TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor–mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A3 receptor (A3R)–mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A3R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A3R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A3R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A3Rs.

Toll-like receptors are evolutionary conserved pattern recognition receptors expressed by different cell lineages of the immune system (1). In myeloid cells, agonist recognition by TLRs initiates a cascade of intracellular signaling events that ultimately culminate in the activation of transcription factors such as NF-κB and AP-1 (24). These transcription factors induce the expression of inflammatory soluble mediators like TNF-α, IL-12, and CCL2, as well as of molecules involved in Ag presentation (1, 5).

The purine nucleoside adenosine is a known regulator of inflammatory responses (6, 7). During inflammatory conditions, intracellular and extracellular adenosine levels rapidly rise. Extracellular adenosine interacts with four G protein–coupled adenosine receptor (ADORA) subtypes. Adenosine A1 and A3 receptors (A1R and A3R) are coupled to Gi proteins and mediate opposite effects to adenosine A2A and A2B receptors (A2AR and A2BR) that are coupled to Gs proteins. The ADORA expression pattern, which is highly dynamic, therefore orchestrates the cellular response to adenosine (8, 9).

ADORA-mediated signaling also modulates innate immune responses induced by myeloid cells. A2AR-mediated signaling inhibits TLR-induced NF-κB activation and concomitant production of cytokines in macrophages and microglia (1013). Recently, our group described that A3R-mediated signaling counteracts A2AR-mediated inhibitory signaling (14). Data from the same study suggested that A3R-mediated signaling was also directly involved in TLR-induced cytokine responses.

In this study, we show that A3R-mediated signaling contributes to IL-12 and CCL2 production as induced by a broad range of TLRs and pathogens and has downstream effects on T cell responses to whole pathogens. Dendritic cell (DC)–CD4+ T cell coincubation experiments demonstrate that bacterial and fungal-induced IFN-γ and IL-17 responses are significantly inhibited when they were evoked in the presence of an A3R antagonist. Next-generation sequencing of mRNA expression profiles revealed that A3R-mediated signaling controls the expression levels of methallothioneins, which in turn can inhibit the phosphorylation of the transcription factor STAT1. These results demonstrate that endogenously produced adenosine provokes A3R-dependent signaling, which essentially contributes to the TLR-induced cytokine response profile.

Adenosine A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A2AR antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261), adenosine A2BR antagonist 8-(4-[([4-cyanophenyl]carbamoylmethyl)oxy]phenyl)-1,3-di(n-propyl)xanthine hydrate (MRS1754), adenosine A3R antagonists 9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)-[1,2,4]triazolo[1,5-c]quinazoline (MRS1220), and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191; all Sigma-Aldrich, Saint Louis, MO) were diluted in DMSO, aliquoted, and stored at −20°C. The 4-methoxy-N-(3-[2-pyridinyl]-1-isoquinolinyl)benzamide (VUF8504) was synthesized in-house. DMSO controls were included in all functional assays.

TLR agonists used were Pam3CSK4 (TLR2), poly(I:C) (TLR3), LPS (TLR4), flagellin (TLR5), and CL075 (TLR8; all Invivogen, San Diego, CA). Heat-killed Candida albicans and heat-killed Staphylococcus aureus were obtained from Invivogen; Haemophilus influenzae strain 86-028NP [nontypeable (15)] was cultured in brain heart infusion medium at 37°C, washed, and heat-killed for 30 min at 56°C. Recombinant human IFN-γ was from Peprotech (London, U.K.), and PMA was from Sigma-Aldrich.

Human PBMCs were isolated from human buffy coats obtained from anonymized healthy donors (Sanquin, the Netherlands) using lymphocyte separation medium gradient centrifugation (Lymphoprep; Axis-Shield, Norway). Monocytes were isolated with anti-CD14, and T cells with anti-CD4 mAb-coated Microbeads using MACS single-use separation columns from Miltenyi Biotec (Bergisch Gladbach, Germany) as described by the manufacturer. CD4+ T cells were resuspended in 10% DMSO (v/v) with heat-inactivated FCS and frozen in liquid nitrogen until further use.

Purified CD14+ cells were resuspended in RPMI 1640 (Life Technologies, Thermo Fisher Scientific, Waltham, MA) containing 10% (v/v) FCS (Life Technologies) and penicillin 100 U/ml and streptomycin 0.1 mg/ml (Life Technologies) supplemented with ≥4 U recombinant human M-CSF/ml or ≥40 U recombinant human GM-CSF and 200 ng of recombinant human IL-4/ml (all Peprotech) to yield macrophages or DC, respectively. Half of the medium was replaced by fresh medium containing new growth factors every 3–4 d. After 6–7 d in culture, cells were used for functional assays. For the Ag presentation assays, donor-matched CD4+ T cells were thawed and added to DC in a ratio of 5:1 together with either VUF8504 or vehicle control. Heat-killed Candida albicans, heat-killed Staphylococcus aureus, or heat-killed Haemophilus influenzae strain 86-028NP (1.25 × 106 CFU/well) or an equivalent volume of medium was added subsequently. Following stimulation, supernatant was isolated 24 h and 5 d later and stored at −20°C until further analysis.

THP-1 cells were cultured in RPMI 1640 supplemented with 10% (v/v) FCS, 2 mM GlutaMAX, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U penicillin/ml, and 0.1 mg streptavidin/ml (all Life Technologies).

THP-1 cells were seeded in a 96-well plate at a concentration of 1 × 104 cells per well and incubated for 24 h before lentiviral short hairpin RNA (shRNA) transduction particles (SHCLNV MISSION pLKO.1-puro nonmammalian shRNA; Sigma-Aldrich) were added to the cells at different multiplicities of infection in the presence of 80 μg polybrene/ml for overnight incubation. Medium containing lentiviral particles was replaced by fresh cell culture medium and incubated for 48 h. At day 3, fresh medium containing 5 μg puromycin/ml (Invivogen) was added, and medium was refreshed 1:1 v/v every 3–4 d. After outgrowth, clones were selected and characterized.

The Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) was used to prepare and process the samples. Briefly, mRNA was isolated from total RNA using oligo(dT) magnetic beads. After fragmentation of the mRNA, cDNA synthesis was performed followed by ligation of sequencing adapters and PCR amplification. The quality and yield after sample preparation was measured with a fragment analyzer (Advanced Analytical, Heidelberg, Germany). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300 and 500 bp, data not shown). Clustering and DNA sequencing using the Illumina NextSeq 500 was performed according to the manufacturer’s protocols. A total of 1.6 pM of DNA was loaded and analyzed using NextSeq control software 2.0.2. Image analysis, base calling, and quality verification were performed with Illumina data analysis pipeline RTA v2.4.11 and Bcl2fastq v2.17. The reads were mapped to reference sequence Homo_sapiens.GCRh37.75 using a short-read aligner based on Burrows-Wheeler transform (mismatch rate of maximum 2%). For the 16 samples, the number of reads obtained was between 22,537,666 and 32,979,730. The % reads of reads aligned to the reference was between 95.4 and 95.7%. Reads that aligned to multiple locations were between 7.5 and 9.4%. Reads that aligned to genes were between 60.0 and 87.8%. SAMtools 0.1.18 was used to sort and index the BAM files. The number of times a read aligned to a gene and normalized read counts in fragments per kb million were determined using in-house–developed scripts. The R package DESeq 1.10.1 was used to calculate false discovery rate–corrected p values for the genes differentially expressed in the tested samples.

