The neutrophil chemoattractant proline-glycine-proline (PGP) is generated from collagen by matrix metalloproteinase-8/9 (MMP-8/9) and prolyl endopeptidase (PE), and it is concomitantly degraded by extracellular leukotriene A4 hydrolase (LTA4H) to limit neutrophilia. Components of cigarette smoke can acetylate PGP, yielding a species (AcPGP) that is resistant to LTA4H-mediated degradation and can, thus, support a sustained neutrophilia. In this study, we sought to elucidate if an antiinflammatory system existed to degrade AcPGP that is analogous to the PGP-LTA4H axis. We demonstrate that AcPGP is degraded through a previously unidentified action of the enzyme angiotensin-converting enzyme (ACE). Pulmonary ACE is elevated during episodes of acute inflammation, as a consequence of enhanced vascular permeability, to ensure the efficient degradation of AcPGP. Conversely, we suggest that this pathway is aberrant in chronic obstructive pulmonary disease (COPD) enabling the accumulation of AcPGP. Consequently, we identify a potentially novel protective role for AcPGP in limiting pulmonary fibrosis and suggest the pathogenic function attributed to ACE in idiopathic pulmonary fibrosis (IPF) to be a consequence of overzealous AcPGP degradation. Thus, AcPGP seemingly has very divergent roles: it is pathogenic in its capacity to drive neutrophilic inflammation and matrix degradation in the context of COPD, but it is protective in its capacity to limit fibrosis in IPF.
Philip J. O’Reilly, Qiang Ding, Samia Akthar, Guoqiang Cai, Kristopher R. Genschmer, Dhiren F. Patel, Patricia L. Jackson, Liliana Viera, Mojtaba Roda, Morgan L. Locy, Ellen A. Bernstein, Clare M. Lloyd, Kenneth E. Bernstein, Robert J. Snelgrove, J. Edwin Blalock
Despite initial remission after successful treatments, B lymphoma patients often encounter relapses and resistance causing high mortality. Thus, there is a need to develop therapies that prevent relapse by providing long-term protection and, ultimately, lead to functional cure. In this study, our goal was to develop a simple, clinically relevant, and easily translatable therapeutic vaccine that provides durable immune protection against aggressive B cell lymphoma and identify critical immune biomarkers that are predictive of long-term survival. In a delayed-treatment, aggressive, murine model of A20 B lymphoma that mimics human diffuse large B cell lymphoma, we show that therapeutic A20 lysate vaccine adjuvanted with an NKT cell agonist, α-galactosylceramide (α-GalCer), provides long-term immune protection against lethal tumor challenges and the antitumor immunity is primarily CD8 T cell dependent. Using experimental and computational methods, we demonstrate that the initial strength of germinal center reaction and the magnitude of class-switching into a Th1 type humoral response are the best predictors for the long-term immunity of B lymphoma lysate vaccine. Our results not only provide fundamentally insights for successful immunotherapy and long-term protection against B lymphomas, but also present a simple, therapeutic vaccine that can be translated easily due to the facile and inexpensive method of preparation.
Pallab Pradhan, Jardin Leleux, Jiaying Liu, Krishnendu Roy
A role of B cells in multiple sclerosis (MS) is well established, but there is limited understanding of their involvement during active disease. Here, we examined cerebrospinal fluid (CSF) and peripheral blood (PB) B cells in treatment-naive patients with MS or high-risk clinically isolated syndrome. Using flow cytometry, we found increased CSF lymphocytes with a disproportionate increase of B cells compared with T cells in patients with gadolinium-enhancing (Gd+) lesions on brain MRI. Ig gene heavy chain variable region (Ig-VH) repertoire sequencing of CSF and PB B cells revealed clonal relationships between intrathecal and peripheral B cell populations, which could be consistent with migration of B cells to and activation in the CNS in active MS. In addition, we found evidence for bystander immigration of B cells from the periphery, which could be supported by a CXCL13 gradient between CSF and blood. Understanding what triggers B cells to migrate and home to the CNS may ultimately aid in the rational selection of therapeutic strategies to limit progression in MS.
Erica L. Eggers, Brady A. Michel, Hao Wu, Sheng-zhi Wang, Carolyn J. Bevan, Aya Abounasr, Natalie S. Pierson, Antje Bischof, Max Kazer, Elizabeth Leitner, Ariele L. Greenfield, Stanislas Demuth, Michael R. Wilson, Roland G. Henry, Bruce A.C. Cree, Stephen L. Hauser, H.-Christian von Büdingen
Although recent evidence has pointed to the role of organ- and pathogenesis-specific macrophage subsets, it is still unclear which subsets are critically involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Using melanocortin-4 receptor–deficient (MC4R-KO) mice fed Western diet (WD), which exhibit liver phenotypes similar to those of human NASH, we found a histological structure, termed hepatic crown-like structure (hCLS), in which CD11c+ macrophages surround dead/dying hepatocytes, a prominent feature of NASH. Here, we demonstrate that hCLS-constituting macrophages could be a novel macrophage subset that drives hepatocyte death-triggered liver fibrosis. In an “inducible NASH model,” hepatocyte death induces hCLS formation and liver fibrosis sequentially in the short term. In combination with the long-term WD feeding model, we also showed that resident macrophages are a major cellular source of CD11c+ macrophages constituting hCLS, which exhibited gene expression profiles distinct from CD11c– macrophages scattered in the liver. Moreover, depletion of CD11c+ macrophages abolished hCLS formation and fibrogenesis in NASH. Our clinical data suggest the role of CD11c+ macrophages in the disease progression from simple steatosis to NASH. This study sheds light on the role of resident macrophages, in addition to recruited macrophages, in the pathogenesis of NASH.
Michiko Itoh, Takayoshi Suganami, Hideaki Kato, Sayaka Kanai, Ibuki Shirakawa, Takeru Sakai, Toshihiro Goto, Masahiro Asakawa, Isao Hidaka, Hiroshi Sakugawa, Koji Ohnishi, Yoshihiro Komohara, Kenichi Asano, Isao Sakaida, Masato Tanaka, Yoshihiro Ogawa
While the prevalence of osteoporosis is growing rapidly with population aging, therapeutic options remain limited. Here, we identify potentially novel roles for CaV1.2 L-type voltage–gated Ca2+ channels in osteogenesis and exploit a transgenic gain-of-function mutant CaV1.2 to stem bone loss in ovariectomized female mice. We show that endogenous CaV1.2 is expressed in developing bone within proliferating chondrocytes and osteoblasts. Using primary BM stromal cell (BMSC) cultures, we found that Ca2+ influx through CaV1.2 activates osteogenic transcriptional programs and promotes mineralization. We used Prx1-, Col2a1-, or Col1a1-Cre drivers to express an inactivation-deficient CaV1.2 mutant in chondrogenic and/or osteogenic precursors in vivo and found that the resulting increased Ca2+ influx markedly thickened bone not only by promoting osteogenesis, but also by inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts. Activating the CaV1.2 mutant in osteoblasts at the time of ovariectomy stemmed bone loss. Together, these data highlight roles for CaV1.2 in bone and demonstrate the potential dual anabolic and anticatabolic therapeutic actions of tissue-specific CaV1.2 activation in osteoblasts.
Chike Cao, Yinshi Ren, Adam S. Barnett, Anthony J. Mirando, Douglas Rouse, Se Hwan Mun, Kyung-Hyun Park-Min, Amy L. McNulty, Farshid Guilak, Courtney M. Karner, Matthew J. Hilton, Geoffrey S. Pitt
Three Akt isoforms, encoded by 3 separate genes, are expressed in mammals. While the roles of Akt1 and Akt2 in metabolism are well established, it is not yet known whether Akt3 plays a role in metabolic diseases. We now report that Akt3 protects mice from high-fat diet–induced obesity by suppressing an alternative pathway of adipogenesis via with no lysine protein kinase-1 (WNK1) and serum/glucocorticoid-inducible kinase 1 (SGK1). We demonstrate that Akt3 specifically phosphorylates WNK1 at T58 and promotes its degradation via the ubiquitin-proteasome pathway. A lack of Akt3 in adipocytes increases the WNK1 protein level, leading to activation of SGK1. SGK1, in turn, promotes adipogenesis by phosphorylating and inhibiting transcription factor FOXO1 and, subsequently, activating the transcription of PPARγ in adipocytes. Akt3-deficient mice have an increased number of adipocytes and, when fed a high-fat diet, display increased weight gain, white adipose tissue expansion, and impaired glucose homeostasis. Pharmacological blockade of SGK1 in high-fat diet–fed Akt3-deficient mice suppressed adipogenesis, prevented excessive weight gain and adiposity, and ameliorated metabolic parameters. Thus, Akt3/WNK1/SGK1 represents a potentially novel signaling pathway controlling the development of obesity.
