A recent study has uncovered a potential mechanism through which Helicobacter pylori infections may exacerbate rheumatoid arthritis. 

In the study, published in Annals of the Rheumatic Diseases, researchers compared disease activity and anticitrullinated protein antibody (ACPA) levels between H pylori–positive (n = 39) and H pylori–negative (n = 42) patients with rheumatoid arthritis.

The study included 81 patients with rheumatoid arhtirits. Disease activity was assessed using DAS-28. Serum ACPA and anti–Cit-K1 antibody levels were measured by ELISA.

In vitro experiments utilized the human gastric epithelial cell line GES-1 and the synovial cell line MH7A. Cells were infected with H pylori strain NCTC 11637 at various multiplicities of infection (MOI = 0, 10, 20, 50, 100, 200).

Protein citrullination was detected by western blot using an antimodified citrulline antibody. PAD4 expression and activity were assessed by RT-qPCR, western blot, and an antibody-based assay for peptidylarginine deiminase activity (ABAP).

HIF-1α binding to the PADI4 promoter was confirmed using chromatin immunoprecipitation and luciferase reporter assays. Mass spectrometry was employed to identify PAD4 interacting proteins.

Statistical analyses were performed using Student's t-test, Mann-Whitney U test, and one-way or two-way ANOVA as appropriate. P-values < .05 were considered statistically significant.

The researchers found that H pylori upregulated the expression of the PAD4 protein, leading to increased citrullination of proteins and production of ACPAs.

Among the key findings were:

• H pylori–positive patients had significantly higher Disease Activity Score 28 (DAS-28) values compared with H pylori–negative patients (exact values not provided, P < .05).
• Serum ACPA levels were markedly elevated in H pylori–positive patients (P < .01).
• A positive correlation was observed between serum ACPA levels and DAS-28 scores (r = 0.3971, P < .001).

In vitro experiments demonstrated that ACPA IgG (2.5 μg/mL) derived from H pylori–positive patients promoted proliferation of MH7A synovial cells and induced secretion of inflammatory cytokines. After 48 hours of stimulation:

• Interleukin (IL)-6 levels increased from approximately 50 pg/mL to 150 pg/mL (P < .001).
• IL-8 levels increased from approximately 100 pg/mL to 250 pg/mL (P < .001).

The researchers identified a novel mechanism whereby H pylori infections led to upregulation of PAD4 expression through a reactive oxygen species (ROS)/hypoxia-inducible factor 1α (HIF-1α) signaling pathway.

Specifically:

• H pylori infection (MOI = 100) increased intracellular ROS levels in GES-1 gastric epithelial cells by approximately threefold at 6 hours postinfection (P < .001).
• Elevated ROS stabilized HIF-1α protein, with expression increasing in an MOI-dependent manner.
• HIF-1α directly bound to the PADI4 gene promoter to induce transcription, confirmed by ChIP assay (approximately eightfold enrichment, P < .001).
• PADI4 mRNA levels increased by over 10-fold at 48 and 72 hours after CoCl2 treatment (P < .001).
• PAD4 protein levels increased three- to fourfold at 24, 48, and 72 hours after CoCl2 treatment.

Using mass spectrometry and immunoprecipitation, the study identified keratin 1 (K1) as a novel target of PAD4-mediated citrullination. Levels of citrullinated K1 (Cit-K1) were significantly higher in sera from H pylori-positive patients with rheumatoid arthritis compared with H pylori-negative patients (P < .05).

Furthermore, the researchers detected anti–Cit-K1 antibodies in both serum and synovial fluid of patients with rheumatoid arthritis, with higher levels observed in H pylori–positive patients

Additional findings included:

• Serum anti–Cit-K1 antibody levels (OD450nm): H pylori–positive = 0.6–0.8, H pylori–negative = 0.4–0.6 (P < .05)
• Synovial fluid anti–Cit-K1 antibody levels (OD450nm): H pylori–positive (n = 3) = approximately 0.8, H pylori–negative (n = 4) = approximately 0.5 (P < .05).

Serum anti–Cit-K1 antibody levels positively correlated with:

• Cit-K1 expression (r = 0.3755, P < .05)
• DAS-28 scores (r = 0.4149, P < .01)
• ACPA levels (r = 0.5755, P < .001).

This comprehensive study provided quantitative data on the potential role of H pylori infections in exacerbating rheumatoid arthritis pathogenesis, offering insights into possible new therapeutic approaches for managing rheumatoid arthritis in infected patients.

The authors declared having no competing interests.