Abstract
Multiple myeloma (MM) is an incurable plasma cell malignancy with frequent treatment failures and relapses, suggesting the existence of pathogenic myeloma stem/progenitor populations. However, the identity of MM stem cells remains elusive. We used a murine model of MM with transgenic overexpression of the unfolded protein response sensor X-box binding protein 1 (XBP1s) in the B cell compartment to define MM stem cells. We herein report that a post–germinal center, pre–plasma cell population significantly expands as MM develops. This population has the following characteristics: (a) cell surface phenotype of B220+CD19+IgM–IgD–CD138–CD80+sIgG–AA4.1+FSChi; (b) high expression levels of Pax5 and Bcl6 with intermediate levels of Blimp1 and XBP1s; (c) increased expression of aldehyde dehydrogenase, Notch1, and c-Kit; and (d) ability to efficiently reconstitute antibody-producing capacity in B cell–deficient mice in vivo. We thus have defined a plasma cell progenitor population that resembles myeloma stem cells in mice. These results provide potentially novel insights into MM stem cell biology and may contribute to the development of novel stem cell–targeted therapies for the eradication of MM.
Authors
Joshua Kellner, Caroline Wallace, Bei Liu, Zihai Li
Abstract
Alveolar type 2 (AT2) cell endoplasmic reticulum (ER) stress is a prominent feature in adult and pediatric interstitial lung disease (ILD and ChILD), but in vivo models linking AT2 cell ER stress to ILD have been elusive. Based on a clinical ChILD case, we identified a critical cysteine residue in the surfactant protein C gene (SFTPC) BRICHOS domain whose mutation induced ER stress in vitro. To model this in vivo, we generated a knockin mouse model expressing a cysteine-to-glycine substitution at codon 121 (C121G) in the Sftpc gene. SftpcC121G expression during fetal development resulted in a toxic gain-of-function causing fatal postnatal respiratory failure from disrupted lung morphogenesis. Induced SftpcC121G expression in adult mice resulted in an ER-retained pro-protein causing AT2 cell ER stress. SftpcC121G AT2 cells were a source of cytokines expressed in concert with development of polycellular alveolitis. These cytokines were subsequently found in a high-dimensional proteomic screen of bronchoalveolar lavage fluid from ChILD patients with the same class of SFTPC mutations. Following alveolitis resolution, SftpcC121G mice developed spontaneous pulmonary fibrosis and restrictive lung impairment. This model provides proof of concept linking AT2 cell ER stress to fibrotic lung disease coupled with translationally relevant biomarkers.
Authors
Jeremy Katzen, Brandie D. Wagner, Alessandro Venosa, Meghan Kopp, Yaniv Tomer, Scott J. Russo, Alvis C. Headen, Maria C. Basil, James M. Stark, Surafel Mulugeta, Robin R. Deterding, Michael F. Beers
Abstract
Idiopathic intracranial hypertension (IIH) is a condition of unknown etiology, characterized by elevated intracranial pressure frequently manifesting with chronic headaches and visual loss. Similar to polycystic ovary syndrome (PCOS), IIH predominantly affects obese women of reproductive age. In this study, we comprehensively examined the systemic and cerebrospinal fluid (CSF) androgen metabolome in women with IIH in comparison with sex-, BMI-, and age-matched control groups with either simple obesity or PCOS (i.e., obesity and androgen excess). Women with IIH showed a pattern of androgen excess distinct to that observed in PCOS and simple obesity, with increased serum testosterone and increased CSF testosterone and androstenedione. Human choroid plexus expressed the androgen receptor, alongside the androgen-activating enzyme aldoketoreductase type 1C3. We show that in a rat choroid plexus cell line, testosterone significantly enhanced the activity of Na+/K+-ATPase, a surrogate of CSF secretion. We demonstrate that IIH patients have a unique signature of androgen excess and provide evidence that androgens can modulate CSF secretion via the choroid plexus. These findings implicate androgen excess as a potential causal driver and therapeutic target in IIH.
