An Exclusive NEMO B Mutation Leads to NF-κB-linked Recurrent Pneumonia by Disrupting the NEMO/IKK Complex

doi:10.21203/rs.3.rs-1764128/v1

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Research Article

An Exclusive NEMO B Mutation Leads to NF-κB-linked Recurrent Pneumonia by Disrupting the NEMO/IKK Complex

https://doi.org/10.21203/rs.3.rs-1764128/v1

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Purpose

A number of NEMO mutations in the shared sequences of NEMO isoform A and B have been associated with NF-κB-linked diseases. Here, we first report an exclusive NEMO B mutation as the genetic etiology of a male case with recurrent bacterial pneumonia and explore the underlying mechanism.

Methods

The genomic protein-coding regions of genes were analyzed using the whole exome sequencing (WES) technique, and the rare gene variant was confirmed by Sanger sequence analysis; activation of NF-kB signaling pathway was assessed by co-immunoprecipitation, western blotting, and single-cell RNA-seq analysis; glycolytic changes of peripheral immune cells were evaluated by extracellular acidification rate assay; protein-protein binding affinity and stability were estimated by structure modeling and molecular docking analysis.

Results

A c.20T > C mutation in exon 1 of the IKBKG germline sequence (NM_001099856.2) resulted in a Val7Ala replacement in the exclusive N-terminal sequence of NEMO B. This mutation significantly decreased the binding affinity and stability of NEMO B with IKKα/β, leading to the failure to form functional NEMO/IKK complex and the subsequent inhibition of RelA phosphorylation and nuclear translocation to initiate pro-inflammatory TNF-α and IL-1β gene transcription and metabolic support by glycolysis in response to the TLR4 agonist LPS. Single-cell sequencing analysis revealed abnormalities in monocyte and T-lymphocyte function and B-cell hyperplasia.

Conclusions

The exclusive N-terminal sequence of NEMO B is essential for a functional NEMO/IKKa/IKKb complex to activate NF-κB-dependent antibacterial immunity.

Exclusive NEMO isoform B mutation

Anti-bacterial immunodeficiency

NF-kB-linked disease

Recurrent bacterial pneumonia.

The NF-kB transcription factor family is evolutionarily conserved during the development of innate immunity. Infection informs Toll-like receptors (TLR) to activate the NF-kB-dependent signaling pathway, which triggers a pro-inflammatory response to eliminate pathogens and restore physiological homeostasis (1). Loss of function of the NF-kB-dependent pathway leads to recurrent and chronic infections (2).

RelA (p65) is a central transactivator of the NF-κB pathway. It is retained in the cytoplasm in a resting state by forming a complex with IκBα. TLR4 signaling phosphorylates IκBα to free RelA, resulting in subsequent RelA phosphorylation, nuclear translocation, and recruitment onto specific sequences in the promoter or enhancer regions of target genes to initiate their transcription (3). Both IκBα and RelA phosphorylation are catalyzed by the upstream IκB kinase (IKK) α and β, which are scaffolded by a regulatory non-enzymatic protein NF-κB essential modulator (NEMO or IKKγ) to form the IKKα/IKKβ/NEMO complex (4).

NEMO is encoded by the X-chromosome IKBKG gene (5), which encodes ten NEMO transcript variants using two alternative promoters, the liver-specific promoter A and the housekeeping promoter B (6). The transcript variant 1 translates NEMO isoform A in humans (NP_001093327.1) and transcript variant 2 translates NEMO isoform B in primates (NP_001093326.2). Both NEMO isoforms share C-terminal amino acid sequences, but NEMO B contains an additional 68 amino acids in its far N-terminal sequence. Following TLR4 activation, the inflammatory signaling cascade causes posttranslational modifications including K63-linked non-degradative polyubiquitination and phosphorylation on NEMO, which subsequently forms a heterotrimeric complex with IKKα/β and recruits TAK1, which, in turn, phosphorylates IKKα/β. The phosphorylation of IKKα/β activates kinase activity that catalyzes the downstream phosphorylation cascade of NEMO, IκBα, and RelA to trigger RelA-dependent gene transcription (4). Failure of K63-linked polyubiquitination and phosphorylation of NEMO prevents the formation of the IKKα/IKKβ/NEMO heterotrimer, disrupts RelA activation, and increases the occurrence of recurrent bacterial infections that are associated with NF-κB-linked genetic diseases (7–10), such as Familial incontinentia pigmenti (IP) and anhidrotic ectodermal dysplasia associated with immunodeficiency (EDA-ID) (11, 12), immunodeficiency without ectodermal dysplasia (ID without EDA) (13), recurrent invasive pneumococcal disease (IPD) (14), and Behcet-like disease (15). These diseases are associated with NEMO mutations in functional domains or intron sequences (16, 17); however, the loss of function of exclusive NEMO B mutations has not been reported to date.

