Intestinal microbiome analysis revealed dysbiosis in sickle cell disease
Authors: Seah H. Lim, Alison Morris, Kelvin Li, Adam C. Fitch, Loren Fast, Laura Goldberg, Matthew Quesenberry, Philippa Sprinz, Barbara Methé
Journal: American Journal of Hematology (2017)

Vaso-occlusive crisis (VOC) that affects individuals with sickle cell disease (SCD) is caused by the interplay between sickled erythrocytes (SEs), activated neutrophils, inflammatory cytokines, monocytes, and endothelium. Under physiologic stresses, such as dehydration, infections, and hypoxia, hemoglobin S polymerizes and causes erythrocytes to sickle. However, the clinical observations of the dissociation between the number of circulating SEs and symptoms of VOC in patients with SCD argue against SEs being solely responsible for inducing VOC. A mouse model of SCD showed that SEs more commonly adhered to activated neutrophils than endothelium during VOC,1 supporting the notion that activated neutrophils are pivotal in the pathogenesis of VOC. At baseline, SCD patients have increased levels of activated neutrophils that could adhere to endothelium.2 The immobilized neutrophils act as niduses for SEs to attach to and cause VOC. The kinetics of these activated/aged neutrophils has been found, in gene-knockout mice, to be regulated by the intestinal microbiota through interaction with Toll-like receptor 2/4 and myeloid differentiation factor 88 (Myd88).3 Depletion of the microbiota with broad-spectrum antibiotics reduced the number of aged neutrophils and protected against Lipopolysaccharides (LPS)-induced fatal VOC. If intestinal microbes regulate aged neutrophils, abnormalities in either the integrity of the intestinal barriers or a chronic disequilibrium of the intestinal microbiota, or both are likely to occur in SCD patients. We previously hypothesized that, due to recurrent segmental or global subclinical intestinal ischemia that occurs due to VOC in the splanchnic vasculature, both the intestinal barrier and the ability of the mucosa to support a normal intestinal microbiota in SCD patients are compromised.4 The compromised intestinal barrier renders SCD patients more susceptible to recurrent translocation by bacterial inocula not high enough to cause overt infections, but sufficient to activate neutrophils. To investigate the baseline intestinal microbiome, we carried out an analysis of 16S rRNA gene sequences from stool samples from 35 subjects with SCD [20 were collected by The Biocollective (CO) for this study]. Possible variation due to race was reduced by using 15 individuals with sickle trait (AS) as controls. This study was approved by the Institution Review Board and registered with ClinicalTrials.gov [NCT03007849]. All stool samples were obtained in steady state and not during hospitalization or VOC. No subjects were on prophylactic penicillin. To control for differences in sex and age, 43 subjects in whom the data were available (33 females, 10 males; age range: 10–67 years, median 31) were used in the statistical modeling as was the sample origin. We did not observe significant differences between the microbiota diversity of the SCD and AS control groups (Shannon: P = .11, Tail: P = .22) (Figure 1A) or when microbiota composition was examined at the phylum level. However, differences in microbiota composition between SCD and AS were observed when we modeled the top 15 genera that accounted for 84% of the taxonomic abundance across all samples. Abundances were log-ratio transformed and treated as responses.5 Six genera were identified based on their greatest potential for associating with SCD: Pseudobutyrivibrio (P = .05), Faecalibacterium (P = .11), Subdoligranulum (P = .16) (all phylum Firmicutes), Prevotella 9 (P = .07), and Alistipes (P = .04) (both phylum Bacteroidetes) were lower in abundance, while Escherichia-Shigella (P = 0.11) (phylum Proteobacteria) was higher in abundance in SCD. Alterations in taxonomic membership and abundance of Firmicutes and Bacteroidetes, along with increases in Proteobacteria abundance have been associated with some disorders.6, 7 Microbiome profiles in subjects with SCD compared to controls with sickle trait. A, Subjects with SCD showed the comparable diversity as those with sickle trait. B, The association of LDH with the microbiota represented by the 6 genera: Subdoligranulum, Prevotella 9, Faecalibacterium, Escherichia-Shigella, Pseudobutyrivibrio, and Alistipes. Next, the six identified genera were used as predictors of a set of clinically variables as univariate responses: hemoglobin, fetal hemoglobin, lactate dehydrogenase (LDH), white cell count, neutrophil count, erythrocyte sedimentation rate, and C-reactive protein. These variables were measured for 11 SCD individuals in whom the full set of these clinical parameters were available. Trends could be identified based on the abridged model consisting of the six genera. The most significant association was found with LDH (adjusted R2 = 0.81, P = .03) (Figure 1B). LDH was positively correlated with Subdoligranulum, Prevotella_9, Faecalibacterium, and Escherichia-Shigella, and negatively correlated with Pseudobutyrivibrio and Alistipes. Although not statistically significant, directionality of these genera was reversed in relation to hemoglobin (adjusted R2 = 0.47, P = .20), which is consistent with the general physiological relationship between these two variables, where hemoglobin would be expected to decrease with elevated LDH levels. Recent published findings have reported associations between activated neutrophil counts and microbiota composition.3 The abridged model suggested an association with neutrophil counts may exist, although the relationship was not conclusive (adjusted R2 = 0.45, P = .21). This result may reflect the lack of information on neutrophil activation state, highlighting the importance of including in future studies careful evaluation of activated neutrophils instead of only total neutrophil counts. We chose to not include in the correlation studies the frequency of hospitalization for VOC since the need for hospitalization is highly subjective, depending on multiple factors, including pain threshold and social circumstances. Among the group of 11 SCD subjects with laboratory parameters available, higher levels of Lachnoclostridium were positively correlated with higher baseline hemoglobin (P = .01) and fetal hemoglobin (P = .01), and lower baseline C-reactive protein (P = .05) and total white cell count (P =.05). These are parameters of less severe disease, although higher levels of Lachnoclostridium were also positively correlated to neutrophil counts (P = .05). Similarly, Parabacteroides were found to be positively correlated with baseline hemoglobin level (P = .05). Our results intriguingly suggest that a broader interplay may exist between the gut microbiome with both erythrocytes and white blood cells through potential mechanisms such as physical translocation, inflammatory processes, and production or conversion of metabolites.8, 9 Our preliminary findings are in keeping with the hypothesis that we have previously proposed.4 These findings provide the rationale for future studies to further characterize and define the microbiome to determine the effects of the specific bacterial genera taxa in inducing VOC or protecting the host from the detrimental effects of VOC. All authors declare no conflict of interest