3.1. Changes of clinical indicators of RA patients in different treatment groups and HCs
The study included 50 patients with RA and 25 healthy people. RA patients were divided into three groups, including 10 untreated RA patients, 20 RA patients treated with csDMARDs, and 20 RA patients treated with csDMARDs + bDMARDs. The clinical information and clinical characteristics of the subjects are shown in Table 1. In this study, Disease duration was elevated in the csDMARD + bDMARDs treatment group (P < 0.05), whereas DAS28-ESR (P < 0.0001) and ESR (P < 0.001) were significantly lower in the csDMARD + bDMARDs treatment group compared with the untreated group. DAS28-ESR (P < 0.0001) and ESR (P < 0.001) were significantly lower in the csDMARD + bDMARDs treatment group compared with the csDMARD + bDMARDs treatment group, whereas CRP was slightly lower in the csDMARD + bDMARDs treatment group (P < 0.05). However, there was no significant variability in other clinical indicators of RA patients, such as Anti-CCP antibody, RF, IL-6, C3, IgG, IgA, and IgM.
Table 1
Clinical characteristics of RA patients and health controls.
Subject Characteristics | Untreated (n = 10) | csDMARDs (n = 20) | csDMARD + bDMARDs (n = 20) | HCs (n = 25) | P -value |
Male/Female (N) | 4/6 | 6/14 | 5/15 | 15/10 | |
Age # | 45.1 ± 6.4 | 58.3 ± 2.8 | 57.7 ± 3.7 | 53.3 ± 3.1 | ns |
Disease duration (years) # | 4.8 ± 2.3 | 7.5 ± 1.8 | 13.1 ± 3.1 | | * |
DAS28-ESR# | 6.12 ± 0.4 | 6.3 ± 0.2 | 3.2 ± 0.4 | | **** |
Anti-CCP antibody (RU/ml) # | 65.6 ± 29.0 | 78.8 ± 18.2 | 36.1 ± 16.0 | | ns |
RF (IU/Ml) # | 170.8 ± 100.8 | 948.6 ± 663.9 | 267.3 ± 124.0 | | ns |
IL-6 (pg/ml) # | 30.6 ± 16.3 | 49.4 ± 12.3 | 16.0 ± 6.8 | | ns |
ESR (mm/h) # | 52.3 ± 6.4 | 41.2 ± 3.9 | 16.9 ± 3.6 | | *** |
CRP (mg/L) # | 20.1 ± 5.3 | 32.6 ± 8.3 | 9.1 ± 3.4 | | * |
C3 (g/L) # | 1.0 ± 0.2 | 0.9 ± 0.1 | 0.8 ± 0.2 | | ns |
IgG (IU/mL) # | 14.2 ± 5.2 | 13.3 ± 3.0 | 13.1 ± 1.1 | | ns |
IgA (IU/mL) # | 4.1 ± 0.6 | 4.1 ± 1.3 | 2.8 ± 0.4 | | ns |
IgM (IU/mL) # | 1.9 ± 0.4 | 1.4 ± 0.3 | 1.5 ± 0.2 | | ns |
#: mean ± standard error of the mean; DAS28-ESR: disease activity score on 28 joints with erythrocyte sedimentation rate; Anti-CCP antibody: Anti-cyclic citrullinated peptide antibody; RF: Rheumatoid factor; IL-6: interleukin-6; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; C3: complement3; IgG: immunoglobulin G; IgA: immunoglobulin A; IgM: immunoglobulin M; csDMARDs: conventional synthetic disease-modifying antirheumatic drugs; bDMARDs: biological disease-modifying antirheumatic drugs; ns: not significant (*P < 0.05, ***P < 0.001, ****P < 0.0001).
3.2. Changes Of Intestinal Flora Of Hcs And Untreated Ra Patients
To determine the diversity of intestinal flora and the species composition in HCs and RA patients who didn't receive treatment, we analyzed the stools of 25 healthy individuals and 10 untreated RA patients by 16SrRNA gene sequencing. We performed a cluster analysis using a Venn diagram, and the results showed that there were 9100 ASV/OUT in the HCs group and 1743 ASV/OUT in the Untreated group, for a total of 741 ASV/OUT in both groups (Fig. 1a). Chao1 (P < 0.01), Observed species (P < 0.01), Shannon (P < 0.05), and Faiths P'D (P < 0.01) indices were lower in the Untreated group, while Good's coverage (P < 0.05) index was increased compared to the HCs group (Fig. 1b). These results suggest that the diversity and richness of the gut microbiota decreased, while the coverage increased in treatment-naïve RA patients.
Based on Jaccard distance, Bray-curtis distance, and unweighted UniFrac distance algorithms, we evaluated the β diversity of intestinal flora in the HCs group and RA patients with not receiving treatment (Fig. 1c). Overall, the β diversity of the intestinal flora also changed significantly in the HCs and untreated groups. We also found that the species composition of the gut flora changed significantly (Fig. 2a,2b). Notably, at the phylum level, Relative abundance of Firmicutes was elevated in the Untreated group compared to HCs (P < 0.05; Fig. 2c), while Relative abundance of Acidobacteria was decreased in the untreated group (P < 0.05; Fig. 2c). At the genus level, the relative abundance of Prevotella (P < 0.01; Fig. 2d), Lachnospiraceae_Clostridium (P < 0.01; Fig. 2d), and Megamonas (P < 0.01; Fig. 2d) were significantly lower in RA patients who did not receive treatment.
