ORIGINAL RESEARCH

Correlating Interleukin-12 Stimulated Interferon-γ Productionand 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

AbstractObjective Patients with hypomorphic mutations in NuclearFactor-κB Essential Modulator (NEMO) are immunodeficient(ID) and most display ectodermal dysplasia and anhidrosis(EDA). We compared cytokine production by NEMO-IDpatients with and without EDA.Methods PBMCs of NEMO-ID patients, four with EDA car-rying E315A, C417R, D311N and Q403X, and three withoutEDA carrying E315A, E311_L333del and R254G, were cul-tured with PHA, PHA plus IL-12p70, LPS, LPS plus IFN-γ,TNF and IL-1β. The production of various cytokines wasmeasured in the supernatants. Fifty-nine healthy individualsserved as controls.Results PBMCs of NEMO-ID patients without EDA producesubnormal amounts of IFN-γ after stimulation with PHA, butnormal amounts of IFN-γ after PHA plus IL-12p70. In con-trast, IFN-γ production by patients with EDAwas low in bothcases. Patients with EDA also generate lower PHA-stimulated

IL-10 and IL-1β than controls, whereas the production ofthese cytokines by patients without EDAwas normal.Conclusion Responses of PBMCs in NEMO-ID patients withEDA to PHA with and without IL-12p70 appear less robustthan in NEMO-ID patients without EDA. This possibly indi-cates a better preserved NEMO function in our patients with-out EDA.

Keywords NF-κB essential modulator (NEMO) . ectodermaldysplasia and anhidrosis (EDA) . nontuberculousmycobacteria (NTM) . cytokines

Introduction

Many cell activation pathways converge on the transcriptionfactor NF-κB, the full activation of which requires NF-κBessential modulator (NEMO) [1]. At rest, NF-κB is bound toIκBα and kept inactive in the cytoplasm (Fig. 1). Uponactivation by bacterial components or pro-inflammatory cyto-kines, IκBα is phosphorylated by IκB-kinase (IKK). Thelatter is a heterotrimer, consisting of IKK-α, IKK-β and itsscaffold protein IKK-γ, better known as NEMO. While IκBαis subsequently ubiquitinated and degraded by theproteosome, NF-κB translocates to the nucleus [1].

The gene that encodes NEMO, IKBKG, is located on theX-chromosome and consists of ten exons (Fig. 2). Mutationsin IKBKG are either amorphic or hypomorphic with 40 %missense mutations [2]. Amorphic mutations are lethal inmales. In females who often display skewed X-inactivation,they can lead to Incontinentia Pigmenti (OMIM #308300)with “overfloating skin-pigmentation” and nail- and toothdysplasia [3]. In contrast, hypomorphic mutations in

M. H. Haverkamp (*) : E. van de VosseDepartment of Infectious Diseases, Leiden University MedicalCenter, Albinusdreef 2, 2333 ZA Leiden, The Netherlandse-mail: m.h.haverkamp@lumc.nl

M. H. Haverkamp : B. E. Marciano : S. M. HollandLaboratory of Clinical Infectious Diseases, National Institutes ofHealth, Bethesda, MD, USA

D. M. FruchtDivision of Monoclonal Antibodies, Center for Drug Evaluation andResearch, U.S. Food and Drug Administration, Bethesda, MD, USA

A. JainLaboratory of Host Defenses, National Institutes of Health, Bethesda,MD, USA

J Clin ImmunolDOI 10.1007/s10875-014-9998-2

IL-18

I B , TNF, IL-1, IL-6, IL-12, IL-18

Monocyte

T-Lymphocyte

TNF

CD40

IFN- R

IL-12R

Ubiquitination,Degradation

TNF, IFN γ-

IFN γ-

IL-12

NF- B

Nucleus

Nucleus

I B

pp

NEMOp

IKKIKK

TIR superfamily

TIR superfamily

TNFR superfamily

NF- B

IKK

I B

NF- B

+

Activation of I Bα by the IKK complex, degradation of I B

CD40-L

IL-1EDA

γ

LPS

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 thenucleus by the binding of numerous cytokines and pathogen-associatedmolecules like LPS to receptors belonging to the TIR and TNFR super-families on the cell surface of many cell types. For this to happen, IκBαhas to be phosphorylated by the IκB-kinase (IKK)-complex. NF-κB

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 excretionby monocytes stimulates T-cells to produce IFN-γ. This in turn activatesthe IFN-γ receptor on monocytes leading to the translocation of Interfer-on-gamma Activating Factor (GAF) to the nucleus for activation oftranscription

