Rheumatoid Arthritis (RA) is a chronic autoimmune disease associated with inflammation and joint remodeling. Adenosine deaminase (ADA), a risk factor in RA, degrades adenosine, an anti-inflammatory molecule, resulting in an inflammatory bias. We present an integrative analysis of clinical data, cytokines, serum metabolomics in RA patients and mechanistic studies on ADA-mediated effects on in vitro cell culture models. ADA activity differentiated patients into low and high ADA sets. The levels of the cytokines TNFα, IFNγ, IL-10, TGFβ and sRANKL were elevated in RA and more pronounced in high ADA sets. Serum metabolomic analysis shows altered metabolic pathways in RA which were distinct between low and high ADA sets. Comparative analysis with previous studies shows similar pathways are modulated by DMARDs and biologics. Random forest analysis distinguished RA from control by methyl-histidine and hydroxyisocaproic acid, while hexose-phosphate and fructose-6-phosphate distinguished high ADA from low ADA. The deregulated metabolic pathways of High ADA datasets significantly overlapped with high ADA expressing PBMCs GEO transcriptomics dataset. ADA induced the death of chondrocytes, synoviocyte proliferation, both inflammation in macrophages and their differentiation into osteoclasts and impaired differentiation of mesenchymal stem cells to osteoblasts and mineralization. PBMCs expressing elevated ADA had increased expression of cytokines and P2 receptors compared to synovial macrophages which has low expression of ADA. Our data demonstrates increased cytokine levels and distinct metabolic signatures of RA based on the ADA activity, suggests an important role for ADA in the pathophysiology of RA joints and as a potential marker and therapeutic target in RA patients.

The Asian tiger mosquito, Aedes albopictus, is an important vector for the transmission of arboviruses such as dengue virus (DENV). Adenosine deaminase (ADA) is a well-characterized metabolic enzyme involved in facilitating blood feeding and (or) arbovirus transmission in some hematophagous insect species. We previously reported the immunologic function of ADA by investigating its effect on mast cell activation and the interaction with mast cell tryptase and chymase. The 2-D gel electrophoresis and mass spectrometry analysis in the current study revealed that ADA is present and upregulated following mosquito blood feeding, as confirmed by qRT-PCR and western blot. In addition, the recombinant ADA efficiently converted adenosine to inosine. Challenging the Raw264.7 and THP-1 cells with recombinant ADA resulted in the upregulation of IL-1β, IL-6, TNF-α, CCL2, IFN-β, and ISG15. The current study further identified recombinant ADA as a positive regulator in NF-κB signaling targeting TAK1. It was also found that recombinant Ae. albopictus ADA facilitates the replication of DENV-2. Compared with cells infected by DENV-2 alone, the co-incubation of recombinant ADA with DENV-2 substantially increased IL-1β, IL-6, TNF-α, and CCL2 gene transcripts in Raw264.7 and THP-1 cells. However, the expression of IFN-β and ISG15 were markedly downregulated in Raw264.7 cells but upregulated in THP-1 cells. These findings suggest that the immunomodulatory protein, Ae. albopictus ADA is involved in mosquito blood feeding and may modulate DENV transmission via macrophage or monocyte-driven immune response.

Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid- to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPR-induced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing.

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

Deficient insulin signaling is a key event mediating diabetic glomerulopathy. Additionally, diabetic kidney disease has been related to increased levels of adenosine. Therefore, we tested a link between insulin deficiency and dysregulated activity of the equilibrative nucleoside transporters (ENTs) responsible for controlling extracellular levels of adenosine. In ex vivo glomeruli, high D-glucose decreased nucleoside uptake mediated by ENT1 and ENT2 transporters, resulting in augmented extracellular levels of adenosine. This condition was reversed by exposure to insulin. Particularly, insulin through insulin receptor/PI3K pathway markedly upregulated ENT2 uptake activity to restores the extracellular basal level of adenosine. Using primary cultured rat podocytes as a cellular model, we found insulin was able to increase ENT2 maximal velocity of transport. Also, PI3K activity was necessary to maintain ENT2 protein levels in the long term. In glomeruli of streptozotocin-induced diabetic rats, insulin deficiency leads to decreased activity of ENT2 and chronically increased extracellular levels of adenosine. Treatment of diabetic rats with adenosine deaminase attenuated both the glomerular loss of nephrin and proteinuria. In conclusion, we evidenced ENT2 as a target of insulin signaling and sensitive to dysregulation in diabetes, leading to chronically increased extracellular adenosine levels and thereby setting conditions conducive to kidney injury.

Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens.

