A single bacterium restores the microbiome dysbiosis to protect bones from destruction in a rat model of rheumatoid arthritis

Abstract

Background: Early treatment is key for optimizing the therapeutic success of drugs, and the current initiating treatment that blocks the progression of bone destruction during the pre-arthritic stages remains unsatisfactory. The microbial disorder in rheumatoid arthritis (RA) patients is significantly reversed with effective treatment. Modulating aberrant gut microbiomes into a healthy state is a potential therapeutic approach for preventing bone damage.

Results: By using metagenomic shotgun sequencing and a metagenome-wide association study, we assessed the effect of Lactobacillus casei (L. casei) on the induction of arthritis as well as on the associated gut microbiota and immune disorders in adjuvant-induced arthritis (AIA) rats. Treatment of AIA rats with L. casei inhibited joint swelling, lowered arthritis scores, and prevented bone destruction. Along with the relief of arthritis symptoms, dysbiosis in the microbiome of arthritic rats was significantly reduced after L. casei intervention. The relative abundance of AIA-decreased Lactobacillus strains, including Lactobacillus hominis, Lactobacillus reuteri, and Lactobacillus vaginalis, were restored to normal and Lactobacillus acidophilus was upregulated by the administration of L. casei to the AIA rats. Moreover, L. casei downregulated the expression of pro-inflammatory cytokines, which are closely linked to the effect of the L. casei treatment-associated microbes. Functionally, the maintenance of the redox balance of oxidative stress was involved in the improvement in the L. casei-treated AIA rats.

Conclusion: A single bacterium, L. casei (ATCC334), was able to significantly suppress the induction of AIA and protect bones from destruction in AIA rats by restoring the microbiome dysbiosis in the gut, indicating that using probiotics may be a promising strategy for treating RA, especially in the early stage of the disease.

Fig. 1

L. casei alleviates the adjuvant-induced arthritis of rats. Effects of L. casei on arthritis score (a) and increased hind paw volume (b) are shown (n = 7 for each group). Data in a, b are shown as mean ± s.e.m. Differences between groups are analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 VS model). The photographs, X-ray, and micro-CT images of ankles are shown in c. Representative images of pathological sections of knees in rats in different groups are shown in d. The pathological improvements are assessed by pathological score e. Radiological score and micro-CT score are evaluated using the micro-CT image and micro-CT analyzer, respectively (f, g). Data in e, f, and g are shown as mean ± s.e.m. Differences among groups are analyzed by one-way ANOVA. (*P < 0.05, **P < 0.01, ***P < 0.001 VS model). The integral assessments of the bone destruction levels are shown in (h). Data are shown as mean and classified into several levels. 0–0.2: normal; 0.2–0.4: light (Lig); 0.4–0.6: moderate (Mod); 0.6–0.8: severe (Sev); 0.8 and above: very severe. Normal, normal control; model, disease control; MTX, methotrexate

Fig. 2

Dynamic changes of the gut microbiota composition in the L. casei/MTX-treated rats over time. Distances from healthy plane (HP) and model plane (DP) for each sample of the L. casei-/MTX-treated rats on the five time points are shown. The colors of lines correspond to different samples and the thickness reflects the severity of arthritis. Difference between HP and DP is analyzed by paired t tests

Fig. 3

Log₁₀ fold change of the relative abundance of arthritis-correlated species at TP5 in comparison with samples of TP1. Boxes represent the median and interquartile ranges (IQRs) between the first and third quartiles; whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. Circles represent samples. Significant fold change is marked with an asterisk

Fig. 4

L. casei inhibits pro-inflammatory cytokines expression via resurrection of L. acidophilus. a The expressions of cytokines (IL-17, IL-1β, TNFα, IL-6, IFN-γ, IL-2) in serum are assessed using ELISA. Data are shown as mean ± s.e.m and min, max. Differences between groups are analyzed by one-way ANOVA. (*P < 0.05, **P < 0.01, ***P < 0.001 VS model). b Associations of the abundance of L. acidophilus with plasmatic cytokines. cc, Spearman’s correlation coefficient after adjustment for weight, group, and arthritis score

Fig. 5

L. casei maintains the redox balance of oxidative stress. Mean log₂ fold changes in the abundance of immune-related KEGG modules at TP5 in comparison with samples of TP1 from MTX, L. casei, and model group. The KEGG orthology group modules and groups are ordered by unsupervised hierarchical clustering. Cyan, reduced modules; red, increased modules. Modules missing from one or more groups are not plotted