1. Volume 23, Issue 5, Pages 465-478 (November 2005)
Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses 
Kun Yang, Anne Puel, Shenying Zhang, Céline Eidenschenk, Cheng-Lung Ku, Armanda Casrouge, Capucine Picard, Horst von Bernuth, Brigitte Senechal, Sabine Plancoulaine, Sami Al-Hajjar, Abdulaziz Al-Ghonaium, László Maródi, Donald Davidson, David Speert, Chaim Roifman, Ben-Zion Garty, Adrian Ozinsky, Franck J. Barrat, Robert L. Coffman, Richard L. Miller, Xiaoxia Li, Pierre Lebon, Carlos Rodriguez-Gallego, Helen Chapel, Frédéric Geissmann, Emmanuelle Jouanguy, Jean-Laurent Casanova 
Immunity 
Volume 23, Issue 5, Pages (November 2005)
DOI: /j.immuni
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Production of IFN-α/β in Response to TLR Agonists, HSV-1, and VSV in Blood Cells from Healthy Controls and IRAK-4-Deficient Patients
PBMCs from nine healthy adults and two healthy children used as controls and from IRAK-4-deficient patients P1, P2, and P3 were left unstimulated, stimulated with TLR agonists, or infected with HSV-1 and VSV in the following conditions: medium (Med), poly(I:C) (50 μg/ml, an agonist of TLR-3), LPS (100 ng/ml, TLR-4), flagellin (10 ng/ml, TLR-5), 3M-13 (3 μg/ml, TLR-7), 3M-2 (3 μg/ml, TLR-8), R-848 (5 μg/ml, TLR-7/8), CpG-C274 (5 μg/ml, TLR-9), HSV-1 (MOI: 1), VSV (MOI: 1). Experiments were carried out twice for each patient.
(A and B) IFN-α secretion (A) and IFN-β secretion (B) were measured by ELISA after 24 hr of stimulation.
(C) IFN-β mRNA levels were analyzed by reverse transcription and real-time quantitative PCR, 2 hr after stimulation for TLR-3, -4, -5, -7, and -8, 6 hr after stimulation for TLR-9, and 24 hr after infection for HSV-1 and VSV, corresponding to the respective peaks of induction. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from 11 independent individuals, each tested once.
(D) PBMCs from a healthy control and from IRAK-4-deficient patient P7 were left unstimulated or stimulated with poly(I:C) (50 μg/ml), IFN-β (8 × 104 U/ml), or both for 36 hr. IFN-α was measured by ELISA. The experiment shown is representative of two independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2
Flow Cytometry Analysis of Blood Monocyte Subsets, Plasmacytoid, and Myeloid Dendritic Cells in IRAK-4 Deficient Patients
Monocyte subsets, plasmacytoid dendritic cells (PDCs), and myeloid dendritic cells (MDCs) in PBMC from an IRAK-4-deficient patient (P2), as determined by FACS.
(A) HLA-DR+, CD14+/16− (magenta), CD14low CD16high (green), and CD14+/16+ (blue), monocyte subsets.
(B) HLA-DR+, BDCA-2+, CD123+ plasmacytoid dendritic cells.
(C) HLA-DR+, lin−, BDCA-1+, CD11c+ myeloid dendritic cells. All subsets were present at the expected frequency.
(D) Percentage of monocytes, PDCs, and MDCs, among PBMC, in IRAK-4-deficient patients P2 and P3 and healthy donors.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Induction of the CD40, CD80, and CD86 Costimulatory Molecules and MX1 mRNA in Response to TLR-3, -4, -7, -8, and -9 Stimulation in IRAK-4-Deficient Patients
(A) Monocytes and PBMCs isolated from a control and from patient P3 were untreated or stimulated with poly(I:C) (50 μg/ml, TLR-3), LPS (1 μg/ml, TLR-4), R-848 (5 μg/ml, TLR-7/8), or CpG-C274 (5 μg/ml, TLR-9) for 24 hr. The expression of CD40, CD80, and CD86 was analyzed by flow cytometry in poly(I:C)-, LPS-, or R-848-stimulated (shaded bar) or unstimulated (white bar) CD14+ monocytes and in CpG-C274-stimulated (shaded bar) or unstimulated (white bar) CD19+ B lymphocytes. This experiment is representative of two independent experiments and was also performed on three other patients, once each.
(B) PBMCs isolated from two controls and from patients P3 and P4 were left unstimulated or were stimulated with TLR agonists for 6 hr (in the conditions described in [A]) or infected with HSV-1 and VSV for 24 hr (see Supplemental Data for the multiplicities of infection, moi). Total RNA was extracted and analyzed by reverse transcription and real-time quantitative PCR to determine MX1 mRNA levels. Means and standard deviations (SD) were calculated from two experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
Production of IFN-α/β, IFN-λ, and TNF-α after Stimulation with Viruses in IRAK-4-Deficient Blood Cells
PBMCs from controls and/or from IRAK-4-deficient patients P2, P3, or P7 were left unstimulated or stimulated with various intact and UV-inactivated viruses.
