Development of a new humanized mouse model to study acute inflammatory arthritis

Abstract

Background: Substantial advances have been generated in understanding the pathogenesis of rheumatoid arthritis (RA). Current murine models of RA-like disease have provided great insights into the molecular mechanism of inflammatory arthritis due to the use of genetically deficient or transgenic mice. However, these studies are limited by differences that exist between human and murine immune systems. Thus, the development of an animal model that utilizes human immune cells, will afford the opportunity to study their function in the initiation and propagation of inflammatory arthritis.

Methods: One to two-day old irradiated NOD-scid IL2rγ(null) (NSG) mice were reconstituted with human CD34+ cord blood stem cells. Leukocytes were analyzed by flow cytometry and circulating antibodies were determined by ELISA. Arthritis was induced by injecting complete Freund's adjuvant into knee or ankle joints. Mice were also treated with the TNF inhibitor, Etanercept, or PBS and joints were analyzed histologically.

Results: Humanized mice were established with high reconstitution rates and were able to spontaneously produce human immunoglobulins as well as specific IgG in response to immunization. Intraperitoneal injection of thioglycolate or injection of complete Freund's adjuvant into joints resulted in migration of human immune cells to the injected sites. Arthritic humanized mice treated with Etanercept had markedly less inflammation, which was associated with decreased total numbers of human CD45+ cells, including human lymphocytes and neutrophils.

Conclusions: The humanized mouse model is a new model to study inflammatory arthritis disease using human leukocytes without rejection of engrafted tissue. Future studies may adapt this system to incorporate RA patient cord blood and develop a chimeric animal model of inflammatory arthritis using genetically predisposed immune cells.

Figure 1

Multilineage development of the human immune system in humanized mice. Samples were prepared for flow cytometry as described in the Materials and Methods. Dead cells and debris were excluded on FSC-A vs. SSC-A dot plot, doublets were excluded on FSC-A vs. FSC-H dot plots (not shown). (A) Multilineage development in bone marrow of a 12-week old humanized mouse. Both myeloid and lymphoid lineages were present. (B) Peripheral blood of a 12-week old “humanized” mouse. Mature forms of B cells (CD19+), CD4 and CD8 T cells, monocytes (CD14+) and neutrophils (CD15) can be easily identified. (C) Spleen of a 12-week old “humanized” mouse: CD4+ and CD8+ T cells, CD19 B cells, macrophages (CD19-CD68+) and NK cells (CD56+) are shown. (D) Immunohistochemical staining for human T cell markers, CD4 and CD8, B cell marker, CD20, and macrophage marker, CD68, in the spleen of 12-week old humanized mouse. Magnification 20x. Data are representative of >20 mice.

Figure 2

Characterization of the humanized mouse. (A) Spontaneous production of immunoglobulins by 16-week old “humanized” mice. Serum from a non-reconstituted NSG mouse was used as a negative control. (B) Production of the anti-HIB immunoglobulin 4 weeks after the third immunization with Act-HIB. (C) Human immune cells were found in the peritoneal cavity 72 hours after intraperitoneal injection of thioglycollate. All mature cell types, including T and B cells and macrophages were present.

Figure 3

Histopathological changes in joints of humanized mice 7 days after the initiation of the CFA-induced arthritis. (A) H&E staining showing CFA-induced infiltration of the soft tissues of humanized mice treated with Etanercept (left) and PBS (right), magnification 20x. SL = synovial lining. Arrow denotes invasion site. (B) Histopathological scores for pannus formation, inflammation, and bone erosion in CFA-treated mice. (C) Immunohistochemistry for human CD45, human CD68, mouse CD45, and mouse F4/80 in humanized mice treated with PBS (upper row) and Etanercept (lower row). Magnification 40x. (D) Immunohistopathological scores for infiltration of human CD45+ cells, human CD68+ cells, mouse CD45+ cells, mouse F4/80+ cells, neutrophils, and lymphocytes in humanized mice treated with PBS or Etanercept. Data represents 5 (Etanercept) and 3 (PBS) mice/group.