Namit Sharma

SH2 Domain-Containing Phosphatase-2 Protein-Tyrosine Phosphatase Promotes FcεRI-Induced Activation of Fyn and Erk Pathways Leading to TNFα Release from Bone Marrow-Derived Mast Cells

Clustering of the high affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Initiation of FcεRI signaling involves rapid tyrosine phosphorylation of FcεRI and membrane-localized adaptor proteins that recruit additional SH2 domain-containing proteins that dynamically regulate downstream signaling. SH2 domain-containing phosphatase-2 (SHP2) is a protein-tyrosine phosphatase implicated in FcεRI signaling, but whose function is not well defined. In this study, using a mouse model allowing temporal shp2 inactivation in bone marrow-derived mast cells (BMMCs), we provide insights into SHP2 functions in the FcεRI pathway. Although no overt defects in FcεRI-induced tyrosine phosphorylation were observed in SHP2 knock-out (KO) BMMCs, several proteins including Lyn and Syk kinases displayed extended phosphorylation kinetics compared with wild-type BMMCs. SHP2 was dispensable for FcεRI-induced degranulation of BMMCs, but was required for maximal activation of Erk and Jnk mitogen-activated protein kinases. SHP2 KO BMMCs displayed several phenotypes associated with reduced Fyn activity, including elevated phosphorylation of the inhibitory pY531 site in Fyn, impaired signaling to Grb2-associated binder 2, Akt/PKB, and IκB kinase, and decreased TNF-α release compared with control cells. This is likely due to elevated Lyn activity in SHP2 KO BMMCs, and the ability of Lyn to antagonize Fyn activity. Overall, our study identifies SHP2 as a positive effector of FcεRI-induced activation of Fyn/Grb2-associated binder 2/Akt and Ras/Erk pathways leading to TNF-α release from mast cells.

SH2 Domain-Containing Phosphatase 2 Is a Critical Regulator of Connective Tissue Mast Cell Survival and Homeostasis in Mice

ABSTRACT Mast cells require KIT receptor tyrosine kinase signaling for development and survival. Here, we report that SH2 domain-containing phosphatase 2 (SHP2) signaling downstream of KIT is essential for mast cell survival and homeostasis in mice. Using a novel mouse model with shp2 deletion within mature mast cells (MC-shp2 knockout [KO]), we find that SHP2 is required for the homeostasis of connective tissue mast cells. Consistently with the loss of skin mast cells, MC-shp2 KO mice fail to mount a passive late-phase cutaneous anaphylaxis response. To better define the phenotype of shp2-deficient mast cells, we used an inducible shp2 knockout approach in bone marrow-derived mast cells (BMMCs) or cultured peritoneal mast cells and found that SHP2 promotes mast cell survival. We show that SHP2 promotes KIT signaling to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and downregulation of the proapoptotic protein Bim in BMMCs. Also, SHP2-deficient BMMCs failed to repopulate mast cells in mast cell-deficient mice. Silencing of Bim partially rescued survival defects in shp2-deficient BMMCs, consistent with the importance of a KIT → SHP2 → Ras/ERK pathway in suppressing Bim and promoting mast cell survival. Thus, SHP2 is a key node in a mast cell survival pathway and a new potential therapeutic target in diseases involving mast cells.

Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe

Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis.

Aberrant mast cell activation promotes chronic recurrent multifocal osteomyelitis

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease. While some patients exhibit bone lesions at single sites, most patients develop chronically active or recurrent bone inflammation at multiple sites, and are then diagnosed with recurrent multifocal osteomyelitis (CRMO). Chronic multifocal osteomyelitis (CMO) mice develop IL-1β-driven sterile bone lesions reminscent of severe CRMO. Mechanistically, CMO disease arises due to loss of PSTPIP2, a negative regulator of macrophages, osteoclasts and neutrophils. The goal of this study was to evaluate the potential involvement of mast cells in CMO/CRMO disease pathophysiology. Here, we show that mast cells accumulate in the inflamed tissues from CMO mice, and mast cell protease Mcpt1 was detected in the peripheral blood. The role of mast cells in CMO disease was investigated using a transgenic model of connective tissue mast cell depletion (Mcpt5-Cre:Rosa26-Stopfl/fl-DTa) that was crossed with CMO mice. The resulting CMO/MC- mice showed a significant delay in disease onset compared to age-matched CMO mice. At 5-6 months of age, CMO/MC- mice had fewer bone lesions and immune infiltration in the popliteal lymph nodes that drain the affected tail and paw tissues. To test the relevance of mast cells to human CRMO, we tested serum samples from a cohort of healthy controls or CRMO patients at diagnosis. Interestingly, mast cell chymase was elevated in CRMO patients as well as patients with oligoclonal juvenile arthritis. Tryptase-positive mast cells were also detected in bone lesions from CRMO patients as well as patients with bacterial osteomyelitis. Taken together, our results identify mast cells as cellular contributors to bone inflammation in CMO/CRMO. Observations of this study promise potential for mast cells and derived mediators as future biomarkers and/or therapeutic targets.Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease. While some patients exhibit bone lesions at single sites, most patients develop chronically active or recurrent bone inflammation at multiple sites, and are then diagnosed with recurrent multifocal osteomyelitis (CRMO). Chronic multifocal osteomyelitis (CMO) mice develop IL-1β-driven sterile bone lesions reminscent of severe CRMO. Mechanistically, CMO disease arises due to loss of PSTPIP2, a negative regulator of macrophages, osteoclasts and neutrophils. The goal of this study was to evaluate the potential involvement of mast cells in CMO/CRMO disease pathophysiology. Here, we show that mast cells accumulate in the inflamed tissues from CMO mice, and mast cell protease Mcpt1 was detected in the peripheral blood. The role of mast cells in CMO disease was investigated using a transgenic model of connective tissue mast cell depletion (Mcpt5-Cre:Rosa26-Stopfl/fl-DTa) that was crossed with CMO mice. The resulting CMO/MC-mice showed a significant delay in disease onset compared to age-matched CMO mice. At 5-6 months of age, CMO/MC- mice had fewer bone lesions and immune infiltration in the popliteal lymph nodes that drain the affected tail and paw tissues. To test the relevance of mast cells to human CRMO, we tested serum samples from a cohort of healthy controls or CRMO patients at diagnosis. Interestingly, mast cell chymase was elevated in CRMO patients as well as patients with oligoclonal juvenile arthritis. Tryptase-positive mast cells were also detected in bone lesions from CRMO patients as well as patients with bacterial osteomyelitis. Taken together, our results identify mast cells as cellular contributors to bone inflammation in CMO/CRMO. Observations of this study promise potential for mast cells and derived mediators as future biomarkers and/or therapeutic targets.

