Tricia R. Bhatti

Three Linked Vasculopathic Processes Characterize Kawasaki Disease: A Light and Transmission Electron Microscopic Study

Background Kawasaki disease is recognized as the most common cause of acquired heart disease in children in the developed world. Clinical, epidemiologic, and pathologic evidence supports an infectious agent, likely entering through the lung. Pathologic studies proposing an acute coronary arteritis followed by healing fail to account for the complex vasculopathy and clinical course. Methodology/Principal Findings Specimens from 32 autopsies, 8 cardiac transplants, and an excised coronary aneurysm were studied by light (n=41) and transmission electron microscopy (n=7). Three characteristic vasculopathic processes were identified in coronary (CA) and non-coronary arteries: acute self-limited necrotizing arteritis (NA), subacute/chronic (SA/C) vasculitis, and luminal myofibroblastic proliferation (LMP). NA is a synchronous neutrophilic process of the endothelium, beginning and ending within the first two weeks of fever onset, and progressively destroying the wall into the adventitia causing saccular aneurysms, which can thrombose or rupture. SA/C vasculitis is an asynchronous process that can commence within the first two weeks onward, starting in the adventitia/perivascular tissue and variably inflaming/damaging the wall during progression to the lumen. Besides fusiform and saccular aneurysms that can thrombose, SA/C vasculitis likely causes the transition of medial and adventitial smooth muscle cells (SMC) into classic myofibroblasts, which combined with their matrix products and inflammation create progressive stenosing luminal lesions (SA/C-LMP). Remote LMP apparently results from circulating factors. Veins, pulmonary arteries, and aorta can develop subclinical SA/C vasculitis and SA/C-LMP, but not NA. The earliest death (day 10) had both CA SA/C vasculitis and SA/C-LMP, and an “eosinophilic-type” myocarditis. Conclusions/Significance NA is the only self-limiting process of the three, is responsible for the earliest morbidity/mortality, and is consistent with acute viral infection. SA/C vasculitis can begin as early as NA, but can occur/persist for months to years; LMP causes progressive arterial stenosis and thrombosis and is composed of unique SMC-derived pathologic myofibroblasts.

Sirtuin-1 Targeting Promotes Foxp3+ T-Regulatory Cell Function and Prolongs Allograft Survival

ABSTRACT Sirtuin 1 (Sirt1), a class III histone/protein deacetylase, is central to cellular metabolism, stress responses, and aging, but its contributions to various host immune functions have been little investigated. To study the role of Sirt1 in T cell functions, we undertook targeted deletions by mating mice with a floxed Sirt1 gene to mice expressing CD4-cre or Foxp3-cre recombinase, respectively. We found that Sirt1 deletion left conventional T-effector cell activation, proliferation, and cytokine production largely unaltered. However, Sirt1 targeting promoted the expression of Foxp3, a key transcription factor in T-regulatory (Treg) cells, and increased Treg suppressive functions in vitro and in vivo. Consistent with these data, mice with targeted deletions of Sirt1 in either CD4+ T cells or Foxp3+ Treg cells exhibited prolonged survival of major histocompatibility complex (MHC)-mismatched cardiac allografts. Allografts in Sirt1-targeted recipients showed long-term preservation of myocardial histology and infiltration by Foxp3+ Treg cells. Comparable results were seen in wild-type allograft recipients treated with Sirt1 inhibitors, such as EX-527 and splitomicin. Hence, Sirt1 may inhibit Treg functions, and its targeting may have therapeutic value in autoimmunity and transplantation.

Genotype and Phenotype Correlations in 417 Children With Congenital Hyperinsulinism

CONTEXT Hypoglycemia due to congenital hyperinsulinism (HI) is caused by mutations in 9 genes. OBJECTIVE Our objective was to correlate genotype with phenotype in 417 children with HI. METHODS Mutation analysis was carried out for the ATP-sensitive potassium (KATP) channel genes (ABCC8 and KCNJ11), GLUD1, and GCK with supplemental screening of rarer genes, HADH, UCP2, HNF4A, HNF1A, and SLC16A1. RESULTS Mutations were identified in 91% (272 of 298) of diazoxide-unresponsive probands (ABCC8, KCNJ11, and GCK), and in 47% (56 of 118) of diazoxide-responsive probands (ABCC8, KCNJ11, GLUD1, HADH, UCP2, HNF4A, and HNF1A). In diazoxide-unresponsive diffuse probands, 89% (109 of 122) carried KATP mutations; 2% (2 of 122) had GCK mutations. In mutation-positive diazoxide-responsive probands, 42% were GLUD1, 41% were dominant KATP mutations, and 16% were in rare genes (HADH, UCP2, HNF4A, and HNF1A). Of the 183 unique KATP mutations, 70% were novel at the time of identification. Focal HI accounted for 53% (149 of 282) of diazoxide-unresponsive probands; monoallelic recessive KATP mutations were detectable in 97% (145 of 149) of these cases (maternal transmission excluded in all cases tested). The presence of a monoallelic recessive KATP mutation predicted focal HI with 97% sensitivity and 90% specificity. CONCLUSIONS Genotype to phenotype correlations were most successful in children with GLUD1, GCK, and recessive KATP mutations. Correlations were complicated by the high frequency of novel missense KATP mutations that were uncharacterized, because such defects might be either recessive or dominant and, if dominant, be either responsive or unresponsive to diazoxide. Accurate and timely prediction of phenotype based on genotype is critical to limit exposure to persistent hypoglycemia in infants and children with congenital HI.

