Inflammatory bowel diseases and primary immunodeficiency diseases

Abstract Recent advances in molecular biology have provided important insights into the genetic background of various inflammatory diseases. In particular, genome-wide association studies of inflammatory diseases have revealed genetic loci that play critical roles in the pathology of inflammation. Whole-exome and whole-genome sequencing analyses have also identified more than 300 causative genes for primary immunodeficiency diseases (PIDs). Some genetic loci that are associated with inflammatory diseases are mutated in PIDs, suggesting close relationships between inflammation and PIDs. Inflammatory diseases for which genetic associations have been described include inflammatory bowel disease (IBD), multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus, and systemic lupus erythematosus. Herein, I discuss about the genetic interactions between IBD and PIDs.


Introduction
Most diseases are associated with imbalances between intrinsic genetic factors and extrinsic environmental factors. Whereas inflammatory diseases have mainly been considered consequences of heterogenous environmental factors, recent advances in gene analysis have led to the identification of genetic components of inflammatory diseases. Among technology advances, genome-wide association studies (GWAS) of inflammatory diseases have revealed genetic loci that play central roles in pathological inflammation. Concomitantly, whole-exome and whole-genome sequencing studies have identified more than 300 genes that are causative for primary immunodeficiency diseases (PIDs) [1] and some of these are genetic loci that have been associated with inflammatory diseases. Hence, inflammatory diseases such as inflammatory bowel disease (IBD), multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus, and systemic lupus erythematosus may be related to PIDs. In this review, genetic interactions between IBD and PIDs are discussed.

Genes associated with IBD
IBD encompasses all inflammatory diseases that affect the gastrointestinal tract. Crohn's disease (CD) and uncreative colitis (UC) are the major IBD and symptoms associated with gut inflammation include abdominal pain, fever, diarrhea, rectal bleeding, anemia, and weight loss. Although patients with IBD cannot be fundamentally cured, the symptoms can be managed using anti-inflammatory steroids or immunosuppressant, or through dietary changes and surgery. CD and UC share several clinical features, but are distinguished by incidence patterns, disease localization, histopathology, and endoscopic features, and these differences may reflect underlying pathological mechanism [2,3]. The prevalence of IBD is highest in Europe and North America [4], but is rising in Japan due to increasing consumption of Westernized diets.
IBD is characterized by dysregulated immune responses to unknown environmental triggers, and it is thought to occur in genetically susceptible individuals. Twin studies revealed that coincidence of IBD is higher in monozygotic twins than in dizygotic twins, suggesting genetic contributor to IBD risk [5]. Linkage analyses also linked chromosome 16 with CD [6], and the candidate gene was finally identified as NOD2, which plays an important role in the recognition of bacterial peptidoglycans, hence, immune responses [7,8]. Yet NOD2 did not play a significant role in the pathogenesis of CD in a Japanese population [9]. GWAS showed that the genes IL23R and ATG16L1 are related to CD [10,11]. IL23R encodes a receptor protein that is presented on cell membranes of many different immune types, whereas ATG16L1 encodes a protein that is involved in autophagy Meta-analyses revealed a total number of 163 IBD loci [12], and previously described associations of these with innate immune responses, activation of adaptive immune responses, and regulation of adaptive immune responses, implicate these pathways in the pathogenesis of IBD (Table 1). Immunochip is a low-cost contemporary GWAS chip, and includes 70% (113 of 163) of the identified IBD loci. In further meta-analysis and trans-ancestry studies, more than 200 IBD-associated loci were identified ( Figure 1). Some of these IBD-associated loci include genes that are involved PIDs, including ADA, CD40, TAP1, TAP2, NBN, BLM, DNMT3B, STAT3, SP110, and STAT5B.

