Overview
Spontaneous mutations that cause defects in immunological function in mice have provided important research tools with which to investigate the development and regulation of the immune system. A number of these mutations serve as models for specific human diseases and provide critical tools to increase the understanding of human immunodeficiency diseases, autoimmunity and leukemia. Spontaneous as well as targeted immunological mutations also provide mouse models that support engraftment with human stem cells and with human peripheral blood lymphocytes. These "humanized" mice have provided critical models for experimental investigation of human immune diseases without putting individuals at risk. Our recent research has leveraged these mouse models for studies on diabetes, bone development, autoimmunity, malignancy, anemia, thrombocytopenia, infectious diseases, transplantation tolerance and asthma.
Research details
Development of Humanized SCID Mice and Studies of Single Gene Models for Human Hematological Diseases
Advances in our understanding of the development and regulation of the immune system in normal and pathologic states rely heavily on studies of mice with genetically determined immunodeficiency and autoimmunity. Research in our laboratory encompasses (1) basic studies focused on elucidating the mechanisms underlying immunodeficiency, autoimmunity, and hematological diseases; and (2) applied studies focused on the development of effective immunodeficient mouse models that support heightened engraftment with human hematopoietic stem cells. These "humanized SCID mice" will facilitate studies of human immunity, regenerative medicine, and neoplasia. Examples of the studies carried out over the last year are summarized below:
Development of Humanized Mice for Translational Biomedical Research
Complex biological processes often require in vivo analysis, and important research advances have been obtained using mice as a model system for the study of many biological systems. However, mice are not humans, and the study of human biology in vivo is severely limited by ethical and technical constraints. There is a growing need for animal models to carry out in vivo studies on human cells, tissues, and organs without putting individuals at risk. Humanized mice, or mouse–human chimeras, have been developed to overcome these limitations and have become an important research tool for the in vivo study of human cells and tissues. We have developed a NOD-Prkdcscid (NOD/SCID) mouse strain harboring a null mutation of the common cytokine receptor γ chain (Il2rgtm1Wjl) (NOD/SCID/IL2rγnull) (full name NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). In collaboration with Dale Greiner and his colleagues at The University of Massachusetts Medical School along with Fumihiko Ishikawa and his colleagues at The Riken Research Center for Allergy and Immunology, we have shown that these mice can efficiently support development of all myelo-erythroid components and functional lymphoid cells following human hematopoietic stem cell (HSC) engraftment. Development of mice that are ‘humanized’ by engraftment of human tissues, HSCs, and peripheral-blood mononuclear cells (PBMCs) provides an opportunity to study human biological processes in vivo that would otherwise not be possible. The new generations of humanized mice are proving to be powerful tools in preclinical testing and in the investigation of many human biological processes. The culmination of decades of research on humanized mice is leading to advances in our understanding of human hematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, and regenerative medicine. The potential for new advances in our understanding of human biological systems provided by studies in humanized mice remains promising. The development of humanized mice based on immunodeficient IL2rγnull hosts has overcome many of the limitations and constraints of previously available models.
The Mouse Mutation "Thrombocytopenia and Cardiomyopathy" (trac) is within the Abcg5 Gene: A Single Gene Model for Human Mediterranean Macrothrombocytopenia
Homozygosity for a new spontaneous mouse mutation named "thrombocytopenia and cardiomyopathy" (trac) results in thrombocytopenia, dilated cardiomyopathy, and infertility. A/J-trac/trac mice show a precipitous drop in platelet numbers and increases in platelet volume by 4 weeks of age. By 2-3 months of age, trac/trac mice have a 20-fold decrease in platelet number, a 3-fold increase in platelet volume, and a greatly increased bleeding time. Blood smears showed abnormally large platelets and megakaryocytoid cells. The trac/trac mice also developed mild microcytic anemia accompanied by the presence of stomatocytes, a doubling of reticulocyte numbers, and a two-fold decrease in WBC counts. Increased numbers of megakaryocytes were present in bone marrow, spleen, and lungs. Ultrastructural studies of trac/trac megakaryocytes showed a poorly developed demarcation system and a failure to form platelet territories. The trac/trac platelets were enlarged, spherical, and contained numerous small alpha granules. The thrombocytopenia was not associated with defects intrinsic to bone marrow progenitor cells. Although thrombopoietin (TPO) levels were decreased, TPO treatment failed to reverse the thrombocytopenia. To identify the responsible gene, we produced a fine-structure genetic map of a 5-megabase interval containing the trac locus on mouse chromosome 17. The human syntenic region is Chr 2p21-p22. Analyses of 1100 F2 progeny from intercross matings of (A/J x C57BL/6J) F1 +/trac mice narrowed the interval to 0.3 Mb containing 4 genes. Sequencing of these genes revealed a G-to-A mutation at base 1435 (refseq nm031884) of Abcg5 (ATP-binding cassette sub-family G, member 5). This G>A base change results in a tryptophan codon (UGG) at amino acid position 463(uniprot) being changed to a premature stop codon (UAG). The transmembrane helices prediction program, TMHMM, predicts that the premature stop codon would truncate the last four of the six transmembrane domains of the ABCG5 protein. No DNA alterations were found in any of the other candidate genes. Genetic crosses of +/trac mice with mice doubly transgenic for the closely linked human ABCG5 and ABCG8 genes (stock B6SJL-Tg(ABCG5/ABCG8)14-2Hobb/J) showed that the transgenes normalized platelet counts and volumes in trac/trac mice. ABCG5 (sterolin-1) functions as part of a heterodimer, with ABCG8, that regulates plant sterol uptake. The trac/trac mutant mice have greatly elevated plasma levels of plant sterols. When placed on a phytosterol-free diet, the thrombocytopenia was reversed. Recent studies have shown that Mediterranean Macrothrombocytopenia is caused by mutations in ABCG5 or ABCG8. Identification of the molecular basis of the mouse Abcg5trac mutation provides a new model for studying the role of phytosterols in pathogenic changes in the hematopoietic, cardiovascular, and reproductive systems.
Lab staff
Principal Investigator: Leonard D. Shultz, Ph.D.Postdoctoral Fellows: Thomas H. Chase, D.V.M., Melissa L. Cox
Research Assistant II: Lisa Carney, B.S.
Research Assistant I: Bruce Gott
RAF Primary Room Technician: Allison Ingalls
Research Administrative Assistant: Norma Buckley
Publication listings
Dash Y, Ramesh M, Greiner D, Shultz LD, Klei TR, Rajan TV. 2007. Determinants of memory in experimental filarial infections in mice. Parasite Immunol 29:567-574.
Ishikawa F, Niiro H, Iino T, Yoshida S, Saito N, Onohara S, Miyamoto T, Minagawa H, Fujii SI, Shultz LD, Harada M, Akashi K. 2007. The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways. Blood 110: 3591-3600.
Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, Nakamura R, Tanaka T, Tomiyama H, Saito N, Fukata M, Miyamoto T, Lyons B, Ohshima K, Uchida N, Taniguchi S, Ohara O, Akashi K, Harada M, Shultz LD. 2007. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 25: 1315-1321.
King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, Atkinson M, Wasserfall C, Herold K, Mordes JP, Rossini AA, Greiner DL. 2007. Development of new generation HU-PBMC-NOD/SCID mice to study human islet alloreactivity. Ann N Y Acad Sci 1103: 90-93.
King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, Atkinson MA, Wasserfall C, Herold KC, Woodland RT, Schmidt MR, Woda BA, thompson MJ, Rossini AA, Greiner DL. 2007. A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene. Clin Immunol, doi: 10.1016/j.clim.2007.11.001.
