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. 2022 Mar 8;219(4):e20211273. doi: 10.1084/jem.20211273

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© 2022 Martin-Fernandez et al.

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Figure 5. — Impairment in IL-12 production by monocytes in USP18 I60N–carrying patients is due to uncontrolled IFN-I inflammation. (A) WT or USP18^KO THP1 cells were primed with 1,000 IU/ml IFN-α2b for 12 h and stimulated with BCG or BCG + IFN-γ. 24 h later, RNA was isolated, and relative IL12B mRNA levels were assessed. The results are calculated relative to the peak of induction (BCG + IFN-γ). A representative experiment is shown. (B) WT, USP18^KO, or TREX1^KO THP1 cells were primed with 1,000 IU/ml IFN-α2b for 12 h and stimulated with BCG or BCG + IFN-γ. 24 h later, RNA was isolated, and relative IL12B mRNA levels were assessed. The results are calculated relative to the peak of induction (BCG + IFN-γ). A representative experiment is shown. (C) USP18^KO THP1 cells were transduced with Luc-RFP, WT USP18, or USP18 I60N, sorted, treated with 1,000 IU/ml IFN-α2b for 12 h, and stimulated with BCG or BCG + IFN-γ. 24 h later, RNA was isolated, and relative IL12B mRNA levels were assessed. A representative experiment is shown. (D) WT or USP18^KO THP1 cells were primed with 1,000 IU/ml IFN-α2b for 12 h and stimulated with BCG or Pam3CSK, with or without IFN-γ. 24 h later, RNA was isolated, and the relative IL12B mRNA levels were assessed. A representative experiment is shown. (E) WT or USP18^KO THP1 cells were primed with 1,000 IU/ml IFN-α2b for 12 h and stimulated with BCG or LPS, with or without IFN-γ. 24 h later, RNA was isolated, and relative IL12B mRNA levels were assessed. A representative experiment is shown. (F) WT or USP18^KO THP1 cells were pretreated with blocking antibodies for IFNAR2 and IFN-β for 12 h, primed with 1,000 IU/ml IFN-α2b for 12 h, and stimulated with LPS or LPS + IFN-γ. 24 h later, RNA was isolated, and relative IL12B mRNA levels were assessed. A representative experiment is shown. (G) WT or USP18^KO THP1 cells were primed with 1,000 IU/ml IFN-α2b for 12 h and stimulated with BCG or BCG + IFN-γ. 24 h later, RNA was isolated, and relative TNF mRNA levels were assessed. A representative experiment is shown. (H) WT or USP18^KO THP1 cells were primed with 1,000 IU/ml IFN-α2b for 12 h and stimulated with BCG or BCG + IFN-γ. 24 h later, RNA was isolated, and relative IL6 mRNA levels were assessed. A representative experiment is shown. (I and J) WT or USP18^KO THP1 cells were primed with 1,000 IU/ml IFN-α2b for 12 h and stimulated with Pam3CSK or LPS with or without IFN-γ. 24 h later, RNA was isolated, and the relative expression of the indicated genes were assessed. All results in the figure are representative of at least two independent experiments. RU, relative units.