Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of
Susanna Choi, Sungyong You, Donghyun Kim, Soo Youn Choi, H. Moo Kwon, Hyun-Sook Kim, Daehee Hwang, Yune-Jung Park, Chul-Soo Cho, Wan-Uk Kim
An intracellular complement system (ICS) has recently been described in immune and nonimmune human cells. This system can be activated in a convertase-independent manner from intracellular stores of the complement component C3. The source of these stores has not been rigorously investigated. In the present study, Western blotting identified a band corresponding to C3 in freshly isolated human peripheral blood cells that was absent in corresponding cell lines. One difference between native cells and cell lines was the time absent from a fluid-phase complement source; therefore, we hypothesized that loading C3 from plasma was a route of establishing intracellular C3 stores. We found that many types of human cells specifically internalized C3(H2O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H2O), indicating a specific mechanism of loading. Under steady-state conditions, approximately 80% of incorporated C3(H2O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3(H2O). The loaded C3(H2O) represents a source of C3a, and its uptake altered the cytokine profile of activated CD4+ T cells. Importantly, these results indicate that the impact of soluble plasma factors should be considered when performing in vitro studies assessing cellular immune function.
Michelle Elvington, M. Kathryn Liszewski, Paula Bertram, Hrishikesh S. Kulkarni, John P. Atkinson
Myocardial infarction (MI) results in the generation of dead cells in the infarcted area. These cells are swiftly removed by phagocytes to minimize inflammation and limit expansion of the damaged area. However, the types of cells and molecules responsible for the engulfment of dead cells in the infarcted area remain largely unknown. In this study, we demonstrated that cardiac myofibroblasts, which execute tissue fibrosis by producing extracellular matrix proteins, efficiently engulf dead cells. Furthermore, we identified a population of cardiac myofibroblasts that appears in the heart after MI in humans and mice. We found that these cardiac myofibroblasts secrete milk fat globule-epidermal growth factor 8 (MFG-E8), which promotes apoptotic engulfment, and determined that serum response factor is important for MFG-E8 production in myofibroblasts. Following MFG-E8–mediated engulfment of apoptotic cells, myofibroblasts acquired antiinflammatory properties. MFG-E8 deficiency in mice led to the accumulation of unengulfed dead cells after MI, resulting in exacerbated inflammatory responses and a substantial decrease in survival. Moreover, MFG-E8 administration into infarcted hearts restored cardiac function and morphology. MFG-E8–producing myofibroblasts mainly originated from resident cardiac fibroblasts and cells that underwent endothelial-mesenchymal transition in the heart. Together, our results reveal previously unrecognized roles of myofibroblasts in regulating apoptotic engulfment and a fundamental importance of these cells in recovery from MI.
Michio Nakaya, Kenji Watari, Mitsuru Tajima, Takeo Nakaya, Shoichi Matsuda, Hiroki Ohara, Hiroaki Nishihara, Hiroshi Yamaguchi, Akiko Hashimoto, Mitsuho Nishida, Akiomi Nagasaka, Yuma Horii, Hiroki Ono, Gentaro Iribe, Ryuji Inoue, Makoto Tsuda, Kazuhide Inoue, Akira Tanaka, Masahiko Kuroda, Shigekazu Nagata, Hitoshi Kurose
Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from the rupture of a blood vessel in the brain, leading to a mass of blood within the brain parenchyma. The injury causes a rapid inflammatory reaction that includes activation of the tissue-resident microglia and recruitment of blood-derived macrophages and other leukocytes. In this work, we investigated the specific responses of microglia following ICH with the aim of identifying pathways that may aid in recovery after brain injury. We used longitudinal transcriptional profiling of microglia in a murine model to determine the phenotype of microglia during the acute and resolution phases of ICH in vivo and found increases in TGF-β1 pathway activation during the resolution phase. We then confirmed that TGF-β1 treatment modulated inflammatory profiles of microglia in vitro. Moreover, TGF-β1 treatment following ICH decreased microglial
Roslyn A. Taylor, Che-Feng Chang, Brittany A. Goods, Matthew D. Hammond, Brian Mac Grory, Youxi Ai, Arthur F. Steinschneider, Stephen C. Renfroe, Michael H. Askenase, Louise D. McCullough, Scott E. Kasner, Michael T. Mullen, David A. Hafler, J. Christopher Love, Lauren H. Sansing
Fibrotic disease is associated with matrix deposition that results in the loss of organ function. Pericytes, the precursors of myofibroblasts, are a source of pathological matrix collagens and may be promising targets for treating fibrogenesis. Here, we have shown that pericytes activate a TLR2/4- and MyD88-dependent proinflammatory program in response to tissue injury. Similarly to classic immune cells, pericytes activate the NLRP3 inflammasome, leading to IL-1β and IL-18 secretion. Released IL-1β signals through pericyte MyD88 to amplify this response. Unexpectedly, we found that MyD88 and its downstream effector kinase IRAK4 intrinsically control pericyte migration and conversion to myofibroblasts. Specific ablation of MyD88 in pericytes or pharmacological inhibition of MyD88 signaling by an IRAK4 inhibitor in vivo protected against kidney injury by profoundly attenuating tissue injury, activation, and differentiation of myofibroblasts. Our data show that in pericytes, MyD88 and IRAK4 are key regulators of 2 major injury responses: inflammatory and fibrogenic. Moreover, these findings suggest that disruption of this MyD88-dependent pathway in pericytes might be a potential therapeutic approach to inhibit fibrogenesis and promote regeneration.
