Identification of Novel Disease-Relevant Genes and Pathways in the Pathogenesis of Type 1 Diabetes: A Potential Defect in Pancreatic Iron Homeostasis
2022; 71 (7): 1490-1507
Multiple pathways contribute to the pathophysiological development of type 1 diabetes (T1D); however, the exact mechanisms involved are unclear. We performed differential gene expression analysis in pancreatic islets of NOD mice versus age-matched congenic NOD.B10 controls to identify genes that may contribute to disease pathogenesis. Novel genes related to extracellular matrix development and glucagon and insulin signaling/secretion were changed in NOD mice during early inflammation. During "respective" insulitis, the expression of genes encoding multiple chemosensory olfactory receptors were upregulated, and during "destructive" insulitis, the expression of genes involved in antimicrobial defense and iron homeostasis were downregulated. Islet inflammation reduced the expression of Hamp that encodes hepcidin. Hepcidin is expressed in β-cells and serves as the key regulator of iron homeostasis. We showed that Hamp and hepcidin levels were lower, while iron levels were higher in the pancreas of 12-week-old NOD versus NOD.B10 mice, suggesting that a loss of iron homeostasis may occur in the islets during the onset of "destructive" insulitis. Interestingly, we showed that the severity of NOD disease correlates with dietary iron intake. NOD mice maintained on low-iron diets had a lower incidence of hyperglycemia, while those maintained on high-iron diets had an earlier onset and higher incidence of disease, suggesting that high iron exposure combined with a loss of pancreatic iron homeostasis may exacerbate NOD disease. This mechanism may explain the link seen between high iron exposure and the increased risk for T1D in humans.
View details for DOI 10.2337/db21-0948
View details for Web of Science ID 000905183700009
View details for PubMedID 35499603
View details for PubMedCentralID PMC9233262
Gene Expression Analysis of the Pre-Diabetic Pancreas to Identify Pathogenic Mechanisms and Biomarkers of Type 1 Diabetes.
Frontiers in endocrinology
2020; 11: 609271
Type 1 Diabetes (T1D) occurs as a result of the autoimmune destruction of pancreatic beta-cells by self-reactive T cells. The etiology of this disease is complex and difficult to study due to a lack of disease-relevant tissues from pre-diabetic individuals. In this study, we performed gene expression analysis on human pancreas tissues obtained from the Network of Pancreatic Organ Donors with Diabetes (nPOD), and showed that 155 genes were differentially expressed by ≥2-fold in the pancreata of autoantibody-positive (AA+) at-risk individuals compared to healthy controls. Only 48 of these genes remained changed by ≥2-fold in the pancreata of established T1D patients. Pathway analysis of these genes showed a significant association with various immune pathways. We were able to validate the differential expression of eight disease-relevant genes by QPCR analysis: A significant upregulation of CADM2, and downregulation of TRPM5, CRH, PDK4, ANGPL4, CLEC4D, RSG16, and FCGR2B was confirmed in the pancreata of AA+ individuals versus controls. Studies have already implicated FCGR2B in the pathogenesis of disease in non-obese diabetic (NOD) mice. Here we showed that CADM2, TRPM5, PDK4, and ANGPL4 were similarly changed in the pancreata of pre-diabetic 12-week-old NOD mice compared to NOD.B10 controls, suggesting a possible role for these genes in the pathogenesis of both T1D and NOD disease. The loss of the leukocyte-specific gene, FCGR2B, in the pancreata of AA+ individuals, is particularly interesting, as it may serve as a potential whole blood biomarker of disease progression. To test this, we quantified FCGR2B expression in peripheral blood samples of T1D patients, and AA+ and AA- first-degree relatives of T1D patients enrolled in the TrialNet Pathway to Prevention study. We showed that FCGR2B was significantly reduced in the peripheral blood of AA+ individuals compared to AA- controls. Together, these findings demonstrate that gene expression analysis of pancreatic tissue and peripheral blood samples can be used to identify disease-relevant genes and pathways and potential biomarkers of disease progression in T1D.
View details for DOI 10.3389/fendo.2020.609271
View details for PubMedID 33424774
Impact of blood collection and processing on peripheral blood gene expression profiling in type 1 diabetes
2017; 18: 636
The natural history of type 1 diabetes (T1D) is challenging to investigate, especially as pre-diabetic individuals are difficult to identify. Numerous T1D consortia have been established to collect whole blood for gene expression analysis from individuals with or at risk to develop T1D. However, with no universally accepted protocol for their collection, differences in sample processing may lead to variances in the results. Here, we examined whether the choice of blood collection tube and RNA extraction kit leads to differences in the expression of genes that are changed during the progression of T1D, and if these differences could be minimized by measuring gene expression directly from the lysate of whole blood.Microarray analysis showed that the expression of 901 genes is highly influenced by sample processing using the PAXgene versus the Tempus system. These included a significant number of lymphocyte-specific genes and genes whose expression has been reported to differ in the peripheral blood of at-risk and T1D patients compared to controls. We showed that artificial changes in gene expression occur when control and T1D samples were processed differently. The sample processing-dependent differences in gene expression were largely due to loss of transcripts during the RNA extraction step using the PAXgene system. The majority of differences were not observed when gene expression was measured in whole blood lysates prepared from blood collected in PAXgene and Tempus tubes.We showed that the gene expression profile of samples processed using the Tempus system is more accurate than that of samples processed using the PAXgene system. Variation in sample processing can result in misleading changes in gene expression. However, these differences can be minimized by measuring gene expression directly in whole blood lysates.
View details for PubMedID 28821222
Self-antigen expression in the peripheral immune system: roles in self-tolerance and type 1 diabetes pathogenesis.