Here, we first reported a loss-of-function point mutation in NEMO B in its far N-terminal non-domain sequence, which is associated with antibacterial immunodeficiency, clinically presenting recurrent pneumonia, hypoimmunoglobulinemia, and splenomegaly. Whole exome sequencing (WES) analysis identified a T to C substitution at nucleotide position 20 of exon 1 of the IKBKG gene that causes a Val7Ala (V7A) missense mutation in NEMO B, leading to the failure of RelA activation in response to TLR-4 activation signaling. Single-cell sequencing analysis further indicated abnormalities in the function of monocytes and T lymphocytes and B-cell hyperplasia.

Molecular genetics

Venous blood samples were collected from a patient with recurrent bacterial pneumonia and the patient’s parents according to a protocol approved by the Ethics Committee of Shanghai Public Health Clinical Center (2021-Y023-01), Fudan University. All participants provided written informed consent prior to inclusion. All participants consented to participate in the study and approved that the results will be published in a peer reviewed journal. Genomic DNA was extracted from white blood cells using a phenol/chloroform extraction method. The NEMO gene sequence was amplified by PCR using a forward primer 5′-AGTGCCAACATCATCATGAC-3′ and a reverse primer 5′-TGATGGCACCTCAGAGTTAC-3′. PCR products were sequenced with the Big Dye Terminator cycle sequencing kit (Applied Biosystems), and analyzed on an ABI Prism 3700 apparatus (Applied Biosystems).

Whole exome sequencing

Genomic DNA (200 ng) from each individual was sheared using a Biorupter (Diagenode, Belgium) into 150–200 bp fragments. The ends of the DNA fragments were repaired and Illumina Adaptors were added (Fast Library Prep Kit, iGeneTech, Beijing, China). After sequencing library construction, the whole exons were captured with an AIExome Enrichment Kit V1 (iGeneTech, Beijing, China) and sequenced on the Illumina platform (Illumina, San Diego, CA) with 150 base-paired-end reads. Raw reads were filtered to remove low-quality reads using FastQC, and the clean reads were mapped to the reference genome, GRCh37, using Bwa. After removing duplicates, SNV and InDel were called and annotated using GATK.

Structure modeling and molecular docking

For structure modeling of NEMO A, NEMO B, NEMO B mut, IKKa, and IKKb, template sequences were selected from the Protein Data Bank (PDB) by BLAST (Basic Local Alignment Search Tool). The top monomer structures of the NEMO variants were modeled using RoseTTAFold (18) with default parameters, and dimer structures were constructed using the Align function of Pymol (version 1.3). The crystal structure of NEMO (PDB id, 6MI3, chain: A and B) was used as the shared template sequence except for one point mutation in NEMO B mut. The structures of the IKKb dimer and IKKα trimer were constructed using Swiss-Model (19) with their crystal structures (PDB id: 4KIK, Chain: A and B, and PDB id: 5EBZ, Chain: A, B and C, respectively) as templates. The molecular docking of protein-protein interactions was performed using the ClusPro web-based server (20). The parameters of ClusPro score and cluster size were introduced to evaluate the binding ability and stability of each structural complex.

TLR4 activation of PBMC

PBMCs were isolated from venous blood samples by gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare) and were activated by LPS (100 ng/mL, tlrl-pb5lps, InvivoGen) for 0 h or 1 h in a 37°C incubator with 5% CO2 in RPMI medium supplemented with 10% FBS (RPMI/10% FBS; Invitrogen). The levels of TNF-α and IL-1b mRNA were compared by quantitative real-time PCR, and the dynamics of RelA translocation between the cytoplasm and the nucleus were imaged by immunofluorescence microscopy.

