FOXP3+ Regulatory T Cells Require TBET to Regulate Activated CD8+ T Cells During Recovery from Influenza Infection.

FOXP3+ regulatory T (Treg) cells are necessary to coordinate resolution of lung inflammation and a return to homeostasis after respiratory viral infections, but the specific molecular requirements for these functions and the cell types governed by Treg cells remain unclear. This question holds significance as clinical trials of Treg cell transfer therapy for respiratory viral infection are being planned and executed. Here, we report causal experiments in mice determining that Treg cells are necessary to control the numbers of activated CD8+ T cells during recovery from influenza infection. Using a genetic strategy paired with adoptive transfer techniques, we determined that Treg cells require the transcription factor TBET to regulate these potentially pro-inflammatory CD8+ T cells. Surprisingly, we found that Treg cells are dispensable for the generation of CD8+ lung tissue resident-memory T (Trm) cells yet similarly influence the transcriptional programming of CD8+ Trm and activated T cells. Our study highlights the role of Treg cells in regulating the CD8+ T cell response during recovery from influenza infection.

AMP-activated protein kinase is necessary for Treg cell functional adaptation to microenvironmental stress.

CD4+FOXP3+ regulatory T (Treg) cells maintain self-tolerance, suppress the immune response to cancer, and protect against tissue injury in the lung and other organs. Treg cells require mitochondrial metabolism to exert their function, but how Treg cells adapt their metabolic programs to sustain and optimize their function during an immune response occurring in a metabolically stressed microenvironment remains unclear. Here, we tested whether Treg cells require the energy homeostasis-maintaining enzyme AMP-activated protein kinase (AMPK) to adapt to metabolically aberrant microenvironments caused by malignancy or lung injury, finding that AMPK is dispensable for Treg cell immune-homeostatic function but is necessary for full Treg cell function in B16 melanoma tumors and during acute lung injury caused by influenza virus pneumonia. AMPK-deficient Treg cells had lower mitochondrial mass and exhibited an impaired ability to maximize aerobic respiration. Mechanistically, we found that AMPK regulates DNA methyltransferase 1 to promote transcriptional programs associated with mitochondrial function in the tumor microenvironment. In the lung during viral pneumonia, we found that AMPK sustains metabolic homeostasis and mitochondrial activity. Induction of DNA hypomethylation was sufficient to rescue mitochondrial mass in AMPK-deficient Treg cells, linking DNA methylation with AMPK function and mitochondrial metabolism. These results define AMPK as a determinant of Treg cell adaptation to metabolic stress and offer potential therapeutic targets in cancer and tissue injury.

Differential roles of regulatory T cells in acute respiratory infections.

Acute respiratory infections trigger an inflammatory immune response with the goal of pathogen clearance; however, overexuberant inflammation causes tissue damage and impairs pulmonary function. CD4+FOXP3+ regulatory T cells (Tregs) interact with cells of both the innate and the adaptive immune system to limit acute pulmonary inflammation and promote its resolution. Tregs also provide tissue protection and coordinate lung tissue repair, facilitating a return to homeostatic pulmonary function. Here, we review Treg-mediated modulation of the host response to respiratory pathogens, focusing on mechanisms underlying how Tregs promote resolution of inflammation and repair of acute lung injury. We also discuss potential strategies to harness and optimize Tregs as a cellular therapy for patients with severe acute respiratory infection and discuss open questions in the field.

Author Correction: TRAF3 regulates the oncogenic proteins Pim2 and c-Myc to restrain survival in normal and malignant B cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TRAF3 regulates the oncogenic proteins Pim2 and c-Myc to restrain survival in normal and malignant B cells.

TRAF3 is a versatile intracellular adapter protein with multiple context-specific roles. Uniquely in B cells, TRAF3 deficiency enhances survival and increases the risk of transformation, as loss of TRAF3 is observed in several types of B cell cancers. Here, we report a new mechanism for TRAF3 in the restraint of B cell survival. We found that TRAF3 deficiency was associated with induction of the pro-survival kinase Pim2 in mouse primary B cells and human malignant B cell lines. The increase in Pim2 was independent of NF-κB2 activation but was ameliorated with inhibition of STAT3 expression or function. TRAF3 deficiency also led to a Pim2-dependent increase in c-Myc protein levels and was associated with reduced c-Myc ubiquitination. TRAF3-deficient primary B cells were less sensitive to cell death induced by the Pim inhibitors SGI-1776 and TP-3654. Interestingly, human malignant B cell lines with low expression of TRAF3 were more sensitive to Pim inhibition-induced cell death. Combination treatment of TRAF3-deficient B cells and B cell tumor lines with c-Myc inhibitors enhanced their sensitivity to Pim inhibition, suggesting a possible therapeutic strategy. TRAF3 thus suppresses a Pim2-mediated B cell survival axis, which can be a potential target for treatment of B cell malignancies.

