Dietary Derived Propionate Regulates Pathogenic Fibroblast Function and Ameliorates Experimental Arthritis and Inflammatory Tissue Priming

Abstract

Short-chain fatty acids are gut-bacteria-derived metabolites that execute important regulatory functions on adaptive immune responses, yet their influence on inflammation driven by innate immunity remains understudied. Here, we show that propionate treatment in drinking water or upon local application into the joint reduced experimental arthritis and lowered inflammatory tissue priming mediated by synovial fibroblasts. On a cellular level, incubation of synovial fibroblasts with propionate or a physiological mixture of short-chain fatty acids interfered with production of inflammatory mediators and migration and induced immune-regulatory fibroblast senescence. Our study suggests that propionate mediates its alleviating effect on arthritis by direct abrogation of local arthritogenic fibroblast function.

Keywords: arthritis; cellular senescence; diet; inflammatory tissue priming; propionate; synovial fibroblasts.

Figure 1

Effects of propionic acid (PA) on arthritis and inflammatory tissue priming: (a) Injection schemes for antigen-induced arthritis (AIA) and MSU crystal-induced arthritis. (b) Time course (left) and area under the curve (AUC, right) of relative thickness of C57BL/6 mouse paws (n = 5) injected with methylated bovine serum albumin (mBSA) with or without PA. * p < 0.05, ANOVA with Sidak´s multiple comparisons test. p = 0.069, Student´s t-test. (c) Inflammatory and bone changes in AIA induced by injection of mBSA with or without PA. Representative H&E staining of sections from mouse paws 8 days after mBSA boost. Black arrowheads indicate inflammatory infiltrates, black arrows areas of bone/cartilage destruction. Scale bars, 200 µm. (d) Time course (left) and AUC (right) of arthritis induced by injection of MSU crystals with/without PA. N = 8. ** p < 0.01, Student´s t-test. (e) Injection scheme for the inflammatory tissue priming model of arthritis upon treatment with PA in the drinking water. (f) Time course of paw thickness (left), AUCs (center) and priming indices (right) during iterated zymosan-induced arthritis in BALB/c mice treated with or without PA in the drinking water (n = 6). Priming index represents the ratio between AUC of the second episode of arthritis and the first episode of arthritis in the contralateral paw. * p < 0.01, ** p < 0.01, *** p < 0.001, ANOVA with Sidak´s multiple comparisons test (AUC); Student´s t-test (priming index). (g) Injection scheme of inflammatory tissue priming by transfer of SFs. (h) Course of arthritis (left and center) and priming indices (right) after transfer of primed PA- or vehicle-treated SFs into the naïve paws of recipient mice (n = 8). * p < 0.05, Student´s t-test.

Figure 2

Treatment with propionic acid (PA) or SCFA blocks arthritogenic fibroblast function: (a,b) Fibroblast migration is reduced by treatment with PA. Representative images (a) of a wound healing/migration assay with murine SFs isolated from C57BL/6 mice. Dashed lines show cell-free space at experiment start. Scale bars, 400 µm (b) Quantification of wound healing/migration (time until confluency) in PA-treated and control murine SFs (n = 5). * p < 0.05, Student´s t-test. (c) Quantitative real-time PCR analysis in cultured naïve and primed mouse SFs stimulated with LPS with or without PA. Shown are expression of Tnf, Il1b and Nlrp3. * p < 0.05, ** p < 0.01, ANOVA with Sidak´s multiple comparisons test. (d) qPCR analysis in cultured naïve SFs stimulated with LPS with/without physiological mixture of short-chain fatty acids (SCFAs; acetate, propionate, butyrate). Plotted are individual values and means of Tnf, Il1b and Nlrp3 expression. (e) qPCR analysis of LPS-stimulated and control SFs treated with PA or SCFA mixture. Shown is expression of C3 and Tnfsf11 (RANKL). * p < 0.05, *** p < 0.001, ANOVA with Sidak´s multiple comparisons test. (f) Representative images and quantification of propionate or vehicle-treated murine synovial fibroblast stained for senescence-associated β-galactosidase (blue). Scale bars, 200 µm. All subfigures show individual values, means and S.E.M. Each symbol represents pooled cells from 2 mice.