Ilaria Tassi

Macrophage colony-stimulating factor induces the proliferation and survival of macrophages via a pathway involving DAP12 and |[beta]|-catenin

Macrophage colony-stimulating factor (M-CSF) influences the proliferation and survival of mononuclear phagocytes through the receptor CSF-1R. The adaptor protein DAP12 is critical for the function of mononuclear phagocytes. DAP12-mutant mice and humans have defects in osteoclasts and microglia, as well as brain and bone abnormalities. Here we show DAP12 deficiency impaired the M-CSF-induced proliferation and survival of macrophages in vitro. DAP12-deficient mice had fewer microglia in defined central nervous system areas, and DAP12-deficient progenitors regenerated myeloid cells inefficiently after bone marrow transplantation. Signaling by M-CSF through CSF-1R induced the stabilization and nuclear translocation of β-catenin, which activated genes involved in the cell cycle. DAP12 was essential for phosphorylation and nuclear accumulation of β-catenin. Our results provide a mechanistic explanation for the many defects of DAP12-deficient mononuclear phagocytes.

Phospholipase C-γ2 Is a Critical Signaling Mediator for Murine NK Cell Activating Receptors

Phospholipase C-γ (PLCγ) is a key regulator of intracellular Ca2+ mobilization. Two isoforms of PLCγ have been identified, PLCγ1 and PLCγ2. Previously, in vitro studies indicated that activating NK cell receptors signal through both isoforms. However, PLCγ2 deficiency alone was sufficient to induce a substantial impairment of NK cell-mediated cytotoxicity in vitro. Why PLCγ2 is more important than PLCγ1 for NK cell activation and whether PLCγ2 is also critical for NK cell development, secretion of IFN-γ, and clearance of viral infections in vivo is not known. In this study, we report that PLCγ2 is the predominant isoform expressed in murine NK cells. PLCγ2 deficiency did not affect NK cell numbers in bone marrow and spleen, but acquisition of Ly49 receptors by NK cells was partially impaired. PLCγ2-deficient NK cells exhibited a dramatic impairment of cytolytic function and IFN-γ production upon ligation of activating receptors, whereas they did secrete IFN-γ in response to cytokines. Consequently, mice lacking PLCγ2 controlled murine CMV infection substantially less effectively than did wild-type animals, and this defect was most evident in the spleen, where viral clearance mostly depends on NK cell lytic function. These results demonstrate that PLCγ2 is crucial for development of the NK cell receptor repertoire and signaling of activating NK cell receptors, mediating optimal NK cell function in vivo.

Differential Requirements for Vav Proteins in DAP10- and ITAM-mediated NK Cell Cytotoxicity

Natural killer (NK) cells express multiple activating receptors that initiate signaling cascades through DAP10- or immunoreceptor tyrosine-based activation motif–containing adapters, including DAP12 and FcRγ. Among downstream signaling mediators, the guanine nucleotide exchange factor Vav1 carries out a key role in activation. However, whether Vav1 regulates only some or all NK cell–activating pathways is matter of debate. It is also possible that two other Vav family molecules, Vav2 and Vav3, are involved in NK cell activation. Here, we examine the relative contribution of each of these exchange factors to NK cell–mediated cytotoxicity using mice lacking one, two, or all three Vav proteins. We found that Vav1 deficiency is sufficient to disrupt DAP10-mediated cytotoxicity, whereas lack of Vav2 and Vav3 profoundly impairs FcRγ- and DAP12-mediated cytotoxicity. Our results provide evidence that these three Vav proteins function specifically in distinct pathways that trigger NK cell cytotoxicity.

Identification of soluble TREM-2 in the cerebrospinal fluid and its association with multiple sclerosis and CNS inflammation