Editorial: leukocyte-targeting toxins as therapeutics in allergic asthma.

Asthma is a prevalent inflammatory disorder of the airway, characterized by immune cell infiltration, epithelial barrier defects, mucous hypersecretion, and obstruction of airflow [1]. In allergic asthma, patients are sensitized to inhaled allergens in a Th2-driven process of allergen-specific IgE production. Subsequent exposures to inhaled allergens result in inflammatory mediator release from mast cells and basophils that enhance blood vessel permeability, leukocyte recruitment, and chronic inflammation [2]. Current therapies largely target the symptoms of this disease and are not effective for all allergic asthma patients [3]. New targets that are fundamentally involved in allergic asthma are being pursued to block more effectively early events in the pathogenesis of allergic asthma. In this issue of the Journal of Leukocyte Biology, Gupta et al. [4] report that LFA-1 is a promising target in human allergic asthma patient samples. They test this by use of a mouse model of allergic asthma and an experimental toxin directed against LFA-1, called LtxA (Leukothera). Integrins form heterodimers on the cell surface that regulate cell adhesion and motility through interactions with ligands expressed on activated endothelium or within the extracellular matrix. Leukocyte integrins play key roles in leukocyte adhesion to blood vessel walls, spreading, and transendothelial migration (Fig. 1) [5]. Relevant integrins include VLA-4 (or a4b1), macrophage 1 antigen (aMb2), and LFA-1 (aLb2, or CD11a/CD18). In asthmatic airways, ligands for these integrins are expressed at high levels, including VCAM-1 and ICAM-1 [6]. This promotes firm adhesion and tissue invasion by immune cells expressing VLA-4 and LFA-1 and has led research into therapeutic blockade of these integrins in asthma and autoimmune disorders. Efalizumab (Raptiva) is an inhibitory LFA-1 antibody that was approved by the U.S. Food and Drug Administration in 2003 for treatment of an autoimmune disease but was withdrawn in 2009 as a result of risk of developing fatal brain infections [7]. One potential drawback with Efalizumab is that it targets all LFA-1-positive cells, whereas targeting only activated LFA-1 would be preferable for therapeutic applications. LtxA is a toxin secreted by the gramnegative bacterium Aggregatibacter actinomycetemcomitans that binds activated LFA-1 on leukocytes leading to cell death [8]. The therapeutic potential of LtxA was first demonstrated in a leukemia model, showing that LtxA preferentially targets leukemic cells expressing activated LFA-1 compared with normal leukocytes [8]. Moreover, LtxA treatment was effective in treating leukemias that had relapsed on standard chemotherapy [9]. In addition, LtxA was tested in a xenograft model of human psoriasis and shown to allow resolution of disease by preferential targeting of proinflammatory leukocytes [10]. In this issue of the Journal of Leukocyte Biology, Gupta et al. [4] have now extended studies of LFA-1 expression in allergic asthma patient samples and effects of LFA-1 blockade with LtxA in a mouse model of allergic asthma [4]. They identified a circulating cell population with elevated levels of LFA-1 (CD11a) in asthma patients that were not found in healthy controls. This population was composed of CD14CD4 CD11a monocytes and CD3CD4 CD11a non-Th cells. The authors went on to show that these CD11a cells undergo apoptosis upon LtxA treatment ex vivo, whereas CD11a cells within the same samples were unaffected. These findings were tested further in mice chronically exposed to HDM extract. The authors validate high expression of LFA-1 in leukocytes from broncheoalveolar lavage fluid of allergic mice compared with control mice. This provides further evidence for the potential targeting of LFA-1 cells in allergic asthma. The authors go on to compare treatment of HDM mice directly with LtxA or dexamethasone, the current gold standard corticosteroid used as a general immunosuppressant. Importantly, the authors demonstrate that LtxA outperforms dexamethasone by several metrics. LtxA-treated mice were protected from airway infiltration of all major leukocyte subtypes, including neutrophils, eosinophils, macrophages, and B and T cells. LtxA-treated mice were protected from airway remodeling induced by HDM exposure and had reduced expression of several cytokines implicated in asthma pathogenesis within the lung. LtxA treatment was more effective than dexamethasone in blocking neutrophil