2 3 4 5 6 7 8 9 10 coding region

1a 1b 1c 2 3 4 5 6 7 8 9 10 exons

1 93 - 194 251 - 299 300 -350 397 -417 amino-acids

N’ CC2

P3:R254G P1a,b:E315A P2:E331_L333del

LZ ZFCC1 C’

3 alternative primary exons, non-coding

IKBKG

NEMO protein

P5:D311N P6:Q403X P4:C417R

Fig. 2 Genomic structure of the X-linked NEMO gene IKBKG. The tenexons of IKBKG span 23 kb of genomic sequence. The first exon can beone of three alternative non-coding exons. The initiating ATG codon islocated in exon 2. The position of R254G, D311N, E315A and

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

J Clin Immunol

NEMO are not lethal for men and only rarely causedisease in women [2].

Hypomorphic mutations in IKBKG lead to a wide varietyof clinical phenotypes (OMIM #300291 and #300584), withimmunodeficiency (NEMO-ID) as a common denominator[2]. Many different bacterial (86 % of patients), mycobacterial(44 % of patients), viral and fungal infections in NEMO-IDpatients result from the central position of NEMO within theNF-κB pathway [2]. Mutations in IKBKG hamper the trans-mission of NF-κB activating signals to the nucleus fromTNFR-, TIR- (among others TLR) and other patternrecognition-receptors (PPRs). Because T- and B-cell receptorsalso signal through NF-κB, NEMO-ID patients can have im-paired innate as well as adaptive immune responses [4], thelatter sometimes manifesting as hypogammaglobulinaemiaand defects in specific antibody responses to polysaccharideantigens [2]. A defective NF-κB pathway can also adverselyaffect the IL-12/IFN-γ cytokine loop. Both pathways arelinked via the CD40/CD40L- and NEMO-dependent produc-tion of IL-12 [5–7], and via IL-18R-stimulated production ofIFN-γ (Fig. 1) [8].

Most, but not all, NEMO-ID patients have ectodermaldysplasia (sparse and/or whorled hair, conical teeth) andanhidrosis (EDA) [2, 9, 10]. A 2008 database of 72 NEMO-ID patients with 32 different hypomorphic mutations showedthat the group of patients without EDA, or with dental abnor-malities alone, is larger than previously thought [2]. Althoughit is difficult to identify clear-cut geno-phenotype relations inNEMO-ID [2], individual mutations in IKBKG have theirspecific impact on NEMO signaling [2].

In this paper, we report ex vivo cytokine production byPBMCs of seven NEMO-ID patients after stimulation of twopathways, namely the NEMO dependent NF-κB pathway andthe IL-12/IFN-γ cytokine loop. We show that NEMO-IDpatients with EDA produce low amounts of IFN-γ after stim-ulation with IL-12p70 and PHA, whereas IFN-γ productionby NEMO-ID patients without EDA in response to thesestimuli was normal. The production of other cytokines afterstimulation with PHA was also slightly higher in NEMO-IDpatients without EDA.

Methods

Subjects

Clinical data of the patients were obtained from medical andresearch records of the National Institutes of Health, Bethesda,and analyzed retrospectively. Cytokine production was deter-mined in seven NEMO-ID patients, of whom three did nothave EDA. Two of the patients were related and carried944A>C (E315A) in the leucin zipper of IKBKG (Patient 1aand 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 hypomorphicNEMO database [2]. Patients 1a [7, 11–14], 1b [7], 2 [12] and3 [15] have been subject of previous reports, most of whichpreceded the publication of their NEMO defect. For an exten-sive description of their clinical history, we refer to thesereports. Blood from 59 healthy individuals was obtained fromthe NIH blood bank, as a control group. Blood samples of thepatients were analyzed simultaneously with controls.

PBMC Stimulation and Cytokine Determination

PBMCs (106 cells per condition) of patients and healthycontrols were stimulated in RPMI-1640, supplemented with20 mM Hepes, 2 mM glutamine, 100 U/ml penicillin,100 μg/ml streptomycin and 10% FCS. Cells were stimulatedfor 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 ofIFN-γ, IL-12p70, IL-6, IL-10, TNF, and IL-1β was measuredwith a 6-plex version of the Bioplex cytokine assay (Bio-Rad).Patients 1a and 1b were assayed several times (three and twotimes respectively), patients 2–6 were assayed once.

Statistical Analysis

The Mann–Whitney U test was used for non-parametric com-parison of cytokine production of unpaired groups. Resultswere considered to be statistically significant when p<0.05(two-sided). For patients 1a and 1b, mean cytokine levels areused for the comparison of cytokine production with healthycontrols.