Inhibition of p300 impairs Foxp3 + T regulatory cell function and promotes antitumor immunity

Forkhead box P3 (Foxp3)+ T regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors. Foxp3+ Treg cells are therefore considered promising targets to enhance antitumor immunity, and approaches for their therapeutic modulation are being developed. However, although studies showing that experimentally depleting Foxp3+ Treg cells can enhance antitumor responses provide proof of principle, these studies lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and nonhistone proteins. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (also known as Ep300 or KAT3B), in Foxp3+ Treg cells increased T cell receptor–induced apoptosis in Treg cells, impaired Treg cell suppressive function and peripheral Treg cell induction, and limited tumor growth in immunocompetent but not in immunodeficient mice. Our data thereby demonstrate that p300 is important for Foxp3+ Treg cell function and homeostasis in vivo and in vitro, and identify mechanisms by which appropriate small-molecule inhibitors can diminish Treg cell function without overtly impairing T effector cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.

Type 2 diabetes and congenital hyperinsulinism cause DNA double-strand breaks and p53 activity in β cells.

β cell failure in type 2 diabetes (T2D) is associated with hyperglycemia, but the mechanisms are not fully understood. Congenital hyperinsulinism caused by glucokinase mutations (GCK-CHI) is associated with β cell replication and apoptosis. Here, we show that genetic activation of β cell glucokinase, initially triggering replication, causes apoptosis associated with DNA double-strand breaks and activation of the tumor suppressor p53. ATP-sensitive potassium channels (KATP channels) and calcineurin mediate this toxic effect. Toxicity of long-term glucokinase overactivity was confirmed by finding late-onset diabetes in older members of a GCK-CHI family. Glucagon-like peptide-1 (GLP-1) mimetic treatment or p53 deletion rescues β cells from glucokinase-induced death, but only GLP-1 analog rescues β cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of β cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of β cell glucotoxicity and suggest pharmacological approaches to enhance β cell survival in diabetes.

Essential role of mitochondrial energy metabolism in Foxp3+ T-regulatory cell function and allograft survival

Conventional T (Tcon) cells and Foxp3+ T‐regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)‐induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild‐type cells but disproportionally affected Treg cells. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg‐dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase‐9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9‐/‐ Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild‐type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg‐dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo.—Beier, U. H., Angelin, A., Akimova, T., Wang, L., Liu, Y., Xiao, H., Koike, M. A., Hancock, S. A., Bhatti, T. R., Han, R., Jiao, J., Veasey, S. C., Sims, C. A., Baur, J. A., Wallace, D. C., Hancock, W. W. Essential role of mitochondrial energy metabolism in Foxp3+ T‐regulatory cell function and allograft survival. FASEB J. 29, 2315‐2326 (2015). www.fasebj.org

Foxp3 Protein Stability Is Regulated by Cyclin-dependent Kinase 2

Background: Foxp3 post-translational regulation remains unclear. Results: Foxp3 is phosphorylated by cyclin-dependent kinase 2 (CDK2) and has increased stability and function without its CDK motifs. Conclusion: CDK2 is a negative regulator of Foxp3 function. Significance: Understanding how Foxp3 is stabilized could lead to new therapeutic measures for autoimmunity and transplantation. Foxp3 is a transcription factor required for the development of regulatory T cells (Treg). Mice and humans with a loss of Foxp3 function suffer from uncontrolled autoimmunity and inflammatory disease. Expression of Foxp3 is necessary for the anti-inflammatory capacity of Treg, but whether Foxp3 activity is further subject to regulation by extracellular signals is unclear. The primary structure of Foxp3 contains four cyclin-dependent kinase (CDK) motifs (Ser/Thr-Pro) within the N-terminal repressor domain, and we show that CDK2 can partner with cyclin E to phosphorylate Foxp3 at these sites. Consistent with our previous demonstration that CDK2 negatively regulates Treg function, we find that mutation of the serine or threonine at each CDK motif to alanine (S/T→A) results in enhanced Foxp3 protein stability in CD4+ T cells. T cells expressing the S/T→A mutant of Foxp3 showed enhanced induction (e.g. CD25) and repression (e.g. IL2) of canonical Foxp3-responsive genes, exhibited an increased capacity to suppress conventional T cell proliferation in vitro, and were highly effective at ameliorating colitis in an in vivo model of inflammatory bowel disease. These results indicate that CDK2 negatively regulates the stability and activity of Foxp3 and implicate CDK-coupled receptor signal transduction in the control of regulatory T cell function and stability.

