Sally A. Litherland

Upregulation of Foxp3 expression in mouse and human Treg is IL-2/STAT5 dependent: implications for the NOD STAT5B mutation in diabetes pathogenesis.

Abstract:  Regulatory T cells (Treg), characterized as CD4+/CD25+hi T cells, are critical for sustaining and promoting immune tolerance. Treg are highly dependent on IL‐2 and IL‐2 signaling to maintain their numbers and function and interruption of this pathway promotes autoimmunity. The transcription factor, Foxp3, is also required for Treg function as defective Foxp3 promotos autoimmunity in both mice and humans. We previously reported a point mutation in the DNA‐binding domain of the NOD STAT5B gene that limits DNA binding when compared to wild‐type STAT5 mice. Based on the presence of five STAT5B consensus sequences in the Foxp3 promotor, we hypothesized a critical linkage between IL‐2 signaling/STAT5B and Foxp3 expression in Treg. Our data show IL‐2 activates long‐form (LF) STAT5 and sustains Foxp3 expression in Treg. In contrast, CD4+/CD25− T cells do not active LF STAT5 and do not express Foxp3 under the same conditions. In addition, blocking LF STAT5 activation with a Jak inhibitor (AG‐490) significantly reduced Foxp3 expression in Treg. Examination of human Treg using flow cytometry and intracellular staining for Foxp3 expression likewise demonstrates that IL‐2 maintains Foxp3 expression through LF STAT5 signaling. These studies reveal a critical link between IL‐2 mediated JAK‐STAT5 signaling and the maintenance of Foxp3 expression in Treg of mice and humans.

Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin-4 receptor knockout mouse

Exercise is a mechanism for maintenance of body weight in humans. Morbidly obese human patients have been shown to possess single nucleotide polymorphisms in the melanocortin‐4 receptor (MC4R). MC4R knockout mice have been well characterized as a genetic model that possesses phenotypic metabolic disorders, including obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia, similar to those observed in humans possessing dysfunctional hMC4Rs. Using this model, we examined the effect of voluntary exercise of MC4R knockout mice that were allowed access to a running wheel for a duration of 8 wk. Physiological parameters that were measured included body weight, body composition of fat and lean mass, food consumption, body length, and blood levels of cholesterol and nonfasted glucose, insulin, and leptin. At the termination of the experiment, hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti‐related protein (AGRP), proopiomelanocortin (POMC), cocaine‐and amphetamine‐regulated transcript (CART), orexin, brain‐derived neurotropic factor (BDNF), phosphatase with tensin homology (Pten), melanocortin‐3 receptor (MC3R), and NPY‐Y1R were determined. In addition, islet cell distribution and function in the pancreas were examined. In the exercising MC4R knockout mice, the pancreatic islet cell morphology and other physiological parameters resembled those observed in the wild‐type littermate controls. Gene expression profiles identified exercise as having a significant effect on hypothalamic POMC, orexin, and MC3R levels. Genotype had a significant effect on AGRP, POMC, CART, and NPY‐Y1R, with an exercise and genotype interaction effect on NPY gene expression. These data support the hypothesis that voluntary exercise can prevent the genetic predisposition of melanocortin‐4 receptor‐associated obesity and diabetes.—Haskell‐Luevano, C., Schaub, J. W., Andreasen, A., Haskell, K. R., Moore, M. C., Koerper, L. M., Rouzaud, F., Baker, H. V., Millard, W. J., Walter, G., Litherland, S. A., Xiang, Z. Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin‐4 receptor knockout mouse. FASEB J. 23, 642–655 (2009)

Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist.