Results

Patients with and without EDA

Patient 1a was a maternal uncle of patient 1b. They belongedto a Caucasian family that included two other members shar-ing the same mutation. These four patients are deceased, allbut one from their infections. Patients 1a, 2 and 3 did not haveEDA as evaluated by an NIH dermatologist. They lacked thetypical hair, dental and sweating characteristics of ectodermaldysplasia, although their teeth were not completely normal.Patient 1 had sparse teeth, patient 2 had five decaying teethand male pattern baldness, as well as flat warts all over hishands and arms. Patient 3 had decay in 11 teeth and flat-topped papules over his extremities and forehead. The absenceof EDA in patient 1a, 2, and 3 significantly delayed thediscovery of the IKBKG mutations. Persistent disseminated

J Clin Immunol

infections with nontuberculous mycobacteria (NTM) sug-gested severe cases of immunodeficiency that eventually re-quired medical attention. In contrast, patient 1b had overtsigns of EDA: conical teeth, hypodontia, hypotrichosis andwhorled hair but no anhidrosis. In addition to patient 1b,patients 4, 5 and 6 all displayed typical signs of EDA: conicalteeth, sparse hair and anhidrosis. The clinical data and immu-nological parameters of all seven NEMO-ID patients aresummarized in Table I.

Cytokine Profiles of the NEMO-ID Patientswith and without EDA in Response to PHA

In order to explore differences in cytokine profiles be-tween NEMO-ID patients with and without EDA, weperformed cytokine stimulation assays in both groupsand compared the results to those of healthy controls.We found low PHA-stimulated IFN-γ production byPBMCs of NEMO-ID patients with EDA (p<0.006),and normal but below average amounts of IFN-γ in

patients without EDA (Fig. 3a). The addition of exoge-nous IL-12p70 enhanced IFN-γ production by patientswithout EDA to control average. In contrast, in patientswith EDA, PHA-stimulated IFN-γ concentrationsremained low (p<0.005) (Fig. 3a).

Similarly, we observed a subtle difference betweenNEMO-ID patients with and without EDA in the response toPHA when measuring other cytokines. Patients with EDAproduced significantly lower levels of IL-10 and IL-1β afterstimulation with PHA than controls (p<0.006 and p<0.03respectively), whereas the PHA-stimulated production of the-se cytokines by patients without EDAwas within the normalcontrol range (Fig. 3b–c). Both EDA phenotypes producednormal amounts of TNF (Fig. 3d), and low amounts of IL-6and IL-12p70 after stimulation with PHA (Fig. 3e–f). Cyto-kine production in non-stimulated conditions by both groupsof NEMO-ID patients was either normal (IL-1β, IL-12p70)(Fig. 3c and f) or low (IFN-γ) (Fig. 3a). Alternatively, cyto-kine production was significantly lower than normal by pa-tients with EDA only (IL-10, TNF, IL-6) (Fig. 3b, and d–e).

Table I Clinical and immune function of patients with mutations in IKBKG compared to patients from Hanson’s Hypomorphic NEMO mutationdatabase

Functional orclinical category

Patient1a

Patient1b

Patient2

Patient3

Patient4

Patient5

Patient6

Thisreport

Databasea Databasea

E315A E315A E331_L333del R254G C417R D311N Q403X % (#/total) No EDA, %(total=12)

All NEMO, %(total=72)

EDA No Yes No No Yes Yes Yes 57 (4/7) 0 (12/12) 77 (40/52)

Autoimmune disease No No No No No No No 0 (0/7) 8 (1/12)b 23 (14/66)

Dead from disease Yes Yes Yes Yes Yes Yes No 86 (6/7) 33 (5/12)c 36 (24/66)

Age (mean) in years 44 19 49 25 25 10 Alive (29) (36) (6.4)

Infectious susceptibility Yes Yes Yes Yes Yes Yes Yes 100 (7/7) 100 (12/12) 98 (60/61)

Bacterial infection Yes Yes Yes Yes Yes Yes Yes 100 (7/7) 83 (10/12) 86 (45/52)

Mycobacterial inf. Yes Yes Yes Yes Yes Yes Yes 100 (7/7) 75 (9/12) 44 (23/52)

Viral infection No No No Yes Yes Yes No 43 (3/7) 25 (3/12) 21 (11/52)

Pneumonia Yes No Yes No No No Yes 43 (3/7) 92 (11/12) 31 (19/16)

Sepsis/bacteremia Yes Yes Yes No No No No 43 (3/7) 50 (6/12) 33 (20/61)

