A 2014 multicentre study (Cleynen et al., Gut) associated Crohn's disease and ulcerative colitis with common variants in ubiquitin–proteasome system (UPS) genes — most strongly the intronic SNP rs12324931 in the deubiquitinase gene CYLD, adjacent to NOD2 — and showed that the Crohn's-associated adherent-invasive E. coli strain LF82 reduces CYLD protein, raises 26S proteasome activity and activates NF-κB in intestinal epithelial cells. The study left open several linked questions: which variants in the CYLD–NOD2 interval are causal and how they act (its Discussion calls for resequencing of the complete region); whether the USP40, 3p21 (USP4/APEH/DAG1/MST1) and USP3 signals reflect independent UPS genes (requiring dense-SNP conditional analysis before firm conclusions); by what molecular mechanism AIEC alters CYLD and proteasome function; and, per the study's clinical-practice outlook, whether proteasome inhibitor therapy targeting the UPS (bortezomib is cited as a specific example) can ameliorate IBD. Later literature advanced CYLD biology in vivo on other axes (SMAD7/TGF-β, NLRP6, IRE1) and showed proteasome inhibition is protective in generic colitis models, but the specific gene–microbe–UPS axis — causal alleles, AIEC effector and host cascade, and UPS-targeted therapy — remains unresolved.

Background

Inflammatory bowel disease (IBD) comprises Crohn's disease (CD) and ulcerative colitis (UC), chronic relapsing inflammatory disorders of the intestinal mucosa arising from interactions between susceptibility genes, the gut microbiota and immune responses. A central signalling node in intestinal inflammation is NF-κB, a transcription factor held inactive in the cytosol by the inhibitor protein IκB-α; phosphorylated IκB-α is tagged with poly-ubiquitin chains and destroyed by the proteasome, releasing NF-κB to enter the nucleus. Ubiquitination is the attachment of the small protein ubiquitin to substrates via E3 ligases, reversed by deubiquitinases (DUBs); together with the 26S proteasome (the multi-subunit ATP-dependent protease that degrades ubiquitinated proteins) this machinery is called the ubiquitin–proteasome system (UPS). Under inflammatory cytokine stimulation, cells replace constitutive proteasome subunits with inducible 'immunoproteasome' subunits such as LMP7. CYLD is a deubiquitinase that preferentially removes lysine-63 (K63)-linked ubiquitin chains, originally identified as a tumour suppressor mutated in familial cylindromatosis, that removes activating ubiquitin chains from NF-κB pathway components (TRAF2/NEMO and others) and thereby restrains NF-κB; in mice, Cyld loss worsens experimental colitis. CYLD lies on chromosome 16q12 immediately adjacent to NOD2, an intracellular bacterial peptidoglycan sensor and the strongest CD susceptibility gene; because nearby variants are inherited together (linkage disequilibrium, LD), assigning an association signal near NOD2 to CYLD rather than to NOD2 requires conditional genetic analysis. Adherent-invasive Escherichia coli (AIEC) are CD-associated E. coli strains, reference strain LF82, that adhere to and invade intestinal epithelial cells and replicate persistently within macrophages; they are enriched in CD ileal mucosa. Against this background, patient tissue had already shown elevated immunoproteasome subunit expression and enhanced proteasomal IκB-α degradation in CD mucosa (Visekruna et al., J Clin Invest 2006), a candidate-gene systematic review had shortlisted protease and UPS genes (Cleynen et al., PLoS ONE 2011), and an LD-mapping analysis of CD genome-wide data had suggested an independent CYLD contribution (Elding et al., Am J Hum Genet 2011). Cleynen et al. (Gut 2014) then genotyped 182 tagging single nucleotide polymorphisms (tagging SNPs — markers chosen to capture common genetic variation across each candidate gene) in 23 protease/UPS genes across 2,320 CD, 2,112 UC and 1,796 control subjects, and followed up the associations functionally. SNPs in CYLD, USP40, APEH and USP3 associated with CD (rs12324931 in CYLD strongest, OR 2.24), and SNPs in USP40, APEH, DAG1 and USP3 with UC. Conditionally on the three common NOD2 coding variants the CYLD signal persisted, but the associations at USP40 (2q37, neighbouring the CD gene ATG16L1) and the 3p21 cluster (containing APEH, DAG1, USP4 and the IBD gene MST1) were never tested for independence from those known loci. Functionally, infection of T84 cells (a human colonic epithelial cell line) with live LF82 (but not heat-killed bacteria or adhesion-deficient mutants) reduced poly-ubiquitin conjugate accumulation and CYLD protein by about 40%, increased 26S chymotrypsin-like activity (one of the proteasome's standard peptide-cleaving activities, measured with fluorogenic substrates), caused IκB-α degradation that was blocked by the proteasome inhibitor MG132 with NF-κB p65 nuclear translocation, and CYLD knockdown or overexpression respectively increased or reduced intracellular LF82 replication. These results proposed a unified model in which inherited UPS-gene variation and CD-associated bacteria cooperatively dysregulate epithelial UPS–NF-κB signalling — and simultaneously exposed what remains unproven at every step.

Problem Statement

The problem, as posed by the unresolved issues of Cleynen et al. (Gut 2014), is to convert their associative gene-by-microbe finding into a validated causal account of UPS modulation in IBD, with one common objective: determine whether and how inherited variation in UPS genes and CD-associated AIEC jointly dysregulate epithelial ubiquitin–proteasome function and NF-κB signalling in inflammatory bowel disease. Concretely, the following open questions are to be addressed. (1) Causal alleles at the CYLD–NOD2 region (16q12): identify the putative functional alleles in the CYLD–NOD2 interval and determine whether and how specific CYLD variants alter CYLD expression or activity, with causal attribution conditioned on all known NOD2 risk alleles (common and rare). (2) Causal genes at the remaining loci: which gene(s) and alleles drive the observed Crohn's disease and ulcerative colitis associations at USP40 (2q37, neighbouring ATG16L1), the 3p21 cluster containing USP4, APEH, DAG1 and MST1, and USP3 (15q22), including allelic heterogeneity, and whether these signals are independent of the known neighbouring loci. (3) Molecular mechanism of microbial modulation: define at the molecular level how AIEC strain LF82 reduces CYLD protein and increases 26S proteasome activity in intestinal epithelial cells — the responsible bacterial determinant(s), beyond the demonstrated dependence on the bacteria's ability to survive and/or replicate within host cells, the host signalling cascades engaged, and whether the changes arise from altered transcription, translation, deubiquitination, proteasome assembly or selective substrate targeting. (4) Therapeutic test: test whether proteasome inhibitor therapy targeting the UPS (Cleynen et al. (2014) cite bortezomib as a specific example for future use in IBD) can ameliorate intestinal inflammation in IBD; the genetic and microbial contexts identified in (1)–(3) motivate this test. In vivo and patient-derived corroboration of the epithelial UPS changes (for example correlation of CYLD protein, 26S activity or poly-ubiquitin conjugates with UPS genotype and AIEC colonisation) may strengthen any of these answers. The four questions are logically independent; each may be resolved independently of the others. Questions (1) and (2) are resolved by a well-supported positive or negative genetic result (for example replication of an association, or non-replication of an association or absence of a functional CYLD variant). Question (3) is resolved by a well-supported mechanistic finding — identification of the responsible bacterial determinant(s) or host signalling cascade, or a definitive determination that the candidate mechanisms enumerated in (3) do not account for the observed effect; a merely unsupported or inconclusive account does not resolve it. Question (4) is resolved by a well-supported positive or negative outcome of the therapeutic test.

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