Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease that is associated with aberrant activation of TGF-β, myofibroblast differentiation, and abnormal extracellular matrix (ECM) production. Proper regulation of protein stability is important for maintenance of intracellular protein homeostasis and signaling. Ubiquitin E3 ligases mediate protein ubiquitination, and deubiquitinating enzymes (DUBs) reverse the process. The role of ubiquitin E3 ligases and DUBs in the pathogenesis of IPF is relatively unexplored. In this review, we provide an overview of how ubiquitin E3 ligases and DUBs modulate pulmonary fibrosis through regulation of both TGF-β–dependent and –independent pathways. We also summarize currently available small-molecule inhibitors of ubiquitin E3 ligases and DUBs as potential therapeutic strategies for the treatment of IPF.
Shuang Li, Jing Zhao, Dong Shang, Daniel J. Kass, Yutong Zhao
Intraocular injections of VEGF-neutralizing proteins provide tremendous benefits in patients with choroidal neovascularization (NV) due to age-related macular degeneration (AMD), but during treatment some patients develop retinal atrophy. Suggesting that VEGF is a survival factor for retinal neurons, a clinical trial group attributed retinal atrophy to VEGF suppression and cautioned against frequent anti-VEGF injections. This recommendation may contribute to poor outcomes in clinical practice from insufficient treatment. Patients with type 3 choroidal NV have particularly high risk of retinal atrophy, an unexplained observation. Herein we show in mouse models that VEGF signaling does not contribute to photoreceptor survival and functioning: (a) neutralization of VEGFR2 strongly suppresses choroidal NV without compromising photoreceptor function or survival; (b) VEGF does not slow loss of photoreceptor function or death in mice with inherited retinal degeneration, and there is no exacerbation by VEGF suppression; and (c) mice with type 3 choroidal NV develop retinal atrophy due to oxidative damage with no contribution from VEGF suppression. Intraocular injections of VEGF-neutralizing proteins, a highly effective treatment in patients with neovascular AMD, should not be withheld or reduced due to concern that they may contribute to long-term visual loss from retinal atrophy.
Da Long, Yogita Kanan, Jikui Shen, Sean F. Hackett, Yuanyuan Liu, Zibran Hafiz, Mahmood Khan, Lili Lu, Peter A. Campochiaro
Excessive hepatic glucose production (HGP) contributes significantly to the hyperglycemia of type 2 diabetes; however, the molecular mechanism underlying this dysregulation remains poorly understood. Here, we show that fasting temporally increases the expression of H19 long noncoding RNA (lncRNA) in nondiabetic mouse liver, whereas its level is chronically elevated in diet-induced diabetic mice, consistent with the previously reported chronic hepatic H19 increase in diabetic patients. Importantly, liver-specific H19 overexpression promotes HGP, hyperglycemia, and insulin resistance, while H19 depletion enhances insulin-dependent suppression of HGP. Using genome-wide methylation and transcriptome analyses, we demonstrate that H19 knockdown in hepatic cells alters promoter methylation and expression of Hnf4a, a master gluconeogenic transcription factor, and that this regulation is recapitulated in vivo. Our findings offer a mechanistic explanation of lncRNA H19’s role in the pathogenesis of diabetic hyperglycemia and suggest that targeting hepatic H19 may hold the potential of new treatment for this disease.
