Integrity of IKK/NF‐κB Shields Thymic Stroma That Suppresses Susceptibility to Autoimmunity, Fungal Infection, and Carcinogenesis

A pathogenic connection between autoreactive T cells, fungal infection, and carcinogenesis has been demonstrated in studies of human autoimmune polyendocrinopathy‐candidiasis‐ectodermal dystrophy (APECED) as well as in a mouse model in which kinase‐dead Ikkα knock‐in mice develop impaired central tolerance, autoreactive T cell–mediated autoimmunity, chronic fungal infection, and esophageal squamous cell carcinoma, which recapitulates APECED. IκB kinase α (IKKα) is one subunit of the IKK complex required for NF‐κB activation. IKK/NF‐κB is essential for central tolerance establishment by regulating the development of medullary thymic epithelial cells (mTECs) that facilitate the deletion of autoreactive T cells in the thymus. In this review, we extensively discuss the pathogenic roles of inborn errors in the IKK/NF‐κB loci in the phenotypically related diseases APECED, immune deficiency syndrome, and severe combined immunodeficiency; differentiate how IKK/NF‐κB components, through mTEC (stroma), T cells/leukocytes, or epithelial cells, contribute to the pathogenesis of infectious diseases, autoimmunity, and cancer; and highlight the medical significance of IKK/NF‐κB in these diseases.


Reduced mTEC numbers Autommunity and systemic inflammation
No [38,42] TRAF6 Severely impaired mTEC development and Aire expression

Severe autoimmunity and systemic inflammation
No [33] CD40 Reduced mTEC numbers and Aire expression Minor phenotypes No [32] RANK Reduced mTEC numbers and Aire expression

AIRE and APECED/Chronic Mucocutaneous Candidiasis (CMC)
APECED belongs to one of the CMC syndromes. CMC patients suffer persistent or recurrent fungal infection at the superficial layers of the skin, mucous membranes, and nails, which is associated with impaired cell-mediated immunity against candida species and autoimmune disorders. [53] Human CMC is inherited with heterogeneous causes, including inborn errors in one of the AIRE, IL-17A, IL-17RA, IL-17RC, IL-22, STAT1, and IKBKG loci [54][55][56][57][58] (Table 2). Notably, AIRE-and STAT1-mutant CMC patients are susceptible to developing oral, esophageal, and stomach carcinomas. [59][60][61][62] APECED is a rare disease but is more prevalent among some populations, such as Finns (1/25 000), Sardinians (1/14 400), and Iranian Jews (1/9000). [63][64][65] More than 60 AIRE mutations have been reported in APECED. [30] This disease is characterized by autoimmunity against endocrine and ectodermal tissues. The hypothalamus, pineal body, pituitary gland, thyroid, digestive system, kidney, adrenal glands, and reproductive system are derived from endocrine, while the nervous system, teeth, hair, and many exocrine glands are derived from the ectoderm. A patient who has more than three of the listed manifestations can be diagnosed as having APECED. These patients suffer from candidiasis and autoimmunity and tend to develop oral and esophageal SCCs. [59,66] Chronic oral fungal infection is closely associated with oral and esophageal SCC development in APECED. [59] For example, a study reported that six out of 92 APECED patients aged 29-44 years develop oral or esophageal SCCs. [59] At infancy or in their early teenage years prior to SCC formation, these cancer patients acquire chronic fungal infection. T cell-expressed IL-17A, IL-22, and IFN-γ cytokines are important for eliminating fungal www.advancedsciencenews.com www.bioessays-journal.com expansion in the body. [54,67,68] IKK/NF-kB regulate expression of these cytokines. [69,70] APECED patients have high titers of neutralizing autoantibodies against these cytokines, [71,72] suggesting that loss of IL-17, IL-22, and IFN-γ may contribute to increased fungal infection. On the other hand, occasional or weak autoantibodies against IL-6, IL-9, IL-12, IL-21, IL-23, IL-26, and IL-29 were found, while no autoantibodies against IL-1α/β, IL-2, IL-4, IL-8, IL-10, IL-18, TGF-β1, and TNF were found in APECED patients, [72] suggesting that the cytokines that are not neutralized may promote inflammation and tumorigenesis. APECED