Endosomal TLR signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus

Abstract

Using the Unc93b1 3d mutation that selectively abolishes nucleic acid-binding Toll-like receptor (TLR) (TLR3, -7, -9) signaling, we show these endosomal TLRs are required for optimal production of IgG autoAbs, IgM rheumatoid factor, and other clinical parameters of disease in 2 lupus strains, B6-Fas(lpr) and BXSB. Strikingly, treatment with lipid A, an autoAb-inducing TLR4 agonist, could not overcome this requirement. The 3d mutation slightly reduced complete Freund's adjuvant (CFA)-mediated antigen presentation, but did not affect T-independent type 1 or alum-mediated T-dependent humoral responses or TLR-independent IFN production induced by cytoplasmic nucleic acids. These findings suggest that nucleic acid-sensing TLRs might act as an Achilles' heel in susceptible individuals by providing a critical pathway by which relative tolerance for nucleic acid-containing antigens is breached and systemic autoimmunity ensues. Importantly, this helps provide an explanation for the high frequency of anti-nucleic acid Abs in lupus-like systemic autoimmunity.

Fig. 1.

Immunopathology of B6-Fas^lpr 3d mice. (A) IgM and IgG polyclonal and autoAbs from 6- to 8-month-old B6-Fas^lpr wild-type (wt/wt), heterozygous (wt/3d), and mutant (3d/3d) mice determined by ELISA. IgM RF was anti-IgG1, 4–22 mice/group. (B) Representative ANA results from 6- to 7-month-old mice (1/100 dilution), 4–7/group. (C) Lymphoid organ weights: spleen and LN (cervical, axillary, inguinal, and mesenteric) weights from 10-month-old mice, 5–7/group. (D) Representative flow cytometry analyses of splenic B cell subsets in young (1 month) or old (15 month) mice with the Fas^lpr and 3d mutations. Follicular (CD21^lo CD23^hi), marginal zone (CD21^hi CD23^lo), and CD21^lo CD23^lo populations are gated (see Table S3). (E) Cumulative survival, 8–11/group. P < 0.04 for 3d/3d versus wt/wt or wt/3d.

Fig. 2.

AutoAbs and survival of 3d BXSB background mice. (A) Serum IgM and IgG anti-nuclear Abs from 3- to 4-month-old mice (mean ± SE, 4–5/group). (B) Serum IgG anti-RNP from 3- to 4-month-old mice, 7–8 mice/group. (C) Cumulative survival, 10–12 mice/group, P < 0.0001.

Fig. 3.

IgM and IgG polyclonal and autoAbs from lipid A-treated B6-Fas^lpr wt and 3d mice. Six-week-old mice were given 50 μg lipid A i.p. 2 times per week for 20 weeks. Ig amounts are by ELISA (mean ± SE for 3–6/group at each time point). *, P < 0.05.

Fig. 4.

B and T cell responses in B6-Fas^lpr 3d mice. (A) TI-1 anti-TNP response. Sera were from 7 days after 50 μg TNP-LPS i.p. Total Ig and Abs to high-density (TNP-15) or low-density (TNP-3) TNP conjugates were measured by ELISA. P > 0.05 for wt vs. 3d in all groups. (B) TD anti-TNP response was measured serially in mice immunized with TNP-KLH on days 0 and 21 either in CFA for the first dose and IFA for the second (CFA/IFA) or in alum for both doses (mean ± SE from 3–11/group). P-values comparing wt and 3d groups are shown below their respective time points: Upper line for CFA/IFA and Lower line for alum-injected mice. (C) Recall of T cell proliferation of wt and 3d T cells to OVA with either wt or 3d APCs. Splenic T cells and APCs were isolated 10 days after immunization with OVA in CFA and proliferation was assessed by thymidine uptake after 4 days. One of 2 independent experiments is shown.

Fig. 5.

TLR-independent activation of DCs. (A) DC activation with cytoplasmic dsRNA and 5′ (3P)-RNA. Bone marrow (BM)-derived wt, 3d, and MyD88^−/−/Trif^−/− double-knockout DCs were transfected with 250 μg/mL poly(I:C) alone or by Lipofectamine with 10 μg/mL poly(dT:dA*dA:dT) or 200 ng 5′ (3P)-dsRNA. Poly(I:C) in high concentrations directly enters cells. P < 0.009 for all wt, 3d, and MyD88^−/−/Trif^−/− double-knockout DCs transfected with RNA versus Lipofectamine-treated (Lipo) DCs. (B) DC activation with cytoplasmic double-stranded mammalian DNA. DCs from wt, 3d, and TLR9^−/− mice were transfected with 10 μg/mL calf thymus DNA. P < 0.05 for dsDNA-transfected wt, 3d, and TLR9^−/− DCs versus Lipo alone DCs. A and B are mean ± SEM of triplicates.