Monogenic IBD
Pediatric-onset IBD is that observed in patients of less than 17 years of age and is further classified into subgroups based on age at diagnosis (Table 2) [13,14]. Patients with early-onset IBD (EOIBD) and very early-onset IBD (VEOIBD) are diagnosed before 10 and 6 years of age, respectively. Diagnoses in infants of less than 2 years and less than 28 days are considered infantile and neonatal IBD, respectively. Many patients with VEOID have low response rates to conventional anti-inflammatory and immunomodulatory therapy and may have monogenic defects. Monogenic IBD has been documented in patients with a diverse spectrum of genetic disorders. Hence, it is important to distinguish between patients with monogenic IBD and conventional IBD, because the former require treatment with allogeneic hematopoietic stem cell transplantation (HSCT). Moreover, monogenic defects were shown to alter intestinal immune homeostasis through several mechanisms, and other single gene defects have been associated with hyperinflammation or autoinflammation, and disruption of T-and B-cell selection and activation.

Diagnosis of monogenic IBD
As described above, the early diagnosis of VEIOD is critical for the design of treatment strategies. Monogenic IBD is primarily suspected in young patients ( Figure 2) [15]. In addition to early onset, diagnoses of monogenic IBD are supported by diagnosis of IBD in multiple family members, consanguinity, autoimmunity, failure to thrive, conventional treatment failure, endocrine abnormality, recurrent infectious or unexplained fevers, severe perianal disease, macrophage activation syndrome and hemophagocytic lymphohistiocytosis, obstruction and atresia of the intestine, skin lesions and dental and hair abnormalities, and the presence of malignancies.
IBD is typically diagnosed from endoscopic and histopathological observations. The histopathology of IBD is classified as CD, UC, or IBD unclassified, and the latter is frequently observed in patients with VEOIBD, especially those with monogenic IBD. Various functional and genetic tests are required to confirm monogenic IBD in patients with VEOID ( Figure 3). These include analyses of Table 1. Pathways implicated in IBD pathogenesis.

Pathway implicated
Pathway genes in IBD-associated loci   Innate immune response  Epithelial barrier function and repair  CDH1, ERRFI1, GNA12, HNF4A, ITLN1, MUC19, NKX2-3, PLA2G2E, PTGER4, REL, STAT3  Innate mucosal defence  CARD9, FCGR2A, IL18RAP, ITLN1, NOD2, REL, SLC11A1  Autophagy  ATG16L1, CUL2, DAP, IRGM, LRRK2, NOD2, PARK7  Apoptosis/necroptosis  DAP, FASLG, MST1, PUS10, THADA  Activation of adaptive immune response  IL23R response pathway  CCR6, IL12B, IL21, IL23R, JAK2, STAT3, STAT4, TYK2  NF-jB  NFKB1, REL, TNFAIP3, TNIP1  Aminopeptidases  ERAP1, ERAP2  IL-2 and IL-21 T-cell activation  IL2, IL21, IL2RA  Regulation of adaptive immune response  TH17 cell differentiation  AHR, CCR6, IL2, IL21, IL23R, IRF4, JAK2, RORC, STAT3, TNFSF15, TYK2  T-cell activation  ICOSLG, IFNG, IL12B, IL2, IL21, IL23R, IL2RA, IL7R, NDFIP1, PIM3, PRDM1, TAGAP, TNFRSF9, TNFSF8  B-cell   neutrophil-mediated oxidative bursts using nitroblue tetrazolium tests or flow cytometry-based assays, measurements of IgG, IgA, IgM, and IgE, and flow cytometric assays of lymphocyte subsets, such as CD3 þ T, CD4 þ T, CD8 þ T, CD19/C20 þ B, and CD16/CD56 þ natural killer (NK) cells. All types of chronic granulomatous disease can be diagnosed using assays of the neutrophil oxidative burst. Patients with common variable immunodeficiency and agammaglobulinemia show reduced levels of all class immunoglobulins. Patients with hyper IgM syndrome generally have normal to elevated levels of IgM but reduced levels of IgG and IgA. Elevated levels of IgE and/or eosinophilia are also observed in patients with monogenic defects in FOXP3, IL2RA, IKBKG, WAS, or DOCK8 genes. Whereas all patients with severe combined immunodeficiency (SCID) lack T cells, the associated impact on B and NK cells vary between genetic defects. For example, X-linked agammaglobulinemia is associated with reduced numbers of circulating B cells. Moreover, FOXP3 expression in CD4 þ CD25 þ T cells is reduced in a proportion of patients with immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome [16,17]. XIAP expression is decreased in lymphocytes and monocytes of some patients with XIAP deficiency (Figure 4(a)) [17,18], and muramyl dipeptide signaling is selectively defective in patients with XIAP deficiency (Figure 4(b)) [19]. IL-10 receptor deficiency can also be detected using assays that determine whether exogenous IL-10 suppresses lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells [20,21]. Following functional screening of these deficiencies, candidate genes are generally sequenced to confirm the suspected genetic condition. Candidate genes for monogenic IBD have recently become more numerous, but exhaustive sequencing of these is costly and time-consuming. Because of the greatly reduced costs of next-generation sequencing, multiplex gene sequencing may be cost-effective, and can be used to detect causative genes. We previously identified PID-associated genes in 35 Japanese patients with pediatric-onset IBD [22], and 27 of these had VEOIBD. In this study, 55 genes that have been associated with PID and/or IBD were selected for targeted gene panel analysis (Table 3). Gene defects were the identified   in 5 of the 35 patients (14.3%), and these included IL10RA in two patients, XIAP in two patients, and CYBB in one patient. Another group also suggested that targeted sequencing of a panel of genes is a fast and effective way to identify monogenic IBD [23].

