LAUSR

Background: Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Both environmental and genetic factors contribute to disease pathogenesis. This complexity is further exacerbated with an uncontrolled immune response to the gut microbiota. Given that immunoglobulin (Ig) G is formed in response to invasive microbes, we anticipated that bacteria bound by IgG are more likely to be virulent; their isolation could help identify such invasive strains and define mechanisms of immune activation. We hypothesized that host IgG immune response can be used to identify bacteria that may be involved in the pathogenesis of IBD. Methods: Aspirate washes from the terminal ileum of pediatric IBD (including Crohn disease [CD] and ulcerative colitis [UC]) and non-IBD controls were collected during endsocopy, stringently washed, and then fixed in paraformaldehyde. An aliquot was stored (presort) and the remaining washes were labeled with propidium iodide and anti-IgG fluorescent antibody to allow for identification of bacteria bound by IgG and those that are not. Using fluorescence-activated cell sorting (FACS), the samples were then fractioned for IgG bound (IgG+) and non-IgG bound (IgG−) bacteria. Some samples were also imaged by image cytometry to validate the FACS procedure. After sorting, DNA was extracted using beads and purified by phenol/chloroform. DNA was then analyzed by 16S sequencing using the Ilumina MiSeq platform. Results: A total of 36 washes from children without IBD (n = 10), and with CD (n = 17), and UC (n = 9), were suitable for analysis with DNA meeting quality control criteria. The ileal mucosa-associated microbiome composition of non-IBD and IBD groups did not differ significantly, with predominance of Firmicutes and Bacteroidetes. When sorted for IgG coated bacteria, the ratio of IgG+/IgG−bacteria (indicating taxa over-represented by IgG binding) were increased in CD and UC patients by 2- and 1.5-fold, respectively. Notably, the IgG+/IgG−ratio of Bacteroidetes and Proteobacteria taxa were increased in CD, Actinobacteria was increased in UC patients, and there was no changes in Firmicutes, the major phyla of the ileal mucosal microbiome. Although there was considerable inter-individual variation, at the family level, IgG binding favored Porphyromonadaceae (Bacteroidetes) and Enterobacteriaceae (Proteobacteria) in children with CD; Bifidobacteriaceae (Actinobacteria) and Barnesiellaceae (Bacteroidetes) in UC; and Clostridiaceae and Veillonellaceae (both Firmicutes) in non-IBD. Conclusions: IgG coating of mucosa associated bacteria in the ileum of IBD patients is elevated and is selective for unique phyla compared to non-IBD controls. Taxa identified by IgG include bacteria that have been associated with disease and virulence. Identification of individual microbes using this method could facilitate development of targeted diagnostic and therapeutic approaches for IBD.