BET proteins are associated with the induction of small airway fibrosis in COPD.

RATIONALE: In COPD, small airway fibrosis occurs due to increased extracellular matrix (ECM) deposition in and around the airway smooth muscle (ASM) layer. Studies of immune cells and peripheral lung tissue have shown that epigenetic changes occur in COPD but it is unknown whether airway mesenchymal cells are reprogrammed. OBJECTIVES: Determine if COPD ASM cells have a unique epigenetic response to profibrotic cytokine transforming growth factor ß1 (TGF-ß1). METHODS: Primary human ASM cells from COPD and non-COPD smoking patients were stimulated with TGF-ß1. Gene array analysis performed to identify differences in ECM expression. Airway accumulation of collagen 15α1 and tenascin-C proteins was assessed. Aforementioned ASM cells were stimulated with TGF-ß1 ± epigenetic inhibitors with qPCR quantification of COL15A1 and TNC. Global histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity were assessed. chromatin immunoprecipitation (ChIP)-qPCR for histone H3 and H4 acetylation at COL15A1 and TNC promoters was carried out. Effects of bromoterminal and extraterminal domain (BET) inhibitor JQ1(+) on expression and acetylation of ECM target genes were assessed. MEASUREMENTS AND MAIN RESULTS: COPD ASM show significantly higher COL15A1 and TNC expression in vitro and the same trend for higher levels of collagen 15α1 and tenascin-c deposited in COPD airways in vivo. Epigenetic screening indicated differential response to HDAC inhibition. ChIP-qPCR revealed histone H4 acetylation at COL15A1 and TNC promoters in COPD ASM only. ChIP-qPCR found JQ1(+) pretreatment significantly abrogated TGF-ß1 induced histone H4 acetylation at COL15A1 and TNC. CONCLUSIONS: BET protein binding to acetylated histones is important in TGF-ß1 induced expression of COL15A1 and TNC and maintenance of TGF-ß1 induced histone H4 acetylation in cell progeny.

TRF2-independent chromosome end protection during pluripotency.

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.

Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stress.

Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.

Lipid-Producing Ciliochoroidal Melanoma with Expression of HMG-CoA Reductase.

Uveal melanoma (UM) is the commonest primary intraocular malignancy in adults. There is limited published data on lipid production in UM. Here, we describe the clinical, histological, immunohistochemical, and molecular findings in a ciliochoroidal melanoma with lipid production and expression of the enzyme HMG-CoA reductase. This case highlights an unusual UM tumour phenotype with a high-risk molecular metastatic profile and discusses tumour lipogenesis and activation of the mevalonate pathway as a potential therapeutic target in managing lipidised ciliochoroidal UM.

CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle.

The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.

Telomere Loop Dynamics in Chromosome End Protection.

Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.

Antitumor cytotoxicity induced by bone-marrow-derived antigen-presenting cells is facilitated by the tumor suppressor protein p53 via regulation of IL-12.

Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c+ APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.

Differential neutrophil activation in viral infections: Enhanced TLR-7/8-mediated CXCL8 release in asthma.

BACKGROUND AND OBJECTIVE: Respiratory viral infections are a major cause of asthma exacerbations. Neutrophils accumulate in the airways and the mechanisms that link neutrophilic inflammation, viral infections and exacerbations are unclear. This study aims to investigate anti-viral responses in neutrophils from patients with and without asthma and to investigate if neutrophils can be directly activated by respiratory viruses. METHODS: Neutrophils from peripheral blood from asthmatic and non-asthmatic individuals were isolated and stimulated with lipopolysaccharide (LPS) (1 µg/mL), f-met-leu-phe (fMLP) (100 nM), imiquimod (3 µg/mL), R848 (1.5 µg/mL), poly I:C (10 µg/mL), RV16 (multiplicity of infection (MOI)1), respiratory syncytial virus (RSV) (MOI1) or influenza virus (MOI1). Cell-free supernatants were collected after 1 h of neutrophil elastase (NE) and matrix metalloproteinase (MMP)-9 release, or after 24 h for CXCL8 release. RESULTS: LPS, fMLP, imiquimod and R848 stimulated the release of CXCL8, NE and MMP-9 whereas poly I:C selectively induced CXCL8 release only. R848-induced CXCL8 release was enhanced in neutrophils from asthmatics compared with non-asthmatic cells (P < 0.01). RSV triggered the release of CXCL8 and NE from neutrophils, whereas RV16 or influenza had no effect. CONCLUSION: Neutrophils release CXCL8, NE and MMP-9 in response to viral surrogates with R848-induced CXCL8 release being specifically enhanced in asthmatic neutrophils. Toll-like receptor (TLR7/8) dysregulation may play a role in neutrophilic inflammation in viral-induced exacerbations.

