Genomic repertoires linked with pathogenic potency of arthritogenic Prevotella copri isolated from the gut of patients with rheumatoid arthritis

Abstract

Objectives: Prevotella copri is considered to be a contributing factor in rheumatoid arthritis (RA). However, in some non-Westernised countries, healthy individuals also harbour an abundance of P. copri in the intestine. This study investigated the pathogenicity of RA patient-derived P. copri (P. copri _RA) compared with healthy control-derived P. copri (P. copri _HC).

Methods: We obtained 13 P. copri strains from the faeces of patients with RA and healthy controls. Following whole genome sequencing, the sequences of P. copri _RA and P. copri _HC were compared. To analyse the arthritis-inducing ability of P. copri, we examined two arthritis models (1) a collagen-induced arthritis model harbouring P. copri under specific-pathogen-free conditions and (2) an SKG mouse arthritis model under P. copri-monocolonised conditions. Finally, to evaluate the ability of P. copri to activate innate immune cells, we performed in vitro stimulation of bone marrow-derived dendritic cells (BMDCs) by P. copri _RA and P. copri _HC.

Results: Comparative genomic analysis revealed no apparent differences in the core gene contents between P. copri _RA and P. copri _HC, but pangenome analysis revealed the high genome plasticity of P. copri. We identified a P. copri _RA-specific genomic region as a conjugative transposon. In both arthritis models, P. copri _RA-induced more severe arthritis than P. copri _HC. In vitro BMDC stimulation experiments revealed the upregulation of IL-17 and Th17-related cytokines (IL-6, IL-23) by P. copri _RA.

Conclusion: Our findings reveal the genetic diversity of P. copri, and the genomic signatures associated with strong arthritis-inducing ability of P. copri _RA. Our study contributes towards elucidation of the complex pathogenesis of RA.

Keywords: arthritis, experimental; cytokines; rheumatoid arthritis.

Figure 1

Structural differences between the genomes of RA patient-derived Prevotella copri (P. copri _RA) and P. copri _HC. (A) A whole-genome phylogenetic tree analysis revealed that P. copri strains, which were isolated in this study, were categorised into three distinct clades. Tree was inferred with FastME 2.1.6.1 from Genome Blast Distance Phylogeny (GBDP) distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 31.2%. The tree was rooted at the midpoint. (B) Comparison of the genomic regions between P. copri _RA and P. copri _HC strains. The presence of each genomic region is represented by colour at the chromosomal position, using strain N001-13 as reference. P. copri _RA and P. copri _HC strains are coloured red and blue, respectively. P. copri _RA and P. copri _HC core regions of density are shown by genomic density of a filled-line plot. The P. copri _RA-specific genomic region is indicated by a grey background. (C) Comparison of the abundance of the P. copri _RA-specific genomic region in faecal DNA between patients with RA and HC from cohorts 1, 2 and 3. Seventeen patients with RA (cohort 1: n=2, cohort 2: n=10, cohort 3: n=5) and 14 HC (cohort 1: n=1, cohort 2: n=6, cohort 3: n=7) of which the relative abundance of P. copri was higher than 1% were selected for analysis. Box and whisker plots show median, 25th and 75th percentiles, and minimum and maximum values. Two-tailed Mann-Whitney U test was performed for statistical analysis. **p<0.01. HC, healthy controls; RA, rheumatoid arthritis.

Figure 2

Conjugative transposon (CTn) identified in the RA patient-derived Prevotella copri (P. copri _RA)-specific region. The gene organisations of CTns identified in the P. copri _RA-specific regions are shown. The coding sequences (CDSs) are depicted by arrows (black arrow, integrase genes; yellow arrow, mobilisation protein; green arrow, DNA primase family protein; blue arrow, genes related to conjugation; grey arrow, other functionally annotated CDSs; and white arrow, genes for hypothetical proteins). The grey shading indicates homologous CDSs with >70% amino acid sequence identity. RA, rheumatoid arthritis.

