Vasoactive intestinal peptide is required in the maintenance of immune regulatory competency of immune regulatory monocytes

Abstract

Dysfunction of the immune regulatory system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Vasoactive intestinal peptide (VIP) has multiple bioactivities. This study aims to investigate the role of VIP in the maintenance of the immune regulatory capacity of monocytes (Mos). Human peripheral blood samples were collected from RA patients and healthy control (HC) subjects. Mos and CD14⁺ CD71^- CD73⁺ CD25⁺ regulatory Mos (RegMos) were isolated from the blood samples and characterized by flow cytometry. A rat RA model was developed to test the role of VIP in the maintenance of the immune regulatory function of Mos. The results showed that RegMos of HC subjects had immune suppressive functions. RegMos of RA patients expressed less interleukin (IL)-10 and showed an incompetent immune regulatory capacity. Serum levels of VIP were lower in RA patients, which were positively correlated with the expression of IL-10 in RegMos. In-vitro experiments showed that the IL-10 mRNA decayed spontaneously in RegMos, which could be prevented by the presence of VIP in the culture. VIP suppressed the effects of tristetraprolin (TTP) on inducing IL-10 mRNA decay in RegMos. Administration of VIP inhibited experimental RA in rats through restoring the IL-10 expression in RegMos. RegMos have immune suppressive functions. VIP is required in maintaining IL-10 expression in RegMos. The data suggest that VIP has translational potential in the treatment of immune disorders such as RA.

Keywords: immune regulation; inflammation; interleukin-10; monocytes; rheumatoid arthritis.

Figure 1

Assessment of peripheral regulatory monocytes (RegMos) of human subjects. Peripheral blood mononuclear cells (PBMCs) were prepared from blood samples of healthy control (HC) subjects (n = 20) and rheumatoid arthritis (RA) patients (n = 20) and analyzed by flow cytometry. (a) Gated dot‐plots show frequency of CD14⁺CD71^– monocytes. (b) Bars show summarized frequency of CD14⁺CD71^– monocytes. (c) Gated dot‐plots show frequency of RegMos. (d) Bars show summarized frequency of RegMos. The data of bars are presented as mean ± standard error of the mean (s.e.m.). Each dot inside bars presents data from an independent experiment.

Figure 2

Rheumatoid arthritis (RA) regulatory monocytes (RegMos) show incompetent immune suppressive function. RegMos were prepared from peripheral blood mononuclear cells (PBMCs) of RA patients (n = 20) and healthy control (HC) subjects (n = 20). Effector CD4⁺CD25^– T cells (T_effs) were prepared from PBMCs of HC subjects and labeled with carboxyfluorescein succinimidyl ester (CFSE). RegMos and T_effs were co‐cultured at a ratio of 1 : 5 with the treatment denoted above each subpanel of (a) for 3 days. (a) Gated histograms show frequency of proliferating T_effs. (b) Bars indicate summarized frequency of proliferating T_effs. AS101: 2·5 μg/ml AS101 in the culture. Data of bars are presented as mean ± standard error of the mean (s.e.m.). Each dot inside bars presents data from an independent experiment.

Figure 3

Expression of interleukin (IL)‐10 in regulatory monocytes (RegMos) is correlated with serum vasoactive intestinal peptide (VIP). RegMos were isolated from peripheral blood mononuclear cells (PBMCs) collected from healthy control (HC) subjects (n = 20) and rheumatoid arthritis (RA) patients (n = 20). RNA was extracted from the RegMos and analyzed by reverse transcription–quantitative polymerase chain reaction (RT–qPCR) and Western blotting. (a) Bars indicate IL‐10 mRNA levels in RegMos. B, proteins were extracted from the RegMos, pooled and analyzed by Western blotting. The immunoblots indicate protein levels of IL‐10 in RegMos (representing three independent experiments). (c) Bars indicate serum levels of VIP. (d) Scatter‐plots show a positive correlation between serum VIP and IL‐10 mRNA levels in RA RegMos. Data of bars are presented as mean ± standard error of the mean (s.e.m.). Each dot inside bars presents data from an independent experiment.

Figure 4

Vasoactive intestinal peptide (VIP) blocks interleukin (IL)‐10 mRNA decay in monocytes (Mos). (a,b) CD14⁺CD16^– Mos were isolated from blood samples collected from healthy control (HC) subjects and exposed to lipopolysaccharide (LPS) (100 ng/ml) in the culture for 48 h. The cells were analyzed by reverse transcription–quantitative polymerase chain reaction (RT–qPCR) and Western blotting. Bars indicate IL‐10 mRNA levels (a); immunoblots indicate IL‐10 protein levels (b). (c) Mos were stimulated with LPS in the culture for 48 h. The cells were washed with fresh medium and recultured with fresh medium in the presence or absence of VIP (10 ng/ml). The cells were collected at indicated time‐points (on the x‐axis) and analyzed by RT–qPCR. Curves indicate IL‐10 mRNA in Mos. Data of bars are presented as mean ± standard error of the mean (s.e.m.). Each dot inside bars presents data from an independent experiment.

Figure 5

Vasoactive intestinal peptide (VIP) prevents tristetraprolin (TTP) from binding interleukin (IL)‐10 mRNA in monocytes (Mos). CD14⁺CD16^– Mos were prepared from blood samples of healthy control (HC) subjects. (a–c) Mos were stimulated with lipopolysaccharide (LPS) for 30 min in the culture, washed and cultured with fresh medium for 48 h. The cells were analyzed by reverse transcription–quantitative polymerase chain reaction (RT–qPCR) and Western blotting. Bars indicate levels of tristetraprolin (TTP) mRNA (a); immunoblots indicate TTP protein levels (b). (c) A portion of the Mos were further treated with the procedures denoted on the x‐axis. Bars indicate IL‐10 mRNA levels in Mos. (d) TTP RNAi results in Mos. (e) Mos were treated with LPS and VIP in the culture for 48 h. The cells were analyzed by immunoprecipitation (IP). Immunoblots indicate a complex of VIP and TTP in Mos. (f) Mos were exposed to LPS or LPS/VIP in the culture for 48 h. The cells were analyzed by RT–qPCR and Western blotting. Bars indicate IL‐10 mRNA (f) and immunoblots indicate TTP protein in a complex in Mos (the β‐actin is from cytosolic extracts serving as a reference). Data of bars are presented as mean ± standard error of the mean (s.e.m.). Each dot inside bars presents data from an independent experiment.

Figure 6

Vasoactive intestinal peptide (VIP) suppresses experimental arthritis through restoring interleukin (IL)‐10 expression in regulatory monocytes (RegMos). Rats (six per group) were treated with procedures denoted with each histology image. (a) Representative images show rat articular joint histology. (b) Bars show serum levels of tumor necrosis factor (TNF) (b), interferon (IFN)‐γ (c), IL‐17 (d) and IL‐10 (e). (f) Bars show frequency of RegMos in the rat spleen. (g) Bars show IL‐10 mRNA levels in isolated RegMos. (h) Immunoblots show IL‐10 protein levels in isolated RegMos. Data of bars are presented as mean ± standard error of the mean (s.e.m.). Each dot inside bars presents data from an independent experiment. *P < 0·01, compared with group (a). VIP: rats were peritoneally injected with VIP (0·166 mg/kg/day in 0·1 ml saline) 1 day before each administration of type II collagen (CII). αCD14 antibody [or isotype immunoglobulin (IgG)]: anti‐CD14 antibody (or isotype IgG; control); rats were peritoneally injected with αCD14 antibody on day 0 (0·5 mg/rat); n.d. = not detectable.