Meyerrose TE, De Ugarte DA, Hofling AA, Herrbrich PE, Cordonnier TD, Shultz LD, Eagon JC, Wirthlin L, Sands MS, Hedrick MA, Nolta JA. 2007. In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models. Stem Cells 25: 220-227.
Ramesh M, Paciorkowski N, Dash Y, Shultz L, Rajan TV. 2007. Acute but not chronic macrophage recruitment in filarial infections in mice is dependent on C-C chemokine ligand 2. Parasite Immunol 29: 395-404.
Seymour RE, Hasham MG, Cox GA, Shultz LD, Hogenesch H, Roopenian DC, Sundberg JP. 2007. Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis. Genes Immun 8: 416-421.
Shultz LD, Ishikawa F, Greiner DL. 2007. Humanized mice in translational biomedical research. Nat Rev Immunol 7: 118-130.
Shultz LD, Pearson T, King M, Giassi L, Carney L, Gott B, Lyons B, Rossini AA, Greiner DL. 2007. Humanized NOD/LtSz-scid IL2 receptor common gamma chain knockout mice in diabetes research. Ann N Y Acad Sci 1103: 77-89.
Thornley TB, Phillips NE, Beaudette-Zlatanova BC, Markees TG, Bahl K, Brehm MA, Shultz LD, Kurt-Jones EA, Mordes JP, Welsh RM, Rossini AA, Greiner DL. 2007. Type 1 IFN mediates cross-talk between innate and adaptive immunity that abrogates transplantation tolerance. J Immunol 179: 6620-6629.
Yamazaki M, Pearson T, Brehm MA, Miller DM, Mangada JA, Markees TG, Shultz LD, Mordes JP, Rossini AA, Greiner DL. 2007. Different mechanisms control peripheral and central tolerance in hematopoietic chimeric mice. Am J Transplant 7: 1710-1721.
Adachi Y, Oyaizu H, Taketani S, Minamino K, Yamaguchi K, Shultz LD, Iwasaki M, Tomita M, Suzuki Y, Nakano K, Koike Y, Yasumizu R, Sata M, Hirama N, Kubota I, Fukuhara S, Ikehara S. 2006. Treatment and transfer of emphysema by a new bone marrow transplantation method from normal mice to Tsk mice and vice versa. Stem Cells 24: 2071-2077.
Ishikawa F, Shimazu H, Shultz LD, Fukata M, Nakamura R, Lyons B, Shimoda K, Shimoda S, Kanemaru T, Nakamura K, Ito H, Kaji Y, Perry AC, Harada M. 2006. Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion. Faseb J 20: 950-952.
Lyons BL, Smith RS, Hurd RE, Hawes NL, Burzenski LM, Nusinowitz S, Hasham MG, Chang B, Shultz LD. 2006. Deficiency of SHP-1 protein-tyrosine phosphatase in "viable motheaten" mice results in retinal degeneration. Invest Ophthalmol Vis Sci 47: 1201-1209.
Masaki H, Appel MC, Leahy L, Leif J, Paquin L, Shultz LD, Mordes JP, Greiner DL, Rossini AA. 2006. Anti-mouse CD154 antibody treatment facilitates generation of mixed xenogeneic rat hematopoietic chimerism, prevents wasting disease and prolongs xenograft survival in mice. Xenotransplantation 13: 224-232.
Mikaelian I, Hovick M, Silva KA, Burzenski LM, Shultz LD, Ackert-Bicknell CL, Cox GA, Sundberg JP. 2006. Expression of terminal differentiation proteins defines stages of mouse mammary gland development. Vet Pathol 43: 36-49.
Taube C, Miyahara N, Ott V, Swanson B, Takeda K, Loader J, Shultz LD, Tager AM, Luster AD, Dakhama A, Gelfand EW. 2006. The leukotriene B4 receptor (BLT1) is required for effector CD8+T cell-mediated, mast cell-dependent airway hyperresponsiveness. J Immunol 176: 3157-3164.
