TLR9 regulates TLR7- and MyD88-dependent autoantibody production and disease in a murine model of lupus

Abstract

Systemic lupus erythematosus is characterized by the production of autoantibodies against nucleic acid-associated Ags. We previously found that Tlr7 was required for anti-Sm and Tlr9 for anti-chromatin autoantibodies. Yet, although Tlr7 deficiency ameliorated disease, Tlr9 deficiency exacerbated it. Despite the mechanistic and clinical implications of this finding, it has yet to be elucidated. In this study, we characterize MRL/lpr lupus-prone mice genetically deficient in Tlr7, Tlr9, both Tlr7 and Tlr9, or Myd88 to test whether Tlr7 and Tlr9 function independently or instead regulate each other. We find that disease that is regulated by Tlr9 (and hence is worse in its absence) depends on Tlr7 for its manifestation. In addition, although Tlr7 and Tlr9 act in parallel pathways on different subsets of autoantibodies, Tlr9 also suppresses the production of Tlr7-dependent RNA-associated autoantibodies, suggesting previously unrecognized cross-regulation of autoantibody production as well. By comparing disease in mice deficient for Tlr7 and/or Tlr9 to those lacking Myd88, we also identify aspects of disease that have Tlr- and Myd88-independent components. These results suggest new models for how Tlr9 regulates and Tlr7 enhances disease and provide insight into aspects of autoimmune disease that are, and are not, influenced by TLR signals.

FIGURE 1

ANAs are absent in mice lacking Myd88 or both Tlr9 and Tlr7. A, Representative HEp-2 ANA staining from serum of 16-wk (Tlr) or 24-wk (Myd88) old MRL/lpr mice of the indicated genotypes. Arrows indicate mitotic chromatin staining. B and C, HEp-2 slides were scored for intensity of nuclear (□) and cytoplasmic (■) staining patterns. Data are represented as mean ± SEM. Statistics were calculated by two-tailed Mann-Whitney U test. *p < 0.05; **p < 0.01; ***p < 0.001. D and E, HEp-2 slides were scored for the presence or absence of mitotic chromatin staining.

FIGURE 2

Tlr7, Tlr9, and Myd88 dependence of autoantibodies differs depending on the nature of the autoantigen. IgG serum autoantibodies were measured by ELISA in mice of the indicated genotypes and ages. A and B, Anti-nucleosome Abs measured in units relative to micrograms per milliliter PL2-3 binding. C and D, Anti-Sm Abs measured in units relative to micrograms per milliliter Y12 binding. E and F, Anti-riboP Abs expressed in OD units. G and H, Anti-RNA Abs measured in OD units. I and J, Total κ⁺ anti-IgG2a RF Abs measured relative to micrograms per milliliter 400tμ23 binding. Statistics were calculated by two-tailed Mann-Whitney U test. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Hypergammaglobulinemia is Tlr and Myd88 dependent. A and B, Total serum IgG was measured by ELISA in mice of the indicated genotypes and ages. C–F, Total serum IgG2a and IgG3 were measured by cytometric bead assay. Statistics were calculated by two-tailed Mann-Whitney U test. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

Splenomegaly and lymphadenopathy are Tlr and Myd88 dependent. Spleen (□) and axillary lymph nodes (■) from MRL/lpr mice of the indicated Tlr (A) or Myd88 (B) genotypes were weighed. Data are represented as mean ± SEM. Statistics were calculated by two-tailed Mann-Whitney U test. ***p < 0.001.

FIGURE 5

Activation of CD4 T cells. A, Representative FACS staining of CD45R⁻CD90.2⁺CD4⁺ T cells from 16-wk-old Myd88^+/− and Myd88^−/− MRL/lpr mice. B and C, Number of CD45R⁻CD90.2⁺CD4⁺ cells with naive (CD44^lowCD62L^high, left), activated (CD44^highCD62L^high, middle) and activated/memory (CD44^highCD62L^low, right) phenotypes. Statistics were calculated by two-tailed Mann-Whitney U test. *p < 0.05; **p < 0.01.

FIGURE 6

Kidney disease is ameliorated in Tlr7/9 and Myd88-deficient mice. Disease parameters were assessed in mice of the indicated genotypes and ages. A and B, Proteinuria. C and D, Severity of glomerulonephritis. E and F, The area of interstitial and perivascular infiltrates expressed as a percentage of the total kidney area. Data are represented as mean ± SEM. Statistics were calculated by two-tailed Mann-Whitney U test. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 7

MRL/lpr mice deficient in Tlr7/9 or Myd88 develop dermatitis. A, Percentage of animals of the indicated genotypes and genders with a dorsal skin lesion of ≥5 mm diameter by 24 wk of age. Statistics were calculated by Fisher’s exact test. *p < 0.05; **p < 0.01. B, Dermatitis scores for female (solid symbols) or male (open symbols) MRL/lpr mice of the indicated genotypes at 24 wk of age. Scores were assigned as described in Materials and Methods.