Friederike Meyer-Wentrup

Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors

Recognition of microbial components by germ‐line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β‐glucan receptor, Dectin‐1, which collaborates through an undefined mechanism with Toll‐like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin‐1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP‐1α and MIP‐2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk‐ and MyD88‐coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

Dectin‐1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages

The β‐glucan receptor dectin‐1 and Toll‐like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin‐1 amplifies TLR2‐dependent induction of cytokines in mouse models. In the present study we hypothesized that dectin‐1 has potent synergistic effects with both TLR2 and TLR4 in human PBMCs and macrophages. Human PBMCs and monocyte‐derived macrophages were stimulated with curdlan, a linear β‐1,3‐glucan‐polymer derived from Alcaligenes faecalis with specific ligand affinity for dectin‐1, in combination with the synthetic TLR2 ligand Pam3Cys and the ultrapure TLR4 ligand LPS. TNF‐α and IL‐10 production was measured in the supernatants with ELISA. Curdlan is a specific dectin‐1 ligand without TLR2‐ or TLR4‐stimulating properties. Human primary monocytes and macrophages express dectin‐1 on the cell membrane. Stimulation of human PBMCs with curdlan in combination with Pam3Cys or LPS leads to synergistic increase in TNF‐α production that was inhibited by GE2, a neutralizing dectin‐1 antibody. Dectin‐1‐dependent synergy between curdlan and TLR agonists was also apparent in human monocyte‐derived macrophages. Conclusively, dectin‐1 synergizes with both TLR2 and TLR4 pathways for the production of TNF‐α in human primary PBMCs and in monocyte‐derived macrophages.

Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-alpha production.

C-type lectin receptors (CLRs) fulfill multiple functions within the immune system by recognition of carbohydrate moieties on foreign or (altered) self-structures. CLRs on myeloid dendritic cells (DCs) have been well characterized as pattern-recognition receptors (PRRs) combining ligand internalization with complex signaling events. Much less is known about CLR expression and function in human plasmacytoid DCs (pDCs), the major type I interferon (IFN) producers. In this study, we demonstrate that, next to the CLR BDCA-2, human pDCs express DC immunoreceptor (DCIR), a CLR with putative immune-inhibitory function, but not dectin-1, mannose receptor, or DC-specific ICAM-3-grabbing nonintegrin. DCIR surface levels are reduced on pDC maturation after TLR9 triggering. Interestingly, DCIR triggering inhibits TLR9-induced IFN-alpha production while leaving up-regulation of costimulatory molecule expression unaffected. Furthermore, DCIR is readily internalized into pDCs after receptor triggering. We show that DCIR internalization is clathrin-dependent because it can be inhibited by hypertonic shock and dominant-negative dynamin. Importantly, antigens targeted to pDCs via DCIR are presented to T cells. These findings indicate that targeting DCIR on pDCs not only results in efficient antigen presentation but also affects TLR9-induced IFN-alpha production. Collectively, the data show that targeting of DCIR can modulate human pDC function and may be applied in disease prevention and treatment.

DCIR is endocytosed into human dendritic cells and inhibits TLR8-mediated cytokine production

C‐type lectin receptors (CLRs) expressed on APCs play a pivotal role in the immune system as pattern‐recognition and antigen‐uptake receptors. In addition, they may signal directly, leading to cytokine production and immune modulation. To this end, some CLRs, like dectin‐1 and dendritic cell immunoreceptor (DCIR), contain intracellular ITIMs or ITAMs. In this study, we explored expression and function of the ITIM‐containing CLR DCIR on professional APCs. DCIR is expressed on immature and mature monocyte‐derived DCs (moDC) but also on monocytes, macrophages, B cells, and freshly isolated myeloid and plasmacytoid DCs. We show that endogenous DCIR is internalized efficiently into human moDC after triggering with DCIR‐specific mAb. DCIR internalization is clathrin‐dependent and leads to its localization in the endo‐/lysosomal compartment, including lysosome‐associated membrane protein‐1+ lysosomes. DCIR triggering affected neither TLR4‐ nor TLR8‐mediated CD80 and CD86 up‐regulation. Interestingly, it did inhibit TLR8‐mediated IL‐12 and TNF‐α production significantly, and TLR2‐, TLR3‐, or TLR4‐induced cytokine production was not affected. Collectively, the data presented characterize DCIR as an APC receptor that is endocytosed efficiently in a clathrin‐dependent manner and negatively affects TLR8‐mediated cytokine production. These data provide further support to the concept of CLR/TLR cross‐talk in modulating immune responses.

Dectin-1 Interaction with Tetraspanin CD37 Inhibits IL-6 Production

C-type lectins are pattern-recognition receptors important for pathogen binding and uptake by APCs. Evidence is accumulating that integration of incoming cellular signals in APCs is regulated by grouping of receptors and signaling molecules into organized membrane complexes, such as lipid rafts and tetraspanin microdomains. In this study, we demonstrate that C-type lectin dectin-1 functionally interacts with leukocyte-specific tetraspanin CD37. Dectin-1 and CD37 colocalize on the surface of human APCs. Importantly, macrophages of CD37-deficient (CD37−/−) mice express decreased dectin-1 membrane levels, due to increased dectin-1 internalization. Furthermore, transfection of CD37 into a macrophage cell line elevated endogenous dectin-1 surface expression. Although CD37 deficiency does not affect dectin-1-mediated phagocytosis, we observed a striking 10-fold increase of dectin-1-induced IL-6 production in CD37−/− macrophages compared with wild-type cells, despite reduced dectin-1 cell surface expression. Importantly, the observed increase in IL-6 production was specific for dectin-1, because signaling via other pattern-recognition receptors was unaffected in CD37−/− macrophages and because the dectin-1 ligand curdlan was used. Taken together, these findings show that tetraspanin CD37 is important for dectin-1 stabilization in APC membranes and controls dectin-1-mediated IL-6 production.

The inhibitory Fc gamma IIb receptor dampens TLR4-mediated immune responses and is selectively up-regulated on dendritic cells from rheumatoid arthritis patients with quiescent disease.

Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcγRs and TLRs is accepted, their precise involvement remains to be elucidated. FcγRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcγRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcγRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcγRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcγRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcγRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcγRIIb in the induction of these phenomena. This TLR4-FcγRIIb interaction was shown to dependent on the PI3K and Akt pathway.

Detection of Fungi by Mannose-based Recognition Receptors

Fungi characteristically display mannose groups on the surface of their cell wall. These mannose residues are crucial for cell wall integrity and fungal viability. They constitute Pathogen-Associated-Molecular-Patterns (PAMPs) that can be detected by specific recognition receptors expressed by mammalian antigen-presenting cells. Fungal mannosylation, the mammalian membrane receptors that have evolved to detect fungal mannose groups and the immune responses resulting from fungal recognition by these receptors are the topics of this chapter. It consists of four parts. First, we give an introduction into fungal mannosylation to lay the ground for understanding mannose-based fungal recognition receptors. In the second part of the chapter we describe common features of mannose receptors. We then focus on two membranous mannose-receptors for which fungal binding has been demonstrated, the dendritic cell specific ICAM-3 grabbing non-integrin receptor (DC-SIGN) and the classic Mannose Receptor (MR). Finally, we give an outlook into potential therapeutic applications by targeting DC-SIGN and MR to prevent and treat fungal infections