Thornley TB, Brehm MA, Markees TG, Shultz LD, Mordes JP, Welsh RM, Rossini AA, Greiner DL. 2006. TLR agonists abrogate costimulation blockage-induced prolongation of skin allografts. J Immunol 176: 1561-1570.
Yoshino M, Yamazaki H, Shultz LD, Hayashi S. 2006. Constant rate of steady-state self-antigen trafficking from skin to regional lymph nodes. Int Immunol 18: 1541-1548.
Zhou J, Chen J, Zhong R, Mokotoff M, Shultz LD, Ball ED. 2006. Targeting gastrin-releasing peptide receptors on small cell lung cancer cells with a bispecific molecule that activates polyclonal T lymphocytes. Clin Cancer Res 12: 2224-2231.
Gordon EJ, Wicker LS, Peterson LB, Serreze DV, Markees TG, Shultz LD, Rossini AA, Greiner DL, Mordes JP. 2005. Autoimmune diabetes and resistance to xenograft transplantation tolerance in NOD mice. Diabetes 54: 107-115.
Helms C, Pelsue S, Cao L, Lamb E, Loffredo B, Taillon-Miller P, Herrin B, Burzenski LM, Gott B, Lyons BL, Keppler D, Shultz LD, Bowcock AM. 2005. The Tetratricopeptide repeat domain 7 gene is mutated in flaky skin mice: a model for psoriasis, autoimmunity, and anemia. Exp Biol Med 230: 659-667.
Huang Z, Coleman JM, Su Y, Mann M, Ryan J, Shultz LD, Huang H. 2005. SHP-1 regulates STAT6 phosphorylation and IL-4 mediated function in a cell type-specific manner. Cytokine 29: 118-124.
Ishikawa F, Yasukawa M, Lyons B, Yoshida S, Miyamoto T, Yoshimoto G, Watanabe T, Akashi K, Shultz LD, Harada M. 2005. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor g chain null mice. Blood 106: 1565-1573.
Jaeschke A, Rincon M, Doran B, Reilly J, Neuberg D, Greiner DL, Shultz LD, Rossini AA, Flavell RA, Davis RJ. 2005. Disruption of the Jnk2 (Mapk9) gene reduces destructive insulitis and diabetes in a mouse model of type I diabetes. Proc Natl Acad Sci U S A 102: 6931-6935.
Kawano N, Ishikawa F, Shimoda K, Yasukawa M, Nagafuji K, Miyamoto T, Baba E, Tanaka T, Yamasaki S, Gondo H, Otsuka T, Ohshima K, Shultz LD, Akashi K, Harada M. 2005. Efficient engraftment of primary adult T-cell leukemia cells in newborn NOD/SCID/b 2-microglobulin null mice. Leukemia 19: 1384-1390.
Macchiarini F, Manz MG, Palucka AK, Shultz LD. 2005. Humanized mice: are we there yet? J Exp Med 202: 1307-1311.
Minamiguchi H, Wingard JR, Laver JH, Mainali ES, Shultz LD, Ogawa M. 2005. An assay for human hematopoietic stem cells based on transplantation into nonobese diabetic recombination activating gene-null perforin-null mice. Biol Blood Marrow Transplant 11: 487-494.
Park IK, Shultz LD, Letterio JJ, Gorham JD. 2005. TGF-b 1 inhibits T-bet induction by IFN-g in murine CD4+ T cells through the protein tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1. J Immunol 175: 5666-5674.
Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M, Mangada J, Greiner DL, Handgretinger R. 2005. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174: 6477-6489.
Ueno M, Lyons BL, Burzenski LM, Gott B, Shaffer DJ, Roopenian DC, Shultz LD. 2005. Accelerated wound healing ofalkali-burned corneas in MRL mice is associated with a reduced inflammatory signature. Invest Ophthalmol Vis Sci 46: 4097-4106.
