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Abstract
Background Spontaneous murine models of lupus-like disease are used to study the pathogenesis and genetic causes of SLE. Environmentally-induced lupus models (i.e. pristine-induced) are also valuable to study potential triggers of SLE. Both require extended observation. Some accelerants, (i.e. Type I IFN) are used to trigger earlier disease onset. We used a TLR 7/8 agonist (R848), previously reported to induce lupus-like disease in WT mice within weeks, as an accelerant in lupus prone NZM2410 mice.
Methods Female and male C57BL/6J mice (n=29) and pre-disease NZM2410 (n=31, 13–15wk) were treated with topical R848 (100 ug) or acetone vehicle 3x weekly for 8 weeks. 24 hour urine collection was performed to assess proteinuria by ELISA. Blood was collected to assess autoantibody levels, blood counts and renal function. Spleen and bone marrow (BM) cells were isolated for flow cytometric analysis of immune cell subsets. Spleen and renal tissue were analyzed histologically.
Results Compared to vehicle-treated mice, R848-treated B6 and NZM mice had profoundly enlarged spleens (p<0.009 and p<0.0001, respectively) and survival was significantly reduced (p<0.009 and p<0.001). Treated B6 mice trended towards a higher ANA (p=0.059), but not anti-dsDNA, while treated NZM mice had higher levels of ANA (p=0.07) and dsDNA (p=0.004). Serum IFN levels were increased in R848-treated NZM mice, correlating with earlier mortality. Albuminuria and renal pathology in treated NZM mice indicated acceleration of nephritis, but not sufficient to cause death. Consistent with this, there was no significant difference in serum BUN or creatinine between treated and untreated groups. Treated NZM mice had significantly reduced number and percent of B cells isolated from spleen compared with vehicle (4% vs. 40%), as well as T cells (8% vs. 31%) and pDCs (0.10% vs. 0.75%). CD11b+cells were significantly expanded (66% vs. 45%) in BM from treated NZM mice. IHC revealed a massive expansion of F4/80+cells in spleens from treated NZM. Pathology of spleens in B6 and NZM mice revealed extramedullary hematopoiesis and changes consistent with histiocytic sarcoma.
Treatment with Resiquimod (RQ) is associated with a marked proliferation of mononuclear cells consistent with histiocytic sarcoma. In acetone treated mice, the normal lymphoid follicular architecture of the spleen is maintained (top, low mag). In these animals, there are rare, scattered cells within the lymphoid follicles that exhibit strong staining for F4/80, consistent with histiocytic cells (bottom). In animals treated with RQ, there is effacement of the normal lymphoid follicles by a proliferation of mononuclear cells. In these mice, 80-90% of spleen cells exhibit strong punctate to diffuse cytoplasmic staining with F4/80 (bottom) consistent with histiocytic cells. The top images are stained with H&E. The bottom images are stained with an F4/80 antibody using a red chromogen and DAB counterstainTreatment of lupus prone mice with R848/resiquimod (RQ) is associated with a marked proliferation of mononuclear cells in spleen consistent with histiocytic sarcoma.
Conclusions Topical TLR7/8 agonist treatment induced mild autoimmunity in B6 mice and accelerated autoimmunity in NZM2410 mice. Both had a severe immunophenotype and early death most consistent with malignant histiocytosis. Renal disease was accelerated in NZM2410 mice but was not the cause of death. Care should be taken in using TLR7/8 as a disease accelerant in NZM2410 mice as data suggest that this strain is vulnerable to death hastened by myeloprolferative disease rather than nephritis.
Funding Source(s): NIH NIAMS K08AR068471