Total cellular RNA was isolated using TriReagent (Sigma-Aldrich) according to the manufacturer’s protocol. Subsequently, mRNA was reverse transcribed into cDNA using the Fermentas kit according to the manufacturer’s protocol (Qiagen Benelux, Venlo, the Netherlands) using 1 μg of mRNA as template and 0.25 μg of oligo(dT)15 primers supplied with the kit. RT-PCRs were performed on the CFX96 Thermal Cycler (Bio-Rad, Hercules, CA) using primer (Life Technologies, Thermo Fisher Scientific) and probe (human Exiqon ProbeLibrary, Roche, Woerden, the Netherlands) combinations listed in Table I. Gene of interest mRNA expression levels were standardized to GAPDH expression levels using the Pfaffl method (16).

IL-12p40 and TNF-α levels were determined by ELISA according to the manufacturer’s protocol (U-Cytech, Utrecht, the Netherlands). IL-17 and IFN-γ levels were determined by ELISA kits (IL-17; R&D Systems, IFN-γ; Sanquin, the Netherlands) according to the manufacturer’s protocol. Multiplex assays were performed using a customized Non-Human Primate MILLIPLEX MAP kit (Millipore, Billerica, MA). CCL2, IL-12, IL-6, IL-8, MIP1α, MIP1β, and TNF-α levels were determined according to the manufacturer’s protocol and analyzed on the Luminex 200 System (Bio-Rad).

NF-κBp65, p–NF-κBp65 (Ser536), p–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185), p-p38 (Thr180/Tyr182), and p-ERK1/2 (Thr202/Tyr204) levels were measured in cell lysates using PathScan ELISA Kit (Cell Signaling Technology, Beverly, MA). Cell lysates were generated according to protocol in lysis buffer provided with the kit, supplemented with protease inhibitors (Roche). Samples were stored at −80°C before use and diluted 1:1 in dilution buffer provided with the kit before incubation for 16 h at 4°C. All following steps were performed according to the manufacturer’s protocol. Nonphosphorylated NF-κBp65 levels were used as reference values for each sample.

NF-κBp65, AP-1, and ATF2 DNA binding activity was analyzed by TransAM ELISA according to the manufacturer’s protocol (Active Motif). Bound transcription factors were visualized by addition of a primary Ab directed against NF-κBp65, AP-1, or ATF2 elements, followed by detection with a HRP-conjugated secondary Ab. Absorbances were read at 450 nm with a reference wavelength of 655 nm. For the NF-κB and AP-1 assays, a protein standard curve was performed using recombinant NF-κBp65 or c-Jun recombinant protein to quantitate transcription factor levels in the protein samples.

Cells were lysed in lysis buffer (Cell Signaling) supplemented with protease inhibitors (Roche), and protein concentrations were determined using the Bradford Protein Assay (Pierce, Rockford, IL) according to the manufacturer’s protocol. Samples were stored at −20°C before use. Proteins (2.5 mg/ml per lane) were separated on 4–12% Bis-Tris gels (Invitrogen) and transferred onto Hybond-ECL nitrocellulose membranes (Amersham, Arlington Heights, IL) via a semidry blotting system (Thermo Fisher Scientific). The membranes were blocked and probed with primary Abs directed against STAT1 phosphorylated at tyrosine 701 (clone: D4A7), STAT1 phosphorylated at serine 727 (polyclonal), or total STAT1 (clone: 42H3; all Cell Signaling), followed by anti-rabbit–IgG-HRP AffiniPure Ab (Jackson ImmunoResearch Laboratories, Suffolk, U.K.) developed using Femto ECL substrate (Pierce) and quantitated with the ChemiDoc MP system (Bio-Rad).

GraphPad Prism 4.0b (GraphPad Software, San Diego, CA) was used for statistical analysis. Principal component and redundancy analysis was performed using Canoco 5.02 (Microcomputer Power, Ithaca, NY) at default settings.

Recently, we published on the complex interplay between A2AR and A3R as modulators of innate immune responses (14). Our data also suggested that A3R-mediated signaling was directly involved in TLR-induced IL-12 production by myeloid cells. To investigate this, human monocytes, monocyte-derived macrophages, and monocyte-derived DC were exposed to the TLR4 agonist LPS in the absence or presence of specific ADORA antagonists. The presence of ADORA antagonists alone did not induce measurable levels of IL-12p40 in any of the cell types, whereas exposure to LPS induced the production of variable amounts of IL-12p40 in different cell types (Supplemental Table I). The presence of an A3R antagonist significantly inhibited LPS-induced IL-12p40 production in monocytes, macrophages, and DC, whereas antagonizing A1R, A2AR, or A2BR did not significantly affect the production of IL-12p40 in any of the cell types (Fig. 1A). An important role for A3R-mediated signaling in LPS-induced IL-12p40 was confirmed by using combinations of different antagonists (Fig. 1A).

FIGURE 1.

LPS-induced IL-12p40 is dependent on A3R-mediated signaling. (A) Monocytes, monocyte-derived macrophages, and monocyte-derived DC were exposed for 16 h to 100 ng LPS/ml in the presence or absence of 1 μM A1R, A2AR, A2BR, or A3R antagonist either alone or in combination. IL-12p40 levels are expressed relative to production after exposure to LPS alone. Different symbols represent different donors. (dotted line = 100%, *p < 0.05, **p < 0.01, ***p < 0.001 paired t tests against LPS-exposed controls, SD are derived from measurements in triplicate). (B) Monocyte-derived DC from three donors were exposed for 16 h to 100 ng LPS/ml in the presence or absence of A3R antagonists VUF8504, MRS1220, or MRS1911. Different symbols represent different donors. Horizontal lines indicate mean values with 95% confidence intervals. IL-12p40 levels are expressed relative to production after exposure to LPS alone (dotted line = 100%, *p < 0.05 paired t tests against LPS-exposed controls). (C) IL-12p40 levels induced by exposure to different concentrations of LPS in THP-1 cells, in THP-1 cells transduced with nontargeting control (ntc) shRNA (THP-1ntc), or in THP-1 cells transduced with A3R-targeting shRNA (THP-1A3) in the presence or absence of 1 or 5 μM VUF8504.

FIGURE 1.

LPS-induced IL-12p40 is dependent on A3R-mediated signaling. (A) Monocytes, monocyte-derived macrophages, and monocyte-derived DC were exposed for 16 h to 100 ng LPS/ml in the presence or absence of 1 μM A1R, A2AR, A2BR, or A3R antagonist either alone or in combination. IL-12p40 levels are expressed relative to production after exposure to LPS alone. Different symbols represent different donors. (dotted line = 100%, *p < 0.05, **p < 0.01, ***p < 0.001 paired t tests against LPS-exposed controls, SD are derived from measurements in triplicate). (B) Monocyte-derived DC from three donors were exposed for 16 h to 100 ng LPS/ml in the presence or absence of A3R antagonists VUF8504, MRS1220, or MRS1911. Different symbols represent different donors. Horizontal lines indicate mean values with 95% confidence intervals. IL-12p40 levels are expressed relative to production after exposure to LPS alone (dotted line = 100%, *p < 0.05 paired t tests against LPS-exposed controls). (C) IL-12p40 levels induced by exposure to different concentrations of LPS in THP-1 cells, in THP-1 cells transduced with nontargeting control (ntc) shRNA (THP-1ntc), or in THP-1 cells transduced with A3R-targeting shRNA (THP-1A3) in the presence or absence of 1 or 5 μM VUF8504.

