Probing the Immunoreceptor Tyrosine-Based Inhibition Motif Interaction Protein Partners with Proteomics.

Phosphorylation of tyrosine is the basic mode of protein function and signal transduction in organisms. This process is regulated by protein tyrosine kinases (PTKs) and protein tyrosinases (PTPs). Immunoreceptor tyrosine-based inhibition motif (ITIM) has been considered as regulating the PTP activity through the interaction with the partner proteins in the cell signal pathway. The ITIM sequences need to be phosphorylated first to active the downstream signaling proteins. To explore potential regulatory mechanisms, the ITIM sequences of two transmembrane immunoglobulin proteins, myelin P0 protein-related protein (PZR) and programmed death 1 (PD-1), were analyzed to investigate their interaction with proteins involved in regulatory pathways. We discovered that phosphorylated ITIM sequences can selectively interact with the tyrosine phosphatase SHP2. Specifically, PZR-N-ITIM (pY) may be critical in the interaction between the ITIM and SH2 domains of SHP2, while PD1-C-ITSM (pY) may play a key role in the interaction between the ITIM and SH2 domains of SHP2. Quite a few proteins were identified containing the SH2 domain, exhibiting phosphorylation-mediated interaction with PZR-ITIM. In this study, 14 proteins with SH2 structural domains were identified by GO analysis on 339 proteins associated to the affinity pull-down of PZR-N-ITIM (pY). Through the SH2 domains, these proteins may interact with PZR-ITIM in a phosphorylation-dependent manner.

Clinical significance of signal regulatory protein alpha and T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain expression in undifferentiated pleomorphic sarcoma.

PURPOSE: Undifferentiated pleomorphic sarcoma (UPS) is associated with poor prognosis. Recently, signal regulatory protein alpha (SIRPα), which is the immune checkpoint of macrophages, and T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), which is the immune checkpoint of T cells and natural killer cells, have been considered as potential targets for cancer immunotherapy. This study aimed to assess the value of SIRPα and TIGIT as prognostic factors of UPS. MATERIALS AND METHODS: The cBio Cancer Genomics Portal was used to analyze mRNA expression data of 50 UPS cases in the Cancer Genome Atlas. We retrieved 49 UPS cases and performed immunohistochemistry (IHC) to detect programmed death ligand 1 (PD-L1), SIRPα, CD68, CD163, TIGIT, CD155, and CD8. RESULTS: SIRPα was positively associated with CD163 (Pearson's r = 0.51, p = 0.0002) as per open access data and IHC of the cohort (p = 0.002), which revealed that SIRPα-positive macrophage infiltration was higher in UPS cells with ≥ 1% PD-L1 expression than that in UPS cells with < 1% PD-L1 expression (p = 0.047). TIGIT was positively correlated with PD-L1 (r = 0.54, p < 0.0001) and CD8A (r = 0.98, p < 0.0001). In 35 of 49 cases, IHC revealed high levels of TIGIT expression on tumor cells. Furthermore, TIGIT expression on tumor cells was negatively correlated with CD155-positive (p = 0.0144) and CD8-positive (p = 0.0487) cell infiltration. Survival analysis showed that the high degree of SIRPα-positive macrophage infiltration was associated with poor overall survival and metastasis (p < 0.0001, p = 0.0006, respectively). CONCLUSION: SIRPα-positive macrophages infiltrated UPS cells, which predicted poor prognosis. High TIGIT expression on tumor cells was associated with decreased levels of tumor-infiltrating macrophages in UPS.

The CEACAM1-derived peptide QLSN impairs collagen-induced human platelet activation through glycoprotein VI.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates collagen-mediated platelet activation through its cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). However, the function of CEACAM1's extracellular cleavage fragments is currently unknown. In the present study, we used mass spectrometry (MS) to identify 9 cleavage fragments shed by matrix metallopeptidase 12 (MMP-12), and then we synthesized peptides with sequences corresponding to the fragments. QLSNGNRTLT (QLSN), a peptide from the A1-domain of CEACAM1, significantly attenuated collagen-induced platelet aggregation. QLSN also attenuated platelet static adhesion to collagen. Additionally, QLSN reduced human platelet secretion and integrin αIIbß3 activation in response to glycoprotein VI (GPVI)-selective agonist, convulxin. Correspondingly, QLSN treatment significantly decreased convulxin-mediated phosphorylation of Src, protein kinase B (Akt), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) in human platelets. These data indicate that the CEACAM1-derived peptide QLSN inhibits GPVI-mediated human platelet activation. QLSN could potentially be developed as a novel antiplatelet agent.

Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells.

PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248+ (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1+CD8+ T cells were identified in human peripheral blood and had impaired effector function. pPD-1+ T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1+ T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.

Signaling Cross-Talk between MHC Class II Molecular Conformers in Resting Murine B Cells.

In addition to functioning as a ligand to engage TCRs and drive TCR signaling, MHC class II molecules are signaling molecules that generate a number of signals within APCs, such as B lymphocytes. Moreover, MHC class II signaling is critical for B cell activation and development of a robust humoral immune response. Murine class II molecules exist in two distinct conformational states, based primarily on the differential pairing of transmembrane domain GxxxG dimerization motifs (i.e., M1- and M2-paired class II). This laboratory has previously reported that the binding of a multimerized form of an anti-class II mAb that selectively recognizes M1-paired I-Ak class II drives intracellular calcium signaling in resting murine B cells and that this signaling is dependent on both src and Syk protein tyrosine kinase activity. In contrast, multimerized forms of two different anti-I-Ak mAbs that bind both M1- and M2-paired class II fail to elicit a response. In this report, a flow cytometry-based calcium flux assay is used to demonstrate that coligation of M1- and M2-paired I-Ak class II results in the active and selective inhibition of M1-paired I-Ak class II B cell calcium signaling by M2-paired class II molecules. Because M1- and M2-paired class II can be loaded with different sets of peptides derived from Ags acquired by distinct pathways of endocytosis, these findings suggest an MHC class II signaling-based mechanism by which CD4 T cells of differing specificities can either enhance or suppress B cell activation.

Molecular mechanisms of immunoreceptors in platelets.

BACKGROUND: The main role of platelets is to control haemostasis when there is a blood vessel injury in order to minimise blood loss at the injury site. Under normal circumstances, platelets flow freely within blood vessels as the endothelial cells provide a non-adhesion surface. Naturally, bioactive mediators are released from endothelial cells to prevent and control platelet activation. However, when the vascular endothelium is ruptured, the local concentration of nitric oxide and prostaglandin is diminished and receptors containing a sequence of amino acids known as, immunoreceptor tyrosine-based inhibition motifs (ITIMs), serve as natural inhibitors within platelets. The main role of ITIMs is to decrease immunoreceptor tyrosine-based activation motif (ITAM) signalling in platelets; however, some studies have revealed their novel role in integrin αIIbß3 activation. This review highlights the main structural and functional features of immunoreceptors in platelets.

CD72 is a Negative Regulator of B Cell Responses to Nuclear Lupus Self-antigens and Development of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease characterized by production of autoantibodies to various nuclear antigens and overexpression of genes regulated by IFN-I called IFN signature. Genetic studies on SLE patients and mutational analyses of mouse models demonstrate crucial roles of nucleic acid (NA) sensors in development of SLE. Although NA sensors are involved in induction of anti-microbial immune responses by recognizing microbial NAs, recognition of self NAs by NA sensors induces production of autoantibodies to NAs in B cells and production of IFN-I in plasmacytoid dendritic cells. Among various NA sensors, the endosomal RNA sensor TLR7 plays an essential role in development of SLE at least in mouse models. CD72 is an inhibitory B cell co-receptor containing an immunoreceptor tyrosine-based inhibition motif (ITIM) in the cytoplasmic region and a C-type lectin like-domain (CTLD) in the extracellular region. CD72 is known to regulate development of SLE because CD72 polymorphisms associate with SLE in both human and mice and CD72−/− mice develop relatively severe lupus-like disease. CD72 specifically recognizes the RNA-containing endogenous TLR7 ligand Sm/RNP by its extracellular CTLD, and inhibits B cell responses to Sm/RNP by ITIM-mediated signal inhibition. These findings indicate that CD72 inhibits development of SLE by suppressing TLR7-dependent B cell response to self NAs. CD72 is thus involved in discrimination of self-NAs from microbial NAs by specifically suppressing autoimmune responses to self-NAs.

