L. casei significantly alleviates experimental arthritis

SD rats were orally gavaged with or without the L. casei strain ATCC334 and subsequently challenged with complete Freund’s adjuvant (CFA) on the same day (day 0) [13]. The animals were orally gavaged with 2 × 108 CFU every day from day 0 to day 30. As the culture medium for L. casei contains lactic acid and other metabolites, we washed off the medium, re-suspended the bacteria in CMC-Na, and then fed the live bacteria to the SD rats within half an hour. The activity of L. casei in CMC-Na was similar to that in MRS broth in such a short time (Additional file 1: Figure S1, Additional file 2: Figure S2). MTX, a widely used DMARD [18], was used as a comparative control. After a 30-day intervention, the rats treated with L. casei or MTX showed less aggravated symptoms, as assessed by the arthritis scores and hind paw volumes (Fig. 1a, b), and rats treated with a combined therapy of L. casei and MTX revealed an even better improvement than those with MTX or L. casei monotherapy (Additional file 3: Figure S3). Moreover, L. casei exerted its anti-arthritic effect without migrating to the places beside the gut as L. casei is facultative anaerobic bacteria and no bacteremia was detected in the L. casei-treated animals.

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 Full size image

Bone and cartilage destruction, which often induce partial or permanent disability in poly-joints, are the major characteristics of pathological alterations in RA patients. Here, we used micro-computed tomography (micro-CT) to evaluate the effect of L. casei on bone remodeling. The bone erosion was diminished in the ankles of the L. casei-treated rats compared to the vehicle-treated animals (Fig. 1c). Consistent with the micro-CT results, histological sections of the knees showed similar improvements after the intervention (Fig. 1d). In general, the pathological scores of the knees and the radiological scores of the ankles were significantly improved in the L. casei-treated animals compared to the vehicle-treated animals (Fig. 1e, g). The micro-CT score, calculated using the trabecular bone mineral density (BMD), bone volume rate (BV/TV), trabecular number (Tb.N), porosity percent (Po × total), and tissue mineral density (TMD), suggested severe osteoporosis in the vehicle-treated animals (Additional file 4: Figure S4). Compared with the vehicle-treated rats, the rats treated with L. casei exhibited reduced bone erosion with a higher micro-CT score (Fig. 1f). From the three bone destruction evaluation scores, we created a classification scale that equally integrated the scores to more precisely assess the degree of damage. Using this classification scale, the L. casei-treated animals showed a significant change in the arthritis level from severe to moderate (Fig. 1h). Moreover, the metabolites produced by L. casei were also used separately in the AIA rats, and results were shown with no significant improvement (Additional file 3: Figure S3). These data indicate that administration of L. casei was able to alleviate experimental arthritis and protect bones from erosion.

L. casei prevents gut dysbiosis caused by the induction of arthritis

We next examined how the gut microbiota changed during the process of arthritis induction and how the administration of L. casei affected these changes. It is important to note that a well-defined RA-associated microbiota signature is lacking, so we collected fecal samples from all four groups at five time points (TPs) and determined the dynamic alterations in the microbiome during the development of arthritis by using shotgun metagenomic sequencing. The five TPs included day 0, before AIA induction (TP1), one pre-arthritis time point on day 7 (TP2), and three TPs, day 14 (TP3), day 21 (TP4), and day 30 (TP5), during arthritis progression in the AIA rats (Additional file 5: Figure S5).

To determine the dynamics of the changes in the gut microbiome composition in the L. casei ATCC334-treated animals, we used a space-based calculation derived from the PCoA of the Bray-Curtis distances of the healthy and vehicle-treated subjects at the species level, which were set as the “healthy plane” (HP) and “disease plane” (DP), respectively. Distance from each sample to the HP and DP revealed dynamic changes in the gut microbiome, and the thickness of the line reflected the severity of arthritis. Samples from the rats treated with L. casei exhibited significant closer distances to the HP than the DP at TP2, while for the samples from the MTX-treated rats, there showed an apparent change at TP2 and a significant closer distance to DP than HP at TP3 (Fig. 2), suggesting that L. casei might remedy volatile gut dysbiosis at the early stage. At TP5, along with the relief of arthritis observed in L. casei group, the composition of the gut microbiomes was significantly closer to HP than DP (Fig. 2), suggesting that dysbiosis in arthritis could be reversed by L. casei, which finally contributed to the amelioration of AIA. Interestingly, the arthritic scores were positively correlated with the distance to HP (Spearman’s correlation coefficient = 0.48, P = 0.015) and negatively correlated with the distance to DP (Spearman’s correlation coefficient = − 0.46, P = 0.019) after adjustment for weight and group, indicating that the alterations in microbiome are closely correlated to the severity of arthritis.

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 Full size image

L. casei remedies the perturbed microbiome in AIA rats

To identify the microbial species or strains along with progression and treatment of arthritis, we next conducted a non-negative elastic-net regularized linear regression of the metagenomic sequencing results at the five TPs for each fecal sample against its related arthritis score. In total, 25 microbes were selected, among which Lachnospiraceae bacterium, Proteus mirabilis, and Corynebacterium urealyticum have been found to be related to RA [7, 19]. The relative abundances of the identified 25 microbes were processed by normalization and then presented as log 10 fold changes at each TPs in comparison to TP 1. In the log 10 fold change of TP2 to TP1, eight microbes were significantly altered in the model group, among which six microbes were changed in the MTX treated group, and only four were altered in L. casei treated rats (Additional file 6: Figure S6), consistent to the result that L. casei effectively inhibited RA progression at the very early stage, while at TP3, the alterations of microbes in L. casei group were much closer to those in model group (Additional file 7: Figure S7).

