Adenosine A2A receptor, a potential valuable target for controlling reoxygenated DCs-triggered inflammation

Molecular Immunology 63 (2015) 559–565

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Adenosine A2A receptor, a potential valuable target for controlling reoxygenated DCs-triggered inflammation Chunmei Liu a,b , Qianwen Shang a , Yang Bai a , Chun Guo a , Faliang Zhu a , Lining Zhang a , Qun Wang a,∗ a b

Department of Immunology, Shandong University School of Medicine, Jinan 250012, Shandong, PR China Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong, PR China

a r t i c l e

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Article history: Received 26 May 2014 Received in revised form 22 September 2014 Accepted 12 October 2014 Available online 25 October 2014 Keywords: Reoxygenation Dendritic cells Adenosine receptor Inflammation Hypoxia

a b s t r a c t Dendritic cells (DCs) exposed to various oxygen tensions under physiopathological conditions are the critical immune cells linking innate and adaptive immunity. We have previously demonstrated that reoxygenation of hypoxia-differentiated DCs triggers complete DCs activation and inflammatory responses, so restraining the activation of reoxygenated DCs is important to suppress inflammatory responses in diseases caused by oxygen redelivery such as ischemia-reperfusion injury. In the current study, we showed that reoxygenation of hypoxia-differentiated DCs led to predominant expression of high levels of adenosine receptor A2A R on reoxygenated DCs as compared to those on hypoxic or normoxic DCs. Agonist CGS21680 targeting A2A R could effectively inhibit the maturation and activation of reoxygenated DCs through downregulating the expression of MHC class II molecules and CD86. In response to CGS21680 treatment, reoxygenated DCs exhibited a decrease in proinflammatory cytokines IL-1␤, IL-6 and TNF-␣, and an increase in immune-regulatory cytokine TGF-␤. These data suggest the critical role of A2A R signaling pathway in inhibiting the maturation and proinflammatory function of reoxygenated DCs, thereby proposing A2A R as a potential valuable target for controlling reoxygenated DCs-triggered inflammation. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Hypoxia resides in ischemic conditions including organ transplantation, trauma, hypovolemic shock, liver surgery and cardiovascular diseases, while the return of blood flow to the ischemic tissues, which is accompanied by the redelivery of oxygen (also known as reoxygenation), absolutely accelerates the damage of hypoxic organ through inducing ischemia reperfusion injury (IRI) (Kutala et al., 2007; Li and Jackson, 2002; Wang et al., 2002; Yamauchi and Kimura, 2008). Considerable data have demonstrated the important roles of innate and adaptive immune responses in IRI (Boros and Bromberg, 2006). As the most potent antigen presenting cells (APCs) linking innate immunity with adaptive immunity, dendritic cells (DCs) play the curial roles in IRI. It has been reported that activated DCs are involved in the pathologic process of some organs’ IRI such as kidney and liver (Castellaneta et al., 2014; Dong et al., 2007; Jang et al., 2009; Zhai et al., 2011). However, there are few data elucidating how DCs are activated in the

∗ Corresponding author. Tel.: +86 531 88382038. E-mail address: [email protected] (Q. Wang). http://dx.doi.org/10.1016/j.molimm.2014.10.012 0161-5890/© 2014 Elsevier Ltd. All rights reserved.

organs of IRI. We have reported in previous study that the change of oxygen tension maybe an important causal factor triggering the tolerance or activation of immune responses through regulating the maturation of DCs. Hypoxic microenvironment drives immune tolerance via suppressing the maturation of DCs; while reoxygenation of hypoxia-differentiated DCs results in complete recovery of their mature phenotype and function, and then drives immune response toward a proinflammatory direction, thus suggesting reoxygenation contributes to DCs activation and subsequent inflammation in IRI (Wang et al., 2010). So it is important to make clear and restrict the activation of reoxygenated DCs with regard to the control of IRI. Previous studies have indicated the accumulation of extracellular adenosine can trigger and translate hypoxia-related signal into immune cells through the adenosine receptors (ARs), which are definitely involved in the regulation of DCs function (Sitkovsky and Lukashev, 2005; Yang et al., 2010). To date, four ARs (A1 R, A2A R, A2B R, and A3 R) have been identified on most immune cells including DCs (Sitkovsky and Lukashev, 2005; Sitkovsky et al., 2004). Regarding the immunoregulatory function of ARs on DCs, studies from different models draw differential conclusions; and also different ARs pathways regulate DCs function in different ways. In normoxic conditions, adenosine upregulates the

