Lysosomes are at the epicenter of cellular processes critical for inflammasome activation in macrophages. Inflammasome activation and IL-1β secretion are implicated in myocardial infarction (MI) and resultant heart failure; however, little is known about how macrophage lysosomes regulate these processes. In mice subjected to cardiac ischemia/reperfusion (IR) injury and humans with ischemic cardiomyopathy, we observed evidence of lysosomal impairment in macrophages. Inducible macrophage-specific overexpression of transcription factor EB (TFEB), a master regulator of lysosome biogenesis (Mϕ-TFEB), attenuated postinfarction remodeling, decreased abundance of proinflammatory macrophages, and reduced levels of myocardial IL-1β compared with controls. Surprisingly, neither inflammasome suppression nor Mϕ-TFEB–mediated attenuation of postinfarction myocardial dysfunction required intact ATG5-dependent macroautophagy (hereafter termed “autophagy”). RNA-seq of flow-sorted macrophages postinfarction revealed that Mϕ-TFEB upregulated key targets involved in lysosomal lipid metabolism. Specifically, inhibition of the TFEB target, lysosomal acid lipase, in vivo abrogated the beneficial effect of Mϕ-TFEB on postinfarction ventricular function. Thus, TFEB reprograms macrophage lysosomal lipid metabolism to attenuate remodeling after IR, suggesting an alternative paradigm whereby lysosome function affects inflammation.
Ali Javaheri, Geetika Bajpai, Antonino Picataggi, Smrithi Mani, Layla Foroughi, Hosannah Evie, Attila Kovacs, Carla J. Weinheimer, Krzystztof Hyrc, Qingli Xiao, Andrea Ballabio, Jin-Moo Lee, Scot J. Matkovich, Babak Razani, Joel D. Schilling, Kory J. Lavine, Abhinav Diwan
Pulmonary drug delivery presents a unique opportunity to target lower airway inflammation, which is often characterized by the massive recruitment of neutrophils from blood. However specific therapies are lacking that can modulate airway neutrophil function, and difficult challenges must be overcome to achieve therapeutic efficacy against pulmonary inflammation, notably drug hydrophobicity, mucociliary and macrophage-dependent clearance, and high extracellular protease burden. Here, we present a multi-stage, aerodynamically favorable delivery platform that uses extracellular proteolysis to its advantage in order to deliver nanoparticle-embedded hydrophobic drugs to neutrophils within the lower airways. Our design consists in a self-regulated nanoparticle-in-microgel system, in which microgel activation is triggered by extracellular elastase (degranulated by inflammatory neutrophils), and nanoparticles are loaded with Nexinhib20, a potent neutrophil degranulation inhibitor. Successful in vivo delivery of Nexinhib20 to the airways and into neutrophils promoted resolution of the inflammatory response by dampening neutrophil recruitment and degranulation, pro-inflammatory cytokine production in both airway and systemic compartments, as well as the presence of neutrophil-derived pathological extracellular vesicles in the lung fluid. Our findings showcase a new platform that overcomes challenges in pulmonary drug delivery and allows customization to match the proteolytic footprint of given diseases.
Joscelyn C Mejías, Osric A Forrest, Camilla Margaroli, David A. Frey Rubio, Liliana Viera, Jindong Li, Xin Xu, Amit Gaggar, Rabindra Tirouvanziam, Krishnendu Roy
Efferocytosis, or phagocytic clearance of dead/dying cells by brain-resident microglia and/or infiltrating macrophages, is instrumental for inflammation resolution and restoration of brain homeostasis after stroke. Here, we identify the signal transducer and activator of transcription 6/arginase1 (STAT6/Arg1) signaling axis as a potentially novel mechanism that orchestrates microglia/macrophage responses in the ischemic brain. Activation of STAT6 was observed in microglia/macrophages in the ischemic territory in a mouse model of stroke and in stroke patients. STAT6 deficiency resulted in reduced clearance of dead/dying neurons, increased inflammatory gene signature in microglia/macrophages, and enlarged infarct volume early after experimental stroke. All of these pathological changes culminated in an increased brain tissue loss and exacerbated long-term functional deficits. Combined in vivo analyses using BM chimeras and in vitro experiments using microglia/macrophage-neuron cocultures confirmed that STAT6 activation in both microglia and macrophages was essential for neuroprotection. Adoptive transfer of WT macrophages into STAT6-KO mice reduced accumulation of dead neurons in the ischemic territory and ameliorated brain infarction. Furthermore, decreased expression of Arg1 in STAT6–/– microglia/macrophages was responsible for impairments in efferocytosis and loss of antiinflammatory modality. Our study suggests that efferocytosis via STAT6/Arg1 modulates microglia/macrophage phenotype, accelerates inflammation resolution, and improves stroke outcomes.