The oncogenic membrane protein LMP1 sequesters TRAF3 in B-cell lymphoma cells to produce functional TRAF3 deficiency.

Loss-of-function mutations in genes encoding the signaling protein tumor necrosis factor receptor-associated factor 3 (TRAF3) are commonly found in human B-cell malignancies, especially multiple myeloma and B-cell lymphoma (BCL). B-cell TRAF3 deficiency results in enhanced cell survival, elevated activation receptor signaling, and increased activity of certain transcriptional pathways regulating expression of prosurvival proteins. A recent analysis of TRAF3 protein staining of ∼300 human BCL tissue samples revealed that a higher proportion of samples expressing the oncogenic Epstein-Barr virus-encoded protein latent membrane protein 1 (LMP1) showed low/negative TRAF3 staining than predicted. LMP1, a dysregulated mimic of the CD40 receptor, binds TRAF3 more effectively than CD40. We hypothesized that LMP1 may sequester TRAF3, reducing its availability to inhibit prosurvival signaling pathways in the B cell. This hypothesis was addressed via 2 complementary approaches: (1) comparison of TRAF3-regulated activation and survival-related events with relative LMP1 expression in human BCL lines and (2) analysis of the impact upon such events in matched pairs of mouse BCL lines, both parental cells and subclones transfected with inducible LMP1, either wild-type LMP1 or a mutant LMP1 with defective TRAF3 binding. Results from both approaches showed that LMP1-expressing B cells display a phenotype highly similar to that of B cells lacking TRAF3 genes, indicating that LMP1 can render B cells functionally TRAF3 deficient without TRAF3 gene mutations, a finding of significant relevance to selecting pathway-targeted therapies for B-cell malignancies.

TRAF3 deficiency promotes metabolic reprogramming in B cells.

The adaptor protein TNF receptor-associated factor 3 (TRAF3) is a critical regulator of B lymphocyte survival. B cell-specific TRAF3 deficiency results in enhanced viability and is associated with development of lymphoma and multiple myeloma. We show that TRAF3 deficiency led to induction of two proteins important for glucose metabolism, Glut1 and Hexokinase 2 (HXK2). This was associated with increased glucose uptake. In the absence of TRAF3, anaerobic glycolysis and oxidative phosphorylation were increased in B cells without changes in mitochondrial mass or reactive oxygen species. Chemical inhibition of glucose metabolism or glucose deprivation substantially attenuated the enhanced survival of TRAF3-deficient B cells, with a decrease in the pro-survival protein Mcl-1. Changes in Glut1 and Mcl-1 levels, glucose uptake and B cell number in the absence of TRAF3 were all dependent upon NF-κB inducing kinase (NIK). These results indicate that TRAF3 deficiency suffices to metabolically reprogram B cells, a finding that improves our understanding of the role of TRAF3 as a tumor suppressor, and suggests potential therapeutic strategies.

Nuclear TRAF3 is a negative regulator of CREB in B cells.

The adaptor protein TNF receptor-associated factor 3 (TRAF3) regulates signaling through B-lymphocyte receptors, including CD40, BAFF receptor, and Toll-like receptors, and also plays a critical role inhibiting B-cell homoeostatic survival. Consistent with these findings, loss-of-function human TRAF3 mutations are common in B-cell cancers, particularly multiple myeloma and B-cell lymphoma. B cells of B-cell-specific TRAF3(-/-) mice (B-Traf3(-/-)) display remarkably enhanced survival compared with littermate control (WT) B cells. The mechanism for this abnormal homeostatic survival is poorly understood, a key knowledge gap in selecting optimal treatments for human B-cell cancers with TRAF3 deficiency. We show here for the first time to our knowledge that TRAF3 is a resident nuclear protein that associates with the transcriptional regulator cAMP response element binding protein (CREB) in both mouse and human B cells. The TRAF-C domain of TRAF3 was necessary and sufficient to localize TRAF3 to the nucleus via a functional nuclear localization signal. CREB protein was elevated in TRAF3(-/-) B cells, without change in mRNA, but with a decrease in CREB ubiquitination. CREB-mediated transcriptional activity was increased in TRAF3-deficient B cells. Consistent with these findings, Mcl-1, an antiapoptotic target of CREB-mediated transcription, was increased in the absence of TRAF3 and enhanced Mcl-1 was suppressed with CREB inhibition. TRAF3-deficient B cells were also preferentially sensitive to survival inhibition with pharmacologic CREB inhibitor. Our results identify a new mechanism by which nuclear TRAF3 regulates B-cell survival via inhibition of CREB stability, information highly relevant to the role of TRAF3 in B-cell malignancies.