Immunoreceptor tyrosine-based inhibitory motif-dependent functions of an MHC class I-specific NK cell receptor.

Natural killer (NK) cells express MHC class I (MHC-I)-specific receptors, such as Ly49A, that inhibit killing of cells expressing self-MHC-I. Self-MHC-I also "licenses" NK cells to become responsive to activating stimuli and regulates the surface level of NK-cell inhibitory receptors. However, the mechanisms of action resulting from these interactions of the Ly49s with their MHC-I ligands, particularly in vivo, have been controversial. Definitive studies could be derived from mice with targeted mutations in inhibitory Ly49s, but there are inherent challenges in specifically altering a single gene within a multigene family. Herein, we generated a knock-in mouse with a targeted mutation in the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Ly49A that abolished the inhibitory function of Ly49A in cytotoxicity assays. This mutant Ly49A caused a licensing defect in NK cells, but the surface expression of Ly49A was unaltered. Moreover, NK cells that expressed this mutant Ly49A exhibited an altered inhibitory receptor repertoire. These results demonstrate that Ly49A ITIM signaling is critical for NK-cell effector inhibition, licensing, and receptor repertoire development.

Enzymatic self-assembly of an immunoreceptor tyrosine-based inhibitory motif (ITIM).

Here we show the first example of an immunoreceptor tyrosine-based inhibitory motif (ITIM), LYYYYL, as well as its enantiomeric or retro-inverso peptide, to self-assemble in water via enzyme-instructed self-assembly. Upon enzymatic dephosphorylation, the phosphohexapeptides become hexapeptides, which self-assemble in water to result in supramolecular hydrogels. This work illustrates a new approach to design bioinspired soft materials from a less explored, but important pool of immunomodulatory peptides.

ITIM receptors: more than just inhibitors of platelet activation.

Since their discovery, immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptors have been shown to inhibit signaling from immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors in almost all hematopoietic cells, including platelets. However, a growing body of evidence has emerged demonstrating that this is an oversimplification, and that ITIM-containing receptors are versatile regulators of platelet signal transduction, with functions beyond inhibiting ITAM-mediated platelet activation. PECAM-1 was the first ITIM-containing receptor identified in platelets and appeared to conform to the established model of ITIM-mediated attenuation of ITAM-driven activation. PECAM-1 was therefore widely accepted as a major negative regulator of platelet activation and thrombosis for many years, but more recent findings suggest a more complex role for this receptor, including the facilitation of αIIbß3-mediated platelet functions. Since the identification of PECAM-1, several other ITIM-containing platelet receptors have been discovered. These include G6b-B, a critical regulator of platelet reactivity and production, and the noncanonical ITIM-containing receptor TREM-like transcript-1, which is localized to α-granules in resting platelets, binds fibrinogen, and acts as a positive regulator of platelet activation. Despite structural similarities and shared binding partners, including the Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, knockout and transgenic mouse models have revealed distinct phenotypes and nonredundant functions for each ITIM-containing receptor in the context of platelet homeostasis. These roles are likely influenced by receptor density, compartmentalization, and as-yet unknown binding partners. In this review, we discuss the diverse repertoire of ITIM-containing receptors in platelets, highlighting intriguing new functions, controversies, and future areas of investigation.

Regulation of neutrophil functions through inhibitory receptors: an emerging paradigm in health and disease.

Neutrophils are the most abundant subset of leukocytes and play a crucial role in the immune responses against the daily pathogen attacks faced by the host. Neutrophils exhibit several functions for fighting microbes, including the release of granules containing highly toxic molecules, the production of reactive oxygen species and inflammatory cytokines as well as NETosis. Therefore, immune responses mediated by neutrophils must be tightly regulated to protect the host from pathogen assaults without inducing detrimental inflammation and tissue damage. There is now compelling evidence showing that neutrophils express various inhibitory receptors that specifically control their functions. Some of these inhibitory receptors are contained in the membrane of granules and rapidly move to the cell surface upon neutrophil stimulation. This fast upregulation of inhibitory receptors is an efficient way to rapidly enhance inhibitory signals and increase the neutrophil activation threshold. However, because of their ability to attenuate the immune responses of neutrophils, the inhibitory receptors are attractive target for pathogens. This review discusses these various aspects with a particular emphasis on the regulation of neutrophil behavior through immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing inhibitory receptors belonging to LILR and SIGLEC multi-gene families in humans and animal models.

