TLR7 Activation Accelerates Cardiovascular Pathology in a Mouse Model of Lupus

Abstract

We report a novel model of lupus-associated cardiovascular pathology accelerated by the TLR7 agonist R848 in lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice. R848-treated TC mice but not non-autoimmune C57BL/6 (B6) controls developed microvascular inflammation and myocytolysis with intracellular vacuolization. This histopathology was similar to antibody-mediated rejection after heart transplant, although it did not involve complement. The TC or B6 recipients of serum or splenocytes from R848-treated TC mice developed a reactive cardiomyocyte hypertrophy, which also presents spontaneously in old TC mice as well as in TC.Rag^-/- mice that lack B and T cells. Each of these cardiovascular lesions correspond to abnormalities that have been reported in lupus patients. Lymphoid and non-lymphoid immune cells as well as soluble factors contribute to lupus-associated cardiovascular lesions in TC mice, which can now be dissected using this model with and without R848 treatment.

Keywords: TLR7; autoimmunity; cardiovascular disease; lupus; mouse models.

Figure 1

TLR7 activation induces cardiovascular pathology in TC mice. Representative H&E-stained heart sections of untreated and R848-treated B6 and TC mice at 20X (A) and 40X (B) magnification (scale bars: 50 um and 20 um, respectively) at week 3. (C) Representative H&E staining from R848-treated B6 and TC mouse at 40X showing in the TC mouse: vascular congestion (black arrow); vascular dilatation (green arrow); myocytolysis with vacuolization (yellow arrow); and capillary injury with enlarged endothelial cell (red arrow). Capillary injury (D), vascular congestion, vascular dilatation and myocytolysis scores respectively, R848-treated B6 mice were used as a control. (E) CD45 staining with representative sections on the left (20X magnification, scale bar: 200 um) and quantitation of the right (PPC: pixel per cell). n = 1 - 5 untreated mice, n = 5 - 12 treated mice per strain. t tests and 1-way ANOVA with multiple comparison tests, * P < 0.05, ** P < 0.01, *** P < 0.001; **** P < 0.001.

Figure 2

TLR7 activation induces immune cell infiltration in the heart of TC mice. (A) Representative heart sections stained for CD11b, CD3 and IgG2a (20X scale bars: 200 um). (B) Quantification of CD11b⁺ and CD3⁺ cells as well as IgG2a deposits (MFI, mean florescence intensity). (C) Representative heart sections co-stained with CD11b and CD43 (40X, scale bars: 50 um). (D) Quantification of CD11b⁺ and CD43⁺ infiltrates (MFI). Arrows indicate co-staining. n = 5 - 12 treated mice per strain. t tests, ** P < 0.01, **** P < 0.001.

Figure 3

Splenocytes or serum from R848-treated TC mice induced a reactive cardio-hypertrophy in B6 and TC recipients. (A) Representative H&E-stained heart sections of the indicated groups (top: 4X, scale bars 200um, and bottom: 40X, scale bars 20 um). (B) Myocardial hypertrophy scores. (C) Representative heart sections from TC controls as well as TC recipients of serum or splenocytes, and B6 recipients of splenocytes stained for IgG2a, CD11b and CD3 (20X, scale bars: 200 um). Quantification (MFI) of CD11b⁺ (D) and CD3⁺ (E) cells and IgG2a deposits (F). n = 3 - 4 per group. 1-way ANOVA with multiple comparison tests, * P < 0.05, ** P < 0.01, *** P < 0.001; **** P < 0.001.

