Background/Purpose

Various studies of the relationship between Epstein-Barr virus (EBV) and rheumatoid arthritis (RA) have not produced convincing evidence. Many human viruses do not infect mice; thus, it is difficult to conduct biomedical research. At this congress, we previously reported that EBV infection induces erosive arthritis that resembles RA in humanized NOD/Shi-scid/IL-2Rγ^null (NOG) mice. However, the mechanisms underlying arthritis in this mouse model are unknown.

In this mouse model, osteoclast-like cells are observed during bone erosion. To determine whether the human or mouse immune system activates bone erosion, we analyzed the origin of osteoclasts in this mouse model.

Methods

The NOG mouse is a highly immunodeficient mouse strain. Seven-week-old female NOG mice were intravenously injected with human CD34⁺ stem cells from cord blood (1.0 × 10⁵ cells/mouse). Characterization of human hematoimmune system reconstitution (we termed it humanization) was then performed. Human CD4⁺, CD8⁺, and CD45⁺ cells in peripheral blood of these humanized mice were quantified every week using flow cytometery. The engraftment rate of human cells and characteristics of lymphocytes were determined. After 3 months of humanization, these mice were intravenously infected with EBV (1.0 × 10¹ TD50/mouse). EBV was purified from EBV-producing cells (AKATA or B95-8). After 8-10 weeks of EBV infection, these mice were sacrificed. The joint tissue samples were stained with hematoxylin-eosin, stained for tartrate-resistant acid phosphatase (TRAP) with an immunoenzyme method, and immunostained for human cathepsin K (specific to humans and dogs). To let osteoclasts differentiate with progenitor cells, bone marrow cells from EBV-infected humanized NOG mice were cultured with human receptor activator of nuclear factor κB ligand (RANKL) and human macrophage colony-stimulating factor (M-CSF) in slide chambers. These stimulated multinucleated cells were subjected to TRAP staining and immunostaining for human cathepsin K and human mitochondria.

Results

After humanization, >40% of peripheral-blood lymphocytes in these mice were human CD45⁺ cells. When the number of human CD8⁺ T-cells increased and surpassed the number of human CD4⁺ T-cells in peripheral blood, erosive arthritis was observed histologically at a high rate (approximately 90%). Multinucleated cells present in the bone erosion zone were positive for human cathepsin K and TRAP staining. Multinucleated giant cells resembling osteoclasts were observed among cultured bone marrow cells stimulated with human RANKL and M-CSF. Human cathepsin K, mitochondrial, and TRAP staining were all positive in these multinucleated cells.

Conclusion

In this mouse model, we achieved a high engraftment rate. The relationship between the degree of arthritis and the ratio of CD4⁺ to CD8⁺ T-cell numbers in peripheral blood was evident. Osteoclasts present in the bone erosion zone originated from human cells. In addition, observed that multinucleated giant cells among bone marrow cells cultured with human RANKL and M-CSF were osteoclasts and originated from human cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/human-osteoclasts-are-mobilized-in-erosive-arthritis-of-epstein-barr-virus-infected-humanized-nodshi-scidil-2r%ce%b3null-mice/