The Effect And Predictive Value Of Intestinal Microbiota On AGVHD After Allo-HSCT

Background and ObjectivesAllogeneic hematopoietic cell transplantation(allo-HSCT)is a curative therapy for hematological malignancies and nonmalignant diseases,and some non-hematologic disorders.Graft-versus-host disease(GVHD)is a major complication and cause of death after allo-HSCT,which is one of the most important factors that restrict the efficacy of allo-HSCT.Many factors affect the development of GVHD,such as HLA matching,donor source,stem cell source,patient and donor age,sex,conditioning regimen,and so on.In recent years,more and more studies have demonstrated that intestinal microbiota is associated with the development of acute GVHD(aGVHD).A number of studies have demonstrated that intestinal microbiota composition influnences the development of inflammatory diseases and aGVHD.In animal experiments,the mechanism of the effects of the microbiota on immune homeostasis has been widely studied.These studies indicate that different microbiota drive or inhibit the activation of the immune system via their metabolites,such as short chain fatty acids(SCFAs),histone deacetylase inhibitors(HDACis)or lipopolysaccharide(LPS),a potent inflammatory activator.The experiments showed that SCFAs,the metabolites of anaerobic bacteria(such as Clostrida),can improve the level of acetylated histone and promote Foxp3 histone acetylation,mediate histone modification of regulatory T cells(Treg),maintain immune balance and reduce the occurrence of GVHD.Whereas LPS,the metabolites of aerobic bacteria(such as pathogenic Gammaproteobacteria),can activate immune cells,destroy the immune balance,promote the occurrence of GVHD.However,the mechanism underlying the effects of microbiota on immune homeostasis in recipients undergoing allo-HSCT has not been determined,and the specific bacterial as the risk marker of aGVHD has not been clear.To explore the mechanism of the effects of intestinal microbiota on immune homeostasis prior to aGVHD in patients post-transplantation and determine the prediction of microbiota on aGVHD,we performed a retrospective analysis of a prospective study of recipients undergoing allo-HSCT.Methods1.Samples collection:From January 2015 to December 2016,Samples were collected from adult patients who underwent allo-HSCT in our hospital.Stool samples were collected at the time of pre-conditioning and engraftment and stored in a bio specimen bank.At the same time,blood samples were directly examined or stored in another biospecimen bank.For each subject,biospecimens were collected during 3-4 days of stem cell engraftment(absolute neutrophil count:0.5*107/L for 3 consecutive days)after allo-HSCT.According to the occurrence of aGVHD,the patients were divided into 2 groups:aGVHD group(?-? aGVHD)and non-aGVHD group(0-I aGVHD).2.Intestinal flora detection:For each fecal specimen,DNA was extracted and purified,and the V3 to V4 region of the 16S rRNA gene was polymerase chain reaction(PCR)-amplified using modified universal bacterial primers.Purified PCR products were sequenced with the Hiseq2500 PE250 platform.Sequence data were analyzed according to microbiota diversity and Linear discriminant analysis(LDA)effect size(LEfSe)analysis using LEfSe software.3.Detection of T cell subsets:The proportions of T cell subsets(Treg,Th17 and Thl)in peripheral blood,and the levels of acetylated histone 3(ace-H3)and ace-H4(median fluorescence intensity,MFI)were examined by flow cytometry.4.Cytokine detection:The expressions of interleukin-4(IL-4),IL-10,IL-17A and interferon-gamma(IFN-?)were detected by liquid chip technology.5.Correlation analysis:The relationships among intestinal microbiota and T cell subsets and the level of acetylated histone in CD4+T cells were analyzed by Spearman correlation analysis.6.Prediction for aGVHD:Based on the characteristics of intestinal microbiota markers after allo-HSCT,the predictive value of bacteria for aGVHD was evaluated by ROC(receiver operator characteristic,ROC)curve area(AUC).The accumulated intestinal microbiota(AIM)score was also used to predict both aGVHD and the grades.Results1.The clinical and transplant characteristics of the two groups:In the 81 patients undergoing allo-HSCT,51 males and 30 females were included,and the median age was 33 years(16-55 years old).The patients were categorized into aGVHD group(n=32)and non-aGVHD group(n = 49).Except more patients