Abstract
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 modulation of 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 multistage, aerodynamically favorable delivery platform that uses extracellular proteolysis to its advantage to deliver nanoparticle-embedded hydrophobic drugs to neutrophils within the lower airways. Our design consists of 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, proinflammatory 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.
Authors
Joscelyn C. Mejías, Osric A. Forrest, Camilla Margaroli, David A. Frey Rubio, Liliana Viera, Jindong Li, Xin Xu, Amit Gaggar, Rabindra Tirouvanziam, Krishnendu Roy
Abstract
Mitophagy, by maintaining mitochondrial quality control, plays a key role in maintaining kidney function and is impaired in pathologic states. Macrophages are well known for their pathogenic role in kidney fibrosis. Here, we report that PINK1/Parkin-mediated mitophagy in macrophages is compromised in experimental and human kidney fibrosis. We demonstrate downregulation of mitophagy regulators mitofusin-2 (MFN2) and Parkin downstream of PINK1 in kidney fibrosis. Loss of either Pink1 or Prkn promoted renal extracellular matrix accumulation and frequency of profibrotic/M2 macrophages. Pink1–/– or Prkn–/– BM-derived macrophages (BMDMs) showed enhanced expression of rictor. Mitochondria from TGF-β1–treated Pink1–/– BMDMs exhibited increased superoxide levels, along with reduced respiration and ATP production. In addition, mitophagy in macrophages involves PINK1-mediated phosphorylation of downstream MFN2, MFN2-facilitated recruitment of Parkin to damaged mitochondria, and macrophage-specific deletion of Mfn2 aggravates kidney fibrosis. Moreover, mitophagy regulators were downregulated in human CKD kidney and TGF-β1–treated human renal macrophages, whereas Mdivi1 treatment suppressed mitophagy mediators and promoted fibrotic response. Taken together, our study is the first to our knowledge to demonstrate that macrophage mitophagy plays a protective role against kidney fibrosis via regulating the PINK1/MFN2/Parkin-mediated pathway.
Authors
Divya Bhatia, Kuei-Pin Chung, Kiichi Nakahira, Edwin Patino, Michelle C. Rice, Lisa K. Torres, Thangamani Muthukumar, Augustine M.K. Choi, Oleh M. Akchurin, Mary E. Choi
Abstract
The purpose of this study was to define the mechanism by which cardiac neuraxial decentralization or spinal cord stimulation (SCS) reduces ischemia-induced ventricular fibrillation (VF). Direct measurements of norepinephrine (NE) levels in the left ventricular interstitial fluid (ISF) by microdialysis, in response to transient (15-minute) coronary artery occlusion (CAO), were performed in anesthetized canines. Responses were studied in animals with intact neuraxes and were compared with those in which the intrathoracic component of the cardiac neuraxes (stellate ganglia) or the intrinsic cardiac neuronal (ICN) system was surgically delinked from the central nervous system and those with intact neuraxes with preemptive SCS (T1–T3). With intact neuraxes, animals with exaggerated NE release due to CAO were at increased risk for VF. During CAO, there was a 152% increase in NE when the neuraxes were intact compared with 114% following stellate decentralization and 16% following ICN decentralization. During SCS, CAO NE levels increased by 59%. Risk for CAO-induced VF was 38% in controls, 8% following decentralization, and 11% following SCS. These data indicate that ischemia-related afferent neuronal transmission differentially engages central and intrathoracic sympathetic reflexes and amplifies sympathoexcitation. Differences in regional ventricular NE release are associated with increased risk for VF. Surgical decentralization or SCS reduced NE release and VF.
Authors
Jeffrey L. Ardell, Robert D. Foreman, J. Andrew Armour, Kalyanam Shivkumar
Abstract
Diabetic foot ulcers (DFUs) are a life-threatening disease that often results in lower limb amputations and a shortened life span. Current treatment options are limited and often not efficacious, raising the need for new therapies. To investigate the therapeutic potential of topical statins to restore healing in patients with DFUs, we performed next-generation sequencing on mevastatin-treated primary human keratinocytes. We found that mevastatin activated and modulated the EGF signaling to trigger an antiproliferative and promigratory phenotype, suggesting that statins may shift DFUs from a hyperproliferative phenotype to a promigratory phenotype in order to stimulate healing. Furthermore, mevastatin induced a migratory phenotype in primary human keratinocytes through EGF-mediated activation of Rac1, resulting in actin cytoskeletal reorganization and lamellipodia formation. Interestingly, the EGF receptor is downregulated in tissue biopsies from patients with DFUs. Mevastatin restored EGF signaling in DFUs through disruption of caveolae to promote keratinocyte migration, which was confirmed by caveolin-1 (Cav1) overexpression studies. We conclude that topical statins may have considerable therapeutic potential as a treatment option for patients with DFUs and offer an effective treatment for chronic wounds that can be rapidly translated to clinical use.
