Research Article
Abstract
Cardiac Nav1.5 and Kir2.1–2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems — rat ventricular cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) — demonstrated that endoplasmic reticulum (ER) trafficking–defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking–defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking–defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.
Authors
Marta Pérez-Hernández, Marcos Matamoros, Silvia Alfayate, Paloma Nieto-Marín, Raquel G. Utrilla, David Tinaquero, Raquel de Andrés, Teresa Crespo, Daniela Ponce-Balbuena, B. Cicero Willis, Eric N. Jiménez-Vazquez, Guadalupe Guerrero-Serna, Andre M. da Rocha, Katherine Campbell, Todd J. Herron, F. Javier Díez-Guerra, Juan Tamargo, José Jalife, Ricardo Caballero, Eva Delpón
Abstract
Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A– through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.
Authors
Fumiyuki Sasaki, Tomoaki Koga, Mai Ohba, Kazuko Saeki, Toshiaki Okuno, Keijiro Ishikawa, Takahito Nakama, Shintaro Nakao, Shigeo Yoshida, Tatsuro Ishibashi, Hamid Ahmadieh, Mozhgan Rezaei Kanavi, Ali Hafezi-Moghadam, Josef M. Penninger, Koh-Hei Sonoda, Takehiko Yokomizo
Abstract
The pathogenetic mechanisms underlying the pathologic fibrosis in diseases such as idiopathic pulmonary fibrosis (IPF) are poorly understood. To identify genetic factors affecting susceptibility to IPF, we analyzed a murine genetic model of IPF in which a profibrotic cytokine (TGF-β1) was expressed in the lungs of 10 different inbred mouse strains. Surprisingly, the extent of TGF-β1–induced lung fibrosis was highly strain dependent. Haplotype-based computational genetic analysis and gene expression profiling of lung tissue obtained from fibrosis-susceptible and -resistant strains identified laminin α1 (Lama1) as a genetic modifier for susceptibility to IPF. Subsequent studies demonstrated that Lama1 plays an important role in multiple processes that affect the pulmonary response to lung injury and susceptibility to fibrosis, which include: macrophage activation, fibroblast proliferation, myofibroblast transformation, and the production of extracellular matrix. Also, Lama1 mRNA expression was significantly increased in lung tissue obtained from IPF patients. These studies identify Lama1 as the genetic modifier of TGF-β1 effector responses that significantly affects the development of pulmonary fibrosis.
Authors
Chang-Min Lee, Soo Jung Cho, Won-Kyung Cho, Jin Wook Park, Jae-Hyun Lee, Augustine M. Choi, Ivan O. Rosas, Ming Zheng, Gary Peltz, Chun Geun Lee, Jack A. Elias
Abstract
Chronic obstructive pulmonary disease (COPD), associated with cigarette smoke–induced (CS-induced) emphysema, contributes significantly to the global health care burden of disease. Although chronic kidney disease (CKD) may occur in patients with COPD, the relationship between COPD and CKD remains unclear. Using a murine model of experimental COPD, we show that chronic CS exposure resulted in marked kidney injury and fibrosis, as evidenced by histological and ultrastructural changes, altered macrophage subpopulations, and expression of tissue injury, fibrosis, and oxidative stress markers. CS induced mitochondrial dysfunction, and increased autophagic flux in kidney tissues and in kidney tubular epithelial (HK-2) cells, as determined by LC3B turnover assays. Mice heterozygous for Beclin-1 (Becn1+/–) were protected from the development of kidney tissue injury and renal fibrosis in response to CS exposure, and displayed impaired basal and inducible mitochondrial turnover by mitophagy. Interestingly, CS caused a reduction of Beclin-1 expression in mouse kidneys and kidney tubular epithelial cells, attributed to increased autophagy-dependent turnover of Beclin-1. These results suggest that Beclin-1 is required for CS-induced kidney injury and that reduced levels of Beclin-1 may confer renoprotection. These results identify the kidney as a target for CS-induced injury in COPD and the Beclin-1–dependent autophagy pathway as a potential therapeutic target in CKD.