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To validate that the observed effects were attributable to A3R-mediated signaling, DC were stimulated with LPS in the presence of different concentrations of three structurally unrelated A3R antagonists (Table II). All A3R antagonists significantly inhibited LPS-induced IL-12p40 production (Fig. 1B). Finally, we silenced A3R expression in THP-1 cells (THP-1 cells transduced with A3R-targeting shRNA [THP-1A3]) with lentiviral shRNA and measured LPS-induced IL-12p40 production. A3R knockdown was >90% as confirmed by quantitative RT-PCR (Supplemental Fig. 1). LPS-induced IL-12p40 levels in THP-1A3 cells were decreased compared with those in THP-1 cells or in THP-1 cells transduced with nontargeting control (ntc) shRNA (THP-1ntc; Fig. 1C). In contrast to THP-1 and THP-1ntc cells, exposure of THP-1A3 cells to an A3R antagonist had no effects on LPS-induced IL-12p40 production (Fig. 1C), confirming the specific involvement of A3R-mediated signaling.

Table I.
Primer and probe sequences
TargetForward Primer (5′-3′)Reverse Primer (5′-3′)Probe
TNF-α 5′-CAGCCTCTTCTCCTTCCTGAT-3′ 5′-GCCAGAGGGCTGATTAGAGA-3′ GGCAGAAG 
IL-12p40 5′-CCCTGACATTCTGCGTTCA-3′ 5′-AGGTCTTGTCCGTGAAGACTCTA-3′ CCAGGGCA 
IL-12p35 5′-CACTCCCAAAACCTGCTGAG-3′ 5′-TCTCTTCAGAAGTGCAAGGGTA-3′ TCTGGAGC 
IL-12p19 5′-TGTTCCCCATATCCAGTGTG-3′ 5′-TCCTTTGCAAGCAGAACTGA-3′ CACAGCCA 
MT1A 5′-GCAAATGCAAAGAGTGCAAAT-3′ 5′-GCACACTTGGCACAGCTC-3′ CTGCTCCT 
MT1H 5′-TGGGAACTCCAGTCTCACCT-3′ 5′-TGCATTTGCACTTTTTGCAC-3′ CTGCTCCT 
MT2A 5′-AACCTGTCCCGACTCTAGCC-3′ 5′-GCAGGTGCAGGAGTCACC-3′ CTGCTCCT 
MT3 5′-AAGTGCGAGGGATGCAAAT-3′ 5′-CTGCACTTCTCTGCTTCTGC-3′ CTGCCTCT 
GAPDH 5′-AGCCACATCGCTCAGACAC-3′ 5′-GCCCAATACGACCAAATCC-3′ CTTCCCCA 
TargetForward Primer (5′-3′)Reverse Primer (5′-3′)Probe
TNF-α 5′-CAGCCTCTTCTCCTTCCTGAT-3′ 5′-GCCAGAGGGCTGATTAGAGA-3′ GGCAGAAG 
IL-12p40 5′-CCCTGACATTCTGCGTTCA-3′ 5′-AGGTCTTGTCCGTGAAGACTCTA-3′ CCAGGGCA 
IL-12p35 5′-CACTCCCAAAACCTGCTGAG-3′ 5′-TCTCTTCAGAAGTGCAAGGGTA-3′ TCTGGAGC 
IL-12p19 5′-TGTTCCCCATATCCAGTGTG-3′ 5′-TCCTTTGCAAGCAGAACTGA-3′ CACAGCCA 
MT1A 5′-GCAAATGCAAAGAGTGCAAAT-3′ 5′-GCACACTTGGCACAGCTC-3′ CTGCTCCT 
MT1H 5′-TGGGAACTCCAGTCTCACCT-3′ 5′-TGCATTTGCACTTTTTGCAC-3′ CTGCTCCT 
MT2A 5′-AACCTGTCCCGACTCTAGCC-3′ 5′-GCAGGTGCAGGAGTCACC-3′ CTGCTCCT 
MT3 5′-AAGTGCGAGGGATGCAAAT-3′ 5′-CTGCACTTCTCTGCTTCTGC-3′ CTGCCTCT 
GAPDH 5′-AGCCACATCGCTCAGACAC-3′ 5′-GCCCAATACGACCAAATCC-3′ CTTCCCCA 
Table II.
IL-12p40 production values in picograms per milliliter per donor
+ 100 ng LPS/mlDonor 1Donor 2Donor 3
μMpg/mlSDpg/mlSDpg/mlSD
VUF8504 79,436 22,939 106,007 26,507 54,163 5,941 
0.1 60,980 36,049 134,561 44,785 42,202 1,694 
0.5 48,134 29,284 119,011 17,470 39,546 344 
29,440 4,873 99,952 16,915 34,026 2,160 
11,316 4,198 85,177 23,751 12,865 2,480 
10 7,361 1,294 19,794 6,849 9,532 368 
MRS1191 94,776 39,350 102,135 18,130 54,163 5,941 
0.1 79,206 11,822 110,306 19,756 43,261 589 
0.5 37,929 2,364 80,687 9,873 31,387 1,228 
40,010 4,726 46,819 3,052 18,038 246 
28,295 12,329 58,197 898 13,160 638 
10 25,627 7,262 51,231 5,206 8,282 2,676 
MRS1220 79,436 22,939 106,007 26,507 43,119 2,485 
0.1 33,076 916 78,734 11,554 29,837 1,814 
0.5 40,191 7,182 47,498 13,639 30,981 2,005 
20,762 5,056 49,623 10,885 29,233 6,664 
35,090 1,733 44,056 4,484 24,199 2,540 
10 30,642 5,956 48,476 5,161 27,145 734 
+ 100 ng LPS/mlDonor 1Donor 2Donor 3
μMpg/mlSDpg/mlSDpg/mlSD
VUF8504 79,436 22,939 106,007 26,507 54,163 5,941 
0.1 60,980 36,049 134,561 44,785 42,202 1,694 
0.5 48,134 29,284 119,011 17,470 39,546 344 
29,440 4,873 99,952 16,915 34,026 2,160 
11,316 4,198 85,177 23,751 12,865 2,480 
10 7,361 1,294 19,794 6,849 9,532 368 
MRS1191 94,776 39,350 102,135 18,130 54,163 5,941 
0.1 79,206 11,822 110,306 19,756 43,261 589 
0.5 37,929 2,364 80,687 9,873 31,387 1,228 
40,010 4,726 46,819 3,052 18,038 246 
28,295 12,329 58,197 898 13,160 638 
10 25,627 7,262 51,231 5,206 8,282 2,676 
MRS1220 79,436 22,939 106,007 26,507 43,119 2,485 
0.1 33,076 916 78,734 11,554 29,837 1,814 
0.5 40,191 7,182 47,498 13,639 30,981 2,005 
20,762 5,056 49,623 10,885 29,233 6,664 
35,090 1,733 44,056 4,484 24,199 2,540 
10 30,642 5,956 48,476 5,161 27,145 734 

Detection level limit was 100 pg/ml.

