Introduction
Chronic inflammatory disorders are often associated with ongoing damage to epithelial tissue, resulting in ulcers and immune disorders. Epithelial tissue is a thin sheet of tightly packed cells—creating order in the body which would otherwise be an unorganized and unarticulated mixture of cell types. The epithelium covers all exposed surfaces of the body and lines body cavities. Epithelial cells form glandular tissue, the outer layer of skin, and lines the tracts of the respiratory, digestive, urinary, reproductive and nervous systems. The endothelium covers the components of the cardiovascular system. Due to the wide function of epithelial tissue, it is unsurprising that epithelial cells derive from all three major embryonic layers. Differentiated epithelial cells comes from the segregation of organelles across the cell along with their shape.
A single layer of tall epithelial cells, referred to as a simple columnar epithelium, is present in the lower digestive tract. The distribution of organelles is such that efficient uptake of nutrients is achieved by numerous folds in the plasma membrane, or villi. Inflammatory bowel disease (IBD) is a group of disorders that affects the epithelial lining primarily of the large intestine and rectum. The most common symptoms of IBD are diarrhea, abdominal pain, bloody stool, weight loss, and fever (1). IBD has a complex pathogenesis, as IBD is used as a general term for many disorders. Genome-wide association studies (GWAS) have identified over 230 loci linked to polygenic IBD. Monogenic, or Mendelian, disorders present with IBD-like symptoms in rare cases (2).
X-linked lymphoproliferative syndrome (XLP) is a recessive condition that negatively affects the immune system and often presents with IBD-like symptoms. The two types of XLP are distinguished by two genetic causes. Mutations in the SH2D1A gene, coding for the signaling lymphocyte activation molecule (SLAM) associated protein (SAP), is responsible for XLP type 1 (XLP-1). SAP is a protein associated with the immune system and is encoded for in natural killer (NK), T, and invariant NKT (iNKT) cells (3). SAP binds to SLAM to enable downstream activation of genes necessary for immune response. XLP type 2 (XLP-2) is caused by mutations in the BIRC4 gene encoding for the X-linked inhibitor of apoptosis protein (XIAP). In people with XIAP deficiency, lymphocytes are subject to more incidence of apoptosis, thereby decreasing their immune response (4).
XIAP deficiency affects the NOD-like receptors (NLRs) signaling pathways of the innate immune response. NOD-like receptors are proteins involved in recognizing pathogens and directing defense responses (2). Because epithelial cells of the digestive system work as a primary line of defense against many pathogens, NLRs are present in high numbers within the epithelium. Disruptions in the innate immune response from the NOD pathway leads to epithelial defects and proinflammatory responses, often causing ulcers. Both XLP-1 and XLP-2 are associated with high susceptibility to Epstein-Barr virus (EBV), and frequently results in the life-threatening condition, hemophagocytic lymphohistiocytosis (HLH) (6).
HLH is the primary cause for the IBD-like symptoms present in patients with XLP. It involves highly activated macrophages engulfing hematopoietic stem cells. Clinical findings include fever, low NK-cell activity, diarrhea, and ulcers (5). Cytotoxic cell function combined with high activity of macrophages leads to immune dysregulation and epithelial tissue damage, consistent with IBD. HLH is more common in children than in adults and is likely largely underdiagnosed (6). Hematopoietic stem cell transplantation (HSCT) is the most common treatment for HLH (2), but long-term survival is estimated to be as low as 4% (5).
Indeed, as this review will explore, HLH is oftentimes the final outcome of mutations in genes associated with the NLR signaling pathway. XIAP deficiency, therefore, leads towards decreased innate immune response in epithelial cells, characteristic of XLP-2. In this chronic condition, repeated ulceration with lack of immune attack on many digestive pathogens causes hemophagocytosis when left unchecked.
XIAP deficiency interrupts the NLR signaling pathway
XIAP deficiency leads towards chronic ulceration and epithelial defects characteristic of XLP-2 and IBD due to its role in the NOD-like receptor (NLR) signaling pathway to direct an innate immune response against pathogens. The role of XIAP in the NLR signaling pathway has been defined through experiments (7) with human colonic epithelial tumor cells with the XIAP gene ablated. This cell line was exposed to NLR ligands for 24 hours, and immune cell production was measured. In wild-type cells that produce XIAP, interleukin-8 (IL-8) would be produced as the NLRs mediated an innate immune response. XIAP deficient cell lines did not produce IL-8, confirming that XIAP is required for NLR signaling pathways. RIP2 is the protein found to be the link between XIAP and NLRs through testing with cell lines deficient in IL-8 production but not in XIAP nor NLRs, as confirmed by SDS/PAGE/immunoblotting. The researchers hypothesized that RIP2 binds to caspase activation recruitment domains (CARDs), which are protein domains characteristic of inflammatory and apoptotic responses, of NLRs. Due to the known active kinase domain (KD) of XIAP, the researchers hypothesized that RIP2 binds to XIAP at its KD and not its CARD (7). Therefore, a mutation causing XIAP deficiency in colonic epithelial cells was found to lead towards a reduction in innate immune response by not binding to RIP2 proteins and playing an important role in the NLR signaling pathway.
