A NOVEL AND IMPROVED MOUSE SYSTEM FOR STUDYING MACROPHAGE SPECIFIC GENE DELETION
R Harris, Assistant Professor
Northwestern Universitycity: Chicago country: United States (us)
Grant 1R21AI092490-01 from National Institute Of Allergy And Infectious Diseases
Abstract: Macrophages are crucial for tissue homeostasis and play essential roles in inflammation, immunity and cancer, and therefore understanding macrophage biology is fundamental for understanding homeostasis and disease. Currently available mice that target Cre expression for the deletion of floxed genes to macrophages have significant and severe restrictions, primarily based on the promoter driving Cre expression. 1) all of the available promoters currently driving Cre in macrophages are active in the entire myeloid lineage and even other cell types, which is frequently stronger than the one in macrophages, and therefore lack macrophage specificity; 2) currently available promoters only target a subset of macrophages; and 3) the two most commonly used Cre lines driven by F4/80 and LysM, are also knocked in one endogenous allele resulting in weak expression and prevent breeding of homozygous mice. The goal of this study is to develop and characterize a novel and improved transgenic mouse line to specifically target expression of Cre to macrophages. We propose to use the human CD68 promoter in combination with a macrophage-specific enhancer to drive expression of an activity-improved Cre and the dtTomato fluorescent protein from a bicistronic transcript for in vivo tracking and sorting. Transgenic mice will be extensively characterized with reporter genes and endogenous floxed genes and compared to the currently available Cre-expressing mice. Therefore our proposal to develop a novel true macrophage selective and specific Cre expressing mouse line in the C57BL/6 strain, and to make this mouse widely available to the research community, will significantly impact our current ability to study macrophages and will have significant ramifications for researchers studying macrophages in homeostasis, immunity and cancer, and will therefore enable the field to progress forward. Macrophages are essential for tissue homeostasis and contribute to disease. Therefore understanding macrophage biology is fundamental to advance current knowledge of these processes, which requires macrophage specific deletion of genes using the Cre recombinase. Because currently available mice expressing Cre in macrophages have severe disadvantages, our study will develop and characterize a new and substantially improved transgenic mouse with macrophage specific expression of an improved version of Cre, which will be shared with the scientific community
Keywords: 21+ years old; Ablation; Adult; adult human (21+); Affect; Alleles; Allelomorphs; amebocyte; Animals; Assay; Automobile Driving; Autoregulation; bacteriophage P1 recombinase Cre; base; Bioassay; Biologic Assays; Biological; Biological Assay; Biology; Blood granulocytic cell; Body Tissues; Bone Marrow; Breeding; Cancers; CD11b; cell type; Cells; Communicable Diseases; Communities; CR3A; CRE Recombinase; Cre recombinase, Enterobacteria phage P1; Crossmatching, Tissue; Cytofluorometry, Flow; cytokine; Development; Disadvantaged; Disease; disease/disorder; Disorder; DNA Molecular Biology; DNA Recombination; DNA recombination (naturally occurring); driving; Drivings, Automobile; Engineering; Engineerings; Enhancers; Enterobacteria phage P1 Cre recombinase; Event; Flow Cytofluorometries; Flow Cytometry; flow cytophotometry; Flow Microfluorimetry; Fluorescence; Gene Deletion; gene deletion mutation; gene function; gene product; Genes; Genetic Recombination; Goals; Granular Leukocytes; granulocyte; Granulocytic cell; Histocompatibility Testing; histocompatibility typing; Homeostasis; host response; Human; Human, Adult; Human, General; Immune; Immune response; Immunity; immunoresponse; improved; in vivo; Infection; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Inflammation; Inflammatory; Inflammatory Response; INFLM; Internal Ribosome Entry Segment; Internal Ribosome Entry Site; Investigators; IRES; ITGAM; ITGAM gene; Knock-in; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Lysozyme; MAC-1; MAC1A; macrophage; magnetic beads; malignancy; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Masks; Mediating; Messenger RNA; Mice; Mice, Knock-out; Mice, Knockout; Microfluorometry, Flow; MO1A; Molecular; Molecular Biology; mRNA; Muramidase; Murine; Mus; Myelogenous; Myeloid; N-Acetylmuramide Glycanhydrolase; neoplasm/cancer; novel; Null Mouse; pathway; Pathway interactions; Peptidoglycan N-acetylmuramoylhydrolase; peripheral blood; Peritonitis; Phagocytes; Phagocytic Cell; Phenotype; Physiological Homeostasis; Play; Population; prevent; preventing; Process; Production; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteins; public health relevance; recombinase; Recombination; Recombination, Genetic; Reporter; Reporter Genes; Research; Research Personnel; Researchers; response; Reticuloendothelial System, Bone Marrow; Ribosome Entry Site; RNA, Messenger; Role; Site; social role; sorting; Sorting - Cell Movement; Specificity; System; System, LOINC Axis 4; Thioglycolates; Thioglycollates; Time; Tissue Crossmatchings; Tissue Typing; Tissues; Tomatoes; tool; Transcript; transgenic; Transgenic Mice; Transgenic Organisms; Validation; Work