We next characterized the involvement of A3R-mediated signaling on IL-12p40 production as induced by other TLRs that are expressed by DC. Engagement of TLR2, 3, 4, 5, and 8 on DC from four donors induced the production of variable levels of IL-12p40 (Table III). Exposure of DC to TLR ligands in the presence of an A3R antagonist significantly reduced TLR2, TLR3, TLR4, TLR5, and TLR8-induced IL-12p40 levels (Fig. 2A), demonstrating that A3R-mediated signaling is broadly involved in TLR-induced IL-12p40 production. It should, however, be noted that although the qualitative aspect of the contribution of A3R-mediated signaling to TLR responses is clear, effect sizes vary considerably between different TLR as well as between different donors.

Table III.
IL-12p40 production values in picograms per milliliter per donor
Donor 1Donor 2Donor 3Donor 4
Stimuluspg/mlSDpg/mlSDpg/mlSDpg/mlSD
+Pam3CSK4 1,292 105 1,906 307 17,984 1,545 5,304 1,889 
+Pam3CSK4+VUF 364 108 1,496 972 6,073 1,223 125 68 
+Poly(I:C) 2,918 311 1,574 525 4,082 372 1,140 378 
+Poly(I:C)+VUF 983 234 890 165 2,402 224 759 276 
+LPS 47,745 3,508 172,130 5,853 45,866 3,580 74,871 10,338 
+LPS+VUF 6,417 493 84,546 22,682 26,547 2,318 7,359 481 
+Flagellin 8,179 429 3,229 746 5,499 1,683 1,748 168 
+Flagellin+VUF 1,647 1,050 930 213 3,461 1,044 110 
+CL075 30,970 9,750 76,674 17,068 117,767 9,501 39,910 15,462 
+CL075+VUF 16,862 4,383 21,397 8,437 64,751 14,621 4,444 1,910 
Donor 1Donor 2Donor 3Donor 4
Stimuluspg/mlSDpg/mlSDpg/mlSDpg/mlSD
+Pam3CSK4 1,292 105 1,906 307 17,984 1,545 5,304 1,889 
+Pam3CSK4+VUF 364 108 1,496 972 6,073 1,223 125 68 
+Poly(I:C) 2,918 311 1,574 525 4,082 372 1,140 378 
+Poly(I:C)+VUF 983 234 890 165 2,402 224 759 276 
+LPS 47,745 3,508 172,130 5,853 45,866 3,580 74,871 10,338 
+LPS+VUF 6,417 493 84,546 22,682 26,547 2,318 7,359 481 
+Flagellin 8,179 429 3,229 746 5,499 1,683 1,748 168 
+Flagellin+VUF 1,647 1,050 930 213 3,461 1,044 110 
+CL075 30,970 9,750 76,674 17,068 117,767 9,501 39,910 15,462 
+CL075+VUF 16,862 4,383 21,397 8,437 64,751 14,621 4,444 1,910 

Detection level limit was 100 pg/ml. DC were exposed to different TLR ligands in the absence or presence of the A3R antagonist VUF8504.

FIGURE 2.

A3R-mediated signaling is broadly involved in the TLR-induced production of IL-12p40 and CCL2. (A) Monocyte-derived DC of four different donors were exposed for 16 h to different TLR ligands in the absence or presence of 5 μM VUF8504. For each TLR ligand, IL-12p40 levels in the presence of VUF8504 are expressed relative to production after exposure to each TLR ligand alone (dotted line = 100%, dots represent different donors, bars represent mean values (+ and − SD, calculated from four different means) of normalized response levels. TLR ligands used were 100 ng PAM3CSK4/ml (TLR2), 20 ng poly(I:C)/ml (TLR3), 100 ng LPS/ml (TLR4), 100 ng flagellin/ml (TLR5), or 1 μg CL075/ml (TLR8). *p < 0.05, **p < 0.01 paired t tests against TLR-exposed controls. (B) Luminex analysis of cell culture supernatants of DC from three donors exposed to 100 ng LPS/ml (TLR4, left panel) or 1 μg CL075/ml (TLR8, middle panel) in the absence or presence of 5 μM VUF8504. Bars represent mean values (+ and − SD, calculated from data from three different donors). In the right panels, IL-12p40 levels (upper panel) and CCL2 levels (lower panel) in the presence of VUF8504 are expressed relative to production after exposure to each TLR ligand alone (dotted line = 100%, dots represent different donors, bars indicate mean values of normalized response levels, SD were calculated from three different means, *p < 0.05, **p < 0.01 paired t tests against TLR-exposed controls). (C) Monocytes, monocyte-derived macrophages, and monocyte-derived DC of four different donors were exposed for 16 h to whole pathogens (multiplicity of infection of 10) in the absence or presence of 5 μM VUF8504. Different symbols represent different donors. IL-12p40 levels are expressed relative to production after exposure to corresponding pathogens alone (dotted line = 100%, *p < 0.05, **p < 0.01 paired t tests against controls). (D) Monocyte-derived DC of five different donors (represented by different symbols) were exposed to different pathogens in the absence (closed symbols) or presence (open symbols) of 5 μM VUF8504 and cocultured with donor-matched CD4+ T cells in a ratio of 5:1. IFN-γ levels were measured after 24 h, and IL-17 levels were measured after 5 d. Horizontal bars indicate the means (*p < 0.05, **p < 0.01, ***p < 0.001, paired t tests on 10log-transformed data). bd, below detection levels.

FIGURE 2.

A3R-mediated signaling is broadly involved in the TLR-induced production of IL-12p40 and CCL2. (A) Monocyte-derived DC of four different donors were exposed for 16 h to different TLR ligands in the absence or presence of 5 μM VUF8504. For each TLR ligand, IL-12p40 levels in the presence of VUF8504 are expressed relative to production after exposure to each TLR ligand alone (dotted line = 100%, dots represent different donors, bars represent mean values (+ and − SD, calculated from four different means) of normalized response levels. TLR ligands used were 100 ng PAM3CSK4/ml (TLR2), 20 ng poly(I:C)/ml (TLR3), 100 ng LPS/ml (TLR4), 100 ng flagellin/ml (TLR5), or 1 μg CL075/ml (TLR8). *p < 0.05, **p < 0.01 paired t tests against TLR-exposed controls. (B) Luminex analysis of cell culture supernatants of DC from three donors exposed to 100 ng LPS/ml (TLR4, left panel) or 1 μg CL075/ml (TLR8, middle panel) in the absence or presence of 5 μM VUF8504. Bars represent mean values (+ and − SD, calculated from data from three different donors). In the right panels, IL-12p40 levels (upper panel) and CCL2 levels (lower panel) in the presence of VUF8504 are expressed relative to production after exposure to each TLR ligand alone (dotted line = 100%, dots represent different donors, bars indicate mean values of normalized response levels, SD were calculated from three different means, *p < 0.05, **p < 0.01 paired t tests against TLR-exposed controls). (C) Monocytes, monocyte-derived macrophages, and monocyte-derived DC of four different donors were exposed for 16 h to whole pathogens (multiplicity of infection of 10) in the absence or presence of 5 μM VUF8504. Different symbols represent different donors. IL-12p40 levels are expressed relative to production after exposure to corresponding pathogens alone (dotted line = 100%, *p < 0.05, **p < 0.01 paired t tests against controls). (D) Monocyte-derived DC of five different donors (represented by different symbols) were exposed to different pathogens in the absence (closed symbols) or presence (open symbols) of 5 μM VUF8504 and cocultured with donor-matched CD4+ T cells in a ratio of 5:1. IFN-γ levels were measured after 24 h, and IL-17 levels were measured after 5 d. Horizontal bars indicate the means (*p < 0.05, **p < 0.01, ***p < 0.001, paired t tests on 10log-transformed data). bd, below detection levels.