patients show diverse phenotypes. It is possible that oral and esophageal organs become the major targets of autoreactive T cells, resulting in tissue injury, autoinflammation, increased fungal infection, and tumorigenesis. We have shown that increased inflammation and the epithelial epidermal growth factor receptor (EGFR) pathway are required for maintaining chronic fungal infection in Ikkα KA/KA mice. [25] Consistently, Ikkα KA/KA autoreactive CD4 T cells had a defect in the induction of IL-17A, IL-22, and IFN-γ expression; however, the Ikkα KA/KA esophageal epithelium and macrophages highly expressed many other cytokines and chemokines compared to the wild-type cells. [25] Therefore, CD4 T cell-mediated immunity is the threshold for preventing or allowing fungal colonization and expansion, while an impaired epithelium and inflammation are required for maintaining chronic fungal infections that cause diseases. Of note, Aire À/À mice do not completely recapitulate APECED disease, suggesting differences between Aire's functions in human and mouse systems. [73,74] 4. STAT, IFN Signaling, and CMC STAT1 upregulates the expression of genes in response to a signal mediated by a type I, II, or III IFN. [75,76] Type I IFNs have 17 subtypes, including IFN-α/β, that bind to IFNAR1 and IFNAR2. IFN-γ, a type II IFN, signals through the IFN-γR1 and IFN-γR2 receptor complex. Type III IFNs include various forms of IFN-λ. STAT1 forms homodimers or heterodimers with STAT3 that bind to the IFN-γ-activated sequence promoter element in response to IFN-γ stimulation. In response to IFN-α or IFN-β stimulation, STAT1 forms a heterodimer with STAT2 that can bind to the interferon-stimulated response promoter element. In either case, STAT binding to the promoter elements of the interferon target genes leads to the increased expression of a broad range of genes. The STAT1 protein contains the coiled-coil, DNA-binding, SH2, and transactivation functional domains. [76] Interestingly, only the gain-of-function (GOF) mutations in the STAT1 locus were found in CMC patients. These mutations include D165G, D165H, Y170N, F172S, C174R, N179K, M202V, M202T, A267V, R274Q, R274W, R274Q, K286I, Q285R, T288A, P329L, N357D, T385M, K388E, and T437I, which inactivate the DNA-binding and coiledcoil functions of STAT1 but increase its phosphorylation. [55,56,61,62,77,78] These patients develop autoimmunity, persistent, or recurrent fungal infection, and gastrointestinal tract (GI) cancers found in the oral cavity, esophagus, and stomach ( Table 2).
Furthermore, transgenic K5.STAT3 that is controlled by the keratin 5 (K5) promoter enhances the proliferation of a subset (CD80 Hi MHCII Low Aire -) of mTECs, but it does not alter TRA expression. [82] On the other hand, the ablation of the Stat3 gene in K5-expressing mTECs reduces the number of immature and mature mTEC subsets in mice. [82,83] The Stat3 f/f ;K5.Cre mice do not develop systemic inflammation upon birth to 9 months. It remains to be examined whether challenges can induce the autoinflammation phenotypes and whether the regulatory effect of STAT3 on mTEC development requires STAT1. STATs and NF-kB cross-talk in different types of cells. [84] The detailed mechanisms underlying STAT1 mutation-induced CMC remain to be further elucidated.

IKBKB Inborn Errors, Severe Combined Immunodeficiency (SCID), and Impaired mTEC
SCID, the most severe primary immunodeficiency, comprises a heterogeneous group of heritable deficiencies in humoral and cell-mediated immunity. [69,85] Affected infants are usually identified in the first months of life with Pneumocystis jiroveci pneumonia, bacterial sepsis, cytomegalovirus, oral candida infection, persistent respiratory, or gastrointestinal viral infection that is often associated with protracted diarrhea and failure to thrive. SCID involves a defective antibody response due to either direct involvement with B cells or improper B-cell activation caused by nonfunctional T helper cells. These infants, if untreated, usually die within 1 year because of severe, recurrent infections unless they have undergone successful hematopoietic stem cell transplantation.
