Background/Purpose: Although collagen-induced arthritis (CIA) in mice is a useful tool for studying inflammatory arthritis, it is a highly variable model with inconsistent disease severity and penetrance across studies. Previous studies have shown that CIA severity and penetrance vary by mouse strain, sex, and source of type II collagen. Furthermore, we and others have shown that CIA development is microbiome-dependent. Despite these findings, the extent to which distinct microbiome compositions affect CIA outcomes has not been well studied. As such, microbiome variation between mouse vendors, housing facilities, and even over time has the potential to negatively impact the reproducibility of CIA studies. We therefore performed a head-to-head comparison of CIA in DBA/1 mice obtained from three different vendors and compared CIA outcomes to microbiome composition.

Methods: 7-week-old male DBA/1 mice were purchased from The Jackson Laboratory (Jax), Envigo, or Taconic. Mice from the three vendors simultaneously were injected intradermally at the base of the tail with 200µg bovine CII (Elastin) emulsified in 50µl complete Freund’s adjuvant (CFA, Sigma) on days 0 and 21. Disease severity was assessed every other day starting at day 21. Serum and fecal pellets were collected weekly throughout the experiment. Serum cytokines and autoantibodies were measured by ELISA; 16S rRNA sequencing was performed by the CU Anschutz Mucosal Immunobiology Core.

Results: CIA severity was significantly higher in Envigo and Taconic mice compared to Jax mice (Figure 1). While Taconic and Envigo mice had nearly identical mean CIA severity scores, there was a higher degree of score variability within the Taconic mice. Despite significant differences in CIA severity between the Jax, Envigo, and Taconic groups, there were no significant differences in total CII-IgG levels at day 28, nor CII-IgG isotypes (IgG1, IgG2a, IgG2b). We also observed significant differences in microbiome alpha and beta diversity between the three groups. Finally, we identified 7 genera that significantly correlated with CIA score (Figure 2). Candidatus arthromitus (also known as segmented filamentous bacteria) had the strongest positive correlation with CIA severity, while butyrate-producing Butyricoccaceae had a significant negative correlation with CIA severity.

Conclusion: Our findings reveal that the vendor source of mice significantly impacts CIA severity, independent of systemic autoantibody and cytokine levels. Several bacterial taxa (e.g. Candidatus arthromitus, Butyrococcaceae) may play a key role in disease severity differences. This highlights a critical but often overlooked source of variability that may contribute to irreproducibility across preclinical arthritis studies. Moving forward, intentional selection of mouse source—and consideration of microbiota as a key experimental variable—will be essential for enhancing the reproducibility of the CIA model.

Figure 1. CIA was induced in 7-week old male DBA/1 mice from Envigo, Jax, and Taconic. Arthritis severity was scored every other day starting at day 21. Data shown are represented as mean (symbol) ± SEM (error bars). Two-way ANOVA with Tukey correction for multiple comparisons were performed.

Figure 2. Fecal microbiome on CIA day 35 was assessed by 16S rRNA sequencing. The top 25 genera that correlate (spearman rank) with CIA severity were plotted. Bars represent correlation coefficient, with darker shading representing increased absolute value of the correlation efficient. * denotes FDR < 0.1.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/same-model-different-results-vendor-and-microbiome-influence-reproducibility-in-collagen-induced-arthritis/