J Clin Immunol DOI 10.1007/s10875-014-9998-2

ORIGINAL RESEARCH

Correlating Interleukin-12 Stimulated Interferon-γ Production and the Absence of Ectodermal Dysplasia and Anhidrosis (EDA) in Patients with Mutations in NF-κB Essential Modulator (NEMO) Margje H. Haverkamp & Beatriz E. Marciano & David M. Frucht & Ashish Jain & Esther van de Vosse & Steven M. Holland

Received: 22 July 2013 / Accepted: 7 February 2014 # Springer Science+Business Media New York 2014

Abstract Objective Patients with hypomorphic mutations in Nuclear Factor-κB Essential Modulator (NEMO) are immunodeficient (ID) and most display ectodermal dysplasia and anhidrosis (EDA). We compared cytokine production by NEMO-ID patients with and without EDA. Methods PBMCs of NEMO-ID patients, four with EDA carrying E315A, C417R, D311N and Q403X, and three without EDA carrying E315A, E311_L333del and R254G, were cultured with PHA, PHA plus IL-12p70, LPS, LPS plus IFN-γ, TNF and IL-1β. The production of various cytokines was measured in the supernatants. Fifty-nine healthy individuals served as controls. Results PBMCs of NEMO-ID patients without EDA produce subnormal amounts of IFN-γ after stimulation with PHA, but normal amounts of IFN-γ after PHA plus IL-12p70. In contrast, IFN-γ production by patients with EDA was low in both cases. Patients with EDA also generate lower PHA-stimulated

M. H. Haverkamp (*) : E. van de Vosse Department of Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands e-mail: [email protected] M. H. Haverkamp : B. E. Marciano : S. M. Holland Laboratory of Clinical Infectious Diseases, National Institutes of Health, Bethesda, MD, USA D. M. Frucht Division of Monoclonal Antibodies, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, USA A. Jain Laboratory of Host Defenses, National Institutes of Health, Bethesda, MD, USA

IL-10 and IL-1β than controls, whereas the production of these cytokines by patients without EDA was normal. Conclusion Responses of PBMCs in NEMO-ID patients with EDA to PHA with and without IL-12p70 appear less robust than in NEMO-ID patients without EDA. This possibly indicates a better preserved NEMO function in our patients without EDA. Keywords NF-κB essential modulator (NEMO) . ectodermal dysplasia and anhidrosis (EDA) . nontuberculous mycobacteria (NTM) . cytokines

Introduction Many cell activation pathways converge on the transcription factor NF-κB, the full activation of which requires NF-κB essential modulator (NEMO) [1]. At rest, NF-κB is bound to IκBα and kept inactive in the cytoplasm (Fig. 1). Upon activation by bacterial components or pro-inflammatory cytokines, IκBα is phosphorylated by IκB-kinase (IKK). The latter is a heterotrimer, consisting of IKK-α, IKK-β and its scaffold protein IKK-γ, better known as NEMO. While IκBα is subsequently ubiquitinated and degraded by the proteosome, NF-κB translocates to the nucleus [1]. The gene that encodes NEMO, IKBKG, is located on the X-chromosome and consists of ten exons (Fig. 2). Mutations in IKBKG are either amorphic or hypomorphic with 40 % missense mutations [2]. Amorphic mutations are lethal in males. In females who often display skewed X-inactivation, they can lead to Incontinentia Pigmenti (OMIM #308300) with “overfloating skin-pigmentation” and nail- and tooth dysplasia [3]. In contrast, hypomorphic mutations in

J Clin Immunol TNF, IFN-γ Nucleus

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Fig. 1 The NF-κB signaling pathway and the IL-12/IFNγ cytokine loop. NF-κB, a heterodimer, is bound to IκBα in the cytoplasm of resting cells. This transcription factor can be indirectly activated and released to the nucleus by the binding of numerous cytokines and pathogen-associated molecules like LPS to receptors belonging to the TIR and TNFR superfamilies on the cell surface of many cell types. For this to happen, IκBα has to be phosphorylated by the IκB-kinase (IKK)-complex. NF-κB

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Essential Modulator protein (NEMO) is the scaffold of this complex. After activation, NF-κB ensures the production of its own shepherd, IκBα, and of various cytokines, among others IL-12. IL-12 excretion by monocytes stimulates T-cells to produce IFN-γ. This in turn activates the IFN-γ receptor on monocytes leading to the translocation of Interferon-gamma Activating Factor (GAF) to the nucleus for activation of transcription

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P5:D311N Fig. 2 Genomic structure of the X-linked NEMO gene IKBKG. The ten exons of IKBKG span 23 kb of genomic sequence. The first exon can be one of three alternative non-coding exons. The initiating ATG codon is located in exon 2. The position of R254G, D311N, E315A and

