Objectives: Vasoactive intestinal peptide (VIP) is an immunoregulatory peptide present in periodontal tissues which is known to inhibit inflammatory immune responses in myeloid cells. Our objective was to investigate the effect of VIP on Toll-like receptor 2 (TLR2) and TLR4 expression in monocytes exposed to lipopolysaccharide (LPS) from P.gingivalis and E.coli and to investigate the role of the myeloid cell transcription factor PU.1 in this effect.
Methods: Human promonocytic THP1 cells (1x106 cells/ml) were differentiated to monocytes by addition of vitamin D3 (confirmed by increased CD14 expression), and exposed to LPS (P.gingivalis W50 LPS, E.coli 0111:B4 LPS, 100ng/ml) or LPS + 10-8M VIP. Controls included unstimulated cells and cells exposed to PMA (1μg/ml). Fluorescence activated cell sorting (FACS) analyses measured TLR2 and TLR4 expression 24h after stimulation with P.gingivalis or E.coli LPS, respectively. Nuclear translocation of PU.1 was investigated 90 min after stimulation using confocal laser scanning microscopy (CLSM). Expression of M-CSFR (a gene target of PU.1) on the surface of CD14high/FSChigh adherent THP1 cells was measured by FACS analyses, 48h post-stimulation.
Results: VIP inhibited TLR2- and TLR4-dominant adherent cells (macrophages) induced by 24h culture with P.gingivalis and E.coli LPS, respectively. Both P.gingivalis and E.coli LPS stimulated nuclear translocation of PU.1 in THP1 cells after 90 min, and this was comparable to the effect of PMA. In cells co-cultured with LPS+VIP, VIP inhibited nuclear translocation of PU.1, which, at 90 min, had a peri-nuclear localisation similar to unstimulated controls. VIP inhibited M-CSFR in the CD14high/FSChigh adherent THP1 cells.
Conclusion: VIP decreases monocyte differentiation to macrophages and overall TLR2 and TLR4 expression following exposure to P.gingivalis and E.coli LPS, respectively. VIP inhibits nuclear translocation of PU.1, and this may be important in regulating LPS-induced monocyte differentiation and induction of TLR.
Supported by a UK Department of Health Clinician Scientist award: DHCS/030G121/46.