Erythropoietin-induced serine 727 phosphorylation of STAT3 in erythroid cells is mediated by a MEK-, ERK-, and MSK1-dependent pathway

Objective. Erythropoietin (EPO) is a key regulator of erythropoiesis, playing a role in both the proliferation and differentiation of erythroid cells. One of the signal transduction molecules activated upon EPO stimulation is signal transducer and activator of transcription (STAT) 3. Besides tyrosine 705 phosphorylation of STAT3, serine 727 phosphorylation has been described upon EPO stimulation. In the present study, we investigated which molecular pathways mediate the STAT3 serine 727 phosphorylation and the functional implications of this phosphorylation. Methods. The EPO-dependent erythroid cell line ASE2 was used to investigate which signaling routes were involved in the STAT3 serine 727 phosphorylation. Western blotting using phosphospecific antibodies was used to assess the phosphorylation status of STAT3 molecules. Transfection analysis was performed to investigate the transactivational potential of STAT3, and quantitative RT-PCR was used to study the in vivo gene expression of STAT3-responsive genes. Results. Western blotting of extracts of cells exposed to various chemical inhibitors revealed that the MEK inhibitors PD98059 and U0126 abrogated the EPO-mediated STAT3 serine 727 phosphorylation without an effect on tyrosine phosphorylation. Further analysis showed that MSK1 is activated downstream of ERK, and retroviral transductions with kinase-inactive MSK1 revealed that MSK1 is necessary for STAT3 serine phosphorylation. Furthermore, the STAT3-mediated transactivation was reduced by blocking the STAT3 serine phosphorylation with the MEK inhibitor U0126 or by expression of kinase-inactive MSK1. Conclusions. The EPO-induced STAT3 serine 727 phosphorylation is mediated by a pathway involving MEK, ERK, and MSK1. Furthermore, serine phosphorylation of STAT3 augments the transactivational potential of STAT3.

Role of the Fractalkine Receptor in CNS Autoimmune Inflammation: New Approach Utilizing a Mouse Model Expressing the Human CX3CR1

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation. © 2018 Cardona et al

Late-Life Exercise Mitigates Skeletal Muscle Epigenetic Aging

There are functional benefits to exercise in muscle, even when performed late in life, but the contributions of epigenetic factors to late-life exercise adaptation are poorly defined. Using reduced representation bisulfite sequencing (RRBS), ribosomal DNA (rDNA) and mitochondrial-specific examination of methylation, targeted high-resolution methylation analysis, and DNAge™ epigenetic aging clock analysis with a translatable model of voluntary murine endurance/resistance exercise training (progressive weighted wheel running, PoWeR), we provide evidence that exercise may mitigate epigenetic aging in skeletal muscle. Late-life PoWeR from 22–24 months of age modestly but significantly attenuates an age-associated shift toward promoter hypermethylation. The epigenetic age of muscle from old mice that PoWeR-trained for eight weeks was approximately eight weeks younger than 24-month-old sedentary counterparts, which represents ~8% of the expected murine lifespan. These data provide a molecular basis for exercise as a therapy to attenuate skeletal muscle aging

Generation of T Follicular Helper Cells Is Mediated by Interleukin-21 but Independent of T Helper 1, 2, or 17 Cell Lineages

SummaryAfter activation, CD4+ helper T (Th) cells differentiate into distinct effector subsets. Although chemokine (C-X-C motif) receptor 5-expressing T follicular helper (Tfh) cells are important in humoral immunity, their developmental regulation is unclear. Here we show that Tfh cells had a distinct gene expression profile and developed in vivo independently of the Th1 or Th2 cell lineages. Tfh cell generation was regulated by ICOS ligand (ICOSL) expressed on B cells and was dependent on interleukin-21 (IL-21), IL-6, and signal transducer and activator of transcription 3 (STAT3). However, unlike Th17 cells, differentiation of Tfh cells did not require transforming growth factor β (TGF-β) or Th17-specific orphan nuclear receptors RORα and RORγ in vivo. Finally, naive T cells activated in vitro in the presence of IL-21 but not TGF-β signaling preferentially acquired Tfh gene expression and promoted germinal-center reactions in vivo. This study thus demonstrates that Tfh is a distinct Th cell lineage

