In this study we evaluated the role of hyaluronan (HA) in reactive adipogenesis, a local expansion of preadipocytes that provides host defense by release of antimicrobial peptides. We observed that HA accumulated during maturation of adipocytes in vitro and was associated with increased expression of preadipocyte factor 1, zinc finger protein 423, and early B cell factor 1. Although HA is normally abundant in the extracellular matrix, a further increase in HA staining occurred in mice at sites of reactive adipogenesis following injury of colon by dextran sodium sulfate or injury of skin from infection with Staphylococcus aureus. HA also abundantly accumulated around adipocytes seen in the colons of patients with inflammatory bowel disease. This HA was necessary for adipocyte maturation because digestion of HA by administration of soluble hyaluronidase or transgenic expression of hyaluronidase 1 inhibited adipogenesis in vitro and in vivo. Furthermore, hyaluronidase also suppressed inflammation of both skin and colon and decreased antimicrobial peptide expression by developing preadipocytes. This resulted in increased bacterial transit across the epithelial barrier despite decreased tissue injury from inflammation. These observations suggest HA plays an important role in reactive adipogenesis and host defense after injury.
Tatsuya Dokoshi, Ling-juan Zhang, Teruaki Nakatsuji, Christopher A. Adase, James A. Sanford, Rudolph D. Paladini, Hiroki Tanaka, Mikihiro Fujiya, Richard L. Gallo
TGF-β signals through a receptor complex composed of 2 type I and 2 type II (TGF-βRII) subunits. We investigated the role of macrophage TGF-β signaling in fibrosis after AKI in mice with selective monocyte/macrophage TGF-βRII deletion (macrophage TGF-βRII–/– mice). Four weeks after injury, renal TGF-β1 expression and fibrosis were higher in WT mice than macrophage TGF-βRII–/– mice, which had decreased renal macrophages. The in vitro chemotactic response to f-Met-Leu-Phe was comparable between bone marrow–derived monocytes (BMMs) from WT and macrophage TGF-βRII–/– mice, but TGF-βRII–/– BMMs did not respond to TGF-β. We then implanted Matrigel plugs suffused with either f-Met-Leu-Phe or TGF-β1 into WT or macrophage TGF-βRII–/– mice. After 6 days, f-Met-Leu-Phe induced similar macrophage infiltration into the Matrigel plugs of WT and macrophage TGF-βRII–/– mice, but TGF-β induced infiltration only in WT mice. We further determined the number of labeled WT or TGF-βRII–/– BMMs infiltrating into WT kidneys 20 days after ischemic injury. There were more labeled WT BMMs than TGF-βRII–/– BMMs. Therefore, macrophage TGF-βRII deletion protects against the development of tubulointerstitial fibrosis following severe ischemic renal injury. Chemoattraction of macrophages to the injured kidney through a TGF-β/TGF-βRII axis is a heretofore undescribed mechanism by which TGF-β can mediate renal fibrosis during progressive renal injury.
Sungjin Chung, Jessica M. Overstreet, Yan Li, Yinqiu Wang, Aolei Niu, Suwan Wang, Xiaofeng Fan, Kensuke Sasaki, Guan-Nan Jin, Stellor Nlandu Khodo, Leslie Gewin, Ming-Zhi Zhang, Raymond C. Harris
Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.
Temo Barwari, Seda Eminaga, Ursula Mayr, Ruifang Lu, Paul C. Armstrong, Melissa V. Chan, Mahnaz Sahraei, Marta Fernández-Fuertes, Thomas Moreau, Javier Barallobre-Barreiro, Marc Lynch, Xiaoke Yin, Christian Schulte, Ferheen Baig, Raimund Pechlaner, Sarah R. Langley, Anna Zampetaki, Peter Santer, Martin Weger, Roberto Plasenzotti, Markus Schosserer, Johannes Grillari, Stefan Kiechl, Johann Willeit, Ajay M. Shah, Cedric Ghevaert, Timothy D. Warner, Carlos Fernández-Hernando, Yajaira Suárez, Manuel Mayr
IQ motif–containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffolding protein that integrates multiple cellular processes, including motility, adhesion, and proliferation, but its role in metabolism is unknown. Here, we show that IQGAP1 is induced upon fasting and regulates β-oxidation of fatty acids and synthesis of ketone bodies in the liver. IQGAP1-null (Iqgap1–/–) mice exhibit reduced hepatic PPARα transcriptional activity, as evidenced during fasting, after ketogenic diet, and upon pharmacological activation. Conversely, we found that the activity of fed-state sensor mTORC1 is enhanced in Iqgap1–/– livers, but acute inhibition of mTOR in Iqgap1–/– mice was unable to rescue the defect in ketone body synthesis. However, reexpressing IQGAP1 in the livers of Iqgap1–/– mice was sufficient to promote ketone body synthesis, increase PPARα signaling, and suppress mTORC1 activity. Taken together, we uncover what we believe to be a previously unidentified role for IQGAP1 in regulating PPARα activity and ketogenesis.
