Nonalcoholic fatty liver disease (NAFLD), a metabolic predisposition for development of hepatocellular carcinoma (HCC), represents a disease spectrum ranging from steatosis to steatohepatitis to cirrhosis. Acox1, a rate-limiting enzyme in peroxisomal fatty acid β-oxidation, regulates metabolism, spontaneous hepatic steatosis, and hepatocellular damage over time. However, it is unknown whether Acox1 modulates inflammation relevant to NAFLD pathogenesis or if Acox1-associated metabolic and inflammatory derangements uncover and accelerate potential for NAFLD progression. Here, we show that mice with a point mutation in Acox1 (Acox1Lampe1) exhibited altered cellular metabolism, modified T cell polarization, and exacerbated immune cell inflammatory potential. Further, in context of a brief obesogenic diet stress, NAFLD progression associated with Acox1 mutation resulted in significantly accelerated and exacerbated hepatocellular damage via induction of profound histological changes in hepatocytes, hepatic inflammation, and robust upregulation of gene expression associated with HCC development. Collectively, these data demonstrate that β-oxidation links metabolism and immune responsiveness and that a better understanding of peroxisomal β-oxidation may allow for discovery of mechanisms central for NAFLD progression.
Maria E. Moreno-Fernandez, Daniel A. Giles, Traci E. Stankiewicz, Rachel Sheridan, Rebekah Karns, Monica Cappelletti, Kristin Lampe, Rajib Mukherjee, Christian Sina, Anthony Sallese, James P. Bridges, Simon P. Hogan, Bruce J. Aronow, Kasper Hoebe, Senad Divanovic
A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) — an IL-6 receptor blocker — would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.
Ciriana Orabona, Giada Mondanelli, Maria T. Pallotta, Agostinho Carvalho, Elisa Albini, Francesca Fallarino, Carmine Vacca, Claudia Volpi, Maria L. Belladonna, Maria G. Berioli, Giulia Ceccarini, Susanna M.R. Esposito, Raffaella Scattoni, Alberto Verrotti, Alessandra Ferretti, Giovanni De Giorgi, Sonia Toni, Marco Cappa, Maria C. Matteoli, Roberta Bianchi, Davide Matino, Alberta Iacono, Matteo Puccetti, Cristina Cunha, Silvio Bicciato, Cinzia Antognelli, Vincenzo N. Talesa, Lucienne Chatenoud, Dietmar Fuchs, Luc Pilotte, Benoît Van den Eynde, Manuel C. Lemos, Luigina Romani, Paolo Puccetti, Ursula Grohmann
While the treatment of inflammatory disorders is generally based on inhibiting factors that drive onset of inflammation, these therapies can compromise healing (NSAIDs) or dampen immunity against infections (biologics). In search of new antiinflammatories, efforts have focused on harnessing endogenous pathways that drive resolution of inflammation for therapeutic gain. Identification of specialized pro-resolving mediators (SPMs) (lipoxins, resolvins, protectins, maresins) as effector molecules of resolution has shown promise in this regard. However, their action on inflammatory resolution in humans is unknown. Here, we demonstrate using a model of UV-killed Escherichia coli–triggered skin inflammation that SPMs are biosynthesized at the local site at the start of resolution, coinciding with the expression of receptors that transduce their actions. These include receptors for lipoxin A4 (ALX/FPR2), resolvin E1 (ChemR23), resolvin D2 (GPR18), and resolvin D1 (GPR32) that were differentially expressed on the endothelium and infiltrating leukocytes. Administering SPMs into the inflamed site 4 hours after bacterial injection caused a reduction in PMN numbers over the ensuing 6 hours, the phase of active resolution in this model. These results indicate that in humans, the appearance of SPMs and their receptors is associated with the beginning of inflammatory resolution and that their therapeutic supplementation enhanced the resolution response.