3.3. Changes of intestinal flora in different treatment groups of RA patients
50 RA patients were divided into the untreated group, the csDMARDs-treated group, and the csDMARDs + bDMARDs-treated group. The variability of intestinal flora in RA patients treated with different DMARDs was analyzed. The Venn diagram showed that 1288 ASV/OUT in the untreated group, 2510 ASV/OUT in the csDMARDs treatment group, and 2151 ASV/OUT in the csDMARDs + bDMARDs treatment group, for a total of 741 ASV/OUT in the three groups (Fig. 3a). Similarly, we analyzed the diversity and richness of intestinal flora in various treatment groups of RA patients. The results showed that there was no statistical difference in the alpha and beta diversity of the intestinal flora in the three groups (Fig. 3b, 3c; P > 0.05). At the phylum level, the intestinal flora in the different treatment groups of RA patients was mainly dominated by Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria (Fig. 3d). However, the relative abundance of these florae was not statistically significant in the three groups (Fig. 3d; P > 0.05). At the genus level, the relative abundance of Ruminococcaceae_Ruminococcus was decreased in the csDMARDs and csDMARDs + bDMARDs treatment group compared to the untreated group, while the relative abundance of Faecalibacterium was decreased in the csDMARDs treatment group (Fig. 3e,3f; P < 0.05). However, Ruminococcaceae_Ruminococcus and Faecalibacterium were not statistically significant in the csDMARDs and csDMARDs + bDMARDs treatment groups (P > 0.05).
3.4. Changes of Treg and memory Tfh cells in the different treatment groups of RA patients
To clarify the changes of Treg and memory Tfh cells before and after treatment with csDMARDs and csDMARDs + bDMARDs treatment in RA patients, we examined the number and function of these cells using flow cytometry. The results showed that FOXP3+ Treg cells were significantly higher in the csDMARDs-treated and csDMARDs + bDMARDs-treated groups compared with the untreated group (Fig. 4a; P < 0.001). Compared with the untreated group, CD4+CD45RO+CCR7+CXCR5+ central memory Tfh cells (Fig. 4b; P < 0.001) and CD4+CD45RO+CCR7−CXCR5+ effector memory Tfh cells (Fig. 4c; P < 0.001) were significantly lower in the csDMARDs + bDMARDs treatment group, whereas CD4+CD45RO+CCR7+CXCR5+central memory Tfh cells were somewhat lower in the csDMARDs treatment group (Fig. 4b; P < 0.05).
3.5. Changes Of Memory B-cell Subpopulations In Each Treatment Group Of Ra Patients
To explore the changes of memory B-cell subsets before and after treatment with csDMARDs and csDMARDs + bDMARDs in RA patients, we examined the number and function of memory B-cell subsets in each treatment group of RA patients using flow cytometry. Compared with the untreated group, CD19+CD27+IgD+ pre-switched memory B (PSMB) cells were elevated in the csDMARDs and csDMARDs + bDMARDs treatment group (Fig. 5a; P < 0.05), whereas CD19+CD27+IgD− switched memory B (SMB)cells were significantly lower (Fig. 5b; P < 0.01). CD19+CD27−IgD− double negative memory B cells were not statistically different in the three treatment groups of RA patients (Fig. 5c; P > 0.05).
3.6. Serum Levels Of Cytokines In Varied Treatment Groups Of Patients With Ra Patients
The serum levels of cytokines IL-15, IL-21, IL-33, and IL-35 were measured by ELISA. We found that serum levels of IL-15 were significantly lower in the csDMARDs and csDMARDs + bDMARDs treatment group compared with the untreated group (Fig. 6a; P < 0.0001). Furthermore, serum levels of IL-21 were decreased in the csDMARDs + bDMARDs treatment group compared with the untreated group (Fig. 6b; P < 0.05), whereas serum levels of IL-35 were increased in the csDMARDs + bDMARDs treatment group (Fig. 6d; P < 0.05). Interestingly, serum levels of IL-21 (Fig. 6b; P < 0.05) and IL-33 (Fig. 6c; P < 0.05) were reduced in the csDMARDs treatment group compared with the csDMARDs + bDMARDs treatment group.
3.7. Correlation of intestinal flora with clinical indicators, immune cells, and cytokines in various treatment groups of RA patients
For the purpose of evaluating the correlation of intestinal flora with clinical parameters, immune cells, and cytokines in RA patients before and after treatment with DMARDs, we did Spearman correlation analysis of genus-level differential intestinal flora with clinical indicators, immune cells, and cytokines. The results showed that Ruminococcaceae_Ruminococcus was positively correlated with DAS28-ESR and ESR (Fig. 7a; P < 0.05). Interestingly, we found that Faecalibacterium was negatively correlated with Treg cells, in contrast to positive correlation with CD19+CD27+IgD−switched memory B cells (Fig. 7b; P < 0.05). Ruminococcaceae_Ruminococcus was negatively correlated with Treg and CD19+CD27+IgD+ pre-switched memory B cells, while positively correlated with effector memory Tfh and CD19+CD27+IgD−switched memory B cells (Fig. 7b; P < 0.05). Correlation analysis of intestinal flora and cytokines revealed a positive correlation between Lactobacillus and IL-35 (Fig. 7c; P < 0.05). Ruminococcaceae_Ruminococcus was positively correlated with IL-17A, IFN-γ, TNF-α (Fig. 7d; P < 0.05), and Lactobacillus was negatively correlated with IFN-γ and TNF-α (Fig. 7d; P < 0.05). There is also intestinal flora associated with lymphocytes, cytokines, and clinical parameters.