Abscess No No No Yes No No No 14 (1/7) 50 (6/12) 30 (18/61)

Hyper-IgM No No No nd No No No 0 (0/6) 7 (2/12) 15 (6/40)

Hypogammaglobulinemia Nod No No nd Yesd Yesd Yesd 0 (0/6) 8 (1/12) 59 (24/41)

Hyper-IgA Yes Yes Yes nd Yes Yes Yes 100 (6/6) 50 (6/12) 37 (13/35)

Pneumococcal antibodies Yes Yes Yes Yes nd nd nd 100 (4/4) 55 (6/11) 19 (3/16)

CD4+ lymphopenia No nd Yes Yes No nd No 40 (2/5) 20 (2/10) 21 (6/28)

We compared the patients in this study to the data of 72NEMO-ID patients from theHypomorphic NEMOMutationDatabase (Hanson’s Table 1.). Fromthe latter report, we singled out the information on 12 NEMO-ID patients without EDA, including Patient 1a, 2 and 3 [2]. We completed this table withdata from our own research records and the individual case reports [2, 7, 12, 18–20, 22]a Percentage of patients of whom information is available or in whom tests are performedbOne patient had an autoimmune hemolytic anaemiac The patient carrying E315Awho died at age 10 in a car accident (brother of patient 1b in this study) is not taken into accountd These patients were on gamma-globulin suppletion therapy

J Clin Immunol

Cytokine Profiles of the NEMO-ID Patientswith and without EDA in Response to LPS

The signal triggered by the binding of LPS to the Toll-likereceptor 4 (TLR4) is also transduced via NEMO (Fig. 1). Inorder to detect a possible difference in cytokine productionbetween LPS-stimulated PBMCs from NEMO-ID patientswith and without EDA, we measured this production in pa-tients and controls in response to LPS with and withoutIFN-γ. Although patient 1b, an EDA-patient carrying theE315A mutation, was capable of normal IL-12p70 productionboth at rest and in PHA- and LPS-stimulated conditions(Figs. 3f and 4a), he could not produce more IL-12p70 whenIFN-γ was added to the LPS stimulus (Fig. 4a). The onlypatient producing normal amounts of IL-12p70 after stimula-tion with LPS and IFN-γ was a patient without EDA carryingthe E331_L333del mutation (Patient 2, Fig. 4a). Furthermore,this patient generated the most IL-6 (data not shown), TNFand IL-1β (Fig. 4b) when stimulated with a combination ofIFN-γ and LPS.

LPS alone made little difference in cytokine production inpatients with and without EDA (Fig. 4c–g). LPS-stimulatedPBMCs in both groups produced low amounts of IL-6 and IL-10 (Fig. 4c–d), and normal amounts of TNF (Fig. 4f). Theproduction of IFN-γ and IL-1βwas low by patients with EDAonly (p<0.004 and p<0.009 respectively) (Fig. 4e and g).

Cytokine induction by TNF and IL-1β, which are bothNEMO dependent cytokines, was normal, at least in the tworelated patients (data not shown).

Discussion

NEMO-ID patients are known to have abnormal cytokineproduction profiles that account for their immunodeficiency.Recent research has shown that only two-thirds of NEMO-IDpatients have EDA [2]. This phenotype originates from defec-tive, NEMO-dependent signaling by the ectodysplasin recep-tor EDAR, affecting NF-κB activation [16]. EDAR is mainlyexpressed during the development of ectodermal appendages

C EDA C EDA1

10

100

1000

10000

100000

1000000

noEDA

noEDA

NS PHA

IL-6

(p

g/m

l)

n.s.

P<0.006

P<0.007

P<0.03

a b

d e fc

Healthy Controls

Patient 1a, NEMO-ID no EDA

Patient 3, NEMO-ID no EDA

Patient 4, NEMO-ID with EDA

Patient 1b, NEMO-ID with EDA

Patient 2, NEMO-ID no EDA

Patient 5, NEMO-ID with EDA

Patient 6, NEMO-ID with EDAC EDA C EDA

1

10

100

1000

10000

noEDA

noEDA

n.s.

P<0.01

n.s.

P<0.006

NS PHA

IL-1

0 (p

g/m

l)

C EDA C EDA1

10

100

1000

10000

noEDA

noEDA

NS PHA

IL-1

(pg

/ml)

n.s.

n.s.

n.s.

P<0.03

C EDA C EDA0.1

1

10

100

1000

noEDA

noEDA

NS PHA

IL-1

2p70

(p

g/m

l)

n.s.

n.s.