Wei Cai, Xuejiao Dai, Jie Chen, Jingyan Zhao, Mingyue Xu, Lili Zhang, Boyu Yang, Wenting Zhang, Marcelo Rocha, Toshimasa Nakao, Julia Kofler, Yejie Shi, R. Anne Stetler, Xiaoming Hu, Jun Chen
Platelet inositol hexakisphosphate kinase 1 (IP6K1) has been shown to control systemic inflammation. Herein, we examined if platelets and IP6K1 regulate pancreatic tissue injury via formation of NETs in experimental models of acute pancreatitis (AP) in mice. By use of electron microscopy abundant NET formation was observed in the inflamed pancreas. These NETs contained numerous microparticles (MP) expressing CD41 or Mac-1. Platelet depletion reduced deposition of NET-MP complexes in the inflamed pancreas. Circulating platelet-neutrophil aggregates (PNA) were increased and inhibition of P-selectin not only disrupted PNA formation but also reduced NETs formation in the inflamed pancreas. NETs depleted of MPs had lower capacity to provoke amylase secretion and STAT-3 phosphorylation in acinar cells. Taurocholate-induced NETs formation, inflammation and tissue damage in the pancreas were decreased in IP6K1-deficient mice. Thrombin stimulation of mixtures of wild-type platelets and neutrophils resulted in NETs formation but not when IP6K1-deficient platelets were incubated with wild-type neutrophils. Polyphosphate rescue restored thrombin-induced NET formation in mixtures of IP6K1-deficient platelets and wild-type neutrophils. Platelet IP6K1 regulates NET-MP complex formation in the pancreas of mice during induction of AP. Targeting platelet IP6K1 might useful to decrease NET-dependent pancreatic tissue inflammation and tissue injury in patients with AP.
Raed Madhi, Milladur Rahman, Dler Taha, Johan Linders, Mohammed Merza, Yongzhi Wang, Matthias Mörgelin, Henrik Thorlacius
Islet transplantation can restore lost glycemic control in type 1 diabetes subjects, but is restricted in its clinical application by limiting supplies of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signalling thresholds. Here we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for approximately 45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon regulatory T cells, was antigen-specific and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signalling, graft hyper-inflammation and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators with no impact on glucose stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signalling thresholds to favour immune tolerance and promotes islet allogeneic survival. Clinically this would allow for reduced immunosuppression and support the use of alternate islet sources.
Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey
Dysregulated proinflammatory cytokine release has been implicated in the pathogenesis of several life-threatening acute lung illnesses such as pneumonia, sepsis, and acute respiratory distress syndrome. Suppressors of cytokine signaling proteins, particularly SOCS2, have recently been described as antiinflammatory mediators. However, the regulation of SOCS2 protein has not been described. Here we describe a mechanism of SOCS2 regulation by the action of the ubiquitin E3 ligase KIAA0317. KIAA0317-mediated degradation of SOCS2 exacerbated inflammation in vitro, and depletion of KIAA0317 in vivo ameliorated pulmonary inflammation. KIAA0317-knockout mice exhibited resistance to LPS-induced pulmonary inflammation, while KIAA03017 reexpression mitigated this effect. We uncovered a small molecule inhibitor of KIAA0317 protein (BC-1365) that prevented SOCS2 degradation and attenuated LPS- and P. aeruginosa–induced lung inflammation in vivo. These studies show KIAA0317 to be a critical mediator of pulmonary inflammation through its degradation of SOCS2 and a potential candidate target for therapeutic inhibition.
Travis B. Lear, Alison C. McKelvey, John W. Evankovich, Shristi Rajbhandari, Tiffany A. Coon, Sarah R. Dunn, James D. Londino, Bryan J. McVerry, Yingze Zhang, Eleanor Valenzi, Christine L. Burton, Rachael Gordon, Sebastien Gingras, Karina C. Lockwood, Michael J. Jurczak, Robert Lafyatis, Mark J. Shlomchik, Yuan Liu, Bill B. Chen
miR-511-3p, encoded by CD206/Mrc1, was demonstrated to reduce allergic inflammation and promote alternative (M2) macrophage polarization. Here, we sought to elucidate the fundamental mechanism by which miR-511-3p attenuates allergic inflammation and promotes macrophage polarization. Compared with wild-type mice, the allergen-challenged Mrc1-/- mice showed increased airway hyper-responsiveness (AHR) and inflammation. However, this increased AHR and inflammation were significantly attenuated when these mice were pre-transduced with adeno-associated virus (AAV)-miR-511-3p. Gene expression profiling of macrophages identified Ccl2 as one of the major genes that was highly expressed in M2 macrophages but antagonized by miR-511-3p. The interaction between miR-511-3p and Ccl2 was confirmed by in silico analysis and mRNA-miRNA pull-down assay. Further evidence for the inhibition of Ccl2 by miR-511-3p was given by reduced levels of Ccl2 in supernatants of miR-511-3p transduced macrophages and in bronchoalveolar lavage fluids of AAV-miR-511-3p-infected Mrc1-/- mice. Mechanistically, we demonstrated that Ccl2 promotes M1 macrophage polarization by activating RhoA signaling through Ccr2. The interaction between Ccr2 and RhoA was also supported by co-immunoprecipitation assay. Importantly, inhibition of RhoA signaling suppressed cockroach allergen-induced AHR and lung inflammation. These findings suggest a novel mechanism by which miR-511-3p regulates allergic inflammation and macrophage polarization by targeting Ccl2 and its downstream Ccr2/RhoA axis.
