Adrenergic β2 receptor activation stimulates anti-inflammatory properties of dendritic cells in vitro

Vagal nerve efferent activation has been shown to ameliorate the course of many inflammatory disease states. This neuromodulatory effect has been suggested to rest on acetylcholine receptor (AChR) activation on tissue macrophages or dendritic cells (DCs). In more recent studies, vagal anti-inflammatory activity was shown involve adrenergic, splenic, pathways. Here we provide evidence that the adrenergic, rather than cholinergic, receptor activation on bone marrow derived DCs results in enhanced endocytosis uptake, enhanced IL-10 production but a decreased IL-6, IL-12p70 and IL-23 production. In antigen specific T cell stimulation assays, adrenergic β2 receptor activation on bone marrow DCs led to an enhanced potential to induce Foxp3 positive suppressive Treg cells. These effects were independent of IL10-R activation, TGFβ release, or retinoic acid (RA) secretion. Hence, adrenergic receptor β2 activation modulates DC function resulting in skewing towards anti-inflammatory T cell phenotypes

Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile

BACKGROUND: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients. METHODS: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array. RESULTS: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12. CONCLUSIONS: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profil

Incubation of vehicle (veh), epinephrine (epine), and salbutamol (sal) of immature Ovalbumin loaded BMDC in a T cell proliferation assay.

<p>Cells were incubated during LPS maturation o/n. The cells were washed and freshly isolated naïve CD4 OT-II cells were stained with CFSE. Cells were co-cultured for 3 days and CFSE dilution was determined by flow cytometry. Overlays shown are representative of 3 independent experiments.</p

The effect of adrenergic agonists on BMDC potential to skew Tcells.

<p>Panel A. FACS plots of intracellular IFNγFand IL-4 in -OT-II T cells co-cultured with BMDC pre-treated with vehicle (Veh), epinephrine (Epine) or salbutamol (Sal) (all at 1 µM). Intracellular IFNγ FNr salarerer affected, while IL-4 production is increased after AR-βaffected, while IL-4 productB, histograms of % IFNγ positive T cells by FACS and by ELISA of day 4 of co-culture supernatant. Panel C, histograms of % IL-4 positive T cells by FACS and by ELISA. Data are expressed as % positive of CD4 gated T cells or as pg/ml and represent the mean ± SEM of three independent experiments representative of 4 experiments. ***p<0.001 (ANOVA).</p

Effect of adrenergic and cholinergic activation on cytokine production in maturing BMDC after 24

<p>Panel A, IL-10, IL-12p70 and IL-23 concentrations in ACh and Nicotine (Nic; 1 µM) pre-treated BMDC. Panel B, IL-10, IL-12p70 and IL-23 concentrations in epinephrine (epine), salbutamol (sal) and/or propranolol (prop) (all at 1 µM) pre-treated BMDC. The values are expressed in pg/ml and represent the mean ± SEM of three independent experiments representative of 5 experiments. * p<0.05, ** p<0.01, ***p<0.001 (ANOVA).</p

The relative mRNA expression levels of the AR-βe r<i>adrb1</i>) and AR-β an<i>adrb2</i>) in immature BMDC, and matured BMDC matured in the presence of epinephrine or salbutamol (Panel A).

<p>AR-β3 receptors are not expressed in BMDC. Panel B, the expression of the enzyme for catecholamine production, Tyrosine hydroxylase (TH) expression is not affected by epinephrine in matured BMDC. Panel C, blocking of endogenous TH activity by AMPT does not affect the potential of epinephrine or salbutamol to modulate IL-10 and IL-12p70 production. The data represent the mean ± SEM of three independent experiments representative of 5 experiments. * p<0.05, ** p<0.01, ***p<0.001 (ANOVA).</p

Adrenergic agonists exposure stimulates skewing of a Treg population.

<p>Panel A shows flow cytometry analyses of intracellular staining of Foxp3 positive T cells skewed by BMDC treated with indicated AR agonist. Panel B, histograms of Foxp3 positive T cells (left) and IL-10 (right) concentrations in supernatant representative of three independent experiments. Panel C, FACS analyses and, panel D histograms of T cells skewed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085086#pone-0085086-g007" target="_blank">fig. 7A</a> with added TGF-β, to stimulate Foxp3 differentiation. Panel E shows the concentrations of TGF-β analyses of intracellular staining of Foxp3 posVeh), epinephrine (Epine) and salbutamol (Sal). Data are expressed in % positive of CD4 gated T cells (left) or as pg/ml (right) and represent the mean ± SEM of three independent experiments representative of 4 experiments. *p<0.05 **p<0.01 (ANOVA).</p

The effects of ACh, epinephrine, salbutamol and nicotine on BMDC endocytosis of FITC-Dextran.

<p>Panel A shows a time course of endocytosis in treated BMDCs. CD11c gated immature BMDC endocytosis was assessed using flow cytometry. Results are expressed as mean fluorescence intensity and are the mean ± SEM of three experiments. Statistical significance was calculated using one way ANOVA. * p<0.05, ***p<0.001 versus control (37°C). Panel B: overlay histograms of BMDC stained for MHCII and co-stimulatory molecules CD40, CD80 and CD86 after 24 hours of LPS stimulation were determined by flow cytometry. Cell populations in the grey area indicate the specific isotype control. Both MHCII and co-stimulatory molecules are up-regulated by immature BMDC compared to LPS stimulated matured BMDC. Incubation with the various neurotransmitters before or during maturation has no effect on the levels of maturation markers. Numbers on each histogram indicate the geometric mean fluorescence intensity of cell population for each molecule from representative data of three experimental groups.</p