Relevance: Macrophages are essential for tissue homeostasis and contribute to disease. Therefore understanding macrophage biology is fundamental to advance current knowledge of these processes, which requires macrophage specific deletion of genes using the Cre recombinase. Because currently available mice expressing Cre in macrophages have severe disadvantages, our study will develop and characterize a new and substantially improved transgenic mouse with macrophage specific expression of an improved version of Cre, which will be shared with the scientific community
Project start date: 2010-12-15
Project end date: 2012-11-30
Budget start date: 15-DEC-2010
Budget end date: 30-NOV-2011
PFA/PA: PA-07-336
1R21AI092490-01 (2011): $228750
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to R Harris
THERAPEUTIC ROLE FOR P21 IN SUPPRESSING IL-1 BETA MEDIATED PATHOLOGIES
R Harris, Associate Professor
Northwestern Universitycity: Chicago country: United States (us)
Grant 5R01AR050250-07 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that leads to destruction of bone and cartilage and is associated with increased morbidity and mortality. The mechanism responsible for the persistent inflammation of the synovium is not clear. We were the first to demonstrate that expression of the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) is reduced in synovial tissue from RA patients compared to osteoarthritis patients. Although one of the major functions of p21 is to arrest the cell cycle, we recently uncovered a novel role for p21 in the suppression of the inflammatory response in macrophages. Macrophages lacking p21 (p21-/-) secrete elevated levels of pro-inflammatory cytokines and display increased markers of activation following ligation by toll like receptor (TLR) agonists. Further, CDK2, a target for p21, is constitutively active in p21-/- macrophages while CDK4 is induced in Wt and p21-/- macrophages following TLR stimulation despite the fact that these cells are terminally differentiated and are withdrawn from the cell cycle. Mice lacking p21 exhibit an enhanced and sustained development of experimental inflammatory arthritis which is associated with markedly increased numbers of macrophages and severe articular destruction. Further, administration of a p21 peptide mimetic suppresses activation of macrophages and prevents development of experimental arthritis. Mechanistically, the increased activation in p21-/- macrophages is associated with the reduction in the activity of the serine/threonine kinase Akt and an increase in the activation of the mitogen-activated protein kinase, p38. Treatment with the p21-peptide mimetic restores Akt and reduces p38 activity in macrophages. Based on these data, we hypothesize that decreased expression of p21 in macrophages leads to the enhanced production of pro-inflammatory molecules mediated by reduced levels of active Akt and increased phosphorylated Ask1 and p38, independent of CDK activation. Taken together, the data anticipated from these studies will be the first to reveal that in macrophages, p21 is apical in determining the fate of the TLR intracellular signaling cascade. Thus, the elucidation of the molecular mechanism governing p21 in macrophages is crucial for understanding the development and persistence of RA and for potential new therapeutic targets for ameliorating RA. Inflammatory diseases, including rheumatoid arthritis, are responsible for significant mortality and morbidity. Because treatments are often ineffective or must be discontinued due to side effects, a greater understanding of the mechanisms involved in the progression of disease is crucial to the development of more efficacious treatments. Analysis of synovial tissue sections from rheumatoid arthritis patients revealed a decrease in expression of a cell cycle inhibitor protein, p21. Further studies using a mouse model of arthritis showed that increasing the level of p21 reduces the severity of disease. These data indicate that p21 may be an inhibitor of arthritis and other inflammatory diseases. Therefore, our goal with these studies is to uncover the mechanism for p21-mediated inhibition of inflammation