Authors
Michael W. O’Reilly, Connar S.J. Westgate, Catherine Hornby, Hannah Botfield, Angela E. Taylor, Keira Markey, James L. Mitchell, William J. Scotton, Susan P. Mollan, Andreas Yiangou, Carl Jenkinson, Lorna C. Gilligan, Mark Sherlock, James Gibney, Jeremy W. Tomlinson, Gareth G. Lavery, David J. Hodson, Wiebke Arlt, Alexandra J. Sinclair
Abstract
B cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B cells exist in the cerebrospinal fluid (CSF), meninges, and CNS parenchyma of MS patients. We sought to investigate the presence of clonally related B cells over time by performing Ig heavy chain variable region repertoire sequencing on B cells from longitudinally collected blood and CSF samples of MS patients (n = 10). All patients were untreated at the time of the initial sampling; the majority (n = 7) were treated with immune-modulating therapies 1.2 (±0.3 SD) years later during the second sampling. We found clonal persistence of B cells in the CSF of 5 patients; these B cells were frequently Ig class-switched and CD27+. Specific blood B cell subsets appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B cell persistence in CSF and blood, even in patients on immune-modulating therapy. Our findings support the concept that peripheral B cell activation and CNS-compartmentalized immune mechanisms can in part be therapy resistant.
Authors
Ariele L. Greenfield, Ravi Dandekar, Akshaya Ramesh, Erica L. Eggers, Hao Wu, Sarah Laurent, William Harkin, Natalie S. Pierson, Martin S. Weber, Roland G. Henry, Antje Bischof, Bruce A.C. Cree, Stephen L. Hauser, Michael R. Wilson, H.-Christian von Büdingen
Abstract
Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all 3 Munc18 isoforms. Using conditional airway epithelial cell–deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.
Authors
Ana M. Jaramillo, Lucia Piccotti, Walter V. Velasco, Anna Sofia Huerta Delgado, Zoulikha Azzegagh, Felicity Chung, Usman Nazeer, Junaid Farooq, Josh Brenner, Jan Parker-Thornburg, Brenton L. Scott, Christopher M. Evans, Roberto Adachi, Alan R. Burns, Silvia M. Kreda, Michael J. Tuvim, Burton F. Dickey
Abstract
About one-third of dilated cardiomyopathy (DCM) cases are caused by mutations in sarcomere or cytoskeletal proteins. However, treating the cytoskeleton directly is not possible because drugs that bind to actin are not well tolerated. Mutations in the actin binding protein CAP2 can cause DCM and KO mice, either whole body (CAP2-KO) or cardiomyocyte-specific KOs (CAP2-CKO) develop DCM with cardiac conduction disease. RNA sequencing analysis of CAP2-KO hearts and isolated cardiomyocytes revealed overactivation of fetal genes, including serum response factor–regulated (SRF-regulated) genes such as Myl9 and Acta2 prior to the emergence of cardiac disease. To test if we could treat CAP2-KO mice, we synthesized and tested the SRF inhibitor CCG-1423-8u. CCG-1423-8u reduced expression of the SRF targets Myl9 and Acta2, as well as the biomarker of heart failure, Nppa. The median survival of CAP2-CKO mice was 98 days, while CCG-1423-8u–treated CKO mice survived for 116 days and also maintained normal cardiac function longer. These results suggest that some forms of sudden cardiac death and cardiac conduction disease are under cytoskeletal stress and that inhibiting signaling through SRF may benefit DCM by reducing cytoskeletal stress.
Authors
Yao Xiong, Kenneth Bedi, Simon Berritt, Bennette K. Attipoe, Thomas G. Brooks, Kevin Wang, Kenneth B. Margulies, Jeffrey Field
Abstract
Acute respiratory distress syndrome is an often fatal disease that develops after acute lung injury and trauma. How released tissue damage signals, or alarmins, orchestrate early inflammatory events is poorly understood. Herein we reveal that IL-33, an alarmin sequestered in the lung epithelium, is required to limit inflammation after injury due to an unappreciated capacity to mediate Foxp3+ Treg control of local cytokines and myeloid populations. Specifically, Il33–/– mice are more susceptible to lung damage–associated morbidity and mortality that is typified by augmented levels of the proinflammatory cytokines and Ly6Chi monocytes in the bronchoalveolar lavage fluid. Local delivery of IL-33 at the time of injury is protective but requires the presence of Treg cells. IL-33 stimulates both mouse and human Tregs to secrete IL-13. Using Foxp3Cre × Il4/Il13fl/fl mice, we show that Treg expression of IL-13 is required to prevent mortality after acute lung injury by controlling local levels of G-CSF, IL-6, and MCP-1 and inhibiting accumulation of Ly6Chi monocytes. Our study identifies a regulatory mechanism involving IL-33 and Treg secretion of IL-13 in response to tissue damage that is instrumental in limiting local inflammatory responses and may shape the myeloid compartment after lung injury.
