Studies of microbial diversity in experimental models of arthritis

Administering harmless antigens orally can induce immune tolerance, leveraging the gastrointestinal tract's capacity to withstand exposure to dietary components and commensal microbiota without eliciting inflammatory responses. Repetitive administration of type II collagen via oral route establishes oral tolerance, thereby impeding the onset of arthritis, a chronic inflammatory condition affecting the joints. While some mechanisms underlying oral tolerance are elucidated, the impact of dysregulated gut immune networks on extra-intestinal inflammation, such as in the joints, remains ambiguous. Employing undenatured type II collagen in a prophylactic regimen, we investigated the microbiome diversity associated with protective oral immune-therapy in ileum and colon during experimental Collagen-Induced Arthritis (CIA).We defined three experimental groups: 1) non-treated mice (Naive), 2) mice undergoing experimental Collagen-Induced Arthritis and 3) animals undergoing arthritis with prophylactic undenatured type II collagen oral immunotherapy. Immunotherapy started 2 weeks prior to CIA induction, and oral gavage of undenatured type II collagen (7.33 mg/kg) was applied 3 times per week. Group 2 was further divided into mice with high clinical scores, and low clinical scores. Group 3 included mice completed protected by immunotherapy and mice that did not respond to the intervention. At day 34, animals were culled, and faecal matter was collected from ileum and colon. Genomic DNA from the faecal matter was purified using QIAamp DNA Stool Mini Kit (Qiagen, Germany). All samples were subjected to paired-end sequencing by Novogene (UK) Company Limited. Specific 16S target hypervariable regions (amplicons) were amplified by PCR. Amplified DNA fragments were end-repaired, and A-tailed. The sequencing adapters were ligated to the ends of the DNA fragments using DNA-binding enzyme, and the DNA fragments were purified using AMpure PB magnetic beads to construct a SMRTbell library. Finally, sequencing primer was annealed to the SMRTbell templates, followed by binding of the sequence polymerase to the annealed template. Quantified libraries were pooled and sequenced on PacBio Sequel II/IIe systems. For sequencing data processing, the PacBio BAM file was split according to barcode and filtered to get clean data, used to generate Amplicon Sequence Variants (ASVs).