Na Zhang, Tingting Geng, Zhangsheng Wang, Ruling Zhang, Tiefeng Cao, Joao Paulo Camporez, Shi-Ying Cai, Ya Liu, Luisa Dandolo, Gerald I. Shulman, Gordon G. Carmichael, Hugh S. Taylor, Yingqun Huang
Extensive kidney fibrosis occurs in several types of chronic kidney diseases. PBI-4050, a potentially novel first-in-class orally active low–molecular weight compound, has antifibrotic and antiinflammatory properties. We examined whether PBI-4050 affected the progression of diabetic nephropathy (DN) in a mouse model of accelerated type 2 diabetes and in a model of selective tubulointerstitial fibrosis. eNOS–/– db/db mice were treated with PBI-4050 from 8–20 weeks of age (early treatment) or from 16–24 weeks of age (late treatment). PBI-4050 treatment ameliorated the fasting hyperglycemia and abnormal glucose tolerance tests seen in vehicle-treated mice. In addition, PBI-4050 preserved (early treatment) or restored (late treatment) blood insulin levels and increased autophagy in islets. PBI-4050 treatment led to significant improvements in lifespan in the diabetic mice. Both early and late PBI-4050 treatment protected against progression of DN, as indicated by reduced histological glomerular injury and albuminuria, slow decline of glomerular filtration rate, and loss of podocytes. PBI-4050 inhibited kidney macrophage infiltration, oxidative stress, and TGF-β–mediated fibrotic signaling pathways, and it also protected against the development of tubulointerstitial fibrosis. To confirm a direct antiinflammatory/antifibrotic effect in the kidney, further studies with a nondiabetic model of EGFR-mediated proximal tubule activation confirmed that PBI-4050 dramatically decreased the development of the associated tubulointerstitial injury and macrophage infiltration. These studies suggest that PBI-4050 attenuates development of DN in type 2 diabetes through improvement of glycemic control and inhibition of renal TGF-β–mediated fibrotic pathways, in association with decreases in macrophage infiltration and oxidative stress.
Yan Li, Sungjin Chung, Zhilian Li, Jessica M. Overstreet, Lyne Gagnon, Brigitte Grouix, Martin Leduc, Pierre Laurin, Ming-Zhi Zhang, Raymond C. Harris
Transmission-blocking vaccines (TBVs) are considered an integral element of malaria eradication efforts. Despite promising evaluations of Plasmodium falciparum Pfs25-based TBVs in mice, clinical trials have failed to induce robust and long-lived Ab titers, in part due to the poorly immunogenic nature of Pfs25. Using nonhuman primates, we demonstrate that multiple aspects of Pfs25 immunity were enhanced by antigen encapsulation in poly(lactic-co-glycolic acid)–based [(PLGA)-based] synthetic vaccine particles (SVP[Pfs25]) and potent TLR-based adjuvants. SVP[Pfs25] increased Ab titers, Pfs25-specific plasmablasts, circulating memory B cells, and plasma cells in the bone marrow when benchmarked against the clinically tested multimeric form Pfs25-EPA given with GLA-LSQ. SVP[Pfs25] also induced the first reported Pfs25-specific circulating Th1 and Tfh cells to our knowledge. Multivariate correlative analysis indicated several mechanisms for the improved Ab responses. While Pfs25-specific B cells were responsible for increasing Ab titers, T cell responses stimulated increased Ab avidity. The innate immune activation differentially stimulated by the adjuvants revealed a strong correlation between type I IFN polarization, induced by R848 and CpG, and increased Ab half-life and longevity. Collectively, the data identify ways to improve vaccine-induced immunity to poorly immunogenic proteins, both by the choice of antigen and adjuvant formulation, and highlight underlying immunological mechanisms.
Elizabeth A. Thompson, Sebastian Ols, Kazutoyo Miura, Kelly Rausch, David L. Narum, Mats Spångberg, Michal Juraska, Ulrike Wille-Reece, Amy Weiner, Randall F. Howard, Carole A. Long, Patrick E. Duffy, Lloyd Johnston, Conlin P. O’Neil, Karin Loré
Allogeneic stem cell transplantation (allo-SCT) can cure some patients with hematopoietic malignancy, but this relies on the development of a donor T cell alloreactive immune response. T cell activity in the first 2 weeks after allo-SCT is crucial in determining outcome, despite the clinical effects of the early alloreactive immune response often not appearing until later. However, the effect of the allogeneic environment on T cells is difficult to study at this time point due to the effects of profound lymphopenia. We approached this problem by comparing T cells at week 2 after allograft to T cells from autograft patients. Allograft T cells were present in small numbers but displayed intense proliferation with spontaneous cytokine production. Oligoclonal expansions at week 2 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation, including OX40L, TWEAK, and CD70. These findings reveal that recognition of alloantigen drives naive T cells toward a unique phenotype. Moreover, they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of targets for potential therapeutic immunomodulation.