Authors
Quan Liu, Gaelen K. Dwyer, Yifei Zhao, Huihua Li, Lisa R. Mathews, Anish Bhaswanth Chakka, Uma R. Chandran, Jake A. Demetris, John F. Alcorn, Keven M. Robinson, Luis A. Ortiz, Bruce R. Pitt, Angus W. Thomson, Ming-Hui Fan, Timothy R. Billiar, Hēth R. Turnquist
Abstract
While anti-VEGF drugs are commonly used to inhibit pathological retinal and choroidal neovascularization, not all patients respond in an optimal manner. Mechanisms underpinning resistance to anti‑VEGF therapy include the upregulation of other proangiogenic factors. Therefore, therapeutic strategies that simultaneously target multiple growth factor signaling pathways would have significant value. Here, we show that Ca2+/calmodulin-dependent kinase II (CAMKII) mediates the angiogenic actions of a range of growth factors in human retinal endothelial cells and that this kinase acts as a key nodal point for the activation of several signal transduction cascades that are known to play a critical role in growth factor–induced angiogenesis. We also demonstrate that endothelial CAMKIIγ and -δ isoforms differentially regulate the angiogenic effects of different growth factors and that genetic deletion of these isoforms suppresses pathological retinal and choroidal neovascularization in vivo. Our studies suggest that CAMKII could provide a novel and efficacious target to inhibit multiple angiogenic signaling pathways for the treatment of vasoproliferative diseases of the eye. CAMKIIγ represents a particularly promising target, as deletion of this isoform inhibited pathological neovascularization, while enhancing reparative angiogenesis in the ischemic retina.
Authors
Sadaf Ashraf, Samuel Bell, Caitriona O’Leary, Paul Canning, Ileana Micu, Jose A. Fernandez, Michael O’Hare, Peter Barabas, Hannah McCauley, Derek P. Brazil, Alan W. Stitt, J. Graham McGeown, Tim M. Curtis
Abstract
Anthracyclines are among the most effective chemotherapeutics ever developed, but they produce grueling side effects and serious adverse events, and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular prion protein (PrPC), which blocks their cytotoxic activity. This effect was dose dependent using either cell line–conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pretreatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly among doxorubicin-treated patients with residual disease at surgery. These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by IHC, particularly soluble isoforms released into the extracellular environment by shedding. These findings have important potential clinical implications for frontline regimen decision making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemosensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.
Authors
Adrian P. Wiegmans, Jodi M. Saunus, Sunyoung Ham, Richard Lobb, Jamie R. Kutasovic, Andrew J. Dalley, Mariska Miranda, Caroline Atkinson, Simote T. Foliaki, Kaltin Ferguson, Colleen Niland, Cameron N. Johnstone, Victoria Lewis, Steven J. Collins, Sunil R. Lakhani, Fares Al-Ejeh, Andreas Möller
Abstract
Recovery from acute lung injury (ALI) is an active process. Foxp3+ Tregs contribute to recovery from ALI through modulating immune responses and enhancing alveolar epithelial proliferation and tissue repair. The current study investigates Treg transcriptional profiles during resolution of ALI in mice. Tregs from either lung or splenic tissue were isolated from uninjured mice or mice recovering from ALI and then examined for differential gene expression between these conditions. In mice with ALI, Tregs isolated from the lungs had hundreds of differentially expressed transcripts compared with those from the spleen, indicating that organ specificity and microenvironment are critical in Treg function. These regulated transcripts suggest which intracellular signaling pathways modulate Treg behavior. Interestingly, several transcripts having no prior recognized function in Tregs were differentially expressed by lung Tregs during resolution. Further investigation into 2 identified transcripts, Mmp12 and Sik1, revealed that Treg-specific expression of each plays a role in Treg-promoted ALI resolution. This study provides potentially novel information describing the signals that may expand resident Tregs, recruit or retain them to the lung during ALI, and modulate their function. The results provide insight into both tissue- and immune microenvironment–specific transcriptional differences through which Tregs direct their effects.
Authors
Jason R. Mock, Catherine F. Dial, Miriya K. Tune, Dustin L. Norton, Jessica R. Martin, John C. Gomez, Robert S. Hagan, Hong Dang, Claire M. Doerschuk
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