Extracellular adenosine, generated via the concerted action of CD39 and CD73, contributes to T-cell differentiation and function. Adenosine concentrations are furthermore influenced by adenosine deaminase binding protein CD26. Because aberrant T-cell phenotypes had been reported in anti-neutrophil cytoplasmic auto-antibody (ANCA)-associated vasculitis (AAV) patients, an impaired expression of these molecules on T-cells of AAV patients was hypothesized in the present study. While in AAV patients (n = 29) CD26 was increased on CD4+ lymphocytes, CD39 and CD73 were generally reduced on patients' T-cells. In CD4+ cells significant differences in CD73 expression were confined to memory CD45RA- cells, while in CD4- lymphocytes differences were significant in both naïve CD45RA+ and memory CD45RA- cells. The percentage of CD4-CD73+ cells correlated with micro-RNA (miR)-31 expression, a putative regulator of factor inhibiting hypoxia-inducible factor 1 alpha (FIH-1), inversely with serum C-reactive protein (CRP) and positively with estimated glomerular filtration rate (eGFR). No correlation with disease activity, duration, and ANCA profile was found. It remains to be assessed if a decreased CD73 and CD39 expression underlies functional impairment of lymphocytes in AAV patients. Likewise, the relations between frequencies of CD4-CD73+ cells and serum CRP or eGFR require further functional elucidation.

AMPD1 is an adenosine monophosphate deaminase that catalyzes the deamination of AMP to IMP. To understand the physiological function of AMPD1, we obtained a strain of Ampd1 mutant mice from KOMP repository, which was generated by a knockout-first strategy. An elevated AMP level and almost complete lack of IMP was detected in the skeletal muscle of E18.5 Ampd1tm1a/tm1a mice. However, Ampd1tm1a/tm1a mice died in 2 days postnatally, which was contradicting to previous reports. After removal of the knockout-first cassette and critical exon, mice homozygous for the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d alleles survived to adulthood. RNA-seq analysis indicated that the expression of two neighboring genes, Man1a2 and Nras, were disrupted in the Ampd1tm1a/tm1a mice, but normal in the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d mice. The neonatal lethality phenotype in the Ampd1tm1a/tm1a mice was consistent with the Man1a2-deficient mice. Our results indicated the knockout-first cassette may cause off-target effect by influence the expression of neighboring genes. This study, together with other reports, strongly suggests that removal of targeting cassette by site-specific recombinases is very important for the accurate phenotypic interpretation on mice generated by target mutations.

Adenosine-to-inosine RNA editing is a process mediated by adenosine deaminases that act on the RNA (ADAR) gene family. It has been discovered recently as an epigenetic modification dysregulated in human cancers. However, the clinical significance of RNA editing in patients with liver metastasis from colorectal cancer (CRC) remains unclear. The current study aimed to systematically and comprehensively investigate the significance of adenosine deaminase acting on RNA 1 (ADAR1) expression status in 83 liver metastatic tissue samples collected from 36 patients with CRC. The ADAR1 expression level was significantly elevated in liver metastatic tissue samples obtained from patients with right-sided, synchronous, or RAS mutant-type CRC. ADAR1-high liver metastasis was significantly correlated with remnant liver recurrence after hepatic metastasectomy. A high ADAR1 expression was a predictive factor of remnant liver recurrence (area under the curve = 0.72). Results showed that the ADAR1 expression level could be a clinically relevant predictive indicator of remnant liver recurrence. Patients with liver metastases who have a high ADAR1 expression requires adjuvant chemotherapy after hepatic metastasectomy.

As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.

Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We examined the pH-sensitivity of nucleoside influx and efflux by hENT4 using a recombinant transfection model that lacks the confounding influences of other nucleoside transporters (PK15-NTD). We established that [3H]2-chloroadenosine, which is resistant to metabolism by adenosine deaminase, is a substrate for hENT4. Transport of [3H]2-chloroadenosine at a pH of 6.0 in PK15-NTD cells stably transfected with SLC29A4 was biphasic, with a low capacity (Vmax ~ 30 pmol/mg/min) high-affinity component (Km ~ 50 µM) apparent at low substrate concentrations, which shifted to a high capacity (Vmax ~ 500 pmol/mg/min) low affinity system (Km > 600 µM) displaying positive cooperativity at concentrations above 200 µM. Only the low affinity component was observed at a neutral pH of 7.5 (Km ~ 2 mM). Efflux of [3H]2-chloroadenosine from these cells was also enhanced by more than 4-fold at an acidic pH. Enhanced influx and efflux of nucleosides by hENT4 under acidic conditions supports its potential as a therapeutic target in pathologies such as ischaemia-reperfusion injury.