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P6:Q403X P4:C417R

E331_333del are given, all four within the CC2-LZ domain. The mutations of patient 4 (C417R) and 6 (Q403X) are located within the Zinc Finger domain. CC1 coiled-coil domain 1; CC2 coiled-coil domain 2; LZ Leucine Zipper; ZF Zinc Finger. Domains according to Lo, 2009 [29]

J Clin Immunol

NEMO are not lethal for men and only rarely cause disease in women [2]. Hypomorphic mutations in IKBKG lead to a wide variety of clinical phenotypes (OMIM #300291 and #300584), with immunodeficiency (NEMO-ID) as a common denominator [2]. Many different bacterial (86 % of patients), mycobacterial (44 % of patients), viral and fungal infections in NEMO-ID patients result from the central position of NEMO within the NF-κB pathway [2]. Mutations in IKBKG hamper the transmission of NF-κB activating signals to the nucleus from TNFR-, TIR- (among others TLR) and other pattern recognition-receptors (PPRs). Because T- and B-cell receptors also signal through NF-κB, NEMO-ID patients can have impaired innate as well as adaptive immune responses [4], the latter sometimes manifesting as hypogammaglobulinaemia and defects in specific antibody responses to polysaccharide antigens [2]. A defective NF-κB pathway can also adversely affect the IL-12/IFN-γ cytokine loop. Both pathways are linked via the CD40/CD40L- and NEMO-dependent production of IL-12 [5–7], and via IL-18R-stimulated production of IFN-γ (Fig. 1) [8]. Most, but not all, NEMO-ID patients have ectodermal dysplasia (sparse and/or whorled hair, conical teeth) and anhidrosis (EDA) [2, 9, 10]. A 2008 database of 72 NEMOID patients with 32 different hypomorphic mutations showed that the group of patients without EDA, or with dental abnormalities alone, is larger than previously thought [2]. Although it is difficult to identify clear-cut geno-phenotype relations in NEMO-ID [2], individual mutations in IKBKG have their specific impact on NEMO signaling [2]. In this paper, we report ex vivo cytokine production by PBMCs of seven NEMO-ID patients after stimulation of two pathways, namely the NEMO dependent NF-κB pathway and the IL-12/IFN-γ cytokine loop. We show that NEMO-ID patients with EDA produce low amounts of IFN-γ after stimulation with IL-12p70 and PHA, whereas IFN-γ production by NEMO-ID patients without EDA in response to these stimuli was normal. The production of other cytokines after stimulation with PHA was also slightly higher in NEMO-ID patients without EDA.

Methods Subjects Clinical data of the patients were obtained from medical and research records of the National Institutes of Health, Bethesda, and analyzed retrospectively. Cytokine production was determined in seven NEMO-ID patients, of whom three did not have EDA. Two of the patients were related and carried 944A>C (E315A) in the leucin zipper of IKBKG (Patient 1a and 1b) (Fig. 2). Five other patients had unique mutations,

namely 991del9 (E331_L333del3) (Patient 2), 760C>G (R254G) (Patient 3), 1249T>C (C417R) (Patient 4), 931G>A (D311N) (Patient 5), 1207C>T (Q403X) (Patient 6) (Fig. 2). All patients were briefly mentioned in the 2008 hypomorphic NEMO database [2]. Patients 1a [7, 11–14], 1b [7], 2 [12] and 3 [15] have been subject of previous reports, most of which preceded the publication of their NEMO defect. For an extensive description of their clinical history, we refer to these reports. Blood from 59 healthy individuals was obtained from the NIH blood bank, as a control group. Blood samples of the patients were analyzed simultaneously with controls. PBMC Stimulation and Cytokine Determination PBMCs (106 cells per condition) of patients and healthy controls were stimulated in RPMI-1640, supplemented with 20 mM Hepes, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 % FCS. Cells were stimulated for 48 h with either PHA (1 %) with and without IL-12p70 (1 ng/ml); LPS (200 ng/ml) with and without IFN-γ (1,000 U/ ml); TNF (20 ng/ml) or IL-1β (10 ng/ml). The production of IFN-γ, IL-12p70, IL-6, IL-10, TNF, and IL-1β was measured with a 6-plex version of the Bioplex cytokine assay (Bio-Rad). Patients 1a and 1b were assayed several times (three and two times respectively), patients 2–6 were assayed once. Statistical Analysis The Mann–Whitney U test was used for non-parametric comparison of cytokine production of unpaired groups. Results were considered to be statistically significant when p

Correlating interleukin-12 stimulated interferon-γ production and the absence of ectodermal dysplasia and anhidrosis (EDA) in patients with mutations in NF-κB essential modulator (NEMO).

Patients with hypomorphic mutations in Nuclear Factor-κB Essential Modulator (NEMO) are immunodeficient (ID) and most display ectodermal dysplasia and...
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