The Biology and Disease Relevance of CD300a, an Inhibitory Receptor for Phosphatidylserine and Phosphatidylethanolamine.

The CD300a inhibitory receptor belongs to the CD300 family of cell surface molecules that regulate a diverse array of immune cell processes. The inhibitory signal of CD300a depends on the phosphorylation of tyrosine residues embedded in ITIMs of the cytoplasmic tail. CD300a is broadly expressed on myeloid and lymphoid cells, and its expression is differentially regulated depending on the cell type. The finding that CD300a recognizes phosphatidylserine and phosphatidylethanolamine, two aminophospholipids exposed on the outer leaflet of dead and activated cells, has shed new light on its role in the modulation of immune functions and in its participation in the host response to several diseases states, such as infectious diseases, cancer, allergy, and chronic inflammatory diseases. This review summarizes the literature on CD300a expression, regulation, signaling pathways, and ligand interaction, as well as its role in fine tuning immune cell functions and its clinical relevance.

Termination of immune activation: an essential component of healthy host immune responses.

The ideal immune response is rapid, proportionate and effective. Crucially, it must also be finite. An inflammatory response which is disproportionate or lasts too long risks injury to the host; chronic un-regulated inflammation in autoimmune diseases is one example of this. Thus, mechanisms to regulate and ultimately terminate immune responses are central to a healthy immune system. Despite extensive knowledge of what drives immune responses, our understanding of mechanisms of immune termination remains relatively sparse. It is clear that such processes are more complex than a one-dimensional homeostatic balance. Recent discoveries have revealed ever more nuanced mechanisms of signal termination, such as intrinsically self-limiting signals, multiple inhibitory mechanisms acting in tandem and activating proteins behaving differently in a variety of contexts. This review will summarise some important mechanisms, including termination by immunoreceptor tyrosine-based inhibitory motifs (ITIM), inhibition by soluble antagonists, receptor endocytosis or ubiquitination, and auto-inhibition by newly synthesised intracellular inhibitory molecules. Several recent discoveries showing immunoreceptor tyrosine-based activation motifs transducing inhibitory signals, ITIM mediating activating responses and the possible roles of immunoreceptor tyrosine-based switch motifs will also be explored.

Targeting glycoprotein VI and the immunoreceptor tyrosine-based activation motif signaling pathway.

Coronary artery thrombosis and ischemic stroke are often initiated by the disruption of an atherosclerotic plaque and consequent intravascular platelet activation. Thus, antiplatelet drugs are central in the treatment and prevention of the initial, and subsequent, vascular events. However, novel pharmacological targets for platelet inhibition remain an important goal of cardiovascular research because of the negative effect of existing antiplatelet drugs on primary hemostasis. One promising target is the platelet collagen receptor glycoprotein VI. Blockade or antibody-mediated depletion of this receptor in circulating platelets is beneficial in experimental models of thrombosis and thrombo-inflammatory diseases, such as stroke, without impairing hemostasis. In this review, we summarize the importance of glycoprotein VI and (hem)immunoreceptor tyrosine-based activation motif signaling in hemostasis, thrombosis, and thrombo-inflammatory processes and discuss the targeting strategies currently under development for inhibiting glycoprotein VI and its signaling.

IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform.