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To expand our analyses to other TLR-induced soluble factors, we choose to evaluate the effects of A3R-mediated signaling on the robust cytokine responses induced by TLR4 and TLR8 ligands on DC from three different donors using Luminex technology. Exposure to TLR4 and TLR8 ligands triggered the production of multiple soluble factors (Fig. 2B). When the inhibition of A3R-mediated signaling was analyzed against normalized TLR-induced cytokine production from different donors, only IL-12p40 and CCL2 were significantly affected (Fig. 2B, right panels, Table IV). Exposure of DC to TLR2, -3, and -5 ligands in the presence of an A3R antagonist also reduced CCL2 levels (data not shown), demonstrating that the effects were not limited to TLR4- and TLR8-induced responses.

Table IV.
Cytokine production values in picograms per milliliter per donor as determined by Luminex
Donor 1Donor 2Donor 3
IL12p40    
+LPS 9,211 2,707 7,791 
+LPS +VUF8504 4,730 1,546 1,960 
+CL075 4,708 10,065 3,605 
+CL075+VUF8504 1,076 1,349 991 
CCL2    
+LPS 1,798 2,699 2,619 
+LPS +VUF8504 272 1,676 316 
+CL075 293 1,900 3,396 
+CL075+VUF8504 49 315 229 
Donor 1Donor 2Donor 3
IL12p40    
+LPS 9,211 2,707 7,791 
+LPS +VUF8504 4,730 1,546 1,960 
+CL075 4,708 10,065 3,605 
+CL075+VUF8504 1,076 1,349 991 
CCL2    
+LPS 1,798 2,699 2,619 
+LPS +VUF8504 272 1,676 316 
+CL075 293 1,900 3,396 
+CL075+VUF8504 49 315 229 

Detection level limit was 30 pg/ml.

Subsequently, we investigated the contribution of A3R-mediated signaling to responses induced by whole pathogens that contain multiple ligands for innate immune receptors. We exposed monocytes, monocyte-derived macrophages, and monocyte-derived DC of four different donors to the fungus C. albicans as well as to Gram-negative H. influenzae and Gram-positive S. aureus bacteria in the absence or presence of an A3R antagonist and measured IL-12p40 production. Monocytes produced relatively low levels (Supplemental Table II) of IL-12p40 in response to C. albicans and even lower levels when A3R-mediated signaling was antagonized (Fig. 2C). Although exposure to H. influenzae potently triggered the production of IL-12p40 in all four donors, and S. aureus triggered detectable production of IL-12p40 in three donors, these responses were not significantly affected when A3R-mediated signaling was inhibited. Macrophages of all four donors produced detectable levels of IL-12p40 in response to H. influenzae only (Supplemental Table II), which were significantly lower when A3R-mediated signaling was inhibited. In DC, C. albicans and H. influenzae potently induced IL-12p40 production, whereas exposure to S. aureus did not induce detectable levels of IL-12p40 (Supplemental Table II). Both C. albicans– and H. influenzae–induced IL-12p40 responses were significantly inhibited when A3R-signaling was antagonized (Fig. 2C).

The IL-12p40 subunit can heterodimerize with p35 or p19 to form bioactive IL-12 or IL-23 respectively. IL-12 is an important factor in the polarization of T cells to Th1 cells and the consequent production of IFN-γ, whereas IL-23 is involved in the polarization to Th17 cells and the consequent production of IL-17 (17). To examine whether blocking A3R-mediated signaling could affect downstream T cell cytokine production, DC from five different donors were exposed to C. albicans and H. influenzae in the presence or absence of A3R antagonist and coincubated with autologous CD4+ T cells for 5 d. Analysis of cell culture supernatants reveals that exposure to H. influenzae potently induced IFN-γ, whereas exposure to C. albicans mainly induced the production of IL-17 (Fig. 2D). There was considerable donor-donor variation, and it is interesting to note that the two donors that were poor IFN-γ producers were the ones that produced the highest levels of IL-17. When A3R signaling was antagonized, both IFN-γ and IL-17 responses were significantly lower with larger effect sizes on IL-17 responses (Fig. 2D). Future experiments will further focus on the implications of these differences.

A3R-mediated signaling affected IL-12 and CCL2 production at the transcription level, as LPS-induced IL-12p40– and CCL2-encoding mRNA levels in DC in the presence of an A3R antagonist were 5- and 39-fold lower, respectively (Fig. 3A, Table I). In addition, LPS-induced mRNA levels of IL-12p35 and IL-23p19 subunits were both ∼5-fold lower when A3R-mediated signaling was antagonized. Interestingly, exposure of cells to A3R antagonist alone already decreased basal mRNA expression levels of the IL-12p40 (to a mean of 23%) and p35 (to a mean of 65%) subunits as well as of CCL2 (to a mean of 14%), suggesting a role for homeostatic A3R-mediated signaling in DC. To delineate how A3R-mediated signaling affects TLR-induced intracellular signaling cascades, we first assessed LPS-induced phosphorylation levels of the NF-κB at serine 536 by PathScan ELISA, an important TLR-induced modulator of transcriptional activity (18). This was not affected by antagonizing A3R-mediated signaling, and in accordance, NF-κB binding to its consensus site as assessed by TransAM ELISA was similar in nuclear extracts of DC exposed to LPS both in the absence or presence of an A3R antagonist (Fig. 3B, 3C). In addition to activation of NF-κB (19), TLR-induced signaling activates several MAPKs. We therefore used PathScan ELISA to evaluate phosphorylation levels of SAPK/JNK, ERK1/2, and p38 in cellular lysates of DC exposed to LPS in the absence or presence of an A3R antagonist. Whereas LPS-induced levels of p-ERK1/2 and p-p38 were unaffected, LPS-induced p-SAPK/JNK levels were decreased at early time points in the presence of an A3R antagonist (Fig. 3D). SAPK/JNK induces activation of the transcription factor AP-1. However, LPS-induced c-Jun, c-Fos, and ATF2 binding levels were not significantly different in nuclear extracts of DC exposed to LPS in the presence of an A3R antagonist (Fig. 3E), rendering it unlikely that differences in SAPK/JNK phosphorylation underlie the observed effects on IL-12 and CCL2 production.

FIGURE 3.

