Research ArticleINFECTIOUS DISEASES

The probacterial effect of type I interferon signaling requires its own negative regulator USP18

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Science Immunology  28 Sep 2018:
Vol. 3, Issue 27, eaau2125
DOI: 10.1126/sciimmunol.aau2125
  • Fig. 1 Usp18 is an important IFN-I–induced gene that promotes L.m. infection.

    (A) C57BL/6 WT mice were treated with 1 mg of anti–IFN-αR1 or isotype control antibody. The next day, mice were infected with 4000 colony-forming units (CFU) L.m. for 24 hours. DCs and macrophages were sorted by fluorescence-activated cell sorting (FACS), and indicated genes were measured with quantitative reverse transcription PCR (qRT-PCR). RQs were determined with the equation RQ = 2−ΔΔCt (n = 4). Data are representative of at least two independent experiments. (B) Ifnarfl/fl CD11c-Cre mice and littermate controls were infected with 4000 CFU L.m. After 4 days, bacterial titers were measured in the liver, spleen, kidney, and lung (n = 7). Data are pooled from two independent experiments. (C) DCs and macrophages were sorted by FACS from naive spleens of C57BL/6 mice. Usp18 mRNA expression was measured by qRT-PCR. RQs were determined with the equation RQ = 2−ΔΔCt relative to DCs’ average value (n = 4). Data are representative of at least two independent experiments. (D) WT or Usp18−/− mice were infected with 4000 CFU L.m. After 4 days, bacterial titers were measured in the indicated organs (n = 4). Data are representative of at least two independent experiments. Statistical significance was determined by Student’s t test (A to D). n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  • Fig. 2 Deletion of USP18 in CD11c-Cre+ cells enhances the resistance to L.m.

    (A) Usp18fl/fl CD11c-Cre mice and littermate controls were infected with 4000 CFU L.m. After 4 days, titers of bacteria were measured in the indicated organs (n = 6 to 7). (B) Survival curve of Usp18fl/fl CD11c-Cre and littermate control mice infected with 7000 CFU L.m. (n = 8). (C) Usp18fl/fl LysM-Cre mice and littermate controls were infected with 4000 CFU L.m. After 4 days, bacterial titers were measured in the indicated organs (n = 5 to 6). (D) Usp18fl/fl CD11c-Cre mice and littermate controls were treated intraperitoneally with 1 mg of anti–IFN-αR1 or isotype control antibody. The next day, mice were infected with 4000 CFU L.m. After 4 days, titers of bacteria were measured in the indicated organs (n = 4 to 5). Data are representative of at least two independent experiments. (E) C57BL/6 WT mice were treated with 1 mg of anti–IFN-αR1 or isotype control antibody. The next day, mice were infected with 2 × 108 CFU S. aureus. After 2 days, titers of bacteria were measured in the lungs (n = 4). (F) Ifnarfl/fl CD11c-Cre mice or (G) Usp18fl/fl CD11c-Cre mice and littermate controls were infected with 2 × 108 CFU S. aureus. After 2 days, titers of bacteria were measured in the lungs (n = 9 to 10). Statistical significance was determined by Student’s t test (A and C to G) and log-rank (Mantel-Cox) test (B). n.s., not significant; *P < 0.05; **P < 0.01. Data are pooled from two independent experiments (A to C, F, and G).

  • Fig. 3 Probacterial effect of USP18 is independent of its isopeptidase activity or cytokine production.

    (A) WT and Usp18C61A/C61A mice were infected with 4000 CFU L.m. After 4 days, titers of bacteria were measured in the indicated organs (n = 6 to 7). (B) WT and Ube1L−/− mice were infected with 4000 CFU L.m. After 4 days, titers of bacteria were measured in the indicated organs (n = 7 to 8). (C) Usp18fl/fl CD11c-Cre mice and littermate controls were infected with 4000 CFU L.m. IFN-γ and TNF-α production were measured in the serum at the indicated time points (n = 4 to 6). Statistical significance was determined by Student’s t test (A and B) and two-way ANOVA (C). n.s., not significant; *P < 0.05; **P < 0.01. Data are pooled from two independent experiments (A to C).

  • Fig. 4 USP18 inhibits the antibacterial effect of TNF-α.