Current diabetes reports
2014; 14 (9): 525-?
Type 1 diabetes (T1D) may result from a breakdown in peripheral tolerance that is partially controlled by the ectopic expression of peripheral tissue antigens (PTAs) in lymph nodes. Various subsets of lymph node stromal cells and certain hematopoietic cells play a role in maintaining T cell tolerance. These specialized cells have been shown to endogenously transcribe, process, and present a range of PTAs to naive T cells and mediate the clonal deletion or inactivation of autoreactive cells. During the progression of T1D, inflammation leads to reduced PTA expression in the pancreatic lymph nodes and the production of novel islet antigens that T cells are not tolerized against. These events allow for the escape and activation of autoreactive T cells and may contribute to the pathogenesis of T1D. In this review, we discuss recent findings in this area and propose possible therapies that may help reestablish self-tolerance during T1D.
View details for DOI 10.1007/s11892-014-0525-x
View details for PubMedID 25030265
Diminished Adenosine A1 Receptor Expression in Pancreatic a-Cells May Contribute to the Pathology of Type 1 Diabetes.
2013; 62 (12): 4208-4219
Prediabetic NOD mice exhibit hyperglucagonemia, possibly due to an intrinsic α-cell defect. Here, we show that the expression of a potential glucagon inhibitor, the adenosine A1 receptor (Adora1), is gradually diminished in α-cells of NOD mice, autoantibody-positive (AA(+)) and overtly type 1 diabetic (T1D) patients during the progression of disease. We demonstrated that islet inflammation was associated with loss of Adora1 expression through the alternative splicing of Adora1. Expression of the spliced variant (Adora1-Var) was upregulated in the pancreas of 12-week-old NOD versus age-matched NOD.B10 (non-diabetes-susceptible) control mice and was detected in the pancreas of AA(+) patients but not in control subjects or overtly diabetic patients, suggesting that inflammation drives the splicing of Adora1. We subsequently demonstrated that Adora1-Var expression was upregulated in the islets of NOD.B10 mice after exposure to inflammatory cytokines and in the pancreas of NOD.SCID mice after adoptive transfer of activated autologous splenocytes. Adora1-Var encodes a dominant-negative N-terminal truncated isoform of Adora1. The splicing of Adora1 and loss of Adora1 expression on α-cells may explain the hyperglucagonemia observed in prediabetic NOD mice and may contribute to the pathogenesis of human T1D and NOD disease.
View details for DOI 10.2337/db13-0614
View details for PubMedID 24264405
View details for PubMedCentralID PMC3837064
Reduced DEAF1 function during type 1 diabetes inhibits translation in lymph node stromal cells by suppressing Eif4g3.
Journal of molecular cell biology
2013; 5 (2): 99-110
The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.
View details for DOI 10.1093/jmcb/mjs052
View details for PubMedID 22923498
View details for PubMedCentralID PMC3604916
Deaf1 isoforms control the expression of genes encoding peripheral tissue antigens in the pancreatic lymph nodes during type 1 diabetes
2009; 10 (9): 1026-U107
Type 1 diabetes may result from a breakdown in peripheral tolerance that is partially controlled by the expression of peripheral tissue antigens (PTAs) in lymph nodes. Here we show that the transcriptional regulator Deaf1 controls the expression of genes encoding PTAs in the pancreatic lymph nodes (PLNs). The expression of canonical Deaf1 was lower, whereas that of an alternatively spliced variant was higher, during the onset of destructive insulitis in the PLNs of nonobese diabetic (NOD) mice. We identified an equivalent variant Deaf1 isoform in the PLNs of patients with type 1 diabetes. Both the NOD mouse and human Deaf1 variant isoforms suppressed PTA expression by inhibiting the transcriptional activity of canonical Deaf1. Lower PTA expression resulting from the alternative splicing of DEAF1 may contribute to the pathogenesis of type 1 diabetes.
View details for DOI 10.1038/ni.1773
View details for Web of Science ID 000269120900017
View details for PubMedID 19668219
View details for PubMedCentralID PMC2752139
How GRAIL controls Treg function to maintain self-tolerance.