Charlotte F. Inman, Suzy A. Eldershaw, Joanne E. Croudace, Nathaniel J. Davies, Archana Sharma-Oates, Tanuja Rai, Hayden Pearce, Mirjana Sirovica, Y.L. Tracey Chan, Kriti Verma, Jianmin Zuo, Sandeep Nagra, Francesca Kinsella, Jane Nunnick, Rasoul Amel-Kashipaz, Charles Craddock, Ram Malladi, Paul Moss
Myeloid leukocytes are essentially involved in both tumor progression and control. We show that neo-adjuvant treatment of mice with an inhibitor of CSF1 receptor (CSF1R), a drug that is used to deplete tumor-associated macrophages, unexpectedly promoted metastasis. CSF1R blockade indirectly diminished the number of NK cells due to a paucity of myeloid cells that provide the survival factor IL-15 to NK cells. Reduction of the number of NK cells resulted in increased seeding of metastatic tumor cells to the lungs but did not impact on progression of established metastases. Supplementation of mice treated with CSF1R-inhibitor with IL-15 restored numbers of NK cells and diminished metastasis. Our data suggest that CSF1R blockade should be combined with administration of IL-15 to reduce the risk of metastasis.
Michal Beffinger, Paulino Tallón de Lara, Sònia Tugues, Marijne Vermeer, Yannick Montagnolo, Isabel Ohs, Virginia Cecconi, Giulia Lucchiari, Aron Gagliardi, Nikola Misljencevic, James Sutton, Roman Spörri, Burkhard Becher, Anurag Gupta, Maries van den Broek
The excessive production of type I IFNs is a hallmark and a main pathogenic mechanism of many autoimmune diseases, including systemic lupus erythematosus (SLE). In these pathologies, the sustained secretion of type I IFNs is dependent on the improper activation of plasmacytoid DCs (pDCs) by self–nucleic acids. However, the nature and origin of pDC-activating self–nucleic acids is still incompletely characterized. Here, we report that exosomes isolated from the plasma of SLE patients can activate the secretion of IFN-α by human blood pDCs in vitro. This activation requires endosomal acidification and is recapitulated by microRNAs isolated from exosomes, suggesting that exosome-delivered microRNAs act as self-ligands of innate single-stranded endosomal RNA sensors. By using synthetic microRNAs, we identified an IFN induction motif that is responsible for the TLR7-dependent activation, maturation, and survival of human pDCs. These findings identify exosome-delivered microRNAs as potentially novel TLR7 endogenous ligands able to induce pDC activation in SLE patients. Therefore, microRNAs may represent novel pathogenic mediators in the onset of autoimmune reactions and potential therapeutic targets in the treatment of type I IFN–mediated diseases.
Valentina Salvi, Veronica Gianello, Sara Busatto, Paolo Bergese, Laura Andreoli, Ugo D’Oro, Alessandra Zingoni, Angela Tincani, Silvano Sozzani, Daniela Bosisio
Estimating the size of the viral reservoir is critical for HIV cure strategies. Biomarkers in peripheral circulation may give insights into the establishment of the viral reservoir in compartments not easily accessible. We therefore measured systemic levels of 84 soluble biomarkers belonging to a broad array of immune pathways in acute HIV infection in both antiretroviral therapy–naive (ART-naive) individuals as well as individuals who began ART upon early detection of HIV infection. These biomarkers were measured longitudinally during acute and chronic infection and their relationship to viral reservoir establishment and persistence was assessed. We observed several distinct biomarker pathways induced following HIV infection such as IFN-γ–signaled chemokines, proinflammatory markers, and TNF-α–family members. Levels of several of these factors directly correlated with contemporaneous viral loads and/or frequency of peripheral blood mononuclear cells harboring HIV DNA during acute HIV infection. MCP-1, MIP-3β, sTNFR-II, and IL-10 levels prior to ART associated with HIV DNA levels after 96 weeks of treatment, suggesting a link between early immune signaling events and the establishment and persistence of the viral reservoir during ART. Furthermore, they offer potentially novel tools for gaining insight into relative reservoir size in acutely infected individuals and the potential of associated risks of treatment interruption.