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expression of CD80, CD86 and HLA-DR on DCs, but inhibits the release of TNF-␣, IL-12, as well as the capacity to induce Th1 polarization (Panther et al., 2003). While Sergey et al. show that adenosine is an important factor affecting differentiation of myeloid DCs, signaling through A2B R skews DCs differentiation toward a distinct DCs population that produces high levels of angiogenic factors and Th2-type cytokines, phenotype associated with promotion of angiogenesis, tumor growth, immune suppression, and tolerance (Novitskiy et al., 2008). Some in vivo animal models points out the anti-inflammatory effects of ARs signaling in IRI, which provide potential therapeutic targets for the control of IRI. Injection of A2A R agonist ATL146e reduced the infarct size in B6 mice after 24 h of reperfusion, but had no effect in A2A R knock-out mice (Yang et al., 2005). More interestingly, mice with A2A R-deficient DCs were more susceptible to kidney IRI and were not protected from injury by A2A R agonists (Li et al., 2012). Furthermore, Thiel found that the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2A R-mediated lung tissueprotecting pathway (Thiel et al., 2005). All these suggested the important roles of ARs pathways in DCs function or in IRI. However, it remains largely unknown about the function of ARs pathway in the metergasis of reoxygenated DCs, which is important for exploring the effective strategy to regulate the immune inflammation mediated by reoxygenated DCs. In this study, we investigated the expression and function of four ARs on the reoxygenated DCs. The results have demonstrated that reoxygenated DCs express high levels of A2A R compared to normoxic or hypoxic DCs; of note, A2A R is the most predominant receptor on reoxygenated DCs as compared to other ARs. A2A R agonist CGS21680 significantly inhibits the upregulation of costimultory molecules CD86 and MHC class II molecules (MHC-II) in reoxygenated DCs. More importantly, in response to CGS21680 treatment, reoxygenated DCs exhibited marked decrease in proinflammatory cytokines IL-1␤, IL-6 and TNF-␣, but increase in immunoregulatory cytokine TGF-␤, suggesting that A2A R pathway maybe a potential target to control the inflammation mediated by reoxygenated DCs. 2. Materials and methods 2.1. Mice Female C57BL/6 mice (6–8 weeks) used in the experiments were purchased from Shanghai SLAC Laboratory Animal co. Ltd (Shanghai, China) and maintained in the specific pathogen-free animal facility at Shandong University (Jinan, China). All animal studies were approved by the Animal Care and Utilization Committee of the Shandong University. 2.2. Reagents and antibodies Recombinant murine GM-CSF and IL-4 were obtained from PeproTech Inc. (New Jersey, USA), 7-AAD viability staining solution, FITC-conjugated anti-mouse CD11c, PE-conjugated antimouse MHC-II, CD80, CD86 and anti-mouse CD16/CD32 purified mAb were purchased from eBioscience (San Diego, CA, USA). Lipopolysaccharides (LPS), adenosine, and different ARs agonists including N6 -cyclopentyladenosine (CPA, A1 R agonist), 2-p-(2Carboxyethyl)phenethylamino-5 -N-ethylcarboxamidoadenosine (CGS21680, A2A R agonist), N-ethylcarboxamidoadenosine, (NECA, A2B R agonist), N6 -(3-Iodobenzyl)-9-[5-(methylcarbamoyl)-␤-dribofuranosyl] adenine, (IB-MECA, A3 R agonist) were purchased from Sigma-Aldrich (Saint Louis, USA). All the ARs agonists were dissolved with DMSO (Solarbio, Shanghai, China) follow the