HABP2 is a Novel Regulator of Hyaluronan-Mediated Human Lung Cancer Progression.

BACKGROUND: Lung cancer is a devastating disease with limited treatment options. Many lung cancers have changes in their microenvironment including upregulation of the extracellular matrix glycosaminoglycan, hyaluronan (HA), which we have previously demonstrated can regulate the activity of the extracellular serine protease, hyaluronan binding protein 2 (HABP2). This study examined the functional role of HABP2 on HA-mediated human lung cancer dynamics. METHODS: Immunohistochemical analysis was performed on lung cancer patient samples using anti-HABP2 antibody. Stable control, shRNA, and HABP2 overexpressing human lung adenocarcinoma cells were evaluated using immunoblot analysis, migration, extravasation, and urokinase plasminogen activator (uPA) activation assays with or without high-molecular weight HA or low-molecular weight HA (LMW-HA). In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs. RESULTS: We provide evidence that HABP2 is an important regulator of lung cancer progression. HABP2 expression was increased in several subtypes of patient non-small cell lung cancer samples. Further, HABP2 overexpression increased LMW-HA-induced uPA activation, migration, and extravasation in human lung adenocarcinoma cells. In vivo, overexpression of HABP2 in human lung adenocarcinoma cells increased primary tumor growth rates in nude mice by ~2-fold and lung metastasis by ~10-fold compared to vector control cells (n = 5/condition). CONCLUSION: Our data suggest a possible direct effect of HABP2 on uPA activation and lung cancer progression. Our observations suggest that exploration of HABP2 in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

Transactivation of the receptor-tyrosine kinase ephrin receptor A2 is required for the low molecular weight hyaluronan-mediated angiogenesis that is implicated in tumor progression.

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression.

The novel role of the mu opioid receptor in lung cancer progression: a laboratory investigation.

BACKGROUND: The possibility that µ opioid agonists can influence cancer recurrence is a subject of recent interest. Epidemiologic studies suggested that there were differences in cancer recurrence in breast and prostate cancer contingent on anesthetic regimens. In this study, we identify a possible mechanism for these epidemiologic findings on the basis of µ opioid receptor (MOR) regulation of Lewis lung carcinoma (LLC) tumorigenicity in cell and animal models. METHODS: We used human lung tissue and human non-small cell lung cancer (NSCLC) cell lines and evaluated MOR expression using immunoblot and immunohistochemical analysis. LLC cells were treated with the peripheral opioid antagonist methylnaltrexone (MNTX) or MOR shRNA and evaluated for proliferation, invasion, and soft agar colony formation in vitro and primary tumor growth and lung metastasis in C57BL/6 and MOR knockout mice using VisEn fluorescence mediated tomography imaging and immunohistochemical analysis. RESULTS: We provide several lines of evidence that the MOR may be a potential target for lung cancer, a disease with high mortality and few treatment options. We first observed that there is ∼5- to 10-fold increase in MOR expression in lung samples from patients with NSCLC and in several human NSCLC cell lines. The MOR agonists morphine and [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin (DAMGO) increased in vitro LLC cell growth. Treatment with MNTX or silencing MOR expression inhibited LLC invasion and anchorage-independent growth by 50%-80%. Injection of MOR silenced LLC lead to a ∼65% reduction in mouse lung metastasis. In addition, MOR knockout mice do not develop significant tumors when injected with LLC in comparison with wild-type controls. Finally, continuous infusion of the peripheral opioid antagonist MNTX attenuates primary LLC tumor growth and reduces lung metastasis. CONCLUSIONS: Taken together, our data suggest a possible direct effect of opiates on lung cancer progression, and provide a plausible explanation for the epidemiologic findings. Our observations further suggest a possible therapeutic role for opioid antagonists.

High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness.