Glycolysis assay

Cellular glycolytic capacity was evaluated by measuring the ECAR with a glycolysis stress kit using the XFe-24 Extracellular Flux Analyzer (Seahorse Biosciences). In brief, PBMCs were stimulated with or without LPS (100 ng/ml) for 1 h, and 2 × 10⁵ cells/well in basal assay medium (pH 7.4, Invitrogen) were seeded into a Cell-Tak (Corning) pre-treated microplate and pre-incubated for 1 h at 37°C in a CO₂-free incubator. Ten-fold concentrated glucose, oligomycin, and 2-deoxy-D-glucose (2-DG) were then added to a sensor cartridge to produce a final concentration of 10 mM, 1 μM, and 50 mM, respectively. After a 30-min calibration with the XFe sensor and the pre-incubated sensor cartridge, the plate was loaded into the XFe-24 Analyzer. The glycolytic background was determined by incubating cells in a stress medium without glucose or pyruvate. The basal ECAR was measured after adding glucose and the glycolytic capacity was calculated by subbstraction of the basal ECAR from that after the addition of oligomycin. Finally, the hexokinase inhibitor, 2-DG, was added to define the glycolytic reserve by subtracting the 2-DG-conducted ECAR from oligomycin-induced ECAR.

RT-PCR

Total RNA was extracted from PBMCs using an RNA extraction kit (cat# 9767, Takara, Japan) and was reverse-transcribed into cDNA with an oligo (dT) primer using the PrimeScript RT reagent Kit (cat# RR037A, Takara, Japan). cDNAs were subjected to quantitative PCR using GoTaq qPCR Master Mix (cat# A6001, Promega, USA) and the FQD-96A Fluorescent Quantitative Detection System (Bioer Technology). The gene-specific primer sequences were as follows: TNF-α forward 5′-GAGGCCAAGCCCTGGTATG-3′ and reverse 5′-CGGGCCGATTGATCTCAGC-3′; NEMO isoform A forward 5′-AACTGGGACTTTCTCGGAGC-3′ and reverse 5′-ATGGCATCTCGGAGCTCTTG-3′; NEMO isoform B forward 5′-GACCGCGAAACTGGGACTTT-3′ and reverse 5′-CCGCAGAATCTGGTTGCTCT-3′; GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′. GAPDH mRNA transcription was used as an internal loading control.

Immunofluorescence microscopy

Cells were cytospun onto slides, fixed with 4% paraformaldehyde (PFA) for 20 min, and permeabilized for 1 h at room temperature with 0.3% Triton-X-100 in phosphate-buffered saline (PBS-T). The cells were then incubated with primary rabbit anti-RelA antibody (cat# sc-372, Santa Cruz, 1:50 in PBS-T) overnight at 4°C. After three washes with PBS-T, the cells were incubated for 1 h at room temperature with rhodamine (TRITC)-conjugated goat anti-rabbit IgG (Cat# 111-025-003, 1:2,000 in PBS-T, Jackson Immunoresearch). After nuclear counterstaining for 5 min with DAPI (5μg/ml, Sigma), the slides were mounted with glycerol and analyzed using a Leica TCS SP5 microscope (Leica) with LAS AF Lite 4.0 image browser software.

Western blotting and co-immunoprecipitation

Total protein extracts were prepared from PBMCs using RIPA lysis buffer. The proteins were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes, which were then probed with antibody against phosphorylated RelA, RelA (Sigma-Aldrich), NEMO, IKKa, IKKb (Cell Signaling Technology), or GAPDH (Santa Cruz Biotechnology, Inc.).

For co-immunoprecipitation, cell lysates were precleared using protein A/G plus Sepharose beads (Santa Cruz) and incubated overnight with mouse anti-human NEMO antibody (Santa Cruz). The NEMO bound proteins were pulled down by incubating the lysates for 1 h with protein A/G plus Sepharose beads. After three washes with RIPA lysis buffer, the immunoprecipitates were probed with rabbit anti-human IKKb, mouse anti-human IKKa, or rabbit anti-NEMO antibodies (Cell Signaling Technology).