A3R-mediated signaling is necessary to induce phosphorylation of STAT1 tyrosine 701. (A) Monocyte-derived DC were exposed for 6 h to 500 ng LPS/ml in the presence or absence of 5 μM VUF8504, and mRNA levels encoding for IL-12p40, CCL2, IL-12p35, and IL-23p19 were quantified by real-time RT-PCR and standardized to GAPDH mRNA expression levels. Different symbols represent different donors. In the graph, relative expression levels are given on a 10log scale (*p < 0.05, **p < 0.01 paired t tests on the 10log-transformed data). (B) Phosphorylated NF-κB protein levels in cellular lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 0, 5, 15, 30, or 60 min in the presence or absence of 5 μM VUF8504. Samples were standardized to total NF-κB levels. A representative example of three different donors is shown. (C) Bound NF-κB levels in nuclear lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 1 h in the presence or absence of 5 μM VUF8504. Different symbols represent different donors. (D) Phosphorylated ERK1/2, p38, SAPK/JNK protein levels in cellular lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 0, 5, 15, 30, or 60 min in the presence or absence of 5 μM VUF8504. Samples were standardized to NF-κB levels. A representative example of three different donors is shown. (E) Different symbols represent different donors. Bound c-Jun, c-Fos, and ATF2 levels in nuclear lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 1 h in the presence or absence of 5 μM VUF8504 (*p < 0.05 paired t tests for A3R-mediated effects). (F) Western blot analysis of phosphorylated STAT1 serine 727 and tyrosine 701 in monocyte-derived DC exposed to 500 ng LPS/ml for 0, 5, 15, 30, or 60 min in the presence or absence of 5 μM VUF8504. Samples were standardized to total STAT1 levels and quantitated (lower panels). A representative example of three different donors is shown. (G) A representative example of three different donors is shown. Monocyte-derived DC were exposed to 500 ng LPS/ml for 16 h in the presence or absence of 5 μM VUF8504 and of increasing concentrations of IFN-γ (*p < 0.05, **p < 0.01 paired t tests for A3R-mediated effects).

FIGURE 3.

A3R-mediated signaling is necessary to induce phosphorylation of STAT1 tyrosine 701. (A) Monocyte-derived DC were exposed for 6 h to 500 ng LPS/ml in the presence or absence of 5 μM VUF8504, and mRNA levels encoding for IL-12p40, CCL2, IL-12p35, and IL-23p19 were quantified by real-time RT-PCR and standardized to GAPDH mRNA expression levels. Different symbols represent different donors. In the graph, relative expression levels are given on a 10log scale (*p < 0.05, **p < 0.01 paired t tests on the 10log-transformed data). (B) Phosphorylated NF-κB protein levels in cellular lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 0, 5, 15, 30, or 60 min in the presence or absence of 5 μM VUF8504. Samples were standardized to total NF-κB levels. A representative example of three different donors is shown. (C) Bound NF-κB levels in nuclear lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 1 h in the presence or absence of 5 μM VUF8504. Different symbols represent different donors. (D) Phosphorylated ERK1/2, p38, SAPK/JNK protein levels in cellular lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 0, 5, 15, 30, or 60 min in the presence or absence of 5 μM VUF8504. Samples were standardized to NF-κB levels. A representative example of three different donors is shown. (E) Different symbols represent different donors. Bound c-Jun, c-Fos, and ATF2 levels in nuclear lysates of monocyte-derived DC exposed to 500 ng LPS/ml for 1 h in the presence or absence of 5 μM VUF8504 (*p < 0.05 paired t tests for A3R-mediated effects). (F) Western blot analysis of phosphorylated STAT1 serine 727 and tyrosine 701 in monocyte-derived DC exposed to 500 ng LPS/ml for 0, 5, 15, 30, or 60 min in the presence or absence of 5 μM VUF8504. Samples were standardized to total STAT1 levels and quantitated (lower panels). A representative example of three different donors is shown. (G) A representative example of three different donors is shown. Monocyte-derived DC were exposed to 500 ng LPS/ml for 16 h in the presence or absence of 5 μM VUF8504 and of increasing concentrations of IFN-γ (*p < 0.05, **p < 0.01 paired t tests for A3R-mediated effects).

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Next to binding sites for NF-κB and AP-1 elements, the promoter regions of IL-12p40 and CCL2 also share binding sites for STAT1. Full activation of STAT1 requires phosphorylation at serine 727 and tyrosine 701. Whereas LPS-induced phosphorylation of STAT1 at S727 was unaffected in the presence of an A3R antagonist, LPS-induced phosphorylation of STAT1 at Y701 was strongly decreased after 60 min of stimulation (Fig. 3F). To confirm the involvement of STAT1 tyrosine 701 phosphorylation, we tested whether the reduced levels of LPS-induced IL-12p40 in the presence of an A3R antagonist could be rescued by the addition of IFN-γ, a well-known inducer of STAT1 tyrosine 701 phosphorylation (20). Confirming our earlier results, IFN-γ partially restored LPS-induced IL-12p40 levels in the presence of an A3R antagonist (Fig. 3G).

Finally, we submitted mRNA samples of DC exposed to LPS in the presence or absence of an A3R antagonist to total transcriptome analysis by RNA sequencing (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE125747). Principal component analysis of the samples (Fig. 4A) demonstrates a clear separation of the different sample classes according to stimulation conditions, which was confirmed by redundancy analysis (p < 0.002; data not shown). Venn diagrams illustrate the overlap between different sample groups (Fig. 4B). Differential gene expression analysis demonstrates that exposure to LPS significantly (adjusted p < 0.01) altered the expression levels of 4,893 gene transcripts (GSE125747), whereas exposure to an A3R antagonist altered the expression levels of 381 gene transcripts only (Table V). Expression levels of 146 transcripts were significantly different between cells exposed to LPS alone or in combination with an A3R antagonist (GSE125747). Analysis of the individual transcripts reveals that exposure to an A3R antagonist, even in the absence of LPS activation, leads to a strong increase of metallothionein mRNA levels (the top 13 genes with the lowest p values are all metallothionein genes). These findings were confirmed for four metallothionein isoforms by quantitative RT-PCR (Fig. 4C, Table I). In addition, the data confirm that LPS-induced CCL2 and IL-12 (NS) mRNA levels were lower in the presence of an A3R antagonist (GSE125747).

FIGURE 4.

Analysis of the mRNA transcriptome reveals the involvement of metallothioneins in A3R-mediated signaling. (A) Principal component analysis of mRNA transcriptomes from monocyte-derived DC from four human donors exposed for 6 h to 500 ng LPS/ml in the presence or absence of 5 μM VUF8504. The x-axis explains 65% of the variation, and the y-axis explains 7% of the variation. (B) Venn diagrams demonstrating overlaps in differentially expressed genes between the sample groups. (C) Monocyte-derived DC were exposed for 6 h to 500 ng LPS/ml in the presence or absence of 5 μM VUF8504, and MT1A-, MT1H-, MT2A-, and MT3-encoding mRNA expression levels were quantified by real-time RT-PCR and standardized to GAPDH mRNA expression levels. Different symbols represent different donors. In the graph, relative expression levels are given on a 10log scale (*p < 0.05, **p < 0.01, ***p < 0.001 paired t tests on the 10log-transformed data).

FIGURE 4.

Analysis of the mRNA transcriptome reveals the involvement of metallothioneins in A3R-mediated signaling. (A) Principal component analysis of mRNA transcriptomes from monocyte-derived DC from four human donors exposed for 6 h to 500 ng LPS/ml in the presence or absence of 5 μM VUF8504. The x-axis explains 65% of the variation, and the y-axis explains 7% of the variation. (B) Venn diagrams demonstrating overlaps in differentially expressed genes between the sample groups. (C) Monocyte-derived DC were exposed for 6 h to 500 ng LPS/ml in the presence or absence of 5 μM VUF8504, and MT1A-, MT1H-, MT2A-, and MT3-encoding mRNA expression levels were quantified by real-time RT-PCR and standardized to GAPDH mRNA expression levels. Different symbols represent different donors. In the graph, relative expression levels are given on a 10log scale (*p < 0.05, **p < 0.01, ***p < 0.001 paired t tests on the 10log-transformed data).

