Transplantation from a symptomatic carrier sister restores host defenses but does not prevent colitis in NEMO deficiency

Abstract

NF-κB essential modulator (NEMO) deficiency causes ectodermal dysplasia with immunodeficiency in males, while manifesting as incontinentia pigmenti in heterozygous females. We report a family with NEMO deficiency, in which a female carrier displayed skewed X-inactivation favoring the mutant NEMO allele associated with symptoms of Behçet’s disease. Haematopoietic stem cell transplantation of an affected boy from this donor reconstituted an immune system with retained skewed X-inactivation. After transplantation no more severe infections occurred, indicating that an active wild-type NEMO allele in only 10% of immune cells restores host defense. Yet he developed inflammatory bowel disease (IBD). While gut infiltrating immune cells stained strongly for nuclear p65 indicating restored NEMO function, this was not the case in intestinal epithelial cells – in contrast to cells from conventional IBD patients. These results extend murine observations that epithelial NEMO-deficiency suffices to cause IBD. High anti-TNF doses controlled the intestinal inflammation and symptoms of Behçet’s disease.

1. Introduction

NF-κB essential modulator (NEMO), encoded by the X-linked IKBKG gene, constitutes an essential activator of the transcription factor NF-κB [1]. As many Toll-like and IL1R-family receptors signal via NF-κB, defects in NEMO impair the activation of immune responses, thereby compromising the early constraint of infections [2]. Thus, impaired NEMO function is associated with a severe immunodeficiency in hemizygous males [3].

Downstream of Tumor necrosis factor (TNF) receptor 1 canonical NF-κB-signaling via the NEMO/IKKα/IKKβ complex inhibits cell death [4–6]. Accordingly, murine models of epithelial NEMO deficiency exhibit increased TNF-mediated cell death, which may facilitate bacterial translocation and cause inflammatory bowel disease (IBD) [7,8]. Indeed, intestinal inflammation has been observed in approximately 20% of male NEMO-deficient patients, frequently associated with intractable diarrhea and/or failure to thrive [9][10]. While hematopoetic stem cell transplantation (HSCT) can correct the immunodeficiency [11,12], it often fails to improve the IBD [13,14]. The few reports describing conservative treatment options for NEMO-associated intestinal inflammation also report poor responses to steroids or other immunosuppressive drugs [14–17]. In a single patient NEMO-associated IBD has been successfully treated with the anti-TNF antibody infliximab [15].

Heterozygous female carriers of NEMO mutations may display incontinentia pigmenti. The variability of manifestations in females is attributed to different ratios of X-chromosome inactivation, leading to variable silencing of the wild-type or the mutated allele [16]. Here we provide the first report on HSCT of a NEMO-deficient patient from a symptomatic female carrier with skewed X-inactivation favoring an active mutant allele.

2. Methods

• 2.1Informed consent was obtained in accordance with local ethics committee guidelines (282/11_150561).
• 2.2Sequencing: 100 ng genomic DNA or cDNA corresponding to 25 ng total RNA of PBMC was amplified, followed by sequencing using Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany). Sequencing products were separated on an Applied Biosystems 3130xl Genetic Analyzer. Primer sequences are available upon request (ulrich.pannicke@uni-ulm.de).
• 2.3Western Blot: Cells were lysed using a buffer containing 50 mmol/L HEPES pH 7, 250 mmol/L NaCl, 5 mmol/L EDTA, 1% NP40 and Complete Protease Inhibitor. Lysates were first incubated with a rabbit anti-human IKKγ primary antibody (FL-419: sc-8330, Santa Cruz Biotechnology, Inc.) in PBS/5% BSA/0.1% Tween, followed by a goat anti-rabbit horse-radish peroxidase-secondary antibody (Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L), cat. no. 111-035-045, Jackson Immuno Research Laboratories). Finally, membranes were incubated in ECL buffer (Thermo Scientific).
• 2.4X-inactivation analysis: The inactivation status of the mutant IKBKG allele was analysed by assessing differential methylation of Hha1 sites in the androgen receptor (AR) gene [17]. We quantified the degree of skewing by analyzing 6-Fam-labelled PCR products on an ABI 3100 sequencer, followed by analysis using GeneScan software (both from Applied Biosystems, Foster city, CA). DNA from a healthy male was included as a control for HhaI digestion. Since it was apparent that the patient and his sister had inherited different alleles for the AR locus (Xq21) from their mother, haplotyping using X-chromosomal microsatellite markers located between the AR and IKBKG loci and six common SNPs in the FLNA gene was performed in the parents, proband and his sister in order to identify the IKBKG mutant allele in the patient’s sister (Supplementary Fig. 1).
• 2.5NK cell function was assessed as reported [18].
• 2.6Histology: Immunostaining for p65 was performed using rabbit polyclonal anti-p65 antibody (C-20, SC372, Santa Cruz) on paraffin embedded tissue sections after heat induced epitope retrieval in citrate buffer at pH 6.0. Secondary biotinylated anti-rabbit antibody was purchased from Perkin Elmer. Staining was visualized with ABC Kit Vectastain Elite and DAB substrate (both Vector Laboratories) followed by haematoxylin counterstaining [19].

