A Tie2-Notch1 signaling axis regulates regeneration of the endothelial bone marrow niche.

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.We thank Dr. Warren Pear for invaluable advice and for sharing the Notch1+/ΔTAD murine model system. We also thank Dr. Kishore Wary for sharing the Cdh5-CreERT2 mouse model. Drs. Jon Aster and Stephen Blacklow for advice and thoughtful discussion, Dr. Dawson Gerhardt for her help in generating the Notch1- ΔTAD plasmids and vector constructs, Dr. Jan Kitejewski for helpful advice on Notch mutant mice and Drs. Fotini Gounari and Linda Dagenstein of the University of Chicago transgenic mouse facility for help in maintaining the transgenic mouse colonies. The following cores at the University of Illinois at Chicago contributed to this study: RRC Histology Core and RRC Flow Cytometry Core. This study was funded by NIH grants 1R01HL134971 to KVP and 1R01HL136529 to DL.S

Externalized glycolytic enzymes are novel, conserved, and early biomarkers of apoptosis

The intriguing cell biology of apoptotic cell death results in the externalization of numerous autoantigens on the apoptotic cell surface, including protein determinants for specific recognition, linked to immune responses. Apoptotic cells are recognized by phagocytes and trigger an active immunosuppressive response (“innate apoptotic immunity” [IAI]) even in the absence of engulfment. IAI is responsible for the lack of inflammation associated normally with the clearance of apoptotic cells; its failure also has been linked to inflammatory and autoimmune pathology, including systemic lupus erythematosis (SLE) and rheumatic diseases. Apoptotic recognition determinants underlying IAI have yet to be identified definitively; we argue that these molecules are surface-exposed (during apoptotic cell death), ubiquitously-expressed, protease-sensitive, evolutionarily-conserved, and resident normally in viable cells (“SUPER”). Taking independent and unbiased quantitative proteomic approaches to characterize apoptotic cell surface proteins and identify candidate SUPER determinants, we made the surprising discovery that components of the glycolysis pathway are enriched on the apoptotic cell surface. Our data demonstrate that glycolytic enzyme externalization is a common and early aspect of cell death in different cell types triggered to die with distinct suicidal stimuli. Exposed glycolytic enzyme molecules meet the criteria for IAI-associated SUPER determinants. In addition, our characterization of the apoptosis-specific externalization of glycolytic enzyme molecules may provide insight into the significance of previously reported cases of plasminogen binding to α-enolase on mammalian cells, as well as mechanisms by which commensal bacteria and pathogens maintain immune privilege

CCL25 and CCR9 is a unique pathway that potentiates pannus formation by remodeling RA macrophages into mature osteoclasts

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL‐1β and IL‐6. CCR9 is also presented on IL‐1β and IL‐6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3‐month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25‐stimulated RA FLS secrete potentiated levels of IL‐8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL‐8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25‐induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25‐induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF‐α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.Rheumatoid arthritis synovial fluid (RA SF) express markedly higher levels of CCL25 compared to osteoarthritis (OA) SF. We found that CCL25 is secreted from RA fibroblast like synoviocytes (FLS) and macrophages in response to IL‐1β and IL‐6 activation. Moreover, RA FLS and monocyte infiltration is potentiated by CCL25 through ERK and p38 phosphorylation. Extending these observations, inhibition of ERK and p38 pathways interferes with CCL25‐induced inflammatory phenotype in RA FLS and macrophages as well as its ability to promote osteoclastogenesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167430/1/eji4976.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167430/2/eji4976-sup-0001-SuppMat.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167430/3/eji4976_am.pd

CCL25 and CCR9 is a unique pathway that potentiates pannus formation by remodeling RA macrophages into mature osteoclasts

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL‐1β and IL‐6. CCR9 is also presented on IL‐1β and IL‐6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3‐month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25‐stimulated RA FLS secrete potentiated levels of IL‐8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL‐8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25‐induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25‐induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF‐α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.Rheumatoid arthritis synovial fluid (RA SF) express markedly higher levels of CCL25 compared to osteoarthritis (OA) SF. We found that CCL25 is secreted from RA fibroblast like synoviocytes (FLS) and macrophages in response to IL‐1β and IL‐6 activation. Moreover, RA FLS and monocyte infiltration is potentiated by CCL25 through ERK and p38 phosphorylation. Extending these observations, inhibition of ERK and p38 pathways interferes with CCL25‐induced inflammatory phenotype in RA FLS and macrophages as well as its ability to promote osteoclastogenesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167430/1/eji4976.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167430/2/eji4976-sup-0001-SuppMat.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167430/3/eji4976_am.pd

Interleukin‐34 Reprograms Glycolytic and Osteoclastic Rheumatoid Arthritis Macrophages via Syndecan 1 and Macrophage Colony‐Stimulating Factor Receptor

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170983/1/art41792.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170983/2/art41792_am.pd

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing Notch1 mutant alleles in mice fails to efficiently induce the develo