Liang Ding, Lifang Zhang, Sudipta Biswas, Rebecca C. Schugar, J. Mark Brown, Tatiana Byzova, Eugene Podrez
Expansion of novel therapeutics to all patients with cystic fibrosis (CF) requires personalized CFTR modulator therapy. We have developed nasospheroids, a primary cell culture–based model derived from individual CF patients and healthy subjects by a minimally invasive nasal biopsy. Confocal microscopy was utilized to measure CFTR activity by analyzing changes in cross-sectional area over time that resulted from CFTR-mediated ion and fluid movement. Both the rate of change over time and AUC were calculated. Non-CF nasospheroids with active CFTR-mediated ion and fluid movement showed a reduction in cross-sectional area, whereas no changes were observed in CF spheroids. Non-CF spheroids treated with CFTR inhibitor lost responsiveness for CFTR activation. However, nasospheroids from F508del CF homozygotes that were treated with lumacaftor and ivacaftor showed a significant reduction in cross-sectional area, indicating pharmacologic rescue of CFTR function. This model employs a simple measurement of size corresponding to changes in CFTR activity and is applicable for detection of small changes in CFTR activity from individual patients in vitro. Advancements of this technique will provide a robust model for individualized prediction of CFTR modulator efficacy.
Jennifer S. Guimbellot, Justin M. Leach, Imron G. Chaudhry, Nancy L. Quinney, Susan E. Boyles, Michael Chua, Inmaculada Aban, Ilona Jaspers, Martina Gentzsch
Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.
Shih-Yu Chang, Elijah J. Weber, Viktoriya S. Sidorenko, Alenka Chapron, Catherine K. Yeung, Chunying Gao, Qingcheng Mao, Danny Shen, Joanne Wang, Thomas A. Rosenquist, Kathleen G. Dickman, Thomas Neumann, Arthur P. Grollman, Edward J. Kelly, Jonathan Himmelfarb, David L. Eaton
IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that, under steady-state conditions, IL-7 signaling is principally mediated by activation of signal transducers and activators of transcription 5 (STAT5). In contrast, under lymphopenic conditions, there is a modulation of STAT1 expression resulting in an IL-7–dependent STAT1 and STAT5 activation. Consequently, the IL-7–induced transcriptome is altered with enrichment of IFN-stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4+ T cells undergoing lymphopenia-induced proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7–dependent program. This mechanism could be a physiological process to regulate the expansion and size of the CD4+ T cell pool. During HIV infection, the virus could exploit this pathway, leading to the homeostatic dysregulation of the T cell pools observed in these patients.
Cecile Le Saout, Megan A. Luckey, Alejandro V. Villarino, Mindy Smith, Rebecca B. Hasley, Timothy G. Myers, Hiromi Imamichi, Jung-Hyun Park, John J. O’Shea, H. Clifford Lane, Marta Catalfamo
Restoring blood flow after myocardial infarction (MI) is essential for survival of existing and newly regenerated tissue. Endogenous vascular repair processes are deployed following injury but are poorly understood. We sought to determine whether developmental mechanisms of coronary vessel formation are intrinsically reactivated in the adult mouse after MI. Using pulse-chase genetic lineage tracing, we establish that de novo vessel formation constitutes a substantial component of the neovascular response, with apparent cellular contributions from the endocardium and coronary sinus. The adult heart reverts to its former hypertrabeculated state and repeats the process of compaction, which may facilitate endocardium-derived neovascularization. The capacity for angiogenic sprouting of the coronary sinus vein, the adult derivative of the sinus venosus, may also reflect its embryonic origin. The quiescent epicardium is reactivated and, while direct cellular contribution to new vessels is minimal, it supports the directional expansion of the neovessel network toward the infarcted myocardium. Thymosin β4, a peptide with roles in vascular development, was required for endocardial compaction, epicardial vessel expansion, and smooth muscle cell recruitment. Insight into pathways that regulate endogenous vascular repair, drawing on comparisons with development, may reveal novel targets for therapeutically enhancing neovascularization.
Karina N. Dubé, Tonia M. Thomas, Sonali Munshaw, Mala Rohling, Paul R. Riley, Nicola Smart
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