instructions, using DMSO with the same dilution as vehicle control in the experiments. SYBR Premix Ex TaqTM Real time PCR test kit was purchased from TaKaRa Biotechnology CO.LTD (Dalian, China). 2.3. Generation and treatment of bone marrow-derived DCs DCs were generated from the bone marrow (BM) precursors of C57BL/6 mice as described in references (Inaba et al., 1992; Lutz et al., 1999) under normoxic, hypoxic or reoxygenated conditions, respectively. In brief, BM cells were flushed from femurs and tibias with RPMI 1640 medium and treated with RBC lysis buffer for 5 min, washed twice with phosphate buffered saline (PBS), then cultured in complete RPMI 1640 medium supplemented with 50 ng/ml GMCSF and 20 ng/ml IL-4. Half of the medium was replaced every two days. At day 7, LPS (1 ␮g/ml) was administrated to induce maturation for 24 h. DCs morphology and phenotypes (CD11c) were determined by light microscopy and flow cytometry (FCM). For different culture conditions, the cells were maintained in a humidified incubator (HERAcell, Germany) at 37 ◦ C under normoxic (21% O2 , 5% CO2 , 74% N2 ) or hypoxic (1% O2 , 5%CO2 , 94% N2 ) conditions, respectively. Hypoxia/reoxygenation was performed by transferring hypoxia-differentiated DCs to normoxic condition during the last 24 h of culture in the absence or presence of different ARs agonists. In some experiments, normoxic DCs were treated with different ARs agonists for 24 h. To clarify the toxic effects of ARs agonists on DCs, cell viability were tested by 7-AAD staining, which showed more than 90% viability of DCs. 2.4. Real time PCR Total cellular RNA was extracted from cells using a modified Trizol one-step extraction method, and reverse-transcribed into cDNA using Reverse Transcription System (Promega, USA) according to the manufacturer’s protocol. The real time PCR was performed to determine the relative mRNA levels of interested genes. Primers used in the experiments includes: A1 R, sense: 5 -CAT TGG GCC ACA GAC CTA CT-3 , antisense: 5 -ACC GGA GAG GGA TCT TGA CT-3 ; A2A R, sense: 5 -AAC CTG CAG AAC GTC AC-3 , antisense: 5 -GTC ACC AAG CCA TTG TAC CG-3 ; A2B R, sense: 5 -CAT TAC AGA CCC CCA CCA AC-3 , antisense: 5 -AGG ACC CAG AGG ACA GCA AT-3 ; A3 R, sense: 5’-ATA CCA GAT GTC GCA ATG TGC-3’, antisense: 5’-GCA GGC GTA GAC AAT AGG GTT-3, ␤-actin, sense: 5 -TGC GTG ACA TCA AAG AGA AG-3 , antisense: 5 -TCC ATA CCC AAG AAG GAA GG-3 . 2.5. Flow cytometry For the staining of surface molecules, DCs were blocked with anti-CD16/CD32 mAb and stained with FITC-conjugated antiCD11c, PE-conjugated anti-MHC class II, CD80, and CD86 mAbs. Flow cytometry (FCM) data acquisition and analysis were performed on a Cytomics FC500 Flow Cytometer (Beckman Coulter, CA, USA). 2.6. RT-PCR Total cellular RNA was extracted from cells using a modified Trizol one-step extraction method, and reverse-transcribed into cDNA using Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. PCR was performed using specific primers. Primers for amplification of each gene are as follows: IL-1␤, sense: 5 -GCA ACT GTT CCT GAA CTC A-3 , antisense: 5 -CTC GGA GCC TGT AGT GCA G-3 ; IL-6, sense: 5 -TTC TTG GGA CTG ATG CTG-3 , antisense: 5 -CTG GCT TTG TCT TTC TTG TT-3 ; TNF-␣, sense: 5 -ATG AGC ACA GAA AGC ATG ATC-3 , antisense: 5 TAC AGG CTT GTC ACT CGA ATT-3 ; TGF-␤, sense: 5 -GGC GGT GCT CGC TTT GTA-3 , antisense: 5 -CGT GGA GTT TGT TAT CTT TGC T-3 ;

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which may play a critical role in the regulation of DCs function via adenosine pathway. Notably, A2A R might be used as a potential anti-inflammatory target on reoxygenated DCs.

