Figure 4.

Unc93b1^R336C knock-in mice develop systemic autoimmune pathology. (A) Sanger chromatograms depicting C1006T nucleotide substitution in the endogenous murine Unc93b1 locus, resulting in amino acid substitution R336C. (B) Observed percentages of pups of the indicated genotypes surviving to weaning age; expected Mendelian percentages are indicated in parentheses. (C–J) Phenotyping of Unc93b1^+/+, Unc93b1^+/R336C, and Unc93b1^R336C/R336C mice: (C) Body weight of 5-wk-old mice. (D) Representative image of 13–15-wk-old-mice upon sacrifice. (E) Normalized spleen weight of 13–15-wk-old mice. (F and G) Representative flow cytometry plots, frequency among live splenocytes, and absolute number of splenic ABCs (F) and plasma cells (G). (H) Frequency and absolute number of splenic monocytes. (I) Serum CXCL1 and CXCL10 concentration. (J) Serum ANA IgG level by ELISA. (K) Representative hematoxylin/eosin (H&E) staining (top panels) and periodic acid-Schiff (PAS) staining (bottom panels) from 13–15-wk-old Unc93b1^R336C mice. Interstitial leukocyte accumulation, mesangial matrix expansion, and endocapillary hypercellularity are highlighted by red, yellow, and blue arrowheads, respectively. (L) Blinded pathologic scoring of kidney sections from 13–15-wk-old Unc93b1^R336C mice. Symbols overlying bars represent values from individual mice (C, E–J, and L). P values were obtained using a one-way ANOVA with Sidak’s (C) or Dunnett’s (E–I) correction for multiple comparisons, or a Kruskal–Wallis test with Dunn’s correction for multiple comparisons (J and L). Data are pooled from at least nine litters (B and C); pooled from four independent experiments using mice from two independently generated knockin lines (E–H); are plotted from one and representative of two independent experiments (I and J); or are pooled from three independent experiments (L). (M and N) BMMs were prepared from mice of the indicated genotypes and stimulated overnight with PolyIC (20 µg ml⁻¹), R848 (4 ng ml⁻¹), ssPolyU complexed with DOTAP (12.5 µg ml⁻¹), CpG-B (50 nM), or LPS (2 ng ml⁻¹). Cytokine production was measured in supernatants by LEGENDPlex assay. Data are mean ± SD of triplicate technical replicates, representative of at least two independent experiments, and P values were determined using an unpaired two-tailed Student’s t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.