3. Case reports, results and discussion

We report on a family in which 3 females suffered from several autoimmune phenomena including uveitis, erythema nodosum, polyarthritis, hepatitis and recurrent oral ulcers. As two patients were HLA-B51 positive, they were diagnosed with familial Behçet´s disease. Two displayed incontinentia pigmenti (Fig. 1A). One boy of this family was diagnosed with neonatal-onset Crohn’s disease and experienced recurrent septic episodes believed to be a consequence of his immunosuppressive treatment. At 4 years of age he died from peritonitis. His younger brother, the index patient, developed severe staphylococcal scrotal cellulitis and necrotizing fasciitis at 3 weeks of age. In infancy, he also suffered from an interstitial pneumonia and two episodes of enterobacter cloacae meningitis. While these two episodes of meningitis caused by enteric bacteria may indicate intestinal bacterial translocation, he did not have any gastrointestinal symptoms.

Figure 1. Identification of a mutation in IKBKG in a family with female Behçet’s disease and male immunodeficiency and intestinal inflammation.

(A) Pedigree of the family with IKBKG gene mutation. Diagnosis of familial Behçet’s disease was made in patients 1, 2 and 3. Patients 1 and 3 were HLA-B51 positive. Patient (Pt.) 5 (index patient) underwent HSCT from his sister (patient 3). (B) Western blot analysis of PBMC using a NEMO specific antibody. The 48kDa band representing full length NEMO protein is absent in the patient (black arrow). Instead, a truncated protein of approximately 40 kD is more prominent than in the control. (C) Gel electrophoresis of RT-PCR products upon amplification with primers flanking exon 3 to 7 of IKBKG. The approximately 600 bp product corresponds to the wildtype sequence of IKBKG, while in the patient a shorter, truncated product lacking exon 5 dominates (black arrow). (D) NK cell degranulation as assessed by CD107a expression of fresh NK cells before and after stimulation with K562 cells. ΔCD107 represents the increase in the percentage of CD107 positive cells after stimulation; the normal range (grey area) starts above 10%. The assay was performed before (left plot) and after HSCT (right plot). (E) X-chromosomal inactivation pattern in the index patient (pt. 5) before and after HSCT, his mother (pt. 1) and sister (pt. 3, HSCT donor). The AR allele carrying the IKBKG mutation is depicted in red, wt alleles are depicted in black. Active (unmethylated) alleles are reduced after HhaI digestion (lower row). Please note: while the 276 bp fragment in pt. 3 corresponds to the paternally-inherited wt X-chromosome, the 284 bp fragment has the same fragment length as the maternal X-chromosome harboring the wild-type IKBKG-allele. However, sequencing the IKBKG-gene revealed that the sister was heterozygous for the same mutation present in pt. 1 and pt. 5. Further analysis of microsatellite markers revealed a X-chromosomal genetic recombination event as a cause of this unexpected finding in the sister (pt. 3) (supplementary figure 1).