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, a perturbation observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that enhance EC barrier integrity have important therapeutic implications. We observed that binding of high-molecular-weight hyaluronan (HMW-HA) to its cognate receptor CD44 within caveolin-enriched microdomains (CEM) enhances human pulmonary EC barrier function. Immunocytochemical analysis indicated that HMW-HA promotes redistribution of a significant population of CEM to areas of cell-cell contact. Quantitative proteomic analysis of CEM isolated from human EC demonstrated HMW-HA-mediated recruitment of cytoskeletal regulatory proteins (annexin A2, protein S100-A10, and filamin A/B). Inhibition of CEM formation [caveolin-1 small interfering RNA (siRNA) and cholesterol depletion] or silencing (siRNA) of CD44, annexin A2, protein S100-A10, or filamin A/B expression abolished HMW-HA-induced actin cytoskeletal reorganization and EC barrier enhancement. To confirm our in vitro results in an in vivo model of inflammatory lung injury with vascular hyperpermeability, we observed that the protective effects of HMW-HA on LPS-induced pulmonary vascular leakiness were blocked in caveolin-1 knockout mice. Furthermore, targeted inhibition of CD44 expression in the mouse pulmonary vasculature significantly reduced HMW-HA-mediated protection from LPS-induced hyperpermeability. These data suggest that HMW-HA, via CD44-mediated CEM signaling events, represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.

Methylnaltrexone potentiates the anti-angiogenic effects of mTOR inhibitors.

BACKGROUND: Recent cancer therapies include drugs that target both tumor growth and angiogenesis including mammalian target of rapamycin (mTOR) inhibitors. Since mTOR inhibitor therapy is associated with significant side effects, we examined potential agents that can reduce the therapeutic dose. METHODS: Methylnaltrexone (MNTX), a peripheral mu opioid receptor (MOR) antagonist, in combination with the mTOR inhibitors temsirolimus and/or rapamycin, was evaluated for inhibition of VEGF-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration as well as in vivo angiogenesis (mouse Matrigel plug assay). RESULTS: MNTX inhibited VEGF-induced EC proliferation and migration with an IC50 of approximately 100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation and migration from approximately 10 nM to approximately 1 nM and from approximately 50 to approximately 10 nM respectively. We observed similar effects with rapamycin. On a mechanistic level, we observed that MNTX increased EC plasma membrane-associated tyrosine phosphate activity. Inhibition of tyrosine phosphatase activity (3,4-dephostatin) blocked the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR Complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR Complex 1 (rapamycin and temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events. CONCLUSIONS: Our data indicate that MNTX exerts a synergistic effect with rapamycin and temsirolimus on inhibition of VEGF-induced human EC proliferation and migration and in vivo angiogenesis. Therefore, addition of MNTX could potentially lower the dose of mTOR inhibitors which could improve therapeutic index.

Hyaluronic Acid binding protein 2 is a novel regulator of vascular integrity.

OBJECTIVE: The disruption of the endothelial cell barrier is a critical feature of inflammation and an important contributing factor to acute lung injury (ALI), an inflammatory condition that is a major cause of morbidity and mortality in critically ill patients. We evaluated the role of the extracellular serine protease, hyaluronic acid binding protein 2 (HABP2), in vascular barrier regulation. METHODS AND RESULTS: By using immunoblot and immunohistochemical analysis, we observed that lipopolysaccharide (LPS) induces HABP2 expression in murine lung endothelium in vivo and in human pulmonary microvascular endothelial cells (ECs) in vitro. High-molecular-weight hyaluronan (HMW-HA, approximately 1x10(6) Da) decreased HABP2 protein expression in human pulmonary microvascular ECs and decreased purified HABP2 enzymatic activity, whereas low-molecular-weight HA (LMW-HA, approximately 2500 Da) increased these activities. The effects of LMW-HA, but not HMW-HA, on HABP2 activity were inhibited with a peptide of the polyanion-binding domain of HABP2. Silencing (small interfering RNA) HABP2 expression augmented HMW-HA-induced EC barrier enhancement and inhibited LPS and LMW-HA-mediated EC barrier disruption, results that were reversed with overexpression of HABP2. Silencing protease-activated receptor 1 and 3, RhoA, or Rho kinase expression attenuated LPS-, LMW-HA-, and HABP2-mediated EC barrier disruption. By using murine models of acute lung injury, we observed that LPS- and ventilator-induced pulmonary vascular hyperpermeability was significantly reduced with vascular silencing (small interfering RNA) of HABP2. CONCLUSIONS: HABP2 negatively regulates vascular integrity via activation of protease-activated receptor/RhoA/Rho kinase signaling and represents a potentially useful therapeutic target for syndromes of increased vascular permeability.

Dynamin 2 and c-Abl are novel regulators of hyperoxia-mediated NADPH oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.

Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.