Toyoshi Yanagihara

The Rac activator DOCK2 regulates natural killer cell–mediated cytotoxicity in mice through the lytic synapse formation

Natural killer (NK) cells play an important role in protective immunity against viral infection and tumor progression, but they also contribute to rejection of bone marrow grafts via contact-dependent cytotoxicity. Ligation of activating NK receptors with their ligands expressed on target cells induces receptor clustering and actin reorganization at the interface and triggers polarized movement of lytic granules to the contact site. Although activation of the small GTPase Rac has been implicated in NK cell-mediated cytotoxicity, its precise role and the upstream regulator remain elusive. Here, we show that DOCK2, an atypical guanine nucleotide exchange factor for Rac, plays a key role in NK cell-mediated cytotoxicity. We found that although DOCK2 deficiency in NK cells did not affect conjugate formation with target cells, DOCK2-deficienct NK cells failed to effectively kill leukemia cells in vitro and major histocompatibility complex class I-deficient bone marrow cells in vivo, regardless of the sorts of activating receptors. In DOCK2-deficient NK cells, NKG2D-mediated Rac activation was almost completely lost, resulting in a severe defect in the lytic synapse formation. Similar results were obtained when the Rac guanine nucleotide exchange factor activity of DOCK2 was selectively abrogated. These results indicate that DOCK2-Rac axis controls NK cell-mediated cytotoxicity through the lytic synapse formation.

DOCK2 and DOCK5 Act Additively in Neutrophils To Regulate Chemotaxis, Superoxide Production, and Extracellular Trap Formation

Neutrophils are highly motile leukocytes that play important roles in the innate immune response to invading pathogens. Neutrophils rapidly migrate to the site of infections and kill pathogens by producing reactive oxygen species (ROS). Neutrophil chemotaxis and ROS production require activation of Rac small GTPase. DOCK2, an atypical guanine nucleotide exchange factor (GEF), is one of the major regulators of Rac in neutrophils. However, because DOCK2 deficiency does not completely abolish fMLF-induced Rac activation, other Rac GEFs may also participate in this process. In this study, we show that DOCK5 acts with DOCK2 in neutrophils to regulate multiple cellular functions. We found that fMLF- and PMA-induced Rac activation were almost completely lost in mouse neutrophils lacking both DOCK2 and DOCK5. Although β2 integrin–mediated adhesion occurred normally even in the absence of DOCK2 and DOCK5, mouse neutrophils lacking DOCK2 and DOCK5 exhibited a severe defect in chemotaxis and ROS production. Similar results were obtained when human neutrophils were treated with CPYPP, a small-molecule inhibitor of these DOCK GEFs. Additionally, we found that DOCK2 and DOCK5 regulate formation of neutrophil extracellular traps (NETs). Because NETs are involved in vascular inflammation and autoimmune responses, DOCK2 and DOCK5 would be a therapeutic target for controlling NET-mediated inflammatory disorders.

Intronic regulation of Aire expression by Jmjd6 for self-tolerance induction in the thymus

The thymus has spatially distinct microenvironments, the cortex and the medulla, where the developing T-cells are selected to mature or die through the interaction with thymic stromal cells. To establish the immunological self in the thymus, medullary thymic epithelial cells (mTECs) express diverse sets of tissue-specific self-antigens (TSAs). This ectopic expression of TSAs largely depends on the transcriptional regulator Aire, yet the mechanism controlling Aire expression itself remains unknown. Here, we show that Jmjd6, a dioxygenase that catalyses lysyl hydroxylation of splicing regulatory proteins, is critical for Aire expression. Although Jmjd6 deficiency does not affect abundance of Aire transcript, the intron 2 of Aire gene is not effectively spliced out in the absence of Jmjd6, resulting in marked reduction of mature Aire protein in mTECs and spontaneous development of multi-organ autoimmunity in mice. These results highlight the importance of intronic regulation in controlling Aire protein expression.

Probucol attenuates hyperoxia-induced lung injury in mice

Hyperoxic lung injury is pathologically characterized by alveolar edema, interlobular septal edema, hyaline membrane disease, lung inflammation, and alveolar hemorrhage. Although the precise mechanism by which hyperoxia causes lung injury is not well defined, oxidative stress, epithelial cell death, and proinflammatory cytokines are thought to be involved. Probucol—a commercially available drug for treating hypercholesterolemia—has been suggested to have antioxidant and antiapoptotic effects. This study aimed to assess whether probucol could attenuate hyperoxic lung injury in mice. Mice were exposed to 95% O2 for 72 h, with or without pre-treatment with 130 μg/kg probucol intratracheally. Probucol treatment significantly decreased both the number of inflammatory cells in the bronchoalveolar lavage fluid and the degree of lung injury in hyperoxia-exposed mice. Probucol treatment reduced the number of cells positive for 8-hydroxyl-2′-deoxyguanosine or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and suppressed NF-κB activation, Bax expression, and caspase-9 activation in lung tissues from hyperoxia-exposed mice. These results suggest that probucol can reduce oxidative DNA damage, apoptotic cell death, and inflammation in lung tissues. Intratracheal administration of probucol may be a novel treatment for lung diseases induced by oxidative stress, such as hyperoxic lung injury and acute respiratory distress syndrome.

