Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells

The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1β, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1β, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000’s small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR. Supplementary Information The online version contains supplementary material available at 10.1007/s11302-024-09992-z.


Introduction
Adenosine is a modulator of numerous functions by acting on the four adenosine receptor (AR) subtypes [1][2][3].One of the mechanisms of adenosine-promoted tissue protection and repair has been ascribed to its anti-inflammatory effect [4].In this context, A 3 AR modulation has been suggested as a promising approach for treating inflammatory and ischemic conditions.Additionally, A 3 AR agonists have progressed in clinical trials [2,[5][6][7] despite A 3 AR having both pro-and anti-inflammatory effects in various cell types [8][9][10][11][12].Furthermore, Cohen et al. [13] have demonstrated that the A 3 AR positive allosteric modulator (PAM) N- (3,4dichloro-phenyl)-2-cyclohexyl-1H-imidazo [4,5-c]quinolin-4-amine (LUF6000) also induced anti-inflammatory effects in three experimental rodent models, by a mechanism related to decreased levels of the transcription factor nuclear factor-κ (NF-κB) p65 protein.Moreover, LUF6000 induced a slight stimulatory effect on the number of normal white blood cells and neutrophils.
The A 3 AR has generated tremendous interest in antiinflammatory drug development [7].Many early studies used Cl-IB-MECA and IB-MECA as selective A 3 AR agonists, currently used in clinical trials for liver conditions and psoriasis [5][6][7]14].However, in light of the only moderate A 3 AR-selectivity of Cl-IB-MECA and IB-MECA [14], a concentration of ≤10 nM is often advisable for A 3 AR-specific analysis.Alternatively, A 1 and A 2A AR antagonists can be combined with Cl-IB-MECA to isolate A 3 AR's role.For example, Wagner et al. [9] pre-administered A 2A AR antagonist ZM241385 (1 mg/kg body weight) before Cl-IB-MECA treatment to reveal potential unspecific effects of Cl-IB-MECA on the A 2A AR.With AR subtype selectivity in mind, Lillo et al. [15] performed RNAseq using striatal primary microglial cultures activated and treated with Cl-IB-MECA (200 nM) and/or A 2A AR antagonist SCH58261 (200 nM).These results ultimately demonstrated that the chronic (24 h) A 3 AR agonist treatment regulated more genes negatively than positively.The associated gene ontology enrichment analysis showed the regulation of genes that participate in immune-related events.Analysis of protein-protein interactions showed that Smad3 and Sp1 genes are regulated by Cl-IB-MECA [15].Under the conditions of cell activation with an agonist treatment regimen, Cl-IB-MECA tended not to favor gene expression related to neuroprotective microglia.Microarray or RNA-Seq has been used to explore antior pro-inflammatory functions of AR modulation in several cell types, including macrophages and microglia [15][16][17].However, to our knowledge, the role of A 3 AR in neutrophils has not been explored using genetic approaches such as microarray or RNAseq.
Neutrophils are immune cells that are an important part of the host defense system against infection and tissue injury [18].In response to these challenges, neutrophils migrate from peripheral blood toward the site of infection through the endothelial cell layer and into tissues [19].Thus, the pharmacological modulation of neutrophil functions could enhance neutrophil capability against infections and protect tissues during inflammation.In the present study, we studied differentiated HL-60 cells, a commonly used cell line to model neutrophil functions such as cytokine release and migration [20][21][22][23].
The human (h) A 3 AR endogenously expressed in HL-60 cells has been characterized functionally and using radioligand binding [24,25].It was suggested that A 3 AR expression in HL-60 cells is like that in neutrophils, thus providing a practical model for investigating A 3 AR's role.It is noted that A 2A AR is also expressed in HL-60 cells [25].Through studying both human neutrophils and HL-60 cells, Chen et al. [22] suggested that A 3 AR appears as the crucial AR for regulating neutrophil chemotaxis by controlling cell migration.Both A 3 AR agonists [26] and A 3 AR PAMs [13] are of interest as anti-inflammatory agents.
In the present study, we investigated gene regulation following the acute treatment of HL-60 cells with the recently available and highly selective A 3 AR agonist MRS5698, which we reported to be > 3000-fold receptor subtype selective at both human and rat A 3 AR [27], and the heterocyclic A 3 AR-selective PAM LUF6000.In addition to genetic modulation by the exogenous agonist, we also studied the allosteric enhancement by LUF6000 of the endogenously expressed HL-60 cell A 3 AR, which lacks previous exploration.It remains to be seen if chronic administration of MRS5698 and LUF6000 will be similar to or different than the chronic effects of Cl-IB-MECA, as reported by Lillo et al. [15].

Cell culture, drug treatment, and cell differentiation
CHO-A 3 cells were grown in DMEM/F12 medium with 10% fetal bovine serum and 1% penicillin-streptomycin. HL-60 cells were cultured in RPMI-1640 medium (ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Differentiation of HL-60 cells was accomplished by incubating cells with 1.3% DMSO for seven days before experiments.Our microarray analysis utilized four experimental groups, including a control group, MRS5698 group (1 µM, 20 min treatment), LUF6000 group (3 µM, 20 min treatment), MRS5698 + LUF6000 group (LUF6000 treatment for 20 min, followed by MRS5698 for 20 min).DMSO (5 mM) was used to dissolve the drugs before dilution in aqueous medium.

RNA extraction and real-time PCR
Total RNA was extracted from 10 7 differentiated HL-60 cells using a RNeasy kit (Qiagen, Redwood City, CA, USA) and was reversed-transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol.The cDNA was then amplified with gene-specific primers for each of the four AR subtypes, two cytokines IL-1± and IL-1², and GAPDH on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol.The TaqMan gene expression assays were from Applied Biosystems (Foster City, CA, USA).Values were normalized to GAPDH and then expressed as relative expression levels.The ΔΔCt method was used to conduct quantitative analysis of data.

Intracellular calcium mobilization
The measurement of calcium mobilization was essentially described previously [29,30].Briefly, CHO cells expressing the recombinant hA 3 AR were grown in a set of 96-well black-wall, clear-bottom plates overnight before the day of the experiment and maintained at 37 °C/5% CO 2 .Then, we added 100 µl of dye (Calcium 6) to each well.Afterward, the plates were subsequently maintained at room temperature for 60 min.Finally, 50 µl of each compound or a control agonist was added into the respective assay plate wells during the determination of intracellular Ca 2+ using a FLIPR (Molecular Devices, San Jose, CA, USA).We utilized a calcium assay kit as directed without washing cells and with probenecid added to the loading dye at a final concentration of 2.5 mM to increase dye retention.The compound plate was prepared using dilutions of various compounds in Hank's Buffer (pH 7.4).Samples were run in duplicate at room temperature.Cell fluorescence (excitation = 485 nm; emission = 525 nm) was monitored following compound exposure.Increases in intracellular Ca 2+ are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure.

Measurement of cyclic AMP levels
CHO-A 3 cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 µmol/ml glutamine.For the assay of 3′,5′-cyclic adenosine monophosphate (cAMP), cells were plated in 96-well plates in 100 µl of medium overnight.An AlphaScreen cAMP kit was used for the measurement of cAMP levels as described previously [29,30].HL-60 cells 4 × 10 4 cells/well were suspended in 80 µl Hank's buffer containing 20 mM HEPES.Where applicable, the A 3 AR allosteric enhancer LUF6000 (3 µM) was added, and the incubation was continued for 20 min in the presence of phosphodiesterase inhibitor rolipram (10 µM), followed by the addition of A 3 AR agonist MRS5698 plus 10 µM forskolin to the mixture and incubation for 10 min.The reaction was terminated by centrifugation at 250 g for 5 min at 4°C upon the addition of 100 µL cold 0.3% Tween-20 to each well.Cells were then shaken at room temperature for 10 min.Finally, the reaction was terminated by supernatant removal, and the cells were lysed with the addition of 50 µl of lysis buffer (0.3% Tween-20).For the determination of cAMP production, an AlphaScreen cAMP kit was used according to the manufacturer's instructions (PerkinElmer, Waltham, MA).