Single-cell RNA-seq analysis of PBMC

The anticoagulant peripheral blood was collected and PBMCs were isolated as previously described (21). Single-cell RNA-sequencing libraries were constructed with the Chromium Single Cell 3′ Reagent Kits according to the manufacturer’s instructions. Sequencing was performed with a BGI’s DNBSEQ-T7 sequencer.

Raw sequencing reads were mapped to a reference genome (hg38) using Cellranger software (version 4.0). The generated expression matrix was then processed with Seurat software (version 4.0) (22). Principal component analysis and manifold approximation and projection (UMAP) were used for dimensionality reduction and visualization. Each cluster of cells was identified using known cell markers, including CD3D, CD3E, CD4, CD8A, CD8B, CD14, EPCAM, CCR7, GZMK, CD19, MS4A1, IGHG1, IGHG4, BCL11B, GYPC, ITGA2B, NRGN, and NCAM1. Cluster-specific genes were identified by the FindMarkers program in the Seurat suite. Differentially expressed genes in each cluster between different groups were identified by the MAST algorithm. Gene ontology analyses were conducted with the topGO package (23).

Statistical analyses

Differences in gene expression between two related genotypes were analyzed by Student’s t-test using GraphPad Prism version 6 (San Diego, CA). P values less than 0.05 were considered statistically significant. Data points represent the mean ± SEM of replicates.

Case description

A 30-year-old man with an unknown parental consanguinity complained of recurrent fever and cough since the age of 21-year-old. Ectodermal dysplasia-related signs were absent, but streptococcus pneumonia was diagnosed by next-generation sequencing analysis of bronchoalveolar lavage fluid. A physical exam indicated a significantly enlarged spleen and its inferior margin reached the umbilicus level. A computerized tomography (CT) scan revealed multiple patches in both the left and right lungs (Figure 1A) along with splenomegaly (Figure 1B), PET-CT showed increased metabolic lesions in the lung (Figure 1C), and PET-MRI revealed uneven signals with multiple, small patchy signal foci in the spleen (Figure 1D). A laboratory exam indicated low levels of serous IgM, IgG, and IgA and low counts of CD3+, CD4+, CD19+, and CD56+ cells (Supplement Table S1). The family members of the patient were healthy.

Identification of a c20T>C missense mutation in the IKBKG transcript variant 2 gene

To explore the genetic etiology of this disease, WES analysis on genomic DNA from the patient and his parents found dominant presentation of the full-length exon sequence of NEMO B. The patient displayed a T to C substitution at nucleotide position 20 in exon 1 of the IKBKG gene (NM_001099856.2:1/10:c.20T>C), resulting in the substitution of a valine residue with an alanine residue at amino acid position 7 (V7A) of the NEMO B amino acid sequence. The c.20T>C mutation was further confirmed by Sanger sequence analysis and has not yet been reported in any public databases including the Human Gene Mutation Database, Ensembl, NHLBI GO Exome Sequencing Project [ESP], 1000 Genomes Project, and the Genome Aggregation Database (gnomAD). No rare coding mutations were found in other NF-κB transcription factor family members including RelA (p65), p50, c-Rel, RelB, and p52 (data not shown). The patient’s mother was a heterozygote for the mutant allele, whereas his father showed no coding error in the IKBKG gene (Figure 2A). The pedigree suggests that the trait was of X-linked recessive inheritance (Figure 2B).

By searching the NCBI database, ten NEMO transcript variants have been described. Of these, NEMO B is encoded by the transcript variant 2 and has been identified only in primates thus far (Figure 2C). In contrast to other transcript variants, the transcript variant 2 translates a protein that is 68 amino acids longer at the N-terminus compared with other variants. This V7A mutation occurs in a highly conserved position in primate NEMO and was not found in other NEMO isoforms (Figure 2D).