Do we really need to keep redesigning ß2-agonists for the management of asthma?

There is an enormous drive to refine therapeutic designs and delivery systems, but in this review we ask if this is always the right direction? We choose to play devil's advocate, and argue that refining drug design is not always needed, and what is actually needed is a greater understanding of the biology of the disease. Here we focus on asthma and the ß2-agonist group of bronchodilators as an example of how a class of therapeutic has been developed and continues to be developmentally refined. In this review, we define viral-induced exacerbations as the greatest cause of lung attacks and the most crucial time ß2-agonist therapy is needed. We explore the reasons why ß2-agonist therapy fails in patients with rhinovirus-induced exacerbations, and explain why further "engineered" ß2-agonist therapies are likely to continue to fail in this subset of asthmatic population. We justify our perspective by returning to the biology that underlies the cause of disease and highlight the need for "more research" into alternative therapies for this population of asthmatic patients.

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated, but not rhinovirus infected, primary human airway smooth muscle.

BACKGROUND: Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE4) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations. METHODS: In the present study we investigated whether the PDE4 inhibitor piclamilast (10(-6) M) could alter RV or viral mimetic (5 µg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E2 and cAMP production were assayed by ELISA and RV replication was assayed by viral titration. RESULTS: We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines. CONCLUSION: By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE4 inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.

Characterising the mechanism of airway smooth muscle ß2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells.

Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to ß2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced ß2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused ß2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent ß2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause ß2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC ß2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that ß2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and ß2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused ß2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which ß2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2 induced prostaglandins.

Prostaglandins but not leukotrienes alter extracellular matrix protein deposition and cytokine release in primary human airway smooth muscle cells and fibroblasts.

Eicosanoids are lipid-signaling mediators released by many cells in response to various stimuli. Increasing evidence suggests that eicosanoids such as leukotrienes and prostaglandins (PGs) may directly mediate remodeling. In this study, we assessed whether these substances could alter extracellular matrix (ECM) proteins and the inflammatory profiles of primary human airway smooth muscle cells (ASM) and fibroblasts. PGE(2) decreased both fibronectin and tenascin C in fibroblasts but only fibronectin in ASM. PGD(2) decreased both fibronectin and tenascin C in both ASM and fibroblasts, whereas PGF(2α) had no effect on ECM deposition. The selective PGI(2) analog, MRE-269, decreased fibronectin but not tenascin C in both cell types. All the PGs increased IL-6 and IL-8 release in a dose-dependent manner in ASM and fibroblasts. Changes in ECM deposition and cytokine release induced by prostaglandins in both ASM and fibroblasts were independent of an effect on cell number. Neither the acute nor repeated stimulation with leukotrienes had an effect on the deposition of ECM proteins or cytokine release from ASM or fibroblasts. We concluded that, collectively, these results provide evidence that PGs may contribute to ECM remodeling to a greater extent than leukotrienes in airway cells.

Effects of ß(2) Agonists, Corticosteroids, and Novel Therapies on Rhinovirus-Induced Cytokine Release and Rhinovirus Replication in Primary Airway Fibroblasts.

Rhinovirus-(RV-) induced asthma exacerbations account for high asthma-related health costs and morbidity in Australia. The cellular mechanism underlying this pathology is likely the result of RV-induced nuclear-factor-kappa-B-(NF-κB-) dependent inflammation. NF-κB may also be important in RV replication as inhibition of NF-κB inhibits replication of other viruses such as human immunodeficiency virus and cytomegalovirus. To establish the role of NF-κB inhibitors in RV-induced IL- 6 and IL-8 and RV replication, we used pharmacological inhibitors of NF-κB, and steroids and/or ß(2) agonists were used for comparison. Primary human lung fibroblasts were infected with RV-16 in the presence of NF-κB inhibitors: BAY-117085 and dimethyl fumarate; ß(2) agonist: salmeterol; and/or corticosteroids: dexamethasone; fluticasone. RV-induced IL-6 and IL-8 and RV replication were assessed using ELISAs and virus titration assays. RV replicated and increased IL-6 and IL-8 release. Salmeterol increased, while dexamethasone and fluticasone decreased RV-induced IL-6 and IL-8 (P<0.05). The NF-κB inhibitor BAY-117085 inhibited only RV-induced IL-6 (P<0.05) and dimethyl fumarate did not alter RV-induced IL-6 and IL-8. Dimethylfumarate increased RV replication whilst other drugs did not alter RV replication. These data suggest that inhibition of NF-κB alone is unlikely to be an effective treatment compared to current asthma therapeutics.