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. 2023 Dec 1;42(23):e113279.
doi: 10.15252/embj.2022113279. Epub 2023 Oct 26.

Systematic P2Y receptor survey identifies P2Y11 as modulator of immune responses and virus replication in macrophages

Line Lykke Andersen  1 , Yiqi Huang  1 , Christian Urban  1 , Lila Oubraham  1 , Elena Winheim  2 , Che Stafford  3 , Dennis Nagl  3 , Fionan O'Duill  3 , Thomas Ebert  3 , Thomas Engleitner  4 , Søren Riis Paludan  5   6 , Anne Krug  2 , Roland Rad  4 , Veit Hornung  3 , Andreas Pichlmair  1   6   7
Affiliations

Systematic P2Y receptor survey identifies P2Y11 as modulator of immune responses and virus replication in macrophages

Line Lykke Andersen et al. EMBO J. .

Abstract

The immune system is in place to assist in ensuring tissue homeostasis, which can be easily perturbed by invading pathogens or nonpathogenic stressors causing tissue damage. Extracellular nucleotides are well known to contribute to innate immune signaling specificity and strength, but how their signaling is relayed downstream of cell surface receptors and how this translates into antiviral immunity is only partially understood. Here, we systematically investigated the responses of human macrophages to extracellular nucleotides, focusing on the nucleotide-sensing GPRC receptors of the P2Y family. Time-resolved transcriptomic analysis showed that adenine- and uridine-based nucleotides induce a specific, immediate, and transient cytokine response through the MAPK signaling pathway that regulates transcriptional activation by AP-1. Using receptor trans-complementation, we identified a subset of P2Ys (P2Y1, P2Y2, P2Y6, and P2Y11) that govern inflammatory responses via cytokine induction, while others (P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14) directly induce antiviral responses. Notably, P2Y11 combined both activities, and depletion or inhibition of this receptor in macrophages impaired both inflammatory and antiviral responses. Collectively, these results highlight the underappreciated functions of P2Y receptors in innate immune processes.

Keywords: P2YR; antiviral immunity; cytokine induction; innate immunity; nucleotide sensing.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. P2RY expression levels in various cell lines
  1. A, B

    Total RNA from HeLa, SKN‐BE2, A549, HEK293‐R1, Huh7.5, THP‐1, and BLaER1 cells and differentiated THP‐1 and BLaER1 cells (A) or differentiated BLaER1 cells untreated or treated with 250 U/ml IFN‐α for 8 h (B) were analyzed for the endogenous P2RY levels by RT–qPCR. The P2RY levels were normalized to GAPDH and the mean of three independent experiments shown as a heat map (A) or as individual values together with the mean and ****P < 0.0001, ***P < 0.001, **P < 0.01 (two‐way ANOVA with Šídák's multiple comparison test) (B).

Source data are available online for this figure.
Figure 2
Figure 2. Nucleotides induce changes to the transcriptome over time
Transcriptomic analysis of differentiated BLaER1 cells treated with 500 μM Ap4A, ATP, ADP, UTP, or UDP for 1, 2, 3, 4, or 6 h.

  1. Principal component analysis (PCA) on global gene expression profiles of individual biological replicates. Colors represent different treatment groups, and axes represent the first two components.

  2. The number of distinct transcripts that are significantly regulated (adjusted P‐value ≤ 0.01, log2 fold change ≥ 1) at given time points after treatment relative to untreated cells at the same time point.

  3. Gene set enrichment analysis of the regulated transcripts from (B) using Reactome database (Fisher's exact test unadjusted P‐value ≤ 0.001).

Figure 3
Figure 3. Adenine‐derived nucleotides induce interleukin gene expression
  1. A

    Heat map of the log2 fold change gene expression (treatment vs. mock within a time point) for transcripts within the interleukin signaling Reactome term from Fig 2C.

  2. B, C

    Differentiated BLaER1 cells treated with 500 μM Ap4A, ATP, ADP, UTP, or UDP, 0.1 ng/ml LPS or 200 U/ml IFN‐α for the indicated time points (B) or various concentrations of Ap4A, ATP, ADP, UTP, or UDP for 2 h (C) and the levels of the indicated cytokines analyzed by RT–qPCR. The cytokine levels were normalized to GAPDH and the mean ± SD of three biological replicates shown. The data presented are a representative of two independent experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 (two‐way ANOVA with Dunnett's multiple comparison test).

Source data are available online for this figure.
Figure 4
Figure 4. Adenine‐derived nucleotides induce interleukin release

  1. Differentiated BLaER1 cells treated with 500 μM Ap4A, ATP, ADP, UTP, or UDP for 2, 4, 6, or 10 h and analyzed for cytokine release by cytometric bead array. The graph shows the cytokine levels from three biological replicates together with mean ± SD. *P < 0.0001, § P < 0.001, $ P < 0.01, # P < 0.05 (two‐way ANOVA with Dunnett's multiple comparison test, comparison to mock).

  2. PBMCs isolated from six donors treated with 500 μM Ap4A or 10 ng/ml LPS for 8 h with protein secretion blocked after the initial 4 h. The cells were stained for viability using a fluorescent cell permeability dye and intracellular IL‐8 and surface CD14 (monocytes) using specific fluorescently coupled antibodies and analyzed by flow cytometry. The percentage of IL‐8+ CD14+ cells of the total CD14+ cells is shown for each donor together with the mean ± SD and P‐value (one‐way repeated measures ANOVA with Dunnett's multiple comparison test).

