TLR7 Agonism Accelerates Disease and Causes a Fatal Myeloproliferative Disorder in NZM 2410 Lupus Mice

Abstract

Murine models of lupus, both spontaneous and inducible, are valuable instruments to study SLE pathogenesis. Accelerants such as Type I IFN are often used to trigger earlier disease onset. We used a topical TLR7 agonist, previously reported to induce lupus-like disease in WT mice within weeks, to validate this data in C57BL/6j mice, and to test TLR7 agonism as an accelerant in lupus-prone NZM2410 mice. We found that TLR7-stimulated B6 and NZM2410 mice had significantly reduced survival and exhibited profound splenomegaly with significantly reduced B cells (4 vs. 40%), and T cells (8 vs. 31%). Spleen pathology and IHC revealed massive expansion of F4/80+ cells in TLR7-treated mice consistent with histiocytosis. While resiqimod treatment caused mild autoimmunity in B6 mice and accelerated autoimmunity in NZM2410 mice, it did not cause significant nephritis or proteinuria in either strain (renal function intact at death). Given the macrophage expansion, cytopenias, and disruption of normal splenic lymphoid follicle architecture, histiocytic sarcoma is favored as the cause of death. An alternative etiology is a macrophage activation syndrome (MAS)-like syndrome, since the mice also had a transaminitis and histologic hemophagocytosis in the setting of their rapid mortality. For investigators who are focused on murine models of lupus nephritis, this model is not ideal when utilizing B6 mice, however topical resiqimod may prove useful to accelerate autoimmunity and nephritis in NZM2410 mice, or potentially to investigate secondary complications of lupus such as histiocytic diseases or macrophage activation like syndromes.

Keywords: histiocytosis; interferon; lupus; mouse models; toll-like receptor (TLR)-7.

Figure 1

Survival of WT C57BL/6 and NZM2410 mice after treatment with a topical TLR7/8 agonist (R848, RQ). (A,B) TLR7/8 stimulation in both B6 and NZM mice resulted in significantly reduced survival. Median time to death in TLR7-stimulated NZM mice was also significantly accelerated vs. treated B6 WT mice. (C,D) TLR7-treated mice lost significant weight and (E) spleens were profoundly enlarged, with significant increases in spleen cell counts.

Figure 2

Autoimmunity and IFNα levels in TLR7-stimulated WT and NZM2410 mice. (A,B) We observed low +ANAs, but not anti-dsDNA Abs, in TLR7-stimulated B6 mice. NZM mice had both +ANAs and +anti-dsDNA Abs, as would be expected for age (16–23 weeks); serum levels of anti-dsDNA were significantly increased in TLR7-stimulated mice. (C,D) Female B6 and NZMs trended toward higher ANAs and anti-dsDNA Abs, respectively. (E,F) IFNα levels were also significantly increased in NZM mice and correlated with earlier death.

Figure 3

Despite accelerated death after TLR7/8 agonism, both B6 and NZM mice had minimal proteinuria. (A–C) TLR7-treated NZM mice had significantly increased 24 h albuminuria vs. vehicle-treated NZM and TLR7-treated B6, however, these levels were not elevated to the degree typically seen in lupus prone mice that succumb from renal failure (*terminal 24 h proteinuria in NZM2410 mice is usually >1–2 mg); albuminuria also did not correlate with earlier death. (D,E) Renal pathology demonstrated significantly higher glomerular scores (GS) for nephritis (proliferative lesions, mesangial expansion, etc.) in TLR7-treated NZM, but not to the degree seen in terminally nephritic mice (GS typically ~13–18), and mice that died earlier had milder nephritis.

Figure 4

TLR7-stimulated NZM2410 mice have normal serum BUN and creatinine, but have hematologic derangements. (A) We observed normal metabolic panels in TLR7-treated mice, except for significant hypoalbuminemia, and transaminitis in a subset of TLR7-treated mice. (B) TLR7-treated NZM mice were severely anemic, and had a leukocytosis, more consistent with malignancy, or chronic infection than lupus nephritis/ESRD (above). Most TLR-7 treated mice were also thrombocytopenic.

Figure 5

Spleen immunophenotype of topical TLR7-treated B6 and NZM mice. Flow cytometry revealed profound decreases in (A,C) CD19+ B cells and CD3+ T cells as well as (B,C) MHCII+ cells and pDCs from spleens from TLR7-stimulated B6 and NZM mice. Similar decreases in B cell counts, MHCII+ cells and pDCs were seen in bone marrow (BM) from these animals, while CD11b+ cells were significantly increased in BM (Supplemental Figure 2).

Figure 6

Topical treatment with TLR7/8 agonist is associated with marked proliferation of mononuclear cells in the spleen, consistent with histiocytic sarcoma. (A) In vehicle-treated mice, the normal lymphoid follicular architecture of the spleen is maintained whether B6 or NZM (top, low magnification) and only rare, scattered cells within the lymphoid follicles exhibit strong staining for F4/80, consistent with histiocytes/macrophages (middle). In TLR7(RQ)-stimulated mice, there is effacement of the normal lymphoid follicles by mononuclear cells (top, low magnification). In these mice, 80–90% of spleen cells exhibit strong staining with F4/80 (middle) consistent with histiocytic cells. These cells frequently exhibit hemophagocytosis (bottom image, arrows). The top and bottom images are stained with H&E. The middle images are stained with an F4/80 antibody using a red chromogen and DAB counterstain. (B) Semi-quantitation of spleen histiocytic infiltrate, extramedullary hematopoiesis, and hemophagocytosis between animal groups.