#METABOLOMICS WORKBENCH amat_20181105_073530 DATATRACK_ID:1559 STUDY_ID:ST001089 ANALYSIS_ID:AN001774 PROJECT_ID:PR000728 VERSION 1 CREATED_ON November 5, 2018, 1:43 pm #PROJECT PR:PROJECT_TITLE Host NLRP6 exacerbates graft-versus-host disease independent of microbial PR:PROJECT_TITLE diversity PR:PROJECT_SUMMARY Host NLRP6 regulates innate immune responses and gastrointestinal (GI) PR:PROJECT_SUMMARY homeostasis. It plays a protective role in pathogenic processes such as PR:PROJECT_SUMMARY intestinal colitis and tumorigenesis in a microbiome dependent manner. Host PR:PROJECT_SUMMARY innate immunity and changes in microbial diversity also play a role in the PR:PROJECT_SUMMARY severity of allo-immune-mediated gastrointestinal pathogenic process, namely PR:PROJECT_SUMMARY graft-versus-host disease (GVHD), the principal toxicity after allogeneic bone PR:PROJECT_SUMMARY marrow transplantation (allo-BMT). Herein, we examined the role of NLRP6 in PR:PROJECT_SUMMARY multiple murine models of allo-BMT. In contrast to its role in intestinal PR:PROJECT_SUMMARY colitis, host NLRP6 aggravated GI GVHD. NLRP6-deficient animals showed improved PR:PROJECT_SUMMARY intestinal barrier function, increased levels of tissue repair associated PR:PROJECT_SUMMARY proteins and preserved Goblet and Paneth cell numbers in the GI tract after PR:PROJECT_SUMMARY allo-BMT. The impact of host NLRP6 deficiency in mitigating GVHD was observed PR:PROJECT_SUMMARY regardless of co-housing, antibiotic treatment, or colonizing littermate germ PR:PROJECT_SUMMARY free wild type (WT) and NLRP6 deficient hosts with fecal microbial PR:PROJECT_SUMMARY transplantation from SPF WT and Nlrp6-/- animals. Chimera studies were performed PR:PROJECT_SUMMARY to assess the role of NLRP6 expression on host hematopoietic and PR:PROJECT_SUMMARY non-hematopoietic cells. The allogeneic [B6Ly5.2→Nlrp6-/-] animals PR:PROJECT_SUMMARY demonstrated significantly improved survival compared to the allogeneic PR:PROJECT_SUMMARY [B6Ly5.2→B6] animals, demonstrating that the absence of NLRP6 in host PR:PROJECT_SUMMARY non-hematopoietic cells is crucial for the protection against GVHD, but did not PR:PROJECT_SUMMARY alter the therapeutic graft-versus-tumor effects after BMT. Our results unveil a PR:PROJECT_SUMMARY novel role for NLRP6 and demonstrate a pathogenic role in GVHD that is PR:PROJECT_SUMMARY independent of variations in its intestinal microbiome in contrast to its PR:PROJECT_SUMMARY well-appreciated microbiome-dependent protective role in intestinal colitis and PR:PROJECT_SUMMARY tumorigenesis. PR:INSTITUTE University of Michigan PR:LAST_NAME Mathew PR:FIRST_NAME Anna PR:ADDRESS 5112 Brehm Tower PR:EMAIL amat@umich.edu PR:PHONE 7342328228 #STUDY ST:STUDY_TITLE Host NLRP6 exacerbates graft-versus-host disease independent of microbial ST:STUDY_TITLE diversity ST:STUDY_SUMMARY Host NLRP6 regulates innate immune responses and gastrointestinal (GI) ST:STUDY_SUMMARY homeostasis. It plays a protective role in pathogenic processes such as ST:STUDY_SUMMARY intestinal colitis and tumorigenesis in a microbiome dependent manner. Host ST:STUDY_SUMMARY innate immunity and changes in microbial diversity also play a role in the ST:STUDY_SUMMARY severity of allo-immune-mediated gastrointestinal pathogenic process, namely ST:STUDY_SUMMARY graft-versus-host disease (GVHD), the principal toxicity after allogeneic bone ST:STUDY_SUMMARY marrow transplantation (allo-BMT). Herein, we examined the role of NLRP6 in ST:STUDY_SUMMARY multiple murine models of allo-BMT. In contrast to its role in intestinal ST:STUDY_SUMMARY colitis, host NLRP6 aggravated GI GVHD. NLRP6-deficient animals showed improved ST:STUDY_SUMMARY intestinal barrier function, increased levels of tissue repair associated ST:STUDY_SUMMARY proteins and preserved Goblet and Paneth cell numbers in the GI tract after ST:STUDY_SUMMARY allo-BMT. The impact of host NLRP6 deficiency in mitigating GVHD was observed ST:STUDY_SUMMARY regardless of co-housing, antibiotic treatment, or colonizing littermate germ ST:STUDY_SUMMARY free wild type (WT) and NLRP6 deficient hosts with fecal microbial ST:STUDY_SUMMARY transplantation from SPF WT and Nlrp6-/- animals. Chimera studies were performed ST:STUDY_SUMMARY to assess the role of NLRP6 expression on host hematopoietic and ST:STUDY_SUMMARY non-hematopoietic cells. The allogeneic [B6Ly5.2→Nlrp6-/-] animals ST:STUDY_SUMMARY demonstrated significantly improved survival compared to the allogeneic ST:STUDY_SUMMARY [B6Ly5.2→B6] animals, demonstrating that the absence of NLRP6 in host ST:STUDY_SUMMARY non-hematopoietic cells is crucial for the protection against GVHD, but did not ST:STUDY_SUMMARY alter the therapeutic graft-versus-tumor effects after BMT. Our results unveil a ST:STUDY_SUMMARY novel role for NLRP6 and demonstrate a pathogenic role in GVHD that is ST:STUDY_SUMMARY independent of variations in its intestinal microbiome in contrast to its ST:STUDY_SUMMARY well-appreciated microbiome-dependent protective role in intestinal