Statistical and data analyses
The microarray RNA normalized data sets were loaded into the commercial bioinformatics software Partek Flow and were aligned with human genome Hg38 alongside the aligner BWA.The gene ANOVA result lists three treatment groups (Group 1, MRS5698, final concentration (1 µM); Group 2 LUF6000, final concentration (3 µM); Group 3, MRS5698 + LUF6000) in comparison to a corresponding control group, which were filtered by both P value of ≤0.05 and an absolute value of fold change ≥1.8.These filtered gene lists were then both used to create a Heat map using Partek Genomic Suite (Chesterfield, MO, USA) and a gene pathway analysis using the commercial software IPA (Qiagen, Redwood City, CA, USA).Functional parameters were calculated using Prism 10.0.0 software (GraphPAD, San Diego, CA, USA).Data was expressed as mean ± standard upon stimulation [31].NFκB1A is an NF-κB response suppressor that attenuates inflammation, aging, and cancer [32].TNFAIP3, the tumor necrosis factor α-induced protein 3, has been reported as a key molecule controlling NF-κB activation and has been linked to the development of multiple inflammation-related conditions in humans [33].CCL4 has been shown to enhance preosteoclast migration alongside its receptor [34].Additionally, CCR5 downregulation by RANKL promotes osteoclastogenesis [34].Neutrophilproduced CCL4L2 was negatively related to inhaled corticosteroid (ICS) responsiveness in asthmatics and has been suggested as a potential therapeutic target for ICS-refractory asthma [35].CCL3L3 has been suggested to be involved in susceptibility to systemic lupus erythematosus, an autoimmune disease [36].Of the two most upregulated genes IL1A and IL1B, we confirmed the result using quantitative realtime PCR analysis.MRS5698 upregulated IL-1α and IL-1β mRNA expression by 19.1-and 16.4-fold, respectively, which is consistent with the data from microarray analysis.
The changes in the LUF6000 (3 µM, 20 min) group were modest in comparison to the MRS5698 group (Table 1).The MRS5698 + LUF6000 group (LUF6000 was added 20 min before MRS5698 and incubated for an additional 20 min) showed overall larger upregulation compared with the MRS5698 group.This pattern of gene expression by LUF6000 is consistent with its functional enhancement [30].The complete list of genes with significant upregulation or downregulation is listed in Table S1 (Supporting Information).
Notably, a similar number of genes were upregulated and downregulated upon MRS5698 treatment (20 min) (Fig. 1).These results contrast with a recent report by Lillo et al. [15], reporting that chronic treatment (24 h) of activated mouse microglia with another A 3 AR agonist Cl-IB-MECA resulted in mostly downregulated genes.The upregulation by LUF6000 and MRS5698 + LUF6000 is generally consistent with that in the MRS5698 alone group.
It is also interesting to note that the heatmap shows that LUF6000 enhanced both the down-and upregulation of genes regulated by the selective A 3 AR agonist MRS5698 (Fig. 1).Furthermore, even in the absence of an exogenous agonist, the allosteric enhancer LUF6000 alone shows an effect in the regulation of many genes in the same direction as the agonist MRS5698, which is consistent with an early report that another A 3 AR PAM, LUF6096, induced anti-ischemic cardioprotective effect in the absence of an exogenous agonist [37].
error.A Student's t-test (between two conditions) or a One-Way Analysis of Variance (ANOVA) followed by Bonferroni's multiple comparison tests (between multiple conditions) was used to compare statistically significant differences.Differences yielding P values < 0.05 are considered statistically significant.

Genetic modulation by A 3 AR selective agonist MRS5698 and allosteric enhancer LUF6000
Table 1 shows the top ten genes upregulated (the complete list of genes is listed in Table S1).Following the treatment of MRS5698 (1 µM) for 20 min, many inflammation-associated genes were found upregulated 3-fold or more.For example, IL1A and IL1B, which are known for their inflammatory functions, are among the most highly upregulated genes.Interestingly, the anti-inflammatory IL-1RN, which reduces the inflammatory effects of cytokines IL-1α and IL-1β and aids the preservation of cell functions, was also highly upregulated.Both NFKBIA and NFKBIZ were also upregulated.NFKBIZ is a regulator of NFKB that inhibits NF-κB activity without affecting its nuclear translocation 20.4 a HL-60 cells were cultured in RPMI-1640 medium (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Differentiation of HL-60 cells was accomplished by incubating cells with 1.3% DMSO for seven days before experiments.Four groups were divided for microarray analysis.Control group; MRS5698 group (1 µM, 20 min treatment); LUF6000 group (3 µM, 20 min treatment); MRS5698 + LUF6000 group (LUF6000 treatment for 20 min followed by MRS5698 for 20 min).Total RNA was extracted from 10 7 differentiated HL-60 cells using a RNeasy kit (Qiagen, Redwood City, CA, USA).The microarray RMA normalized data sets were loaded into commercial bioinformatics software Partek Flow and were aligned with human genome Hg38 with aligner BWA.The complete list of genes regulated by A 3 AR activation is shown in Table S1 1 3

Selective A 3 AR agonist MS5698: functional characterization at four AR subtypes
The moderately selective A 3 AR agonists IB-MECA and Cl-IB-MECA have been used in many previous pharmacological studies.As mentioned above, at higher concentrations both agonists may activate other AR subtypes.Thus, we chose a more highly selective A 3 AR agonist in the present study.MRS5698 has been shown in radioligand binding assays to be a selective agonist for both human and mouse A 3 AR compared to A 1 AR and A 2A AR [27].However, its activation profile at four ARs has not been compared.To ensure that the effect of MRS5698 used in the genetic regulation is solely via the A 3 AR, we determined the effect of MRS5698 on cAMP accumulation in CHO cells expressing the recombinant human A 3 AR.Figure 5 shows that MRS5698 is potent in inhibiting forskolin-stimulated cAMP accumulation (EC 50 = 2.52 nM) in hA 3 AR-expressing cells, and the nucleoside was largely inactive as an agonist at hA 1 , A 2A , and A 2B ARs at concentrations up to 1 µM (Fig. 5).Thus, we selected a concentration of 1 µM to treat HL-60 cells.
The complete list of pathways from IPA analysis is included in the Supplementary Information (Figure S1).Canonical peroxisome proliferator-activated receptor (PPAR) and adrenergic receptor signaling pathways are strongly downregulated in both agonist groups and somewhat downregulated even in the LUF6000 group.Retinoic acid receptor (RAR) activation and liver X receptor-retinoid X receptor (LXR/RXR) gene expression is also downregulated in both agonist groups.
The IPA analysis summary of the MRS5698 + LUF6000 group is shown in Fig. 4. Two partially overlapping pathway nodes are evident: downstream of PPAR, which is highly downregulated by an A 3 AR agonist, and an anti-inflammatory node.The modulation of eicosanoid/arachidonic acid pathways downstream of PPAR would demonstrate mixed effects on inflammation and metabolism.Fig. 1 List of genes were upregulated and downregulated in differentiated HL-60 cells comparing all three groups following acute A 3 AR modulator treatment: MRS5698 (G1), LUF6000 (G2), and MRS5698 + LUF6000 (G3) also enhanced the agonist efficacy of MRS5698 by ~ 20% (Fig. 7).

MRS5698-induced intracellular calcium mobilization
It has not been previously characterized whether MRS5698 is a full agonist or a partial agonist.The relatively small enhancement by LUF6000 on MRS5698-induced gene upregulation alongside the smaller effect of LUF6000 on the E max of MRS5698 in the cAMP assay in comparison to the relatively larger effect of LUF6000 on another known partial agonist Cl-IB-MECA [30] suggests that MRS5698 is likely a nearly full A 3 AR agonist.Thus, in the present study, we compared MRS5698 with a known full A AR agonist NECA [30] in a calcium mobilization assay in CHO-A3 cells.Figure 8 shows that MRS5698 is as efficacious as NECA as an A 3 AR agonist but 4.0-fold more potent.

Enhancement of A 3 AR function in HL-60 cells by LUF6000
LUF6000 at a concentration of 3 µM has demonstrated a substantial enhancement of the A 3 AR agonist E max without affecting agonist potency [30].It has also been shown that LUF6000 is more efficacious in enhancing the E max of partial agonists than full agonists [30].At a concentration of 3 µM, LUF6000 enhanced the E max of Cl-IB-MECA by 41% in an assay of cAMP accumulation [30], while it enhanced the E max of the full agonist NECA by less than 20%.Here we show that it enhanced the E max of MRS5698 by 19%, which suggests that MRS5698 is probably a nearly full agonist (Fig. 6).
We further tested the function of MRS5698 and its enhancement by LUF6000 in differentiated HL-60 cells.Similar to CHO cells expressing the recombinant hA 3 AR, HL-60 cells endogenously expressing the hA 3 AR LUF6000 Fig. 3 UPS analysis of regulators from all three HL-60 cell treatment groups, MRS5698 (G1-S-1-ipa), LUF6000 (G2-S-ipa), and MRS5698 + LUF6000 (G3-S-ipa), showing only the top genes.The complete list is found in Figure S2 (Supporting Information) Fig. 2 Pathway analysis for genes were upregulated and downregulated in differentiated HL-60 cells upon acute treatment with A 3 AR modulators: MRS5698 (G1-S-1-ipa), LUF6000 (G2-S-ipa), and MRS5698 + LUF6000 (G3-S-ipa) the A 3 AR PAM showing changes in parallel to those of the A 3 AR agonist further supported this nature of regulation.Also, a study of off-target activities of a congeneric series of nucleoside/nucleobase derivatives, including MRS5698, indicated only weak µM affinity at a few other receptors [38].