Novel monogenic IBD
Recent advances in genetic analyses including WES and whole-genome sequencing have allowed identification of novel mutations even in single patients. Below, I describe novel monogenic IBD types that were identified recently.

TRIM22
In a previous WES study, mutations in the tripartite motif containing 22 (TRIM22) gene were identified in three patients with VEOIBD [34]. TRIM22 is a RING finger E3 ubiquitin ligase that is expressed in intestinal cells and macrophages [35], and exhibits antiviral activity and activate nuclear factor-kB (NF-jB) signaling [36]. Because all three VEOIBD patients had distinct granulomatous colitis and severe perianal disease, it was concluded that the TRIM22-NOD2 network functions as a key antiviral and mycobacterial regulator.

NPC1
Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder that is associated with defects in lysosomal calcium homeostasis and lipid   trafficking, and is caused by mutations in NPC1 and NPC2 genes [37]. Although patients with NPC1 often present with IBD, the associated functional mechanisms remain unclear. Nonetheless, mutation in NPC1 led to defects in autophagosome functions and abolished NOD2-mediated autophagy of bacteria [38]. Similar functional defects were observed in CD patients associated with NOD2 variants and XIAP deficiency. Taken together, these observations suggest that antibacterial autophagy that is initiated by the NOD2-RIPK2-XIAP pathway is a key defect in patients with granulomatous intestinal inflammation.

NOX1
In a study using whole-genome sequencing analyses, a patient with UC-like VEOIBD was shown to carry a novel hemizygous mutation in the NOX1 gene [39], and subsequent WES analyses identified other pediatric IBD patients with rare NOX1 variants. NOX1 is the catalytic subunit of superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex 1, and comprises NOX1, p22 phox , NOXA1, NOXO1, and Rac1-GTP protein subunits [40]. NOX1 is a close structural homolog of the NADPH oxidase complex 2 component NOX2 (gp91 phox , CYBB) and in phagocytes, NADPH complex 2 is responsible for the microbicidal respiratory burst. NOX1 is located in membranes of intestinal epithelial cells [41] and was shown to constitutively generate high levels of reactive oxygen species (ROS) in the crypt lumen. A missense mutation abrogated ROS production by NOX1, suggesting that NOX1 variants change brush border ROS levels within colonic crypts at the interface between the epithelium and luminal microbes.