Frontiers in immunology
2022; 13: 1046631
Regulatory T cells (Tregs) normally maintain self-tolerance. Tregs recognize "self" such that when they are not working properly, such as in autoimmunity, the immune system can attack and destroy one's own tissues. Current therapies for autoimmunity rely on relatively ineffective and too often toxic therapies to "treat" the destructive inflammation. Restoring defective endogenous immune regulation (self-tolerance) would represent a paradigm shift in the therapy of these diseases. One recent approach to restore self-tolerance is to use "low dose IL-2" as a therapy to increase the number of circulating Tregs. However, studies to-date have not demonstrated that low-dose IL-2 therapy can restore concomitant Treg function, and phase 2 studies in low dose IL-2 treated patients with autoimmune diseases have failed to demonstrate significant clinical benefit. We hypothesize that the defect in self-tolerance seen in autoimmunity is not due to an insufficient number of available Tregs, but rather, due to defects in second messengers downstream of the IL-2R that normally control Treg function and stability. Previous studies from our lab and others have demonstrated that GRAIL (a ubiquitin E3 ligase) is important in Treg function. GRAIL expression is markedly diminished in Tregs from patients with autoimmune diseases and allergic asthma and is also diminished in Tregs of mice that are considered autoimmune prone. In the relevant pathway in Tregs, GRAIL normally blocks cullin ring ligase activity, which inhibits IL-2R desensitization in Tregs and consequently promotes Treg function. As a result of this defect in GRAIL expression, the Tregs of patients with autoimmune diseases and allergic asthma degrade IL-2R-associated pJAK1 following activation with low dose IL-2, and thus cannot maintain pSTAT5 expression. pSTAT5 controls the transcription of genes required for Treg function. Additionally, the GRAIL-mediated defect may also allow the degradation of the mTOR inhibitor, DEP domain-containing mTOR interacting protein (Deptor). This can lead to IL-2R activation of mTOR and loss of Treg stability in autoimmune patients. Using a monoclonal antibody to the remnant di-glycine tag on ubiquitinated proteins after trypsin digestion, we identified a protein that was ubiquitinated by GRAIL that is important in Treg function, cullin5. Our data demonstrate that GRAIL acts a negative regulator of IL-2R desensitization by ubiquitinating a lysine on cullin5 that must be neddylated to allow cullin5 cullin ring ligase activity. We hypothesize that a neddylation inhibitor in combination with low dose IL-2 activation could be used to substitute for GRAIL and restore Treg function and stability in the Tregs of autoimmune and allergic asthma patients. However, the neddylation activating enzyme inhibitors (NAEi) are toxic when given systemically. By generating a protein drug conjugate (PDC) consisting of a NAEi bound, via cleavable linkers, to a fusion protein of murine IL-2 (to target the drug to Tregs), we were able to use 1000-fold less of the neddylation inhibitor drug than the amount required for therapeutically effective systemic delivery. The PDC was effective in blocking the onset or the progression of disease in several mouse models of autoimmunity (type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis) and a mouse model of allergic asthma in the absence of detectable toxicity. This PDC strategy represents targeted drug delivery at its best where the defect causing the disease was identified, a drug was designed and developed to correct the defect, and the drug was targeted and delivered only to cells that needed it, maximizing safety and efficacy.
View details for DOI 10.3389/fimmu.2022.1046631
View details for PubMedID 36569931
A Common Druggable Defect in Regulatory T Cells from Patients with Autoimmunity.
Critical reviews in immunology
2020; 40 (3): 185–93
We identified a druggable defect in IL-2 receptor (IL-2R) signaling by comparing the response of regulatory T cells (Tregs) of autoimmune disease patients to that of healthy controls. This defect was in the inhibition of Treg desensitization and was shared across various autoimmune diseases. Low-dose IL-2 stimulation results in maintained pSTAT5 expression for > 4 h, allowing the Treg transcriptome for "function" to be transcribed. Tregs of autoimmune Tregs of autoimmune disease patients more rapidly terminate IL-2R signaling through STAT5. Prolonged pSTAT5 expression following IL-2R activation is mediated by blocking proteasomal degradation of pJAKl, which is associated with the IL-2RP chain. In Tregs of controls, this is accomplished by inhibiting a requisite-activating post-translational modification (neddylation) of the SOCS3/Cul5 cullin ring ligase (CRL), which normally ubiquitinates pJAKl. Many receptor-associated tyrosine kinases are desensitized by a CRL. Tregs uniquely constitutively express an E3 ligase known as the gene related to anergy in lymphocytes (GRAIL), which ubiquinates the exact lysine on the Cul5 protein that needs to be neddylated as a condition for the activation and consequent ubiquitination of pJAKl. There is a defect in this GRAIL-associated pathway of competitive inhibition of neddylation in the Tregs of autoimmune disease patients. This defect can be mitigated by the application of a small-molecule drug known as a neddylation activating enzyme inhibitor (NAEi). Low-dose IL-2 and an NAEi as a protein-drug conjugate was found to be much more effective than simply using low-dose IL-2 or a combination of low-dose IL-2 and an NAEi systemically in treating animal models of autoimmune diseases.
View details for DOI 10.1615/CritRevImmunol.2020034631
View details for PubMedID 33389883
Reversal of Hyperglycemia and Suppression of Type 1 Diabetes in the NOD Mouse with Apoptotic DNA Immunotherapy™ (ADi™), ADi-100.
2020; 8 (3)
The antigen-specific apoptotic DNA immunotherapeutic, ADi-100, is designed to suppress type 1 diabetes and consists of two DNA plasmids encoding genetic sequences of the apoptosis-inducing molecule, BAX, and the secreted form of the autoantigen, glutamic acid decarboxylase 65, that is CpG hyper-methylated to avoid inflammatory signaling (msGAD55). Upon a four-day treatment with ADi-100 of young female non-obese diabetic (NOD) mice, the frequency of various tolerogenic dendritic cell populations increased in draining lymph nodes; these cells lost the capacity to stimulate glutamic acid decarboxylase (GAD)-specific CD4+ T lymphocytes and were associated with the previously demonstrated enhancement of GAD-specific regulatory T cells. The efficacy of two ADi-100 formulations containing different proportions of BAX and msGAD55, 1:4 (10/40 µg) and 1:2 (17/33 µg), was evaluated in mildly hyperglycemic pre-diabetic NOD female mice. Both formulations suppressed the incidence of diabetes by 80% in an antigen-specific manner, while all untreated mice developed diabetes. However, treatment of pre-diabetic mice with significantly higher hyperglycemia, denoting progressive disease, showed that ADi-100 1:2 strongly suppressed diabetes incidence by 80% whereas the ADi-100 1:4 was less effective (50%). As an antigen-specific monotherapy, ADi-100 is highly efficacious in reversing elevated hyperglycemia to prevent diabetes, in which increasing apoptosis-inducing BAX content is a promising immune tolerance feature.
