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Science 337 (6102): 1684-1688

Copyright © 2012 by the American Association for the Advancement of Science

Mycobacterial Disease and Impaired IFN-{gamma} Immunity in Humans with Inherited ISG15 Deficiency

Dusan Bogunovic1, Minji Byun1, Larissa A. Durfee2,*, Avinash Abhyankar1,*, Ozden Sanal3,*, Davood Mansouri4,*, Sandra Salem5,*, Irena Radovanovic5, Audrey V. Grant6, Parisa Adimi4, Nahal Mansouri1,4, Satoshi Okada1, Vanessa L. Bryant1, Xiao-Fei Kong1, Alexandra Kreins1, Marcela Moncada Velez1, Bertrand Boisson1, Soheila Khalilzadeh4, Ugur Ozcelik3, Ilad Alavi Darazam4, John W. Schoggins7, Charles M. Rice7, Saleh Al-Muhsen8,9, Marcel Behr10, Guillaume Vogt1,6, Anne Puel6, Jacinta Bustamante6,11,{dagger}, Philippe Gros5,{dagger}, Jon M. Huibregtse2,{dagger}, Laurent Abel1,6,{dagger}, Stéphanie Boisson-Dupuis1,6, and Jean-Laurent Casanova1,6,12,{ddagger}

1 St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA.
2 Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
3 Immunology Division and Pediatric Chest Disease Department, Hacettepe University Children’s Hospital, 06100 Ankara, Turkey.
4 Division of Infectious Diseases and Clinical Immunology, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Teheran, Iran.
5 Department of Biochemistry, McGill University, Montreal, Canada.
6 Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Institut National de la Santé et de la Recherche Médicale, U980, University Paris Descartes, Necker Medical School, 75015 Paris, France.
7 Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA.
8 Prince Naif Center for Immunology Research, Department of Pediatrics, College of Medicine, King Saud University, Riyadh, 11211, Saudi Arabia.
9 Department of Pediatrics, King Faisal Specialist Hospital and Research Center, Riyadh, 11211, Saudi Arabia.
10 Research Institute, McGill University Health Center, Montreal, Canada.
11 Center for the Study of Primary Immunodeficiencies, Assistance Publique–Hopitaux de Paris, Necker Hospital, Paris, France.
12 Pediatric Hematology-Immunology Unit, Necker Hospital, 75015 Paris, France.

Figure 1
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Fig. 1. The familial segregation and expression pattern of the ISG15 allele indicates recessive inheritance and an absence of protein production. (A) Familial segregation in a family from Turkey (Kindred A) and a family from Iran (Kindred B). (B) A graphical representation of the proISG15 protein is shown. The LRLRGG ISGylation domain, the eight–amino acid sequence (black) cleaved to yield active ISG15, and the putative proteins synthesized in the patients are shown. (C) EBV-B cells from control 1 (C1), control 2 (C2), a STAT1–/– patient (negative control), and P1 were left untreated or treated with IFN-α. The cells were then lysed, and the lysates were subjected to Western blotting. (D) EBV-B cells from C1, a STAT1–/– patient, and P1 were stained with ISG15 and STAT1 antibodies and analyzed by flow cytometry. (E) SV-40–fibroblasts from C1, a STAT1–/– patient, P1, and P2 were analyzed as in (C). All experiments were performed at least three times.


Figure 2
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Fig. 2. ISGylation and viral susceptibility in cell lines derived from patients with mutations in ISG15. (A) Control SV-40–immortalized fibroblasts (C1) and fibroblasts derived from P1, P2, or a STAT1–/– patient were either left untreated or treated with IFN-β for 24 hours. Cell extracts were analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotting with antibodies against ISG15, IFIT3, or α tubulin. (B) SV-40 fibroblast cell lines from patients P1 and P2 were mock-transfected (M), transfected with a plasmid encoding 3XFLAG-ISG15 (WT), or transfected with a plasmid encoding a form of ISG15 unable to conjugate with proteins (AA). Eighteen hours after transfection, we treated the cells with IFN-β for an additional 18 hours. Cell extracts were analyzed by SDS-PAGE and immunoblotting with FLAG, IFIT3, or tubulin antibodies. (A) and (B) are representative of at least three independent experiments. (C and D) Herpes simplex virus (HSV)–1 replication was monitored by assessing the fluorescence of green fluorescent protein (GFP) fused to a viral capsid protein in SV-40 fibroblasts from a healthy control C1, P1, P2, and a STAT1–/– patient, infected with HSV-1 at a multiplicity of infection of 0.2 for the times indicated. Cells were treated with either medium alone (C) or with IFN-α (D) for 24 hours before infection. The results shown are the means of four independent experiments. Error bars indicate SEM.


Figure 3
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Fig. 3. ISG15 secretion and the induction of IFN-{gamma} production in leukocytes. Control and P1 leukocytes were left unstimulated (-) or were stimulated with BCG and IFN-α2b. After 0 and 12 hours, cells and supernatants were harvested and subjected to Western blotting (A) or TNF-α enzyme-linked immunosorbent assay (ELISA) (B). (C) Resting control and P1 leukocytes were labeled extracellularly with CD16 and CD3 antibodies and intracellularly with ISG15 antibody and were subjected to ImageStreamX (Amnis, Seattle, WA) analysis with bright field (BF) and side scatter (SSC) also shown. (D and E) IFN-{gamma} secretion was measured by ELISA in PBMCs stimulated with vehicle (-), various doses of recombinant human ISG15 (including boiled recombinant human ISG15 to exclude LPS contamination) (D), recombinant human ISG15, and IL-12, alone and in combination (E). The IFN-{gamma} secretion results shown in the figure are representative means of at least three independent experiments. Error bars indicate SEM.


Figure 4
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Fig. 4. Impaired IFN-{gamma} production in ISG15–/– patients and rescue by exogenous ISG15. (A to C) Cytokine production in the supernatants of whole-blood cells from local controls (n = 29), travel controls (n = 9), ISG15–/– (n = 3), and IL12RB1–/– patients (n = 58), left unstimulated or stimulated with BCG alone or BCG plus cytokine (indicated), as detected by ELISA. (D) Alternatively, recombinant human ISG15 was added at the same time or 24 hours before whole-blood activation by BCG and IL-12. The amounts of cytokine secreted are normalized for 106 PBMCs on a logarithmic scale, and medians are indicated by solid bars. Differences in log-transformed IFN-{gamma} levels after stimulation with BCG and IL-12 were assessed (i) between ISG15–/– patients and travel controls in Student’s t test (A) and (ii) between ISG15–/– patients before and after adding recombinant human ISG15 by two-way analysis of variance, to account for both activation by recombinant ISG15 and activation time (D). (E) IFN-{gamma} secretion was measured in whole blood stimulated with vehicle (-), BCG, or BCG plus IL-12, in the presence of vehicle, a blocking antibody against ISG15, or an immunoglobulin G (IgG) 1 isotype control.


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