Maintaining stemness of leukemic stem cells (LSCs) and reciprocal interactions between leukemia and stromal cells support leukemic progression and resistance to chemotherapy. Targeting the niche-based microenvironment is thus a new approach for leukemia therapy. Cordycepin is an analogue of adenosine and has been suggested to possess anti-leukemia properties. However, whether cordycepin influences association of leukemia and mesenchymal stromal cells has never been investigated. Here we show that cordycepin reduces CD34+CD38- cells in U937 and K562 cells and induces Dkk1 expression via autocrine and paracrine regulation in leukemia and mesenchymal stromal/stem cells (MSCs). Cordycepin suppresses cell attachment of leukemia with MSCs and downregulates N-cadherin in leukemia and VCAM-1 in MSCs. Moreover, incubation with leukemic conditioned media (CM) significantly induces IL-8 and IL-6 expression in MSCs, which is abrogated by cordycepin. Suppression of leukemic CM-induced VCAM-1 and IL-8 by cordycepin in MSCs is mediated by impairing NFκB signaling. Finally, cordycepin combined with an adenosine deaminase inhibitor prolongs survival in a leukemic mouse model. Our results indicate that cordycepin is a potential anti-leukemia therapeutic adjuvant via eliminating LSCs and disrupting leukemia-stromal association.

Mesenchymal stromal cells (MSCs) represent a key component of bone marrow (BM) microenvironment and display immune-regulatory properties. We performed a detailed analysis of biological/functional properties of BM-MSCs derived from 33 pediatric patients affected by primary immune-deficiencies (PID-MSCs): 7 Chronic Granulomatous Disease (CGD), 15 Wiskott-Aldrich Syndrome (WAS), 11 Severe Combined Immunodeficiency (SCID). Results were compared with MSCs from 15 age-matched pediatric healthy-donors (HD-MSCs). Clonogenic and proliferative capacity, differentiation ability, immunophenotype, immunomodulatory properties were analyzed. WB and RT-qPCR for CYBB, WAS and ADA genes were performed. All PID-MSCs displayed clonogenic and proliferative capacity, morphology and immunophenotype comparable with HD-MSCs. PID-MSCs maintained the inhibitory effect on T- and B-lymphocyte proliferation, except for decreased inhibitory ability of SCID-MSCs at MSC:PBMC ratio 1:10. While HD- and CGD-MSCs were able to inhibit monocyte maturation into immature dendritic cells, in SCID- and WAS-MSCs this ability was reduced. After Toll-like Receptor priming, PID-MSCs displayed in vitro an altered gene expression profile of pro- and anti-inflammatory soluble factors. PID-MSCs displayed lower PPARγ levels and WAS- and SCID-MSCs higher levels of key osteogenic markers, as compared with HD-MSCs. Our results indicate that PID-MSCs may be defective in some functional abilities; whether these defects contribute to disease pathophysiology deserves further investigation.

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme intrinsic to the purine salvage pathway, leads to severe combined immunodeficiency (SCID) both in humans and mice. Lack of ADA results in the intracellular accumulation of toxic metabolites which have effects on T cell development and function. While untreated ADA-SCID is a fatal disorder, there are different therapeutic options available to restore ADA activity and reconstitute a functioning immune system, including enzyme replacement therapy (ERT). Administration of ERT in the form of pegylated bovine ADA (PEG-ADA) has proved a life-saving though non-curative treatment for ADA-SCID patients. However, in many patients treated with PEG-ADA, there is suboptimal immune recovery with low T and B cell numbers. Here, we show reduced thymus cellularity in ADA-SCID mice despite weekly PEG-ADA treatment. This was associated with lack of effective adenosine (Ado) detoxification in the thymus. We also show that thymocyte development in ADA-deficient thymi is arrested at the DN3-to-DN4 stage transition with thymocytes undergoing dATP-induced apoptosis rather than defective TCRβ rearrangement or β-selection. Our studies demonstrate at a detailed level that exogenous once-a-week enzyme replacement does not fully correct intra-thymic metabolic or immunological abnormalities associated with ADA deficiency.

We recently showed that spinal cord injury (SCI) leads to a decrease in mRNA editing of serotonin receptor 2C (5-HT2CR) contributing to post-SCI spasticity. Here we study post-SCI mRNA editing and global gene expression using massively parallel sequencing. Evidence is presented that the decrease in 5-HT2CR editing is caused by down-regulation of adenosine deaminase ADAR2 and that editing of at least one other ADAR2 target, potassium channel Kv1.1, is decreased after SCI. Bayesian network analysis of genome-wide transcriptome data indicates that down-regulation of ADAR2 (1) is triggered by persistent inflammatory response to SCI that is associated with activation of microglia and (2) results in changes in neuronal gene expression that are likely to contribute both to post-SCI restoration of neuronal excitability and muscle spasms. These findings have broad implications for other diseases of the Central Nervous System and could open new avenues for developing efficacious antispastic treatments.