3.2. A2A R agonist CGS21680 inhibits the phenotypic maturation of reoxygenated DCs

Fig. 1. The expression of adenosine receptors on different DCs. Murine bone marrow progenitor cells from C57BL/6 were cultured in different conditions for 5–7 days in the presence of GM-CSF and IL-4 at 3 × 106 ml−1 in 6 wells-culture plate and stimulated for 24 h with or without LPS (1 ␮g/ml). mRNA levels of A1 R, A2A R, A2B R and A3 R were analyzed by real time PCR. Similar results were obtained in three separate experiments. The results are presented as the mean ± SEM. * p < 0.05. Nim: normoxic DCs; Nm: LPS-treated normoxic DCs; H: LPS-treated hypoxic DCs; H/R: hypoxic/reoxygenated DCs.

␤ -actin, sense: 5 -TGC GTG ACA TCA AAG AGA AG-3 , antisense: 5 -TCC ATA CCC AAG AAG GAA GG-3 . RT-PCR was performed in at least three independent experiments. 2.7. ELISA Culture supernatants were harvested and analyzed for the presence of cytokines IL-1␤, IL-6, TNF-␣, IL-12p70, IL-10, and TGF-␤ using commercial ELISA kits (eBioscience, San Diego, CA) according to the manufacturer’s protocols. A standard curve was generated using recombinant cytokine for each assay. 2.8. Statistical analysis The Student’s t test or one-way ANOVA was used to assess the differences between the groups. The analysis was performed by Windows SPSS 21.0 software. p < 0.05 was considered statistically significance. 3. Results 3.1. Reoxygenation causes upregulation of A2A R mRNA levels on DCs DCs generated under both normoxic and hypoxic conditions expressed more than 80% of CD11c, indicating the differentiation of DCs was not influenced by different oxygen tensions (data not shown). Since adenosine pathway serves as a major sensor of hypoxia and adenosine receptors have been shown to be involved in the regulation of DCs function in different manner, the expression levels of different ARs (A1 R, A2A R, A2B R and A3 R) were evaluated on DCs resided in different oxygen tensions. As shown in Fig. 1, both A2A R and A2B R showed higher mRNA levels than A1 R and A3 R on mature DCs under normoxic conditions. When DCs were stimulated to maturation under hypoxic or reoxygenated conditions, the mRNA levels of A2A R showed a remarked elevation compared with A1 R, A2B R and A3 R, which displayed no obvious alteration in expression mode. Compared with hypoxic DCs, reoxygenated DCs showed an increased trend on A2A R mRNA levels, although no significant difference was observed. These data suggest that A2A R was the predominant receptor on both hypoxic and reoxygenated DCs,

To clarify the effects of ARs pathway on the phenotypic maturation of reoxygenated DCs, different agonists for A1 R, A2A R, A2B R and A3 R were administrated when reoxygenation was performed on hypoxic DCs, and the phenotypes of DCs were detected by FCM. Consistent with our previous results, reoxygenation significantly reversed the phenotypic maturation of hypoxic DCs as suggested by increased expression of MHC-II, CD80 and CD86 in response to LPS (Wang et al., 2010). When DCs were treated with different agonists during the process of reoxygenation, only A2A R specific agonist CGS21680 dramatically inhibited the upregulation of MHC-II and CD86, as evidenced by decreased mean fluorecence intensity (MFI) on reoxygenated DCs (p < 0.05); the inhibitory effect on CD80 upregulation was not significantly detected (Figs. 2 and 3). Although other agonists including CPA, NECA and IB-MECA showed a slight increase in MFI of CD80 and CD86 on reoxygenated DCs, no significant difference was observed (p > 0.05). The adenosine displayed no effect on reoxygenated DCs (Fig. 2), indicating that CGS21680 could partially restrain the phenotypic maturation of DCs elicited by reoxygenation and has potential suppressive effects on function of reoxygenated DCs.