Hanna L. Erickson, Sayeepriyadarshini Anakk
Immune checkpoint blockade has achieved significant therapeutic success for a subset of cancer patients; however, a large portion of cancer patients do not respond. Unresponsive tumors are characterized as being immunologically “cold,” indicating that these tumors lack tumor antigen-specific primed cytotoxic T cells. Sitravatinib is a spectrum-selective tyrosine kinase inhibitor targeting TAM (TYRO3, AXL, MerTK) and split tyrosine-kinase domain–containing receptors (VEGFR and PDGFR families and KIT) plus RET and MET, targets that contribute to the immunosuppressive tumor microenvironment. We report that sitravatinib has potent antitumor activity by targeting the tumor microenvironment, resulting in innate and adaptive immune cell changes that augment immune checkpoint blockade. These results suggest that sitravatinib has the potential to combat resistance to immune checkpoint blockade and expand the number of cancer patients that are responsive to immune therapy.
Wenting Du, Huocong Huang, Noah Sorrelle, Rolf A. Brekken
Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma.
Valerie Fong, Austin Hsu, Esther Wu, Agnieszka P. Looney, Previn Ganesan, Xin Ren, Dean Sheppard, Sarah A. Wicher, Michael A. Thompson, Rodney D. Britt Jr., Y.S. Prakash, Mallar Bhattacharya
Pulmonary fibrosis and emphysema are irreversible chronic events after inhalation injury. However, the mechanism(s) involved in their development remain poorly understood. Higher levels of plasma and lung heme have been recorded in acute lung injury associated with several insults. Here, we provide the molecular basis for heme-induced chronic lung injury. We found elevated plasma heme in chronic obstructive pulmonary disease (COPD) (GOLD stage 4) patients and also in a ferret model of COPD secondary to chronic cigarette smoke inhalation. Next, we developed a rodent model of chronic lung injury, where we exposed C57BL/6 mice to the halogen gas, bromine (Br2) (400 ppm, 30 minutes), and returned them to room air resulting in combined airway fibrosis and emphysematous phenotype, as indicated by high collagen deposition in the peribronchial spaces, increased lung hydroxyproline concentrations, and alveolar septal damage. These mice also had elevated pulmonary endoplasmic reticulum (ER) stress as seen in COPD patients; the pharmacological or genetic diminution of ER stress in mice attenuated Br2-induced lung changes. Finally, treating mice with the heme-scavenging protein, hemopexin, reduced plasma heme, ER stress, airway fibrosis, and emphysema. This is the first study to our knowledge to report elevated heme in COPD patients and establishes heme scavenging as a potential therapy after inhalation injury.
Saurabh Aggarwal, Israr Ahmad, Adam Lam, Matthew A. Carlisle, Changzhao Li, J. Michael Wells, S. Vamsee Raju, Mohammad Athar, Steven M. Rowe, Mark T. Dransfield, Sadis Matalon
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited ataxia caused by expansion of a translated CAG repeat encoding a glutamine tract in the ataxin-1 (ATXN1) protein. Despite advances in understanding the pathogenesis of SCA1, there are still no therapies to alter its progressive fatal course. RNA-targeting approaches have improved disease symptoms in preclinical rodent models of several neurological diseases. Here, we investigated the therapeutic capability of an antisense oligonucleotide (ASO) targeting mouse Atxn1 in Atxn1154Q/2Q-knockin mice that manifest motor deficits and premature lethality. Following a single ASO treatment at 5 weeks of age, mice demonstrated rescue of these disease-associated phenotypes. RNA-sequencing analysis of genes with expression restored to WT levels in ASO-treated Atxn1154Q/2Q mice was used to demonstrate molecular differences between SCA1 pathogenesis in the cerebellum and disease in the medulla. Finally, select neurochemical abnormalities detected by magnetic resonance spectroscopy in vehicle-treated Atxn1154Q/2Q mice were reversed in the cerebellum and brainstem (a region containing the pons and the medulla) of ASO-treated Atxn1154Q/2Q mice. Together, these findings support the efficacy and therapeutic importance of directly targeting ATXN1 RNA expression as a strategy for treating both motor deficits and lethality in SCA1.