Keywords: Adoptive Transfer; Adverse effects; Agonist; Apical; Arthritis; Autoimmune Diseases; base; biomarker; bone; Bone Marrow; Cartilage; CDC2 Protein Kinase; CDK2 gene; CDK4 gene; Cell Cycle; Cell Cycle Arrest; Cells; Collaborations; Cyclin-Dependent Kinases; cytokine; Data; Degenerative polyarthritis; Development; Disease; Disease Progression; Exhibits; Experimental Arthritis; Flow Cytometry; Goals; Immunoblot Analysis; Inflammation; Inflammatory; Inflammatory Response; inhibitor/antagonist; Interleukin-1; Knockout Mice; Ligation; macrophage; Macrophage Activation; MAPK14 gene; Mediating; mimetics; mitogen-activated protein kinase p38; Molecular; Morbidity - disease rate; Mortality Vital Statistics; mouse model; Mus; new therapeutic target; novel; oncoprotein p21; Pathogenesis; Pathology; Patients; Pattern; Peptides; Phenotype; Phosphorylation; Play; prevent; Production; Protein-Serine-Threonine Kinases; public health relevance; response; Rheumatoid Arthritis; Role; Severity of illness; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; small molecule; Synovial Fluid; Synovial Membrane; Testing; Therapeutic; Tissues; Toll-like receptors; Translating; upstream kinase
Relevance: . Inflammatory diseases, including rheumatoid arthritis, are responsible for significant mortality and morbidity. Because treatments are often ineffective or must be discontinued due to side effects, a greater understanding of the mechanisms involved in the progression of disease is crucial to the development of more efficacious treatments. Analysis of synovial tissue sections from rheumatoid arthritis patients revealed a decrease in expression of a cell cycle inhibitor protein, p21. Further studies using a mouse model of arthritis showed that increasing the level of p21 reduces the severity of disease. These data indicate that p21 may be an inhibitor of arthritis and other inflammatory diseases. Therefore, our goal with these studies is to uncover the mechanism for p21-mediated inhibition of inflammation
Project start date: 2003-07-07
Project end date: 2015-04-30
Budget start date: 1-MAY-2011
Budget end date: 30-APR-2012
PFA/PA: PA-07-070
5R01AR050250-07 (2011): $329400
ROLE OF BH3-DOMAIN PROTEINS IN THE EFFECTOR PHASE OF RA
R Harris, Associate Professor
Northwestern Universitycity: Chicago country: United States (us)
Grant 5R01AR054796-05 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: Rheumatoid arthritis (RA) is an autoimmune disease characterized by hyperplasia of the synovial lining, inflammation, and destruction of cartilage and bone. In RA, the balance between anti- and pro-apoptotic members of the Bcl-2 family may be shifted towards survival. While we demonstrated that the anti-apoptotic proteins, Bcl-2 and Mcl-1 are increased in RA synovial tissue as compared to controls, no study has examined the therapeutic potential of antagonizing Bcl-2 anti-apoptotic members in arthritis. The study of deficiencies in anti-apoptotic members of the Bcl-2 family is complicated by embryonic lethality or early post-natal death. In contrast, mice deficient in pro-apoptotic Bcl-2 members such as Bak, Bax, and Bim survive and reach adulthood. Since Bim is a critical activator of apoptosis by virtue of its sequestration of Bcl-2/Mcl- 1 and/or its activation of Bak and Bax, it is a potential target for treatment of RA. We demonstrated that expression of the apoptotic initiator Bim is markedly reduced in synovial tissue from RA compared to controls. Further, mice lacking Bim but not the downstream effectors Bak or Bax develop a more severe form of inflammatory arthritis. This exacerbated disease in Bim-/- mice is associated with decreased apoptosis, increased expression of pro-inflammatory molecules, and more macrophages in pannus. Bim-/- macrophages display elevated levels of IL-6, TNF1, and IL-12, enhanced expression of CD40 and CD69, and increased and sustained level of active p38 in response to stimulation with LPS. Based on these data, we hypothesize that the ratio of Bim to Bcl-2 and/or Mcl-1 serves as molecular rheostat that determines the extent of hyperplasia and activation of macrophages in the joint. We will use a pharmacological approach, a whole animal approach, and a cell-specific approach to identify how deficiency in Bim exacerbates inflammatory arthritis. These studies will potentially lead to novel therapeutic approaches to RA. A.1. Examine the effect of altering the association of Bcl-2 family members in the development of inflammatory arthritis. A.2. Determine the effect of loss of function in Bak, Bax, or Bcl-2 on the development of inflammatory arthritis in Bim-/- mice. A.3. Determine the effect of targeted deletion of Bim in monocytes and macrophages on the development of inflammatory arthritis. A.4. Determine the mechanism of Bim-mediated suppression of macrophage activation. The regulation of cell death and growth is vital for