Identification of a dendritic cell receptor that couples sensing of necrosis to immunity.

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair. In addition, antigens present in necrotic cells can sometimes provoke a specific immune response and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection. In the mouse, the CD8alpha+ subset of dendritic cells phagocytoses dead cell remnants and cross-primes CD8+ T cells against cell-associated antigens. Here we show that CD8alpha+ dendritic cells use CLEC9A (also known as DNGR-1), a recently-characterized C-type lectin, to recognize a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair the uptake of necrotic cell material by CD8+ dendritic cells, but specifically reduces cross-presentation of dead-cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue in its intracellular tail that allows the recruitment and activation of the tyrosine kinase SYK, which is also essential for cross-presentation of dead-cell-associated antigens. Thus, CLEC9A functions as a SYK-coupled C-type lectin receptor to mediate sensing of necrosis by the principal dendritic-cell subset involved in regulating cross-priming to cell-associated antigens.

Internalization of Dectin-1 terminates induction of inflammatory responses.

Dectin-1 is a pattern-recognition receptor recognizing beta-(1,3)-glucans found on fungal cell walls. Dectin-1 plays an important role in immunity to fungi by mediating phagocytic clearance of fungal particles and inducing transcription of innate response genes. We show here that the two processes are linked and that Dectin-1 signalling for inflammation is attenuated by phagocytosis. Blocking Dectin-1 ligand-dependent internalization using either actin polymerization or dynamin inhibitors, large non-phagocytosable beta-glucan particles or poorly phagocytic cells leads in all cases to enhanced and sustained activation of downstream signalling pathways and culminates in production of high levels of pro-inflammatory cytokines. These findings establish the importance of phagocytosis not only in the clearance of pathogens, but also in the modulation of pattern-recognition receptor signalling and strongly suggest that internalization is the first step to attenuation of Dectin-1-mediated pro-inflammatory responses.

Syk-dependent ERK activation regulates IL-2 and IL-10 production by DC stimulated with zymosan.

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL-2 and IL-10 response in DC and emphasises the importance of Syk-coupled pattern recognition receptors such as dectin-1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors.

A framework for computational and experimental methods: identifying dimerization residues in CCR chemokine receptors.

Solving relevant biological problems requires answering complex questions. Addressing such questions traditionally implied the design of time-consuming experimental procedures which most of the time are not accessible to average-sized laboratories. The current trend is to move towards a multidisciplinary approach integrating both theoretical knowledge and experimental work. This combination creates a powerful tool for shedding light on biological problems. To illustrate this concept, we present here a descriptive example of where computational methods were shown to be a key aspect in detecting crucial players in an important biological problem: the dimerization of chemokine receptors. Using evolutionary based sequence analysis in combination with structural predictions two CCR5 residues were selected as important for dimerization and further validated experimentally. The experimental validation of computational procedures demonstrated here provides a wealth of valuable information not obtainable by any of the individual approaches alone.

Identification of amino acid residues crucial for chemokine receptor dimerization.

Chemokines coordinate leukocyte trafficking by promoting oligomerization and signaling by G protein-coupled receptors; however, it is not known which amino acid residues of the receptors participate in this process. Bioinformatic analysis predicted that Ile52 in transmembrane region-1 (TM1) and Val150 in TM4 of the chemokine receptor CCR5 are key residues in the interaction surface between CCR5 molecules. Mutation of these residues generated nonfunctional receptors that could not dimerize or trigger signaling. In vitro and in vivo studies in human cell lines and primary T cells showed that synthetic peptides containing these residues blocked responses induced by the CCR5 ligand CCL5. Fluorescence resonance energy transfer showed the presence of preformed, ligand-stabilized chemokine receptor oligomers. This is the first description of the residues involved in chemokine receptor dimerization, and indicates a potential target for the modification of chemokine responses.

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor.

The identification of chemokine receptors as HIV-1 coreceptors has focused research on developing strategies to prevent HIV-1 infection. We generated CCR2-01, a CCR2 receptor-specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T-tropic (X4) HIV-1 strains. This effect is explained by the ability of CCR2-01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV-1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells.

Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responses.

The JAK/STAT (Janus kinase / signaling transducer and activator of transcription) signaling pathway is implicated in converting stationary epithelial cells to migratory cells. In mammals, migratory responses are activated by chemoattractant proteins, including chemokines. We found that by binding to seven-transmembrane G-protein-coupled receptors, chemokines activate the JAK/STAT pathway to trigger chemotactic responses. We show that chemokine-mediated JAK/STAT activation is critical for G-protein induction and for phospholipase C-beta dependent Ca(2+) flux; in addition, pharmacological inhibition of JAK or mutation of the JAK kinase domain causes defects in both responses. Furthermore, G alpha(i) association with the receptor is dependent on JAK activation, and the chemokine-mediated Ca(2+) flux that requires phospholipase C-beta activity takes place downstream of JAK kinases. The chemokines thus employ a mechanism that links heterologous signaling pathways--G proteins and tyrosine kinases--in a network that may be essential for mediating their pleiotropic responses.

Functional inactivation of CXC chemokine receptor 4-mediated responses through SOCS3 up-regulation.

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca(2+) flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Galpha(i) pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.