Authors
Andrew P. Sawaya, Ivan Jozic, Rivka C. Stone, Irena Pastar, Andjela N. Egger, Olivera Stojadinovic, George D. Glinos, Robert S. Kirsner, Marjana Tomic-Canic
Abstract
Angiogenesis is a key process that allows nutrient uptake and cellular trafficking and is coopted in cancer to enable tumor growth and metastasis. Recently, extracellular vesicles (EVs) have been shown to promote angiogenesis; however, it is unclear what unique features EVs contribute to the process. Here, we studied the role of EVs derived from head and neck squamous cell carcinoma (HNSCC) in driving tumor angiogenesis. Small EVs (SEVs), in the size range of exosomes (50–150 nm), induced angiogenesis both in vitro and in vivo. Proteomic analysis of HNSCC SEVs revealed the cell-to-cell signaling receptor ephrin type B receptor 2 (EPHB2) as a promising candidate cargo to promote angiogenesis. Analysis of patient data further identified EPHB2 overexpression in HNSCC tumors to be associated with poor patient prognosis and tumor angiogenesis, especially in the context of overexpression of the exosome secretion regulator cortactin. Functional experiments revealed that EPHB2 expression in SEVs regulated angiogenesis both in vitro and in vivo and that EPHB2 carried by SEVs stimulates ephrin-B reverse signaling, inducing STAT3 phosphorylation. A STAT3 inhibitor greatly reduced SEV-induced angiogenesis. These data suggest a model in which EVs uniquely promote angiogenesis by transporting Eph transmembrane receptors to nonadjacent endothelial cells to induce ephrin reverse signaling.
Authors
Shinya Sato, Suhas Vasaikar, Adel Eskaros, Young Kim, James S. Lewis, Bing Zhang, Andries Zijlstra, Alissa M. Weaver
Abstract
Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case-control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first 2 years of life, and 129 healthy matched controls, we found the cytomegalovirus-stimulated (CMV-stimulated) IFN-γ response to be associated with CD8+ T cell activation (Spearman’s rho, P = 6 × 10–8). A CMV-specific IFN-γ response was also associated with increased risk of developing TB disease (conditional logistic regression; P = 0.043; OR, 2.2; 95% CI, 1.02–4.83) and shorter time to TB diagnosis (Log Rank Mantel-Cox, P = 0.037). CMV+ infants who developed TB disease had lower expression of NK cell–associated gene signatures and a lower frequency of CD3–CD4–CD8– lymphocytes. We identified transcriptional signatures predictive of TB disease risk among CMV ELISpot–positive (area under the receiver operating characteristic [AUROC], 0.98, accuracy, 92.57%) and –negative (AUROC, 0.9; accuracy, 79.3%) infants; the CMV– signature was validated in an independent infant study (AUROC, 0.71; accuracy, 63.9%). A 16-gene signature that previously identified adolescents at risk of developing TB disease did not accurately classify case and control infants in this study. Understanding the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.
Authors
Julius Müller, Rachel Tanner, Magali Matsumiya, Margaret A. Snowden, Bernard Landry, Iman Satti, Stephanie A. Harris, Matthew K. O’Shea, Lisa Stockdale, Leanne Marsay, Agnieszka Chomka, Rachel Harrington-Kandt, Zita-Rose Manjaly Thomas, Vivek Naranbhai, Elena Stylianou, Stanley Kimbung Mbandi, Mark Hatherill, Gregory Hussey, Hassan Mahomed, Michele Tameris, J. Bruce McClain, Thomas G. Evans, Willem A. Hanekom, Thomas J. Scriba, Helen McShane, Helen A. Fletcher
Abstract
Dietary salt consumption leads to cutaneous Na+ storage and is associated with various disorders, including osteopenia. Here, we explore the impact of Na+ and the osmoprotective transcription factor nuclear factor of activated T cell 5 (NFAT5) on bone density and osteoclastogenesis. Compared with treatment of mice with high-salt diet, low-salt diet (LSD) increased bone density, decreased osteoclast numbers, and elevated Na+ content and Nfat5 levels in the BM. This response to LSD was dependent on NFAT5 expressed in myeloid cells. Simulating in vivo findings, we exposed osteoclast precursors and osteoblasts to elevated Na+ content (high-salt conditions; HS¢), resulting in increased NFAT5 binding to the promotor region of RANKL decoy receptor osteoprotegerin (OPG). These data not only demonstrate that NFAT5 in myeloid cells determines the Na+ content in BM, but that NFAT5 is able to govern the expression of the osteoprotective gene OPG. This provides insights into mechanisms of Na+-induced cessation of osteoclastogenesis and offers potentially new targets for treating salt-induced osteopenia.
