Delayed onset of autoreactive antibody production and M2-skewed macrophages contribute to improved survival of TACI deficient MRL-Fas/Lpr mouse

Abstract

Anti-B cell activating factor belonging to TNF-family (BAFF) antibody therapy is indicated for the treatment of patients with active systemic lupus erythematosus (SLE). We hypothesized that the BAFF receptor, transmembrane activator and calcium-modulator and cyclophilin interactor (TACI) may be responsible for the generation of antibody secreting plasma cells in SLE. To test this hypothesis, we generated TACI deficient MRL-Fas/Lpr (LPR-TACI-/-) mouse. TACI deficiency resulted in improved survival of MRL-Fas/Lpr mice and delayed production of anti-dsDNA and anti-SAM/RNP antibodies. There was also a delay in the onset of proteinuria and the accumulation of IgG and inflammatory macrophages (Mϕs) in the glomeruli of young LPR-TACI-/- mice compared to wild-type mice. Underscoring the role of TACI in influencing Mϕ phenotype, the transfer of Mϕs from 12-week-old LPR-TACI-/- mice to age-matched sick wild-type animals led to a decrease in proteinuria and improvement in kidney pathology. The fact that, in LPR-TACI-/- mouse a more pronounced delay was in IgM and IgG3 autoreactive antibody isotypes and the kinetics of follicular helper T (Tfh) cell-development was comparable between the littermates suggest a role for TACI in T cell-independent autoantibody production in MRL-Fas/Lpr mouse prior to the onset of T cell-dependent antibody production.

Figure 1

TACI deficient MRL-Fas/Lpr mice manifest decreased lymphadenopathy and prolonged survival. (A) Representative histograms as well as mean percentages and mean fluorescence intensities (MFI) of TACI expression on splenic B cells (CD19+), plasmablasts (CD19 + CD138+) and plasma cells (PC) (CD19 − CD138+) of 6 to 8-week-old LPR-TACI+/+, LPR-TACI+/− and LPR-TACI+/− mice are shown. The data shown are from 5 female mice per group. (B) Lymphadenopathy assessed as enlarged lymph nodes over 40 weeks. LPR-TACI−/− mice had reduced lymphadenopathy compared to LPR-TACI+/+ and LPR-TACI+/− mice. (C) Percent survival rates over 42 months. The cumulative survival of female LPR-TACI+/+, LPR-TACI+/−, and LPR-TACI−/− mice was monitored daily for 42 weeks. TACI deficient MRL-Fas/Lpr mice exhibited significantly better survival curve compared with the two other groups. **p < 0.01 and ***p < 0.001 indicate statistical differences between LPR-TACI+/+ and LPR-TACI−/− mice. ^##p < 0.01 indicates statistical difference between LPR-TACI+/− and LPR-TACI−/− mice.

Figure 2

Serum autoreactive antibody development is delayed in TACI deficient MRL-Fas/Lpr mice. Titers of serum anti-dsDNA total IgG antibodies (A), anti-RNP/Sm antibodies (B), and anti-dsDNA IgM, IgG2a, IgG2b, IgG3 antibodies isotypes (C) were determined by ELISA. Titer is defined by the serum dilution exhibiting an OD reading 2 times higher than background. Mean titers ± SD from 5 to 7 mice in each group were plotted. Anti-dsDNA antibodies (D) and Antinuclear antibodies (E) were assessed by indirect immunofluorescence measurement. Serum anti-dsDNA antibodies were detected using C. luciliae slides and ANAs were detected by HEp-2 slides. The fluorescence intensity of single cell was quantified with ImageJ and plotted. The data shown are from 5 female mice per age group. *p < 0.05 and **p < 0.01 indicate statistical differences between LPR-TACI+/+ and LPR-TACI−/− mice. ^#p < 0.05 and ^##p < 0.01 indicate statistical differences between LPR-TACI+/− and LPR-TACI−/− mice.