NEMO B V7A mutation disrupts the NF-kB-dependent inflammatory pathway

We next determined whether the V7A mutation of NEMO B affects the pro-inflammatory response. Peripheral blood mononuclear cells (PBMCs) from the patient and healthy control were stimulated with bacterial endotoxin lipopolysaccharides (LPS), the representative pro-inflammatory cytokine, TNF-a and IL-1b, gene expression was measured by RT-PCR analysis. Control rather than patient PBMCs responded well to LPS stimulation and expressed over 20-fold more TNF-a and IL-1b mRNA compared with unstimulated cells (Figure 3A and 3B), indicating that the NEMO V7A mutation minimizes the pro-inflammatory response following TLR4 signaling.

To explore the underlying mechanism of NF-kB pathway dysfunction resulting from NEMO B V7A mutation, we examined the phosphorylation of RelA in response to LPS stimulation. PBMCs were stimulated with LPS, and the levels of NEMO, RelA, and phosphorylated RelA protein were assessed by western blot analysis. The control PBMCs expressed higher basal NEMO and RelA protein levels compared with patient PBMCs, whereas phosphorylated RelA was observed in the control, but not in patient PBMCs following LPS treatment (Figure 3C). As the phosphorylation of RelA determines its nuclear translocation, the defect in RelA phosphorylation in patient PBMCs was further confirmed by its failure to translocate from the cytosol to the nucleus in contrast to the rapid nuclear accumulation of phosphorylated RelA in control PBMCs after LPS stimulation (Figure 3D).

RelA phosphorylation depends on the NEMO-mediated IKKa/IKKb/NEMO heterotrimer. Therefore, we determined whether the NEMO B V7A mutation disrupts the formation of the IKKa/IKKb/NEMO complex. Patient and control PBMCs were stimulated with LPS, cell lysates were immunoprecipitated with an anti-NEMO antibody and the resulting immunoprecipitates were respectively probed using anti-IKKa and anti-IKKb antibodies to detect the presence of the IKKa/IKKb/NEMO complex. In basal status, NEMO and IKKa/b were not co-immunoprecipitated in either the patient or control PBMCs; however, upon LPS stimulation, they co-immunoprecipitated in the control, but not in the patient PBMCs (Figure 3E). Thus, the NEMO B V7A mutation disrupts NEMO scaffold function, thereby interrupting the formation of the IKKa/IKKb/NEMO complex and the downstream phosphorylation, nuclear translocation, and transactivation of RelA.

NEMO B V7A mutation decreases its binding ability with IKKs

To further confirm the inability of mutant NEMO B to form a NEMO/IKK complex, we performed a structure modeling and molecular docking analysis of the protein-protein interactions between IKKα and IKKβ with wild-type or mutant NEMO B or NEMO A (Figure 4). The ClusPro score and cluster size represent the binding affinity and stability, respectively. The lower the ClusPro score, the higher the binding affinity, whereas the larger the cluster size, the more stable the complex. The respective ClusPro score for wild-type NEMO B against the IKKα homotrimer and IKKβ homodimer reached −1338.3 and −1391.6, which is in contrast to −1245.9 and −1264.8 of the mutant NEMO B. Consistent with the ClusPro score, wild-type NEMO B generated 21 and 24 clusters for IKKα and IKKβ, respectively, which was significantly greater compared with the 11 and 17 generated by mutant NEMO B (Supplement Table S2). Remarkably, molecular docking analysis of NEMO A with IKKs obtained ClusPro scores and cluster sizes comparable with that of mutant NEMO B with IKKs (Figure 4, Supplement Table S2). Thus, the V7A mutation may change the structural conformation of NEMO B homodimer to affect its binding with IKKα and IKKβ, and the far N-terminal sequence of NEMO B appears to be essential for high-affinity binding between NEMOs and IKKs, which is consistent with the findings of the biochemistry analyses.

NEMO B V7A mutation attenuates the glycolytic response of immune cells

The activation of innate immune response depends on glycolysis for energy and building materials (24, 25). We thus compared the real-time kinetics of glycolysis in patient and control PBMCs in response to LPS stimulation by measuring the extracellular acidification rate (ECAR) using a glycolytic stress assay. Both PBMCs showed comparable basal glycolytic activity; however, LPS stimulation significantly increased basal glycolysis in the control PBMCs (0.69 ± 0.028 mpH/min vs. 1.89 ± 0.177 mpH/min), but not in patient PBMCs (0.74 ± 0.104 mpH/min vs. 0.84 ± 0.033 mpH/min). Furthermore, LPS stimulation significantly increased the glycolytic capacity and glycolytic reserve in the control rather than in patient PBMCs (Figure 5A and 5B). The idle glycolytic response mirrors the failure of NF-kB activation by the TLR4 signal in patient PBMCs.