Inborn errors in the IKBKB locus, which encodes IKKβ, were found in SCID [69,[86][87][88] (Table 2). Pannicke et al. [69] reported four SCID cases that carried a homozygous duplication of the frameshift mutation in IKBKB that leads to a premature stop codon, resulting in a complete loss of the IKKβ protein. The peripheral blood B cells and T cells were almost exclusively of a naïve phenotype; Tregs and γδT cells were absent; and TNFα-and PMA-induced NF-kB signaling for IkBα phosphorylation was impaired in these patients. Lipopolysaccharide (LPS)-or flagellin-induced IL-6 expression was reduced in patients' T cells, and the expression of IL-17 and IFN-γ was impaired in the SCID patients. A 6-week-old infant developed severe oropharyngeal candidiasis in the upper airway, as well as bacterial septicemia. Her cerebrospinal fluid contained Listeria monocytogenes. She died from this infection 2.5 months after birth. The autopsy analysis revealed a small spleen, thymus, and lymph nodes in the neck and mesentery. Other nonsense IKBKB mutations, including R286X and R272X, that cause the absence of IKKβ were also reported in human SCID. [86,87] These findings indicate that IKKβ is required for the expression of important cytokines in leukocytes, including T cells, that protect the human body from bacterial, viral, and fungal infections. Consistently in mice, the canonical NF-kB pathway of IKKβ and p65 and c-Rel NF-kB has been demonstrated to regulate CD4 T-cell functions and Treg development. [89][90][91] Tnfr deletion-rescued p65 À/À mice develop severe infections, leading to failure to thrive. [92,93] Furthermore, Hassall's corpuscles, which are differentiated from mTECs due to a lack of AIRE expression, [94] were observed in the medulla of human SCID patients, and a scant lymphoid population was seen in the thymus but was largely located in the medulla, [69] indicating that IKKβ mutations impair AIRE expression and mTEC development in SCID patients. A germline mutation delivers the inborn error to most cell populations in humans. Therefore, IKBKB mutation-induced defects in both leukocytes and mTECs contribute to disease development. Overall, leukocyte IKKβ plays an essential role in preventing infection, and mTEC IKKβ contributes to maintaining immune homeostasis.

GOF IKBA Mutations and Loss-of-Function (LOF) IKBKG Mutations in Human Anhidrotic Ectodermal Dysplasia With Immune Deficiency (EDA-ID)
Ectodermal dysplasia is a group of more than 150 different heritable syndromes, all of which are derived from abnormalities of the ectodermal structures. [57,[95][96][97] Individuals with two or more ectodermal structural abnormalitiessuch as in the hair, teeth, nails, sweat glands, salivary glands, cranial-facial structure, digits, and other parts of the bodycan be diagnosed as having this disease. The signs and symptoms of ectodermal dysplasia are evident soon after birth. EDA-ID is a form of ectodermal dysplasia with varying degrees of immune deficiency. Children with EDA-ID often produce abnormally reduced antibodies or immunoglobulins, immune T cells with a decreased ability to recognize foreign invaders (such as bacteria, viruses, and fungi), and other impaired components of the immune system. EDA-ID patients commonly obtain infections in the lungs (pneumonia), ears (otitis media), sinuses (sinusitis), lymph nodes (lymphadenitis), skin, bones, and GI tract. Some patients show inflammatory and CMC manifestations. Due to the severity of the immune-system problems, most patients survive only into childhood. There are two forms of EDA-ID that have similar symptoms but are distinguished by the modes of inheritance: X-linked recessive (such as NEMO mutations) and autosomal (such as IKBA mutations) forms. [57,98] Ectodermal dysplasia is susceptible to developing tumorigenic potential.
