Upregulation of H3K27 Demethylase KDM6 During Respiratory Syncytial Virus Infection Enhances Proinflammatory Responses and Immunopathology.

Severe disease following respiratory syncytial virus (RSV) infection has been linked to enhanced proinflammatory cytokine production that promotes a Th2-type immune environment. Epigenetic regulation in immune cells following viral infection plays a role in the inflammatory response and may result from upregulation of key epigenetic modifiers. In this study, we show that RSV-infected bone marrow-derived dendritic cells (BMDC) as well as pulmonary dendritic cells (DC) from RSV-infected mice upregulated the expression of Kdm6b/Jmjd3 and Kdm6a/Utx, H3K27 demethylases. KDM6-specific chemical inhibition (GSK J4) in BMDC led to decreased production of chemokines and cytokines associated with the inflammatory response during RSV infection (i.e., CCL-2, CCL-3, CCL-5, IL-6) as well as decreased MHC class II and costimulatory marker (CD80/86) expression. RSV-infected BMDC treated with GSK J4 altered coactivation of T cell cytokine production to RSV as well as a primary OVA response. Airway sensitization of naive mice with RSV-infected BMDCs exacerbate a live challenge with RSV infection but was inhibited when BMDCs were treated with GSK J4 prior to sensitization. Finally, in vivo treatment with the KDM6 inhibitor, GSK J4, during RSV infection reduced inflammatory DC in the lungs along with IL-13 levels and overall inflammation. These results suggest that KDM6 expression in DC enhances proinflammatory innate cytokine production to promote an altered Th2 immune response following RSV infection that leads to more severe immunopathology.

Histone Methylation Directs Myeloid TLR4 Expression and Regulates Wound Healing following Cutaneous Tissue Injury.

Myeloid cells are critical for orchestrating regulated inflammation during wound healing. TLRs, particularly TLR4, and its downstream-signaling MyD88 pathway play an important role in regulating myeloid-mediated inflammation. Because an initial inflammatory phase is vital for tissue repair, we investigated the role of TLR4-regulated, myeloid-mediated inflammation in wound healing. In a cutaneous tissue injury murine model, we found that TLR4 expression is dynamic in wound myeloid cells during the course of normal wound healing. We identified that changes in myeloid TLR4 during tissue repair correlated with increased expression of the histone methyltransferase, mixed-lineage leukemia 1 (MLL1), which specifically trimethylates the histone 3 lysine 4 (H3K4me3) position of the TLR4 promoter. Furthermore, we used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+ ) to determine MLL1 drives Tlr4 expression during wound healing. To understand the critical role of myeloid-specific TLR4 signaling, we used mice deficient in Tlr4 (Tlr4-/- ), Myd88 (Myd88 -/-), and myeloid-specific Tlr4 (Tlr4f/fLyz2Cre+) to demonstrate delayed wound healing at early time points postinjury. Furthermore, in vivo wound myeloid cells isolated from Tlr4-/- and Myd88 -/- wounds demonstrated decreased inflammatory cytokine production. Importantly, adoptive transfer of monocyte/macrophages from wild-type mice trafficked to wounds with restoration of normal healing and myeloid cell function in Tlr4-deficient mice. These results define a role for myeloid-specific, MyD88-dependent TLR4 signaling in the inflammatory response following cutaneous tissue injury and suggest that MLL1 regulates TLR4 expression in wound myeloid cells.

Postnatal ß2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle.

KEY POINTS: Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity. Although whole-body glucose clearance is normal, 1-month-old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation. Impaired glucose-stimulated insulin secretion in IUGR lambs is due to lower intra-islet insulin availability and not from glucose sensing. We investigated adrenergic receptor (ADR) ß2 desensitization by administering oral ADRß modifiers for the first month after birth to activate ADRß2 and antagonize ADRß1/3. In IUGR lambs ADRß2 activation increased whole-body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies. IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose-stimulated insulin secretion and insulin-stimulated glucose oxidation, providing new insights into potential mechanisms for this risk. ABSTRACT: Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated ß adrenergic receptor (ADRß) desensitization. Our objectives were to measure insulin-sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRß2 agonist and ADRß1/ß3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose-stimulated insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR-AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole-body GUR were not different from controls. Of importance, ADRß2 stimulation with ß1/ß3 inhibition increases both insulin sensitivity and whole-body glucose utilization in IUGR lambs. In IUGR and IUGR-AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates and ex vivo skeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR-AR skeletal muscle than in controls but GLUT1 was greater in IUGR-AR. ADRß2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR-AR lambs heart rates were greater, which was independent of cardiac ADRß1 activation. We conclude that targeted ADRß2 stimulation improved whole-body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch-up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes.