in the aGVHD group undergoing intensified conditioning,the other characters between the two groups were similar,including donor type,stem cell source,TBI conditioning,and so on.2.The intestinal microbiota prior to conditioning was compared between the two groups.There was no significant difference in the diversity and the constitution of bacteria between the groups.3.The differences of antibiotics before engraftment between the groups:More patients in the aGVHD group were administered beta-lactam antibiotics(3 days or more than 3 days administration were continuous from conditioning to engraftment).4.The difference of intestinal microbiota at engraftment between the groups:The diversity of microbiota in the aGVHD group was lower than that in the non-aGVHD group.In terms of bacterial constitution,at Class level,the abundance of Clostridia was less in the aGVHD group compared with the non-aGVHD group,while the abundance of Gammaproteobacteria in the aGVHD group was more than that in the non-aGVHD group.At the family level,several bacterials' abundance,such as Lachnospiraceae and Ruminococcaceae,was less in the aGVHD group than that in the non-aGVHD group,whereas the abundance of Enterobacteriaceae was higher in the aGVHD group than that in the non-aGVHD group.5.The intestinal microbiota was affected by antibiotics and Conditioning.The diversity of microbiota was affected by beta-lactam antibiotics.Furthermore,beta-lactam antibiotics can lead to less abundance of Lachnospiraceae,while vancomycin can lead to more abundance of Enterobacteriaceae.Additionally,intensified conditioning can lead to lower diversity and less abundance of Lachnospiraceae and Ruminococcaceaee.No other factors was found association with microbiota.6.The risk factors for aGVHD:Cox regression model showed that beta-lactam antibiotics and low bacterial diversity post-transplantation were independent risk factors for aGVHD.The patient's age,donor type and vancomycin(intravenous)were not independent risk factors for aGVHD.7.The T subsets cells and cytokins between the groups:The proportion of Treg cells(account for CD4+ cells)in the aGVHD group was less than that in the non-aGVHD group.Whereas,the proportion of Th17 cells in the aGVHD group was more than that in the non-aGVHD group.There was no significant difference in the proportion of Thl cells.The concentration of IL-17A cytokine in plasma was higher in the aGVHD group compared with the non-aGVHD group,but there was no significant difference in the concentration of IL-4,IL-10 and IFN-gamma.8.The relationship between the level of acetylated histone in CD4+ T cells and aGVHD and the subsets of T cells.The MFI of ace-H3 was lower in the aGVHD group than that in the non-aGVHD.The MFI of ace-H4 was not significantly different between the groups.The MFI of ace-H3 correlated with the Treg/Th17 cell ratio positively.No correlation was found between ace-H4 and the Treg/Th17 cell ratio.9.The relationship between intestinal microbiota and immune balance:The abundance of Lachnospiraceae,Ruminococcaceae and Enterobacteriaceae correlated with the Treg/Th17 cell ratio positively,positively and negatively,respectively.The abundance of Lachnospiraceae and Ruminococcaceae also correlated with the MFI of ace-H3 positively.10.The predictive value of intestinal microbiota for aGVHD:Exploring the predictive value of microbiota for aGVHD,we found that the abundance of four kinds of bacteria----Lachnospiraceae,Erysipelotrichaceae,Enterobacteriaceae and Peptostreptococcaceae could predict the occurrence of aGVHD.The AIM Score of microbiota could not only predict aGVHD(AUC for ?-? aGVHD = 0.73;AUC for?-? aGVHD = 0.85),but also correlate with the grades of aGVHD closely(r =0.560,P<0.001).Conclusions1,During allo-HSCT,both the diversity and the constitution of intestinal microbiota at engraftment are closely associated with the occurrence of aGVHD.The alteration of microbiota is influnenced by some special antibiotics and Conditioning.2.The intestinal microbiota at engraftment is closely related to the balance of Treg/Thl7 cell ratio and the level of ace-H3 in CD4+T cells.It could be speculated that the intestinal microbiota might modulate the balance of Treg/Thl7 by regulating the level of ace-H3 in CD4+ T cells,and further affect the development of aGVHD.3.The combination of microbiota score at engraftment can predict not only the occurrence of aGVHD but also the grades of aGVHD.