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Table V.
Selection of significantly differentially expressed transcripts
Gene IDControlVUF8504VUF8504/Controlp ValueProtein
Upregulated 
 ENSG00000205358 7.12 21,350.49 2,997.39 4.17 × 10−281 MT1H 
 ENSG00000125144 25.88 68,870.43 2,660.90 1.16 × 10−248 MT1G 
 ENSG00000187193 29.30 22,074.37 753.32 6.58 × 10−244 MT1X 
 ENSG00000233929 5.88 6,135.09 1,042.68 9.62 × 10−219 MT1XP1 
 ENSG00000125148 104.41 54,901.37 525.81 4.82 × 10−205 MT2A 
 ENSG00000260549 2,535.10 INF 1.62 × 10−125 MT1L 
 ENSG00000244020 0.52 988.00 1,906.61 5.21 × 10−115 MT1HL1 
 ENSG00000162840 10.28 7,672.82 746.13 2.20 × 10−100 MT2P1 
 ENSG00000205364 0.73 26,587.48 36,440.65 7.38 × 10−76 MT1M 
 ENSG00000169715 9.76 20,576.90 2,108.37 4.20 × 10−66 MT1E 
 ENSG00000229230 0.26 3,222.95 12,272.09 3.21 × 10−56 MT1P3 
 ENSG00000198417 12.49 8,660.24 693.20 5.69 × 10−45 MT1F 
 ENSG00000205362 9,797.37 INF 5.01 × 10−33 MT1A 
 ENSG00000197358 70.77 758.76 10.72 5.72 × 10−33 BNIP3P1 
 ENSG00000177181 1.76 181.56 103.39 9.76 × 10−33 RIMKLA 
 ENSG00000176171 105.96 957.69 9.04 3.81 × 10−32 BNIP3 
 ENSG00000196739 4.62 197.23 42.70 5.50 × 10−32 COL27A1 
 ENSG00000109762 107.88 1,322.26 12.26 1.48 × 10−30 SNX25 
 ENSG00000108352 22.38 1,087.54 48.61 6.25 × 10−27 RAPGEFL1 
 ENSG00000135766 1,143.40 5,965.38 5.22 3.31 × 10−25 EGLN1 
Downregulated 
 ENSG00000196950 838.97 243.35 0.29 5.54 × 10−11 SLC39A10 
 ENSG00000141655 5,982.98 2,691.38 0.45 5.71 × 10−7 TNFRSF11A 
 ENSG00000171659 170.90 35.11 0.21 7.05 × 10−7 GPR34 
 ENSG00000168906 7,886.25 3,700.90 0.47 4.88 × 10−6 MAT2A 
 ENSG00000163563 1,851.51 784.99 0.42 6.66 × 10−6 MNDA 
 ENSG00000100644 4,385.27 2,124.05 0.48 1.31 × 10−5 HIF1A 
 ENSG00000164163 1,143.55 524.27 0.46 4.56 × 10−5 ABCE1 
 ENSG00000095585 958.15 343.88 0.36 5.24 × 10−5 BLNK 
 ENSG00000134242 717.17 306.51 0.43 6.58 × 10−5 PTPN22 
 ENSG00000179041 1,604.57 773.89 0.48 1.79 × 10−4 RRS1 
 ENSG00000145569 1,454.94 663.16 0.46 2.24 × 10−4 FAM105A 
 ENSG00000187037 457.64 193.28 0.42 3.52 × 10−4 GPR141 
 ENSG00000197442 2,218.70 1,171.86 0.53 4.37 × 10−4 MAP3K5 
 ENSG00000164125 2,894.64 1,551.32 0.54 7.06 × 10−4 FAM198B 
 ENSG00000168918 4,673.92 2,535.44 0.54 8.17 × 10−4 INPP5D 
 ENSG00000075303 1,303.48 666.43 0.51 8.34 × 10−4 SLC25A40 
 ENSG00000104549 1,598.28 825.70 0.52 8.87 × 10−4 SQLE 
 ENSG00000164933 480.59 219.03 0.46 9.25 × 10−4 SLC25A32 
 ENSG00000178105 260.35 106.77 0.41 1.02 × 10−3 DDX10 
 ENSG00000196814 499.07 231.36 0.46 1.08 × 10−3 MVB12B 
Gene IDControlVUF8504VUF8504/Controlp ValueProtein
Upregulated 
 ENSG00000205358 7.12 21,350.49 2,997.39 4.17 × 10−281 MT1H 
 ENSG00000125144 25.88 68,870.43 2,660.90 1.16 × 10−248 MT1G 
 ENSG00000187193 29.30 22,074.37 753.32 6.58 × 10−244 MT1X 
 ENSG00000233929 5.88 6,135.09 1,042.68 9.62 × 10−219 MT1XP1 
 ENSG00000125148 104.41 54,901.37 525.81 4.82 × 10−205 MT2A 
 ENSG00000260549 2,535.10 INF 1.62 × 10−125 MT1L 
 ENSG00000244020 0.52 988.00 1,906.61 5.21 × 10−115 MT1HL1 
 ENSG00000162840 10.28 7,672.82 746.13 2.20 × 10−100 MT2P1 
 ENSG00000205364 0.73 26,587.48 36,440.65 7.38 × 10−76 MT1M 
 ENSG00000169715 9.76 20,576.90 2,108.37 4.20 × 10−66 MT1E 
 ENSG00000229230 0.26 3,222.95 12,272.09 3.21 × 10−56 MT1P3 
 ENSG00000198417 12.49 8,660.24 693.20 5.69 × 10−45 MT1F 
 ENSG00000205362 9,797.37 INF 5.01 × 10−33 MT1A 
 ENSG00000197358 70.77 758.76 10.72 5.72 × 10−33 BNIP3P1 
 ENSG00000177181 1.76 181.56 103.39 9.76 × 10−33 RIMKLA 
 ENSG00000176171 105.96 957.69 9.04 3.81 × 10−32 BNIP3 
 ENSG00000196739 4.62 197.23 42.70 5.50 × 10−32 COL27A1 
 ENSG00000109762 107.88 1,322.26 12.26 1.48 × 10−30 SNX25 
 ENSG00000108352 22.38 1,087.54 48.61 6.25 × 10−27 RAPGEFL1 
 ENSG00000135766 1,143.40 5,965.38 5.22 3.31 × 10−25 EGLN1 
Downregulated 
 ENSG00000196950 838.97 243.35 0.29 5.54 × 10−11 SLC39A10 
 ENSG00000141655 5,982.98 2,691.38 0.45 5.71 × 10−7 TNFRSF11A 
 ENSG00000171659 170.90 35.11 0.21 7.05 × 10−7 GPR34 
 ENSG00000168906 7,886.25 3,700.90 0.47 4.88 × 10−6 MAT2A 
 ENSG00000163563 1,851.51 784.99 0.42 6.66 × 10−6 MNDA 
 ENSG00000100644 4,385.27 2,124.05 0.48 1.31 × 10−5 HIF1A 
 ENSG00000164163 1,143.55 524.27 0.46 4.56 × 10−5 ABCE1 
 ENSG00000095585 958.15 343.88 0.36 5.24 × 10−5 BLNK 
 ENSG00000134242 717.17 306.51 0.43 6.58 × 10−5 PTPN22 
 ENSG00000179041 1,604.57 773.89 0.48 1.79 × 10−4 RRS1 
 ENSG00000145569 1,454.94 663.16 0.46 2.24 × 10−4 FAM105A 
 ENSG00000187037 457.64 193.28 0.42 3.52 × 10−4 GPR141 
 ENSG00000197442 2,218.70 1,171.86 0.53 4.37 × 10−4 MAP3K5 
 ENSG00000164125 2,894.64 1,551.32 0.54 7.06 × 10−4 FAM198B 
 ENSG00000168918 4,673.92 2,535.44 0.54 8.17 × 10−4 INPP5D 
 ENSG00000075303 1,303.48 666.43 0.51 8.34 × 10−4 SLC25A40 
 ENSG00000104549 1,598.28 825.70 0.52 8.87 × 10−4 SQLE 
 ENSG00000164933 480.59 219.03 0.46 9.25 × 10−4 SLC25A32 
 ENSG00000178105 260.35 106.77 0.41 1.02 × 10−3 DDX10 
 ENSG00000196814 499.07 231.36 0.46 1.08 × 10−3 MVB12B 