Irina A. Leaf, Shunsaku Nakagawa, Bryce G. Johnson, Jin Joo Cha, Kristen Mittelsteadt, Kevin M. Guckian, Ivan G. Gomez, William A. Altemeier, Jeremy S. Duffield
Tumor-associated macrophages (TAMs) can influence ovarian cancer growth, migration, and metastasis, but the detailed mechanisms underlying ovarian cancer metastasis remain unclear. Here, we have shown a strong correlation between TAM-associated spheroids and the clinical pathology of ovarian cancer. Further, we have determined that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in an established mouse model for epithelial ovarian cancer. M2 macrophage–like TAMs were localized in the center of spheroids and secreted EGF, which upregulated αMβ2 integrin on TAMs and ICAM-1 on tumor cells to promote association between tumor cells and TAM. Moreover, EGF secreted by TAMs activated EGFR on tumor cells, which in turn upregulated VEGF/VEGFR signaling in surrounding tumor cells to support tumor cell proliferation and migration. Pharmacological blockade of EGFR or antibody neutralization of ICAM-1 in TAMs blunted spheroid formation and ovarian cancer progression in mouse models. These findings suggest that EGF secreted from TAMs plays a critical role in promoting early transcoelomic metastasis of ovarian cancer. As transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our findings uncover a mechanism for TAM-mediated spheroid formation and provide a potential target for the treatment of ovarian cancer and other transcoelomic metastatic cancers.
Mingzhu Yin, Xia Li, Shu Tan, Huanjiao Jenny Zhou, Weidong Ji, Stefania Bellone, Xiaocao Xu, Haifeng Zhang, Alessandro D. Santin, Ge Lou, Wang Min
Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20–like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation.
Angela R.M. Kurz, Monika Pruenster, Ina Rohwedder, Mahalakshmi Ramadass, Kerstin Schäfer, Ute Harrison, Gabriel Gouveia, Claudia Nussbaum, Roland Immler, Johannes R. Wiessner, Andreas Margraf, Dae-Sik Lim, Barbara Walzog, Steffen Dietzel, Markus Moser, Christoph Klein, Dietmar Vestweber, Rainer Haas, Sergio D. Catz, Markus Sperandio
Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.
Christian C. Yost, Hansjörg Schwertz, Mark J. Cody, Jared A. Wallace, Robert A. Campbell, Adriana Vieira-de-Abreu, Claudia V. Araujo, Sebastian Schubert, Estelle S. Harris, Jesse W. Rowley, Matthew T. Rondina, James M. Fulcher, Curry L. Koening, Andrew S. Weyrich, Guy A. Zimmerman
Data from preclinical and clinical studies have demonstrated that granulocyte macrophage colony-stimulating factor (GM-CSF) can function as a key proinflammatory cytokine. However, therapies that directly target GM-CSF function could lead to undesirable side effects, creating a need to delineate downstream pathways and mediators. In this work, we provide evidence that GM-CSF drives CCL17 production by acting through an IFN regulatory factor 4–dependent (IRF4-dependent) pathway in human monocytes, murine macrophages, and mice in vivo. In murine models of arthritis and pain, IRF4 regulated the formation of CCL17, which mediated the proinflammatory and algesic actions of GM-CSF. Mechanistically, GM-CSF upregulated IRF4 expression by enhancing JMJD3 demethylase activity. We also determined that CCL17 has chemokine-independent functions in inflammatory arthritis and pain. These findings indicate that GM-CSF can mediate inflammation and pain by regulating IRF4-induced CCL17 production, providing insights into a pathway with potential therapeutic avenues for the treatment of inflammatory diseases and their associated pain.
Adrian Achuthan, Andrew D. Cook, Ming-Chin Lee, Reem Saleh, Hsu-Wei Khiew, Melody W.N. Chang, Cynthia Louis, Andrew J. Fleetwood, Derek C. Lacey, Anne D. Christensen, Ashlee T. Frye, Pui Yeng Lam, Hitoshi Kusano, Koji Nomura, Nancy Steiner, Irmgard Förster, Stephen L. Nutt, Moshe Olshansky, Stephen J. Turner, John A. Hamilton
ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol accumulation and alters T cell homeostasis, which may contribute to progression of atherosclerosis. Here, we investigated how the selective loss of ABCG1 in T cells impacts atherosclerosis in LDL receptor–deficient (LDLR-deficient) mice, a model of the disease. In LDLR-deficient mice fed a high-cholesterol diet, T cell–specific ABCG1 deficiency protected against atherosclerotic lesions. Furthermore, T cell–specific ABCG1 deficiency led to a 30% increase in Treg percentages in aorta and aorta-draining lymph nodes (LNs) of these mice compared with animals with only LDLR deficiency. When
Hsin-Yuan Cheng, Dalia E. Gaddis, Runpei Wu, Chantel McSkimming, LaTeira D. Haynes, Angela M. Taylor, Coleen A. McNamara, Mary Sorci-Thomas, Catherine C. Hedrick
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