P<0.03

P<0.03

C EDA C EDA C EDA1

10

100

1000

10000

100000

noEDA

noEDA

noEDA

NS PHA PHA+IL-12p70

IFN

-(p

g/m

l)

n.s.

P<0.006

n.s.

P<0.005

C EDA C EDA1

10

100

1000

10000

noEDA

noEDA

NS PHA

TN

F (

pg

/ml)

n.s. n.s.

n.s.

Fig. 3 PBMCs of NEMO-ID patients without EDA have a more robustresponse to stimulation with PHA and IL-12p70 than cells of patientswith EDA. PBMCs of NEMO-ID patients with and without EDA and of ahealthy control group (n=59) were stimulated for 48 h with 1 % PHA orwere left unstimulated (NS). The amounts of IFN-γ, IL-10, TNF, IL-1β,IL-6 and IL-12p70 were measured in the supernatants by a Bioplex

cytokine assay. The concentration of IFN-γ was also measured after theaddition of IL-12p70 (1 ng/ml) to the PHA-stimulus. Patients 1a and 1bwere assayed several times (three and two times respectively). Their meanvalues are depicted. Bars indicate the median cytokine production bypatients and healthy controls

J Clin Immunol

such as hair and teeth [17]. Fifteen NEMO-ID patients withoutEDA have been published [2, 7, 12, 18–24]. To date, E315A isthe only NEMOmutation with a variable penetrance for EDA.Families with defects in both the gene for ectodysplasin-A(EDA) and IKBKG have been described [25], but wewere ableto exclude mutations in EDA and in the gene for its receptorEDAR (EDAR) in our patients with E315A. In this report, wedetermined cytokine production in NEMO-ID patients withand without EDA and noticed that patients without EDA havea more robust response to various stimuli than patients withEDA.

Previous research on cytokine production in NEMO-IDpatients described the impairment of innate signaling throughTLR (64 % of patients), IL-1R (86 %), TNFR (82 %) andCD40 (94 %) [2, 21, 23], as well as a failure to secrete normalamounts of IFN-γ and IL-12 in response to PHA [2, 7, 12, 13].It also showed that PHA-stimulated TNF and IL-10 produc-tion [7, 13], as well as LPS-stimulated IL-12 [7] and TNF [21,26] production were normal. However, these results werecontingent upon the specific IKBKG mutation tested. Bydefinition, all hypomorphic IKBKG mutations impair NF-κBsignaling partially but not fully [2].

We confirm low PHA-stimulated IFN-γ and IL-12p70production in seven NEMO-ID patients with various muta-tions, including E315A. The E315A mutation has been rec-ognized as a potential cause of Mendelian Susceptibility toMycobacterial Disease (MSMD) [7]. MSMD-patients areprone to NTM infections because of a defective IFN-γ/IL-12 cytokine loop. Therefore, we assume that the abnormalproduction of IFN-γ and IL-12p70 explains the incidence ofNTM infections in all our patients.

Importantly, we found that by adding IL-12p70, thePBMCs of patients without EDA were capable of bringingback levels of PHA-stimulated IFN-γ to control average. Incontrast, exogenous IL-12p70 could not normalize the pro-duction of IFN-γ in patients with EDA. Apparently, in pa-tients without EDA, exogenous IL-12 can compensate fordefective in vivo IL-12 production, as has previously beenshown for NEMO patients with MSMD carrying the E315Amutation [7, 13].

Partial normalization of PHA-stimulated IFN-γ productionby exogenous IL-12p70 indicates that in NEMO-ID patientswith EDA, other mechanisms are at play than simply thedefective in vivo IL-12 production. For example, the

Healthy Controls

Patient 1a, NEMO-ID no EDA

Patient 3, NEMO-ID no EDA

Patient 4, NEMO-ID with EDA

Patient 1b, NEMO-ID with EDA

Patient 2, NEMO-ID no EDA

Patient 5, NEMO-ID with EDA

Patient 6, NEMO-ID with EDA

a b c

d e f g

C EDA C EDA1

10

100

1000

10000

noEDA

noEDA

NS LPS

IL-1

0 (p

g/m

l)

n.s.

P<0.01

P<0.006

P<0.002

C EDA C EDA1

10

100

1000

10000

100000

TNF IL-1

noEDA

noEDA

LPS + IFN-

n.s.

P<0.03

n.s.

P<0.003

cyto

kin

e (p

g/m

l)

C EDA C EDA1

10

100

1000

10000

100000

noEDA

noEDA

NS LPS

IL-1

(pg

/ml)

n.s.

n.s.

n.s.