View details for DOI 10.3390/biomedicines8030053
View details for PubMedID 32143316
Expression-Based Genome-Wide Association Study Links Vitamin D-Binding Protein With Autoantigenicity in Type 1 Diabetes
2016; 65 (5): 1341-1349
Type 1 diabetes (T1D) is caused by autoreactive T cells that recognize pancreatic islet antigens and destroy insulin-producing β-cells. This attack results from a breakdown in tolerance for self-antigens, which is controlled by ectopic antigen expression in the thymus and pancreatic lymph nodes (PLNs). The autoantigens known to be involved include a set of islet proteins, such as insulin, GAD65, IA-2, and ZnT8. In an attempt to identify additional antigenic proteins, we performed an expression-based genome-wide association study using microarray data from 118 arrays of the thymus and PLNs of T1D mice. We ranked all 16,089 protein-coding genes by the likelihood of finding repeated differential expression and the degree of tissue specificity for pancreatic islets. The top autoantigen candidate was vitamin D-binding protein (VDBP). T-cell proliferation assays showed stronger T-cell reactivity to VDBP compared with control stimulations. Higher levels and frequencies of serum anti-VDBP autoantibodies (VDBP-Abs) were identified in patients with T1D (n = 331) than in healthy control subjects (n = 77). Serum vitamin D levels were negatively correlated with VDBP-Ab levels in patients in whom T1D developed during the winter. Immunohistochemical localization revealed that VDBP was specifically expressed in α-cells of pancreatic islets. We propose that VDBP could be an autoantigen in T1D.
View details for DOI 10.2337/db15-1308
View details for Web of Science ID 000375028000023
View details for PubMedID 26983959
View details for PubMedCentralID PMC4839207
Mitochondrial Dysfunction, Depleted Purinergic Signaling, and Defective T Cell Vigilance and Immune Defense.
journal of infectious diseases
2016; 213 (3): 456-464
T cell suppression in sepsis is a well-known phenomenon; however, the underlying mechanisms are not fully understood. Previous studies have shown that T cell stimulation up-regulates mitochondrial adenosine triphosphate (ATP) production to fuel purinergic signaling mechanisms necessary for adequate T cell responses. Here we show that basal mitochondrial ATP production, ATP release, and stimulation of P2X1 receptors represent a standby purinergic signaling mechanism that is necessary for antigen recognition. Inhibition of this process impairs T cell vigilance and the ability of T cells to trigger T cell activation, up-regulate mitochondrial ATP production, and stimulate P2X4 and P2X7 receptors that elicit interleukin 2 production and T cell proliferation. T cells of patients with sepsis lack this standby purinergic signaling system owing to defects in mitochondrial function, ATP release, and calcium signaling. These defects impair antigen recognition and T cell function and are correlated with sepsis severity. Pharmacological targeting of these defects may improve T cell function and reduce the risk of sepsis.
View details for DOI 10.1093/infdis/jiv373
View details for PubMedID 26150546
View details for PubMedCentralID PMC4704665
- Mitochondrial Dysfunction, Depleted Purinergic Signaling, and Defective T Cell Vigilance and Immune Defense JOURNAL OF INFECTIOUS DISEASES 2016; 213 (3): 456-464
Inflammation and Hyperglycemia Mediate Deaf1 Splicing in the Pancreatic Lymph Nodes via Distinct Pathways During Type 1 Diabetes.
2015; 64 (2): 604-617
Peripheral tolerance is partially controlled by the expression of peripheral tissue antigens (PTAs) in lymph node stromal cells (LNSCs). We previously identified a transcriptional regulator, deformed epidermal autoregulatory factor 1 (Deaf1), that can regulate PTA expression in LNSCs of the pancreatic lymph nodes (PLNs). During the pathogenesis of type 1 diabetes (T1D), Deaf1 is spliced to form the dominant-negative isoform Deaf1-Var1. Here we show that Deaf1-Var1 expression correlates with the severity of disease in NOD mice and is reduced in the PLNs of mice that do not develop hyperglycemia. Inflammation and hyperglycemia independently drive Deaf1 splicing through activation of the splicing factors Srsf10 and Ptbp2, respectively. Inflammation induced by injection of activated splenocytes increased Deaf1-Var1 and Srsf10, but not Ptbp2, in the PLNs of NOD.SCID mice. Hyperglycemia induced by treatment with the insulin receptor agonist S961 increased Deaf1-Var1 and Ptbp2, but not Srsf10, in the PLNs of NOD.B10 and NOD mice. Overexpression of PTBP2 and/or SRSF10 also increased human DEAF1-VAR1 and reduced PTA expression in HEK293T cells. These data suggest that during the progression of T1D, inflammation and hyperglycemia mediate the splicing of DEAF1 and loss of PTA expression in LNSCs by regulating the expression of SRSF10 and PTBP2.
View details for DOI 10.2337/db14-0803
View details for PubMedID 25187368
View details for PubMedCentralID PMC4303971
- Self-antigen expression in the peripheral immune system: roles in self-tolerance and type 1 diabetes pathogenesis. Current diabetes reports 2014; 14 (9): 525-?
Type 1 diabetes in mice and men: gene expression profiling to investigate disease pathogenesis.
2014; 58 (2-3): 340-350
Type 1 diabetes (T1D) is a complex polygenic disease that is triggered by various environmental factors in genetically susceptible individuals. The emphasis placed on genome-wide association studies to explain the genetics of T1D has failed to advance our understanding of T1D pathogenesis or identify biomarkers of disease progression or therapeutic targets. Using the nonobese diabetic (NOD) mouse model of T1D and the non-disease prone congenic NOD.B10 mice, our laboratory demonstrated striking tissue-specific and age-dependent changes in gene expression during disease progression. We established a "roadmap" of differential gene expression and used this to identify candidate genes in mice (and human orthologs) that play a role in disease pathology. Here, we describe two genes, Deformed epidermal autoregulatory factor 1 (Deaf1) and Adenosine A1 receptor (Adora1), that are differentially expressed and alternatively spliced in the pancreatic lymph nodes or islets of NOD mice and T1D patients to form dominant-negative non-functional isoforms. Loss of Deaf1 function leads to reduced peripheral tissue antigen expression in lymph node stromal cells and may contribute to a breakdown in peripheral tolerance, while reduced Adora1 function results in an early intrinsic alpha cell defect that may explain the hyperglucagonemia and resulting beta cell stress observed prior to the onset of diabetes. Remarkably, both genes were also alternatively spliced in the same tissues of auto-antibody positive prediabetic patients, and these splicing events resulted in similar downstream effects as those seen in NOD mice. These findings demonstrate the value of gene expression profiling in studying disease pathogenesis in T1D.