Madhur P. Motwani, Romain A. Colas, Marc J. George, Julia D. Flint, Jesmond Dalli, Angela Richard-Loendt, Roel P.H. De Maeyer, Charles N. Serhan, Derek W. Gilroy
Alterations in ectopic lipid deposition and circulating lipids are major risk factors for developing cardiometabolic diseases. Angiopoietin-like protein 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL), controls fatty acid (FA) uptake in adipose and oxidative tissues and regulates circulating triacylglycerol-rich (TAG-rich) lipoproteins. Unfortunately, global depletion of ANGPTL4 results in severe metabolic abnormalities, inflammation, and fibrosis when mice are fed a high-fat diet (HFD), limiting our understanding of the contribution of ANGPTL4 in metabolic disorders. Here, we demonstrate that genetic ablation of ANGPTL4 in adipose tissue (AT) results in enhanced LPL activity, rapid clearance of circulating TAGs, increased AT lipolysis and FA oxidation, and decreased FA synthesis in AT. Most importantly, we found that absence of ANGPTL4 in AT prevents excessive ectopic lipid deposition in the liver and muscle, reducing novel PKC (nPKC) membrane translocation and enhancing insulin signaling. As a result, we observed a remarkable improvement in glucose tolerance in short-term HFD-fed AT-specific Angptl4-KO mice. Finally, lack of ANGPTL4 in AT enhances the clearance of proatherogenic lipoproteins, attenuates inflammation, and reduces atherosclerosis. Together, these findings uncovered an essential role of AT ANGPTL4 in regulating peripheral lipid deposition, influencing whole-body lipid and glucose metabolism and the progression of atherosclerosis.
Binod Aryal, Abhishek K. Singh, Xinbo Zhang, Luis Varela, Noemi Rotllan, Leigh Goedeke, Balkrishna Chaube, Joao-Paulo Camporez, Daniel F. Vatner, Tamas L. Horvath, Gerald I. Shulman, Yajaira Suárez, Carlos Fernández-Hernando
Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr–/– mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.
Wen Ding, Keyvan Yousefi, Stefania Goncalves, Bradley J. Goldstein, Alfonso L. Sabater, Amy Kloosterboer, Portia Ritter, Guerline Lambert, Armando J. Mendez, Lina A. Shehadeh
Hypertrophic cardiomyopathy (HCM) stems from mutations in sarcomeric proteins that elicit distinct biophysical sequelae, which in turn may yield radically different intracellular signaling and molecular pathologic profiles. These signaling events remain largely unaddressed by clinical trials that have selected patients based on clinical HCM diagnosis, irrespective of genotype. In this study, we determined how two mouse models of HCM differ, with respect to cellular/mitochondrial function and molecular biosignatures, at an early stage of disease. We show that hearts from young R92W-TnT and R403Q-αMyHC mutation–bearing mice differ in their transcriptome, miRNome, intracellular redox environment, mitochondrial antioxidant defense mechanisms, and susceptibility to mitochondrial permeability transition pore opening. Pathway analysis of mRNA-sequencing data and microRNA profiles indicate that R92W-TnT mutants exhibit a biosignature consistent with activation of profibrotic TGF-β signaling. Our results suggest that the oxidative environment and mitochondrial impairment in young R92W-TnT mice promote activation of TGF-β signaling that foreshadows a pernicious phenotype in young individuals. Of the two mutations, R92W-TnT is more likely to benefit from anti–TGF-β signaling effects conferred by angiotensin receptor blockers and may be responsive to mitochondrial antioxidant strategies in the early stage of disease. Molecular and functional profiling may therefore serve as aids to guide precision therapy for HCM.
Styliani Vakrou, Ryuya Fukunaga, D. Brian Foster, Lars Sorensen, Yamin Liu, Yufan Guan, Kirubel Woldemichael, Roberto Pineda-Reyes, Ting Liu, Jill C. Tardiff, Leslie A. Leinwand, Carlo G. Tocchetti, Theodore P. Abraham, Brian O’Rourke, Miguel A. Aon, M. Roselle Abraham
Ischemic stroke is a leading cause of morbidity and mortality in the US; however, there currently exists only one effective acute pharmacological therapeutic intervention. Purinergic signaling has been shown to regulate vascular function and pathological processes, including inflammation and arterial myogenic reactivity, and plays a role in ischemic stroke outcome. Purinergic signaling requires extracellular purines; however, the mechanism of purine release from cells is unclear. Pannexin1 (Panx1) channels are potentially novel purine release channels expressed throughout the vascular tree that couples regulated purine release with purinergic signaling. Therefore, we examined the role of smooth muscle and endothelial cell Panx1, using conditional cell type–specific transgenic mice, in cerebral ischemia/reperfusion injury outcomes. Deletion of endothelial cell Panx1, but not smooth muscle cell Panx1, significantly reduced cerebral infarct volume after ischemia/reperfusion. Infiltration of leukocytes into brain tissue and development of cerebral myogenic tone were both significantly reduced when mice lacked endothelial Panx1. Panx1 regulation of myogenic tone was unique to the cerebral circulation, as mesenteric myogenic reactivity and blood pressure were independent of endothelial Panx1. Overall, deletion of endothelial Panx1 mitigated cerebral ischemic injury by reducing inflammation and myogenic tone development, indicating that endothelial Panx1 is a possible novel target for therapeutic intervention of ischemic stroke.