P<0.009

C EDA C EDA1

10

100

1000

10000

100000

1000000

noEDA

noEDA

NS LPS

IL-6

(p

g/m

l)

n.s.

P<0.006

P<0.02

P<0.004

C NEMO C NEMO C NEMO0.1

1

10

100

1000

NS LPS LPS+IFN-

IL-1

2p70

(p

g/m

l)

n.s. n.s. P<0.0007

C EDA C EDA1

10

100

1000

10000

noEDA

noEDA

NS LPS

IFN

-(p

g/m

l)

n.s.

P<0.004

C EDA C EDA1

10

100

1000

10000

noEDA

noEDA

NS LPS

TN

F (

pg

/ml)

n.s. n.s.

n.s.

Fig. 4 No difference in LPS-stimulated cytokine production betweenNEMO-ID patients with and without EDA. PBMCs of NEMO-ID pa-tients with and without EDA and of a healthy control group (n=59) werestimulated for 48 h with LPS (200 ng/ml) or were left unstimulated (NS).The amounts of IL-12p70, IL-6, IL-10, IFN-γ, TNF and IL-1β weremeasured in the supernatants by a Bioplex cytokine assay. The

concentration of IL-12p70 was also measured after the addition of IFN-γ(1,000 U/ml) to the LPS-stimulus. Patients 1a and 1b were assayedseveral times (three and two times respectively). Their mean values aredepicted. Bars indicate the median cytokine production by patients andhealthy controls

J Clin Immunol

upregulation of IL-12Rβ2 via NEMO-dependent IL-18R sig-naling could be blocked [8]. Indeed, PBMCs of a patient withEDA produced lower amounts of IFN-γ after stimulation witha combination of IL-12 and IL-18 compared to healthy controlcells [27].

In our assays, LPS did not result in different levels of mostcytokines between NEMO patients with and without EDA.When a difference was observed, patients with EDA producedsignificantly lower cytokines than normal, whereas cytokinelevels in patients without EDA were similar to those in con-trols. The absence of a discriminating effect means that apotential difference in NEMO function in patients with andwithout EDA depends on the stimulus used. Low cytokineproduction in our patients, mostly in those with EDA, afterstimulation with LPS suggests an impairment of NF-κB sig-naling through TLR in NEMO-ID. This is not in line with aprevious report on E315A [7]. The inconsistencymight be dueto a different selection of the control group; the former pub-lication did not use as many healthy controls as we did andtherefore could have misinterpreted the patient’s cytokineproduction as normal.

Furthermore, it is noteworthy that patients 4 and 6, bothpatients with EDA and mutations in the Zinc Finger domain,produced consistently lower amounts of PHA- and LPS-stimulated cytokines than the other five NEMO-ID patients.Mutations in the Zinc Finger domain are known to cause amore severe clinical, developmental and cellular NEMO phe-notype than other mutations [2, 16].

Milder defects in cytokine responses do not translate into alower susceptibility to NTM infections in NEMO-ID patientswithout EDA. Indeed, all three patients without EDA wetested, as well as 75 % of patients without EDA in thehypomorphic NEMO mutation database, had severe myco-bacterial infections, while only 44 % of NEMO-ID patientswith EDA did [2]. Potential reasons include selection andexposure bias. Patients lacking both EDA and mycobacterialinfections may go undetected, while patients with EDA maydie too young to acquire sufficient mycobacterial exposure.The high frequency of NTM infections in patients withoutEDA explains the higher incidence of hyper-IgA in this group(50 vs 37% in patients with EDA, at least 75% in our patientswithout EDA). IgA antibodies are formed in response tovarious mycobacterial products, including components ofthe cell wall [28].

Our cytokine stimulation assay results show subtle differ-ences in cytokine production between NEMO-ID patientswith and without EDA. This leads us to conjecture that theabsence of EDA parallels a better preserved function ofNEMO in the patients we studied, but this has yet to beconfirmed in a larger group of NEMO-ID/EDA patients. Themilder cellular phenotype in our patients without EDA issupported by a much higher mean age at death compared topatients with EDA (36 vs 6 years, see Table I). The

E331_L333del3 mutation appears to affect NEMO functionthe least of all tested mutations in this report. It did not causeEDA and produced stimulated cytokine levels closest to con-trol average. The relevant patient died at age 49. Salt et al.compared cytokine production in one patient with EDA andone without, and their findings support our conclusions. TLRstimulated-TNF production by PBMCs of a patient withL153R and overt EDA was clearly depressed, while it wasnormal in a patient without EDA and a D113N mutation [22].At any rate, a differential preserved NEMO function requiresconfirmation by in vitro data on NF-κB activation by NEMO,for example NEMO protein expression, IκBα degradation,NF-κB translocation and stimulated DNA-binding.