View details for DOI 10.1007/s12026-014-8501-8
View details for PubMedID 24682832
Involvement of Adenosine Signaling in Controlling the Release of Ghrelin from the Mouse Stomach
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2011; 336 (1): 77-86
Ghrelin, a potent orexigenic hormone released from the stomach, is important in regulating energy metabolism. Abnormal ghrelin levels are associated with eating disorders and metabolic diseases. However, factors involved in the regulation of ghrelin release remain unclear. Here, we examined the involvement of adenosine signaling in the control of ghrelin release from the perfused mouse stomach. Adenosine stimulated ghrelin release concentration-dependently, and the A(2A) receptor-selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) and 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 58261) abolished the increased release. The A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) augmented ghrelin release concentration-dependently, whereas the A(1) receptor-selective agonist 2-chloro-N(6)-cyclopentyladenosine inhibited ghrelin release. In A(2A) receptor knockout mice, adenosine inhibited ghrelin release, and the A(1) receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked this inhibition. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride increased ghrelin release in wild-type and A(1) receptor knockout mice but not in A(2A) receptor knockout mice. Colocalization of ghrelin immunoreactivity with A(1) and A(2A) receptor immunoreactivities in the gastric nerve fibers were observed. Colocalization was also detected for ghrelin and A(1) receptor immunoreactivities in the gastric mucosa. Blockade of neural activities with tetrodotoxin abolished the stimulatory effect of adenosine on ghrelin release. In conclusion, adenosine exerts predominantly a tonic A(2A) receptor-mediated stimulatory action on gastric ghrelin release, whereas an A(1) receptor-mediated inhibitory action is also apparent when the tonic excitatory effect was removed.
View details for DOI 10.1124/jpet.110.171280
View details for Web of Science ID 000285338400011
View details for PubMedID 20876230
Hypertonic stress regulates T cell function via pannexin-1 hemichannels and P2X receptors
JOURNAL OF LEUKOCYTE BIOLOGY
2010; 88 (6): 1181-1189
Hypertonic saline (HS) resuscitation increases T cell function and inhibits posttraumatic T cell anergy, which can reduce immunosuppression and sepsis in trauma patients. We have previously shown that HS induces the release of cellular ATP and enhances T cell function. However, the mechanism by which HS induces ATP release and the subsequent regulation of T cell function by ATP remain poorly understood. In the present study, we show that inhibition of the gap junction hemichannel pannexin-1 (Panx1) blocks ATP release in response to HS, and HS exposure triggers significant changes in the expression of all P2X-type ATP receptors in Jurkat T cells. Blocking or silencing of Panx1 or of P2X1, P2X4, or P2X7 receptors blunts HS-induced p38 MAPK activation and the stimulatory effects of HS on TCR/CD28-induced IL-2 gene transcription. Moreover, treatment with HS or agonists of P2X receptors overcomes T cell suppression induced by the anti-inflammatory cytokine IL-10. These findings indicate that Panx1 hemichannels facilitate ATP release in response to hypertonic stress and that P2X1, P2X4, and P2X7 receptor activation enhances T cell function. We conclude that HS and P2 receptor agonists promote T cell function and thus, could be used to improve T cell function in trauma patients.
View details for DOI 10.1189/jlb.0410211
View details for Web of Science ID 000285867500016
View details for PubMedID 20884646
View details for PubMedCentralID PMC2996895
Pannexin-1 hemichannel-mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse
2010; 116 (18): 3475-3484
Engagement of T cells with antigen-presenting cells requires T-cell receptor (TCR) stimulation at the immune synapse. We previously reported that TCR stimulation induces the release of cellular adenosine-5'-triphosphate (ATP) that regulates T-cell activation. Here we tested the roles of pannexin-1 hemichannels, which have been implicated in ATP release, and of various P2X receptors, which serve as ATP-gated Ca(2+) channels, in events that control T-cell activation. TCR stimulation results in the translocation of P2X1 and P2X4 receptors and pannexin-1 hemichannels to the immune synapse, while P2X7 receptors remain uniformly distributed on the cell surface. Removal of extracellular ATP or inhibition, mutation, or silencing of P2X1 and P2X4 receptors inhibits Ca(2+) entry, nuclear factors of activated T cells (NFAT) activation, and induction of interleukin-2 synthesis. Inhibition of pannexin-1 hemichannels suppresses TCR-induced ATP release, Ca(2+) entry, and T-cell activation. We conclude that pannexin-1 hemichannels and P2X1 and P2X4 receptors facilitate ATP release and autocrine feedback mechanisms that control Ca(2+) entry and T-cell activation at the immune synapse.
View details for DOI 10.1182/blood-2010-04-277707
View details for Web of Science ID 000283853200017
View details for PubMedID 20660288
Autocrine regulation of T-cell activation by ATP release and P2X(7) receptors
2009; 23 (6): 1685-1693
T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X(7) receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4(+) T cells express abundant P2X(7) receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 microM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X(7) receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X(7) receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X(7) receptors, compared to control BALB/c mice, which express fully functional P2X(7) receptors. We conclude that ATP release and autocrine, positive feedback through P2X(7) receptors is required for the effective activation of T cells.