Authors
Maria A. Pabón, Edwin Patino, Divya Bhatia, Jocelyn Quintero, Kevin C. Ma, Eli J. Finkelsztein, Juan C. Osorio, Faryal Malick, Francesca Polverino, Caroline A. Owen, Stefan W. Ryter, Augustine M.K. Choi, Suzanne M. Cloonan, Mary E. Choi
Abstract
Stroke triggers a complex inflammatory process in which the balance between pro- and antiinflammatory mediators is critical for the development of the brain infarct. However, systemic changes may also occur in parallel with brain inflammation. Here we demonstrate that administration of recombinant IL-33, a recently described member of the IL-1 superfamily of cytokines, promotes Th2-type effects following focal ischemic stroke, resulting in increased plasma levels of Th2-type cytokines and fewer proinflammatory (3-nitrotyrosine+F4/80+) microglia/macrophages in the brain. These effects of IL-33 were associated with reduced infarct size, fewer activated microglia and infiltrating cytotoxic (natural killer–like) T cells, and more IL-10–expressing regulatory T cells. Despite these neuroprotective effects, mice treated with IL-33 displayed exacerbated post-stroke lung bacterial infection in association with greater functional deficits and mortality at 24 hours. Supplementary antibiotics (gentamicin and ampicillin) mitigated these systemic effects of IL-33 after stroke. Our findings highlight the complex nature of the inflammatory mechanisms differentially activated in the brain and periphery during the acute phase after ischemic stroke. The data indicate that a Th2-promoting agent can provide neuroprotection without adverse systemic effects when given in combination with antibiotics.
Authors
Shenpeng R. Zhang, Marius Piepke, Hannah X. Chu, Brad R.S. Broughton, Raymond Shim, Connie H.Y. Wong, Seyoung Lee, Megan A. Evans, Antony Vinh, Samy Sakkal, Thiruma V. Arumugam, Tim Magnus, Samuel Huber, Mathias Gelderblom, Grant R. Drummond, Christopher G. Sobey, Hyun Ah Kim
Abstract
Colon cancer is a devastating illness that is associated with gut inflammation. Here, we explored the possible role of lipin-1, a phosphatidic acid phosphatase, in the development of colitis-associated tumorigenesis. Azoxymethane and dextran sodium sulfate–treated (DSS-treated) animals deficient in lipin-1 harbored fewer tumors and carcinomas than WT animals due to decreased cellular proliferation, lower expression of antiapoptotic and protumorigenic factors, and a reduced infiltration of macrophages in colon tumors. They also displayed increased resistance to DSS-induced colitis by producing less proinflammatory cytokines and experiencing less immune infiltration. Lipin-1–deficient macrophages from the colon were less activated and displayed lower phosphatidic acid phosphatase activity than WT macrophages isolated from DSS-treated animals. Transference of WT macrophages into lipin-1–deficient animals was sufficient to increase colitis burden. Furthermore, treatment of lipin-1–deficient mice with IL-23 exacerbated colon inflammation. Analysis of human databases from colon cancer and ulcerative colitis patients showed that lipin-1 expression is increased in those disorders and correlates with the expression of the proinflammatory markers CXCL1 and CXCL2. And finally, clinically, LPIN1 expression had prognostic value in inflammatory and stem-cell subtypes of colon cancers. Collectively, these data demonstrate that lipin-1 is a critical regulator of intestinal inflammation and inflammation-driven colon cancer development.