Conclusion

NEMO-ID is a complex disease whose phenotypic range isstill to a large extent undetermined. In this article, we havehighlighted subtle differences in PHA-stimulated cytokineproduction between NEMO patients with and withoutmalformations of ectodermal structures that are worth explor-ing in a larger patient sample.

Acknowledgments We thank Amy Hsu for sequencing EDA andEDAR in patient 1a and 1b.

M.H. has been supported by grants from the Fulbright/NetherlandAmerica Foundation, the intramural Research Program of the NIH,National Institute of Allergy and Infectious Diseases, the NetherlandsOrganisation for Scientific Research (NWO), the Stichting DoctorCatharine van Tussenbroek Fonds, the Prins Bernhard Cultuurfonds, theLeids Universiteits Fonds, the Stichting Dr Hendrik Muller’sVaderlandsch Fonds, the Stichting Fundatie Vrijvrouwe van Renswoudeand the Stichting Algemeen Studiefonds.

References

1. Karin M, Ben-Neriah B. Phosphorylation meets ubiquitination: thecontrol of NF-κB activity. Ann Rev Immunol. 2002;18:621–63.

2. Hanson EP, Monaco-Shawver L, Solt LA, Madge LA, Banerjee PP,May MJ, et al. Hypomorphic nuclear factor-kappaB essential modu-lator mutation database and reconstitution system identifies pheno-typic and immunologic diversity. J Allergy Clin Immunol. 2008;122:1169–77.

3. Smahi A, Courtois G, Rabia SH, Doffinger R, Bodemer C, MunnichA, et al. The NF-kappaB signaling pathway in human diseases: fromincontinentia pigmenti to ectodermal dysplasia and immune-deficiency syndromes. Hum Mol Genet. 2002;11:2371–5.

4. Vallabhapurapu S, Karin M. Regulation and function of NF-kappaBtranscription factors in the immune system. Ann Rev Immunol.2009;27:693–733.

5. Shu U, KiniwaM,Wu CY,Maliszewski C, Vezzio N, Hakimi J, et al.Activated T cells induce interleukin-12 production by monocytes viaCD40-CD40 ligand interaction. Eur J Immunol. 1995;25:1125–8.

6. Temmerman ST, Ma CA, Borges L, Kubin M, Liu S, Derry JM, et al.Impaired dendritic-cell function in ectodermal dysplasia with

J Clin Immunol

immune deficiency is linked to defective NEMO ubiquitination.Blood. 2006;108:2324–31.

7. Filipe-Santos O, Bustamante J, Haverkamp MH, Vinolo E, Ku CL,Puel A, et al. X-linked susceptibility to mycobacteria is caused bymutations in NEMO impairing CD40-dependent IL-12 production. JExp Med. 2006;203:1745–59.

8. Xu D, Chan WL, Leung BP, Hunter D, Schulz K, Carter RW, et al.Selective expression and functions of interleukin-18 receptor on T-helper (Th) type 1 but not Th2 cells. J Exp Med. 1998;188:1485–92.

9. Zonana J, Elder ME, Schneider LC, Orlow SJ, Moss C, Golabi M,et al. A novel X-linked disorder of immune deficiency andhypohidrotic ectodermal dysplasia is allelic to incontinentia pigmentiand due to mutations in IKK-gamma (NEMO). Am J Hum Genet.2000;67:1555–62.

10. Courtois G, Smahi A, Reichenbach J, Doffinger R, Cancrini C,Bonnet M, et al. A hypermorphic IκBα mutation is associated withautosomal dominant anhidrotic ectodermal dysplasia and T cell im-munodeficiency. J Clin Invest. 2003;112:1108–15.

11. Nedorost ST, Elewski B, Tomford JW, Camisa C. Rosacea-likelesions due to familial Mycobacterium avium-intracellulare infec-tion. Int J Dermatol. 1991;30:491–7.

12. Holland SM, Eisenstein EM, Kuhns DB, Turner ML, Fleisher TA,StroberW, et al. Treatment of refractory disseminated nontuberculousmycobacterial infection with interferon gamma. N Engl J Med.1994;330:1348–55.

13. Frucht DM, Holland SM. Defective monocyte costimulation for IFN-γ production in familial disseminated Mycobacterium avium com-plex infection. J Immunol. 1996;157:411–6.

