Soluble mannose receptor induces proinflammatory macrophage activation and metaflammation.

Proinflammatory activation of macrophages in metabolic tissues is critically important in the induction of obesity-induced metaflammation. Here, we demonstrate that the soluble mannose receptor (sMR) plays a direct functional role in both macrophage activation and metaflammation. We show that sMR binds CD45 on macrophages and inhibits its phosphatase activity, leading to an Src/Akt/NF-κB-mediated cellular reprogramming toward an inflammatory phenotype both in vitro and in vivo. Remarkably, increased serum sMR levels were observed in obese mice and humans and directly correlated with body weight. Importantly, enhanced sMR levels increase serum proinflammatory cytokines, activate tissue macrophages, and promote insulin resistance. Altogether, our results reveal sMR as regulator of proinflammatory macrophage activation, which could constitute a therapeutic target for metaflammation and other hyperinflammatory diseases.

Monitoring Intracellular Routing of Internalized Antigens by Immunofluorescence Microscopy.

Antigen-presenting cells (APCs), especially macrophages and dendritic cells (DCs), are important for the induction of an adaptive immune response through their phagocytic capacity. APCs internalize extracellular antigens and, dependent on their intracellular localization, antigen-derived peptides are presented on MHC I or MHC II molecules. In context of antigen presentation and T cell activation tracking of internalized antigens is of high interest. In this article, we provide an immunofluorescence protocol and illustrate the analysis of intracellular routing of internalized antigens using the example of the model-antigen ovalbumin (OVA) in bone marrow-derived dendritic cells (BM-DCs). This protocol describes a procedure to stain such cells with an antibody against EEA-1, a marker for early endosomes, which can be easily adapted to other endosome markers, antigen-presenting cells, or antigens.

Current Concepts of Antigen Cross-Presentation.

Dendritic cells have the ability to efficiently present internalized antigens on major histocompatibility complex (MHC) I molecules. This process is termed cross-presentation and is important role in the generation of an immune response against viruses and tumors, after vaccinations or in the induction of immune tolerance. The molecular mechanisms enabling cross-presentation have been topic of intense debate since many years. However, a clear view on these mechanisms remains difficult, partially due to important remaining questions, controversial results and discussions. Here, we give an overview of the current concepts of antigen cross-presentation and focus on a description of the major cross-presentation pathways, the role of retarded antigen degradation for efficient cross-presentation, the dislocation of antigens from endosomal compartment into the cytosol, the reverse transport of proteasome-derived peptides for loading on MHC I and the translocation of the cross-presentation machinery from the ER to endosomes. We try to highlight recent advances, discuss some of the controversial data and point out some of the major open questions in the field.

Hyperosmolarity impedes the cross-priming competence of dendritic cells in a TRIF-dependent manner.

Tissue osmolarity varies among different organs and can be considerably increased under pathologic conditions. Hyperosmolarity has been associated with altered stimulatory properties of immune cells, especially macrophages and dendritic cells. We have recently reported that dendritic cells upon exposure to hypertonic stimuli shift their profile towards a macrophage-M2-like phenotype, resulting in attenuated local alloreactivity during acute kidney graft rejection. Here, we examined how hyperosmotic microenvironment affects the cross-priming capacity of dendritic cells. Using ovalbumin as model antigen, we showed that exposure of dendritic cells to hyperosmolarity strongly inhibits activation of antigen-specific T cells despite enhancement of antigen uptake, processing and presentation. We identified TRIF as key mediator of this phenomenon. Moreover, we detected a hyperosmolarity-triggered, TRIF-dependent clustering of MHCI loaded with the ovalbumin-derived epitope, but not of overall MHCI molecules, providing a possible explanation for a reduced T cell activation. Our findings identify dendritic cells as important players in hyperosmolarity-mediated immune imbalance and provide evidence for a novel pathway of inhibition of antigen specific CD8+ T cell response in a hypertonic micromilieu.

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.

Mannose receptor polyubiquitination regulates endosomal recruitment of p97 and cytosolic antigen translocation for cross-presentation.

The molecular mechanisms regulating noncanonical protein transport across cellular membranes are poorly understood. Cross-presentation of exogenous antigens on MHC I molecules by dendritic cells (DCs) generally requires antigen translocation from the endosomal compartment into the cytosol for proteasomal degradation. In this study, we demonstrate that such translocation is controlled by the endocytic receptor and regulated by ubiquitination. Antigens internalized by the mannose receptor (MR), an endocytic receptor that targets its ligands specifically toward cross-presentation, were translocated into the cytosol only after attachment of a lysin48-linked polyubiquitin chain to the cytosolic region of the MR. Furthermore, we identify TSG101 as a central regulator of MR ubiquitination and antigen translocation. Importantly, we demonstrate that MR polyubiquitination mediates the recruitment of p97, a member of the ER-associated degradation machinery that provides the driving force for antigen translocation, toward the endosomal membrane, proving the central role of the endocytic receptor and its ubiquitination in antigen translocation.