Inflammatory arthritis disrupts gut resolution mechanisms, promoting barrier breakdown by Porphyromonas gingivalis
Magdalena B. Flak, Romain A. Colas, Estefanía Muñoz-Atienza, Michael A. Curtis, Jesmond Dalli, Costantino Pitzalis
Magdalena B. Flak, Romain A. Colas, Estefanía Muñoz-Atienza, Michael A. Curtis, Jesmond Dalli, Costantino Pitzalis
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Research Article Immunology Inflammation

Inflammatory arthritis disrupts gut resolution mechanisms, promoting barrier breakdown by Porphyromonas gingivalis

Abstract

Rheumatoid arthritis is linked with altered host immune responses and severe joint destruction. Recent evidence suggests that loss of gut homeostasis and barrier breach by pathobionts, including Porphyromonas gingivalis, may influence disease severity. The mechanism(s) leading to altered gut homeostasis and barrier breakdown in inflammatory arthritis are poorly understood. In the present study, we found a significant reduction in intestinal concentrations of several proresolving mediators during inflammatory arthritis, including downregulation of the gut-protective mediator resolvin D5n-3 DPA (RvD5n-3 DPA). This was linked with increased metabolism of RvD5n-3 DPA to its inactive 17-oxo metabolite. We also found downregulation of IL-10 expression in the gut of arthritic mice that was coupled with a reduction in IL-10 and IL-10 receptor (IL-10R) in lamina propria macrophages. These changes were linked with a decrease in the number of mucus-producing goblet cells and tight junction molecule expression in the intestinal epithelium of arthritic mice when compared with naive mice. P. gingivalis inoculation further downregulated intestinal RvD5n-3 DPA and Il-10 levels and the expression of gut tight junction proteins. RvD5n-3 DPA, but not its metabolite 17-oxo-RvD5n-3 DPA, increased the expression of both IL-10 and IL-10R in macrophages via the upregulation of the aryl hydrocarbon receptor agonist l-kynurenine. Administration of RvD5n-3 DPA to arthritic P. gingivalis–inoculated mice increased intestinal Il-10 expression, restored gut barrier function, and reduced joint inflammation. Together, these findings uncover mechanisms in the pathogenesis of rheumatoid arthritis, where disruption of the gut RvD5n-3 DPA–IL-10 axis weakens the gut barrier, which becomes permissive to the pathogenic actions of the pathobiont P. gingivalis.

Authors

Magdalena B. Flak, Romain A. Colas, Estefanía Muñoz-Atienza, Michael A. Curtis, Jesmond Dalli, Costantino Pitzalis

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Figure 5

Upregulation of 15-PGDH in the lamina propria during inflammatory arthritis promotes the inactivation of RvD5n-3 DPA.

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Upregulation of 15-PGDH in the lamina propria during inflammatory arthri...
Inflammatory arthritis was initiated as detailed in Figure 1, and small intestines were collected on day 8 after initiation. (A) mRNA expression of 15-PGDH. (B) Immunofluorescence analysis of 15-PGDH expression in the small intestine. Original magnification, ×60. (C) Bone marrow macrophages were incubated with immune complexes (IC) or control IgG (16 hours, 37°C), and the expression of 15-PGDH was assessed using flow cytometry. Results for A and C are mean ± SEM. For A, n = 7 mice per group; for C, n = 4 mice per group from 2 independent experiments. Results for B are representative of n = 4 mice per group from 2 independent experiments. (D–F) RvD5n-3 DPA was incubated with hr15-PGDH (0.5 U, room temperature, 45 minutes). Products were extracted and lipid mediators were identified using liquid chromatography tandem mass spectrometry and reversed-phase UV-HPLC. (D) MRM chromatograms of 361 > 199 (RvD5n-3 DPA) and 359 > 215 (17-oxo-RvD5n-3 DPA). (E) Online UV chromatogram for RvD5n-3 DPA and 17-oxo-RvD5n-3 DPA. (F) MS/MS spectrum employed in the identification of 17-oxo-RvD5n-3 DPA. Results are representative of n = 4 incubations. (G) Arthritis was initiated as in Figure 1, small intestines harvested on day 8 after initiation, products were extracted, and the concentration of 17-oxo-RvD5n-3 DPA was determined using LC-MS/MS. (H) Bone marrow–derived macrophages were incubated with vehicle or immune complexes (37°C, 16 hours) and 17-oxo-RvD5n-3 DPA concentrations were determined using LC-MS/MS. Results are mean ± SEM n = 4–5 mice per group from 2 independent experiments. (I–K) Bone marrow–derived macrophages were incubated with vehicle or U0126 (20 μM, 37°C, 1 hour), then with either vehicle or immune complexes (37°C, 16 hours), and the expression of (I) 15-PGDH and (J) ELK1 and was investigated using flow cytometry. (K) Concentrations of 17-oxo-RvD5n-3 DPA were determined using lipid mediator profiling. (L) Bone marrow–derived macrophages were incubated with RvD5n-3 DPA (10 nM), 17-oxo-RvD5n-3 DPA (10 nM), or vehicle (37°C, 2 hours), and concentrations of l-kynurenine determined using LC-MS/MS. (M) Cells were incubated as in K for 16 hours (37°C), and expression of IL-10R and IL-10 was assessed using flow cytometry. Results for I–M are mean ± SEM. n = 4 mice per group from 2 independent experiments. *P < 0.05 using Kruskall-Wallis test followed by Dunn’s post hoc test.