Stress responses, M2 macrophages, and a distinct microbial signature in fatal intestinal acute graft-versus-host disease

Transcriptome sequencing was applied to 44 colon Graft vs Host Disease (GVHD) biopsies from 22 patients to determine differences in immunology and microbial profiles, along with general differences in somatic gene expressions by determining trends of mapped somatic alignments. Read analysis of somatic alignments were subjected to enrichment analysis of cellular pathways, expression comparisons amongst classes, and immune activity characterization based on outputs of deconvulation alogorithms. Unmapped alignments were mapped to microbiome reference assemblies to determine non-somatic profiles. Classes were determined based on healthy vs diseased patients, steriod refractory paired with onset biopsies, and a survival cohort as well. Distinct differences between classes have been noticed in all measures of interest. Characterizing the biopsies consisted of the following steps. RNA extraction was performed on FFPE rectosigmoid biopsies using FFPE biopsies using PureLink® FFPE RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared using Illumina HiSeq 2500® platform. nCounter® Human Immunology panel (NanoString, Seattle, WA) was used to compare immunology findings in comparison to the Cibersort cell deconvulation algorithm. Alignments were pefromed using Tophat and Cufflinks for somatic cells. Microbial reads were isolated and mapped using hisat2 and Pathseq. Gene Enrichment Analysis and Enrichr were used to analyze signal transduction pathways based on gene expressions. Limma was then used for comparison of signal gene expression. Wilcoxin rank sum was used to compare signature microbes. Signature genes and microbes were discovered indicative of survivability, along with enriched pathways discovered as well.