3.3. A2A R agonist CGS21680 inhibits the phenotypic maturation of both normoxic and reoxygenated DCs To confirm the suppressive effects of CGS21680 on the phenotypic maturation of DCs, we further determined the effects of CGS21680 with different doses on the phenotype of normoxic or reoxygenated DCs. Fig. 4 showed that MFIs of MHC-II, CD80 and CD86 on the normoxic DCs were gradually decreased with increased doses of CGS21680. Consistently, increased doses of CGS21680 also showed inhibitory effects on the expression of MHCII and CD86, but not on CD80; unexpectedly, CGS21680 had no enhanced effects on the expression of MHC-II and CD86 at the concentration of 10 ␮m compared with the concentration of 1 ␮m, which exhibited a dose-independent manner (Fig. 4). These data indicate the suppressive effects of A2A R signaling pathway on the phenotypic maturation of reoxygenated DCs and help us to determine the optimal doses of A2A R agonist CGS21680.

3.4. A2A R agonist CGS21680 inhibits the proinflammatory cytokines secreted by reoxygenated DCs To clarify the effects of CGS21680 on inflammatory responses of reoxygenated DCs, cytokines profiles were assayed by PCR and ELISA. As shown in Fig. 5A, on mRNA levels, CGS21680 treatment led to a decrease of pro-inflammatory cytokines IL-1␤, IL-6 and TNF-␣, but an increase of anti-inflammatory cytokines TGF-␤ on reoxygenated DCs. Accordingly on protein levels, CGS21680 inhibited the secretion of IL-1␤, IL-6 and TNF-␣, which was enhanced by reoxygenation; while the levels of TGF-␤ were significantly increased after treatment with CGS21680 (Fig. 5B). No obvious change was observed in the production of IL-10, IL-12 when CGS21680 was administrated to reoxygenated DCs (data not shown). These data suggest that CGS21680 could suppress the pro-inflammatory effects of reoxygenated DCs through A2A R pathway.

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Fig. 2. The differential effects of adenosine receptors pathways on the phenotypic maturation of reoxygenated DCs. Hypoxia/reoxygenation was performed by transferring hypoxia-differentiated DCs to normoxic condition during the last 24 h of culture in the absence or presence of 1 ␮M different ARs agonists. The expression of MHC-II, CD80, CD86 on DCs were analyzed by FCM and mean fluorecence intensity (MFI) was presented as mean ± SEM. * p < 0.05. Hypoxic DCs (H) were shown as control.

4. Discussion In our previous study, we have demonstrated that the change of oxygen tensions modulates the maturation and function of DCs and further drives the direction of immune responses. Hypoxia induces immune tolerance via suppressing the maturation of DCs, while reoxygenation completely restores the mature phenotype and function of hypoxic DCs, and further induces Th1 and Th17mediated inflammatory immune responses (Wang et al., 2010). Since the curial roles of DCs-mediated immune responses in IRI have been demonstrated by recent studies (Dong et al., 2005; Loi et al., 2004; Zhou et al., 2005), the activation of DCs by reoxygenation is very likely an important contributor to the inflammatory responses of IRI, thus providing a potential cellular target for the control of IRI. It has been demonstrated that different ARs pathways are involved in the regulation of DCs function, though several conclusions remain controversial. In LPS-differentiated human DCs, A2A R agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited IL-12 production (Panther et al., 2001). Panther et al. (2003) reported that, in normoxic condition, activation of A2 R on mDCs increased the expression of CD54, CD80, MHC class I, and HLA-DR, but inhibited the release of TNF-␣ and IL12, enhanced the secretion of IL-10, and reduced capacity to induce Th1 polarization. However, recent studies reported that the activation of A2B R on LPS-induced DCs reduced the expression of MHC class II and CD86, and inhibits the production of proinflammatory

