Interleukin-33 Contributes Toward Loss of Tolerance by Promoting B-Cell-Activating Factor of the Tumor-Necrosis-Factor Family (BAFF)-Dependent Autoantibody Production

Abstract

Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.

Keywords: B cell; BAFF; IL-33; autoantibodies; germinal center; immune tolerance; radiation resistant.

Figure 1

IL-33 induced autoantibody formation. C57BL/6 mice (n = 10 mice/group) were injected i.p. with PBS or 500 ng IL-33 daily for four consecutive days. Serum was collected at 1, 2, and 3 weeks after the first IL-33 injection then total Ig, IgM, and IgG anti-DNA titers in PBS (open symbols, dashed line) and IL-33 (closed symbols, solid line) injected mice were determined. Data are representative of two independent experiments. Area under curve (AUC) was determined, comparing PBS to IL-33 injection by two-tailed unpaired t-test, *p < 0.05, ns, not significant.

Figure 2

BAFF is necessary for B cell expansion and autoantibody formation. C57BL/6 mice (n = 5 mice/group) were injected i.p. with PBS or 500 ng IL-33 daily for four consecutive days. Serum was collected and spleens were harvested 24 h after the last injection then processed for flow cytometry. (A) serum BAFF concentrations in PBS (white bar) and IL-33 (black bar) injected mice were determined and (B) total (CD19⁺) B cell numbers (*p < 0.05 by two-tailed unpaired t-test). (C) Total B cell numbers in C57BL/6 mice (n = 5 mice/group) treated 1 day prior to four consecutive i.p. injections of PBS or 500 ng IL-33 with mouse IgG1 isotype antibody (Control Ab) or BAFF antibody (BAFF Ab) (*p < 0.05, ns, not significant; One-way ANOVA with Tukey test and four levels of factor). (D) Total Ig anti-DNA titers were quantified at different serum dilutions and AUC was determined (*p < 0.05 by one-way ANOVA with Tukey test and four levels of factor). All data are representative of two independent experiments.

Figure 3

IL-33 induces BAFF production by radiation resistant cells rather than dendritic cells and neutrophils. (A) BMDCs from C57BL/6 mice were stimulated with selected concentrations of IFNγ (open symbols, dashed line) or IL-33 (closed symbols, solid line) for 24 h then BAFF concentrations were determined (*p < 0.05 AUC by two-tailed unpaired t-test). (B) C57BL/6 mice (n = 5 mice/group) were pre-treated with rat IgG1 isotype antibody (Control Ab) or IFNγ antibody (IFNγ Ab) injected i.p. 1 day prior to and with the third dose of daily PBS or 500 ng IL-33 injected i.p. for four consecutive days. Serum was collected and BAFF concentrations were determined (*p < 0.05 by one-way ANOVA with Tukey test and four levels of factor). (C) Neutrophils isolated from C57BL/6 mice were stimulated with selected concentrations of G-CSF (open symbols, dashed line) or IL-33 (closed symbols, solid line) for 24 h then BAFF concentrations were determined (*p < 0.05 AUC by two-tailed unpaired t-test). (D) C57BL/6 mice (n = 5 mice/group) were pre-treated with rat IgG1 isotype antibody (Control Ab) or G-CSF antibody (G-CSF Ab) injected i.p. 1 day prior and with the third dose of daily PBS or 500 ng IL-33 injected i.p. for four consecutive days. Serum was collected and BAFF concentrations were determined (*p < 0.05 by one-way ANOVA with Tukey test and four levels of factor). (E) Six weeks after transplantation of BAFF KO bone marrow into WT and WT bone marrow into BAFF KO mice (n = 5 mice/group), mice were injected i.p. with PBS or 500 ng IL-33 daily for four consecutive days. WT mice were used as the control. Serum was collected and BAFF concentrations were determined. No significance (n.s.) donor WT → recipient KO/PBS compared with donor WT → recipient KO/IL-33; *p < 0.05 Control compared with donor WT → recipient KO/PBS and donor KO → recipient WT/PBS compared with donor KO → recipient WT/IL-33 by one-way ANOVA with Tukey test and five levels of factor. (F) Total Ig anti-DNA titers were determined at various serum dilutions (comparison of AUC *p < 0.05, ns, not significant by one-way ANOVA with Tukey test five levels of factor). Data are representative of two independent experiments.

Figure 4

Chronic exposure to IL-33 induces BAFF, B cell expansion, class-switching to IgG anti-DNA antibodies, and increase in GC B cell and T_FH cell numbers. C57BL/6 mice (n = 5 mice/group/time point) were inoculated with LacZ AAV or IL-33 AAV. Serum was collected and spleens were harvested at 1, 2, and 4 weeks after inoculation. (A) BAFF concentrations in LacZ AAV (open symbols, dashed line) and IL-33 AAV (closed symbols, solid line) mice were determined (*p < 0.05 comparing AUC by two-tailed unpaired t-test). (B) Total B cell counts in the spleen show elevated cell numbers in IL-33 AAV compared with LacZ AAV at all time points (*p < 0.05 by one-way ANOVA with Tukey test and six levels of factor). (C) Total Ig, IgM, and IgG anti-DNA titers in LacZ AAV (open symbols, dashed line) and IL-33 AAV (closed symbols, solid line) mice were determined at various serum dilutions. AUC for IL-33 AAV mice was significantly greater where indicated (*p < 0.05 AUC by two-tailed unpaired t-test). (D) Total CD95⁺GL7⁺ GC B cell counts and (E) total CD4⁺CXCR5⁺ T_FH cell counts in the spleens were quantified via flow cytometry (*p < 0.05 by one-way ANOVA with Tukey test and six levels of factor). Each symbol represents an individual mouse.