NEMO B V7A mutation defects immune response pathways

Next, we conducted a single-cell RNA-seq (scRNA-seq) analysis of the patient and healthy PBMCs. A total of 45,540 cells and 12 cell-type clusters were identified and denominated as C0 to C11 (Figure 6A). Compared with the healthy control, the patient exhibited a significant decrease in monocyte count, but a marked increase in B-cell and plasma cell counts (Figure 6B), which suggests potential disorders in monocyte and B-cell development by NEMO B V/7 mutation.

We further compared the immune gene expression profiles of the various cell types between the patient and control. Genes associated with TNF-α and TLR4-mediated signaling were significantly downregulated in C2 (monocytes) and C8 (BCL11B+ T cells) and moderately downregulated in the C0 (CD4 T cells) and C6 (plasma cells) cell types of the patient. Moreover, genes related to the adaptive immune response were markedly downregulated in most V7A cell types including C0 (CD4 T cells), C1 (CD8 T cells), C4 (GZMK+ CD8 T cells), C6 (plasma cells), C8 (BCL11B+ T cells), and C11 (NRGN+ T cells). Genes associated with the interferon γ-mediated signaling pathway were downregulated in patient C1 (CD4 T cells), C2 (CD8 T cells), C5 (B cells), and C6 (plasma cells) cell types (Figure 6C, Supplement Table S3). Collectively, these data indicate potential defects in both the innate and adaptive immune response by NEMO B V7A mutation, which is further supported by the dysregulation of cell-cell interactions and their communication-related signaling pathways (Figure 6D).

NEMO B V7A mutation disturbs the monocyte and B-cell response to LPS stimulation

To further examine the defects of immune cells harboring a NEMO B V7A mutation in response to TLR4 activation signaling, we compared the transcriptome changes between patient and control PBMCs in response to LPS. LPS dramatically altered gene expression in all cell types of the patient, including 263 upregulated and 83 downregulated genes (Figure 7A, Supplement Table S4 and S5). A subsequent gene ontology analysis suggested that these differentially expressed genes were involved in different signaling pathways of innate and adaptive immunity, The downregulated genes by NEMO B V7A mutation were associated with TLR4-, NF-kB-, IL-6-, and chemokine-mediated innate immune signaling pathways, but upregulated genes were associated with the inflammatory response and cellular LPS signaling pathways (Figure 7B). These altered genes by NEMO B V7A mutation represent chemokines of innate immune cells (CCL2, CCL4, CCL3L1, and CXCR4), key components of the NF-kB inflammatory signaling pathway (NFKBIA, NFKBIB, and TNFAIP3), gene transactivators (JUNB, JUND, KDM6B, NR4A1, NR4A2, and SPI1), inflammatory mediators (IFIT2, IFIT2, IL-6, and CXCL8), regulators of cell-cell interactions (THBS and CD83) and the cell cycle (BTG2), and a metabolic enzyme (ACSL1) (Figure 7C and 7D). They communicate with each other to regulate the inflammatory response to infections (Figure 7E). Remarkably, SPI1 and BTG2 participate in B-cell proliferation, differentiation, and activation, and their alteration may contribute to B-cell hyperplasia.

A panel of human NEMO mutations have been identified within its functional domain regions and introns. These mutations affect NEMO phosphorylation and/or ubiquitination and have been predominantly associated with IP or EDA-ID (16, 17). IP is an X-linked dominant genodermatotic syndrome that is usually lethal prenatally in males probably due to hyper-apoptosis in the liver (26). IP develops from the loss of NEMO A function by amorphic mutations in the IKBKG gene, and over 85% of the patients share the same deletion mutations that encode for an inactive NEMO A truncation product. This results in a complete loss of downstream RelA activation in response to most known stimuli (4). Over 800 IP cases have been reported (27). In contrast, EDA-ID is an X-linked recessive disorder in males, and is resulted from hypomorphic mutations of IKBKG that cause missense mutations or short truncations of NEMO A and partial loss of RelA function. Patients with EDA-ID present with features of ectodermal dysplasia and immunodeficiency that contributes to a poor antibody response to vaccination and recurrent severe infections in early life, with particular susceptibility to opportunistic infections of bacteria, pneumococci, mycobacteria, and fungi (28, 29).