Figure 3

Severe collagen-induced arthritis in RA patient-derived Prevotella copri (P. copri _RA)-colonised DBA/1 J mice (A, B) Arthritis scores (A) and incidence (B) of P. copri-inoculated DBA/1 J mice in collagen-induced arthritis (CIA) models under specific-pathogen-free (SPF) conditions. Data are pooled from two independent experiments and total numbers of mice are as follows: P. copri _RA (RA-N001 strain)-inoculated mice (RA), n=16; P. copri _HC (HC-H012)-inoculated mice (HC), n=12. Each symbol and vertical line represents the mean±SD. (C) Photographic images of the ankle joint of P. copri-inoculated mice at day 63 after primary immunisation with type II collagen. (D, E) Histopathology (D) and histological score (E) of the lower ankle joint at day 63 after primary immunisation (RA; n=6, HC; n=4). One representative histological image is shown. H&E staining. (F) The concentration of type II collagen-specific IgG in sera at day 63 after primary immunisation was determined by ELISA (RA; n=6 and HC; n=4). Bar graphs show data as the mean±SD. (G) Representative flow cytometry plots of popliteal lymph node (PLN) cells of RA or HC in CIA models under SPF conditions. (H) Percentage and number of IL17A⁺ CD4⁺ cells and IFNγ⁺ CD4⁺ cells in PLN cells. These cells were analysed by flow cytometry after stimulation with phorbol myristate acetate (PMA) and ionomycin at day 77 after primary immunisation (RA; n=9 and HC; n=7). Bar graphs show data as the mean±SD. One-way ANOVA followed by a Tukey’s multiple comparisons test (A), χ²-test (B), and two-tailed Mann-Whitney U tests (E, F, H) were performed for statistical analyses. *p<0.05, **p<0.01, ***p<0.001, N.S., not significant. (E–H) Data were reproduced in three independent experiments with similar results. ANOVA, analysis of variance; HC, healthy controls; RA, rheumatoid arthritis.

Figure 4

Severe arthritis in RA patient-derived Prevotella copri (P. copri _RA)-monocolonised SKG mice. (A, B) Arthritis score (A) and incidence (B) of P. copri-monocolonised SKG mice after the injection of zymosan (RA-N001-monocolonised (RA); n=7, HC-H012-monocolonised (HC); n=7). Each symbol and vertical line represents the mean±SD. (C) Photographic images of the ankle joint of P. copri-monocolonised mice at day 56 after the injection of zymosan. (D, E) Histopathology (D) and histological score (E) of the lower ankle joint at day 56 after the injection of zymosan (RA; n=7, HC; n=7). One representative histological image is shown. H&E staining. (F) Representative flow cytometry plots of PLN cells of RA-N001 or HC-H012-monocolonised mice in the SKG arthritis model. (G) Percentage and number of IL17A⁺ CD4⁺ cells and IFNγ⁺ CD4⁺ cells in PLN cells. These cells were analysed by flow cytometry after stimulation with phorbol myristate acetate (PMA) and ionomycin at day 56 after the injection of zymosan (RA; n=3 and HC; n=3). Bar graphs show data as the mean±SD. Two-tailed Student’s t-test (A, E, G) and χ²-test (B) were performed for statistical analysis. *p<0.05. N.S.=not significant. All data were reproduced in another independent experiment with similar results. HC, healthy controls; RA, rheumatoid arthritis.

Figure 5

Activation of T cells by RA patient-derived Prevotella copri (P. copri _RA)-treated dendritic cells. (A) Expression levels of major histocompatibility complex (MHC) class II, CD80 and CD86 molecules in bone marrow-derived dendritic cells (BMDCs) stimulated with heat-killed P. copri _RA (RA-N001: RA) or P. copri _HC (HC-H012: HC) or PBS for 72 hours. Expression of MHC class II, CD80 and CD86 in the BMDCs was determined as the mean fluorescence intensity (MFI) by flow cytometric analysis. (B) Q-PCR analysis of Il6 and Il23a mRNA expression in BMDCs stimulated with heat-killed P. copri _RA (RA-N001: RA) or P. copri _HC (HC-H012: HC). (C, D) Concentrations of IL-23 (C) and IL-6 (D) were measured by ELISA in the supernatants of BMDCs stimulated with the indicated heat-killed P. copri _RA (RA-N001: RA) or P. copri _HC (HC-H012: HC). (E) The concentration of IL-17A was measured by ELISA in the supernatants of naïve CD4⁺ cells co-cultured with BMDCs stimulated by P. copri _RA (RA-N001: RA) or P. copri _HC (HC-H012: HC). Cells were stimulated with or without bovine type II collagen (ⅡC). Bar graphs show data as the mean±SD. of triplicate measurements. One-way ANOVA followed by a Tukey’s multiple comparison tests (A, E) and two-tailed Mann-Whitney U tests (B–D) were performed for statistical analyses. *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. N.D.=not detected. All data are representative of at least three independent experiments. ANOVA, analysis of variance; HC, healthy controls; RA, rheumatoid arthritis.