Danh C. Do, Jie Mu, Xia Ke, Karan Sachdeva, Zili Qin, Mei Wan, Faoud T. Ishmael, Peisong Gao
Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVβ3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVβ3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVβ3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVβ3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVβ3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVβ3 and CD47 signaling in OA pathogenesis and suggest that activation of αVβ3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVβ3, CD47, and their signaling pathways as a disease-modifying therapy.
Qian Wang, Kazuhiro Onuma, Changhao Liu, Heidi Wong, Michelle S. Bloom, Eileen E. Elliott, Richard R.L. Cao, Nick Hu, Nithya Lingampalli, Orr Sharpe, Xiaoyan Zhao, Dong Hyun Sohn, Christin M. Lepus, Jeremy Sokolove, Rong Mao, Cecilia T. Cisar, Harini Raghu, Constance R. Chu, Nicholas J. Giori, Stephen B. Willingham, Susan S. Prohaska, Zhen Cheng, Irving L. Weissman, William H. Robinson
Dendritic cells (DCs) are crucial to balance protective immunity and autoimmune inflammatory processes. Expression of CD83 is a well-established marker for mature DCs although its physiological role is still not completely understood. Using a DC-specific CD83 conditional KO mouse (CD83ΔDC) we provide new insights into the function of CD83 within this cell type. Interestingly, CD83-deficient DCs produced drastically increased IL-2 levels and displayed higher expression of the co-stimulatory molecules CD25 and OX40L, which causes superior induction of antigen-specific T cell responses and compromises Treg suppressive functions. This also directly translates into accelerated immune responses in vivo. Upon Salmonella typhimurium and Listeria monocytogenes infection, CD83ΔDC mice cleared both pathogens more efficiently, and CD83-deficient DCs expressed increased IL-12 levels after bacterial encounter. Using the experimental autoimmune encephalomyelitis (EAE) model, autoimmune inflammation was dramatically aggravated in CD83ΔDC mice, while resolution of inflammation was strongly reduced. This phenotype was associated with increased cell influx into the CNS accompanied by elevated Th17 cell numbers. Concomitantly, CD83ΔDC mice had reduced Treg numbers in peripheral lymphoid organs. In summary, we show that CD83 ablation on DCs results in enhanced immune responses by dysregulating tolerance mechanisms and thereby impairing resolution of inflammation, which also demonstrates high clinical relevance.
Andreas B. Wild, Lena Krzyzak, Katrin Peckert, Lena Stich, Christine Kuhnt, Alina Butterhof, Christine Seitz, Jochen Mattner, Niklas Grüner, Maximilian Gänsbauer, Martin Purtak, Didier Soulat, Thomas H. Winkler, Lars Nitschke, Elisabeth Zinser, Alexander Steinkasserer
Background: Epicardial adipose tissue (EAT) is the visceral fat depot of the heart. Inflammation of EAT is thought to contribute to coronary artery disease (CAD). Therefore, we hypothesized that the EAT of patients with CAD would have increased inflammatory gene expression compared to controls without CAD. Methods: 26 patients referred for cardiac surgery with (n=13) or without CAD (n=13) were consented. Samples of EAT and subcutaneous adipose tissue (SAT) were obtained at the time of surgery. Gene expression analysis was performed using Affymetrix Human Gene 1.0 ST arrays. Differential regulation was defined as a 1.5 fold change (ANOVA p<0.05). Results: When comparing SAT and EAT of controls, 693 genes were differentially expressed. 805 genes were differentially expressed between SAT and EAT in cases. Expression of 326 genes was different between EAT of cases and controls; expression of 14 genes was increased in cases, while 312 were increased in controls. qRT-PCR confirmed that there was no difference in expression of major inflammatory genes (CCL2, CCR2, TNFα, IL6, IL8, PAI1) between cases and controls. Immunohistochemistry demonstrated that there were more macrophages in EAT than SAT, but that there was no difference in the number or activation state between cases and controls. Conclusion: In contrast to prior studies, we did not find increased inflammatory gene expression in the EAT of patients with CAD in comparison to controls without CAD. We conclude that specific adipose tissue organ, rather than CAD status, is responsible for the majority of differential gene expression.
Timothy P. Fitzgibbons, Nancy Lee, Khanh-Van Tran, Sara Nicoloro, Mark Kelly, Stanley K.C. Tam, Michael P. Czech
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