View details for DOI 10.1096/fj.08-126458
View details for Web of Science ID 000266652400008
View details for PubMedID 19211924
A3 and P2Y2 receptors control the recruitment of neutrophils to the lungs in a mouse model of sepsis
2008; 30 (2): 173-177
We have recently shown that A3 adenosine receptors and P2Y2 purinergic receptors play an important role in neutrophil chemotaxis. Chemotaxis of neutrophils to sites of infections is critical for immune defense. However, excessive accumulation of neutrophils in the lungs can cause acute lung tissue damage. Here we assessed the role of A3 and P2Y2 receptors in neutrophil sequestration to the lungs in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) using adult male C57BL/6J mice (wild type [WT]), homozygous A3 receptor knockout (A3KO) mice, and P2Y2 receptor knockout (P2Y2KO) mice. Animals were killed 2, 4, 6, or 8 h after CLP, and peritoneal lavage fluid and blood were collected. Lungs were removed, and neutrophil infiltration was evaluated using elastase as a marker. Leukocyte and bacterial counts in peritoneal lavage fluid and blood samples were determined. Survival after sepsis was determined in a separate group. Leukocyte counts in the peritoneum were lower in A3KO and P2Y2KO mice than in WT mice. Conversely, initial leukocyte counts in the peripheral blood were higher in KO mice than in WT mice. Neutrophil sequestration to the lungs reached a maximum 2 h after CLP and remained significantly higher in WT mice compared with A3KO and P2Y2KO mice (P < 0.001). Survival after 24 h was significantly lower in WT mice (37.5%) than in A3KO or P2Y2KO mice (82.5%; P < 0.05). These data suggest that A3 and P2Y2 receptors are involved in the influx of neutrophils into the lungs after sepsis. Thus, pharmaceutical approaches that target these receptors might be useful to control acute lung tissue injury in sepsis.
View details for DOI 10.1097/SHK.0b013e318160dad4
View details for Web of Science ID 000257878500010
View details for PubMedID 18091570
Hypertonic stress regulates T-cell function by the opposing actions of extracellular adenosine triphosphate and adenosine
2007; 27 (3): 242-250
Hypertonic saline (HS) treatment promotes interleukin (IL)-2 production and enhances T-cell activation by the release of cellular adenosine triphosphate (ATP) that activates P2 nucleotide receptors. Released ATP can be hydrolyzed to adenosine, which inhibits T-cell activation. We examined if adenosine affects the response of T cells to HS treatment, and found that the amount of ATP released from T cells is a function of the HS concentration and duration of HS exposure. Physiologically relevant HS concentrations (<40 mmol/L) induced rapid ATP release, with the highest ATP concentrations released within 1 min. The released ATP was converted to adenosine, which opposed the enhancing effects of HS on IL-2 production. We found that Jurkat and CD4+ primary human T cells express most abundantly the A2A and A2B adenosine receptor subtypes, which mediate the suppressive effects of adenosine, as the A2 receptor agonist CGS 21680 suppressed IL-2 production, whereas the A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine augmented the enhancing effect of HS on T-cell function. Elimination of extracellular adenosine by adding exogenous adenosine deaminase also increased the enhancing effects of HS. These data suggest that the effect of HS treatment on T-cell function can be modulated with pharmacological agents that abolish the suppressive effects of adenosine formed from the ATP that is released in response to HS treatment.
View details for DOI 10.1097/01.shk.0000245014.96419.3a
View details for Web of Science ID 000244475300005
View details for PubMedID 17304104
Hypertonic saline resuscitation: Efficacy may require early treatment in severely injured patients
63rd Annual Meeting of the American-Association-for-the-Surgery-of-Trauma/Japanese-Association-for-Acute-Medicine
LIPPINCOTT WILLIAMS & WILKINS. 2007: 299–306
Activation of polymorphonuclear neutrophils (PMN) is a critical event leading to host tissue injury and organ damage after trauma. Hypertonic saline (HS) resuscitation prevents PMN activation in vitro and in animal models. Here, we studied how clinical parameters and timing requirements influence the efficacy of HS in suppressing PMN activation.Twenty-six injured patients and 16 healthy volunteers were included as study subjects. To study how clinical parameters affect the efficacy of HS, whole blood samples from patients were collected 24 hours after admission, treated with HS and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and PMN oxidative burst and degranulation were measured using flow cytometry. We studied the effect of timing on the ability of HS to inhibit PMN function by exposing blood of healthy volunteers to plasma samples from trauma patients before or after the addition of fMLP and HS.Age and gender did not significantly influence the effect of HS on PMN function. The suppressive effect of clinically relevant HS concentrations (20 mmol/L) on PMN oxidative burst correlated weakly with Sepsis Severity Score (SSS) and Acute Physiology and Chronic Health Evaluation II (APACHE II) score but not with the Injury Severity Score (ISS) or Multiple Organ Failure score (MOF). There was no correlation between any of these clinical scores and degranulation. HS was significantly less effective in suppressing oxidative burst of PMN from patients with ISS >10, APACHE II >5, MOF >0, or SSS >1 compared with patients with ISS < or =10, APACHE II < or =5, MOF = 0, or SSS < or =1. HS more effectively suppressed PMN activation when PMN were pretreatment with HS, whereas it was less effective on PMN previously primed in vivo or in vitro by adding trauma plasma. HS was ineffective on PMN previously stimulated in vitro with fMLP.Our data suggest that HS resuscitation may prevent PMN activation most effectively when patients are treated with HS early in the field.
