Gut microbial dysbiosis in patients with Cushing's disease in long-term remission. Relationship with cardiometabolic risk

Abstract

Background: Patients with Cushing's disease (CD) in remission maintain an increased cardiovascular risk. Impaired characteristics of gut microbiome (dysbiosis) have been associated with several cardiometabolic risk factors.

Methods: Twenty-eight female non-diabetic patients with CD in remission with a mean ± SD) age of 51 ± 9 years, mean ( ± SD) BMI, 26 ± 4, median (IQR) duration of remission, 11(4) years and 24 gender-, age, BMI-matched controls were included. The V4 region of the bacterial 16S rDNA was PCR amplified and sequenced to analyse microbial alpha diversity (Chao 1 index, observed number of species, Shannon index) and beta diversity analysis through the Principal Coordinates Analysis (PCoA) of weighted and unweighted UniFrac distances. Inter-group difference in microbiome composition was analysed using MaAsLin2.

Results: The Chao 1 index was lower in CD as compared with controls (Kruskal-Wallis test, q = 0.002), indicating lower microbial richness in the former. Beta diversity analysis showed that faecal samples from CS patients clustered together and separated from the controls (Adonis test, p<0.05). Collinsella, a genus form of the Actinobacteria phylum was present in CD patients only, whereas Sutterella, a genus from Proteobacteria phylum, was scarcely detectable/undetectable in CD patients as well as Lachnospira, a genus of the Lachnospiraceae family of the Firmicutes phylum. In CS, the Chao 1 index was associated with fibrinogen levels and inversely correlated with both triglyceride concentrations and the HOMA-IR index (p<0.05).

Conclusions: Patients with CS in remission have gut microbial dysbiosis which may be one of the mechanisms whereby cardiometabolic dysfunctions persist after "cure".

Keywords: Cushing's disease; Cushing's syndrome; cardiovascular risk; gut microbiota.

Figure 1

Patient recruitment flow-chart. CD, Cushing’s disease; GM, gut microbiome; GC, glucocorticoids; GI, gastrointestinal The gray bar includes 27 excluded patients; *Of 15 patients, 9 were on proton-pomp inhibitors; 8 on metformin and 6 on antidepressannts/anxiolytics; some patients were taking more than one of different classes; **Of 10 patients taking propbiotics, three were also on treatment with some medications potentially interfering with GM composition and, therefore, included in the corresponding group of 15 patients; # patients with type 2 diabetes were 9, of whom 6 on metformin and, therefore, included in the group of 15 patients on medications potentially interfering with GM composition.

Figure 2

Alpha-diversity as assessed by Chao1 and Shannon indexes. The Chao1 index gives weight to the low-abundant bacterial species, while the Shannon index gives weight to high-abundant species. Kruskal-Wallis test was applied to compare the diversity of healthy controls (HC) and Cushing disease patients, **q < 0.005.

Figure 3

Beta diversity analysis, using the Principal Coordinates Analysis (PCoA) of weighted and unweighted UniFrac distances, showed that fecal samples from patients with Cushing’s disease (red circles) clustered together and separated from HC samples (blue circles). The plot is based on the distance analysis between samples. The closer the samples are to each other, the more similar their microbiome composition in terms of richness (for unweighted UniFrac metrics) and in terms of evenness, based on the most abundant taxa (for weighted UniFrac metrics).

Figure 4

Bacterial genera significantly different between healthy controls (HC) and Cushing’s disease (CD). The relative abundance of bacterial genera was compared between HC and CD using the MaAsLin2 (Microbiome Multivariable Association with Linear Models) statistical tool, with medications and disease status as fixed effects and age and BMI as random effects. Corrected p-values were displayed: *q < 0.05; **q < 0.005; ***q < 0.005.