maintaining a balance in the human body. However, during the initiation and/or progression of the autoimmune disease, rheumatoid arthritis (RA), this balance is disrupted. In RA there is an increase in cellular growth and a concomitant decrease in cell death leading to an abnormal increase in the tissue that attaches to the cartilage/bone junction, the synovial lining. During RA, the synovial lining invades and destroys the adjacent cartilage and bone. Analysis of tissue from joints of patients with RA revealed that the number of macrophages, correlated with a worse prognosis. We demonstrated that the death signaling cascade mediated by the pro-death protein Bim is dysfunctional in macrophages from patients with RA. Additionally, we have shown that mice lacking Bim in all cell types develop a worse form of arthritis and that the macrophages from these mice are highly activated, meaning they produce significant amounts of deleterious factors that exacerbate the inflammation. We have developed a potential therapeutic molecule that consists of a portion of Bim, termed BH3 domain. We have now demonstrated that systemic delivery of BH3 peptides to mice prevents the development of inflammatory arthritis with no toxicity to the mice. These studies are the first to show the efficacy of BH3 peptides as a potential molecule for treatment of RA. Our immediate goal is to develop the BH3 peptide therapy as a front line treatment for RA and other rheumatic diseases
Keywords: Adult; Animals; Ankle; Apoptosis; Apoptotic; Arthritis; Autoimmune Diseases; base; Bax protein (53-86); BH3 Domain; bone; Cartilage; Cell Death; cell growth; cell type; Cells; Cessation of life; Collaborations; cytokine; Data; Development; Disease; Dominant-Negative Mutation; Edema; Embryo; Equilibrium; Family; Family member; Goals; Human body; Hyperplasia; Inflammation; Inflammatory; Injection of therapeutic agent; Interleukin-1; Interleukin-12; Interleukin-6; Invaded; Joints; K/BxN model; Lead; loss of function; macrophage; Macrophage Activation; MAP Kinase Gene; MAPK14 gene; Measures; Mediating; member; Modeling; Molecular; monocyte; mouse model; Mus; novel therapeutic intervention; outcome forecast; Patients; Peptides; Phase; prevent; Production; Protein Family; Proteins; Regulation; response; Rheumatism; Rheumatoid Arthritis; Role; Serum; Severities; Signal Transduction; Stimulus; Therapeutic; Tissues; TNFRSF5 gene; Toxic effect; tumor
Project start date: 2008-06-01
Project end date: 2013-05-31
Budget start date: 1-JUN-2011
Budget end date: 31-MAY-2012
PFA/PA: PA-07-070
5R01AR054796-05 (2011): $318859
A NOVEL AND IMPROVED MOUSE SYSTEM FOR STUDYING MACROPHAGE SPECIFIC GENE DELETION
R Harris
Northwestern University At Chicagocity: Chicago country: United States (us)
Grant 5R21AI092490-02 from National Institute Of Allergy And Infectious Diseases
Keywords: Ablation; Adult; Affect; Alleles; Animals; Automobile Driving; base; Biological; Biological Assay; Biology; Bone Marrow; Breeding; cell type; Cells; Communicable Diseases; Communities; cytokine; Development; Disadvantaged; Disease; Engineering; Enhancers; Enterobacteria phage P1 Cre recombinase; Event; Flow Cytometry; Fluorescence; Gene Deletion; gene function; Genes; Genetic Recombination; Goals; granulocyte; Histocompatibility Testing; Homeostasis; Human; Immune; Immune response; Immunity; improved; in vivo; Infection; Inflammation; Inflammatory; Inflammatory Response; Internal Ribosome Entry Site; ITGAM gene; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; macrophage; magnetic beads; Malignant Neoplasms; Masks; Mediating; Messenger RNA; Molecular; Molecular Biology; Muramidase; Mus; Myelogenous; novel; Pathway interactions; peripheral blood; Peritonitis; Phagocytes; Phenotype; Play; Population; prevent; Process; Production; Promotor (Genetics); Proteins; public health relevance; recombinase; Reporter; Reporter Genes; Research; Research Personnel; response; Role; Site; Sorting - Cell Movement; Specificity; System; Thioglycolates; Time; Tissues; Tomatoes; tool; Transcript; Transgenic Mice; Transgenic Organisms; Validation; Work
Relevance: Macrophages are essential for tissue homeostasis and contribute to disease. Therefore understanding macrophage biology is fundamental to advance current knowledge of these processes, which requires macrophage specific deletion of genes using the Cre recombinase. Because currently available mice expressing Cre in macrophages have severe disadvantages, our study will develop and characterize a new and substantially improved transgenic mouse with macrophage specific expression of an improved version of Cre, which will be shared with the scientific community
Project start date: 2010-12-15
Project end date: 2012-11-30
Budget start date: 1-DEC-2011
Budget end date: 30-NOV-2012
5R21AI092490-02 (2012): $171563