#METABOLOMICS WORKBENCH gardinassi_20190926_105923 DATATRACK_ID:1827 STUDY_ID:ST001255 ANALYSIS_ID:AN002084 PROJECT_ID:PR000841 VERSION 1 CREATED_ON September 30, 2019, 1:35 pm #PROJECT PR:PROJECT_TITLE Immunomodulatory activity of hyaluronidase is associated with metabolic PR:PROJECT_TITLE adaptations during acute inflammation PR:PROJECT_TYPE Untargeted MS PR:PROJECT_SUMMARY Objective and design: Investigate survival outcomes, and immunological and PR:PROJECT_SUMMARY metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute PR:PROJECT_SUMMARY inflammation and sepsis. Methods: Survival of C57Bl/6 mice was monitored after PR:PROJECT_SUMMARY lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture PR:PROJECT_SUMMARY (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged PR:PROJECT_SUMMARY with LPS and treated with Hz for leukocyte counting, cytokine quantification and PR:PROJECT_SUMMARY determination of metabolomic profiles in the peritoneal fluid. Results: Hz PR:PROJECT_SUMMARY treatment improved survival outcomes after lethal challenge with LPS or PR:PROJECT_SUMMARY CLPinduced sepsis. LPS challenge promoted acute neutrophil accumulation and PR:PROJECT_SUMMARY production of interleukin-1β (IL-1β) and IL-6 in the peritoneum, whereas Hz PR:PROJECT_SUMMARY treatment suppressed neutrophil infiltration and cytokine production. We further PR:PROJECT_SUMMARY characterized the metabolomic alterations caused by LPS challenge, which PR:PROJECT_SUMMARY predicted activity of metabolic pathways related to fatty acids and eicosanoids. PR:PROJECT_SUMMARY Hz treatment had a profound effect over the metabolic response, reflected by PR:PROJECT_SUMMARY reductions of the relative levels of fatty acids. Conclusion: Collectively, PR:PROJECT_SUMMARY these data demonstrate that Hz treatment is associated with metabolic PR:PROJECT_SUMMARY reprogramming of pathways that sustain the inflammatory response. PR:INSTITUTE Sao Paulo University PR:DEPARTMENT Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirao Preto PR:LAST_NAME Gardinassi PR:FIRST_NAME Luiz Gustavo PR:ADDRESS Av do Café s/n - Ribeirão Preto - SP PR:EMAIL gustavogardinassi@usp.br PR:PHONE 551633154189 PR:FUNDING_SOURCE FAPESP; CNPq; CAPES #STUDY ST:STUDY_TITLE Immunomodulatory activity of hyaluronidase is associated with metabolic ST:STUDY_TITLE adaptations during acute inflammation ST:STUDY_SUMMARY Objective and design: Investigate survival outcomes, and immunological and ST:STUDY_SUMMARY metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute ST:STUDY_SUMMARY inflammation and sepsis. Methods: Survival of C57Bl/6 mice was monitored after ST:STUDY_SUMMARY lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture ST:STUDY_SUMMARY (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged ST:STUDY_SUMMARY with LPS and treated with Hz for leukocyte counting, cytokine quantification and ST:STUDY_SUMMARY determination of metabolomic profiles in the peritoneal fluid. Results: Hz ST:STUDY_SUMMARY treatment improved survival outcomes after lethal challenge with LPS or ST:STUDY_SUMMARY CLPinduced sepsis. LPS challenge promoted acute neutrophil accumulation and ST:STUDY_SUMMARY production of interleukin-1β (IL-1β) and IL-6 in the peritoneum, whereas Hz ST:STUDY_SUMMARY treatment suppressed neutrophil infiltration and cytokine production. We further ST:STUDY_SUMMARY characterized the metabolomic alterations caused by LPS challenge, which ST:STUDY_SUMMARY predicted activity of metabolic pathways related to fatty acids and eicosanoids. ST:STUDY_SUMMARY Hz treatment