The Role of Gut Microbiota in the Development of Intestinal Gvhd

The Role of Gut Microbiota in the Development of Intestinal GVHD

Background

Commensal gut microbiota have been implicated in initiating and perpetuating intestinal graft versus host disease (iGVHD), but its role remains controversial.  Recent murine studies have shown that iGVHD results in destruction of intestinal mucosal immune defenses resulting in expansion of pro-inflammatory bacteria (Enterobacteriaceae, ENTERO), and by prophylactically treating mice with oral antibiotics that suppress growth of ENTERO, iGVHD was significantly improved¹.  Furthermore, small-chain fatty acid producing (SCFA) Clostridia have been shown to induce colonic Treg cells, to dampen gut inflammation, and to cure IBD in mice².  But these findings have not been observed or replicated in humans. 

 

Methods

Stool samples were collected on a weekly basis from pediatric allogeneic BMT patients from 7/26/11 – 9/30/13.  Bacterial and fungal gDNA was isolated from fecal specimens.  Amplicons for 16S rRNA V4 variable region and fungal ITS region were generated and sequenced on Roche 454 GS-FLX sequencer and Illumina HiSeq2000 respectively. Sequencing data was analyzed using QIIME software. The abundance of specific intestinal bacterial groups was determined by qPCR using group-specific 16S rRNA gene primers.  For patients undergoing intestinal biopsy for suspicion of iGVHD (abdominal pain, diarrhea), additional pieces of intestine were obtained for transcription profiling experiments using Illumina Human HT12 V4 Expression BeadChips.  Clinical characteristics (i.e. conditioning regimens, specific antibiotic use, immunosuppression, etc) were recorded.

 

Results

Complete bacterial/fungal pyrosequencing and bacterial group qPCR was performed on 10 patients (4 iGVHD, 6 no GVHD).  As evidenced by 16S rRNA sequencing, only patients with iGVHD developed significant expansion of ENTERO and a significant decrease in SCFA Clostridia prior to the diagnosis of iGVHD.  Bacterial group qPCR confirmed these findings: patients with GVHD had significantly higher ENTERO (p< 0.01, Mann Whitney) and significantly lower EREC and CLEPT (subgroups of the SCFA Clostridia) (P<0.01) than non-GVHD counterparts.  Of the clinical characteristics recorded, clindamycin treatment, which is effective against Clostrida spp, was the most strongly associated with the development of iGVHD

 

Conclusion

Expansion of pro-inflammatory ENTERO and decreases in anti-inflammatory Clostridia (CLEPT and EREC) are associated with iGVHD in pediatric BMT patients.  Medical therapies such as chemotherapy and/or antibiotics may disturb the baseline gut microbiota and make certain patients predisposed to the development of iGVHD.  Real-time monitoring of the gut microbiota (bacterial group qPCR) has great potential as a biomarker for iGVHD.

 

1. Eriguchi et al. Blood. Jul 5 2012;120(1):223-231.
2. Atarashi et al Nature. Aug 8 2013;500(7461):232-236