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4Rs and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

Nonobese Diabetic Mouse Congenic Analysis Reveals Chromosome 11 Locus Contributing to Diabetes Susceptibility, Macrophage STAT5 Dysfunction, and Granulocyte-Macrophage Colony-Stimulating Factor Overproduction

Unstimulated monocytes of at-risk/type 1 diabetic humans and macrophages of the NOD mouse have markedly elevated autocrine GM-CSF production and persistent STAT5 phosphorylation. We analyzed the relationship between GM-CSF production and persistent STAT5 phosphorylation in NOD macrophages using reciprocal congenic mouse strains containing either diabetes-susceptible NOD (B6.NODC11), or diabetes-resistant C57L (NOD.LC11) loci on chromosome 11. These intervals contain the gene for GM-CSF (Csf2; 53.8 Mb) and those for STAT3, STAT5A, and STAT5B (Stat3, Stat5a, and Stat5b; 100.4–100.6 Mb). High GM-CSF production and persistent STAT5 phosphorylation in unactivated NOD macrophages can be linked to a region (44.9–55.7 Mb) containing the Csf2 gene, but not the Stat3/5a/5b genes. This locus, provisionally called Idd4.3, is upstream of the previously described Idd4.1 and Idd4.2 loci. Idd4.3 encodes an abundance of cytokine genes that use STAT5 in their macrophage activation signaling and contributes ∼50% of the NOD.LC11 resistance to diabetes.

CapG−/− Mice Have Specific Host Defense Defects That Render Them More Susceptible than CapG+/+ Mice to Listeria monocytogenes Infection but Not to Salmonella enterica Serovar Typhimurium Infection

ABSTRACT Loss of the actin filament capping protein CapG has no apparent effect on the phenotype of mice maintained under sterile conditions; however, bone marrow-derived macrophages from CapG−/− mice exhibited distinct motility defects. We examined the ability of CapG−/− mice to clear two intracellular bacteria, Listeria monocytogenes and Salmonella enterica serovar Typhimurium. The 50% lethal dose of Listeria was 10-fold lower for CapG−/− mice than for CapG+/+ mice (6 × 103 CFU for CapG−/− mice and 6 × 104 CFU for CapG+/+ mice), while no difference was observed for Salmonella. The numbers of Listeria cells in the spleens and livers were significantly higher in CapG−/− mice than in CapG+/+ mice at days 5 to 9, while the bacterial counts were identical on day 5 for Salmonella-infected mice. Microscopic analysis revealed qualitatively similar inflammatory responses in the spleens and livers of the two types of mice. Specific immunofluorescence staining analyzed by fluorescence-activated cell sorting revealed similar numbers of macrophages and dendritic cells in infected CapG−/− and CapG+/+ spleens. However, analysis of bone marrow-derived macrophages revealed a 50% reduction in the rate of phagocytosis of Listeria in CapG−/− cells but a normal rate of phagocytosis of Salmonella. Stimulation of bone marrow-derived dendritic cells with granulocyte-macrophage colony-stimulating factor resulted in a reduction in the ruffling response of CapG−/− cells compared to the response of CapG+/+ cells, and CapG−/− bone-marrowed derived neutrophils migrated at a mean speed that was nearly 50% lower than the mean speed of CapG+/+ neutrophils. Our findings suggest that specific motility deficits in macrophages, dendritic cells, and neutrophils render CapG−/− mice more susceptible than CapG+/+ mice to Listeria infection.

Molecular Mechanism of the Constitutive Activation of the L250Q Human Melanocortin-4 Receptor Polymorphism‡