colitis and ST:STUDY_SUMMARY tumorigenesis. ST:INSTITUTE University of Michigan ST:LAST_NAME Mathew ST:FIRST_NAME Anna ST:ADDRESS 5112 Brehm Tower ST:EMAIL amat@umich.edu ST:PHONE 7342328228 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN B6Ly5.2 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 BMT Type:Naïve | Mice type:NLRP6KO | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 2 BMT Type:Naïve | Mice type:NLRP6KO | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 3 BMT Type:Naïve | Mice type:NLRP6KO | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 4 BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 5 BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 6 BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 7 BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 8 BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 9 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 10 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 11 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 12 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 13 BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 14 BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 15 BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 16 BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 17 BMT Type:Naïve | Mice type:NLRP6KO | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 18 BMT Type:Naïve | Mice type:NLRP6KO | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 19 BMT Type:Naïve | Mice type:NLRP6KO | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 20 BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 21 BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 22 BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 23 BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 24 BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen Time (days)=- SUBJECT_SAMPLE_FACTORS - 25 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 26 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 27 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 28 BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 29 BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 30 BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 31 BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen Time (days)=21 SUBJECT_SAMPLE_FACTORS - 32 BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen Time (days)=21 #COLLECTION CO:COLLECTION_SUMMARY Stool lumen contents CO:SAMPLE_TYPE Intestinal lumen contents #TREATMENT TR:TREATMENT_SUMMARY BMT from naive [B6Ly5.2→B6] and allogenic e allogeneic [B6Ly5.2→Nlrp6-/-] TR:TREATMENT_SUMMARY animals #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To determine targeted taurine quantitation, samples (intestinal fecal content) SP:SAMPLEPREP_SUMMARY from mice 21d post-transplant were harvested, homogenized, and snap-frozen in SP:SAMPLEPREP_SUMMARY liquid N2. Fecal content was weighed at necropsy. Samples were extracted with SP:SAMPLEPREP_SUMMARY acetonitrile spiked with stable isotope-labeled C13 taurine standards. The SP:SAMPLEPREP_SUMMARY supernatant was analyzed by Agilent 6490 mass spectrometer in a positive SP:SAMPLEPREP_SUMMARY electrospray ionization mode after liquid chromatography using a HILIC mode and SP:SAMPLEPREP_SUMMARY isocratic gradient. Quantitation was performed by calibration to internal SP:SAMPLEPREP_SUMMARY standards and expressed in mM after normalization for weights. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Phenomenex Kinetex HILIC 100A (50 x 2.1 mm, 2.6um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6410 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS M/100mg stool MS_METABOLITE_DATA_START Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Factors BMT Type:Naïve | Mice type:NLRP6KO | Site:Large Intestine lumen BMT Type:Naïve | Mice type:NLRP6KO | Site:Large Intestine lumen BMT Type:Naïve | Mice type:NLRP6KO | Site:Large Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Large Intestine lumen BMT Type:Naïve | Mice type:NLRP6KO | Site:Small Intestine lumen BMT Type:Naïve | Mice type:NLRP6KO | Site:Small Intestine lumen BMT Type:Naïve | Mice type:NLRP6KO | Site:Small Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen BMT Type:Naïve | Mice type:WT | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:NLRP6KO | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen BMT Type:Allogenic | Mice type:WT | Site:Small Intestine lumen Taurine 2.694615665 2.273732734 1.104650772 3.007990781 1.824980648 4.546055621 3.593933924 7.04580715 8.709819128 6.091504593 2.59167843 1.28695669 1.921238525 3.76313065 6.959658346 4.42335537 0.639526327 18.90049767 10.67098206 20.16109934 24.99302473 19.98441601 16.9469764 0.275459973 25.44080239 27.01866729 37.42446146 8.757609022 20.16949246 28.18831521 66.53976245 31.78889499 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Moverz Quant Local ID PubChem CID KEGG ID mass spectrum Taurine 2 1123 C00245 METABOLITES_END #END