The Src-family tyrosine kinases play pivotal roles in initiating intracellular signaling from a diverse repertoire of receptors on innate immune leukocytes. The Src-family kinases initiate intracellular signaling from these receptors by phosphorylating specific tyrosine residues within ITAM (Immunoreceptor Tyrosine-based Activation Motifs) domains that are either part of the cytoplasmic domain of the receptor or are located on receptor-associated signaling adapters, such as the FcR? or DAP-12 molecules. Phosphorylation of these ITAM domains leads to recruitment of Syk/Zap70 kinases which in turn initiate downstream signaling, leading to cellular activation. Src-family kinases also phosphorylate inhibitory receptors on specific tyrosines within ITIM (Immunoreceptor Tyrosine-based Inhibitory Motifs) domains that are located within the cytoplasmic regions of each receptor. ITIM phosphorylation leads to recruitment of the tyrosine phosphatase SHP-1, which in turn dephosphorylates both the Src kinases as well as downstream substrates in the signaling cascade. The overall balance between activating and inhibitory signaling determines the cellular response. As a result, the functions of the Src-family kinases are opposed in large part by SHP-1, which serves as a major brake to intracellular signaling in innate cells. Mutation of the SHP-1 gene (Ptpn6) results in severely hyperactive lymphocytes, myeloid cells and platelets. As a result, SHP-1 deficient animals (motheaten (me/me) or motheaten viable (mev/mev)) develop dramatic autoimmunity, inflammation and early mortality due to lung inflammation (~4 weeks for complete SHP-1 loss in me/me mice and ~8-10 weeks in the hypomorphic mev/mev mice). Though these SHP-1 deficient animals have been available for years, the complex interactions and indirect effects between the many hyperactive leukocyte subsets has limited their utility. Thus it remains unclear which specific SHP-1 regulated signaling pathways in each of the different hyperactive leukocyte types is responsible for the different aspects of the autoimmune/inflammatory disease. To address this problem, we have bred the Ptpn6flx/flx (SHP-1flx) mice to a series of Cre expressing animals to achieve lineage-specific deletion of SHP-1. Deletion of SHP-1 specifically in neutrophils (using MRP8-Cre) recapitulates much of the inflammatory disease in present in mev/mev mice but the animals don't get autoimmunity. Deletion of SHP-1 in dendritic cells (using CD11c-Cre) results in autoimmunity, but no inflammation. Deletion of SHP-1 in both neutrophils and macrophages (using LysM-Cre) results in no phenotype at all (despite the presence of activated neutrophils). Finally, deletion of SHP-1 in platelets (using PF4-Cre) results in early mortality due to lung inflammation, without evidence of other disease. By subdividing the overall disease phenotype caused by global SHP-1 deficiency, this set of lineage-specific mutant mice will allow us to characterize the in vivo physiologic functions of SHP-1 regulated signaling in defined leukocytes which has been impossible to do with previous animal models.. We hypothesize that dysregulation of integrin and Toll-like receptor (TLR) signaling are the major drivers to the inflammatory versus autoimmune phenotypes in these different conditional SHP-1 mutant mice. We will examine this using both biochemical and genetic approaches, the latter of which will depend on the generation of double floxed mice that delete specific signaling pathways in each conditional murine line (for example SHP-1flxMyD88flxCD11c-Cre). We will also carry out unbiased screens of signaling pathways in the cells from the various conditional mutant mice. Finally, we will carry out platelet studies and use a novel two-photon imaging method to examine platelet/neutrophil interactions in the inflamed lungs of SHP-1flxPF4-Cre mice, to determine the mechanisms by which dysregulation of platelet function can lead to lethal pulmonary inflammation. Overall, these studies will help define how hyperactivation of specific tyrosine kinase based signaling pathways leads to inflammation and autoimmunity. Since it is well appreciated that alterations in these signaling pathways play a major role in human autoimmune, inflammatory and malignant disease, a more complete in vivo dissection of SHP-1 regulated signaling will help guide therapeutic development.
Immune cells sense pathogens such as bacteria, fungi and viruses through a complex array of cellular receptors. Binding of the pathogen molecules to these receptors activates intracellular enzymes that initiate intracellular signaling pathways that lead to immune cell activation to eliminate the pathogen. Appropriate control of these intracellular signaling pathways is critical to prevent immune cell hyperactivation that can cause damage to host tissues during responses to pathogen invasion. One of the major molecules that downregulates immune cells are the tyrosine phosphatase SHP-1. We have generated a series of mutant mice that lack the SHP-1 protein in defined immune cell types. These conditional SHP-1 mutant animals develop a spectrum of diseases, including severe skin inflammation, autoimmunity or lethal pulmonary inflammation. By studying the specific intracellular signaling pathways that are affected by these different SHP-1 conditional mutations, we will gain a new insight into the regulation of immune cell reactivity. This will lead to better design of therapeutcs to target immune diseases, as well as leukemia and lymphoma.
|Boras, Mark; Volmering, Stephanie; Bokemeyer, Arne et al. (2017) Skap2 is required for ?2integrin-mediated neutrophil recruitment and functions. J Exp Med 214:851-874|
|Abram, Clare L; Lowell, Clifford A (2017) Shp1 function in myeloid cells. J Leukoc Biol 102:657-675|
|Chen, Jun; Zhong, Ming-Chao; Guo, Huaijian et al. (2017) SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin. Nature 544:493-497|
|Sinha, Meenal; Lowell, Clifford A (2017) Efficiency and Specificity of Gene Deletion in Lung Epithelial Doxycycline-Inducible Cre Mice. Am J Respir Cell Mol Biol 57:248-257|
|Clemens, Regina A; Chong, Joshua; Grimes, Derayvia et al. (2017) STIM1 and STIM2 cooperatively regulate mouse neutrophil store-operated calcium entry and cytokine production. Blood 130:1565-1577|
|Poh, Ashleigh R; Love, Christopher G; Masson, Frederick et al. (2017) Inhibition of Hematopoietic Cell Kinase Activity Suppresses Myeloid Cell-Mediated Colon Cancer Progression. Cancer Cell 31:563-575.e5|
|Sinha, Meenal; Lowell, Clifford A (2016) Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting. Bio Protoc 6:|
|Volmering, Stephanie; Block, Helena; Boras, Mark et al. (2016) The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation. Immunity 44:73-87|
|Wang, Huafeng; Li, Mengyi; Hung, Chiung Yu et al. (2016) MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4+ T Cells during the Contraction Phase. PLoS Pathog 12:e1005787|
|Hang, Long; Blum, Arthur M; Kumar, Sangeeta et al. (2016) Downregulation of the Syk Signaling Pathway in Intestinal Dendritic Cells Is Sufficient To Induce Dendritic Cells That Inhibit Colitis. J Immunol 197:2948-57|
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