View details for DOI 10.1097/01.ta.0000222956.88760.33
View details for Web of Science ID 000244333300007
View details for PubMedID 17297316
ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors
2006; 314 (5806): 1792-1795
Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.
View details for DOI 10.1126/science.1132559
View details for Web of Science ID 000242833600064
View details for PubMedID 17170310
Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
2006; 290 (4): C1051-C1059
Hypertonic saline (HS) holds promise as a novel resuscitation fluid for the treatment of trauma patients because HS inhibits polymorphonuclear neutrophil (PMN) activation and thereby prevents host tissue damage and associated posttraumatic complications. However, depending on conditions of cell activation, HS can increase PMN degranulation, which could exacerbate tissue damage in trauma victims. The cellular mechanism by which HS increases degranulation is unknown. In the present study, we tested whether HS-induced ATP release from PMN and feedback via P1 and/or P2 receptors may be involved in the enhancement of degranulation by HS. We found that HS enhances elastase release and ERK and p38 MAPK activation when HS is added after activation of PMN with formyl peptide (fMLP) or phorbol ester (PMA). Agonists of P2 nucleotide and A3 adenosine receptors mimicked these enhancing effects of HS, whereas antagonists of A3 receptors or removal of extracellular ATP with apyrase diminished the response to HS. A1 adenosine receptor antagonists increased the enhancing effect of HS, whereas A1 receptor agonists inhibited elastase release. These data suggest that HS upregulates degranulation via ATP release and positive feedback through P2 and A3 receptors. We propose that these feedback mechanisms can serve as potential pharmacological targets to fine-tune the clinical effectiveness of HS resuscitation.
View details for DOI 10.1152/ajpcell.00216.2005
View details for Web of Science ID 000236573300013
View details for PubMedID 16282197
Surface expression of HSP72 by LPS-stimulated neutrophils facilitates gamma delta T cell-mediated killing
EUROPEAN JOURNAL OF IMMUNOLOGY
2006; 36 (3): 712-721
During inflammation and sepsis, accumulation of activated neutrophils causes lung tissue damage and organ failure. Effective clearance of neutrophils reduces the risk of organ failure; however, its mechanisms are poorly understood. Because lungs are rich in gammadeltaT cells, we investigated the physiological role of these cells in the protection of lung tissue from infiltrating neutrophils. In a mouse model of sepsis, we found that the lungs of survivors contained significantly higher numbers of gammadeltaT cells than those of mice that died from sepsis. The number of gammadeltaT cells correlated inversely with the number of neutrophils in the lungs and with the degree of lung tissue damage. LPS rapidly elicited the expression of heat shock protein (HSP) 72 on the surface of human neutrophils. Inhibitors of transcription, protein synthesis, and intracellular protein transport blocked HSP72 expression, indicating that de novo synthesis is required. gammadeltaT cells targeted and rapidly killed LPS-treated neutrophils through direct cell-to-cell contact. Pre-treatment with neutralizing antibodies to HSP72 diminished neutrophil killing. Our data indicate that HSP72 expression on the cell surface predisposes inflamed neutrophils to killing by gammadeltaT cells. This intercellular exchange may allow gammadeltaT cells to resolve inflammation and limit host tissue damage during sepsis.
View details for DOI 10.1002/eji.200535422
View details for Web of Science ID 000236457400021
View details for PubMedID 16482515
Effect of omeprazole on gastric adenosine A(1) and A(2A) receptor gene expression and function
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2004; 311 (1): 180-189
Adenosine has been shown to inhibit immunoreactive gastrin (IRG) release and to stimulate somatostatin-like immunoreactivity (SLI) release by activating adenosine A(1) and A(2A) receptors, respectively. Since the synthesis and release of gastrin and somatostatin are regulated by the acid secretory state of the stomach, the effect of achlorhydria on A(1) and A(2A) receptor gene expression and function was examined. Omeprazole-induced achlorhydria was shown to suppress A(1) and A(2A) receptor gene expression in the antrum and corporeal mucosa, but not in the corporeal muscle. Omeprazole treatment produced reciprocal changes in A(1) receptor and gastrin gene expression, and parallel changes in A(2A) receptor and somatostatin gene expression. The localization of A(1) and A(2A) receptors on gastrinsecreting G-cells and somatostatin-secreting D-cells, respectively, suggests that changes in adenosine receptor expression may modulate the synthesis and release of gastrin and somatostatin. Thus, the effect of omeprazole on adenosine receptor-mediated changes in IRG and SLI release was also examined in the vascularly perfused rat stomach. After omeprazole treatment, the A(1) receptor-mediated inhibition of IRG and SLI release induced by N(6)-cyclopentyladenosine (A(1) receptor-selective agonist) was not altered, but the A(2A) receptor-mediated augmentation of SLI release induced by 2-p-(2-carboxyethyl-)phenethylamino-5'-N-ethylcarboxamidoadenosine (A(2A)-selective agonist) was significantly attenuated. These findings agree well with the corresponding omeprazole-induced decrease in antral A(2A) receptor mRNA expression. Overall, the present study suggests that adenosine receptor gene expression and function may be altered by omeprazole treatment. Acid-dependent changes in adenosine receptor expression may represent a novel purinergic regulatory feedback mechanism in controlling gastric acid secretion.
View details for DOI 10.1124/jpet.104.069708
View details for Web of Science ID 000223896100022
View details for PubMedID 15155771
Role of adenosine A(1) receptor in the regulation of gastrin release
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2004; 310 (2): 477-487
Adenosine has been demonstrated to inhibit gastric acid secretion. In the rat stomach, this inhibitory effect may be mediated indirectly by the inhibition of gastrin release. Results show that the A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) suppressed immunoreactive gastrin (IRG) release in a concentration-dependent manner. CPA significantly inhibited IRG release at 0.001 microM and maximally inhibited IRG release at 1 microM. At concentrations of 0.001 to 0.1 microM, the A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine and A(3) receptor-selective agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta-d-ribofuranuronamide, had no effect on IRG release, suggesting the involvement of A(1) receptors. In agreement, the A(1) receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine abolished adenosine-induced inhibition of IRG release. Results of immunohistochemistry experiments reveal the presence of A(1) receptor immunoreactivity on mucosal G-cells and D-cells, and the gastric plexi, but not parietal cells, suggesting that adenosine may act directly on G-cells or indirectly on the gastric plexi to modulate IRG release. The structure of the mucosal A(1) receptor was found to be identical to that in the rat brain. Alternative splicing within the coding region of this receptor did not occur. A real-time reverse transcription-polymerase chain reaction assay was developed to measure gastric A(1) receptor gene expression. The highest level of gastric A(1) receptor mRNA was found in the corporeal muscle. However, this level was significantly lower in comparison with the striatum. In conclusion, this study shows that adenosine may suppress IRG release, at least in part, by activating A(1) receptors localized on G-cells and may consequently result in an inhibition of gastric acid secretion.
View details for DOI 10.1124/jpet.104.066654
View details for Web of Science ID 000222728500007
View details for PubMedID 15044554
Role of adenosine A(2A) receptor in the regulation of gastric somatostatin release
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2004; 309 (2): 804-815
Adenosine has been demonstrated to inhibit gastric acid secretion. In the rat stomach, this inhibitory effect may be mediated indirectly by increasing the release of somatostatin-like immunoreactivity (SLI). Results show that adenosine analogs augmented SLI release in the isolated vascularly perfused rat stomach. The rank order of potency of the analogs in stimulating SLI release was 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) approximately 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > R-(-)-N(6)-(2-phenylisopropyl)adenosine >1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta-d-ribofuranuronamide > N(6)-cyclopentyladenosine approximately N(6)-cyclohexyladenosine > S-(+)-N(6)-(2-phenylisopropyl) adenosine, suggesting the involvement of the A(2A) receptor. In agreement, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a] [1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an A(2A) receptor antagonist, was shown to abolish the adenosine- and CGS 21680-stimulated SLI release. Immunohistochemical studies reveal the presence of A(2A) receptor immunoreactivity on the gastric plexi and mucosal D-cells, but not on parietal cells and G-cells, suggesting that adenosine may act directly on D-cells or indirectly on the gastric plexi to augment SLI release. The present study also demonstrates that the structure of the mucosal A(2A) receptor is identical to that in the rat brain, and that alternative splicing of this gene does not occur. A real-time reverse transcription-polymerase chain reaction assay has also been established to quantify the levels of A(2A) receptor mRNA. Results show that gastric tissues contained significantly lower levels of A(2A) receptor mRNA compared with the striatum. The lowest level was detected in the mucosa. In conclusion, adenosine may act on A(2A) receptors to augment SLI release and consequently control gastric acid secretion.
View details for DOI 10.1124/jpet.103.061986
View details for Web of Science ID 000220972900046
View details for PubMedID 14742743
Cellular localization and distribution of neurokinin-1 receptors in the rat stomach
AUTONOMIC NEUROSCIENCE-BASIC & CLINICAL
2003; 104 (2): 95-108
In the stomach, the majority of substance P's effects are mediated by the activation of neurokinin-1 (NK1) receptors. The gastric cellular distribution of these receptors in Wistar and Sprague-Dawley rats was determined using immunocytochemistry. The localization of the NK1 receptors with respect to von Willebrand's factor, protein gene product 9.5, substance P, vasoactive intestinal peptide, and calcitonin gene-related peptide was also determined. Results show that NK1 receptor immunoreactivity was dependent on the duration of fixation. In corpus and antrum tissues that were fixed in 4% paraformaldehyde for 30 min, the presence of NK1 receptor immunoreactivity was demonstrated on nerve fibers throughout the stomach, on the surface and in the cytoplasm of myenteric cell bodies, on circular smooth muscle cells, and on vascular endothelial cells. This was observed in tissues from both rodent strains. Overnight fixation in the same fixative, however, demonstrated the presence of NK1 receptor immunoreactivity only on nerve fibers and cell bodies of the myenteric plexus, and on circular smooth muscle cells. In 30-min fixed tissues, the localization of NK1R immunoreactivity on vascular endothelial cells and nerve fibers was confirmed by co-localization with von Willebrand's factor and protein gene product 9.5 immunoreactivity, respectively. In both rodent strains, NK1 receptor immunoreactivity was co-localized with substance P immunoreactivity on nerve fibers of the longitudinal and circular muscle. In the Wistar rat, NK1 receptor immunoreactivity was co-localized with vasoactive intestinal peptide immunoreactivity or calcitonin gene-related peptide immunoreactivity throughout the stomach. However, in the Sprague-Dawley rat, NK1 receptor immunoreactivity was only co-localized with calcitonin gene-related peptide immunoreactivity in a minority of fibers of the circular muscle. The overall results of this study show that the antigenic epitopes of the NK1 receptor are sensitive to overfixation. When tissues were not overfixed, NK1 receptor immunoreactivity was distributed more extensively throughout the rat stomach than has been described previously. The results of this study provide the anatomical basis for many of the actions of substance P in the rat stomach.
View details for DOI 10.1016/S1566-0702(02)00293-X
View details for Web of Science ID 000181995600004
View details for PubMedID 12648611