Abstract
Introduction
The intestinal microbiota modulates the host immune-system and is particularly relevant in immune-related diseases. Molecular signals from the intestinal microbiota are perceived by the immune system and amplified by the release of cytokines. Depending on the cytokine signal, the immune response may become more pro-inflammatory or more regulatory. Multiple studies have identified the importance of the intestinal microbiota in the outcome of allogeneic hematopoietic cell transplantation (allo-HCT) patients, particularly in graft-versus-host disease (GVHD) incidence. Although specific microbiome features, such as alpha-diversity, Enterococcus expansion and the metabolism of short chain fatty acids (SCFA) have shown association with patient outcomes and causal role in animal models, the interaction between microbiome signatures, cytokine profile and HCT outcomes have been under-investigated.
Methods
The cohort consisted of 30 patients; half received Clostridium butyricum MIYAIRI 588 (CBM588) and half received placebo treatment as part of a pilot clinical trial investigating the safety of CBM588 administration in transplant recipients undergoing reduced intensity conditioning regimen (NCT03922035). Stool and serum samples were collected at baseline and weekly follow-ups until day +28 post-HCT. Shotgun sequencing was performed to evaluate microbiome composition. Alpha diversity, as well as Enterococcus and Clostridium genera abundance, were chosen for downstream evaluation due to their relevance in literature. The genus Phocaeicola was also evaluated due to its high prevalence in the data and fermentative metabolism. Cytokine profiling consisted of the measurement of 23 cytokines or cytokine receptors. Cytokines were normalized from a value of 0 to 1 to enable proper comparisons, where 1 represents the maximum measured value for that cytokine. Eight SCFAs were measured and evaluated from stool samples.
Results We performed hierarchical clustering to investigate the cytokine profiles of patients. We identified five clusters that were associated with different patterns of cytokine concentration. Cluster 1 was particularly high in inflammatory cytokines IL-6, IL-8, and receptors TNF-R1 and IL-2aR. Cluster 2 was high in IL-2aR, the anti-inflammatory cytokine IL-10, and the chemokines ST2 and IP-10. The other three clusters had more diffuse and moderate patterns. We evaluated the association between cytokine patterns and the microbiota and observed that Cluster 1 had the lowest alpha diversity, the highest Enterococcus relative abundance, and the lowest Phocaeicola relative abundance.
Nine patients showed skin or GI GVHD of grade 2 or more. By evaluating the association between cytokine abundance and the incidence of acute GVHD, we noticed that at day 7 after transplant the abundance of cytokines IL-8, IL-6, and the receptors TNF-R1 and IL-2aR were enriched in patients who developed aGVHD. We found that alpha diversity was not associated with the abundance of these cytokines. However, Enterococcus abundance was positively associated (Pearson's correlation ranging from 0.22 to 0.4) with the abundance of each of these cytokines. Interestingly, the genus Phocaeicola was highly negatively correlated with the same cytokines (Pearson's correlation ranging from -0.26 to -0.5). We did not find a significant correlation between aGVHD cytokine markers and the Clostridium genus. Finally, we found that the abundance of SCFA around day 7 and day 14 after allo-HCT were significantly lower in patients that developed aGVHD for all measured SCFAs, with the strongest signal for propionate and butyrate.
Conclusion
The link between intestinal microbiota and the incidence of aGVHD could be related with an increase in pro-inflammatory cytokines, such as IL-8 and IL-6, after allo-HCT. Understanding the molecular mechanisms that link specific taxonomic groups, such as Enterococcus and Phocaeicola species, with the triggering of cytokine release could help identify strategies to mitigate the incidence of aGVHD.