Triggering receptor expressed on myeloid cells 2 (TREM-2) is a membrane-bound receptor expressed by microglia and macrophages. Engagement of TREM-2 on these cells has been reported to reduce inflammatory responses and, in microglial cells, to promote phagocytosis. TREM-2 function is critical within the CNS, as its genetic deficiency in humans causes neurodegeneration with myelin and axonal loss. Blockade of TREM-2 worsened the mouse model for multiple sclerosis. In the present study, a soluble form of TREM-2 protein has been identified by immunoprecipitation and by ELISA. Soluble TREM-2 protein (sTREM-2) was detected in human CSF, and was compared among subjects with relapsing-remitting multiple sclerosis (RR-MS; n = 52), primary progressive multiple sclerosis (PP-MS; n = 21), other inflammatory neurologic diseases (OIND; n = 19), and non-inflammatory neurologic diseases (NIND; n = 41). Compared to NIND subjects, CSF sTREM-2 levels were significantly higher in RR-MS (P = 0.004 by ANOVA) and PP-MS (P < 0.001) subjects, as well as in OIND (P < 0.001) subjects. In contrast, levels of sTREM-2 in blood did not differ among the groups. Furthermore, TREM-2 was detected on a subset of CSF monocytes by flow cytometry, and was also highly expressed on myelin-laden macrophages in eight active demyelinating lesions from four autopsied multiple sclerosis subjects. The elevated levels of sTREM-2 in CSF of multiple sclerosis patients may inhibit the anti-inflammatory function of the membrane-bound receptor suggesting sTREM-2 to be a possible target for future therapies.

The Cytotoxicity Receptor CRACC (CS-1) Recruits EAT-2 and Activates the PI3K and Phospholipase Cγ Signaling Pathways in Human NK Cells

The CD2-like receptor-activating cytotoxic cell (CRACC) is a cell surface receptor of the CD2 family that triggers NK cell-mediated cytotoxicity through an undefined signaling pathway. CRACC contains cytoplasmic tyrosine-based motifs, immunoreceptor tyrosine-based switch motifs, which resemble those found in the NK cell receptor 2B4. In 2B4, these motifs recruit the adaptor signaling lymphocytic activation molecule-associated protein (SAP), which initiates a signaling cascade mediating cytotoxicity. However, CRACC does not recruit SAP. In this study, we demonstrate that, upon activation, CRACC associates with a homolog of SAP, Ewing’s sarcoma’s/FLI1-activated transcript 2 (EAT-2), in human NK cells. We show that association of EAT-2 induces the phosphorylation of CRACC and that this process is partially reduced by a pharmacological inhibitor of Src kinases. We identify PLCγ1, PLCγ2, and PI3K as the major signaling mediators downstream of CRACC/EAT-2 implicated in NK cell-mediated cytotoxicity. Moreover, EAT-2 also associates with 2B4 predominantly in resting NK cells, whereas SAP preferentially binds 2B4 upon activation. These results outline a new signaling pathway that triggers CRACC-mediated cytotoxicity and modulates 2B4-mediated activation.

NK cell–activating receptors require PKC-θ for sustained signaling, transcriptional activation, and IFN-γ secretion

Natural killer (NK) cell sense virally infected cells and tumor cells through multiple cell surface receptors. Many NK cell-activating receptors signal through immunoreceptor tyrosine-based activation motif (ITAM)-containing adapters, which trigger both cytotoxicy and secretion of interferon-gamma (IFN-gamma). Within the ITAM pathway, distinct signaling intermediates are variably involved in cytotoxicity and/or IFN-gamma secretion. In this study, we have evaluated the role of protein kinase C- (PKC-) in NK-cell secretion of lytic mediators and IFN-gamma. We found that engagement of NK-cell receptors that signal through ITAMs results in prompt activation of PKC-. Analyses of NK cells from PKC--deficient mice indicated that PKC- is absolutely required for ITAM-mediated IFN-gamma secretion, whereas it has no marked influence on the release of cytolytic mediators. Moreover, we found that PKC- deficiency preferentially impairs sustained extracellular-regulated kinase signaling as well as activation of c-Jun N-terminal kinase and the transcription factors AP-1 and NFAT but does not affect activation of NF-kappaB. These results indicate that NK cell-activating receptors require PKC- to generate sustained intracellular signals that reach the nucleus and promote transcriptional activation, ultimately inducing IFN-gamma production.

Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse

Summary:  Natural killer (NK) cell cytotoxicity is mediated by multiple germ line‐encoded activating receptors that recognize specific ligands expressed by tumor cells and virally infected cells. These activating receptors are opposed by NK inhibitory receptors, which recognize major histocompatibility complex class I molecules on potential targets, raising the threshold for NK cell activation. Once an abnormal cell has been detected, NK cells are the sentinel source of cytolytic mediators, such as granzymes and perforins, as well as interferon‐γ, which can polarize the immune response to a T‐helper 1 cell type. Activation signals are transmitted by adhesion‐dependent pathways, immunoreceptor tyrosine‐based activation motif (ITAM)‐dependent pathways, DAP10 ITAM‐independent pathways, and by signaling through immunoreceptor tyrosine‐based switch motifs. These pathways activate downstream signaling partners to trigger NK cell cytotoxicity. Some of these downstream molecules are unique to the various pathways, and some of these molecules are shared. Because of the complexity of signals involved in NK cell–target cell interaction, the generation of mice with targeted mutations in signaling molecules involved in adhesion, activation, or inhibition is essential for a precise dissection of the mechanisms regulating NK cell effector functions. Here we review recent advances in the genetic analysis of the signaling pathways that mediate NK cell killing.

Requirement of phospholipase C‐γ2 (PLCγ2) for Dectin‐1‐induced antigen presentation and induction of TH1/TH17 polarization

DC recognize microbial components through an array of receptors known as PRR. PRR initiate intracellular signals, which engender DC with the capacity to stimulate T‐cell responses. Dectin‐1 is a PRR that recognizes β‐glucan, a major constituent of many fungis outer cell wall. Here we show that Dectin‐1 activates DC through phospholipase (PLC)γ2 signaling. PLCγ2‐deficient DC were unable to expand antigen‐specific T cells and induce TH1 and TH17 differentiation in response to β‐glucan. Mechanistically, PLCγ2‐deficiency impaired the capacity of DC to secrete polarizing cytokines following exposure to β‐glucan. Dectin‐1 required PLCγ2 to activate MAPK, AP‐1 and NF‐κB, which induce cytokine gene expression. Moreover, PLCγ2 controlled Dectin‐1‐mediated NFAT activation and induction of NFAT‐dependent genes such as IL‐2, cyclooxigenase‐2 and Egr transcription factors. We conclude that PLCγ2 is a crucial signaling mediator that modifies DC gene expression program to activate DC responses to β‐glucan‐containing pathogens.

DAP10 associates with Ly49 receptors but contributes minimally to their expression and function in vivo.

NK cells recognize target cells through activating receptors, many of which rely on the transmembrane adaptors DAP10, DAP12 and FcR‐γ to deliver intracellular signals. Because these adaptors initiate distinct signaling pathways, they dictate the type of response mediated by receptor engagement. DAP10, for example, primarily triggers cytotoxicity, whereas DAP12 induces both cytotoxicity and IFN‐γ secretion. In mice, NKG2D signals through both DAP10 and DAP12, which broadens and modulates the type of response engendered by encounter with ligand. Although initial studies indicated that Ly49H and Ly49D recruit only DAP12, a recent report suggested that they also associate with DAP10. We asked whether this association occurs and is functionally significant under physiologic conditions. Our data demonstrate that DAP10 does associate with Ly49H and Ly49D in primary NK cells. While this association contributes slightly to cell surface expression of both receptors, it has no significant impact on Ly49H‐mediated control of murine cytomegalovirus infection. Thus, while many activating NK‐cell receptors are promiscuous in terms of adaptor association, our data indicate that the functional consequences of such promiscuity may vary widely and may not be evident in all cases.

The Oncoprotein and Transcriptional Regulator Bcl-3 Governs Plasticity and Pathogenicity of Autoimmune T Cells

Bcl-3 is an atypical member of the IκB family that modulates transcription in the nucleus via association with p50 (NF-κB1) or p52 (NF-κB2) homodimers. Despite evidence attesting to the overall physiologic importance of Bcl-3, little is known about its cell-specific functions or mechanisms. Here we demonstrate a T-cell-intrinsic function of Bcl-3 in autoimmunity. Bcl-3-deficient T cells failed to induce disease in T cell transfer-induced colitis and experimental autoimmune encephalomyelitis. The protection against disease correlated with a decrease in Th1 cells that produced the cytokines IFN-γ and GM-CSF and an increase in Th17 cells. Although differentiation into Th1 cells was not impaired in the absence of Bcl-3, differentiated Th1 cells converted to less-pathogenic Th17-like cells, in part via mechanisms involving expression of the RORγt transcription factor. Thus, Bcl-3 constrained Th1 cell plasticity and promoted pathogenicity by blocking conversion to Th17-like cells, revealing a unique type of regulation that shapes adaptive immunity.