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).

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.

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.

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.

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.

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.

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.

The Histone Methyltransferase MLL1 Directs Macrophage-Mediated Inflammation in Wound Healing and Is Altered in a Murine Model of Obesity and Type 2 Diabetes.

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)-mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+ ) to determine the function of MLL1 in wound healing. Mll1f/fLyz2Cre+ mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from Mll1f/fLyz2Cre+ mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased Mll1 compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.

Notch Regulates Macrophage-Mediated Inflammation in Diabetic Wound Healing.

Macrophages are essential immune cells necessary for regulated inflammation during wound healing. Recent studies have identified that Notch plays a role in macrophage-mediated inflammation. Thus, we investigated the role of Notch signaling on wound macrophage phenotype and function during normal and diabetic wound healing. We found that Notch receptor and ligand expression are dynamic in wound macrophages during normal healing. Mice with a myeloid-specific Notch signaling defect (DNMAMLfloxedLyz2Cre+ ) demonstrated delayed early healing (days 1-3) and wound macrophages had decreased inflammatory gene expression. In our physiologic murine model of type 2 diabetes (T2D), Notch receptor expression was significantly increased in wound macrophages on day 6, following the initial inflammatory phase of wound healing, corresponding to increased inflammatory cytokine expression. This increase in Notch1 and Notch2 was also observed in human monocytes from patients with T2D. Further, in prediabetic mice with a genetic Notch signaling defect (DNMAMLfloxedLyz2Cre+ on a high-fat diet), improved wound healing was seen at late time points (days 6-7). These findings suggest that Notch is critical for the early inflammatory phase of wound healing and directs production of macrophage-dependent inflammatory mediators. These results identify that canonical Notch signaling is important in directing macrophage function in wound repair and define a translational target for the treatment of non-healing diabetic wounds.

Evaluation of Salivary Cytokines for Diagnosis of both Trauma-Induced and Genetic Heterotopic Ossification.

PURPOSE: Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers. METHODS: Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt ). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum. RESULTS: Levels of TNF-α and IL-1ß peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1ß, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05). CONCLUSION: In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.

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.

Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion.

BACKGROUND: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. METHODS: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. RESULTS: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. CONCLUSIONS: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

Systemic Expression of Notch Ligand Delta-Like 4 during Mycobacterial Infection Alters the T Cell Immune Response.

The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4+ T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4+ progenitor cells from infected hosts resulted in an increase DLL4+ myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4+ monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from naïve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mice, and further reveal that expression of DLL4 is upregulated on early hematopoietic progenitors in response to chronic mycobacterial infection. These data suggest that widespread DLL4 expression may occur as a result of mycobacterial infection, and that this expression may alter CD4+ T cell responses to both previously encountered and novel antigens.

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.

Epigenetic regulation of IL-12-dependent T cell proliferation.

It is well established that the cytokine IL-12 and the transcription factor STAT4, an essential part of the IL-12 signaling pathway, are critical components of the Th1 differentiation process in T cells. In response to pathogenic stimuli, this process causes T cells to proliferate rapidly and secrete high amounts of the cytokine IFN-γ, leading to the Th1 proinflammatory phenotype. However, there are still unknown components of this differentiation pathway. We here demonstrated that the expression of the histone methyltransferase Mll1 is driven by IL-12 signaling through STAT4 in humans and mice and is critical for the proper differentiation of a naïve T cell to a Th1 cell. Once MLL1 is up-regulated by IL-12, it regulates the proliferation of Th1 cells. As evidence of this, we show that Th1 cells from Mll1(+/-) mice are unable to proliferate rapidly in a Th1 environment in vitro and in vivo. Additionally, upon restimulation with cognate antigen Mll1(+/-), T cells do not convert to a Th1 phenotype, as characterized by IFN-γ output. Furthermore, we observed a reduction in IFN-γ production and proliferation in human peripheral blood stimulated with tetanus toxoid by use of a specific inhibitor of the MLL1/menin complex. Together, our results demonstrate that the MLL1 gene plays a previously unrecognized but essential role in Th1 cell biology and furthermore, describes a novel pathway through which Mll1 expression is regulated.

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.

Another Perspective on Research as a Measure of High-Quality Practitioner Training: a Response to Dixon, Reed, Smith, Belisle, and Jackson.

Dixon et al. (Behavior Analysis in Practice 8:7-15, 2015) argued that the research productivity of behavior analytic graduate programs may be a reasonable criterion to evaluate training program quality. They reviewed the cumulative publications of graduate programs. From this analysis, they generated a top ten list of graduate programs with the greatest number of faculty publications and, because of the number of these publications, inferred that they may be better training programs than those not on the list. We countered that the quality of graduate training programs is evident in the behavior of those who are trained, and thus, our field's interest should focus on determining the degree to which individual program graduates-and not their faculty-have mastered the research process. Thus, we proposed including student authors' work as an alternative to Dixon et al.'s analysis.

Epigenetic changes in bone marrow progenitor cells influence the inflammatory phenotype and alter wound healing in type 2 diabetes.

Classically activated (M1) macrophages are known to play a role in the development of chronic inflammation associated with impaired wound healing in type 2 diabetes (T2D); however, the mechanism responsible for the dominant proinflammatory (M1) macrophage phenotype in T2D wounds is unknown. Since epigenetic enzymes can direct macrophage phenotypes, we assessed the role of histone methylation in bone marrow (BM) stem/progenitor cells in the programming of macrophages toward a proinflammatory phenotype. We have found that a repressive histone methylation mark, H3K27me3, is decreased at the promoter of the IL-12 gene in BM progenitors and this epigenetic signature is passed down to wound macrophages in a murine model of glucose intolerance (diet-induced obese). These epigenetically "preprogrammed" macrophages result in poised macrophages in peripheral tissue and negatively impact wound repair. We found that in diabetic conditions the H3K27 demethylase Jmjd3 drives IL-12 production in macrophages and that IL-12 production can be modulated by inhibiting Jmjd3. Using human T2D tissue and murine models, we have identified a previously unrecognized mechanism by which macrophages are programmed toward a proinflammatory phenotype, establishing a pattern of unrestrained inflammation associated with nonhealing wounds. Hence, histone demethylase inhibitor-based therapy may represent a novel treatment option for diabetic wounds.

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.