Authors
Agnes Schröder, Patrick Neubert, Jens Titze, Aline Bozec, Wolfgang Neuhofer, Peter Proff, Christian Kirschneck, Jonathan Jantsch
Abstract
Observations in transgenic α-Klotho (Kl) mice (KlTg) defined the antiaging role of soluble Klotho (sKL130). A genetic translocation that elevates sKL levels in humans is paradoxically associated with increased circulating fibroblast growth factor 23 (FGF23) levels and the potential of both membrane KL (mKL135) and sKL130 to act as coreceptors for FGF23 activation of fibroblast growth factor receptors (FGFRs). Neither FGF23 expression nor the contributions of FGF23, mKL135, and sKL130 codependent and independent functions have been investigated in KlTg mice. In the current study, we examined the effects of Kl overexpression on FGF23 levels and functions in KlTg mice. We found that mKL135 but not sKL130 stimulated FGF23 expression in osteoblasts, leading to elevated Fgf23 bone expression and circulating levels in KlTg mice. Elevated FGF23 suppressed 1,25(OH)2D and parathyroid hormone levels but did not cause hypophosphatemic rickets in KlTg mice. KlTg mice developed low aldosterone–associated hypertension but not left ventricular hypertrophy. Mechanistically, we found that mKL135 and sKL130 are essential cofactors for FGF23-mediated ERK activation but that they inhibited FGF23 stimulation of PLC-γ and PI3K/AKT signaling. Thus, increased longevity in KlTg mice occurs in the presence of excess FGF23 that interacts with mKL and sKL to bias FGFR pathways.
Authors
Zhousheng Xiao, Gwendalyn King, Salvatore Mancarella, Undral Munkhsaikhan, Li Cao, Chun Cai, Leigh Darryl Quarles
Abstract
The MEK1/2–ERK1/2 pathway has been implicated in regulating the inflammatory response to lung injury and infection, and pharmacologic MEK1/2 inhibitor compounds are reported to reduce detrimental inflammation in multiple animal models of disease, in part through modulation of leukocyte responses. However, the specific contribution of myeloid MEK1 in regulating acute lung injury (ALI) and its resolution remain unknown. Here, the role of myeloid Mek1 was investigated in a murine model of LPS-induced ALI (LPS-ALI) by genetic deletion using the Cre-floxed system (LysMCre × Mekfl), and human alveolar macrophages from healthy volunteers and patients with acute respiratory distress syndrome (ARDS) were obtained to assess activation of the MEK1/2–ERK1/2 pathway. Myeloid Mek1 deletion results in a failure to resolve LPS-ALI, and alveolar macrophages lacking MEK1 had increased activation of MEK2 and the downstream target ERK1/2 on day 4 of LPS-ALI. The clinical significance of these findings is supported by increased activation of the MEK1/2–ERK1/2 pathway in alveolar macrophages from patients with ARDS compared with alveolar macrophages from healthy volunteers. This study reveals a critical role for myeloid MEK1 in promoting resolution of LPS-ALI and controlling the duration of macrophage proinflammatory responses.
Authors
Matthew E. Long, Ke-Qin Gong, William E. Eddy, Joseph S. Volk, Eric D. Morrell, Carmen Mikacenic, T. Eoin West, Shawn J. Skerrett, Jean Charron, W. Conrad Liles, Anne M. Manicone
Abstract
NK cells contribute to protective antitumor immunity, but little is known about the functional states of NK cells in human solid tumors. To address this issue, we performed single-cell RNA-seq analysis of NK cells isolated from human melanoma metastases, including lesions from patients who had progressed following checkpoint blockade. This analysis identified major differences in the transcriptional programs of tumor-infiltrating compared with circulating NK cells. Tumor-infiltrating NK cells represented 7 clusters with distinct gene expression programs indicative of significant functional specialization, including cytotoxicity and chemokine synthesis programs. In particular, NK cells from 3 clusters expressed high levels of XCL1 and XCL2, which encode 2 chemokines known to recruit XCR1+ cross-presenting DCs into tumors. In contrast, NK cells from 2 other clusters showed a higher level of expression of cytotoxicity genes. These data reveal key features of NK cells in human tumors and identify NK cell populations with specialized gene expression programs.
Authors
Lucas Ferrari de Andrade, Yuheng Lu, Adrienne Luoma, Yoshinaga Ito, Deng Pan, Jason W. Pyrdol, Charles H. Yoon, Guo-Cheng Yuan, Kai W. Wucherpfennig
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