Jeffrey E. Teigler, Louise Leyre, Nicolas Chomont, Bonnie Slike, Ningbo Jian, Michael A. Eller, Nittaya Phanuphak, Eugène Kroon, Suteeraporn Pinyakorn, Leigh Anne Eller, Merlin L. Robb, Jintanat Ananworanich, Nelson L. Michael, Hendrik Streeck, Shelly J. Krebs, RV254/RV217 study groups
Chimeric antigen receptor–modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.
Dongrui Wang, Brenda Aguilar, Renate Starr, Darya Alizadeh, Alfonso Brito, Aniee Sarkissian, Julie R. Ostberg, Stephen J. Forman, Christine E. Brown
In response to collagen stimulation, platelets use a coordinated system of fluid entry to undergo membrane ballooning, procoagulant spreading, and microvesiculation. We hypothesized that water entry was mediated by the water channel aquaporin-1 (AQP1) and aimed to determine its role in the platelet procoagulant response and thrombosis. We established that human and mouse platelets express AQP1 and localize to internal tubular membrane structures. However, deletion of AQP1 had minimal effects on collagen-induced platelet granule secretion, aggregation, or membrane ballooning. Conversely, procoagulant spreading, microvesiculation, phosphatidylserine exposure, and clot formation time were significantly diminished. Furthermore, in vivo thrombus formation after FeCl3 injury to carotid arteries was also markedly suppressed in AQP1-null mice, but hemostasis after tail bleeding remained normal. The mechanism involves an AQP1-mediated rapid membrane stretching during procoagulant spreading but not ballooning, leading to calcium entry through mechanosensitive cation channels and a full procoagulant response. We conclude that AQP1 is a major regulator of the platelet procoagulant response, able to modulate coagulation after injury or pathologic stimuli without affecting other platelet functional responses or normal hemostasis. Clinically effective AQP1 inhibitors may therefore represent a novel class of antiprocoagulant antithrombotics.
Ejaife O. Agbani, Christopher M. Williams, Yong Li, Marion T.J. van den Bosch, Samantha F. Moore, Adele Mauroux, Lorna Hodgson, Alan S. Verkman, Ingeborg Hers, Alastair W. Poole
BACKGROUND. Heat shock protein peptide complex-96 (HSPPC-96) triggers adaptive and innate antitumor immune responses. The safety and efficacy of HSPPC-96 vaccination was examined in patients with newly diagnosed glioblastoma multiforme (GBM). METHODS. In this open-label, single-arm, phase I study, adult patients were vaccinated with HSPPC-96 in combination with the standard treatment for newly diagnosed GBM after surgical resection. Primary endpoints were frequency of adverse events and progression-free survival (PFS) at 6 months. Secondary endpoints included overall survival (OS), PFS, and tumor-specific immune response (TSIR). RESULTS. A total of 20 patients with newly diagnosed GBM were enrolled from September 2013 to February 2015. No grade 3 or 4 vaccine-related adverse events were noted. After a median follow-up of 42.3 months, PFS was 89.5% (95% CI, 66.9%–98.7%) at 6 months, median PFS was 11.0 months (95% CI, 8.2–13.8), and median OS was 31.4 months (95% CI, 14.9–47.9). TSIR was significantly increased by 2.3-fold (95% CI, 1.7–3.2) after vaccination. Median OS for patients with high TSIR after vaccination was >40.5 months (95% CI, incalculable) as compared with 14.6 months (95% CI, 7.0–22.2) for patients with low TSIR after vaccination (hazard ratio, 0.25; 95% CI, 0.071–0.90; P = 0.034). A multivariate Cox regression model revealed TSIR after vaccination as a primary independent predicator for survival. CONCLUSION. The HSPPC-96 vaccination, combined with the standard therapy, is a safe and effective strategy for treatment of newly diagnosed GBM patients. TSIR after vaccination would be a good indicator predicting the vaccine efficacy. TRIAL REGISTRATION. ClinicalTrials.gov NCT02122822. FUNDING. National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2014BAI04B01, 2014BAI04B02), Beijing Natural Science Foundation (7164253), Beijing Talents Fund (2014000021469G257), and Shenzhen Science and Technology Innovation Committee (JSGG20170413151359491).
