Toll-like receptor stimulation differentially regulates vasoactive intestinal peptide type 2 receptor in macrophages

Abstract

Vasoactive intestinal peptide (VIP) was originally isolated as a vasodilator intestinal peptide, then as a neuropeptide. In the immune system, VIP is described as an endogenous macrophage-deactivating factor. VIP exerts its immunological actions in a paracrine and/or autocrine manner, through specific receptors. However, very little is known about the molecular regulation of VIP type 2 receptor (VPAC(2)) in the immune system. We now report that different toll-like receptor (TLR) ligands selectively regulate the VPAC(2) receptor gene and show a gene repression system controlled by key protein kinase signalling cascades in macrophages. VPAC(2) gene expression is regulated by gram-positive (TLR2 ligands) and gram-negative bacteria wall constituents (TLR4 ligands). Moreover, VPAC(2) is tightly regulated: TLR2- or TLR2/6- but not TLR2/1-mediated mechanisms are responsible for the induction of VPAC(2). TLR stimulation by viral or bacterial nucleic acids did not modify the VPAC(2) mRNA levels. Remarkably, imiquimod--a synthetic TLR7 ligand--led to a potent up-regulation of VPAC(2) gene expression. TLR5 stimulation by flagellin present in gram-positive and gram-negative bacteria did not affect VPAC(2) mRNA. The p38 mitogen-activated protein kinase (MAPK) activity accounted for the TLR4-mediated induction of VPAC(2) gene expression. Surprisingly, our data strongly suggest for the first time a tightly repressed control of VPAC(2) mRNA induction by elements downstream of MAPK kinase 1/2, PI3K/Akt, and particularly Jun-NH(2)-terminal kinase signalling pathways.

Figure 1

Differential TLRs regulation of VPAC₂ mRNA in macrophages. (A) Raw 264.7 cells were seeded into 12-well plates in a final volume of 2 ml. At 70–80% confluence (750,000 cells/well), cells were stimulated with LPS (1 μg/ml), CpG⁺ (1 μg/ml, CpG-DNA 1668: 5′-TCCATGACGTTCCTGATGCT-3′), Poly (I: C) acid (50 μg/ml), LTA (10 μg/ml), Pam₃ CSK4 (300 ng/ml), FSL-1 (1 μg/ml), PGN (10 μg/ml), imiquimod (10 μg/ml) or ssRNA40 (0.25 μg/ml). Twenty-four hours, RNA was extracted and qPCR of VPAC₂ was carried out. Results are the mean ± S.D. of five independent experiments performed in triplicate. (Inset) Samples from qPCR samples on 1.7% agarose gel electrophoresis. Lanes showed the DNA molecular size markers (M), reaction performed in the absence of RT (–) and presence (+) of RT from resting Raw 264.7 RNA. (B) Female C57BL6/JIco (n= 20) peritoneal macrophages were isolated and pooled after i.p. injection of thioglycolate medium at day 5 as described in the ‘Materials and methods’ section. For qPCR determinations of VPAC₂ mRNA levels, cDNA (100 ng) was amplified as described in the ‘Materials and methods’ section. Quantitative PCR results were obtained using the ΔΔCt method [25]. The induction of mRNA was calculated as 2^−ΔΔCt (normalized for HPRT as housekeeping gene). For statistical evaluation, Mann-Whitney rank sum tests were performed. Asterisks indicate statistical significance (*, P < 0.05 versus control; **, P < 0.01 versus control; ***, P < 0.001 versus control).

Figure 2

IL-6 production after TLRs stimulation in macrophages. (A) Raw 264.7 cells were seeded into 12-well plates in a final volume of 2 ml. At 70–80% confluence, cells (750,000 cells/well) were stimulated with LPS (1 μg/ml), CpG⁺ (1 μg/ml, CpG-DNA 1668: 5′-TCCATGACGTTCCTGATGCT-3′), Poly (I: C) acid (50 μg/ml), LTA (10 μg/ml), Pam₃ CSK4 (300 ng/ml), FSL-1 (1 μg/ml), PGN (10 μg/ml), imiquimod (10 μg/ml) or ssRNA40 (0.25 μg/ml). Twenty-four hours later, supernatants were harvested for IL-6 ELISA determination. (B) mRNA IL-6 stimulation after ssRNA treatment in Raw 264.7 cells. RNA was extracted for IL-6 gene expression study after ssRNA treatment as described in the ‘Materials and methods’ section. For qPCR determinations of IL-6 mRNA levels, cDNA (100 ng) was amplified as described in the ‘Materials and methods’ section. Quantitative PCR results were obtained using the ΔΔCt method [25]. The induction of mRNA was calculated as 2^−ΔΔCt. Results are the mean ± S.D. of five independent experiments performed in triplicate. (C) Female C57BL6/JIco (n= 20) peritoneal macrophages were isolated and pooled after intraperitoneal injection of 1 ml of 3% thioglycolate at day 5 as described above. Afterwards, non-adherent cells were removed and peritoneal macrophages were treated as indicated above and 24 hrs later, supernatants were harvested for IL-6 ELISA determination.

Figure 3

Effects of protein kinase inhibitors on TLR-mediated VPAC₂ regulation in Raw 264.7 macrophages. Raw 264.7 cells were grown and treated as above. Briefly, at 70–80% confluence, cells (750,000 cells/well) were stimulated for 24 hrs with LPS (1 μg/ml), LTA (10 μg/ml), PGN (10 μg/ml) or imiquimod (10 μg/ml) after 30 min. pre-treatment in the presence or absence of 10 μM SB203580 (a p38 MAPK inhibitor), 25 μM SP600125 (a JNK inhibitor), 50 μM PD98059 (a MEK1/2 inhibitor) and 10 μM LY294002 (a PI3K/Akt inhibitor). Twenty-four hours later, RNA was extracted and qPCR of VPAC₂ was carried out according to the ‘Materials and methods’ section. Results are the mean ± S.D. of three independent experiments performed in triplicate. For statistical evaluation, Mann-Whitney rank sum tests were performed. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.0001 versus the experimental condition in the absence of inhibitor).

Figure 4

TLR-mediated control of VPAC_2. (A) VPAC₂ up-regulation by TLRs represents an additional mechanism to ensure the modulator capabilities of VIP while at the same time a down-regulation of TLRs occurs. (B) Distribution of putative binding sites for transcription factors involved in TLR signalling across the 3000 long upstream region of mouse VPAC₂ gene. Thicker lines correspond to more densely packed putative binding sites.