Identifying Gut Microbiota and Immune Host Factors Associated with Bleeding Risk in Children with Immune Thrombocytopenic Purpura

Background: Immune thrombocytopenia (ITP) may cause clinically significant bleeding episodes that can adversely affect a patient's quality of life. The gut microbiome plays a critical role in host immune system education and response in both healthy and diseased states. Previous studies comparing microbial taxa and cytokine levels in ITP patients have focused on differences between healthy controls and ITP participants. The role of the microbiome and cytokine levels in bleeding risk among ITP patients remains unclear. Methods: In our prospective cohort study, we analyzed stool and blood samples from 30 pediatric ITP participants at platelet levels ≤30,000/uL at time of diagnosis. Participants were divided into two groups, mild or moderate-severe bleeding phenotype, using the Buchanan-Adix bleeding score with mild participants having a score ≤3a and moderate-severe with a score ≥3b. Microbiome data was obtained using 16S rRNA gene sequencing on stool samples and cytokine profiling was obtained using Luminex assays on blood samples. Microbial diversity, microbial abundance, and cytokine levels were then compared between participants with mild and moderate-severe bleeding phenotype. Results: Of the 30 participants, the median age was 5.5 years (IQR 2-9.5). 11 (36.7%) were diagnosed with mild phenotype and 19 (63%) with moderate-severe bleeding phenotype. The median platelet count in the moderate-severe group was 4,000/uL (IQR 4-6) and in the mild group 6,000/uL (IQR 4-15) with no difference found between the two groups (p>0.05). The gut microbiota taxa that were significantly enriched in moderate-severe versus mild bleeders included the bacterial families Erysipelotrichaseae (LDA score 3.75), Ruminococaceae (LDA score 3.16), and Oscillospiraceae (LDA score 2.81), as assessed by LEfSe. Mild phenotype had increased Megamonas (LDA score 4.33) and Fusobacterium (LDA score 3.65) taxa compared to moderate-severe. Systemic cytokine profiling revealed a significant increase in IL-1ar in the moderate-severe group compared to those with mild phenotype (p=0.0447) with a significant positive correlation seen between Ruminococcus gut microbiota and IL-1ar cytokine levels (p=0.0865). Conclusion: Pediatric patients with acute ITP and a moderate-severe bleeding phenotype are enriched in the anti-inflammatory bacteria Ruminococcus and cytokine IL-1ra, while those with a mild phenotype have higher relative amounts of pro-inflammatory bacteria Megamonas and Fusobacterium. These findings suggest that gut microbiota modulates bleeding risk in pediatric ITP. This study provides novel insight into the altered composition of gut microbiota and cytokines in ITP patients with variable bleeding phenotype at similar platelet counts.