Jillian Friedrich, Holly B. Kordasiewicz, Brennon O’Callaghan, Hillary P. Handler, Carmen Wagener, Lisa Duvick, Eric E. Swayze, Orion Rainwater, Bente Hofstra, Michael Benneyworth, Tessa Nichols-Meade, Praseuth Yang, Zhao Chen, Judit Perez Ortiz, H. Brent Clark, Gülin Öz, Sarah Larson, Huda Y. Zoghbi, Christine Henzler, Harry T. Orr
Cancer incidence increases with age, but paradoxically, cancers have been found to grow more quickly in young mice compared with aged ones. The cause of differential tumor growth has been debated and, over time, attributed to faster tumor cell proliferation, decreased tumor cell apoptosis, and/or increased angiogenesis in young animals. Despite major advances in our understanding of tumor immunity over the past 2 decades, little attention has been paid to comparing immune cell populations in young and aged mice. Using mouse colon adenocarcinoma model MC38 implanted in young and mature mice, we show that age substantially influences the number of tumor-infiltrating cytotoxic CD8+ T cells, which control cancer progression. The different tumor growth pace in young and mature mice was abrogated in RAG1null mice, which lack mature T and B lymphocytes, and upon selective depletion of endogenous CD8+ cells. Transcriptome analysis further indicated that young mice have decreased levels of the Itga4 gene (CD49d, VLA-4) in tumor-infiltrating lymphocytes when compared with mature mice. Hypothesizing that VLA-4 can have a tumor-protective effect, we depleted the protein, which resulted in accelerated tumor growth in mature mice. These observations may explain the paradoxical growth rates observed in murine cancers, point to the central role of VLA-4 in controlling tumor growth, and open new venues to therapeutic manipulation.
Juhyun Oh, Angela Magnuson, Christophe Benoist, Mikael J. Pittet, Ralph Weissleder
Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Dietary interventions based on protein restriction (PR) reduce circulating triglycerides (TGs), but underlying mechanisms and clinical relevance remain unclear. Here, we show that 1 week of a protein-free diet without enforced calorie restriction significantly lowered circulating TGs in both lean and diet-induced obese mice. Mechanistically, the TG-lowering effect of PR was due, in part, to changes in very low–density lipoprotein (VLDL) metabolism both in liver and peripheral tissues. In the periphery, PR stimulated VLDL-TG consumption by increasing VLDL-bound APOA5 expression and promoting VLDL-TG hydrolysis and clearance from circulation. The PR-mediated increase in Apoa5 expression was controlled by the transcription factor CREBH, which coordinately regulated hepatic expression of fatty acid oxidation–related genes, including Fgf21 and Ppara. The CREBH-APOA5 axis activation upon PR was intact in mice lacking the GCN2-dependent amino acid–sensing arm of the integrated stress response. However, constitutive hepatic activation of the amino acid–responsive kinase mTORC1 compromised CREBH activation, leading to blunted APOA5 expression and PR-recalcitrant hypertriglyceridemia. PR also contributed to hypotriglyceridemia by reducing the rate of VLDL-TG secretion, independently of activation of the CREBH-APOA5 axis. Finally, a randomized controlled clinical trial revealed that 4–6 weeks of reduced protein intake (7%–9% of calories) decreased VLDL particle number, increased VLDL-bound APOA5 expression, and lowered plasma TGs, consistent with mechanistic conservation of PR-mediated hypotriglyceridemia in humans with translational potential as a nutraceutical intervention for dyslipidemia.
J. Humberto Treviño-Villarreal, Justin S. Reynolds, Alexander Bartelt, P. Kent Langston, Michael R. MacArthur, Alessandro Arduini, Valeria Tosti, Nicola Veronese, Beatrice Bertozzi, Lear E. Brace, Pedro Mejia, Kaspar Trocha, Gustavo S. Kajitani, Alban Longchamp, Eylul Harputlugil, Rose Gathungu, Susan S. Bird, Arnold D. Bullock, Robert S. Figenshau, Gerald L. Andriole, Andrew Thompson, Jöerg Heeren, C. Keith Ozaki, Bruce S. Kristal, Luigi Fontana, James R. Mitchell
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