cytokines such as IL-12, TNF-␣, IL-6 but promotes the secretion of anti-inflammatory cytokines such as TGF-␤, IL-10 (Ben Addi et al., 2008; Hasko et al., 2009; Wilson et al., 2009). These data propose ARs as the potential targets to modulate DCs-mediated inflammatory responses. In the current study, we showed that the expressions of ARs on DCs were modulated by the change of oxygen tension. Normoxic mature DCs expresses higher levels of A2A R and A2B R than A1 R and A3 R, similar to the results from Elisabeth Panther showing the maturation of human DCs by LPS cause the down-regulation of A1 R and A3 R mRNAs, but not A2A R mRNA. While on hypoxic DCs or rexoygenated DCs, only A2A R predominantly expresses compared with A1 R, A3 R and A2B R, indicating the possible distinct roles of A2A R on these DCs. It has been known that the excess accumulation of extracellular adenosine in adaption to hypoxia is enough for active ARs on immune cells (Sitkovsky and Lukashev, 2005; Sitkovsky and Ohta, 2013), which support that the immune tolerance of hypoxic DCs might be the role of adenosine through A2A R pathway; while the immune inflammatory responses of rexoyganated DCs could be the consequence of markedly reduced extracellular adenosine in response to oxygen redelivery. Emerging evidences have demonstrated the anti-inflammatory effects of A2A R through animal experiments. Mice with A2A Rdeficient DCs were more susceptible to kidney IRI and are not protected from injury by A2A R agonists. Injection of a potent and selective agonist of A2A R, ATL146e, could reduce infarct size in B6 mice after 24 h of reperfusion, but had no effect in A2A R knock-out

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Fig. 3. The effects of A2A R agonist CGS21680 on the phenotypic maturation of reoxygenated DCs. Reoxygenated DCs were treated with CGS21680 (1 ␮M), the expression of MHC-II, CD80, and CD86 on DCs were detected by FCM. Representative FCM data was shown.

Fig. 4. A2A R agonist CGS21680 inhibits the phenotypic maturation of both normoxic and reoxygenated DCs. Normoxic or reoxygenated DCs were treated with different concentration of CGS21680, the expression of MHC-II, CD80, and CD86 on DCs were detected by FCM. The results were presented as mean ± SEM; * p < 0.05,** p < 0.01.

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Fig. 5. A2A R agonist CGS21680 affects the cytokines profiles of reoxygenated DCs. Reoxygenated DCs were treated with 1 ␮M CGS21680, cells and supernatants were collected. The levels of IL-1␤, IL-6, TNF-␣ and TGF-␤ was measured by RT-PCR (A) and ELISA (B), each sample was performed in triplicate wells. Data represent three independent experiments. Means ± SEM was shown. * p < 0.05; ** p < 0.01; *** p < 0.001.

mice (Li et al., 2012; Yang et al., 2005). Especially, Thiel found the exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2A R-mediated lung tissue protection (Thiel et al., 2005). Based on the above data, we speculated that the activation of A2A R might be effective approach to control the inflammation mediated by reoxygenated DCs. In the present study, we demonstrated the high affinity agonists of

A2A R, CGS21680, could effectively activate the AR pathway and suppress the upregulation of maturation-related phenotypes on hypoxic DCs in response to reoxygenation. Importantly, CGS21680treated reoxygenated DCs secreted low levels of proinflammatory cytokines IL-1␤, TNF-␣, IL-6, but high levels of anti-inflammatory cytokines TGF-␤. Adenosine also binds to A2A R but has no similar suppressive effects as CGS21680 in the present study, the binding

