Oral mucosal breaks trigger anti-citrullinated bacterial and human protein antibody responses in rheumatoid arthritis

Abstract

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15⁺HLADR^hi and CD48^highS100A2^pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens.

FIG. 1.. Oral mucosal breaks trigger systemic inflammatory responses.

(A) Experimental workflow. (B) Inferred relative bacterial abundances from eight HMP body sites (n=336). (C) Body site inferred relative abundances for timepoints from RA patients with and without periodontal disease, median. (D) Inferred relative oral abundances in blood for one RA donor with and one without periodontal disease. (E) Relative abundances of bacteria genera from oral brushings (left) and blood (right). (F) Log₁₀ adjusted p-values versus log-fold changes of human gene expression relative to bacterial abundances of the three oral body-sites. (G) Enriched GO pathways in differentially expressed human genes from (F) (adjusted p-values). (H) Percent monocytes in blood cell counts compared to inferred relative abundances of oral bacteria, Pearson’s correlation. (I-J) RT-qPCRs of mRNA of inflammatory genes in (I) whole blood, (J) granulocytes, monocytes, lymphocytes (n=4–6) stimulated with oral bacteria vs. unstimulated control. (K) Flow cytometry data showing proportion of ISG15⁺ monocytes in CD14⁺ monocytes incubated with PBS, oral bacteria, and anti-FcγR2a or isotype (n=8). (B), (H), (I), two-tailed Kruskal-Wallis test, Dunnett’s-corrected for multiple comparisons. (C), Mann-Whitney U test. (K), within-subjects ANOVA, Tukey-corrected for multiple comparisons. Mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001 or exact value shown.

FIG. 2.. Inflammatory synovial monocyte genes are enriched in RA blood during flares and in response to oral bacteremia.

(A) Volcano plot of -log₁₀ adjusted p-value versus log fold change of blood samples in flare versus baseline for RA patients without periodontal disease (left) and RA patients with periodontal disease (right). Colored dots indicate FDR < 0.05. (B) For differentially expressed genes (DEGs) during flares in RA patients with periodontal disease, a scatter-plot showing the direction and value of those DEGs during flares of RA patients with and without periodontal disease. (C) Gene ontology (GO) pathway enrichment analysis of upregulated PD⁺ specific flare genes and upregulated common flare genes identified in (B). (D) Gene set enrichment analysis (GSEA) results of synovial macrophage clusters for flares in RA patients with periodontal disease (left, red) and flares in RA patients without periodontal disease (left, blue), and oral-derived bacteremias (right). Dashed line indicates FDR of 0.05. (E) PCA biplot of bacteria–human gene expression with co-occurrence probabilities estimated from MMvec. Distances between points quantify the probability of co-occurrence strength between human genes (points). Distances between arrow tips quantify co-occurrence strength between microbes (arrows). Arrow color indicates strength of association of a bacteria with the oral cavity body site.

FIG. 3.. RA antibodies from plasma, synovium, and gingiva bind both citrullinated human and oral bacteria antigens.

(A) Heatmap of normalized mAb reactivity to citrullinated (cit) and native (nat) bacterial lysates and human recombinant proteins as measured by ELISA. Bars below indicate the mutation load in the V-region and predicted N-glycan motifs. (B) SHM frequency in the V-region in ACPA and unreactive mAbs from (A). (C) Quantification of predicted N-linked glycan motifs in ACPA mAbs and unreactive mAbs from (A). (D) Percent inhibition of mAb binding to citrullinated S. parasanguinis and V. parvula bacterial lysates following preincubation with citrullinated or native human proteins. (E) Reactivity of plasma IgG from RA (n=33) and healthy controls (n=40) against citrullinated and native bacterial lysates. Mean ± SD. (F) Reactivity of synovial fluid IgG from RA (n=25) and OA (n=25) against citrullinated and native bacterial lysates. Mean ± SD. (G) Cyclic citrullinated peptide (CCP) reactivity of IgG and IgA eluted from oral bacteria from RA patients (n=13) and healthy controls (n=13). Mean ± SD. For (B), two-tailed student’s t-test was performed. For (C,G), two-tailed Mann-Whitney test was performed. For (D-F), 2-way ANOVA followed by Dunnett’s multiple comparisons test was performed. *P<0.05, **P<0.01, P***<0.001, ****P<0.0001.

FIG. 4.. Commensal bacteria from multiple mucosal sites are citrullinated in vivo.

(A) Single cell bacteria were analyzed by flow cytometry for citrullination and viability determined by SYTOX BC green, propidium iodine, and anti-peptidyl-citrulline antibody staining. (B) Frequency of live citrullinated bacteria sampled from the mouth (n=18), stool (n=18), and vagina (n=11). (C) Relative abundance of the 13 most abundant families in citrullinated (n=10, left) and unbound (n=10, right) oral sorted fractions. (D) Analysis of composition of microbiomes (ANCOM) of families in citrullinated and unbound sorted oral fractions. (E) Percent of live citrullinated S. parasanguinis after culture with neutrophils (n=7) with or without CaCl₂. (F) ISG15 expression by flow cytometry of blood monocytes stimulated with citrullinated S. parasanguinis and ACPA mAbs, citrullinated S. parasanguinis and hIgG1 isotype mAb, or PBS (n=10). To produce citrullinated S. parasanguinis, S. parasanguinis was incubated in neutrophil conditioned media with calcium and DTT. Results represent means ± SD. Statistical analysis for (D) was performed using ANCOM. One-way ANOVA followed by Dunnett’s multiple comparisons test and paired t-test was used for statistical analysis for (B) and (E) respectively. Repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test was performed for (F). *P<0.05, **P<0.01, P***<0.001.

FIG. 5.. Identification of citrullinated bacterial epitopes targeted by ACPA.

(A) Mass spectrometry spectra and retention times of citrullinated and native S. parasanguinis amylase peptide from saliva. (B) Reactivity of RA mAbs to citrullinated and native bacterial peptides determined by ELISA. (C) Binding kinetics of RA35 and GLRA35 to citrullinated DnaK (87–106) and citrullinated fibrinogen (556–575). (D) KD values derived from Bio-Layer Interferometry of germline (GL) and ACPA mAbs to citrullinated bacterial and human peptides. (E) Lineage tree analysis of ACPA clonal family as predicted by IgTree. KD values derived from Bio-Layer Interferometry of corresponding mAbs to citrullinated bacterial and human peptides. (F-G) IgG reactivity of (F, left) plasma from RA patients (n=46) and healthy patients (n=89), (F, right) synovial fluid from RA patients (n=31) and OA patients (n=34) (right), and (G) plasma from individuals with no or mild periodontal disease (HT, n=20), moderate to severe periodontal disease patients (PD, n=60), and RA patients with moderate to severe periodontal disease (RA PD, n=13) against citrullinated and native AbpA (124–144) by ELISA. Mean ± SD. *P<0.05, **P<0.01, P***<0.001, ****P<0.0001. For (F), 2-way ANOVA followed by Dunnett’s multiple comparisons test was performed. For (G), Kruskal-Wallis test followed by Dunnett’s multiple comparisons test was performed.