Individuals with Sickle Cell Disease Have Decreased Intestinal Microbial Diversity Compared to Healthy Related and Unrelated Black Individuals without Sickle Cell Disease

Alterations in the intestinal microbiota (dysbiosis) are a known driver of chronic inflammation. Dysbiosis leads to a “leaky” intestinal barrier triggering bacterial translocation into the blood, recurrent systemic microbial antigen exposure, and subsequent chronic inflammation. Dysbiosis plays a role in many chronic inflammatory and immune-mediated disorders, however, this process has been minimally explored in sickle cell disease (SCD). Individuals with SCD have disease-related risk factors for dysbiosis such as daily prophylactic antibiotics started in infancy, recurrent opioid therapy, and intestinal ischemia from chronic vaso-occlusion. To date, microbiome studies in SCD have been limited to small sample sizes, do not include related and unrelated Black controls, and have not assessed the impact of covariates on dysbiosis. Thus, we sought to determine intestinal microbiota differences between individuals with SCD and related and unrelated Black controls, while controlling for known covariates present in SCD and controls that can affect dysbiosis.

We conducted a cross-sectional multi-site study. Individuals ≥2 months with SCD in baseline health (no acute care visits for ≥2 weeks) and healthy Black individuals (related and unrelated to those in SCD cohort) were enrolled. Ineligible individuals were those with SCD on chronic transfusions and controls with a known chronic inflammatory disorder. Stool samples were collected, microbial DNA extracted, and 16S rDNA sequencing completed. Alpha diversity (measure of microbial diversity within samples) was compared using a Kruskal Wallis test among 3 groups (SCD, related control, unrelated control) via the Shannon Diversity Index that incorporates evenness and richness of identified organisms; lower index indicates decreased alpha diversity. Beta diversity (measure of microbial diversity between groups) was assessed using Unweighted UniFrac method and the Adonis multivariable permutation test to determine if the covariates of interest explained the observed variability across the 3 groups. Covariates were site of collection, age (years), sex, mode of birth (vaginal, c-section), infant feeding (formula, breastfed), relatedness, area deprivation index (national rank), and participant group (SCD, related control, unrelated control). Significance level was p<0.05.

We enrolled 110 individuals with SCD in baseline health, 53 related and 77 unrelated healthy Black individuals. Mean (SD) age (years) and sex (% female) of the groups were: SCD 16.5 (13.4), 46.4%; related controls 28.7 (12.2), 75.5%; unrelated controls 27.3 (16.9), 71.4%. SCD genotype distribution was HbSS 65.5%, HbSC 27.3%, HbSβ⁰thal 1.8%, HbSβ⁺thal 5.5%. The Shannon Diversity Index (alpha diversity) was significantly different across the 3 groups (0.000000813). Pairwise analyses for Shannon Diversity Index showed SCD was significantly lower than related (p=0.000146) and unrelated (p=1.12E-06) controls; there were no differences between related and unrelated controls (p= 0.820967). Beta diversity was also significantly different between groups as assessed by Unweighted UniFrac. Specifically, participant group was a significant variable that explained the variation between groups (R²=1.1%, p=0.027) after adjusting for site (R²=0.68%, p=0.013), age (R²=1.2%, p=0.001), infant feeding (R²=1.9%, p=0.004), and relatedness (R²=73%, p=0.001). Sex, mode of birth, and area deprivation index were not significant.

In this large cohort study, we found individuals with SCD have lower intestinal microbial diversity. The lack of differences between related and unrelated controls supports SCD-related factors are driving observed intestinal microbial changes. Despite relatedness explaining a significant amount of beta diversity variation, having SCD continued to be significant, providing further evidence that SCD-related factors contribute to intestinal microbial changes. The beta diversity findings support observed microbiota differences in SCD are driven by microbial taxa, not abundance, differences. Further work is needed to assess which taxa are differentially abundant between SCD and controls, whether microbiota changes are related to SCD complications, what SCD-related factors (e.g., prophylactic antibiotics) contribute to these changes, and whether the intestinal microbiome could be a novel target for disease modifying therapy.