TLR7-Mediated Lupus Nephritis Is Independent of Type I IFN Signaling

Abstract

Systemic lupus erythematosus is an autoimmune disease characterized by increased type I IFNs, autoantibodies, and inflammatory-mediated multiorgan damage. TLR7 activation is an important contributor to systemic lupus erythematosus pathogenesis, but the mechanisms by which type I IFNs participate in TLR7-driven pathologic conditions remain uncertain. In this study, we examined the requirement for type I IFNs in TLR7-stimulated lupus nephritis. Lupus-prone NZM2328, INZM (which lack a functional type I IFN receptor), and NZM2328 IL-1β^-/- mice were treated at 10 wk of age on the right ear with R848 (TLR7 agonist) or control (DMSO). Autoantibody production and proteinuria were assessed throughout treatment. Multiorgan inflammation was assessed at the time of decline in health. Renal infiltrates and mRNA expression were also examined after 14 d of treatment. Both NZM2328 and INZM mice exhibited a decline in survival after 3-4 wk of R848 but not vehicle treatment. Development of splenomegaly and liver inflammation were dependent on type I IFN. Interestingly, autoantibody production, early renal infiltration of dendritic cells, upregulation of IL-1β, and lupus nephritis occurred independent of type I IFN signaling. Development of TLR7-driven lupus nephritis was not abolished by the deletion of IL-1β. Thus, although IFN-α is sufficient to induce nephritis acceleration, our data emphasize a critical role for IFN-independent signaling in TLR7-mediated lupus nephritis. Further, despite upregulation of IL-1β after TLR7 stimulation, deletion of IL-1β is not sufficient to reduce lupus nephritis development in this model.

Figure 1. Epicutaneous TLR 7 stimulation leads to type I IFN-independent accelerated decline in survival in lupus-prone mice

10-week-old NZM2328 and INZM mice were treated with 100μg of the TLR7 agonist R848 or control (DMSO) three times weekly. Survival curve for NZM2328 and INZM mice is shown. n=12 each for DMSO and R848 treated NZM mice. n=11 for DMSO and n=12 for R848 treated INZM mice. The p value in blue shows the difference in the survival curves between INZM DMSO vs. INZM R848 treated mice. The p value in red shows the difference in the survival curves between NZM DMSO vs. NZM R848 treated mice. The p value in black shows the difference in the survival curves between NZM R848 and INZM R848 treated mice

Figure 2. TLR 7 stimulation leads to IFN-independent elevated autoantibody production and IFN dependent splenomegaly and liver inflammation

10 week-old NZM2328 and INZM mice treated with R848 or DMSO control were analyzed. A. Total IgG in serum of NZM2328 and INZM mice after 2 weeks of treatment. Each dot represents an individual mouse. B. dsDNA IgG in serum at 0 weeks, 2 weeks, and 4 weeks of treatment. n=13 NZM R848; n=14 NZM DMSO; n= 9 INZM DMSO; n=11 INZM R848 C. Spleen weight of NZM2328 and INZM mice from survival studies. Spleens were harvested when mice were moribund (around 20–40 days of treatment). Littermate DMSO controls were harvested at the same times as the moribund mice. Each dot represents an individual mouse. Representative photographs of DMSO and R848 NZM2328 spleens shown in inset. D. Immune cell populations in the spleen were evaluated by flow cytometry after 2 weeks of R848 or DMSO treatment. n=13 NZM R848; n=11 NZM control; n=8 INZM control; n=9 INZM R848. (E and F) 10 week- old NZM2328 and INZM mice treated with R848 or DMSO control were treated until moribund and analyzed for development of liver inflammation. E. Representative photo of the portal vein. F. Graph represents liver inflammation scoring of NZM2328 and INZM mice. Each symbol represents one mouse.