GOF mutations in the IKBA locus that encodes IkBα have been reported in human EDA-ID disease ( Table 2). [99][100][101][102][103][104] These GOF mutations, including S32I, W11X, E14X, Q9X, M37K, E14X, and E40X at the N-terminal region of IkBα, are heterozygous and generate a dominant-negative IkBα mutant (mIkBα) that is resistant to cytokine-induced degradation. Thus, mIkBα increases its inhibitory activity for NF-kB. Children with these diseases suffer recurrent infections, including hepatitis with cytomegalovirus infection, enteritis with rotavirus, bronchiolitis with respiratory syncytial virus, bacterial pneumonia, urinary tract infection, acute otitis media, and fungal infection (candidiasis, such as CMC), from 1 month to 2 years of age. Some patients suffer autoimmune phenotypes. Despite having a relatively normal number of T and B cells, these patients' T cells show defects in the expression of TNFα, IFN-γ, IL-1β, and IL-6 in response to LPS stimulation. There is increased IgM but reduced globulins and reduced Treg-and natural killer (NK)-cell numbers in EDA-ID patients.
Although EDA-ID and SCID both show defects in the expression of important cytokines against infections in leukocytes, the severity of infectious diseases is less in EDA-ID than in SCID patients because EDA-ID patients retain a wild-type allele. Of note, EDA-ID patients show more phenotypes derived from the ectodermal original compared to SCID patients. Interestingly, transgenic K5.mIkBα mice that express a mIkBα with serine-toalanine mutations at residues 32 and 36 (K32A/K36A) under the control of a K5 promoter develop severe skin inflammation and skin SCCs. [105] K5 is highly and specifically expressed in mTECs and squamous epithelial cells. [25,106] We have shown that reintroduction of K5.IKKα restores NF-kB activities and mTEC numbers and abolishes inflammation and tumors in the skin and esophagus of Ikkα KA/KA mice. [23,25] Given that IKK/NF-kB activities are required for mTEC development, inactivation of NF-kB by overexpressed K5.mIkBα may impair mTEC development, resulting in autoreactive T cells and autoinflammation in mice. Our unpublished results found that kinase-dead IKKα (IKKα KA/KA , IKKα-KA/KA), but not wild-type IKKα, interacts with IkBα because IKKα-KA/KA may not be able to phosphorylate IkBα. Thus, we speculate that mIkBα may bind to IKKα, retaining IKKα in the cytoplasmic compartment in K5.mIkBα mice. The nuclear IKKα suppresses keratinocyte proliferation, inflammatory signaling, and skin tumor formation. [19,23] We therefore propose that K5. mIkBα-mediated IKKα inactivation and impaired central tolerance contribute to skin tumorigenesis in K5.mIkBα mice. On the other hand, K5.mIkBα is not expressed in leukocytes. Thus, there are no reports of increased infections in K5.mIkBα mice. For EDA-ID patients, a GOF IKBA germline mutation is also expressed in leukocytes. A reduction in NF-kB activities can increase infections and downregulate Treg development, [69,89] resulting in autoimmunity, as evidenced in EDA-ID patients.

Fungal Infection and Carcinogenesis
Like bacterial and viral infections, fungal infection can damage cells and organs, leading to inflammation, severe diseases, and death in both humans and animals. On the other hand, epidemiological studies suggest that fungal infection is associated with certain types of human cancers. [115][116][117] Candida albicans is frequently found in the lesion sets of diseases or cancers in humans, [59,118] but many other fungi, including Cladosporium cladosporioides and Candida tropicalis, were also reported in human solid cancers. As discussed above, IKK/NF-kB-regulated immunity plays a decisive role in defending against infections. Consistently, we have reported that IKKα inactivation in T cells contributes to the initiation of fungal infection and that wild-type T-cell replacement prevents fungal infection in Ikkα KA/KA mice. [25] Furthermore, repeated oral administration of C. cladosporioides elevates esophageal tumorigenesis in mice; by contrast, antifungal drug treatment reduced inflammation, DNA damage, and esophageal carcinogenesis, indicating that increased fungal colonization by recurrent fungal infections acts as a tumor promoter. [25] Severe infection is a major phenotype in SCID patients with IKBKB mutations, although most of these patients do not survive to adulthood, suggesting that a defect in IKKβ or NF-kB is not sufficient to result in epithelial malignancies. It has been reported that Als3, a protein of C. albicans, interacts with EGFR and Her2 dimers and induces the tyrosine kinase phosphorylation of