The Melanocortin‐4 Receptor is a G‐protein coupled receptor that has been physiologically linked to participate in the regulation of energy homeostasis. The Melanocortin‐4 Receptor is stimulated by endogenous melanocortin agonists derived from the pro‐opiomelanocortin gene transcript and antagonized by the endogenous antagonist agouti‐related protein. Central administration of melanocortin agonists has been demonstrated to decrease food intake and conversely, treatment with antagonists resulted in increased food intake. Deletion of the Melanocortin‐4 Receptor gene from the mouse genome results in an obese and hyperphagic phenotype. Polymorphisms of the human Melanocortin‐4‐Receptor have been found in severely obese individuals, suggesting that Melanocortin‐4 Receptor malfunction might be involved in human obesity and obesity‐associated diabetes. Herein, we have performed experiments to understand the molecular mechanisms associated with the L250Q human Melanocortin‐4‐Receptor polymorphism discovered in an extremely obese woman. This L250Q human Melanocortin‐4‐Receptor has been pharmacologically characterized to result in a constitutively active receptor. The fact that a constitutively active human Melanocortin‐4‐Receptor mutation was found in an obese person is a physiologic contradiction, as chronic activation of the human Melanocortin‐4‐Receptor and subsequently high cyclic adenosine monophosphate levels should theoretically result in a normal or lean phenotype. In this study, we demonstrated that agouti‐related protein acts as an inverse agonist at this constitutively active receptor, and we propose a mechanism by which agouti‐related protein might contribute to the obese phenotype in the L250Q patient. In addition, using receptor mutagenesis, pharmacology, and computer modeling approaches, we investigated the molecular mechanism by which modification of the L250 residue results in constitutive activation of the human Melanocortin‐4‐Receptor.

Immunopathogenic Interaction of Environmental Triggers and Genetic Susceptibility in Diabetes Is Epigenetics the Missing Link

Autoimmune type 1 diabetes is caused by an interactive combination of genetic and environmental factors, most of which remain unknown. Each individual appears to have a unique combination of these factors that allow for their susceptibility to disease. Investigators have long searched for physiological mechanisms that could link diverse environmental events to inheritable genetic traits and the aberrant gene expression in immune cells. Much of this search has focused on either uncovering elusive gene polymorphisms found in at-risk populations or on finding common elements in the lives of susceptible individuals that “trigger” their immune system toward a self-destructive path (1,2). To date, only a few gene polymorphisms have linked to susceptibility, with none so far proven both essential and sufficient to promote disease (1). The search for environmental etiological agents in this multifactorial disease has been no less difficult, since each individual afflicted appears to have a unique set of factors promoting autoimmunity rather than a single or few common triggers (2). So how can such a diverse set of environmental stimulators rapidly and reversibly trigger a variety of undefined genetic regions to promote the loss of something as basic to normal physiology as tolerance of self? Is there a common mechanism even when the end targets are elusive and environmental slings and arrows are so diverse? Enter epigenetic gene regulation into the tableau, adding a …

GM-CSF induces STAT5 binding at epigenetic regulatory sites within the Csf2 promoter of non-obese diabetic (NOD) mouse myeloid cells.

Myeloid cells from non-obese diabetic (NOD) mouse and human type 1 diabetic (T1D) patients overexpress granulocyte-macrophage colony stimulation factor (GM-CSF). This overproduction prolongs the activation of signal transduction and activator of transcription 5 (STAT5) proteins, involved in GM-CSF-induced control of myeloid cell gene expression. We found that GM-CSF can regulate the binding of STAT5 on the promoter of its own gene, Csf2, within regions previously identified as sites of chromatin epigenetic modification important to the regulation of GM-CSF during myeloid differentiation and inflammation. We found multiple sequence polymorphisms within NOD mouse chromosome 11 Idd4.3 diabetes susceptibility region that alter STAT5 GAS binding sequences within the Csf2 promoter. STAT5 binding at these sites in vivo is increased significantly in GM-CSF-stimulated-bone marrow cells and in unactivated, high GM-CSF-producing macrophages from NOD mice as compared to non-autoimmune C57BL/6 mouse myeloid cells. Thus, GM-CSF overproduction by NOD myeloid cells may be perpetuating a positive epigenetic regulatory feedback on its own gene expression through its induction of STAT5 binding to its promoter. These findings suggest that aberrant STAT5 binding at epigenetic regulatory sites may contribute directly to immunopathology through cytokine-induced gene expression dysregulation that can derail myeloid differentiation and increase inflammatory responsiveness.

