Toll-like receptor 9 suppresses lupus disease in <i>Fas</i>-sufficient MRL Mice

<div><p>Genetic deficiency in TLR9 accelerates pathogenesis in the spontaneous polygenic MRL.<i>Fas</i>^<i>lpr</i> murine model of systemic lupus erythematosus, despite the absence of anti-nucleosome autoantibodies. However, it could be argued that this result was dependent on <i>Fas</i>-deficiency rather than lupus-promoting genes in the MRL genetic background. Here we report the effects of TLR9 deficiency on autoimmune disease independent of the <i>lpr</i> mutation in <i>Fas</i> by characterizing <i>Tlr9</i>^<i>-/-</i> and <i>Tlr9</i>^<i>+/+</i> mice on the <i>Fas</i>-intact MRL/+ genetic background. By 30 weeks of age, <i>Tlr9-</i>deficient MRL/+ had more severe renal disease, increased T cell activation, and higher titers of anti-Sm and anti-RNA autoantibodies than <i>Tlr9</i>-intact animals, as had been the case in the MRL.<i>Fas</i>^<i>lpr</i> model. In addition, <i>Tlr9</i>-deficient MRL/+ mice had increased numbers of germinal center phenotype B cells and an increase in splenic neutrophils and conventional dendritic cell populations. Thus, the disease accelerating effects of <i>Tlr9</i> deficiency are separable from those mediated by the <i>Fas</i> mutation in the lupus-prone MRL genetic background. Nonetheless, disease acceleration in <i>Tlr9</i>-deficient MRL/+ mice was phenotypically distinct from that in <i>Fas</i>-deficient counterparts, which has important implications.</p></div

Renal disease is suppressed by <i>Tlr9</i> in MRL/+ mice.

<p><b>(A)</b> Proteinuria was evaluated in 30 week old mice of the indicated genotypes by dipstick assay. <b>(B)</b> Severity of glomerular disease on H&E stained sections was evaluated by a pathologist on a 0–6 scale. <b>(C)</b> Perivascular and interstitial renal infiltrates were evaluated by a pathologist on a 0–4 scale. <b>(D)</b> Skin lesions were scored based on area with up to an additional 0.5 points for facial rash / loss of whiskers and 0.25 points for dermatitis of each ear. In all graphs, horizontal lines represent medians and each point represents an individual animal. ** p<0.01; **** p<0.0001 by two-tailed Mann-Whitney U-test. Data are pooled from five experimental cohorts.</p

Autoantibody production in <i>Tlr9</i>^<i>-/-</i> MRL/+ mice.

<p><b>(A)</b> Serum anti-nucleosome IgG autoantibodies were measured by ELISA and are expressed relative to a PL2-3 standard. <b>(B)</b> Serum anti-Sm IgG autoantibodies were measured by ELISA and are expressed relative to a Y2 standard. <b>(C)</b> Serum anti-RNA IgG autoantibodies were measured by ELISA and are expressed relative to a BWR4 standard. <b>(D)</b> Serum kappa anti-IgG2a rheumatoid factor autoantibodies were measured by ELISA and are expressed relative to a 400tμ23 standard. * p<0.05; *** p<0.001; **** p<0.0001 by two-tailed Mann-Whitney U-test.</p

HEp-2 antinuclear antibody staining patterns are changed in <i>Tlr9</i>^<i>-/-</i> MRL/+ mice.

<p><b>(A)</b> Representative HEp-2 staining patterns from <i>Tlr9</i>^<i>+/+</i> MRL/+ (<i>top</i>) and <i>Tlr9</i>^<i>-/-</i> MRL/+ (<i>bottom</i>) serum. <b>(B)</b> Proportion of sera with the indicated dominant staining pattern from <i>Tlr9</i>^<i>+/+</i> MRL/+ (<i>left</i>) or <i>Tlr9</i>^<i>-/-</i> MRL/+ (<i>right</i>). <b>(C)</b> Percentage of sera of indicated genotypes which stained positive for mitotic bodies in the HEp-2 ANA assay. **** p<0.0001 by two-tailed Fisher's exact test. <b>(D)</b> Relative intensity of cytoplasmic staining in the HEp-2 ANA assay. * p<0.05 by two-tailed Mann-Whitney U-test.</p

Increased spleen weight and T cell activation in <i>Tlr9</i>^<i>-/-</i> MRL/+ mice.

<p><b>(A)</b> Spleens were weighed. <b>(B)</b> Ly6G⁺CD11b⁺ neutrophils and <b>(C)</b> CD19^-CD11c⁺I-A/I-E⁺ dendritic cells were evaluated by FACS. <b>(D-E)</b> Naive (CD44^-CD62L⁺) cells were evaluated by FACS among TCRß⁺CD4⁺ (B) and TCRß⁺CD8⁺ (C) populations. * p<0.05; ** p<0.01; **** p<0.0001 by two-tailed Mann-Whitney U-test. Data are pooled from five (A) or four (B-E) experimental cohorts.</p