This family history was consistent with a diagnosis of NEMO deficiency and sequencing of the IKBKG-gene in the index patient indeed revealed a hemizygous nucleotide substitution in exon 5 (c.613C>T; NM_001099857.2) leading to a premature stop codon (p.Gln205*; NP_001093327). A western blot showed the virtual absence of the 48kDa band expected for full length NEMO in patient’s cells, but instead displayed a truncated protein of approximately 40kDa (Fig. 1B). RT-PCR followed by sequencing indicated that this shorter isoform is generated from an alternatively spliced mRNA lacking exon 5 (Fig. 1C). While this short isoform could also be detected in control cells, it was much more abundant in patient cells. Consistent with previous reports of NEMO deficiency, NK cell cytotoxic function was markedly impaired underscoring the functional relevance of the mutation identified [20] (Fig. 1D). Mother and sister were heterozygous for the mutation and, strikingly, the sister showed skewed X-inactivation favoring the mutant NEMO allele (Fig. 1E). This observation extends another case report linking incontinentia pigmenti and Behçet’s disease to a mutant NEMO allele in female carriers [21], here associated with skewed X-inactivation. This sister was fully HLA-identical to the index patient, making her an ideal donor for HSCT. Her clinical history of not having had any severe infections indicates that even her low proportion of wild-type Nemo activity was sufficient for protection against severe infections. Based on these considerations we chose her as a HSCT donor, despite the fact that she carried the mutation and experienced autoimmune symptoms. In 2007, the patient was transplanted at the age of 11 months after receiving myeloablative conditioning consisting of busulfan, cyclophosphamide and ATG. The clinical course was complicated by life-threatening veno-occlusive disease and severe mucositis, which eventually resolved. A fatal disease course due to hepatic toxicity after myeloablative conditioning has been reported in another NEMO deficient patient [24]. A third patient developed severe liver dysfunction following GvHD-prophylaxis with methotrexate after HSCT [11]. Together these experiences could suggest that NEMO deficient patients may display an increased vulnerability to hepatic complications upon HSCT. This hypothesis is supported by the observation that in-vitro inhibition of NF-kappaB activity renders cells more susceptible to chemotherapy-induced apoptosis [25,26]. Currently, there is not sufficient data to indicate, whether reduced intensity conditioning (RIC) leads to better outcomes (supplementary table 1) [11,13,14,22–27]. In our patient, HSCT resulted in full donor chimerism and functional immune reconstitution (Fig. 1D) without signs of graft-versus-host-disease. Skewed X-inactivation was even more pronounced in the recipient showing only 10% of cells with an active wild-type allele (Fig. 1E). Apparently, this low level was still sufficient to fully reconstitute anti-microbial defenses as the patient did not experience any severe infections within eight years of follow-up.

Several months after transplantation our patient developed erythema nodosum and severe inflammatory bowel disease (Fig. 2 A&C). Histological features included small intestinal epithelial cell tufting indicating apoptosis and multiple large intestinal ulcerations underneath an intact mucus layer (Fig. 2 A). Spontaneous IBD has been reported in approximately 20% of NEMO patients [9]. Interestingly, our patient developed IBD despite reconstituted immune function suggesting a non-hematopoetic component in disease pathogenesis. Accordingly, in our patient gut-infiltrating immune cells stained strongly for nuclear p65, indicating normal NEMO function in transplant-derived cells. In contrast, his intestinal epithelial cells displayed only weak staining for nuclear p65, whereas epithelial cells from 3 control patients with inflammatory bowel disease stained strongly (Fig. 2 B). These observations are consistent with results from mice in which a conditional deficiency of NEMO in intestinal epithelial cells sufficed to trigger spontaneous IBD development due to an increased apoptosis of gut epithelial cells [7]. In humans this epithelial role of NEMO is underscored by the observation that HSCT usually does not improve preexisting intestinal inflammation [12–14,23].

Figure 2. Severe inflammatory bowel disease after HSCT was successfully treated with Anti-TNF therapy.

(A) Small bowel (upper panel) and colonic (lower panel) pathology in NEMO deficient patient after HSCT (prior to anti-TNF treatment) as indicated by H&E staining. Upper panel: small bowel biopsy displaying irregular epithelial lining and epithelial cell tufting (arrow and inset) indicating apoptosis. Lower panel: epithelial ulcerations (black arrow) in the colon are characterized by severe epithelial cell damage with underlying mononuclear and lymphoid inflammatory infiltration of the lamina propria. The intact mucus layer above the ulcerations excludes a sampling artifact of the biopsy (dashed arrow). (B) Immunohistochemistry of colon tissue from the NEMO deficient patient (lower panel) and a pediatric control patient with Crohn’s disease (upper panel) using anti-p65 antibodies. In the NEMO deficient patient intestinal epithelial cells show largely absent nuclear p65 staining (white arrow), while infiltrating donor-derived immune cells stain strongly for nuclear p65 (black arrows). In the control sample, strong p65 staining is shown in the intestinal epithelial cells and the infiltrating immune cells (black arrows). Sizing bar indicates 100 µm. (C) Photograph illustrating the extended perianal, scrotal and inguinal skin inflammatory lesions, which resolved completely upon escalated anti-TNF treatment.