Erythropoietin (EPO) increases myelin gene expression in CG4 oligodendrocyte cells through the classical EPO receptor

Erythropoietin (EPO) has protective effects in neurodegenerative and neuroinflammatory diseases, including in animal models of multiple sclerosis, where EPO decreases disease severity. EPO also promotes neurogenesis and is protective in models of toxic demyelination. In this study, we asked whether EPO could promote neurorepair by also inducing remyelination. In addition, we investigated whether the effect of EPO could be mediated by the classical erythropoietic EPO receptor (EPOR), since it is still questioned if EPOR is functional in non-hematopoietic cells. Using CG4 cells, a line of rat oligodendrocyte precursor cells, we found that EPO increases the expression of myelin genes (myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP)). EPO had no effect in wild-type CG4 cells, which do not express EPOR, whereas it increased MOG and MBP expression in cells engineered to overexpress EPOR (CG4-EPOR). This was reflected in a marked increase in MOG protein levels, as detected by western blot. In these cells, EPO induced by 10-fold the early growth response gene 2 (Egr2), which is required for peripheral myelination. However, Egr2 silencing with a siRNA did not reverse the effect of EPO, indicating that EPO acts through other pathways. In conclusion, EPO induces the expression of myelin genes in oligodendrocytes and this effect requires the presence of EPOR. This study demonstrates that EPOR can mediate neuroreparative effects

Concentrations of urinary neopterin, but not suPAR, positively correlate with age in rhesus macaques

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData availability statement: The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject, PRJNA715739.Identifying biomarkers of age-related changes in immune system functioning that can be measured non-invasively is a significant step in progressing research on immunosenescence and inflammaging in free-ranging and wild animal populations. In the present study, we aimed to investigate the suitability of two urinary compounds, neopterin and suPAR, as biomarkers of age-related changes in immune activation and inflammation in a free-ranging rhesus macaque (Macaca mulatta) population. We also investigated age-associated variation in gene transcription from blood samples to understand the underlying proximate mechanisms that drive age-related changes in urinary neopterin or suPAR. Neopterin was significantly positively correlated with age, and had a moderate within-individual repeatability, indicating it is applicable as a biomarker of age-related changes. The age-related changes in urinary neopterin are not apparently driven by an age-related increase in the primary signaler of neopterin, IFN-y, but may be driven instead by an age-related increase in both CD14+ and CD14− monocytes. suPAR was not correlated with age, and had low repeatability within-individuals, indicating that it is likely better suited to measure acute inflammation rather than chronic age-related increases in inflammation (i.e., “inflammaging”). Neopterin and suPAR had a correlation of 25%, indicating that they likely often signal different processes, which if disentangled could provide a nuanced picture of immune-system function and inflammation when measured in tandem.National Institutes of Healt

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

Interleukin-26 (IL-26) is a novel anti-microbial peptide produced by T cells in response to staphylococcal enterotoxin

Anti-microbial peptides are produced at outer and inner surfaces by epithelia and innate immune cells in response to bacterial infection. Staphylococcus aureus is an enterotoxin producing, Gram-positive pathogen, which is a major cause of soft tissue infections and life-threatening bacteremia and sepsis. Here we show that (i) skin T cells in chronic wounds infected with S. aureus express interleukin-26 (IL-26) in situ, (ii) staphylococcal enterotoxins (SE) trigger IL-26 expression in T cell lines and primary skin T cells, and (iii) IL-26 triggers death and inhibits biofilm formation and growth of S. aureus. Thus, we provide novel evidence that IL-26 is an anti-microbial peptide produced by T cells in response to SE. Accordingly, we propose that IL-26 producing T cells take part in the innate immune response to SE producing S. aureus and thus play a novel role in the primary innate immune defense in addition to their classical role in adaptive immunity.Published versio