Association of Increased Serum Lipopolysaccharide, But Not Microbial Dysbiosis, With Obesity-Related Osteoarthritis

Abstract

Objective: To test the hypothesis that an altered gut microbiota (dysbiosis) plays a role in obesity-associated osteoarthritis (OA).

Methods: Stool and blood samples were collected from 92 participants with a body mass index (BMI) ≥30 kg/m² , recruited from the Johnston County Osteoarthritis Project. OA patients (n = 50) had hand and knee OA (Kellgren/Lawrence [K/L] grade ≥2 or arthroplasty). Controls (n = 42) had no hand OA and a K/L grade of 0-1 for the knees. Compositional analysis of stool samples was carried out by 16S ribosomal RNA amplicon sequencing. Alpha- and beta-diversity and differences in taxa relative abundances were determined. Blood samples were used for multiplex cytokine analysis and measures of lipopolysaccharide (LPS) and LPS binding protein. Germ-free mice were gavaged with patient- or control-pooled fecal samples and fed a 40% fat, high-sucrose diet for 40 weeks. Knee OA was evaluated histologically.

Results: On average, OA patients were slightly older than the controls, consisted of more women, and had a higher mean BMI, higher mean Western Ontario and McMaster Universities Osteoarthritis Index pain score, and higher mean K/L grade. There were no significant differences in α- or β-diversity or genus level composition between patients and controls. Patients had higher plasma levels of osteopontin (P = 0.01) and serum LPS (P < 0.0001) compared to controls. Mice transplanted with patient or control microbiota exhibited a significant difference in α-diversity (P = 0.02) and β-diversity, but no differences in OA severity were observed.

Conclusion: The lack of differences in the gut microbiota, but increased serum LPS levels, suggest the possibility that increased intestinal permeability allowing for greater absorption of LPS, rather than a dysbiotic microbiota, may contribute to the development of OA associated with obesity.

Figure 1.

Microbial similarity biplot (Principal Coordinate Analysis (PCoA) axes) for study participants with knee and hand OA (red) and without OA (blue). Numbers in parentheses indicate the percent variation explained by the corresponding axis. Each point represents a single sample. The distance between 2 points shows how compositionally different the samples are. Ellipsoids illustrate the 95% CI for the mean location of each group.

Figure 2.

Osteopontin and LPS levels in blood samples from OA cases and controls. Osteopontin was measured in plasma samples by a cytokine array and LPS was measured in serum using an endotoxin assay as detailed in the methods. The units for osteopontin are relative fluorescence units and for LPS are endotoxin units/ml. Data were analyzed using two-sided Student’s t tests.

Figure 3.

Effects of a high fat and high sucrose diet after transfer of human OA case and control fecal samples to germ free mice. A, Body weight of mice starting at time of fecal transplant (−2 weeks) and then bi-weekly while on the high fat and high sucrose diet for 40 weeks. B, %body fat mesured by MRI after 40 weeks on diet. C, Linear mixed-effects model regression of Shannon index from fecal samples collected at the start of the diet (time 0 which is 2 weeks after fecal transplant) and after 6, 22, and 40 weeks on diet. Lines represent linear mixed-effects regression (and 95% confidence interval) of Shannon diversity over time. Each dot represents a mouse and those with the same color indicates they were caged together. D, PCoA Emperor Plot based on Bray-Curtis beta-diversity metric. Points represent samples from the two groups: OA cases (circle) and OA controls (diamond). Time points are colored coded as indicated on the graph. Stars indicate duplicate samples of the pooled human feces analyzed prior to transplant to mice. Numbers in parentheses indicate the percent variation explained by the corresponding axis. The distance between 2 points shows how compositionally different the samples are.

Figure 4.

Results of histologic analysis of knee joints from mice transplanted with case or control fecal pools and placed on a high fat and high sucrose diet for 40 weeks. A, representative images demonstrating histologic findings of OA including cartilage degradation with loss of matrix staining (arrows) and osteophyte formation (arrowhead). B-D, Results of histologic measures of OA including ACS scores (B), Saf-O scores (C) and osteophyte scores (D) presented as means and standard deviation. None of the measures were significantly different between cases and controls (Mann Whitney test). ACS, Articular cartilage structure, Saf-O, safranin-O.