Depicted are the top 20 of upregulated (top) and downregulated (bottom) transcripts (based on adjusted p values) after exposure to VUF8504.

ID, identification; INF, infinite ratio as a result of expression levels below threshold in the reference samples.

In this study, we demonstrate that A3R-mediated signaling is critically involved in LPS-induced IL-12 production by myeloid cells. Further analysis of DC demonstrates that also CCL2 production as induced by a broad range of TLR ligands or whole pathogens was affected. A3R-mediated signaling suppresses metallothionein expression levels and amplifies TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701, demonstrating a complex and novel layer of regulation of innate immune responses. To our knowledge, we are the first to describe a role for endogenously produced extracellular adenosine and A3R-mediated signaling in the initiation of TLR-induced cytokine responses.

Extracellular adenosine can interact with four different ADORA subtypes. Interestingly, A3R expression levels are rapidly downregulated after TLR-mediated activation of myeloid cells, whereas expression levels of A2AR are strongly enhanced (14). Previously, we have hypothesized that this shift in ADORA expression pattern underlies the inhibitory effects of extracellular adenosine on TLR-mediated cytokine production by unleashing the full capacity of A2AR-mediated anti-inflammatory signaling. This study suggests that downregulation of A3R also has direct anti-inflammatory effects by depriving TLR-activated myeloid cells of their capacity to produce IL-12 and CCL2. We did not find evidence for direct effects on Ag presentation capacity, as the LPS-induced increase of cell surface expression levels of receptors involved in Ag presentation by DC was unaffected by exposure to an A3R antagonist (Supplemental Fig. 2).

Our transcriptome data revealed that A3R-mediated effects not only modulated TLR-induced signaling but also affected otherwise unstimulated cells. Most striking was the strong induction of metallothioneins when homeostatic A3R-mediated signaling was inhibited. Metallothioneins are low m.w. cysteine-rich proteins that regulate intracellular zinc homeostasis, detoxify heavy metals, and counteract superoxide stress (21). Their expression is potently induced by metal exposure and oxidative stress but also by cytokine signaling and microbial challenge (2225). In myeloid cells, there is ample evidence for an important role of metallothioneins in the maintenance of intracellular zinc homoeostasis (2628). Manipulation of intracellular and extracellular zinc levels can be used to modulate the phenotype of myeloid cells (2931), and different studies have identified a role for zinc signaling in kinase and phosphatase functions (32, 33). Interestingly, a recent study demonstrated that metallothioneins inhibit the phosphorylation of STAT1 and STAT3 (34). It has been postulated that metallothionein 1 and 2 sequester intracellular zinc, thereby sustaining protein tyrosine phosphatase 1B activity that in turn dephosphorylates STATs (32, 33).

The promoter regions of IL-12p40 and CCL2 share binding sites for STAT1, and we have identified that TLR-induced phosphorylation of STAT1 at tyrosine 701 was strongly reduced when A3R-mediated signaling was inhibited. More pathways may be involved, as IFN-γ–induced phosphorylation of STAT1 tyrosine 701 only partially restored TLR-induced IL-12p40 levels in the presence of an A3R antagonist (Fig. 3G). Although the complete elucidation of intracellular signaling cascades remains to be established, the involvement of the STAT1 pathway is evident. Together, this leads us to propose a novel molecular mechanism that links adenosine-induced signaling via the metallothionein–zinc axis to modulation of innate and adaptive immune responses.

Extracellular adenosine is best known as an anti-inflammatory modulator, and A3R-mediated signaling has previously been associated with the inhibition rather than with the initiation of LPS-induced IL-12 and/or TNF-α in different murine models and cell lines, as well as in human monocytes and U937 cells (3540). Several factors may explain the discrepancies with our findings. First, the described inhibitory effects of A3R-mediated signaling have primarily been observed in murine models and/or cells. Whereas the sequences of A1R, A2AR, and A2BR are well conserved across species, A3R sequences differ considerably. Sequence comparisons between rodent and human show 70–75% gene sequence homology and 85–87% protein sequence homology (6, 14). Second, there is evidence that G protein coupling of A3R is different between rodents and humans. In A3R-humanized mice, bone marrow–derived mast cells were unable to initiate conventional murine A3R-mediated signaling pathways. This was attributed to sequence differences in the intracellular region of human A3R, resulting in the uncoupling of murine G proteins (41). Third, discrepancies with observations in U937 cells might be attributable to the differentiation/transformed status of these cells.

In conclusion, we describe a new mechanism that is important for TLR-induced IL-12 and CCL2 responses. Because all experiments were performed in the absence of exogenous adenosine or other ADORA agonists, the observed effects were attributable to endogenously produced extracellular adenosine contributing to TLR-induced responses in an autocrine or paracrine manner. Interestingly, this mechanism bears resemblance to the mechanism regulating the production of inflammasome-induced IL-1β in which endogenous ATP acts as a similar signal through P2X7 receptors (42). Our data suggest that antagonizing A3R signaling could selectively affect TLR-induced production of IL-12 and CCL2, thereby representing a novel immune modulatory strategy. However, given the widespread distribution of A3R throughout the body (43), specific delivery to the site of inflammation represents an important challenge when considering such an approach.

We thank E. Remarque for expert assistance with the statistical analyses, S.B. Geutskens, M.E. Hoonakker, and B. ‘t Hart for critically reading the manuscript.

The sequences presented in this article have been submitted to the Gene Expression Omnibus under accession number GSE125747.

The online version of this article contains supplemental material.

Abbreviations used in this article:

A2AR

adenosine A2A receptor

A2BR

adenosine A2B receptor

ADORA

adenosine receptor

A1R

adenosine A1 receptor

A3R

adenosine A3 receptor

DC

dendritic cell

ntc

nontargeting control

SAPK

stress-activated protein kinase

shRNA

short hairpin RNA

THP-1A3

THP-1 cell transduced with A3R-targeting shRNA

THP-1ntc

THP-1 cell transduced with ntc shRNA.

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The authors have no financial conflicts of interest.

Supplementary data