had a profound effect over the metabolic response, reflected by ST:STUDY_SUMMARY reductions of the relative levels of fatty acids. Conclusion: Collectively, ST:STUDY_SUMMARY these data demonstrate that Hz treatment is associated with metabolic ST:STUDY_SUMMARY reprogramming of pathways that sustain the inflammatory response. ST:INSTITUTE Sao Paulo University ST:DEPARTMENT School of Pharmaceutical Sciences of Ribeirao Preto ST:LAST_NAME Gardinassi ST:FIRST_NAME Luiz Gustavo ST:ADDRESS Av do Cafe, s/n - Ribeirão Preto - SP ST:EMAIL gustavogardinassi@usp.br ST:PHONE 551633154189 ST:NUM_GROUPS 8 ST:TOTAL_SUBJECTS 40 ST:NUM_MALES 40 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57Bl6 SU:WEIGHT_OR_WEIGHT_RANGE 22–25 g SU:GENDER Male SU:ANIMAL_LIGHT_CYCLE light/dark cycle SU:ANIMAL_FEED Ad libitum SU:ANIMAL_WATER Ad libitum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - PBS_1 Treatment:PBS | Time:0h SUBJECT_SAMPLE_FACTORS - PBS_2 Treatment:PBS | Time:0h SUBJECT_SAMPLE_FACTORS - PBS_3 Treatment:PBS | Time:0h SUBJECT_SAMPLE_FACTORS - PBS_4 Treatment:PBS | Time:0h SUBJECT_SAMPLE_FACTORS - LPS_3h_1 Treatment:LPS | Time:3h SUBJECT_SAMPLE_FACTORS - LPS_3h_2 Treatment:LPS | Time:3h SUBJECT_SAMPLE_FACTORS - LPS_3h_3 Treatment:LPS | Time:3h SUBJECT_SAMPLE_FACTORS - LPS_3h_4 Treatment:LPS | Time:3h SUBJECT_SAMPLE_FACTORS - LPS_3h_5 Treatment:LPS | Time:3h SUBJECT_SAMPLE_FACTORS - LPS_3h_6 Treatment:LPS | Time:3h SUBJECT_SAMPLE_FACTORS - LPS_27h_1 Treatment:LPS | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_27h_2 Treatment:LPS | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_27h_3 Treatment:LPS | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_27h_5 Treatment:LPS | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_27h_6 Treatment:LPS | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_7d_1 Treatment:LPS | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_7d_2 Treatment:LPS | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_7d_3 Treatment:LPS | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_7d_4 Treatment:LPS | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_7d_5 Treatment:LPS | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_7d_6 Treatment:LPS | Time:7d SUBJECT_SAMPLE_FACTORS - Hz_27h_2 Treatment:Hz | Time:27h SUBJECT_SAMPLE_FACTORS - Hz_27h_3 Treatment:Hz | Time:27h SUBJECT_SAMPLE_FACTORS - Hz_27h_4 Treatment:Hz | Time:27h SUBJECT_SAMPLE_FACTORS - Hz_27h_5 Treatment:Hz | Time:27h SUBJECT_SAMPLE_FACTORS - Hz_27h_6 Treatment:Hz | Time:27h SUBJECT_SAMPLE_FACTORS - Hz_7d_1 Treatment:Hz | Time:7d SUBJECT_SAMPLE_FACTORS - Hz_7d_2 Treatment:Hz | Time:7d SUBJECT_SAMPLE_FACTORS - Hz_7d_3 Treatment:Hz | Time:7d SUBJECT_SAMPLE_FACTORS - Hz_7d_5 Treatment:Hz | Time:7d SUBJECT_SAMPLE_FACTORS - Hz_7d_6 Treatment:Hz | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_Hz_27h_1 Treatment:LPS + Hz | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_Hz_27h_2 Treatment:LPS + Hz | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_Hz_27h_3 Treatment:LPS + Hz | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_Hz_27h_4 Treatment:LPS + Hz | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_Hz_27h_5 Treatment:LPS + Hz | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_Hz_27h_6 Treatment:LPS + Hz | Time:27h SUBJECT_SAMPLE_FACTORS - LPS_Hz_7d_1 Treatment:LPS + Hz | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_Hz_7d_2 Treatment:LPS + Hz | Time:7d SUBJECT_SAMPLE_FACTORS - LPS_Hz_7d_3 Treatment:LPS + Hz | Time:7d #COLLECTION CO:COLLECTION_SUMMARY Mice were