Whole gene deletion XIAP coding regions results in VEO-IBD
In a case study from the Hospital of Philadelphia Division of Pediatric Gastroenterology (8), whole gene deletion of XIAP coding regions has been shown to correspond to XLP-2, resulting in very early-onset inflammatory bowel disease (VEO-IBD) and increased susceptibility to HLH. In this case, a male child 3 weeks of life, presented with bloody diarrhea. An initial colonoscopy and endoscopy was performed at 8 weeks, showing gastric and duodenal ulcers, along with severe pancolitis. The child was diagnosed with VEO-IBD. The patient developed a perianal sinus tract at 5 months of age and remained unresponsive to all medications. The child became intolerable for all nutritional methods, and he failed to grow. By age 2, the child’s perianal disease progressed and more upper tract (gastric and duodenal) ulcers were observed. Rectal dilation lead to colonic perforation at age 7, and the child underwent a colectomy at age 10. The patient remained unresponsive to medical and surgical interventions and was enrolled in a VEO-IBD research study at age 17.
The VEO-IBD research study used whole exome sequencing (WES) to identify variants associated with the disease. WES reduces costs of genetic sequencing by analyzing just the protein-coding genes in a genome. Hybridization probes identify the exon locations, which are then sequenced. Copy-number variation (CNV) analysis, using the eXome-Hidden Markov Model, was used to compare the child’s exome to that of many exomes found in several databases. This revealed 1,380 nonsense and missense exome variants in the patient. Variants were restricted to include only rare or novel variants in immunodeficiency associated genes, and a de novo whole gene deletion of XIAP was identified. Analysis of the patient’s XIAP expression in CD4+, CD8+ T, and NK cells led to diagnosis of XLP-2 in the patient. Bone marrow transplantation was performed to prevent HLH, which was ultimately curative for the patient’s disease.
HLH and ulceration can be misdiagnosed for acute appendicitis
HLH has been shown to present as acute appendicitis in a 3-year-old female patient (9). The child was symptomatic for 8 days with bloody diarrhea, abdominal pain, tachycardia, and malaise. Tenderness from palpitation in the lower right-hand quadrant along with enlarged cervical lymph nodes. The patient was admitted to surgical service for observation due to concern for appendicitis and underwent exploratory laparoscopy for suspected perforated appendicitis. Multiple 0.5-1 cm plaques were observed and biopsied, which were found to be epithelial ulceration. The plaques were found to be composed primarily of immune cell fragments, suggestive of HLH.
Postoperatively, the patient remained ill and was admitted to the pediatric intensive care unit. The patient was negative for mutations in genes associated with familial HLH, suggesting the possibility of XLP type 1 or type 2. Future testing for the XIAP associated gene was noted, along with future treatment with unrelated donor hematopoietic stem cell transplant.
Conclusions
Chronic inflammatory disorders associated with epithelial cell defects can be due to innate immune response deficiency caused by genetic mutations. Because the epithelial lining of the digestive tract is the first line of defense against many pathogens, mutations associated with the innate immune response leaves the epithelium at risk for damage. XIAP deficiency, characteristic of XLP-2, results in a disruption in the NLR signaling pathway by not binding to RIP2, therefore inactivating the NOD2 receptor (7). This decrease in innate immune response in the digestive epithelium results in pathogens entering the bloodstream, overloading the immune system. Hemophagocytosis is observed as a response, causing epithelial apoptosis and ultimately ulcerations (8,9) that are symptomatic of HLH.
Genetic analysis in patients allows for appropriate treatment in rare cases that the patient remains unresponsive to conventional therapy. Epithelial defects leading towards ulceration have pathogeneses, with over 230 associated polygenic loci identified associated with IBS alone. Rare monogenic causes, such as XLP types 1 and 2, are well studied, but often remain undiagnosed. WES is an effective tool for clinicians to diagnose these rare disorders, albeit after all other tests have been run due to high costs. WES, when paired with well-established mathematical models such as CNV analysis, provide the diagnosis that can cure patients with decades of ineffective treatments and surgeries (8).