Altered Mucosal Microbiome Diversity and Disease Severity in Sjögren Syndrome

Cintia S de Paiva, Dan B Jones, Michael E Stern, Fang Bian, Quianta L Moore, Shani Corbiere, Charles F Streckfus, Diane S Hutchinson, Nadim J Ajami, Joseph F Petrosino, Stephen C Pflugfelder, Cintia S de Paiva, Dan B Jones, Michael E Stern, Fang Bian, Quianta L Moore, Shani Corbiere, Charles F Streckfus, Diane S Hutchinson, Nadim J Ajami, Joseph F Petrosino, Stephen C Pflugfelder

Abstract

There is mounting evidence that the microbiome has potent immunoregulatory functions. We assessed the effects of intestinal dysbiosis in a model of Sjögren syndrome (SS) by subjecting mice to desiccating stress (DS) and antibiotics (ABX). We characterized the conjunctival, tongue and fecal microbiome profiles of patients with SS. Severity of ocular surface and systemic disease was graded. 16S ribosomal RNA gene sequencing characterized the microbiota. ABX + DS mice had a significantly worse dry eye phenotype compared to controls, a decrease in Clostridium and an increase in Enterobacter, Escherichia/Shigella, and Pseudomonas in stool after ABX + DS for 10 days. Goblet cell density was significantly lower in ABX treated groups compared to controls. Stool from SS subjects had greater relative abundances of Pseudobutyrivibrio, Escherichia/Shigella, Blautia, and Streptococcus, while relative abundance of Bacteroides, Parabacteroides, Faecalibacterium, and Prevotella was reduced compared to controls. The severity of SS ocular and systemic disease was inversely correlated with microbial diversity. These findings suggest that SS is marked by a dysbiotic intestinal microbiome driven by low relative abundance of commensal bacteria and high relative abundance of potentially pathogenic genera that is associated with worse ocular mucosal disease in a mouse model of SS and in SS patients.

Figures

Figure 1. Decreased diversity after antibiotic regimen…
Figure 1. Decreased diversity after antibiotic regimen in mice subjected to desiccating stress.
(A) Schematic of experimental design. Mice were left non-stressed (NS) or subjected to desiccating stress (DS) for 5 or 10 days (DS5 and DS10) while drinking regular water. A separate group of mice received oral antibiotics (ABX: Ampicillin, Gentamicin, Metronidazole, Neomycin, Vancomycin) in water 14 days prior (baseline) and later were randomized to remain non-stressed (ABX + NS) or to be subjected to desiccating stress for 5 or days while still on ABX water (ABX + DS5 and ABX + DS10, respectively). Thick black arrow indicates duration of water treatment. (B) Number of observed operational taxonomic units (OTUs) and Shannon Diversity Index scores in non-stressed mice (NS, baseline) prior to exposure to desiccating stress (DS) with antibiotic cocktail (ABX) for 5 or 10 days (DS5 and DS10, respectively). ****P < 0.0001 compared to baseline group (Kruskall-Wallis test with FDR correction). (C) Principal coordinate analysis (PCoA) plot of unweighted UniFrac distances. Each symbol represents an individual sample from baseline and ABX mice subjected to DS for 5 and 10 days. PERMANOVA test, R2 = coefficient of determination. (D) Comparison of significant relative abundance of different genera among groups. Dotted line divides significant genera that decrease (left) or increase (right) in ABX + DS5 and ABX + DS10 compared to baseline. (Mean ± SEM) *P < 0.05; ****P < 0.0001 (Kruskall-Wallis test with FDR correction).
Figure 2. Gut dysbiosis worsens response to…
Figure 2. Gut dysbiosis worsens response to desiccating stress and increases production of T-cell related cytokines in conjunctival epithelium.
(A–C) Goblet cell density (A), CD4+T cell infiltration (B) and corneal barrier function measured by uptake of Oregon-Green Dextran (OGD, C) in mice prior to (baseline, NS) and after exposure to desiccating stress with and without antibiotic cocktail (ABX) for 5 or 10 days (DS5 and DS10, respectively). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 water vs. ABX comparisons; Kruskall-Wallis followed by Sidak’s multi-comparisons test. (D). Relative fold of expression of IL-17, IFN-γ, IL-13, Foxa2, IL-13/IFN-γ ratio and Integrin alpha 2 (Itga2, CD49b) in conjunctiva from mice prior to (non-stressed, NS) and after exposure to desiccating stress with and without antibiotic cocktail (ABX) for 5 or 10 days (DS5 and DS10, respectively). Data are presented as mean ± SEM of a representative experiment containing 3-4 individual samples/group. Experiment was repeated once with similar results. *p < 0.05; **p < 0.01, ***p < 0.001; ****p < 0.0001 water vs. ABX at each time point, calculated by Kruskall-Wallis followed by Sidak’s multi-comparisons test.
Figure 3. Human stool microbiome in SS.
Figure 3. Human stool microbiome in SS.
(A) Comparison of all significant shifts in the abundance of genera between control subjects and SS patients (Mean ± SEM). Dotted line divides significant genera that decrease (top) or increase (bottom) in SS compared to controls. (B) Inverse Pearson’s correlation of combined severity index and number of intestinal OTUs, R = coefficient of correlation (C). Ocular severity graded (0–4) Dry Eye Workshop (DEWS) criteria, systemic severity graded (0–33) using unweighted 12-domain ESSDAI (European League against rheumatism (EULAR) Sjögren syndrome disease activity index) and combined ocular and systemic severity (sum of ocular and systemic severity scores). NS = non-significant. *p < 0.05; **p < 0.01, ***p < 0.001; ****p < 0.0001 Mann-Whitney test. NP: not performed.

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