Gene expression comparison of the four AR subtypes in differentiated HL-60 cells
Figure 9 shows that the A 3 AR is the highest expressed of the four AR subtypes in differentiated HL-60 cells.The A 2A AR is also at a relatively high level but is only about 40% of the A 3 AR.The A 1 AR and A 2B AR are less than 10% of the A 3 AR expression level.Thus, both the A 3 AR selectivity of MRS5698 and high A 3 AR expression in differentiated HL-60 cells guaranteed that the genetic and functional regulation by A 3 AR agonist occurs via the A 3 AR.The effects of upregulation of IL-1a and IL-1b could be related to the proinflammatory role of the A 3 AR.
The A 3 AR has been reported both pro-and anti-inflammatory depending on cell types [6,7,9,10,39].For example, A 3 AR agonist Cl-IB-MECA (0.1 mg/kg, s.c.) has been found to induce an antiinflammatory and protective effect on the rat liver [39].Wagner et al. [9] characterized the role of A 3 AR in a murine model of lung inflammation and

Discussion
The A 3 AR is known to play an essential role in various physiological processes, and its activation can influence gene regulation and various cellular responses [1,3,6,7).The present study showed that A 3 AR activation can lead to the upregulation of genes associated with both pro-inflammation and anti-inflammation, e.g.NFKB1Z and NFKB1A.The NF-κB response suppressor that also attenuates inflammation, aging, and cancer [32], may at least partially explain the anti-inflammatory effect of A 3 AR agonists.The in monocyte/macrophage, dendritic cells, cytotoxic and helper T cells, and mast cells [6].It has also been suggested that chronic treatment with an agonist-induced A 3 AR downregulation.This is possibly responsible for the suppression of its basal inhibitory effect on cytokine production.However, few if any studies have reported the impact of acute A 3 AR agonist treatment on genetic regulation.
Németh et al. [16] hypothesized that adenosine may exert some of its anti-inflammatory effects by decreasing NF-kB activation because gene expression of most of the proinflammatory cytokines inhibited by adenosine is dependent on NF-kB activation.Microarray analyses revealed that mRNA levels of neither TNF-α nor other cytokines were altered by adenosine (100 µM) in either lipopolysaccharide (LPS)-stimulated, or non-stimulated RAW 264.7 macrophages.Although LPS induced expression of several other, noncytokine genes, such as A 2B AR, adenosine did not affect the expression of these genes.Furthermore, adenosine (100 µM) as well as NECA and other agonists (10 µM) failed to decrease LPS-induced NF-κB DNA binding, NF-κB promoter activity, p65 nuclear translocation, and inhibitory κB degradation.Thus, the authors suggest that the anti-inflammatory effects of adenosine are independent of NF-κB [16].Streitova et al. [43] showed mRNA expression for all four AR subtypes in normal and LPS-activated murine RAW 264.7 macrophages.LPS upregulated A 2A and A 2B mRNA expression, but downregulated A 3 AR.Ehrchen et al. [44] performed microarray analysis on glucocorticoid-treated monocytes and macrophages and identified glucocorticoiddependent regulation of 133 genes, including anti-inflammatory A 3 AR, CD1d, and IL-1 receptor II.Barczyk et al. [45] reported that glucocorticoids promote the survival of antiinflammatory macrophages via A 3 AR stimulation.Baram et al. [46] showed that by coupling to Gi3, the A 3 AR stimulates multiple signaling pathways in human mast cells, leading to found elevated pulmonary A 3 AR expression levels following lipopolysaccharide (LPS) exposure in vivo.LPS inhalation increased the accumulation of polymorphonuclear leukocytes (PMN) in wild-type and whole-body A 3 AR( −/− ) mice in all lung compartments.Pretreatment with the selective A 3 AR agonist Cl-IB-MECA significantly decreased the migration of PMNs into the lung interstitium and alveolar air space of wild-type but not A 3 ( −/− ) mice.Ren et al. [10] showed that treatment of colonic mucosa from patients with ulcerative colitis (UC) with 100 nM Cl-IB-MECA for 24 h downregulated A 3 AR expression and upregulated TNF-α, NF-κB, and IL-1β production.A 3 AR activation by Cl-IB-MECA significantly decreased TNF-α and IL-1β production and attenuated the NF-κB p65 activation in colonic tissues from patients with colonic mucosal inflammation in UC.Hasko et al. [11] reported that in RAW 264.7 macrophages, A 3 AR agonist IB-MECA, at 0.2 and 0.5 mg/kg, potentiated LPS-stimulated IL-10 production and inhibited LPS-induced TNF-a production, thus suggesting antiinflammatory action.Sajjadi et al. [12] reported that A 3 AR stimulation caused a marked decrease in TNF-α mRNA, but not IL-1b, IL-6, or IL-8 mRNA.Thus, the pro-and antiinflammatory effects of adenosine and the A 3 AR are dependent on cell types.
The A 3 AR agonist modulates gene expression and is known to occur through various intracellular signaling pathways.The present study further analyzed pathways potentially involved in the A 3 AR function.Pathway analysis highlighted several important signaling pathways downstream of the A 3 AR, including IL-6, IL-17, and P38 MAPK signaling.UPS analysis of regulators from all three groups MRS5698 (G1-S-1-ipa), LUF6000 (G2-S-ipa), and MRS5698 + LUF6000 (G3-S-ipa) identified important regulatory molecules including IL-1, NFkB, JNK, and ERK.Analysis of known network interactions and regulated genes reveals interactions among many important signaling molecules upon A 3 AR activation, e.g.IL-1b, IL-17, TNF-α, and inflammation response.
IB-MECA and Cl-IB-MECA, which are moderately selective A 3 AR agonists, have been used in many earlier studies [11,12,40].However, at a higher concentration, both agonists may activate other AR subtypes.For example.Kreckler et al. [41] reported that adenosine inhibits tumor necrosis factor-α release from mouse peritoneal macrophages via A 2A and A 2B but not the A 3 AR.Thus, the antiinflammatory effect of adenosine is dependent on both AR subtypes in addition to cell types.Thus, to elucidate the role of the A 3 AR (not to activate other AR subtypes), we chose a more selective A 3 AR agonist MRS5698, and an A 3 AR allosteric enhancer LUF6000 in the present study.
A critical role of the A 3 AR in neutrophil behavior has been suggested [22,42].The A 3 AR is also highly expressed