Clinical Features of Three Girls With Mosaic Genome-Wide Paternal Uniparental Isodisomy

Here we describe three subjects with mosaic genome‐wide paternal uniparental isodisomy (GWpUPD) each of whom presented initially with overgrowth, hemihyperplasia (HH), and hyperinsulinism (HI). Due to the severity of findings and the presence of additional features, SNP array testing was performed, which demonstrated mosaic GWpUPD. Comparing these individuals to 10 other live‐born subjects reported in the literature, the predominant phenotype is that of pUPD11 and notable for a very high incidence of tumor development. Our subjects developed non‐metastatic tumors of the adrenal gland, kidney, and/or liver. All three subjects had pancreatic hyperplasia resulting in HI. Notably, our subjects to date display minimal features of other diseases associated with paternal UPD loci. Both children who survived the neonatal period have displayed near‐normal cognitive development, likely due to a favorable tissue distribution of the mosaicism. To understand the range of UPD mosaicism levels, we studied multiple tissues using SNP array analysis and detected levels of 5–95%, roughly correlating with the extent of tissue involvement. Given the rapidity of tumor growth and the difficulty distinguishing malignant and benign tumors in these GWpUPD subjects, we have utilized increased frequency of ultrasound (US) and alpha‐fetoprotein (AFP) screening in the first years of life. Because of a later age of onset of additional tumors, continued tumor surveillance into adolescence may need to be considered in these rare patients.

FOXP3+ regulatory T cell development and function require histone/protein deacetylase 3

Treg dysfunction is associated with a variety of inflammatory diseases. Treg populations are defined by expression of the oligomeric transcription factor FOXP3 and inability to produce IL-2, a cytokine required for T cell maintenance and survival. FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs). Here, we determined that HDAC3 mediates both the development and function of the two main Treg subsets, thymus-derived Tregs and induced Tregs (iTregs). We determined that HDAC3 and FOXP3 physically interact and that HDAC3 expression markedly reduces Il2 promoter activity. In murine models, conditional deletion of Hdac3 during thymic Treg development restored Treg production of IL-2 and blocked the suppressive function of Tregs. HDAC3-deficient mice died from autoimmunity by 4-6 weeks of age; however, injection of WT FOXP3+ Tregs prolonged survival. Adoptive transfer of Hdac3-deficient Tregs, unlike WT Tregs, did not control T cell proliferation in naive mice and did not prevent allograft rejection or colitis. HDAC3 also regulated the development of iTregs, as HDAC3-deficient conventional T cells were not converted into iTregs under polarizing conditions and produced large amounts of IL-2, IL-6, and IL-17. We conclude that HDAC3 is essential for the normal development and suppressive functions of thymic and peripheral FOXP3+ Tregs.

Resveratrol Rescues Kidney Mitochondrial Function Following Hemorrhagic Shock

ABSTRACT Objective: Hemorrhagic shock may contribute to acute kidney injury (AKI) by profoundly altering renal mitochondrial function. Resveratrol (RSV), a naturally occurring sirtuin 1 (SIRT1) activator, has been shown to promote mitochondrial function and reduce oxidative damage in a variety of aging-related disease states. We hypothesized that RSV treatment during resuscitation would ameliorate kidney mitochondrial dysfunction and decrease oxidative damage following hemorrhagic shock. Methods: Using a decompensated hemorrhagic shock model, male Long-Evans rats (n = 6 per group) were killed prior to hemorrhage (sham), at severe shock, and following either lactated Ringer’s (LR) resuscitation or LR + RSV resuscitation (RSV: 30 mg/kg). At each time point, blood samples were assayed for arterial blood gases, lactate, blood urea nitrogen, and serum creatinine. Mitochondria were also isolated from kidney samples in order to assess individual electron transport complexes (complexes I, II, and IV) using high-resolution respirometry. Total mitochondria reactive oxygen species were measured using fluorometry, and lipid peroxidation was assessed by measuring 4-hydroxynonenal by Western blot. Quantitative polymerase chain reaction was used quantify mRNA from peroxisome proliferator–activated receptor &ggr; coactivator 1-&agr; (PGC1-&agr;) SIRT1, and proteins known to mitigate oxidative damage and promote mitochondrial biogenesis. Results: Resveratrol supplementation during resuscitation restored mitochondrial respiratory capacity and decreased mitochondrial reactive oxygen species and lipid peroxidation. Compared with standard LR resuscitation, RSV treatment significantly increased SIRT1 and PGC1-&agr; expression and significantly increased both superoxide dismutase 2 and catalase expression. Although RSV was associated with decreased lactate production, pH, blood urea nitrogen, and serum creatinine values did not differ between resuscitation strategies. Conclusions: Resuscitation with RSV significantly restored renal mitochondrial function and decreased oxidative damage following hemorrhagic shock.