Figure 3. TLR 7 stimulation leads to IFN-independent increases in secreting cells in the draining lymph nodes and the spleen

Immune cell populations in the spleen were evaluated by flow cytometry after 2 weeks of R848 or DMSO treatment. n= 5 NZM DMSO; n= 5 NZM R848; n=5 INZM DMSO; n=5 INZM R848. A. Gating strategy for Ab-secreting cells. B, C. Graphs displaying changes in B cells: CD4⁻CD8⁻IgH⁺IgL⁺B220⁺ (shown in B) and Ab-secreting cells: CD4⁻CD8⁻ IgH⁺IgL^high (shown in C). D. Total number of cells isolated from indicated lymph nodes for DMSO or R848 treated mice. E, F. Graphs displaying changes in B cells: CD4⁻CD8⁻IgH⁺IgL⁺B220⁺ (shown in E) and Ab-secreting cells: CD4⁻CD8⁻ IgH⁺IgL^high (shown in F) for draining (cervical) and non-draining (inguinal) LN. Data is displayed as mean ± SD. Each symbol represents one mouse. LN=lymph node.

Figure 4. TLR 7- mediated lupus nephritis occurs in an interferon-independent manner

10 week- old NZM2328 and INZM mice treated with R848 or DMSO control were analyzed for development of lupus nephritis. A. Urine alb/cr ratio was measured serially in NZM2328 treated mice n=12 NZM R848; n=15 NZM DMSO B. Urine alb/cr ratio was measured serially in INZM treated mice. n=5 INZM DMSO; n=6 INZM R848 C. Representative photo of the glomeruli in the kidney of NZM2328 and INZM mice following treatment until moribund. Littermate control mice were harvested at the time of illness in R848 treated mice. Scale bar equals 20um. D. Renal activity score for NZM2328 and INZM mice after 2 weeks of treatment or when moribund from R848 treatment (long-term treatment). E. The moribund renal activity score for NZM2328 and INZM mice treated with R848 and DMSO was plotted versus the alb/cr ratio at euthanasia and analyzed via Pearson correlation. F. Renal Chronicity index for NZM2328 and INZM mice after 2 weeks of treatment and when moribund (long-term treatment).

Figure 5. Immune complex deposition in the kidney is interferon-independent

10 week- old NZM2328 and INZM mice treated with R848 or DMSO until moribund and were analyzed for immune complex deposition. A. Representative immunofluorescence microscopy of glomeruli (outlined by white dashed line). Texas Red- IgG, Green- C3, Blue- DAPI. (B–C). Quantification of immune complex staining/area was completed. Littermate DMSO controls were harvested when littermates were ill. B. Quantification of IgG/area. C. Quantification of C3/area. Each dot represents the average fluorescence of 8 glomeruli from a single mouse.

Figure 6. TLR 7-mediated upregulation of IL-1β is not required for lupus nephritis

A–C. RNA was isolated from the kidney of NZM or INZM mice treated for two weeks with DMSO control or R848. Real-time PCR was completed using primers for the genes listed. Graphs display the mean±SD for each gene as compared to the average of b-actin. Each dot represents an individual mouse. D.10 week- old NZM IL-1β KO mice were treated with R848 or DMSO control and survival was plotted. n=9 NZM IL-1β −/− DMSO; n= 8 NZM IL-1β −/− R848 E. Anti-dsDNA IgG in serum at 2 weeks of treatment. n=8 NZM IL-1β −/− R848; n=8 NZM IL-1β −/− DMSO. F. Representative photomicrograph of the glomeruli in the kidney of moribund NZM IL-1β −/− mice treated with R848 or DMSO. Littermate controls were harvested when R848 treated mice were moribund. Scale bar equals 20um. G. Renal activity score for NZM IL-1β−/− mice when moribund. H. Renal chronicity index for mice in G.

Figure 7. Early dendritic cell infiltration is a common feature of TLR7 driven nephritis in NZM and INZM mice

Immune cell population changes in the kidney of NZM and INZM mice following 2 weeks of R848 or DMSO treatment. A. Gating strategy for immune cell populations. B–E. Graphs displaying changes in T cells (B): CD3+, B cells (C): CD19+, Macrophages (D): CDllci^ntCDllb⁺F480⁺, and Dendritic Cells (E): CDllc⁺CDllb⁺F480⁻. n=12 NZM DMSO; n=13 NZM R848; n=8 INZM DMSO; n=8 INZM R848. Data is displayed as mean ± SD.

Figure 8. CCL2 is upregulated in NZM and INZM mice after TLR7 stimulation

NZM and INZM mice were treated with R848 or DMSO for two weeks followed by harvest of RNA from the kidney. Real-time PCR was completed for the genes listed. Graphs display the mean ± SD for each gene as compared to the average of β-actin. Each dot represents a single mouse.