14. Frucht DM, Sandberg DI, BrownMR, Gerstberger SM, Holland SM.IL-12-independent costimulation pathways for interferon-γ produc-tion in familial disseminated Mycobacterium avium complex infec-tion. Clin Immunol. 1999;91:234–41.

15. Tobin E, Rohwedder A, Holland SM, Philips B, Carlson JA.Recurrent ‘sterile’ verrucous cyst abscesses and epidermodysplasiaverruciformis-like eruption associated with idiopathic CD4 lympho-penia. Br J Dermatol. 2003;149:627–33.

16. Doffinger R, Smahi A, Bessia C, Geissmann F, Feinberg J, DurandyA, et al. X-linked anhidrotic ectodermal dysplasia with immunodefi-ciency is caused by impaired NF-κB signaling. Nat Genet. 2001;27:277–85.

17. Laurikkala J, Pispa J, Jung HS, Mikkola M,Wang X, Saarialho-KereU, et al. Regulation of hair follicle development by the TNF signalectodysplasin and its receptor Edar. Development. 2002;129:2541–53.

18. Niehues T, Reichenbach J, Neubert J, Gudowius S, Puel A, HorneffG, et al. Nuclear factor kappaB essential modulator-deficient child

with immunodeficiency yet without anhidrotic ectodermal dysplasia.J Allergy Clin Immunol. 2004;114:1456–62.

19. Orange JS, Levy O, Brodeur SR, Krzewski K, Roy RM, Niemela JE,et al. Human nuclear factor kappaB essential modulator mutation canresult in immunodeficiency without ectodermal dysplasia. J AllergyClin Immunol. 2004;114:650–6.

20. Ku CL, Dupuis-Girod S, Dittrich AM, Bustamante J, Santos OF,Schulze I, et al. NEMO mutations in two unrelated boys with severeinfections and conical teeth. Pediatrics. 2005;115:e615–9.

21. Puel A, Reichenbach J, Bustamante J, Ku CL, Feinberg J, DoffingerR, et al. The NEMO mutation creating the most-upstream prematurestop codon is hypomorphic because of a reinitiation of translation.Am J Hum Genet. 2006;78:691–701.

22. Salt BH, Niemela JE, Pandey R, Hanson EP, Deering RP, QuinonesR, et al. IKBKG (nuclear factor-κB essential modulator) mutation canbe associated with opportunistic infection without impairing Toll-likereceptor function. J Allergy Clin Immunol. 2008;121:976–82.

23. Mooster JL, Cancrini C, Simonetti A, Rossi P, Di Matteo G, RomitiML, et al. Immune deficiency caused by impaired expression ofnuclear factor-κB essential modifier (NEMO) because of a mutationin the 5’untranslated region of the NEMO gene. J Allergy ClinImmunol. 2010;126(1):127.e7–32.e7.

24. Imamura M, Kawai T, Okada S, Izawa K, Takachi T, Iwabuchi H,et al. Disseminated BCG infection mimicking metastatic nasopha-ryngeal carcinoma in an immunodeficient child with a novelhypomorphic NEMO mutation. J Clin Immunol. 2011;31:802–10.

25. Keller MD, Petersen M, Ong P, Church J, Risma K, Burham J, et al.Hypohidrotic Ectodermal Dysplasia and immunodeficiency with co-incident NEMO and EDA mutations. Front Immunol. 2011;2:61.

26. Orange JS, Jain A, Ballas ZK, Schneider LC, Geha RS, Bonilla FA.The presentation and natural history of immunodeficiency caused bynuclear factor kappaB essential modulator mutation. J Allergy ClinImmunol. 2004;113:725–33.

27. Nishikomori R, Akutagawa H, Maruyama K, Nakate-Hizume M,Ohmori K, Mizuno K, et al. X-linked ectodermal dysplasia andimmunodeficiency caused by reversion mosaiscism of NEMO re-veals a critical role for NEMO in human T-cell development and/orsurvival. Blood. 2004;103:4565–72.

28. Mizusawa M, Kawamura M, Takamori M, Kashiyama T, FujitaA, Usuzawa M, et al. Increased synthesis of anti-tuberculousglycolipid immunoglobulin G (IgG) and IgA with cavity for-mation in patients with pulmonary tuberculosis. Clin VaccineImmunol. 2008;15:544–8.

29. Lo Y, Lin S, Rospigliosi CC, Conze DB, Wu C, Ashwell JD, et al.Structural basis for recognition of diubiquitins by NEMO. Mol Cell.2009;33:602–15.

J Clin Immunol