Slow-Myofiber Commitment by Semaphorin 3A Secreted from Myogenic Stem Cells.

Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cell-membrane receptor (neuropilin2-plexinA3) → myogenin-myocyte enhancer factor 2D → slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropilin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fast-myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreERT2 -Sema3Afl °x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease. Stem Cells 2017;35:1815-1834.

Type I Interferon Induced Epigenetic Regulation of Macrophages Suppresses Innate and Adaptive Immunity in Acute Respiratory Viral Infection.

Influenza A virus (IAV) is an airborne pathogen that causes significant morbidity and mortality each year. Macrophages (Mϕ) are the first immune population to encounter IAV virions in the lungs and are required to control infection. In the present study, we explored the mechanism by which cytokine signaling regulates the phenotype and function of Mϕ via epigenetic modification of chromatin. We have found that type I interferon (IFN-I) potently upregulates the lysine methyltransferase Setdb2 in murine and human Mϕ, and in turn Setdb2 regulates Mϕ-mediated immunity in response to IAV. The induction of Setdb2 by IFN-I was significantly impaired upon inhibition of the JAK-STAT signaling cascade, and chromatin immunoprecipitation revealed that both STAT1 and interferon regulatory factor 7 bind upstream of the transcription start site to induce expression. The generation of Setdb2LacZ reporter mice revealed that IAV infection results in systemic upregulation of Setdb2 in myeloid cells. In the lungs, alveolar Mϕ expressed the highest level of Setdb2, with greater than 70% lacZ positive on day 4 post-infection. Silencing Setdb2 activity in Mϕ in vivo enhanced survival in lethal IAV infection. Enhanced host protection correlated with an amplified antiviral response and less obstruction to the airways. By tri-methylating H3K9, Setdb2 silenced the transcription of Mx1 and Isg15, antiviral effectors that inhibit IAV replication. Accordingly, a reduced viral load in knockout mice on day 8 post-infection was linked to elevated Isg15 and Mx1 transcript in the lungs. In addition, Setdb2 suppressed the expression of a large number of other genes with proinflammatory or immunomodulatory function. This included Ccl2, a chemokine that signals through CCR2 to regulate monocyte recruitment to infectious sites. Consistently, knockout mice produced more CCL2 upon IAV infection and this correlated with a 2-fold increase in the number of inflammatory monocytes and alveolar Mϕ in the lungs. Finally, Setdb2 expression by Mϕ suppressed IL-2, IL-10, and IFN-γ production by CD4+ T cells in vitro, as well as proliferation in IAV-infected lungs. Collectively, these findings identify Setdb2 as a novel regulator of the immune system in acute respiratory viral infection.

Numb-deficient satellite cells have regeneration and proliferation defects.

The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.

MDI 301 suppresses myeloid leukemia cell growth in vitro and in vivo without the toxicity associated with all-trans retinoic acid therapy.

MDI 301 is a novel 9-cis retinoic acid derivative in which the terminal carboxylic acid group has been replaced by a picolinate ester. MDI 301, a retinoic acid receptor-α - agonist, suppressed the growth of several human myeloid leukemia cell lines (HL60, NB4, OCI-M2, and K562) in vitro and induced cell-substrate adhesion in conjunction with upregulation of CD11b. Tumor growth in HL60-injected athymic nude mice was reduced. In vitro, MDI 301 was comparable to all-trans retinoic acid (ATRA) whereas in vivo, MDI 301 was slightly more efficacious than ATRA. Most importantly, unlike what was found with ATRA treatment, MDI 301 did not induce a cytokine response in the treated animals and the severe inflammatory changes and systemic toxicity seen with ATRA did not occur. A retinoid with these characteristics might be valuable in the treatment of promyelocytic leukemia, or, perhaps, other forms of myeloid leukemia.

Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres.

Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin-positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA-positive-pax7-positive) than controls (44 ± 2% vs. 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD-positive cells than controls and replicated 20% less (P < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar-sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring.

Metabolic reprogramming and dysregulated IL-17 production impairs CD4 T cell function post sepsis.

Sepsis survivors are at high risk for infection-related rehospitalization and mortality for years following the resolution of the acute septic event. These infection-causing microorganisms generally do not cause disease in immunocompetent hosts, suggesting that the post-septic immune response is compromised. Given the importance of CD4 T cells in the development of long-lasting protective immunity, we analyzed their post-septic function. Here we showed that sepsis induced chronic increased and non-specific production of IL-17 by CD4 T cells, resulting in the inability to mount an effective immune response to a secondary pneumonia challenge. Altered cell function was associated with metabolic reprogramming, characterized by mitochondrial dysfunction and increased glycolysis. This metabolic reprogramming began during the acute septic event and persisted long after sepsis had resolved. Our findings reveal cell metabolism as a potential therapeutic target. Given the critical role of cell metabolism in the physiological and pathophysiological processes of immune cells, these findings reveal a potential new therapeutic target to help mitigate sepsis survivors' susceptibility to secondary infections.

Age-related nitration/dysfunction of myogenic stem cell activator HGF.

Mechanical perturbation triggers activation of resident myogenic stem cells to enter the cell cycle through a cascade of events including hepatocyte growth factor (HGF) release from its extracellular tethering and the subsequent presentation to signaling-receptor c-met. Here, we show that with aging, extracellular HGF undergoes tyrosine-residue (Y) nitration and loses c-met binding, thereby disturbing muscle homeostasis. Biochemical studies demonstrated that nitration/dysfunction is specific to HGF among other major growth factors and is characterized by its locations at Y198 and Y250 in c-met-binding domains. Direct-immunofluorescence microscopy of lower hind limb muscles from three age groups of rat, provided direct in vivo evidence for age-related increases in nitration of ECM-bound HGF, preferentially stained for anti-nitrated Y198 and Y250-HGF mAbs (raised in-house) in fast IIa and IIx myofibers. Overall, findings highlight inhibitory impacts of HGF nitration on myogenic stem cell dynamics, pioneering a cogent discussion for better understanding age-related muscle atrophy and impaired regeneration with fibrosis (including sarcopenia and frailty).

The critical role of Notch ligand Delta-like 1 in the pathogenesis of influenza A virus (H1N1) infection.

Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFNα-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-γ. In addition, we blocked Notch signaling by using γ-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-γ in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-γ levels from CD4(+)and CD8(+)T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection.

Skeletal muscle satellite cell migration to injured tissue measured with 111In-oxine and high-resolution SPECT imaging.

The delivery of adult skeletal muscle stem cells, called satellite cells, to several injured muscles via the circulation would be useful, however, an improved understanding of cell fate and biodistribution following their delivery is important for this goal to be achieved. The objective of this study was to evaluate the ability of systemically delivered satellite cells to home to injured skeletal muscle using single-photon emission computed tomography (SPECT) imaging of (111)In-labeled satellite cells. Satellite cells labeled with (111)In-oxine and green fluorescent protein (GFP) were injected intravenously after bupivicaine-induced injury to the tibialis anterior muscle. Animals were imaged with a high-resolution SPECT system called FastSPECT II for up to 7 days after transplantation. In vivo FastSPECT II imaging demonstrated a three to five-fold greater number of transplanted satellite cells in bupivicaine-injured muscle as compared to un-injured muscle after transplantation; a finding that was verified through autoradiograph analysis and quantification of GFP expression. Satellite cells also accumulated in other organs including the lung, liver, and spleen, as determined by biodistribution measurements. These data support the ability of satellite cells to home to injured muscle and support the use of SPECT and autoradiograph imaging techniques to track systemically transplanted (111)In labeled satellite cells in vivo, and suggest their homing may be improved by reducing their entrapment in filter organs.

Jagged-1 Reduces Th2 Inflammation and Memory Cell Expansion in Allergic Airway Disease.

Notch ligands present during interactions between T cells and dendritic cells (DCs) dictate cell phenotype through a myriad of effects including the induction of T cell regulation, survival, and cytokine response. The presence of Notch ligands on DCs varies with the context of the inflammatory response; Jagged-1 is constitutively expressed, whereas Delta-like 1 and Delta-like 4 are induced in response to pathogen exposure. Although Delta-like and Jagged ligands send different signals through the same Notch receptor, the role of these two ligands in peripheral T cell immunity is not clear. The goal of our studies was to determine the role of Jagged-1 in the pathogen-free inflammation induced by OVA during allergic airway disease in mice. Our studies show that a deletion in DC-expressed Jagged-1 causes a significant increase in cytokine production, resulting in increased mucus production and increased eosinophilia in the lungs of mice sensitized and challenged with OVA. We also observed that a reduction of Jagged-1 expression is correlated with increased expression of the Notch 1 receptor on the surface of CD4+ T cells in both the lung and lymph node. Through transfer studies using OT-II transgenic T cells, we demonstrate that Jagged-1 represses the expansion of CD44+CD62L+CCR7+ memory cells and promotes the expansion of CD44+CD62L- effector cells, but it has no effect on the expansion of naive cells during allergic airway disease. These data suggest that Jagged-1 may have different roles in Ag-specific T cell responses, depending on the maturity of the stimulated T cell.

Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch.

When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 µM (GsMTx-4), 10 µM (nifedipine), or 100 µM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 µM) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade.

Epigenetic regulation of the alternatively activated macrophage phenotype.

Alternatively activated (M2) macrophages play critical roles in diverse chronic diseases, including parasite infections, cancer, and allergic responses. However, little is known about the acquisition and maintenance of their phenotype. We report that M2-macrophage marker genes are epigenetically regulated by reciprocal changes in histone H3 lysine-4 (H3K4) and histone H3 lysine-27 (H3K27) methylation; and the latter methylation marks are removed by the H3K27 demethylase Jumonji domain containing 3 (Jmjd3). We found that continuous interleukin-4 (IL-4) treatment leads to decreased H3K27 methylation, at the promoter of M2 marker genes, and a concomitant increase in Jmjd3 expression. Furthermore, we demonstrate that IL-4-dependent Jmjd3 expression is mediated by STAT6, a major transcription factor of IL-4-mediated signaling. After IL-4 stimulation, activated STAT6 is increased and binds to consensus sites at the Jmjd3 promoter. Increased Jmjd3 contributes to the decrease of H3K27 dimethylation and trimethylation (H3K27me2/3) marks as well as the transcriptional activation of specific M2 marker genes. The decrease in H3K27me2/3 and increase in Jmjd3 recruitment were confirmed by in vivo studies using a Schistosoma mansoni egg-challenged mouse model, a well-studied system known to support an M2 phenotype. Collectively, these data indicate that chromatin remodeling is mechanistically important in the acquisition of the M2-macrophage phenotype.

Effects of transforming growth factor-beta (TGF-ß1) on satellite cell activation and survival during oxidative stress.

The regulation of adult skeletal muscle repair and regeneration is largely due to the contribution of resident adult myogenic precursor cells called satellite cells. The events preceding their participation in muscle repair include activation (exit from quiescence), proliferation, and differentiation. This study examined the effects of transforming growth factor-beta (TGF-ß1) on satellite cell activation, determined whether TGF-ß1 could maintain quiescence in the presence of hepatocyte growth factor (HGF), and whether the regulation of satellite cell activation with TGF-ß1 improves the ability of satellite cells to withstand oxidative stress. The addition of TGF-ß1 during early satellite cell activation (0-48 h) or during the proliferative phase (48-96 h) maintained and induced satellite cell quiescence, respectively, as determined by myogenic differentiation (MyoD) protein expression. TGF-ß1 also attenuated satellite cell activation when used with HGF. Finally, the role of quiescence in protecting cells against oxidative stress was examined. TGF-ß1 treatment and the low pH satellite cell preparation procedure, a technique that forestalls spontaneous activation in vitro, both enhanced survival of cultured satellite cells following hydrogen peroxide treatment. These findings indicate that TGF-ß1 is capable of maintaining and inducing satellite cell quiescence and suggest methods to maintain satellite cell quiescence may improve their transplantation efficiency.

Elevated Notch ligands in serum are associated with HIV/TB coinfection.

OBJECTIVE: There is a clear need for improved biomarkers to diagnose HIV/TB coinfection. Although numerous tests can identify the existence of both of these microbes within the host, a parallel assessment of the host response to HIV/TB coinfection may prove as useful confirmation in cases where microbiological tests are inconclusive. To this end we assessed the levels of Notch ligands found in serum samples of patients with TB, HIV or HIV/TB coinfection. The Notch system is involved in almost every stage of development, including the maturation of the immune response. Upon exposure to a pathogen, the innate immune system will increase expression of Notch ligands Delta-like 1 and Delta-like 4. Previous research has demonstrated that Notch ligand expression is increased on monocytes from patients diagnosed with tuberculosis. We hypothesized that if Notch ligands were present in the peripheral blood of individuals diagnosed with TB, they may serve as a novel marker for infection.Design: Serum samples from patients with HIV, TB or HIV/TB coinfection were compared to serum from uninfected individuals to determine levels of DLL1 and DLL4 in a case controlled study. METHODS: DLL1 and DLL4 were measured by ELISA. Linear regression with post tests were used to determine if levels of DLL1 and DLL4 were increased in individuals with HIV/TB coinfection as compared to individuals infected with either HIV or TB or healthy controls. RESULTS: Delta-like 1 and Delta-like 4 were significantly increased in the serum of patients with HIV and HIV/ M. tuberculosis coinfection compared to other groups. CONCLUSIONS: Assessment of Notch ligands in peripheral blood may enhance the diagnosis of individuals with active TB that are co-infected with HIV. The study will also need to be validated in in a larger cohort.