Miranda E Good, Stephanie A. Eucker, Jun Li, Hannah M. Bacon, Susan M. Lang, Joshua T. Butcher, Tyler J. Johnson, Ronald P. Gaykema, Manoj K. Patel, Zhiyi Zuo, Brant E. Isakson
Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell–derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid–phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.
David A. Slatter, Charles L. Percy, Keith Allen-Redpath, Joshua M. Gajsiewicz, Nick J. Brooks, Aled Clayton, Victoria J. Tyrrell, Marcela Rosas, Sarah N. Lauder, Andrew Watson, Maria Dul, Yoel Garcia-Diaz, Maceler Aldrovandi, Meike Heurich, Judith Hall, James H. Morrissey, Sebastien Lacroix-Desmazes, Sandrine Delignat, P. Vincent Jenkins, Peter W. Collins, Valerie B. O’Donnell
TNF and granulocyte macrophage-colony stimulating factor (GM-CSF) have proinflammatory activity and both contribute, for example, to rheumatoid arthritis pathogenesis. We previously identified a new GM-CSF→JMJD3 demethylase→interferon regulatory factor 4 (IRF4)→CCL17 pathway that is active in monocytes/macrophages in vitro and important for inflammatory pain, as well as for arthritic pain and disease. Here we provide evidence for a nexus between TNF and this pathway, and for TNF and GM-CSF interdependency. We report that the initiation of zymosan-induced inflammatory pain and zymosan-induced arthritic pain and disease are TNF dependent. Once arthritic pain and disease are established, blockade of GM-CSF or CCL17, but not of TNF, is still able to ameliorate them. TNF is required for GM-CSF–driven inflammatory pain and for initiation of GM-CSF–driven arthritic pain and disease, but not once they are established. TNF-driven inflammatory pain and TNF-driven arthritic pain and disease are dependent on GM-CSF and mechanistically require the same downstream pathway involving GM-CSF→CCL17 formation via JMJD3-regulated IRF4 production, indicating that GM-CSF and CCL17 can mediate some of the proinflammatory and algesic actions of TNF. Given we found that TNF appears important only early in arthritic pain and disease progression, targeting a downstream mediator, such as CCL17, which appears to act throughout the course of disease, could be effective at ameliorating chronic inflammatory conditions where TNF is implicated.
Andrew D. Cook, Ming-Chin Lee, Reem Saleh, Hsu-Wei Khiew, Anne D. Christensen, Adrian Achuthan, Andrew J. Fleetwood, Derek C. Lacey, Julia E. Smith, Irmgard Förster, John A. Hamilton
Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E373–374A). Further cleavage by MMPs (N341–342F) releases a 32-amino-acid aggrecan fragment (32-mer). We investigated the role of this 32-mer in driving joint pain. We found that the 32-mer excites dorsal root ganglion nociceptive neurons, both in culture and in intact explants. Treatment of cultured sensory neurons with the 32-mer induced expression of the proalgesic chemokine CCL2. These effects were mediated through TLR2, which we demonstrated was expressed by nociceptive neurons. In addition, intra-articular injection of the 32-mer fragment provoked knee hyperalgesia in WT but not Tlr2-null mice. Blocking the production or action of the 32-mer in transgenic mice prevented the development of knee hyperalgesia in a murine model of osteoarthritis. These findings suggest that the aggrecan 32-mer fragment directly activates TLR2 on joint nociceptors and is an important mediator of the development of osteoarthritis-associated joint pain.
Rachel E. Miller, Shingo Ishihara, Phuong B. Tran, Suzanne B. Golub, Karena Last, Richard J. Miller, Amanda J. Fosang, Anne-Marie Malfait
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