Figure 3

The delayed kidney pathology is accompanied by less IgG and C3 deposition in TACI deficient MRL-Fas/Lpr mice. (A) TACI deficient MRL-Fas/Lpr mice showed a delayed proteinuria onset. Mean ± SD of proteinuria scores are plotted. The data shown are from 3 to 20-week-old mice. Each group contained 7 to 9 female mice. (B) Assessment of kidney pathology. Upper panel shows a representative image of H&E (upper row) and PAS (bottom row) stained kidney specimens from 14-week-old mice. Mesangial cell proliferation and increase of mesangial matrix with inflammatory cell infiltration were more frequently in LPR-TACI+/+ and LPR-TACI+/− kidney tissue samples than TACI-LPR−/− samples. Glomerular changes were scored on a scale of 0 (no pathology) to 3 (severe pathology). (C) SLE related kidney pathology is evaluated by assessment of renal C3 and IgG deposition. Representative immunofluorescence images of IgG (green) and C3 (red) immune deposits in the glomeruli of 12 weeks old mice are shown. Fluorescence intensity ± SD from 5 mice were quantified by imageJ and plotted. x p < 0.05 indicates statistical differences between LPR-TACI+/+ and LPR-TACI+/− mice. *p < 0.05 and **p < 0.01 indicate statistical differences between LPR-TACI+/+ and LPR-TACI−/− mice. ^#Indicates p < 0.05 for statistical difference between LPR-TACI+/− and LPR-TACI−/− mice.

Figure 4

Renal Mϕs from TACI deficient MRL-Fas/Lpr mice exhibit M2-skewed phenotype. (A) Isolation of Mϕs from kidneys. Kidneys from 12-weeks old mice were first minced and then digested with collagenase. The fraction containing immune cells were isolated after Percoll gradient centrifugation. Dead cells were excluded firstly, the Mϕs were gated as F4/80 and CD11b positive cells and quantified. (B) Phenotypes of Mϕs isolated from kidneys were assessed in flow cytometry. The expression of the M2 markers, IL-4Rα, CD206 and M1 marker CD86 in renal Mϕs was measured by FACS. A representative histogram for each marker is shown. Mean fluorescence intensities of IL-4Rα, CD206 and CD86 staining from 5 female mice per group were plotted. (C) Renal Mϕs were sorted from 12-weeks old mice. Gene expression of M1 and M2-associated markers in the sorted cells was assessed by Q-PCR. ^xp < 0.05 and ^xxxp < 0.001 indicate statistical differences between LPR-TACI+/+ and LPR-TACI+/− mice. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistical differences between LPR-TACI+/+ and LPR-TACI−/− mice. ^#p < 0.05 and ^##p < 0.01 indicate statistical differences between LPR-TACI+/− and LPR-TACI−/− mice.

Figure 5

Adoptively transferred TACI deficient Mϕs alleviate glomerulonephritis development in MRL-Fas/Lpr mice. (A) Proteinuria measurement in MRL-Fas/Lpr mice adoptively transferred with LPR-TACI+/+ or LPR-TACI−/− Mϕs. Macrophages isolated from 12-week old LPR-TACI+/+ or LPR-TACI−/− mice were i.v. injected into 10 weeks-old MRL-Fas/Lpr mice on indicated time points. Control MRL-Fas/Lpr mice received equal volume of PBS. Proteinuria measured on every two days from 5 mice from each group and mean proteinuria score ± SD were plotted. *p < 0.05 and **p < 0.01 indicates statistical differences between LPR-TACI+/+ Mϕs transferred groups and LPR-TACI−/− Mϕs transferred groups. ^#p < 0.05 indicates statistical difference between PBS injected and LPR-TACI−/− Mϕs transferred groups. (B) Representative images of H&E (upper row) and PAS (bottom row) stained kidney specimens. Glomerular changes, inflammatory cell infiltration and interstitial fibrosis were semi-quantitatively scored on a scale of 0 (no pathology) to 3 (severe pathology). **p < 0.01 indicates statistical difference between LPR-TACI+/+ Mϕ injected and LPR-TACI−/− Mϕ transferred groups. ^##p < 0.01 indicates statistical difference between PBS injected and LPR-TACI−/− Mϕ transferred groups.

Figure 6

Spontaneous GC, Tfh and Thef accumulations were not altered in TACI deficient MRL-Fas/Lpr mice. Percentages of CD19 + CD3-GL7 + PNA + GC B cells (A), CXCR4-CXCR5+ PD1+ PSGL1loCD62L-CD44+ Tfh cells (B), CXCR4+ CXCR5-PD1+ PSGL1loCD62L-CD44+ Thef cells (C) in spleens of 6 to 12 weeks old LPR-TACI+/+, LPR-TACI+/− and LPR-TACI−/− mice were quantified in flow cytometry. Mean percentage ± SD from 4 to 6 mice in each group were plotted. *p < 0.05 indicates statistical difference between LPR-TACI+/+ and LPR-TACI−/− mice.