EGFR/ Her2 in epithelial cells. [119] Treatment with an EGFR inhibitor significantly reduces the severity of oropharyngeal candidiasis. We also showed that treatment with an inflammatory or an EGFR/ Her2 inhibitor decreases fungal infection in Ikkα KA/KA mice. Increased infiltrating macrophages can activate EGFR. [23] The activated EGFR in epithelial cells may attract fungi and facilitate fungal growth and colonization on the surface of the esophageal, oral, oropharyngeal, and vulvovaginal organs. It is known that IKKα reduction upregulates EGFR activity in keratinocytes. [19,106] Increased levels of inflammatory cytokines downregulate IKKα expression in human SCC cell lines. [23] Furthermore, increased fungal infection is associated with elevated PD-L1 expression in the esophageal epithelial cells and infiltrated macrophages. The increased inflammation, EGFR, fungal infection, and PD-L1 expression are frequently observed in human esophageal SCCs. [25] Most esophageal SCC patients are found in China and South Asia, where damp and rainy weather [120] promotes fungal growth in the environment. Thus, people in those regions have an increased chance of encountering fungi from the environment through their mouths. Once the human body's immune function and epithelial integrity are impaired, the environmental fungi can colonize on the oral cavities and esophagi. Given the pathogenic role of fungal infection in the development of esophageal SCC in mice, the increased fungal infection may promote human esophageal SCC development. A recent report showed that fungi also activate MEK and STAT3 through an ephrin type-A receptor 2 on oral epithelial cells. [121] Whether different types of fungi play different physiological activities and whether and how epithelial IKKα reduction regulates fungal infection remain to be revealed.
It is also known that fungi invade tissues by interacting with other molecules on the cell surface via an endocytosis mechanism. [119] For example, C. albicans can bind to E-cadherin and then recruit clathrin at the C. albicans entry site, which induces pathogen uptake mediated by a rearrangement of the actin cytoskeleton. [122] It has been shown that intracellular calcium plays a regulatory role for clathrin assembly and disassembly. [123] During carcinogenesis, E-cadherin regulates www.advancedsciencenews.com www.bioessays-journal.com cell junction and tumor epithelial-mesenchymal transition, and calcium regulates squamous cell differentiation and proliferation. [14,124] Thus, the impact of the interaction between fungi and epithelial cells in carcinogenesis warrants further investigation. Furthermore, the relationship of fungal infection with commensal fungi and bacteria in disease and cancer development remains to be identified.

Conclusion and Significance
Based on analysis of the pathogenic effects of IKK/NF-kB's inborn errors on the phenotypically related diseases CMC, APECED, EDA-ID, and SCID, we have differentiated the specific roles of IKK/NF-kB in leukocytes and mTECs in the development of infectious diseases, autoimmunity, and tumors. We conclude that IKK/NF-kB activities in leukocytes determine responses to infectious fungi, bacteria, and viruses in humans and mice. In addition, canonical and noncanonical IKK/NF-kB activities through LTβR, RANK, and CD40 are required for immune homoeostasis via their regulation of mTEC development in the thymuses of humans and mice. A defect in mTEC biogenesis causes autoreactive CD4 T helper cell-mediated autoimmunity and autoinflammation. In addition, IKK/NF-kB inactivation in T cells or in mTECs also downregulates Treg numbers and activities, resulting in autoimmunity. The autoinflammation mediated by defective IKK/NF-kB pathways can promote carcinogenesis under certain conditions. For example, although IKKα is part of canonical and noncanonical NF-kB regulation, the loss of nuclear IKKα function accelerates the development of several types of epithelial tumors or results in spontaneous epithelial tumorigenesis independently of canonical and noncanonical NF-kB signaling pathways. The combined NF-kB-dependent and À independent pathways or the combined inflammation and impaired epithelial signaling pathways cause tumorigenesis. Overall, IKK, NF-kB, and their regulators are broadly expressed in many different types of cells and, therefore, have a medical significance in many human diseases. Understanding the detailed mechanisms in the pathogenesis of the diseases associated with impaired IKK/NF-kB activities will provide insight into the prevention and treatment of those human diseases and cancers.