In the log 10 fold change of TP5 to TP1, the relative abundances of Candidatus Arthromitus sp. SFB-rat Yit and Klebsiella pneumoniae were increased, while those of L. hominis, L. reuteri, and L. vaginalis were decreased after the induction of arthritis, indicating that these microbes may play important roles in the induction of experimental arthritis (Fig. 3). We further investigated the alterations of arthritis-correlated species in the L. casei-treated AIA rats. Unlike rats in the model group, a cluster of microbes including Acinetobacter unclassified, Corynebacterium casei, and L. acidophilus was upregulated (Fig. 3), and Corynebacterium urealyticum, Desulfovibrio desulfuricans, and Erysipelotrichaceae bacterium 21-3 were downregulated. Of note, these alterations occurred in the absence of persistent colonization by L. casei ATCC334, as we did not detect an enrichment of L. casei in the fecal samples isolated from the L. casei-treated rats (Additional file 8: Figure S8). However, the relative abundances of other Lactobacillus species were upregulated (such as L. acidophilus) or restored to the normal level ( such as the downregulated microbes in model group: L. hominis, L. reuteri, and L. vaginalis) (Fig. 3), suggesting that the function of L. casei in suppressing arthritis is closely associated with its function in modulating the microbiome, especially other Lactobacillus strains.

Fig. 3 Log 10 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 Full size image

To determine whether the alterations in the microbiota were specific to L. casei, we further compared the differences in microbiome composition between the MTX- and L. casei-treated rats. The abundances of Erysipelotrichaceae bacterium 21-3, Corynebacterium urealyticum, L. hominis, and L. reuteri were partly regulated by intervention with either of these two treatments (Fig. 3), which indicates that these microbes might be useful as biomarkers of therapeutic responses in RA patients. More interestingly, the abundances of Desulfovibrio desulfuricans, L. acidophilus, and L. vaginalis were effectively modulated by the L. casei treatment, but not by the MTX treatment. Thus, L. casei ATCC334 is capable of ameliorating arthritis by normalizing the levels of certain affected microbes in AIA rats.

Inhibition of expression of pro-inflammatory cytokines correlates with the abundance of bacteria affected by administration of L. casei.

Cytokines play critical roles in the pathogenesis of RA, while inhibiting expression or function of cytokines increases the probability of remission of RA and protects bones from destruction [20]. Therefore, we determined the serum levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukins (IL)-1β, IL-17, IL-2, and IL-6 and determined the correlations between the levels of pro-inflammatory cytokines and alterations in the gut microbiota after L. casei treatment in AIA rats. The results showed a significant reduction in the levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β, IL-17, and IL-6 in the L. casei-treated AIA rats compared to the vehicle-treated AIA rats (Fig. 4a). In addition, the levels of IL-17 and IL-1β, which are produced by activated adaptive and innate immune cells [21, 22], respectively, were significantly reduced in the animals treated with L. casei. Collectively, these data indicate that the alleviation of arthritis in the L. casei-treated rats was related to the suppression of pro-inflammatory cytokine expression.

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 Full size image

To better understand the interaction between the abundances of the microbes associated with amelioration of the AIA of rats and the expression levels of cytokines in the L. casei group, we computed covariances to analyze whether the relative abundance of L. casei or MTX treatment-correlated species (Erysipelotrichaceae bacterium 21-3, Corynebacterium urealyticum, Desulfovibrio desulfuricans, and Lactobacillus strains including L. hominis, L. acidophilus, L. reuteri, and L. vaginalis) were correlated with the expression of pro-inflammatory cytokines. Results showed that these bacteria were all significantly correlated to one or more inflammatory cytokines (Fig. 4b, Additional file 9: Figure S9, Additional file 10: Figure S10, Additional file 11: Figure S11, Additional file 12: Figure S12, Additional file 13: Figure S13, Additional file 14: Figure S14). Interestingly, most of the rats with enriched L. acidophilus in the L. casei-treated group showed decreased expression of pro-inflammatory cytokines and significant correlations were detected between the relative abundance of L. acidophilus and the expression level of IL-6, IL-17, and IL-1β (P < 0.05) (Fig. 4b). These results suggest that the effect of L. casei on AIA in rats is associated with the suppressive effect of treatment-correlated microbes on pro-inflammatory cytokine expression.

Maintenance of the redox balance of oxidative stress is involved in the amelioration of RA in the AIA rats induced by treatment with L. casei

To explore the potential mechanisms behind the beneficial effects of administration of L. casei, we conducted a comprehensive analysis of the functional modules in the gut microbiome of the L. casei- and MTX-treated rats using the KEGG database. Consistent with the notion that alterations in the microbiota lead to changes in host metabolism and the redox balance, the functional modules for the pentose phosphate pathway, including the oxidative phase, were found to be enhanced in the AIA rats, which was consistent with the results of previous reports on RA patients [23]. After treatment with L. casei, the abundance of genes involved in the pentose phosphate pathway was reduced, and the abundance of genes involved in the nicotinamide adenine dinucleotide (NADH) quinone oxidoreductase, assimilatory sulfate reduction, NADH ubiquinone oxidoreductase, beta-oxidation, and fatty acid biosynthesis modules were increased, suggesting that L. casei exerts its anti-arthritic effect through modulating immunometabolism and redox capacity (Fig. 5).

Fig. 5 L. casei maintains the redox balance of oxidative stress. Mean log 2 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 Full size image

Moreover, L. acidophilus has been found to be able to improve the antioxidant status of collagen-induced arthritic rats [24]. These data imply that the modulation of the gut microbiome by L. casei, which results in the enrichment of L. acidophilus, is able to alleviate arthritis by maintaining the redox balance of oxidative stress.