    (A) Bone marrow–derived DCs from Usp18fl/fl CD11c-Cre mice and littermate controls were treated with 100 ng of recombinant mouse TNF-α for 24 hours or left untreated. iNOS expression was measured with qRT-PCR. RQs were determined with the equation RQ = 2−ΔΔCt (n = 5 to 6). Data are representative of at least two independent experiments. (B) Bone marrow–derived DCs from Usp18−/− and control mice were treated with 100 ng of recombinant mouse TNF-α for 30 min or left untreated (in vitro experiment), or Usp18−/− and control mice were treated with 50 ng of TNF-α for 30 min or left untreated (in vivo experiment). ROS was measured in DCs (n = 6 in vitro, n = 7 to 9 in vivo). (C) Usp18fl/fl CD11c-Cre mice and littermate controls were infected with 4000 CFU L.m. ROS was measured in DCs on day 1 in blood or day 3 in spleen (n = 4 to 6). Data are representative of at least two independent experiments. (D) Bone marrow–derived DCs from Usp18fl/fl CD11c-Cre mice and littermate controls were infected with L.m. [multiplicity of infection (MOI), 10] in the presence or absence of TNF-α. After 6 hours, intracellular bacterial titers were measured (n = 6). Data are representative of at least two independent experiments. (E) THP-1 control (mip-THP-1) or USP18-overexpressing monocytes (mip-THP-1-USP18) were infected with L.m. (MOI, 10) in the presence or absence of TNF-α. After 6 hours, intracellular bacterial titers were measured (n = 8). (F) Usp18fl/fl CD11c-Cre mice and littermate controls were treated intraperitoneally with 500 μg of anti–TNF-α antibody on day −1, 1, and 2 or left untreated. The next day, mice were infected with 4000 CFU L.m. After 4 days, titers of bacteria were measured in the indicated organs (n = 5). Data are representative of at least two independent experiments. (G) Usp18fl/fl CD11c-Cre mice and littermate controls were treated with 250 μg of anti–IFN-γ antibody on day −1, 1, and 2 or left untreated. The next day, mice were infected with 4000 CFU L.m. After 4 days, titers of bacteria were measured in the indicated organs (n = 4 to 5). Data are representative of at least two independent experiments. Statistical significance was determined by Student’s t test (A to G). n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are pooled from two independent experiments (B and E).

  • Fig. 5 The probacterial role of IFN-I during superinfection is USP18-dependent.

    (A) Ifnarfl/fl CD11c-Cre and littermate control mice or (B) Usp18fl/fl CD11c-Cre mice and littermate controls were either infected with 2 × 106 plaque-forming units LCMV-WE or left uninfected. After 2 days, mice were infected with 4000 CFU L.m. Bacterial titers were measured in the indicated organs on day 4 [n = 5 to 7 (A), n = 5 to 6 (B)]. (C) Usp18fl/fl CD11c-Cre mice and littermate controls were infected with 4000 CFU L.m. with or without 200 μg of poly I:C. Bacterial titers were measured in the indicated organs on day 3 (n = 4 to 6). Statistical significance was determined by Student’s t test (A to C). n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are pooled from two independent experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/27/eaau2125/DC1

    Fig. S1. ISG levels in DCs and MΦ after L.m. infection.

    Fig. S2. The role of IFNAR on MΦ during L.m. infection.

    Fig. S3. Usp18 expression after L.m. infection.

    Fig. S4. ISG levels in WT and Usp18−/− after L.m. infection.

    Fig. S5. The role of USP18 in DCs after low dose (400 CFU) of L.m.

    Fig. S6. DC numbers in Usp18fl/fl CD11c-Cre mice after L.m. infection.

    Fig. S7. Bacterial titers in sorted macrophages and DCs after L.m. infection.

    Fig. S8. The role of USP18 in the uptake and early replication of L.m.

    Fig. S9. The role of USP18 in inhibiting TNF-α signaling.

    Fig. S10. USP18 inhibits ROS production.

    Fig. S11. USP18 inhibits antibacterial effect of TNF-α but not IFN-γ.

    Fig. S12. ISG levels in sorted DCs after LCMV infection.

    Table S1. Mutation sites of murine and human USP18 and the consequences on the functional domains.

    Table S2. Raw data (Excel file).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. ISG levels in DCs and MΦ after L.m. infection.
    • Fig. S2. The role of IFNAR on MΦ during L.m. infection.
    • Fig. S3. Usp18 expression after L.m. infection.
    • Fig. S4. ISG levels in WT and Usp18−/− after L.m. infection.
    • Fig. S5. The role of USP18 in DCs after low dose (400 CFU) of L.m.
    • Fig. S6. DC numbers in Usp18fl/fl CD11c-Cre mice after L.m. infection.
    • Fig. S7. Bacterial titers in sorted macrophages and DCs after L.m. infection.
    • Fig. S8. The role of USP18 in the uptake and early replication of L.m.
    • Fig. S9. The role of USP18 in inhibiting TNF-α signaling.
    • Fig. S10. USP18 inhibits ROS production.
    • Fig. S11. USP18 inhibits antibacterial effect of TNF-α but not IFN-γ.
    • Fig. S12. ISG levels in sorted DCs after LCMV infection.
    • Table S1. Mutation sites of murine and human USP18 and the consequences on the functional domains.

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    Other Supplementary Material for this manuscript includes the following:

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