Yoshida S, Ishikawa F, Kawano N, Shimoda K, Nagafuchi S, Shimoda S, Yasukawa M, Kanemaru T, Ishibashi H, Shultz LD, Harada M. 2005. Human cord blood--derived cells generate insulin-producing cells in vivo. Stem Cells 23: 1409-1416.
Appel MC, Banuelos SJ, Greiner DL, Shultz LD, Mordes JP, Rossini AA. 2004. Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD154 antibody. Transplantation 77: 1341-1349.
Banuelos SJ, Markees TG, Phillips NE, Appel MC, Cuthbert A, Leif J, Mordes JP, Shultz LD, Rossini AA, Greiner DL. 2004. Regulation of skin and islet allograft survival in mice treated with costimulation blockade is mediated by different CD4+ cell subsets and different mechanisms. Transplantation 78: 660-667.
Banuelos SJ, Shultz LD, Greiner DL, Burzenski LM, Gott B, Lyons BL, Rossini AA, Appel MC. 2004. Rejection of human islets and human HLA-A2.1 transgenic mouse islets by alloreactive human lymphocytes in immunodeficient NOD-scid and NOD-Rag1nullPrf1null mice. Clin Immunol 112: 273-283.
Hayashi S, Tsuneto M, Yamada T, Nose M, Yoshino M, Shultz LD, Yamazaki H. 2004. Lipopolysaccharide-induced osteoclastogenesis in Src homology 2-domain phosphatase-1-deficient viable motheaten mice. Endocrinology 145:2721-2729.
Ishikawa F, Yasukawa M, Yoshida S, Nakamura K, Nagatoshi Y, Kanemaru T, Shimoda K, Shimoda S, Miyamoto T, Okamura J, Shultz LD, Harada M. 2004. Human cord blood-and bone marrow-derived CD34+ cells regenerate gastrointestinal epithelial cells. Faseb J 18: 1958-1960.
Kuzin II, Ugine GD, Barth RK, Shultz LD, Nahm MH, Young FM, Bottaro A. 2004. A new murine model of humoral immuno-deficiency specfically affects class switching to T-independent antigens. Eur J Immunol 34: 1807-1816.
Makatsori D, Kourmouli N, Polioudaki H, Shultz LD, McLean K, Theodoropoulos PA, Singh PB, Georgatos SD. 2004. The inner nuclear membrane protein lamin receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope. J Biol Chem 279: 25567-25573.
Markees TG, Pearson T, Cuthbert A, Pearson AL, Shultz LD, Leif J, Phillips NE, Mordes JP, Greiner DL, Rossini AA. 2004. Evaluation of donor-specific transfusion sources: unique failure of bone marrow cells to induce prolonged skin allograft survival with anti-CD154 monoclonal antibody. Transplantation 78: 1601-1608.
Park JW, Taube C, Joetham A, Takeda K, Kodama T, Dakhama A, McConvill G, Allen CB, Sfyroera G, Shultz LD, Lambris JD, Giclas PC, Holers VM, Gelfand EW. 2004. Complement activation is critical to airway hyperresponsiveness after acute ozone exposure. Am J Respir Crit Care Med 169: 726-732.
Pearson T, Weiser P, Markees TG, Serreze DV, Wicker LS, Peterson LB, Cumisky AM, Shultz LD, Mordes JP, Rossini AA, Greiner DL. 2004. Islet allograft survival induced by costimulation blockade in NOD mice iscontrolled by allelic variants of Idd3. Diabetes 53: 1972-1978.
Taube C, Wei X, Swasey CH, Joetham A, Zarini S, Lively T, Takeda K, Loader J, Miyahara N, Kodama T, Shultz LD, Donaldson DD, Hamelmann EH, Dakhama A, Gelfand EW. 2004. Mast cells, Fc epsilon RI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant. J Immunol 172: 6398-6406.
