Enhanced gut bacterial IgG2-coating assists in identifying pathogenic bacteria associated with active disease in Crohn's disease

Crohn’s disease (CD), a relapsing inflammatory bowel disease, exhibits great heterogeneity in disease presentation and treatment responses, where distinct gut bacteria may play a part in theyet unresolved disease aetiology. To investigate temporal immune-mediated processes we first determined the levels of immunoglobulin (Ig) coating of gut bacteria in healthy controls and CD patients from the IBD South Limburg cohort, Netherlands.

Aim & Methods: To examine IgA and IgG coating to the gut microbiota in Crohn’s disease patients we quantified bacterial numbers in stool and identified levels of Ig-coating of gut bacteria in healthy subjects (n = 20) and CD patients (n = 60) from the IBD South Limburg cohort, Netherlands, using multi-color flow cytometry. We identified bacteria tied to disease pathology via 16S rDNA amplicon sequencing of stool samples in addition to specifically IgG2-coated bacteria isolated by fluorescence-activated cell sorting (FACS).

Results: We found a 1.7-fold increased relative IgA-coating in CD patients compared to healthy individuals, but no overall differences in coating frequencies of gut bacteria per gram stool with IgA and IgG1, 2, 3 and 4. Bacterial IgG2-coating set apart some CD patients from other CD and healthy subjects and was found to associate with clinical disease parameters, like fecal calprotectin (FC), disease behavior, Harvey-Bradshaw index (HBI) and intestinal surgery. When separating CD patients based on their gut bacterial IgG2-coating levels (IgG2 Low, Intermediate and High), we found increased IgG2 High levels in patients with active disease.
The gut microbiota of CD patients with an IgG2 High phenotype was significantly different to that of subjects with Low or Intermediate gut bacterial IgG2 coating. Increased disease severity, as represented by enhanced FC levels, HBI and penetrating disease behavior, associated with the microbiota composition of CD patients with an IgG2 High phenotype. Using supervised clustering of bulk stool microbiota, as well as IgG2-specific bacterial sorting, we identified distinct IgG2-coated and uncoated bacterial species to be indicative of the IgG2 High phenotype. Several of these species are highly virulent and holds e.g. Ig-proteases. Two of the uncoated IgG2 High indicator species associated independently of each other to active disease, while not to remitting disease, in the original European-based cohort and in a replication cohort from US. Each of them formed robust networks with distinct coated IgG2 High indicator species. This indicated the existence of two different and IgG2-uncoated pathogenic gut bacteria that may differentially exaggerate disease activity in CD patients with an IgG2 High phenotype.

Conclusion: We identified a subgroup of CD patients with enhanced gut bacterial IgG2-coating, who harbored one of two distinct and IgG2-uncoated pathogenic bacteria each linking individually to severe CD pathology.