What then is the role of the immune system in NEMO-associated IBD? The increased epithelial apoptosis will facilitate bacterial translocation, thereby activating pro-inflammatory immune mechanisms which will further fuel epithelial apoptosis, resulting in a vicious circle. Since NF-κB-signaling is important for the activation of immune responses [28], NEMO deficiency in immune cells may attenuate the inflammatory response. In contrast, after HSCT the bacterial translocation will be met by a reconstituted immune system, which is then capable of mounting a more vigorous inflammatory response than prior to HSCT. This may ultimately lead to inflammatory bowel disease. Thus, while HSCT of NEMO-deficient patients may restore host defenses to prevent life-threatening infections, it may concomitantly increase the susceptibility to inflammatory bowel disease. This hypothesis is supported by observations in humans and mice: First, the presence of a fully functional immune system in the context of NEMO-deficient intestinal epithelial cells in a conditional knock-out mouse model leads to very severe intestinal inflammation [7]. Interestingly, this phenotype was completely abrogated when MyD88, an important adaptor molecule for pathogen-sensing receptors, was also knocked out in these mice [7]. Second, in a NEMO-deficient patient, a genetic reversion of his mutated NEMO-allele in T lymphocytes coincided with an increased severity of gastrointestinal symptoms [15]. Furthermore, in this patient the reverted T cells infiltrating the gut produced high levels of TNF-α, while macrophages still affected by the NEMO mutation showed only very little TNF-α production. Together, these observations may suggest that restoration of NEMO-functionality in immune cells through HSCT, but persistence of NEMO-deficient intestinal epithelium with increased permeability may even aggravate inflammation. Among 11 previously published NEMO-patients 5 had symptoms of intestinal inflammation after HSCT (supplementary table 1). However, from most published reports it remains unclear whether HSCT aggravated pre-existing IBD or whether it occurred de novo only after HSCT. More data on the course of intestinal or other inflammation after HSCT of NEMO-deficient patients could help to corroborate this hypothesis.

Regarding therapeutic options for treatment for NEMO-associated intestinal inflammation there are only few published reports describing a small number of patients: while responses to steroids alone were very variable [14,15], therapies combining steroids with either mesalazine [29] or cyclosporine with [15] or without [27] methotrexate successfully controlled symptoms. However, in all but one patient, intestinal inflammation relapsed upon tapering the steroid therapy [14,15,27,29]. In mice, NEMO-associated intestinal inflammation was markedly ameliorated by abrogating TNFR-signaling [7], providing a rationale for using TNF-blockers in human NEMO deficiency [15]. A single patient responded to infliximab after failure of several other therapies [15]. In our patient complete remission of intestinal inflammation was only achieved upon escalated infliximab therapy (10 mg/kg every 3-4 weeks, trough levels 17 µg/ml (normal target range: 3.0-7.0 µg/ml), no anti-infliximab antibodies detectable) (Fig. 2 C). Prior therapeutic attempts using steroids, azathioprine, cyclosporine and conventional anti-TNF doses had failed to control the disease. Remarkably, also control of the uveitis and oral ulcers in his sister depended on high dose anti-TNF treatment.

In summary, these illustrative case reports reveal that as little as 10% immune cells with an active wild-type NEMO allele suffice to reconstitute immunity to infection. Furthermore, extending murine studies, the data indicates that NEMO deficiency in intestinal epithelial cells may suffice to drive inflammatory bowel disease, which can be controlled by TNF-blockade.

Supplementary Material

Supplementary Figure 1. Haplotyping reveals an X-chromosomal genetic recombination involving the mutant NEMO allele in the HSCT donor sister.

Results of haplotyping using X-chromosomal microsatellite markers located between the AR and IKBKG loci and six common SNPs in the FLNA gene for parents, index patient and his sister in order to identify the IKBKG mutant allele in the patient’s sister. The chromosomal location of the recombination event in the sister is denoted by the red cross.

Supplementary Table 1. Published data on HSCT in NEMO-deficient patients.

Summary of conditioning therapies, engraftment, toxicity, GvHD, outcome, intestinal inflammation after HSCT and follow-up period of published cases of HSCT in NEMO-deficiency.

Abbreviations

AR

androgen receptor

ATG

anti-thymocyte globulin

bp

base pairs

HSCT

hematopoetic stem cell transplantation

IBD

Inflammatory Bowel disease

IP

incontinentia pigmenti

IKBKG

inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma

NEMO

NF-κB essential modulator

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NK cell

Natural killer cell

SNP

Single Nucleotide Polymorphism

TNF

Tumor necrosis factor

wt

wild type