Both TDP-43 pathology and failure of RNA editing of AMPA receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of the majority of patients with amyotrophic lateral sclerosis (ALS). AR2 mice, in which an RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is conditionally knocked out in the motor neurons, exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons through a Ca(2+)-permeable AMPA receptor-mediated mechanism. Therefore, amelioration of the increased Ca(2+) influx by AMPA receptor antagonists may be a potential ALS therapy. Here, we showed that orally administered perampanel, a selective, non-competitive AMPA receptor antagonist significantly prevented the progression of the ALS phenotype and normalized the TDP-43 pathology-associated death of motor neurons in the AR2 mice. Given that perampanel is an approved anti-epileptic drug, perampanel is a potential candidate ALS drug worthy of a clinical trial.

Nuclear dysfunction in motor neurons has been hypothesized to be a principal cause of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we investigated the mechanism by which the nuclear pore complex (NPC) is disrupted in dying motor neurons in a mechanistic ALS mouse model (adenosine deaminase acting on RNA 2 (ADAR2) conditional knockout (AR2) mice) and in ALS patients. We showed that nucleoporins (Nups) that constituted the NPC were cleaved by activated calpain via a Ca2+-permeable AMPA receptor-mediated mechanism in dying motor neurons lacking ADAR2 expression in AR2 mice. In these neurons, nucleo-cytoplasmic transport was disrupted, and the level of the transcript elongation enzyme RNA polymerase II phosphorylated at Ser2 was significantly decreased. Analogous changes were observed in motor neurons lacking ADAR2 immunoreactivity in sporadic ALS patients. Therefore, calpain-dependent NPC disruption may participate in ALS pathogenesis, and inhibiting Ca2+-mediated cell death signals may be a therapeutic strategy for ALS.

Adenosine deaminase acting on RNA 1 (ADAR1), a recently described epigenetic modifier, is believed to play a critical oncogenic role in human cancers. However, its functional role and clinical significance in cervical cancer (CC) remain unclear. ADAR1 knockdown was performed to investigate its oncogenic functions in SiHa (HPV16), HeLa (HPV18), and Yumoto (non-HPV) CC cell lines. Cytoplasmic and nuclear ADAR1 expression were examined to clarify their correlation with clinicopathological parameters and prognosis in patients with CC. This resulted in increased apoptosis and necroptosis in HPV16 -type SiHa, HPV18-type HeLa, and non-HPV-type Yumoto CC cell lines. Progression-free survival (PFS) rates of patients exhibiting high cytoplasmic and nuclear ADAR1 expression were poorer than those in the other groups (P = 0.016). Multivariate analysis indicated that the combination of higher cytoplasmic and nuclear ADAR1 expression was an independent predictor of prognosis in patients with CC (P = 0.017). ADAR1 could be a potential therapeutic target for HPV-positive or HPV-negative CC. The combination of cytoplasmic and nuclear ADAR1 comprises a better prognostic factor for CC.

The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was "proliferation of endothelial cells", indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as "proliferation of endothelial cells" (bta-miR-196 b), "bacteria recognition" (bta-miR-146 b), and "regulation of the inflammatory response" (bta-miR-146 b).

Excessive inflammation resulting from activation of the innate immune system significantly contributes to ischemia/reperfusion injury (IRI). Inflammatory reactions in both IRI and infections share the same signaling pathways evoked by danger/pathogen associated molecular pattern molecules. The cytosolic retinoid-inducible gene I(RIG-I)-like RNA receptor (RLR) RNA sensing pathway mediates type I IFN production during viral infection and the sensing of viral RNA is regulated by adenosine deaminase acting on RNA 1 (ADAR1). Using a model of liver IRI, we provide evidence that ADAR1 also regulates cytosolic RNA-sensing pathways in the setting of ischemic stress. Suppression of ADAR1 significantly enhanced inflammation and liver damage following IRI, which was accompanied by significant increases in type I IFN through cytosolic RNA-sensing pathways. In addition, knocking ADAR1 down in hepatocytes exaggerates inflammatory signaling to dsRNA or endotoxin and results in over production of type I IFN, which could be abolished by the interruption of RIG-I. Therefore, we identified a novel ADAR1-dependent protective contribution through which hepatocytes guard against aberrant cytosolic RLR-RNA-sensing pathway mediated inflammatory reaction in response to acute liver IR. ADAR1 protects against over activation of viral RNA-sensing pathways in non-infectious tissue stress.