A case of immunoglobulin G4-related respiratory disease with multiple lung cysts: A case report

A 48-year-old man was admitted for evaluation of abnormal shadows on chest radiograph. Chest computed tomography (CT) showed cysts, nodules, and cervical and axillary lymphadenopathies. Elevated serum levels of IgG4 and interleukin (IL)-6 suggested IgG4-related disease (IgG4-RD) or multicentric Castlemans disease (MCD). Histologic findings of the cervical lymph node and right lung S6 biopsies revealed numerous IgG4-positive plasma cells. Although CT findings of the lungs were atypical for IgG4-RD, consistent histologic findings, clinical symptoms, and laboratory data made us conclude IgG4-RD. Because histologic findings of IgG4-RD and MCD have similarities, differentiating between the two diseases should consider the clinical presentation.

Bax-inhibiting peptide attenuates bleomycin-induced lung injury in mice

ABSTRACT Bax is a pro-apoptotic member of the Bcl-2 family of proteins, and plays a central role in mitochondria-dependent apoptosis. Several lines of evidence have implied that Bax is involved in both epithelial apoptosis and fibroblast proliferation in idiopathic pulmonary fibrosis; however, the mechanisms remain unknown. Bax-inhibiting peptide V5 (BIP-V5) exhibits membrane permeability and inhibits the activation of Bax. The purpose of this study was to investigate whether the control of Bax activity by BIP-V5 reduces the degree of bleomycin-induced lung injury. C57BL/6J mice were administered bleomycin and BIP-V5 intratracheally on day 0. Bronchoalveolar lavage fluid and lung tissue were obtained on day 7. Human pulmonary alveolar epithelial cells (A549 cells) and mouse pulmonary alveolar epithelial cells (LA-4 cells) were stimulated with bleomycin to induce apoptosis. Administration of BIP-V5 improved the survival rate and degree of bleomycin-induced lung injury by suppressing Bax activation in mice. BIP-V5 treatment decreased bleomycin-induced apoptosis of alveolar epithelial cell lines (A549 cells and LA-4 cells) by suppressing Bax activation. These results indicate that administration of BIP-V5 may constitute a novel therapeutic strategy against lung injury. Summary: The inhibiting peptide for Bax, a pro-apoptotic member of the Bcl-2 family of proteins, ameliorates bleomycin-induced lung injury in mice via apoptosis suppression in epithelial cells.

Recurrent idiopathic pulmonary hemosiderosis after long-term remission presented with Sjogren's syndrome: Idiopathic no more?

We report a case of recurrent idiopathic pulmonary hemosiderosis after a long-term remission presented with Sjögrens syndrome. The patient was diagnosed with IPH due to repeated pneumonia and blood sputum in his childhood. He was admitted to our hospital due to exertional dyspnea and dry cough with bilateral ground-glass opacity in chest computed tomography at the age of 32. Video-assisted thoracoscopic surgery was performed and the specimens showed nonspecific interstitial pneumonia pattern with diffuse, chronic alveolar hemorrhage, suggesting recurrence of IPH. He was also diagnosed with Sjögrens syndrome. Further immunological studies will reveal the pathogenesis of IPH.

Interstitial pneumonia caused by dabigatran

We describe the case of a 73-year-old man who experienced dry cough and exertional dyspnea after dabigatran administration. Chest radiographs revealed the development of bilateral consolidative and ground glass opacity, and transbronchial lung biopsy showed organized materials in the alveolar spaces with moderate inflammatory infiltrate and focal fibrosis. Lung opacity gradually disappeared after discontinuing dabigatran. To date, there has been only one report regarding dabigatran-induced lung injury, except for alveolar hemorrhage and eosinophilic pneumonia. Therefore, we should consider that any drug can cause various types of lung injuries.

An autopsy case of bird-related chronic hypersensitivity pneumonitis presenting with repeated acute exacerbation

A 68-year-old woman was admitted to our hospital with a dry cough in 2010. Chest computed tomography showed the appearance of a nonspecific interstitial pneumonia (NSIP) pattern. Video-assisted thoracoscopic surgery (VATS) was performed, and the specimens prominently showed a usual interstitial pneumonia (UIP) pattern. She was diagnosed with bird-related chronic hypersensitivity pneumonitis (BRCHP) on the basis of the detection of antibodies to pigeon dropping extract in her serum and a history of using feather-filled duvets and indirect exposure to birds in her living environment. Even though she was treated with corticosteroids and immunosuppressants and recommended to avoid bird-related antigens, she had a progressive course with repeated acute exacerbation episodes and died of respiratory failure. The autopsy findings showed diffuse alveolar damage superimposed on UIP. Clinicians should be aware that BRCHP patients especially with histopathologically UIP pattern may experience acute exacerbation.

Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade

Although immune-related adverse events (irAEs) of treatment with immune-checkpoint inhibitors may be due to cellular immunity mediated by T lymphocytes, their pathogenesis has remained unknown. Here we collected bronchoalveolar lavage fluid (BALF) from a cancer patient with nivolumab-induced pneumonitis and isolated mononuclear cells for next-generation sequencing of the complementarity-determining region of the T cell receptor (TCR) β chain. Mononuclear cells in peritumoral pleural effusion isolated from the patient were similarly analyzed, and the results obtained for the two specimens were compared. A substantial number of TCRβ clones in BALF were also identified among lymphocytes in the peritumoral pleural effusion. Such a correlation was not apparent between TCRβ clones in BALF and those in peripheral blood. Moreover, many tumor-associated clones with a read frequency of ≥0.10% were also present in BALF. Our data suggest that irAEs might be induced by drug-activated lymphocytes originating from tumor tissue. Deep sequencing will thus be indispensable for investigations of the immune-based pathogenesis of, and the development of optimal treatments for, irAEs.