Extended Data Fig. 9: UNC93B1PKP knock-in mice develop systemic inflammation. | Nature

Extended Data Fig. 9: UNC93B1PKP knock-in mice develop systemic inflammation.

From: UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity

Extended Data Fig. 9

a. CRISPR–Cas9 strategy to generate UNC93B1PKP knock-in mice. Green line indicates the guide sequence. Red bases indicate the edited codons. A representative sequencing trace of genomic DNA from an edited founder mouse is shown. b, Flow cytometry analysis of the indicated immune cell populations in 6–8-week-old Unc93b1WT/WT, Unc93b1PKP/WT and Unc93b1PKP/PKP mice. Frequencies of dendritic cells (CD11b+CD11c+MHCIIhigh) and inflammatory monocytes (CD11b+Ly6c+Ly6Gneg) in lymph nodes are shown. Data points were pooled from four independent experiments. P values determined by unpaired two-tailed Student’s t-test. c, Unc93b1PKP/PKP mice exhibit signs of emergency granulopoiesis in their bone marrow compartment. Flow cytometry analysis of bone marrow from 6–8-week-old Unc93b1WT/WT, Unc93b1PKP/WT and Unc93b1PKP/PKP mice. Gates representing LSK (CD45+CD3εCD19Ly6cLy6GSca-1highc-Kithigh) and Sca-1highc-Kit cells (CD45+CD3εCD19Ly6cLy6GSca-1highc-Kit) are indicated and compiled frequencies of Sca-1highc-Kit cells are shown on the right. Data are mean ± s.d., n = 3 biological replicates. P value determined by unpaired two-tailed Student’s t-test. d, Representative staining, corresponding to compiled results shown in Fig. 4c, of anti-nuclear antibodies (ANA) using sera from the indicated mouse ages and genotypes. e, f, Flow cytometry analysis showing percentage of TNF-positive cells, measured by intracellular cytokine staining of BMMs and BMDCs derived from the indicated mice after stimulation with CpG-B (150 nM), R848 (10 ng ml−1), ssRNA (1 µg ml−1), poly(I:C) (10 µg ml−1), or LPS (10 ng ml−1). Shaded histograms are unstimulated controls. g, TNF production by BMDCs derived from the indicated mice after stimulation for 8 h with R848, CpG-B (150 nM) or LPS (50 ng ml−1). h, TNF production by BMMs from the indicated mice after stimulation for 8 h with CpG-B (500 nM), LPS (50 ng ml−1), or increasing concentrations of R848. Data in g and h are mean ± s.d., n = 3 biological replicates. P values determined by unpaired two-tailed Student’s t-test. i, B cells from Unc93b1PKP/PKP mice show enhanced proliferation in response to TLR7 stimulation. Proliferation of CFSE-labelled B cells after 3 days stimulation with R848 (8 ng ml−1) or LPS (1.6 µg ml−1) was measured by FACS, pre-gating on live CD19+ cells. The proliferation index is determined by dividing the geometric mean fluorescent intensity (gMFI) of the fluorescent dye CSFE of the unstimulated control by the gMFI of CSFE of the stimulated sample (CSFEunstim/CFSEsample). Data are mean ± s.d., n = 5 mice per group pooled together from three independent experiments. P values determined by unpaired two-tailed Student’s t-test. j, Immunoprecipitation of MyD88 from BMMs from the indicated mice after stimulation with R848 (500 ng ml−1), followed by immunoblot for IRAK2. Input levels of MyD88 and IRAK2 in whole-cell lysates are also shown. k, UNC93B1 protein levels in BMMs from indicated mouse genotypes, measured by immunoblot with polyclonal antibodies against endogenous UNC93B1. All data are representative of three independent experiments, unless otherwise noted.

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