Figure 3. Expression of Tlr7 or Tlr8 is critical for IgG anti-nuclear antibody production in 564Igi.
(A) IgG ANA staining of HEp-2 cells using sera from C57BL/6, 564Igi, and 564Igi Tlr7/8−/− at 100X magnification. Data representative of studies of at least three mice are shown. (B) ANA staining intensity of Hep-2 cells. (C) Anti RNA IgG2a and IgG2b antibodies detected in the sera of mice by ELISA using yeast RNA as capture antigen and detecting binding with AP conjugated anti-IgG2a or anti-IgG2b measures the amount of RNA binding. Sera from C57BL/6, 564Igi, and 564Igi Tlr7/8−/− mice assayed by ELISA (D) Total IgG2a and IgG2b detected in the sera of mice by ELISA using anti-IgG2a or anti-IgG2b capture antibodies and detecting binding with AP conjugated anti-IgG2a or anti-IgG2b antibodies, respectively. (E) Immune complexes containing IgG2a antibody detected in the sera of mice by ELISA with C1q as a capture molecule. (C–E) Data are shown as the means +/− SEM of the number of sera tested indicated on the graph and are representative of many independent experiments. Mice were tested at 3 to 4 months of age. A two tailed Student’s t-test was run, statistically significance at p<.05. * p< .05, ** p< .01, *** p< .001 and **** p< .0001. F) Tlr7 and Tlr8 are up-regulated in B220+ B cells from the bone marrow of 564 Igi mice. Relative gene expression of Tlr7, Tlr8 and Tlr9 to B-actin in BM B220+ cells was measured by real-time PCR (qPCR) from C57BL/6 (n=3), 564Igi-B6 (n=3), 564Igi Tlr7/8−/− and 564Igi Tlr7/9−/− (both female and male), 564Igi Tlr9−/− and 564Igi Tlr8−/−. 3 mice tested for each strain. 3 dilutions of cDNA tested from each mouse, each dilution was tested in triplicate. (F) Data are shown as the mean +/− SEM of n=3 samples and are pooled from 3 independent experiments A two tailed Student’s t-test was run, statistically significance at p<.05. * p< .05, ** p< .01, *** p< .001 and **** p< .0001. Mice were tested at 3 to 4 months of age.(G) Western blots for TLR7 and TLR8 in B cells from the BM. TLR7: and TLR8:β-Actin densitometry ratios are shown.