High concentrations of HGF inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: a possible mechanism for reestablishing satellite cell quiescence in vivo.

Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridine-incorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced by satellite cells and spleen and liver cells in response to muscle damage, local concentrations of HGF bathing satellite cells may reach a threshold sufficient to induce myostatin expression. This time lag may delay action of the quiescence signaling program in proliferating satellite cells during initial phases of muscle regeneration followed by induction of quiescence in a subset of cells during later phases.

TNF-α regulates diabetic macrophage function through the histone acetyltransferase MOF.

A critical component of wound healing is the transition from the inflammatory phase to the proliferation phase to initiate healing and remodeling of the wound. Macrophages are critical for the initiation and resolution of the inflammatory phase during wound repair. In diabetes, macrophages display a sustained inflammatory phenotype in late wound healing characterized by elevated production of inflammatory cytokines, such as TNF-α. Previous studies have shown that an altered epigenetic program directs diabetic macrophages toward a proinflammatory phenotype, contributing to a sustained inflammatory phase. Males absent on the first (MOF) is a histone acetyltransferase (HAT) that has been shown be a coactivator of TNF-α signaling and promote NF-κB-mediated gene transcription in prostate cancer cell lines. Based on MOF's role in TNF-α/NF-κB-mediated gene expression, we hypothesized that MOF influences macrophage-mediated inflammation during wound repair. We used myeloid-specific Mof-knockout (Lyz2Cre Moffl/fl) and diet-induced obese (DIO) mice to determine the function of MOF in diabetic wound healing. MOF-deficient mice exhibited reduced inflammatory cytokine gene expression. Furthermore, we found that wound macrophages from DIO mice had elevated MOF levels and higher levels of acetylated histone H4K16, MOF's primary substrate of HAT activity, on the promoters of inflammatory genes. We further identified that MOF expression could be stimulated by TNF-α and that treatment with etanercept, an FDA-approved TNF-α inhibitor, reduced MOF levels and improved wound healing in DIO mice. This report is the first to our knowledge to define an important role for MOF in regulating macrophage-mediated inflammation in wound repair and identifies TNF-α inhibition as a potential therapy for the treatment of chronic inflammation in diabetic wounds.

Possible implication of satellite cells in regenerative motoneuritogenesis: HGF upregulates neural chemorepellent Sema3A during myogenic differentiation.

Regenerative coordination and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory mechanisms of such coordination remain unclear, although both attractive and repulsive axon guidance molecules may be involved in the signaling pathway. Here we show that expression of a neural secreted chemorepellent semaphorin 3A (Sema3A) is remarkably upregulated in satellite cells of resident myogenic stem cells that are positioned beneath the basal lamina of mature muscle fibers, when treated with hepatocyte growth factor (HGF), established as an essential cue in muscle fiber growth and regeneration. When satellite cells were treated with HGF in primary cultures of cells or muscle fibers, Sema3A message and protein were upregulated as revealed by reverse transcription-polymerase chain reaction and immunochemical studies. Other growth factors had no inductive effect except for a slight effect of epidermal growth factor treatment. Sema3A upregulation was HGF dose dependent with a maximum (about 7- to 8-fold units relative to the control) at 10-25 ng/ml and occurred exclusively at the early-differentiation stage, as characterized by the level of myogenin expression and proliferation (bromodeoxyuridine incorporation) of the cells. Neutralizing antibody to the HGF-specific receptor, c-met, did not abolish the HGF response, indicating that c-met may not mediate the Sema3A expression signaling. Finally, in vivo Sema3A was upregulated in the differentiation phase of satellite cells isolated from muscle regenerating following crush injury. Overall, the data highlight a heretofore unexplored and active role for satellite cells as a key source of Sema3A expression triggered by HGF, hence suggesting that regenerative activity toward motor innervation may importantly reside in satellite cells and could be a crucial contributor during postnatal myogenesis.