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Competing interests
The authors declare no competing interests.
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It is known that acute and chronic administration of A 3 AR agonists produced different functional or behavioral effects [47].Considering that the A 3 AR-related gene downregulation based on chronic administration of Cl-IB-MECA has been recently reported [15], we observed that the total genes are almost equally up and downregulated in HL-60 cells treated with MRS5698 for 20 min.However, it is to be noted that the effects of Cl-IB-MECA were determined in mouse cells, and there are significant species-dependent differences in the pharmacology and role of the A 3 AR [14].
In summary, the present study identified genes and pathways that are regulated by the A 3 AR activation in a neutrophil model HL-60 cells, by using both a recently available and selective A 3 AR agonist, MRS5698, and the allosteric enhancer LUF6000.Based on the selectivity profile of both MRS5698 and LUF6000 together with the AR receptor expression profile of HL-60 cells, we are confident that the regulated genes and pathways are solely via the A 3 ARs.The MRS5698 + LUF6000 group had overall effects larger than MRS5698 alone, consistent with allosteric enhancement.We also observed the allosteric enhancement of the native A 3 AR function by LUF6000 in HL-60 cells.The patterns of transcriptional enhancement by LUF6000 are consistent with those of the functional enhancement.The findings from the present study support the role of A 3 AR in inflammation and cancer [6,7,[48][49][50] and may shed light on the further development of A 3 AR agonists and allosteric enhancers for various conditions.

Fig. 9 Fig. 8 Fig. 7
Fig. 9 Gene expression levels of the four AR subtypes in differentiated HL-60 cells gram (ZIADK031117) to K.A.J.This work was supported by the Intramural Research Program of the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases for support [ZIADK031117 to K.A.J] and the Genomics Core (J.A.H).

Table 1
Fold-change of selected genes that were upregulated by treatment with A 3 AR agonist (MRS5698) alone or with PAM (LUF6000) or both a