Figure 4

TLR7 activation enhanced lymphocyte activation in TC mice. B6 and TC mice were treated with R848 for 1 week and their splenic CD4⁺ T cell and B cell phenotypes were compared to untreated age-matched control mice. (A) Splenocyte numbers. Frequency of CD4⁺ T cells expressing CD69 (B), presenting the CD62L^-CD44⁺ Tem phenotype (C), expressing IFNγ (D) or IL-10 excluding Foxp3⁺ cells, (E). Frequency of PD1^hiCXCR5⁺BCL-6⁺ Tfh cells (F), CD19⁺IgM^-IgD^-class-switched (CS) B cells (G), TBET⁺CD11c⁺ B cells ABCs, (H) CD138⁺B220^lo plasma cells PC, (I). In another experiment, B6 and TC mice were treated with R848 for 2 week and sacrificed at the end of week 3. T cells and myeloid cells phenotypes in the heart were compared to untreated control mice. (J) Number of Tfh cells; (K) Ratio of Tfh to Tfr cells; Numbers of CD11b⁺ cells (L), monocytes (M), neutrophils (N), and macrophages (O). n = 3 - 5 per group. t tests, *: P < 0.05, **: P < 0.01, ***: P < 0.001. ****: P < 0.0001.

Figure 5

TLR7 activation induced the production of anti-cardiovascular autoantibodies in TC mice. Serum IgG in control (n = 3) and R848-treated B6 and TC mice (n = 11 - 16) directed against dsDNA (A), CL (B), APOH (C) and heart tissue (D). Statistics compare treated TC and B6 mice with t test between week 1 values (A), and 2-way ANOVA for all values up to week 3 (B–D) *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 6

TLR7 activation enhanced myeloid cell activation in TC mice. B6 and TC mice were treated with R848 for 1 week and their splenic myeloid cell phenotypes were compared to untreated age-matched control mice. Frequency of neutrophils (A) and macrophages (B) in CD11b⁺ cells. Frequency of cDCs in splenocytes (C) and CD11b⁺ cells in cDCs (D). Expression of CD40 (E) and CD80 (F) on cDCs. Frequency of pDCs (G) and ipDCs (H). n = 4 - 5 per group. 1-way ANOVA, with multiple comparison tests, *: P < 0.05, **: P < 0.01, ****: P < 0.0001.

Figure 7

Both lymphoid and myeloid cells contribute to TC cardiovascular phenotypes. (A) Representative hearts from 9-months old B6 and TC mice and 6-months old B6.Rag^-/- and TC.Rag^-/- mice photographed at the same amplification. (B) Heart to tibia length ratios. (C) Representative H&E-stained heart sections (4X, scale bars: 200 um) of the indicative strain/conditions. (D) Myocardial hypertrophy scores. (E) Representative heart sections from 9 months old B6 and TC mice stained for IgG2a, CD11b and CD3 (20X, scale bars: 200 um). (F) Quantification of CD11b⁺ and CD3⁺ cells and IgG2a deposits (MFI). (G) Representative heart sections of 6-months old B6.Rag^-/- and TC.Rag^-/- mice, treated or not with R848, stained for CD11b (20X, scale bars: 200 um). (H) Quantification of CD11b⁺ cells (MFI). n = 3 - 10 mice per group, 1-way ANOVA, with multiple comparison tests, *: P < 0.05, **: P < 0.01, ***: P < 0.001. ****: P < 0.0001.

Figure 8

CD11b⁺ cell depletion decreased TLR-7-induced vascular injury. (A) Experimental design for pre-treatment with clodronate liposomes (CL) or PBS-loaded control liposomes (PBSL) before R848 treatment of pre-autoimmune TC mice. The time scale is in weeks and each tick corresponds to an injection of liposomes (bottom) or a R848 application (top). (B). Frequency of CD11b⁺ splenocytes. (C) Serum anti-dsDNA IgG starting at the time of CL/PBSL treatment. (D) Cardiac pathology scores. (E) Representative H&E-stained heart sections of TC mice treated with PBSL or CL (10X, scale bars: 100 um). (F) CD45 staining with representative sections on the left (20X magnification, scale bar: 200 um) and quantitation of the right. n = 3 per group, t tests, *: P < 0.05, **: P < 0.01.