The mechanisms underlying rheumatoid arthritis (RA) have remained elusive due to the multitude of factors that influence its development. Previous studies have revealed several loci (mapped to MHC molecules) that confer susceptibility to RA1, but these genetic markers cannot fully explain all incidences of the disease. To expand our understanding of RA pathogenesis recent research has turned to exploring the role of environmental factors, namely gut microbiota composition. The previous Journal Club post highlights the important role that infant gut microbiome composition has in educating the immune system and the consequences that varying microbiota compositions have on that education. This week’s post expands upon this idea with an article that sought to link certain gut microbiota compositions to collagen-induced arthritis (CIA) susceptibility in DBA1 mice.
In their paper “Role of the Gut Microbiome in Modulating Arthritis Progression,” Liu et al. hypothesized that the gut microbiome composition influences an individual’s susceptibility towards collagen-induced arthritis. To test this, mice were immunized, via subcutaneous injections, with bovine type II collagen emulsified with Complete Freund’s Adjuvant (CFA) on day 0 and day 21. Fecal samples were analyzed for bacteria flora, via 16S rRNA sequencing, both before (Day 28) and after (Day 35) the onset of arthritis. Each mouse was classified as either susceptible or resistant to collagen-induced arthritis based on the development of arthritis. Liu et al. then sought to find a correlation between the gut microbiota composition and the mice’s susceptibility to collagen-induced arthritis.
When characterizing the gut microbiota of CIA-susceptible and CIA-resistant mice, Liu et al. found significant differences in their compositions. After immunization and before the onset of arthritis, CIA-susceptible mice housed the largest diversity of phyla, including Deinococcus-Thermus and Verrucomicrobia (not detected in CIA-resistant mice). Significantly, the family Lactobacillaceae, specifically genus Lactobacillus, was notably more abundant in CIA-susceptible mice than CIA-resistant mice. Genus’s that were significantly less abundant in CIA-susceptible mice include Alistipes, Desulfovibrio, and Enterorhabdus. Interestingly, genus Enterorhabdus increased in abundance as arthritis progressed in CIA-susceptible mice. Overall, CIA-susceptible mice exhibited decreased microbial richness and increased diversity when compared to CIA-resistant mice.
It is apparent that there was a distinctive difference in the gut microbiota makeup of CIA-susceptible and CIA-resistant mice, but the more pertinent question is if this difference conferred susceptibility to collagen-induced arthritis. To better elucidate this relationship and to try to move from correlation to causation, Liu et al. conventionalized germ-free (GF) mice, via oral gavage, with the gut microbiota of either CIA-susceptible or CIA-resistant mice (termed sGF-mice or rGF-mice respectively). Fecal samples of these germ-free mice were then characterized to confirm that their gut microbiota composition matched that of the mice they were conventionalized with. Just as in the CIA-susceptible mice, the sGF-mice mice exhibited a relative abundance of genus Lactobacillus when compared to rGF-mice.
After the conventionalization period, the germ-free mice were immunized with bovine type II collagen emulsified with CFA and the development of arthritis was monitored. sGF-mice exhibited increased arthritis incidence and higher arthritis scores than their rGF-mice counterparts. Levels of splenic lymphocytes (Th17 and Treg cells) and serum concentrations of inflammatory cytokines (namely IL-17) were also measured. These cells and their effector molecules were chosen based off of their previously established role in the development of several inflammatory and autoimmune diseases such as RA, systemic lupus erythematosus, asthma and inflammatory bowel disease2. sGF-mice exhibited elevated levels of Th17 cells, decreased levels of Treg cells and increased serum concentrations of IL-17 when compared to rGF-mice. TNF-α and IL-10 also increased in sGF-mice, but not significantly. By conventionalizing germ-free mice with the microbiota of CIA-susceptible and CIA-resistant mice (after arthritis onset) and measuring the difference in arthritis development, Liu et al. were able to show that commensal bacteria in the gut can influence collagen-induced arthritis susceptibility in mice. Furthermore, by sequencing before and after arthritis onset, Liu et al. were able to show specific compositional changes that confer susceptibility to arthritis.
Overall, these results provide valuable insight into how the gut microbiome composition changes throughout the course of the collagen-induced arthritis model. An interesting follow up to this research would be to characterize the gut microbiome composition before administering collagen/CFA injections. Receiving subcutaneous injections of CFA is both a painful and psychologically stressful situation for mice. Previous research has shown that physical and psychological stress can result in increased intestinal permeability that leads to excessive translocation of bacteria and other antigens 3,4. Furthermore, preliminary research performed here at Chondrex indicates that CFA suppresses immune functions, such as T-cell responses to mitogen for six weeks. Given both of these findings, it is likely that there was a drastic change in the gut microbiome composition of the mice between the first immunization (day 0) and the “before arthritis onset” bacterial sequencing (day 28). While this compositional change is not indicative of arthritis susceptibility, sequencing before immunization would illuminate the effect that stress and immune stimulants, like CFA, have on commensal bacteria populations and how those changes can affect arthritis development.
To expand upon this research and further our understanding of how bacteria/bacterial toxins are associated with rheumatoid arthritis development, Chondrex offers the Arthrogen-CIA Monoclonal Antibody Cocktail for the collagen antibody-induced arthritis (CAIA) model. It has been demonstrated previously5,6 that LPS administered by IP injection or oral feeding works synergistically with the Arthrogen-CIA Monoclonal Antibody Cocktail to trigger severe arthritis within 24-48 hours. This protocol can be adapted for a wide variety of bacteria and bacterial toxins, making the CAIA model ideal for studying the association between bacteria/bacterial toxins and arthritis development.
This study by Liu et al. adds to the growing body of research focused on elucidating the relationship between commensal bacteria and immune system function. Although our understanding of this phenomenon remains limited, it is apparent that our microbiota plays a larger role in autoimmune disease pathogenesis, and our overall health, than previously believed. However, accounting for the effects of commensal bacteria and their toxins in animal models of autoimmune diseases could prove to be a difficult and costly endeavor. But, it is a necessary step in order to provide a more comprehensive understanding of autoimmune disease pathogenesis. This new focus on commensal bacteria could open the door to alternative therapeutics for autoimmune diseases, ones that are focused on normalizing gut microbial communities and limiting exposure to certain bacterial toxins.
Liu, X., Zeng, B., Zhang, J., Li, W., Mou, F., Wang, H., Zou, Q., Zhong, B., Wu, L., Wei, H., Fang, Y. Role of the gut microbiome in modulating arthritis progression in mice. Scientific Reports. 2016; 6: 30594.
Keywords: rheumatoid arthritis (RA), collagen-induced arthritis (CIA), DBA1 mice, collagen, microbiota, commensal bacteria, collagen antibody-induced arthritis, Complete Freund's Adjuvant (CFA)