This study identified a V7A missense mutation in the NEMO B isoform rather than other NEMO isoforms. The exclusive NEMO B V7A mutation interrupts the TLR4 signaling-mediated formation of the IKKa/IKKb/NEMO heterotrimer, breaks the downstream signaling cascade of RelA activation, and results in defects in innate immunity and its associated activation of glycolysis that meets the metabolic needs in response to infection. Remarkably, the NEMO B V7A mutation is associated with immunodeficiency only and predominantly manifests as recurrent bacterial pneumonia without EDA. NEMO B shares a C-terminal sequence with NEMO A; however, it contains an exclusive 68 amino acid sequence in its far N-terminal region. Thus, in addition to the C-terminal functional domains, the exclusive N-terminal sequence in NEMO B plays an important role in maintaining the innate immune function. Interestingly, a molecular docking analysis showed significantly lower binding affinity of wild-type NEMO A with IKKs compared with that of wild-type NEMO B with IKKs, but comparable affinity with that of mutant NEMO B and IKKs. Since NEMO A lacks the 68 amino acid sequence in the N-terminus of NEMO B, our data suggest that this sequence is essential for the formation of a functional NEMO/IKK complex. Together with their distinct clinical presentations, this study further suggests a functional difference between NEMO A and B, with the former playing a role in the development of both ectoderm and immunity, whereas the latter predominantly regulating the development of innate immunity.

The defect in immune response resulting from the NEMO B V7A mutation is further evidenced by single-cell sequencing analysis. In general, the NEMO B V7A mutation significantly decreased monocyte count and innate and adaptive immune gene expression in all types of immune cells in response to TLR4 signaling. In particular, the downregulated genes in patient monocytes were involved in TLR4-, NF-kB-, IL-6-, and chemokine-mediated innate immune signaling pathways, whereas the downregulated genes in patient CD4+ cells were involved in the inflammatory response and LPS signaling pathways. These immune changes associated with the NEMO B V7A mutation may collectively account for the recurrent bacterial infection.

In addition, the single-cell RNA sequencing analysis indicated that the NEMO B V7/A mutation markedly increased the number of B cells and plasma cells, which may contribute to splenomegaly, but in contrast to the hypoimmunoglobulinemia phenotype of the patient. Thus, the V7A mutation of NEMO B may potentially affect B-cell developmental function; however, the significance of NEMO B in humoral immunity needs to be defined.

Acknowledgments

We appreciate the patient and his family members for blood donations.

Fundings

This work was supported by the National Natural Science Foundation of China (82072260, 32070946, and 82001680) and Shanghai “Medical Garden Rising Star” Youth Medical Talent Training Funding Program (SHWJRS (2021)-99).

Conflicts of interest/Competing interests: The authors declare no competing financial interests.

Data availability: Data availability is specified in the original study

Author contributions:

LX, JX, XG, YT, and TQ equally contributed to this study. LX, YT, XG, TQ, JT, ST, DT, and YM performed experiments. JX and YL performed the literature search. JL, TFL, and YL accessed and verified all data. YL, DT, and LZ collected clinical samples. HL, PY, and JZ collected clinical information. TFL and YL designed the study, acquired funding, and wrote the manuscript. YH and SO reviewed the manuscript.

Ethics approval

This study was approved by the Ethics Committee of Shanghai Public Health Clinical Center, Fudan University (ID 2021-Y023-01).

Consent to Participate

Informed consent was obtained from all individual participants in the study.

Consent for Publication

All participants consented to participate in the study and approved that the results will be published in a peer reviewed journal.

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An Exclusive NEMO B Mutation Leads to NF-κB-linked Recurrent Pneumonia by Disrupting the NEMO/IKK Complex

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