Nan Ji, Yang Zhang, Yunpeng Liu, Jian Xie, Yi Wang, Shuyu Hao, Zhixian Gao
A dramatic increase in cardiac fatty acid oxidation occurs following birth. However, cardiac hypertrophy secondary to congenital heart diseases (CHDs) delays this process, thereby decreasing cardiac energetic capacity and function. Cardiac lysine acetylation is involved in modulating fatty acid oxidation. We thus investigated what effect cardiac hypertrophy has on protein acetylation during maturation. Eighty-four right ventricular biopsies were collected from CHD patients and stratified according to age and the absence (n = 44) or presence of hypertrophy (n = 40). A maturational increase in protein acetylation was evident in nonhypertrophied hearts but not in hypertrophied hearts. The fatty acid β-oxidation enzymes, long-chain acyl CoA dehydrogenase (LCAD) and β-hydroxyacyl CoA dehydrogenase (βHAD), were hyperacetylated and their activities positively correlated with their acetylation after birth in nonhypertrophied hearts but not hypertrophied hearts. In line with this, decreased cardiac fatty acid oxidation and reduced acetylation of LCAD and βHAD occurred in newborn rabbits subjected to cardiac hypertrophy due to an aortocaval shunt. Silencing the mRNA of general control of amino acid synthesis 5-like protein 1 reduced acetylation of LCAD and βHAD as well as fatty acid oxidation rates in cardiomyocytes. Thus, hypertrophy in CHDs prevents the postnatal increase in myocardial acetylation, resulting in a delayed maturation of cardiac fatty acid oxidation.
Arata Fukushima, Liyan Zhang, Alda Huqi, Victoria H. Lam, Sonia Rawat, Tariq Altamimi, Cory S. Wagg, Khushmol K. Dhaliwal, Lisa K. Hornberger, Paul F. Kantor, Ivan M. Rebeyka, Gary D. Lopaschuk
Studies in human peripheral blood monocyte–derived macrophages in vitro have shown clear evidence that multiple macrophage polarization states exist. The extent to which different alveolar macrophage (AM) polarization states exist in homeostasis or in the setting of severe injury such as acute respiratory distress syndrome (ARDS) is largely unknown. We applied single-cell cytometry TOF (CyTOF) to simultaneously measure 36 cell-surface markers on CD45+ cells present in bronchoalveolar lavage from healthy volunteers, as well as mechanically ventilated subjects with and without ARDS. Visualization of the high-dimensional data with the t-distributed stochastic neighbor embedding algorithm demonstrated wide diversity of cell-surface marker profiles among CD33+CD71+CD163+ AMs. We then used a κ-nearest neighbor density estimation algorithm to statistically identify distinct alveolar myeloid subtypes, and we discerned 3 AM subtypes defined by CD169 and PD-L1 surface expression. The percentage of AMs that were classified into one of the 3 AM subtypes was significantly different between healthy and mechanically ventilated subjects. In an independent cohort of subjects with ARDS, PD-L1 gene expression and PD-L1/PD-1 pathway–associated gene sets were significantly decreased in AMs from patients who experienced prolonged mechanical ventilation or death. Unsupervised CyTOF analysis of alveolar leukocytes from human subjects has potential to identify expected and potentially novel myeloid populations that may be linked with clinical outcomes.
Eric D. Morrell, Alice Wiedeman, S. Alice Long, Sina A. Gharib, T. Eoin West, Shawn J. Skerrett, Mark M. Wurfel, Carmen Mikacenic