Persistent STAT5 Phosphorylation and Epigenetic Dysregulation of GM-CSF and PGS2/COX2 Expression in Type 1 Diabetic Human Monocytes

STAT5 proteins are adaptor proteins for histone acetylation enzymes. Histone acetylation at promoter and enhancer chromosomal regions opens the chromatin and allows access of transcription enzymes to specific genes in rapid response cell signals, such as in inflammation. Histone acetylation-mediated gene regulation is involved in expression of 2 key inflammatory response genes: CSF2, encoding granulocyte-macrophage colony stimulating factor (GM-CSF), and PTGS2, encoding prostaglandin synthase 2/cyclooxygenase 2 (PGS2/COX2). Prolonged CSF2 expression, high GM-CSF production, and GM-CSF activation of PTGS2 gene expression all are seen in type 1 diabetes (T1D) monocytes. Persistent phosphorylation activation of monocyte STAT5 (STAT5Ptyr) is also found in individuals with or at-risk for T1D. To examine whether elevated T1D monocyte STAT5Ptyr may be associated with aberrant inflammatory gene expression in T1D, blood monocytes from non-autoimmune controls and T1D patients were analyzed by flow cytometry for STAT5Ptyr activation, and by chromatin immuno-precipitation (ChIP) analyses for STAT5Ptyr’s ability to bind at CSF2 and PTGS2 regulatory sites in association with histone acetylation. In unstimulated monocytes, STAT5Ptyr was elevated in 59.65% of T1D, but only 2.44% of control subjects (p<0.0001). Increased STAT5Ptyr correlated with T1D disease duration (p = 0.0030, r2 = 0.0784). Unstimulated (p = 0.140) and GM-CSF-stimulated (p = 0.0485) T1D monocytes, had greater STAT5Ptyr binding to epigenetic regulatory sites upstream of CSF2 than control monocytes. Increased STAT5Ptyr binding in T1D monocytes was concurrent with binding at these sites of STAT6Ptyr (p = 0.0283), CBP/P300 histone acetylase, acetylated histones H3, SMRT/NCoR histone deacetylase (p = 0.0040), and RNA Polymerase II (p = 0.0040). Our study indicates that in T1D monocytes, STAT5Ptyr activation is significantly higher and that STAT5Ptyr is found bound to CSF2 promoter and PTGS2 enhancer regions coincident with histone acetylation and RNA polymerase II. These findings suggest that the persistent activation of STAT5 by GM-CSF may be involved in altering the epigenetic regulation of these inflammatory response genes in T1D monocytes.

Csf2 and Ptgs2 Epigenetic Dysregulation in Diabetes-prone Bicongenic B6.NODC11bxC1tb Mice.

In Type 1 diabetic (T1D) human monocytes, STAT5 aberrantly binds to epigenetic regulatory sites of two proinflammatory genes, CSF2 (encoding granulocyte–macrophage colony-stimulating factor) and PTGS2 (encoding prostaglandin synthase 2/cyclooxygenase 2). Bicongenic B6.NOD C11bxC1tb mice re-create this phenotype of T1D monocytes with only two nonobese diabetic (NOD) Idd subloci (130.8 Mb–149.7 Mb, of Idd5 on Chr 1 and 32.08–53.85 Mb of Idd4.3 on Chr11) on C57BL/6 genetic background. These two Idd loci interact through STAT5 binding at upstream regulatory regions affecting Csf2 (Chr 11) and Ptgs2 (Chr 1) expression. B6.NODC11bxC1tb mice exhibited hyperglycemia and immune destruction of pancreatic islets between 8 and 30 weeks of age, with 12%–22% penetrance. Thus, B6.NODC11bxC1tb mice embody NOD epigenetic dysregulation of gene expression in myeloid cells, and this defect appears to be sufficient to impart genetic susceptibility to diabetes in an otherwise genetically nonautoimmune mouse.