BACKGROUND: Interferon regulatory factor-1 (IRF-1) is a master regulator of IFN-γ induced gene transcription. Previously we have shown that IRF-1 transcriptionally induces CEACAM1 via an ISRE (Interferon-Stimulated Response Element) in its promoter. CEACAM1 pre-mRNA undergoes extensive alternative splicing (AS) generating isoforms to produce either a short (S) cytoplasmic domain expressed primarily in epithelial cells or as an ITIM-containing long (L) isoform in immune cells. METHODS: The transcriptional and molecular mechanism of CEACAM1 minigenes AS containing promoter ISREs mutations in the breast epithelial, MDA-MB-468, cell line was detected using flow cytometry. In addition, transcriptome sequencing was utilized to determine whether IRF-1 could direct the AS of other genes as well. Tumor xenografts were used to evaluate CEACAM1 isoform expression on the leading edge of breast tumor cells. RESULTS: In the present study, we provide evidence that CEACAM1's promoter and variable exon 7 cross-talk allowing IRF-1 to direct AS events. Transcriptome sequencing shows that IRF-1 can also induce the global AS of genes involved in regulation of growth and differentiation as well as genes of the cytokine family. Furthermore, MDA-MB-468 cells grown as tumor xenografts exhibit an AS switch to the L-isoform of CEACAM1, demonstrating that an in vivo inflammatory milieu is also capable of generating the AS switch, similar to that found in human breast cancers Mol Cancer 7:46, 2008. CONCLUSIONS: The novel AS regulatory activities attributed to IRF-1 indicate that the IFN-γ response involves a global change in both gene transcription and AS in breast epithelial cells.

Fine specificity and molecular competition in SLAM family receptor signalling.

SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (KD = 0.003 µM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 µM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive dominance of SAP and EAT-2.

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets.

RATIONALE: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor-bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. OBJECTIVE: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. METHODS AND RESULTS: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI-mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2-mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein-coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2-induced G protein-coupled receptor signaling pathways. CONCLUSIONS: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.

An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis.

Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

SHPS-1 and a synthetic peptide representing its ITIM inhibit the MyD88, but not TRIF, pathway of TLR signaling through activation of SHP and PI3K in THP-1 cells.

BACKGROUND: Src homology 2 domain-containing protein tyrosine phosphatase substrate (SHPS)-1 is known to have regulatory effects on myeloid cells. However, its role in macrophage activation is not clearly understood. METHODS AND RESULTS: In order to investigate the role of SHPS-1 in Toll-like receptor (TLR)-mediated activation, human monocytic cell lines were treated with anti-SHPS-1 monoclonal antibody. The triggering of SHPS-1 blocked the expression of IL-8 and TNF-α in cells treated with a TLR4 ligand that induces a signaling pathway involving myeloid differentiation factor 88 (MyD88) and Toll-interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-ß (TRIF). Interestingly, SHPS-1 inhibited TLR9/MyD88-mediated, but not TLR3/TRIF-mediated, expression of IL-8. Accordingly, a synthetic peptide representing the immunoreceptor tyrosine-based inhibition motif (ITIM) of SHPS-1 suppressed only the MyD88 pathway. Utilization of specific inhibitors and Western blot analysis indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatases (SHPs) and phosphoinositide 3-kinase (PI3K). CONCLUSION: SHPS-1 negatively regulates the MyD88-dependent TLR signaling pathway through the inhibition of NF-κB activation.

Microglial activatory (immunoreceptor tyrosine-based activation motif)- and inhibitory (immunoreceptor tyrosine-based inhibition motif)-signaling receptors for recognition of the neuronal glycocalyx.

Microglia sense intact or lesioned cells of the central nervous system (CNS) and respond accordingly. To fulfill this task, microglia express a whole set of recognition receptors. Fc receptors and DAP12 (TYROBP)-associated receptors such as microglial triggering receptor expressed on myeloid cells-2 (TREM2) and the complement receptor-3 (CR3, CD11b/CD18) trigger the immunoreceptor tyrosine-based activation motif (ITAM)-signaling cascade, resulting in microglial activation, migration, and phagocytosis. Those receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif (ITIM)-signaling receptors, such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs). Siglecs recognize the sialic acid cap of healthy neurons thus leading to an ITIM signaling that turns down microglial immune responses and phagocytosis. In contrast, desialylated neuronal processes are phagocytosed by microglial CR3 signaling via an adaptor protein containing an ITAM. Thus, the aberrant terminal glycosylation of neuronal surface glycoproteins and glycolipids could serve as a flag for microglia, which display a multitude of diverse carbohydrate-binding receptors that monitor the neuronal physical condition and respond via their ITIM- or ITAM-signaling cascade accordingly.