Validation of a murine proteome-wide phage display library for identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity.

Myeloid Src-family kinases are critical for neutrophil-mediated autoinflammation in gout and motheaten models.

Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis.

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity.

Neutrophils inhibit γδ T cell functions in the imiquimod-induced mouse model of psoriasis.

Background: Psoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood.

Spleen tyrosine kinase facilitates neutrophil activation and worsens long-term neurologic deficits after spinal cord injury.

Background: Spinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme.

A CD22-Shp1 phosphatase axis controls integrin β display and B cell function in mucosal immunity.

The integrin αβ selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of αβ surface expression and gut immunity. Shp1 selectively inhibited β endocytosis, enhancing surface αβ display and lymphocyte homing to GALT.

Shp1 Loss Enhances Macrophage Effector Function and Promotes Anti-Tumor Immunity.

Shp1, encoded by the gene , is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic.

CARD9 mediates dendritic cell-induced development of Lyn deficiency-associated autoimmune and inflammatory diseases.

CARD9 is an immune adaptor protein in myeloid cells that is involved in C-type lectin signaling and antifungal immunity. CARD9 is implicated in autoimmune and inflammatory-related diseases, such as rheumatoid arthritis, IgA nephropathy, ankylosing spondylitis, and inflammatory bowel disease (IBD). Given that Lyn-deficient ( mice are susceptible to both autoimmunity and IBD, we investigated the immunological role of CARD9 in the development of these diseases using the mouse model.

Shp1 function in myeloid cells.

The mouse was first described in 1975 as a model of systemic inflammation and autoimmunity, as a result of immune system dysregulation. The phenotype was later ascribed to mutations in the cytoplasmic tyrosine phosphatase Shp1. This phosphatase is expressed widely throughout the hematopoietic system and has been shown to impact a multitude of cell signaling pathways.

Leishmania Uses Mincle to Target an Inhibitory ITAM Signaling Pathway in Dendritic Cells that Dampens Adaptive Immunity to Infection.

C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection.

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLβ2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly).

Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice.

Since the first example of conditional gene targeting in mice in 1994, the use of Cre recombinase and loxP flanked sequences has become an invaluable technique to generate tissue and temporal specific gene knockouts. The number of mouse strains expressing floxed-gene sequences, and tissue-specific or temporal-specific Cre-recombinase that have been reported in the literature has grown exponentially. However, increased use of this technology has highlighted several problems that can impact the interpretation of any phenotype observed in these mouse models.

Expression of the TEL-Syk fusion protein in hematopoietic stem cells leads to rapidly fatal myelofibrosis in mice.

The TEL-Syk fusion protein was isolated from a patient with myelodysplasia with megakaryocyte blasts. Expression of TEL-Syk transforms interleukin-3 (IL-3)-dependent Ba/F3 cells in vitro by deregulating STAT5-mediated signal transduction pathways. In vivo, TEL-Syk expression in pre-B cells blocks B cell differentiation, leading to lymphoid leukemia.

Distinct roles for neutrophils and dendritic cells in inflammation and autoimmunity in motheaten mice.

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear.

DAP12 is required for macrophage recruitment to the lung in response to cigarette smoke and chemotaxis toward CCL2.

DAP12 is an adapter protein that associates with several receptors in macrophages. Little is known about the biological role of DAP12 in alveolar macrophages. In genome-wide profiling, we previously found that two DAP12-associated receptors, myeloid DAP12-associated lectin-1 and triggering receptor expressed on myeloid cells 2 (TREM2), were highly induced in alveolar macrophages from habitual smokers.

The ins and outs of leukocyte integrin signaling.

Integrins are the principal cell adhesion receptors that mediate leukocyte migration and activation in the immune system. These receptors signal bidirectionally through the plasma membrane in pathways referred to as inside-out and outside-in signaling. Each of these pathways is mediated by conformational changes in the integrin structure.

PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcR gamma to induce slow leukocyte rolling.

E-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) can activate the beta(2) integrin lymphocyte function-associated antigen-1 by signaling through spleen tyrosine kinase (Syk). This signaling is independent of G alpha(i)-protein-coupled receptors, results in slow rolling, and promotes neutrophil recruitment to sites of inflammation. However, the signaling pathways linking E-selectin engagement of PSGL-1 to Syk activation are unknown.

The diverse functions of Src family kinases in macrophages.

Macrophages are key components of the innate immune response. These cells possess a diverse repertoire of receptors that allow them to respond to a host of external stimuli including cytokines, chemokines, and pathogen-associated molecules. Signals resulting from these stimuli activate a number of macrophage functional responses such as adhesion, migration, phagocytosis, proliferation, survival, cytokine release and production of reactive oxygen and nitrogen species.

Immunoreceptor-like signaling by beta 2 and beta 3 integrins.

Although adhesion to extracellular structures is one of the most fundamental cell biological processes, the intracellular signals triggered by integrins, the most important receptors involved, are incompletely understood. Several recent reports indicate that signaling by beta(2) and beta(3) integrins in various cell types (neutrophils, macrophages, osteoclasts and platelets) use components of the signal transduction machinery of lymphocyte antigen receptors. Central to this immunoreceptor-like signaling is the phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM)-containing adapters (such as DAP12 and the Fc receptor gamma-chain) by Src-family kinases and the concomitant recruitment of the Syk tyrosine kinase through its dual SH2 domains.

Convergence of immunoreceptor and integrin signaling.

A common signaling pathway is known to operate downstream of immunoreceptors, such as the T-cell, B-cell, or Fc receptors, following engagement by their respective ligands. This pathway involves Src family kinase-mediated tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) that recruit and activate spleen tyrosine kinase (Syk) or Zap70 (zeta-associated protein of 70 kDa) kinases, which in turn activate a variety of downstream signals. Evidence has been building from a variety of sources, particularly mouse models, that molecules involved in the immunoreceptor signaling pathway are also required for signals initiated by integrins.

The expanding role for ITAM-based signaling pathways in immune cells.

The immunoreceptor tyrosine-based activation motif (ITAM) is the primary signaling domain used by classical immunoreceptors, such as the antigen receptors on B and T lymphocytes and the Fc receptors (FcRs) on myeloid cells. The ITAM is contained in the intracellular region of subunits associated with these receptors, often in pairs, or is part of the cytoplasmic domain of the receptors themselves. Data from many investigators have demonstrated that ITAMs are both necessary and sufficient for initiation of signaling downstream of all immunoreceptors.

Integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activation motifs.

At sites of inflammation, ligation of leukocyte integrins is critical for the activation of cellular effector functions required for host defense. However, the signaling pathways linking integrin ligation to cellular responses are poorly understood. Here we show that integrin signaling in neutrophils and macrophages requires adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs).