Role of nucleic acid-sensing TLRs in diverse autoantibody specificities and anti-nuclear antibody-producing B cells

Abstract

Nucleic acid (NA)-sensing TLRs (NA-TLRs) promote the induction of anti-nuclear Abs in systemic lupus erythematosus. However, the extent to which other nonnuclear pathogenic autoantibody specificities that occur in lupus and independently in other autoimmune diseases depend on NA-TLRs, and which immune cells require NA-TLRs in systemic autoimmunity, remains to be determined. Using Unc93b1(3d) lupus-prone mice that lack NA-TLR signaling, we found that all pathogenic nonnuclear autoantibody specificities examined, even anti-RBC, required NA-TLRs. Furthermore, we document that NA-TLRs in B cells were required for the development of antichromatin and rheumatoid factor. These findings support a unifying NA-TLR-mediated mechanism of autoantibody production that has both pathophysiological and therapeutic implications for systemic lupus erythematosus and several other humoral-mediated autoimmune diseases. In particular, our findings suggest that targeting of NA-TLR signaling in B cells alone would be sufficient to specifically block production of a broad diversity of autoantibodies.

FIGURE 1

NA-TLRs are required for development of lupus nephritis in MRL-Fas^lpr mice. (A) Protein concentration in urine (left panel) and GN scores (right panel) from 7- to 8-mo-old 3d and WT/Het MRL-Fas^lpr. Urine protein in female B6 mice was 2.5 ± 0.1 mg/ml (n = 4). (B) Representative periodic acid-Schiff–stained kidney sections of 7- to 8-mo-old WT/Het and 3d mice. Glomerular enlargement, deposits (indicated by arrow), and proliferative changes in the mesangium of WT, but not in 3d kidneys, can be observed. (C) IgG immunofluorescence staining and quantification of frozen kidney sections. Fluorescent intensity was normalized to glomerular area. Seven- to 8-mo-old WT/Het and 3d (6–13 mice/group). (D) Kaplan-Meier survival plot of F10 WT and 3d MRL-Fas^lpr mice (6–11 mice/group). Data representative of three independent experiments for (A)–(C). Survival study was carried out once. ⁺p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺p < 0.001.

FIGURE 2

Lupus-associated dermatitis in MRL- Fas^lpr mice is not significantly affected by absence of NA-TLRs. (A) Incidence of dermatitis in mice over time (9–11 mice/group). (B) Surface area of skin lesions at 7 mo (8–18 mice/group). (C) Blinded scores of immune infiltration, basement membrane changes, and follicle loss in skin sections (9–15 mice/ group). (D) Representative H&E sections of skin showing similar epidermal thickening and hyperkeratosis in 7- to 8-mo-old WT/Het and 3d skin compared with B6. IgA anti-Dsg3 levels (E) and IgG3 cryoglobulin levels (F) from 6-mo-old mice. Data representative of three independent experiments. ⁺⁺⁺p < 0.001.

FIGURE 3

Total isotype and subclass Ig concentrations in 3d MRL-Fas^lpr mice. Ig isotype and IgG subclass levels in WT/Het and 3d MRL-Fas^lpr mice and B6 controls at 6 mo (6–17 mice/group). One of three independent experiments is shown. ⁺p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺p < 0.001; p < 0.05 for WT/Het versus B6 for all Ig isotypes and IgG subclasses except IgG2b.

FIGURE 4

ANA specificities and IgM RF in MRL-Fas^lpr 3d mice. (A) Representative ANA staining from 6-mo-old Het and 3d MRL-Fas^lpr mice. (B) ANA scores for WT/Het and 3d MRL-Fas^lpr mice (10–14 mice/group). (C) IgM RF and IgM anti-chromatin (top panel) and IgG anti-chromatin, IgG anti-RNP, and IgG anti-Sm (bottom panel). Each symbol represents an individual mouse. Data representative of at least two independent experiments. ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001; p < 0.05 for WT/Het versus B6 for all autoantibodies in (C).

FIGURE 5

Nonnuclear autoantibody levels in 3d mice. (A) IgM anti-cardiolipin, –β2-GP1, and -MPO (top panel) and IgG anti-cardiolipin, –β2-GP1, -MPO, and -Dsg3 (bottom panel) in 6- to 7-mo-old WT/Het MRL-Fas^lpr, 3d MRL-Fas^lpr, and B6 mice. p < 0.01 for WT/Het versus B6 for all IgM and IgG autoantibodies. (B) Anti-RBC and splenomegaly in NZB WT/ Het and 3d mice. IgG direct Coomb test (n = 11–20 for WT/Het, 4–7 for 3d; p < 0.0001; left panel). Mean fluorescence intensities of IgG anti-RBCs (middle panel). Spleen weights from 10-mo-old mice (right panel). (C) Chromatin competition ELISA. Diluted sera from 6- to 7-mo-old WT/Het or 3d with IgG anti-cardiolipin, anti-MPO, or anti-chromatin specificity were added to ELISA plate wells with or without chromatin (6–17 mice/group). One of three independent experiments is shown. ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001.

FIGURE 6

The effect of NA-TLR deficiency on immune cells in lupus mice. (A) Spleen weights and splenocyte numbers of WT/Het and 3d mice (11–19 mice/group). (B) Percentages and numbers of major spleen cell populations in MRL-Fas^lpr WT/Het (gray bars) and 3d mice (white bars). (C) Percentages and numbers of IFN-γ⁺, IL-4⁺, or IL-17⁺ CD4⁺ T cells. (D) Percentages and numbers of splenic B cell subsets: FO, MZ, and ABCs. (E) Representative flow cytometry plots of splenic CD19⁺CD138⁻ B cell subsets defined by CD21 and CD23 in MRL-Fas^lpr WT and 3d mice at the indicated ages. ABC population is indicated by arrow to demonstrate age-related increase of this subset in WT mice. (F) Percentage of CD40^hi DCs in WT/Het or 3d MRL-Fas^lpr mice. Data from 7-mo-old mice. For (B)–(F), four to seven mice per group. Mean ± SEMs are shown. Data representative of three experiments. ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001.

FIGURE 7

Cell-intrinsic NA-TLR signaling effects on immune cell subsets and autoantibodies in lupus. (A) Proportion (%) of WT cell populations in the bone marrow (BM) and spleen in 1:1 mixed bone marrow chimeras of WT (Ly5a):3d (Ly5b) B6-Fas^lpr donors at 2 (white bars) and 3 (gray bars) mo after reconstitution. Normalized percentage (see Materials and Methods) of Ly5a⁺ WT cells are shown. Mean ± SEM with error bars for line graphs in only one direction to avoid overlap. For spleen B cell subsets, p < 0.05–0.001 for all points except PCs at 2 and 3 mo and FO B cells at 2 mo. (B) Allotype-specific IgM RF (anti-IgG1), total IgM in 1:1 mixed bone marrow chimeras of 7-mo-old WT (IgH^a):3d (IgH^b) B6-Fas^lpr donors. IgG2a anti-chromatin, total IgG2a serum concentrations, and IgG2a ANA scores in 1:1 mixed bone marrow chimeras of 4.5-mo-old WT (IgH^a):3d (IgH^b) B6-Fas^lpr donors. Five to six mice per group. Similar data were obtained from WT and 3d recipients, and results represent combined data from both types of recipients. Data representative of two independent experiments. ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001.