Research ArticleTGF-β-dependent induction of CD4+CD25+Foxp3+ Tregs by liver sinusoidal endothelial cells
Introduction
The microenvironment of the liver greatly favours immune tolerance [1], [2]. Indeed, antigens delivered via the portal vein [3] or allogeneic liver transplants [4], [5] do not provoke inflammatory immune responses. Hepatic tolerance is of importance in preventing and controlling inflammatory or autoimmune diseases [1]; however, it may also contribute to the frequent occurrence of persistent hepatitis virus infections [6] or liver cancer [7], [8]. Beyond regulation of local immune responses within the liver, hepatic tolerance can also act systemically, indicated by the finding that the ectopic expression of a neuroantigen in the liver can prevent autoimmune neuroinflammation [9]. The systemic effect of hepatic tolerance can be explained by the capacity of the liver to generate antigen-specific CD4+ Foxp3+ regulatory T cells (Tregs) that have a profound ability to control immune responses [10], [11]. However, the mechanisms and cell types responsible for hepatic Treg generation have not been elucidated.
Liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs) are liver resident antigen-presenting cells constitutively expressing MHC II molecules and capable of stimulating CD4+ T cells [1], [2]. Therefore, these liver cell types are the main candidate cells facilitating Treg generation. The generation of Tregs in the periphery occurs through conversion of CD4+ Foxp3− non-Tregs into CD4+ Foxp3+ Tregs; this conversion is dependent on T cell stimulation in the presence of TGF-β [11]. TGF-β is produced as a pro-form, which is intracellularly processed by furin into latent TGF-β [12]. Latent TGF-β consists of latency-associated peptide (LAP), the N-terminal part of pro-TGF-β, non-covalently associated with mature TGF-β. LAP/TGF-β cannot bind TGF-β receptors; biological activity requires further processing, which is not well characterised [12], [13]. However, it was found that secreted LAP/TGF-β can be tethered to the outer cell membrane through the receptor glycoprotein-A repetitions predominant (GARP, also known as LRRC32) [14], [15]. Such membrane-bound LAP/TGF-β was shown to exhibit biological activity and induce a tolerogenic phenotype and Foxp3-expression in stimulated T cells [16].
In the present study, we demonstrate that LSECs are the major liver antigen-presenting cell type responsible for TGF-β dependent induction of CD4+ Foxp3+ Tregs. The particular Treg-inducing capacity of LSECs was related to their ability to secrete TGF-β and to tether exogenous LAP/TGF-β to their membrane through GARP. In vivo, antigen-specific Tregs induced by LSECs were potent suppressors of autoimmune inflammation in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) [17], which is induced in susceptible B10.PL mice by immunisation to MBP peptide and marked by ascending paralysis. Our findings indicate that Treg induction by LSECs is instrumental for the control of hepatic and systemic inflammatory immune responses.
Section snippets
Mice
B10.PL mice, C57BL/6 mice, tg4 mice [18], CRP-MBP mice [9], Foxp3gfp.KI mice [19], or hCD2-ΔkTβRII mice [20] were bred and kept in the animal facility of the University Medical Centre Hamburg-Eppendorf under specific pathogen-free conditions. F1 mice were generated by mating of Foxp3gfp.KI mice with tg4 mice or tg4 mice with CRP-MBP mice; hCD2-ΔkTβRII mice were backcrossed to tg4 mice. Mice were 6–12 weeks old at start of experiments. Animal experiments were approved by the institutional review
Results
To assess the capacity of antigen-presenting liver cells for Treg induction, we used primary liver DCs, LSECs, or KCs to stimulate CD4+Foxp3− non-Treg cells from the spleen of (tg4 × Foxp3gfp.KI) F1 mice. These mice feature MBP-specific CD4 T cells [18] and Foxp3 and green fluorescent protein (GFP) co-expressing Tregs (Supplementary Fig. 1). Non-Treg cells were sorted based on the absence of the Foxp3-linked expression of GFP [19]. The stimulation of non-Treg cells was performed in serum-free
Discussion
CD4+Foxp3+ Tregs are important suppressors of inflammation and autoimmunity. Tregs can be generated in the thymus, but also in the periphery through TGF-β dependent conversion from conventional CD4+ T cells [11], [23]. The Tregs generated in the thymus and those generated in the periphery feature distinct complementary T cell receptor repertoires [23]. Peripheral Treg generation is of particular importance for maintaining tolerance to antigens that are not represented in the thymus. We have
Financial support
Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 841; HE3532/2-1) and Wellcome Trust (grant 090175/Z/09/Z).
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Acknowledgements
We are grateful for excellent technical assistance by Marko Hilken, Agnes Malotta, and Christina Trabandt. Cell sorting was performed by the flow cytometry core facility of the University Medical Centre Hamburg-Eppendorf.
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