Alterations of Intestinal Microbiota Correlate with the Emergence of Acute Graft-Versus-Host Disease in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Acute graft-versus-host disease (aGVHD) is one of the most major and severe complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Studies have already shown that the gastrointestinal (GI) tract plays a critical role in the initiation and amplification of aGVHD. Recently, the alterations of intestinal microbiota, a dense and diverse microbioal community which is in symbiosis with the host in GI tract and exerting a considerable influence on the physiological, metabolic, nutritional, and immunological state of the host, have also been proved to be correlated with multiple inflammatory disease in human, including aGVHD. We retrospectively examined the intestinal microbiota in patients treated with allo-HSCT and analyzed the relationship between microbial alterations and the emergence of aGVHD.

Patients and methods: 40 patients who received allo-HSCT at our center from Mar-2013 to Dec-2014 were enrolled in this study. The median age at the time of transplantation was 31 years (range from 14 to 54 years). 17 patients (42.5%) were female, and 23 patients (57.5%) were male. Among them, 17 patients were diagnosed as acute myeloid leukemia (AML), while 4 as acute lymphoblastic leukemia (ALL), 2 as chronic myeloid leukemia (CML), 11 as myelodysplastic/myeloproliferative neoplasms (MDS/MPN), 3 as non-Hodgkin lymphoma (NHL), and 3 as severe aplastic anemia (SAA). Most of the patients (n=38, 95%) were treated with myeloablative conditioning regimen. The donor of the transplantation included HLA-identical siblings (n=13, 32.5%), matched unrelated donors (n=12, 30%), and haplo-identical related donors (n=15, 37.5%). Sulfomethoxazole and norfloxacin were administrated as prophylactic antibiotics in all patients during the conditioning period. Then, cephalosporins with or without teicoplanin were used in patients with no infectious complications before engraftment. But in patients with infections, therapeutic antibiotics, such as imiperem, vancomycin and etc, were used. Fecal specimens were collected from these patients at three different time-points during transplantation period: prior to the administration of conditioning regimen, during the first week after day 0, and after engraftment. After extraction, the bacterial genomic DNA was amplified with the 27F and 533R primers specific for the V1-V3 hypervariable regions of the 16S rRNA gene. PCR products were sequenced on a 454 GS FLX platform. Then, the changes of intestinal microbiota and the clinical manifestations of these patients were evaluated. Statistical difference between groups was determined by the Mann-Whitney U test.

Results: During the follow-up period, 14 patients (35%) had no aGVHD, while 16 patients (40%) developed grade I-II aGVHD and 10 patients (25%) developed grade III-IV aGVHD within 40 days after transplantation. There were 15 patients (37.5%) suffered from intestinal aGVHD, and the other 11 aGVHD patients (27.5%) had skin and/or liver aGVHD without intestinal involvement. Microbial diversity was estimated by calculating the inverse Simpson index. But we did not observe any significant differences among all the groups at different time point during the transplantation. At genus level, after engraftment, Bacteroides was notably decreased in GVHD (p<0.05), GVHD-GI (p<0.01), and grade III-IV GVHD (p<0.01) group (Figure A). Meanwhile, Enterococcus was much higher in these groups compared with non-GVHD group (p<0.05, p<0.01, and p<0.001 respectiverly) (Figure B).

Conclusion: These results suggested that intestinal microbioal alteration in the course of allo-HSCT correlated with the emergence of aGVHD, especially intestinal aGVHD and severe aGVHD. Some intestinal bacteria may play an important role in the development of aGVHD. For example, Bacteroides may have protective effect against aGVHD after engraftment.