Gut Microbiota From Sjögren syndrome Patients Causes Decreased T Regulatory Cells in the Lymphoid Organs and Desiccation-Induced Corneal Barrier Disruption in Mice

Abstract

Sjögren syndrome (SS) is an autoimmune inflammatory disorder characterized by secretory dysfunction in the eye and mouth; in the eye, this results in tear film instability, reduced tear production, and corneal barrier disruption. A growing number of studies show that homeostasis of the ocular surface is impacted by the intestinal microbiome, and several 16S sequencing studies have demonstrated dysbiosis of the intestinal microbiota in SS patients. In this study, we utilized metagenomic sequencing to perform a deeper analysis of the intestinal microbiome using stools collected from sex- and age-matched healthy (n = 20), dry eye (n = 4) and SS (n = 7) subjects. The observed Operational Taxonomic Units (OTUs) and Shannon alpha diversity were significantly decreased in SS compared to healthy controls, and there was a significant inverse correlation between observed OTUs and ocular severity score. We also identified specific bacterial strains that are differentially modulated in SS vs. healthy subjects. To investigate if the differential composition of intestinal microbiome would have an impact on the immune and eye phenotype, we performed functional studies using germ-free mice colonized with human intestinal microbiota from SS patients and healthy controls. Flow cytometry analysis demonstrated reduced frequency of CD4⁺ FOXP3⁺ cells in ocular draining cervical lymph nodes (CLN) in mice colonized with SS patient intestinal microbiota 4 weeks post-colonization. We also found that offspring of SS-humanized mice also have fewer CD4⁺FOXP3⁺ cells in the CLN as well as spleen, demonstrating vertical transmission. SS-humanized mice subjected to desiccating stress exhibited greater corneal barrier disruption as compared to healthy control humanized mice under the same conditions. Taken together, these data support the hypothesis that the intestinal microbiota can modulate ocular surface health, possibly by influencing development of CD4⁺ FOXP3⁺ regulatory T cells (Tregs) in the ocular draining lymph nodes.

Keywords: Sjögren; Tregs; cornea; dry eye; microbiome.

Figure 1

SS patient microbiome alpha diversity is significantly decreased and correlates with disease severity. (A) Number of observed operational taxonomic units (OTUs), Shannon diversity measure, and Chao richness in patient fecal samples. Statistical significance was assessed using non-parametric Mann-Whitney U tests. *P ≤ 0.05. (B) Spearman's correlation of combined severity index and number of intestinal OTUs; R, coefficient of correlation.

Figure 2

Metagenomic sequencing of SS patient stool shows decreased diversity and identifies differential bacterial species that correlate with disease severity. (A) Principal coordinate analysis of the Bray-Curtis distances between healthy, dry eye, and Sjögren syndrome (SS) subjects. (B) Top 25 species ranked based on their relative importance in a Random Forest model trained to separate healthy controls from SS patients. (C) Box plots describe the relative abundance in healthy controls, dry eye patients, and SS patients for the top 5 ranked species selected by the Random Forest model. Box plots show the median, the ranges between the first and the third quantile, and the ranges between the lowest and the highest value. Statistical significance was assessed using non-parametric Mann-Whitney U tests. *P ≤ 0.05, **P ≤ 0.01. (D) Scatter plots for the correlation between severity scores and log-transformed relative abundances for the top 5 species selected by the Random Forest model. Red lines show the predicted severity scores based on simple linear regression models. The goodness of fit is estimated by the coefficient of determination r² shown in the title texts. For ease of visualization in scatter and box plots, a pseudocount of 10⁻⁷ was added to all relative abundances.

Figure 3

Mice colonized with SS fecal material exhibit fewer CD4⁺ FOXP3⁺ cells in cervical lymph nodes (CLN) than mice humanized with normal healthy fecal material. (A,B) 4 weeks after humanization of germ-free mice with patient stools, CLNs were collected for flow cytometry analysis and analyzed for the percentage of CD45⁺ CD4⁺ cells expressing FOXP3. (A) Shows the combined data for animals humanized with healthy vs. SS patient stools (n = 3–5 donors per group). (B) Shows the same data separated by patient donor. (C) Representative flow cytometry data showing CD45⁺ CD4⁺ FOXP3⁺ cell populations in 4 separate experiments using fecal slurries from 5 different SS patients and 4 healthy controls. Exp., Experiment; FOG, fecal oral gavage; SS, Sjögren syndrome.

Figure 4

Mice colonized with SS fecal material had increased corneal barrier disruption after desiccating stress compared with mice colonized with fecal material from healthy patients. (A) Representative images of corneal permeability for animals humanized with either healthy or SS patient stool. Corneal barrier integrity was assessed by measuring the epithelial permeability to Oregon-green-dextran dye (OGD). (B,C) Cumulative OGD intensity measurement data from humanized mice subjected to 5 days of DS. Each data point represents the intensity value for one animal (averaged over the two eyes). (B) Shows the combined data for animals humanized with healthy vs. SS patient stools (n = 3 donors per group). (C) Shows the same data separated by patient donor. Statistical significance was assessed using non-parametric Mann-Whitney U-tests. *P ≤ 0.05.

Figure 5

Offspring of SS-humanized mice also have fewer CD4⁺ FOXP3⁺ cells in the CLN as well as spleen, demonstrating vertical transmission. Cumulative flow cytometry data showing percentage of CD4⁺ FOXP3⁺ cells from CLN, spleen, MLN, and lamina propria of 8-week-old offspring of mice gavaged with patient stools. CLN, cervical draining lymph nodes; MLN, mesenchymal lymph nodes; LP, lamina propria. Statistical significance was assessed using non-parametric Mann-Whitney U-tests. *P ≤ 0.05, **P ≤ 0.01, ns, not significant.