Fig. 6. A2A R agonist CGS21680 might be a potential effective reagent in the therapy of IRI. Reoxygenation of hypoxia-differentiated DCs results in complete recovery of their mature phenotypes and cytokines secretion that were inhibited by hypoxia. Reoxygenated DCs predominantly expressed high levels of A2A R. High affinity agonists of A2A R CGS21680 inhibits the expression of maturation-related phenotypes CD86, MHC-II on reoxygenated DCs, decreases the secretion of proinflammatory cytokines IL-1␤, TNF-␣, IL-6, but increase that of anti-inflammatory cytokines TGF-␤. We propose CGS21680 might be a potential effective reagent in the therapy of IRI.

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of other ARs including A1 R, A2B R and A3 R, which could produce differential effects and counteract the inhibitory role through A2A R pathway, might be an important reason. Taken together, our results suggest that A2A R pathway may play critical roles in the control of inflammation mediated by reoxygenated DCs, and further propose that CGS21680 might be a potential effective reagent in the therapy of IRI, as shown in Fig. 6. More interesting, a recent report by Castellaneta et al. (2014) has demonstrated the pivotal role of plasmacytoid DCs, a newly discovered DCs subtype distinct from classic DCs, in the pathogenesis of liver IRI. This finding proposes another novel cellular target for IRI therapy, and open new research field to explore the possible desirable effects of CGS21680 on this specific type of DCs. Acknowledgements This work was supported in part by the National Natural Science Foundation of China (81102220, 81270923, and 81172863), the National “973” Program of China (2011CB503906) and Shandong Young Scientists Award Fund (2011BSE27110). References Ben Addi, A., Lefort, A., Hua, X., Libert, F., Communi, D., Ledent, C., Macours, P., Tilley, S.L., Boeynaems, J.M., Robaye, B., 2008. Modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the A(2B) receptor. Eur. J. Immunol. 38, 1610–1620. Boros, P., Bromberg, J.S., 2006. New cellular and molecular immune pathways in ischemia/reperfusion injury. Am. J. Transplant. 6, 652–658. Castellaneta, A., Yoshida, O., Kimura, S., Yokota, S., Geller, D.A., Murase, N., Thomson, A.W., 2014. Plasmacytoid dendritic cell-derived IFN-alpha promotes murine liver ischemia/reperfusion injury by induction of hepatocyte IRF-1. Hepatology 60, 267–277. Dong, X., Swaminathan, S., Bachman, L.A., Croatt, A.J., Nath, K.A., Griffin, M.D., 2005. Antigen presentation by dendritic cells in renal lymph nodes is linked to systemic and local injury to the kidney. Kidney Int. 68, 1096–1108. Dong, X., Swaminathan, S., Bachman, L.A., Croatt, A.J., Nath, K.A., Griffin, M.D., 2007. Resident dendritic cells are the predominant TNF-secreting cell in early renal ischemia-reperfusion injury. Kidney Int. 71, 619–628. Hasko, G., Csoka, B., Nemeth, Z.H., Vizi, E.S., Pacher, P., 2009. A(2B) adenosine receptors in immunity and inflammation. Trends Immunol. 30, 263–270. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R.M., 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colonystimulating factor. J. Exp. Med. 176, 1693–1702. Jang, H.R., Ko, G.J., Wasowska, B.A., Rabb, H., 2009. The interaction between ischemiareperfusion and immune responses in the kidney. J. Mol. Med. (Berl.) 87, 859–864. Kutala, V.K., Khan, M., Angelos, M.G., Kuppusamy, P., 2007. Role of oxygen in postischemic myocardial injury. Antioxid. Redox Signal. 9, 1193–1206. Li, C., Jackson, R.M., 2002. Reactive species mechanisms of cellular hypoxia–reoxygenation injury. Am. J. Physiol. Cell Physiol. 282, C227–C241.

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