Method: Mice with caspase 8 flanked by loxP sites (Casp8flox/flox) were crossed with mice expressing Cre under control of either the lysozyme M gene promoter (CreLysM), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage, or the CD11c gene promoter (CreCD11c), which is expressed by dendritic cells. Both CreLysMCasp8flox/flox and CreCD11cCasp8flox/flox mice were verified by RT-PCR. Flow cytometric analysis was employed to characterize both myeloid and lymphoid cell distribution and activation in bone marrow, blood, lymph node, and spleen. Luminex-based assays and ELISAs were used to detect serum cytokine and Ig levels. Immunohistochemical staining revealed kidney pathology.
Result: With age, both CreCD11cCasp8flox/flox and CreLysMCasp8flox/flox mice presented with a break in tolerance, as indicated by splenomegaly, lymphadenopathy, and autoantibody production, though these phenotypes were more exaggerated in CreCD11cCasp8flox/flox mice. While central and peripheral lymphoid organ analysis revealed that this break occurs via peripheral mechanisms, it also strikes early in development. Peripherally, both myeloid cell and DC-specific loss of caspase 8 not only increased circulating granulocytes and Gr-1+ monocytes, as well as splenic and lymph node antigen presenting cells. Additionally, increased peripheral effector T cells coincided with decreased peripheral naïve T cell populations. Moreover, not only did loss of caspase 8 in DCs and myeloid cells intrinsically amplified DC and macrophage activation, respectively, while affecting activation of other immune cells in a paracrine fashion. In an in vitro antigen-specific MLR, CreCD11cCasp8flox/flox DCs induced T cell proliferation, while CreLysMCasp8flox/flox macrophages inhibited proliferation. Elevated serum IL-12, TNFα, sRANKL levels were common to both strains, though CreCD11cCasp8flox/flox mice presented with heightened IgG2b levels and more severe kidney pathology.
Conclusion: These results demonstrate that while loss of caspase 8 in both DCs or myeloid cells initiates inflammatory phenotypes, intact caspase 8 signaling appears to be more crucial in DCs than in myeloid cells to prevent systemic autoimmunity. These data have implications for autoimmunity by elucidating previously unknown functions of a potentially useful target for therapy.
Disclosure: C. M. Cuda, None; J. Chowaniec, None; J. Hutcheson, None; G. K. Haines III, None; C. Mohan, None; H. R. Perlman, None.