Authors
Clara Meana, Ginesa García-Rostán, Lucía Peña, Gema Lordén, África Cubero, Antonio Orduña, Balázs Győrffy, Jesús Balsinde, María A. Balboa
Abstract
IFN-inducible protein 16 (IFI16) is an innate immune sensor that forms filamentous oligomers when activated by double-stranded DNA (dsDNA). Anti-IFI16 autoantibodies occur in patients with Sjögren’s syndrome (SS) and associate with severe phenotypic features. We undertook this study to determine whether the structural and functional properties of IFI16 play a role in its status as an SS autoantigen. IFI16 immunostaining in labial salivary glands (LSGs) yielded striking evidence of filamentous IFI16 structures in the cytoplasm of ductal epithelial cells, representing the first microscopic description of IFI16 oligomerization in human tissues, to our knowledge. Transfection of cultured epithelial cells with dsDNA triggered the formation of cytoplasmic IFI16 filaments with similar morphology to those observed in LSGs. We found that a majority of SS anti-IFI16 autoantibodies immunoprecipitate IFI16 more effectively in the oligomeric dsDNA-bound state. Epitopes in the C-terminus of IFI16 are accessible to antibodies in the DNA-bound oligomer and are preferentially targeted by SS sera. Furthermore, cytotoxic lymphocyte granule pathways (highly enriched in the SS gland) induce striking release of IFI16•dsDNA complexes from cultured cells. Our studies reveal that IFI16 is present in a filamentous state in the target tissue of SS and suggest that this property of DNA-induced filament formation contributes to its status as an autoantigen in SS. These studies highlight the role that tissue-specific modifications and immune effector pathways might play in the selection of autoantigens in rheumatic diseases.
Authors
Brendan Antiochos, Mariusz Matyszewski, Jungsan Sohn, Livia Casciola-Rosen, Antony Rosen
Abstract
Therapeutic strategies that augment antiviral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The coinhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of antiviral CD8+ T cells and B cells. Here, we tested the immunological and virological effects of PD-1 blockade combined with antiretroviral therapy (ART) in rhesus macaques. Administration of anti–PD-1 antibody 10 days prior to ART initiation rapidly enhanced antiviral CD8+ T cell function and diminished IFN-stimulated genes. This resulted in faster viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. PD-1 blockade during ART resulted in lower levels of cell-associated replication-competent virus. Following ART interruption, PD-1 antibody–treated animals showed markedly higher expansion of proliferating CXCR5+perforin+granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in better control of viremia. Our results show that PD-1 blockade can be administered safely with ART to augment antiviral CD8+ T cell function and reduce the viral reservoir, leading to improved control of viral rebound after ART interruption.
Authors
Geetha H. Mylvaganam, Lynette S. Chea, Gregory K. Tharp, Sakeenah Hicks, Vijayakumar Velu, Smita S. Iyer, Claire Deleage, Jacob D. Estes, Steven E. Bosinger, Gordon J. Freeman, Rafi Ahmed, Rama R. Amara
Abstract
Clonal expansion of T cells harboring replication-competent virus has recently been demonstrated in patients on suppressive antiretroviral therapy (ART) regimens. However, there has not been direct evidence of this phenomenon in settings of natural control, including in posttreatment controllers who maintain control of viral replication after treatment when ART is discontinued. We present a case of an individual who has had undetectable viral loads for more than 15 years following the cessation of ART. Using near-full-genome sequence analysis, we demonstrate that 9 of 12 replication-competent isolates cultured from this subject were identical and that this identity was maintained 6 months later. A similar pattern of replication-competent virus clonality was seen in a treatment-naive HLA-B*57 elite controller. In both cases, we show that CD8+ T cells are capable of suppressing the replication of the clonally expanded viruses in vitro. Our data suggest that, while clonal expansion of replication-competent virus can present a barrier to viral eradication, these viral isolates remain susceptible to HIV-specific immune responses and can be controlled in patients with long-term suppression of viral replication.
Authors
Rebecca T. Veenhuis, Abena K. Kwaa, Caroline C. Garliss, Rachel Latanich, Maria Salgado, Christopher W. Pohlmeyer, Christopher L. Nobles, John Gregg, Eileen P. Scully, Justin R. Bailey, Frederic D. Bushman, Joel N. Blankson
Abstract
Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.
Authors
Kandace Gollomp, Minna Kim, Ian Johnston, Vincent Hayes, John Welsh, Gowthami M. Arepally, Mark Kahn, Michele P. Lambert, Adam Cuker, Douglas B. Cines, Lubica Rauova, M. Anna Kowalska, Mortimer Poncz
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