TGFB1
Transforming growth factor (TGF)-b1 (encoded by TGFB1) is the prototypic member of the TGF-b family, which comprises proteins that have been widely associated with embryogenesis, development, and tissue homeostasis [42]. Dysfunctional TGF-b1 signaling is implicated in several human diseases, including cancer, cardiovascular disease, fibrosis, atherosclerosis, and various developmental disorders. In contrast, heterozygous gain-of-function mutations in TGFB1 cause Camurati-Engelmann disease, which is characterized by osteosclerotic lesions in long bones and the skull [43]. Biallelic loss-of-function mutations in TGFB1 were also identified in patients with severe IBD and central nervous system (CNS) diseases such as epilepsy, brain atrophy, and posterior leukoencephalopathy [44]. This study shows that TGF-b1 plays critical roles in the development and homeostasis of intestinal immunity and the CNS.

Treatment of IBD
Patients with IBD are currently treated with antiinflammatory drugs and are encouraged to modify their diet. Surgery is rarely considered and is only performed in severe cases. Conventional drugs for IBD include prednisolone, methylprednisolone, budesonide, 5-aminosalicylic acid, 6-mercaptopurine, and methotrexate (Table 4). Treatment-resistant patients can subsequently be treated with infliximab and adalimumab, which are TNF-a pathway blockade. Alternatively, IL-10R-deficient mice develop spontaneous colitis, and patients with mutations in IL10R develop VEOIBD. These losses of IL-10 signaling lead to intestinal inflammation through increased production of IL-1 by innate immune cells, leading to CD4 þ T cell activation [45]. Therefore, agents that block IL-1 signaling might be used to treat patients with IL10R deficiency-related IBD.
IL-18 blockade may also be effective in some patients with monogenic IBD. Patients with XIAP deficiency often have sustained levels of serum IL-18 during convalescence [46]. The patients are frequently associated with hemophagocytic lymphohistiocytosis (HLH), and HLH susceptibility in XIAP deficiency has been associated with high serum IL-18 levels. Gain-of-function mutations in NLRC4 which activates the inflammasome, result in recurrent macrophage activation syndrome (MAS) with early-onset enterocolitis (NLRC4-MAS) [47,48]. Patients with NLRC4-MAS have extraordinarily high serum IL-18 levels. In accordance, recombinant human IL-18 binding protein (rhIL-18BP) had dramatically favorable effects in a refractory patient with NLRC4-MAS [49]. Patients with XIAP deficiency may be successfully treated with rhIL-18BP.
using allogeneic HSCT ( Figure 5), although transplantation-related morbidities have been observed and remain a risk.
Published case reports suggest that HSCT may also be of benefit to some patients with CD. Accordingly, a randomized clinical trial of HSCT (n ¼ 23) vs. control (n ¼ 22) was conducted with refractory CD patients in Europe from 2007 to 2011 [53]. In this study, autologous HSCT did not significantly improve disease remission rate at 1 year, compared with conventional therapy. In contrast, a recent multicentre retrospective analysis showed that autologous HSCT is relatively safe and effectively controls treatment-resistant CD [54]. Further prospective randomized controlled trials of autologous HSCT vs. the standard of care are warranted.

Conclusion
A proportion of patients with VEOIBD have monogenic IBD and these patients are frequently refractory to conventional treatment. Because patients with monogenic IBD can be cured using allogeneic HSCT, accurate genetic diagnoses are required to determine prognoses and to prescribe appropriate treatment. Further studies of monogenic IBD may also improve the understanding of the more complicated pathogenesis of polygenic IBD.

Disclosure statement
No potential conflict of interest was reported by the author.

XIAP deficiency IPEX syndrome
Pre HSCT Post HSCT Figure 5. Endoscopic findings in patients with primary immunodeficiency diseases before and after hematopoietic stem cell transplantation. HSCT: hematopoietic stem cell transplantation.