(A) IFN-β mRNA levels were analyzed by reverse transcription and real-time quantitative PCR 24 hr after stimulation with intact viruses (see Supplemental Data for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for the controls from three independent individuals, each tested once. The patients were each tested once.
(B and C) IFN-α secretion (B) and IFN-λ secretion (C) were measured by ELISA after 24 hr of stimulation. Means and standard deviations (SD) were calculated for the controls from 14 independent individuals, each tested once.
(D) IFN-α secretion by control PBMCs was measured by ELISA after 24 hr of stimulation by intact or UV-inactivated viruses. Means and standard deviations (SD) were calculated for the controls from seven independent individuals, each tested once.
(E) TNF-α secretion was measured by ELISA after 24 hr of stimulation by two intact viruses. Means and standard deviations (SD) for controls were calculated from two independent individuals.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5
IFN-β, IFN-λ, and IL-6 Induction in IRAK-4-Deficient Fibroblasts upon Stimulation by Poly(I:C), IL-1β, TNF-α, and Viruses
Induction of IFN-β, IFN-λ, and IL-6 in response to poly(I:C) (50 μg/ml), IL-1β (20 ng/ml), TNF-α (10 ng/ml), and viral stimulations in IRAK-4-deficient (P1, P2, or P4) and control fibroblasts.
(A and B) IFN-β mRNA induction, as determined by Q-PCR 2 hr after stimulation (A) and IFN-β, as determined by ELISA 24 hr after stimulation (B). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.
(C) IFN-β and IFN-λ as determined by ELISA 24 hr after stimulation by poly(I:C), following prior treatment with IFN-α2b (105U/ml for 12 hr). This experiment is representative of two independent experiments.
(D) IL-6, as determined by ELISA 24 hr after stimulation. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.
(E) IFN-β mRNA induction 24 hr after stimulation by seven intact viruses (see Supplemental Data for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
IFN-β-Inducing Signaling Pathways in Control and IRAK-4-Deficient Fibroblasts Stimulated with Poly(I:C) and Viruses
SV40-transformed fibroblast lines from a control and from patients P1 and P2 were left unstimulated (labeled “NS”) or stimulated with poly(I:C) (50 μg/ml), IL-1β (20 ng/ml), TNF-α (10 ng/ml), and viruses (see Supplemental Data for the multiplicities of infection).
(A) DNA binding activity of nuclear extracts from fibroblasts left unstimulated or stimulated with poly(I:C) for 2 hr, IL-1β for 20 min, and TNF-α for 40 min was assessed by EMSA, using an NF-κB-specific DNA probe.
(B) IRF-3 dimers and monomers detected by Western blotting in whole-cell extracts from fibroblasts left unstimulated or stimulated with poly(I:C) for 2 hr.
(C) Phospho-p38, phospho-JNK, and p38 detected by Western blotting in cytoplasmic extracts from fibroblasts left unstimulated or stimulated with poly(I:C) for 1 hr and IL-1β and TNF-α for 20 min.
(D) DNA binding activity of nuclear extracts from fibroblasts left unstimulated or stimulated with HSV-1 or VSV for 4, 8, and 12 hr was assessed by EMSA, via an NF-κB-specific DNA probe.
(E) IRF-3 dimers and monomers detected by Western blotting in whole-cell extracts from fibroblasts left unstimulated or stimulated with VSV for 12 hr. These experiments are each representative of two experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7
IFN-α/β-Inducing Signaling Pathways in Control and IRAK-4-Deficient Blood Cells and EBV-B Cells, Stimulated with TLR Agonists
(A) IRAK-1 in EBV-B-cell lines treated with or without R-848 (5 μg/ml) for 2 hr, as determined by Western blotting of whole-cell extracts with an anti-IRAK-1 antibody and normalized with an anti-STAT-2 antibody.
(B) TNF-α secretion, as measured by ELISA, 24 hr after R-848 (5 μg/ml, TLR7/8), 3M-13 (3 μg/ml, TLR7), and 3M-2 (3 μg/ml, TLR8) stimulation. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from three independent individuals, each tested twice.
(C) NF-κB p65 subunit subcellular localization in monocytes from peripheral blood mononuclear cells left unstimulated or stimulated with poly(I:C) (50 μg/ml), LPS (1 μg/ml), or R-848 (5 μg/ml) for 2 hr, as determined by immunofluorescence analysis.
(D) IRAK-1 in freshly prepared monocytes left unstimulated or stimulated for 2 hr in the same conditions, as detected by Western blotting of whole-cell extracts, with an anti-IRAK-1 antibody. IRAK-1 and Stat-2 signals were analyzed and quantified using NIH Image version 1.63 ( IRAK-1 signals were standardized with the corresponding STAT-2 signals, and IRAK-1 degradation was then evaluated in control and patient monocytes. Standardized IRAK-1 signal in control monocytes is 50% lower than that in patient monocytes upon R-848 stimulation, whereas IRAK-1 signals were similar in control and patient monocytes after poly(I:C) stimulation.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions