Shotgun sequencing reveals transkingdom alterations in immunodeficiency associated enteropathy Xiaoxi Dong (Oregon State University), Jialu Hu (Oregon State University), Ekaterina Peremyslova (Oregon State University), Ivan J.Fuss (National Institute of Allergy and Infectious Diseases), Michael Yao (National Institute of Allergy and Infectious Diseases), Warren Strober (National Institute of Allergy and Infectious Diseases) Natalia Shulzhenko (Oregon State University), Andrey Morgun (Oregon State University ) Results 1. Human gene expression of three groups Among ~15000 genes detected by RNA-SEQ, ~1500 genes (F test, FDR < 0.1) up- regulated in CVID enteropathy (GI-CVID). Clustering of samples showed distinct pattern of duodenum gene expression in CVID enteropathy patients compared to the similar pattern between the other two groups. Result for a stool sample from one of the 9 GI-CVID patients is shown on the right side of figure. 4. Relative abundance of bacteria taxa from 16s rRNA sequencing 5. Detection of norovirus in CVID and GI-CVID group 2. Expression of IFNgamma and IFNg-dependent genes and immune cell specific genes in three groups Genes specific for neutrophils, cytotoxic T cells and group of inflammasome-related genes are up-regulated in GI-CVID. Introduction -Common variable immunodeficiency (CVID) is the most common symptomatic primary antibody deficiency syndrome and is characterized by decreased levels of serum immunoglobulins and recurrent respiratory infections. -Up to 50% of patients manifest a non-infectious malabsorption syndrome, also known as CVID enteropathy(Gastrointestinal CVID: GI-CVID) with unclear pathogenesis. -Recent studies in mouse model suggested that it results from an interaction between gut microbiota and intestinal epithelium leading to a hyperactive mucosal immune response characterized by increased interferon-regulated pathway at the expense of decreased expression of metabolic genes. In this study, we extended the investigation into humans by comparing host gene expression and microbiota composition in duodenum between three groups in order to decipher the pathogenesis of CVID enteropathy : healthy volunteers (n=7), CVID patients without enteropathy (nonGI-CVID, n=6) and CVID patients with enteropathy(GI- CVID, n=9). 3. Down regulation of metabolic genes in GI-CVID Down-regulated genes showed enrichment of metabolic genes. P-values are calculated from GO enrichment analysis(Fisher Exact test using human genome as background) Methods Detection of norovirus sequence by PCR in duodenum samples Detection of norovirus sequence by RNA-SEQ shotgun sequencing in a stool sample(S1) and duodenum samples(other samples) Conclusion Transkingdom survey of CVID related enteropathy has identified several alterations in bacterial and viral compartments of duodenal microbiome accompanied by increased IFNg and neutrophilic host responses indicating potential microbes contributing to this syndrome. Further direction - If specific bacteria or fungi strains involve in pathogenesis of GI-CVID - In vivo and in vitro experiments for validation. Reference Shulzhenko, Natalia, et al. "Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut." Nature medicine (2011): Acknowledgements NIAID grant U01 AI Relative abundance Healthy GI-CVID nonGI-CVID Duodenal biopsy, stool sample (host and microbe cells) Total RNA 16s rRNA v4 region amplicon (Illumina MiSeq) cDNA shotgun fragment sequencing (Illumina HiSeq 2000 paired end) Microbe taxa relative abundance table (QIIME) High quality reads(Trimmomatic, prinseq) Human gene abundance table (Tophat, HTSeq,edgeR) Non-human reads General microbiome: (RAPsearch against nr database, evalue ) Taxa, KEGG, SEED annotation(MEGAN5) Virus detection: 1.Blast search against virus genome 2.Assemble read(SOAPdenovo) followed by MetaVir Genus Blautia and family Enterobacteriaceae are enriched in GI-CVID group