculitis have been reported [7]. Segmental zoster paresis is characterized by focal motor weakness that appears in the same segment where the skin eruptions, neuralgia, and sensory symptoms occur, and is a relatively common complication [1, 3, 6, 7]. We report a patient in whom the radiological findings were consistent with aberration of anterior and posterior spinal roots with zoster paresis. A 73-year-old man developed right shoulder pain and herpes zoster eruptions over the C5 dermatome. Two days later, he found it impossible to lift up the right arm. He was admitted to our hospital with 2-week history of shoulder pain and progressive muscle weakness around the right shoulder. Physical examination on admission showed weakness of the upper extremity in the distribution of the right C5 myotome and sensory disturbance in the right C5 dermatome, but no clinical symptoms of myelitis. Laboratory findings showed VZV-IgM level of 4.30 mg/dl (normal \0.8 mg/dl). Examination of cerebrospinal fluid (CSF) showed 102.8 cells/mm, protein content of 66.8 mg/dl, and elevated varicella zoster virus IgG titer (antibody index [4). Polymerase chain reaction (PCR) of CSF fluid was positive for VZV DNA, but no search for the number of copies was performed. Needle electromyography showed neurogenic changes in the right deltoid and biceps brachii muscles. T1-weighted magnetic resonance imaging (MRI) showed contrast enhancement with gadolinium in the right anterior and posterior C5 spinal roots below the 4th cervical vertebra (Fig. 1b), but no abnormal contrast enhancement of the roots of the 4th and 6th spinal nerves (Fig. 1a, c). Furthermore, T2-weighted MRI showed no abnormal intensity in the cervical spinal cord. The patient was treated with intravenous 1,500 mg acyclovir for 7 days together with rehabilitation. Examination about 1 month later showed improvement of muscle weakness and disappearance of the shoulder pain. The MRI findings suggested VZV reactivation in the dorsal root ganglia, which was localized to the anterior and posterior C5 spinal roots, based on the motor and sensory symptoms of C5 dermatome and myotome. The spread of VZV from the posterior spinal root, or anterior and posterior horn, to the anterior spinal root could have resulted from inflammation. CSF analysis indicated pleocytosis and serologically confirmed VZV. Previous studies reported abnormalities of CSF in 61% of patients with VZV and subclinical extension of viral inflammation into the central nervous system [4]. The clinical symptoms and MRI findings in our patient were not confirmative for myelitis. We postulate the spread of VZV either directly or through the CSF, rather than myelitis, from the posterior spinal root to the anterior spinal root in our patient. The cause of the nerve root enhancement was considered to be due to either viral activation itself or associated inflammation. Previous MRI findings in zoster paresis showed myelitis [3], posterior root with anterior horn [9] and posterior horn [8], suggesting myelitis and anterior root [5], suggesting disruption of the blood–brain barrier. Post mortem M. Yoshioka (&) Y. Kurita M. Hashimoto M. Murakami M. Suzuki Department of Neurology, Aoto Hospital, The Jikei University School of Medicine, 6-41-2 Aoto, Katsushika-ku, Tokyo 125-8506, Japan e-mail: m.yoshioka@jikei.ac.jp

Relation between Recurrence of Tuberculosis and Transitional Changes in IFN-γ Release Assays

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