Cyclic adenosine monophosphate and IL-10 coordinately contribute to nTreg cell-mediated suppression of dendritic cell activation
Introduction
The imperfect mode of action of central tolerance in association with the promiscuity of the T cell receptor are indispensable for a preferably broad T cell receptor repertoire but provoke the imminent danger for auto-aggressive immune reactions. Therefore, immune responses have to be tightly regulated by a sophisticated interaction between activating and suppressing mechanisms. Thus, as a third function of the thymus naturally occurring CD4+CD25+FoxP3+ regulatory T cells (nTregs) develop in this organ as a clearly defined population of T cells. nTregs were originally shown to be potent inhibitors of conventional CD4+ and CD8+ T cells [1], [2]. Likewise, they are capable in suppressing the activation and effector functions of other immune cells like natural killer (NK) cells, NKT cells, B cells, and dendritic cells (DC) [3], [4], [5], [6], [7]. Furthermore, recent in vivo studies using two photon microscopy advocate the hypothesis that nTregs directly target DC in vivo, presumably by dampening the ability of DC to efficiently elicit the activation of these T cells [8], [9]. In tumor bearing mice, FoxP3+ Treg cells suppress DC mediated T cell activation by killing DC in an antigen specific and perforin dependent manner [10]. However, the mechanisms of nTreg-mediated suppression are still far from being understood in detail.
In vitro suppression by nTregs was found to be strictly cell-contact-dependent and independent of soluble factors. However, recent in vivo studies clearly vote for a contribution of the immune modulatory cytokines TGF-β and interleukin-10 (IL-10). Particularly, IL-10 has been demonstrated to strongly inhibit antigen presentation and cytokine production of DC [11], [12]. Among others IL-10 is secreted by dendritic cells, induced regulatory T cells (e.g. TR1 cells) and TH2 cells. Regarding nTregs it has been shown that IL-2 enables them to secrete IL-10 [13]. Thus, it is most likely that the suppressive properties of nTregs rely on different mechanisms depending on their site of action and the local conditions.
Cyclic AMP (cAMP) is known to be a general inhibitor of cell activation. With regard to DC it was shown that cAMP-elevators like Forskolin, Prostaglandin E2, cholera toxin and even dibutyryl-cAMP itself considerably counteract their TLR-mediated activation and effector function [14], [15], [16]. Moreover, cAMP not only suppresses DC but also induces a tolerogenic phenotype in these cells via the induction of IL-10 expression [17], [18], [19]. Collectively, a multitude of studies demonstrate the potency of cAMP and IL-10 to impair the accessory functions of DC.
In this study, we show that pre-activated nTregs (preTregs) combine cAMP and IL-10 to exert their inhibitory capacity on DC in a biphasic mechanism. Elevation of the cytosolic cAMP content of DC instantly abrogates the co-stimulatory potency of DC. Moreover, this cAMP-dependent inhibition is subsequently strengthened and further intensified by preTreg-derived IL-10, additionally ensuring the inhibition of cytokine production by DC. Therefore, preTregs use at least two immune suppressive molecules (cAMP, IL-10) in order to inhibit DC on multiple levels ultimately leading to an inert and suppressive DC phenotype.
Section snippets
Mice
C57Bl/6, BALB/c mice, and IL-10-deficient mice (BALB/c) were obtained from Charles River Laboratories and bred in a specific pathogen-free colony in the animal facility of the JGU Mainz.
Generation of bone marrow-derived dendritic cells (DCs)
Mouse immature DCs were generated from bone marrow of C57Bl/6 or BALB/c mice as described [20]. Briefly, bone marrow from 6 to 8 week old mice was cultured in Iscove’s medium supplemented with 5% fetal calve serum (FCS), 1% glutamate, 1% natrium pyruvate and GM-CSF (50 ng/ml). These cells were typically >85% CD11c+
nTregs strongly suppress DC leading to an immunosuppressive DC phenotype
Pre-activated nTregs (preTregs) strongly downregulate several co-stimulatory molecules (CD80, CD86, IL-6, IL-12) of DC while inhibitory molecules B7-H3 on DC and IL-10 in preTreg/DC-supernatants was up-regulated (Fig. 1 and Supplementary Fig. 1). This was in agreement with previously published data [5], [7], [23]. The same held true for the fact that the expression of MHCII and CD70 were additionally reduced strengthening the strongly impaired accessory properties of suppressed DC for CD4+ T
Acknowledgments
We would like to thank S. Gerecht and A. Hobel for excellent technical help. This study was funded by DFG, TR52 TPA1 (T.B. and E.S.), SFB 548 A6 (T.B. and E.S.), SFB 490 E6 (H.S.), KFO 183 (H.S. and M.P.R.), the Carl Zeiss Foundation (T.B.) and the “Mainzer Forschungsförderungsprogramm des Fachbereichs Medizin (MAIFOR (T.B.))”.
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