anesthetized with ketamine (100mg/kg) (Dopaser®, Minas Gerais, CO:COLLECTION_SUMMARY Brazil) and xylazine (10mg/kg) (Agener União, São Paulo, Brazil). Following CO:COLLECTION_SUMMARY euthanasia in CO2 chamber, 3mL of PBS was added into the abdominal cavity, which CO:COLLECTION_SUMMARY was gently massaged for 1 min. The peritoneal fluid was collected with a needle CO:COLLECTION_SUMMARY inserted into the inguinal region. CO:SAMPLE_TYPE Peritoneal fluid CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Endotoxemia was induced in mice by intraperitoneal (i.p.) injection of LPS TR:TREATMENT_SUMMARY dissolved in 0.3 mL of phosphate-buffered saline (PBS). Control groups received TR:TREATMENT_SUMMARY sterile PBS. Tree hours (h) after LPS injection, mice were treated with 16 U of TR:TREATMENT_SUMMARY Hz (dissolved in 0.3 mL of PBS) every 8 h #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Peritoneal fluid samples were spiked with stable isotope internal standards and SP:SAMPLEPREP_SUMMARY metabolites were enriched with solid phase extraction cartridges (Hypersep SP:SAMPLEPREP_SUMMARY C18-500 mg, 3 mL, Thermo Scientific-Bellefonte, PA, USA) and methanol/water as SP:SAMPLEPREP_SUMMARY solvents. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reversed-phase chromatography was accomplished with a 100 × 4.6 mm, 2.7 μm CH:CHROMATOGRAPHY_SUMMARY Ascentis Express C18 column (Supelco - St. Louis, MO, USA) with an CH:CHROMATOGRAPHY_SUMMARY Ultra-High-Performance Liquid Chromatography (UHPLC) system (Nexera X2, CH:CHROMATOGRAPHY_SUMMARY Shimadzu-Kyoto, HO, Japan). The gradient consisted of Phase A, H2O/ACN/acetic CH:CHROMATOGRAPHY_SUMMARY acid (69.98:30:0.02, v/v/v) at pH 5.8 (adjusted with NH4OH), and Phase B, an CH:CHROMATOGRAPHY_SUMMARY ACN/isopropanol (70:30, v/v). Gradient elution was carried out for 25 min at a CH:CHROMATOGRAPHY_SUMMARY flow rate of 0.6 mL. / min. Gradient conditions were as follows: 0 to CH:CHROMATOGRAPHY_SUMMARY 2.0 min, 0% B; 2.0 to 5.0 min, 15% B; 5.0 to 8.0 min, 20% B; 8.0 to CH:CHROMATOGRAPHY_SUMMARY 11.0 min, 35% B; 11.0 to 15.0 min, 70% B; and 15.0 to 19 min, 100% B. At CH:CHROMATOGRAPHY_SUMMARY 19.0 min, the gradient returned to the initial condition of 0% B, and the CH:CHROMATOGRAPHY_SUMMARY column was re-equilibrated until 25.0 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Ascentis Express C18 (100 x 4.6, 2.7) CH:METHODS_FILENAME PR_CH_Methods.pdf #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5600 TripleTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Mass spectral data was acquired with negative electrospray ionization and the MS:MS_COMMENTS full scan of mass-to-charge ratio (m/z) ranged from 100 to 1500. Proteowizard MS:MS_COMMENTS software was used to convert wiff files into mzXML files. Peak peaking, noise MS:MS_COMMENTS filtering, retention time and m/z alignment, and feature quantification were MS:MS_COMMENTS performed with apLCMS. Three parameters define a metabolite feature: MS:MS_COMMENTS mass-to-charge ratio (m/z), retention time and intensity values. Data were log2 MS:MS_COMMENTS transformed and only features detected in at least 80% of samples from one group MS:MS_COMMENTS (5439 m/z features) were used in further analysis. Missing values were imputed MS:MS_COMMENTS using half mean of the feature across all samples. The mummichog software MS:MS_COMMENTS (version 2) was used for metabolic pathway enrichment analysis (mass accuracy MS:MS_COMMENTS under 10 ppm). MS:MS_RESULTS_FILE ST001255_AN002084_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END