Figure 5
Figure 5. P2YRs do not activate NF‐κB signaling

  1. Differentiated BLaER1 WT and TAK1, IKKB, RELA, NIK, IKKA, or RELB KO cells treated with 500 μM Ap4A, ATP, ADP, UTP, or UDP for 2 h and analyzed for IL8 levels using RT–qPCR. The IL8 levels were normalized to GAPDH and the mean ± SD of three biological replicates shown. The data presented are a representative of two independent experiments.

  2. Differentiated BLaER1 cells treated with 500 μM ATP for the indicated times followed by western blotting against IκBα, phosphorylated and total p65 (RELA) and β‐actin.

Source data are available online for this figure.
Figure 6
Figure 6. P2YRs activate MAPK‐AP‐1 signaling
  1. A, B

    Heat maps of the log2 fold change gene expression (treatment vs. mock within a time point) for transcripts within the NGF transcription factor (A) and RAF‐independent MAPK1‐3 signaling (B) Reactome terms from Fig 2C.

  2. C

    HEK293‐R1 cells transfected with individual P2YRs together with AP‐1 promoter Firefly luciferase and EF‐1α promoter Renilla reporter plasmids and then treated with 1 μg/ml doxycycline for 8 h followed by 500 μM Ap4A, ATP, ADP, UTP, or UDP for 16 h. The graph shows the Firefly/Renilla signal from three biological replicates together with mean ± SD. *P < 0.0001, § P < 0.001, $ P < 0.01, # P < 0.05 (two‐way ANOVA with Dunnett's multiple comparison test, comparison to mock). The data presented are a representative of three independent experiments.

  3. D

    Differentiated BLaER1 cells treated with 500 μM ATP for the indicated times followed by western blotting against phosphorylated and total ERK1/2, p38 and SAPK/JNK and β‐actin.

Source data are available online for this figure.
Figure 7
Figure 7. P2Y11 is responsible for the cytokine expression profile in macrophages
  1. A–C

    Differentiated BLaER1 nontargeted control (NTC) and P2RY KO cells treated with 500 μM Ap4A, ATP, or ADP for 2 h (A + C) or differentiated BLaER1 cell pretreated with 100 μM P2Y11 antagonist (NF157) for 30 min, then treated with 10 μM Ap4A for 2 h (B), and analyzed for IL8 (A + B) and TNFA (C) levels using RT–qPCR. The IL8 and TNFA levels were normalized to GAPDH and the mean ± SD of three biological replicates shown. ****P < 0.0001, ***P < 0.001, *P < 0.05 (two‐way ANOVA with Dunnett's (A + C) or Šídák's (B) multiple comparison test). The data presented are a representative of two (A + C) and three (B) independent experiments.

  2. D

    Differentiated BLaER1 NTC and P2RY11 KO cells treated with 500 μM ATP for 10 min followed by western blotting against phosphorylated and total ERK1/2 and p38 and β‐actin.

  3. E–H

    Transcriptomic analysis of differentiated BLaER1 NTC and P2RY11 knockout cells treated with 500 μM Ap4A, ATP, or ADP for 2 h. Principal component analysis (PCA) on global gene expression profiles of individual biological replicates. Colors represent different treatment groups, shapes represent the cell line, and axes represent the first two components (E). The number of distinct transcripts that are significantly regulated (adjusted P‐value ≤ 0.01, log2 fold change ≥ 1) in P2RY11 KO cells relative to NTC cells after treatment (F). Gene set enrichment analysis of the regulated transcripts from (F) using Reactome database (Fisher's exact test unadjusted P‐value ≤ 0.001) (G). Heat map of the log2 fold change gene expression (KO vs. NTC within a nucleotide treatment) for transcripts within the interleukin signaling, NGF transcription factor, and RAF‐independent MAPK1‐3 signaling terms from (G) (H).

Source data are available online for this figure.
Figure 8
Figure 8. P2YRs suppress SFV infection
  1. A, B

    Stable THP‐1 P2YR cells differentiated with 100 ng/ml PMA either with or without 1 μg/ml doxycycline for 24 h and then infected with SFV‐2SG‐mCherry (MOI 2) (A). THP‐1 cells differentiated with 100 ng/ml PMA for 24 h and then pretreated with 100 μM NF157 for 30 min prior to infection with SFV‐mCherry (MOI 2), RVFV‐Katushka (MOI 0.5), HSV‐1‐mCherry (MOI 1), or VSV‐GFP (MOI 0.5) (B). The fluorescent signal and cell confluence were tracked for 24 or 48 hpi using an IncuCyte S3 live imagining platform. The data presented are a representative of three independent experiments, and for each line diagram, the mean ± SD were calculated based on three (A) or four (B) biological replicates. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired two‐tailed t‐test at 15 hpi (A) or 20 hpi SFV‐mCherry, 20 hpi VSV‐GFP, 40 hpi HSV‐1‐mCherry, and 16 hpi RVFV‐RFP (B)).

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