Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Salmonella lipopolysaccharide stimulates uptake of long-chain fatty acids in the small intestine

doi: 10.1073/pnas.2618255123

Figure Lengend Snippet: S . Typhimurium LPS promotes epithelial long-chain fatty acid uptake. ( A ) qPCR analysis of lipid metabolism mRNAs ( Cd36 , Fabp1 , and Apoa4 ) in small intestines of antibiotic-treated mice injected intraperitoneally with 1 µg of PAMPs: LTA, flagellin, or ultra-pure LPS (n = 6 mice per group). ( B ) In vivo uptake of C16:0 BODIPY in small IECs from antibiotic-treated mice injected intraperitoneally with the PAMPs shown in ( A ). Mice were injected daily for 3 d. Mice were gavaged with C16:0 BODIPY 12 h after the final treatment, and IEC fluorescence was quantified by flow cytometry. Analysis was performed on live EpCAM + cells. Representative histograms and quantification of MFI are shown (n = 7 to 8 mice per group). ( C ) Schematic of S . Typhimurium LPS biosynthesis and mutant strains used in ( D – F ). The LPS biosynthesis pathway generates penta-acylated LPS, which is converted to hexa-acylated LPS by the acyltransferase MsbB (LpxM); the Δ msbB mutant retains penta-acylated LPS. ( D ) qPCR analysis of lipid metabolism mRNAs ( Cd36 , Fabp1 , and Apoa4 ) in small intestines of germ-free or antibiotic-treated mice infected by intragastric gavage with parental wild-type (WT) or mutant S . Typhimurium IR715 strains shown in ( C ) (n = 5 to 8 mice per group). ( E ) Mass spectrometry–based lipidomic analysis of IECs from uninfected or WT or mutant S . Typhimurium IR715–infected mice is shown as bar plots. Relative abundance of 14:0, 16:0, and 18:0 fatty acid–containing glycerolipids is shown (n = 3 to 4 mice per group). ( F ) In vivo uptake of C16:0 BODIPY. Antibiotic-treated mice were infected with parental WT or mutant S . Typhimurium IR715 strains and orally gavaged with C16:0 BODIPY. IEC fluorescence was quantified by flow cytometry. Representative histograms and MFI quantification are shown (n = 6 to 11 mice per group). LPS, lipopolysaccharide; PAMPs, pathogen-associated molecular patterns; i.p., intraperitoneal; Abx-treated, antibiotic-treated; qPCR, quantitative real-time PCR; sm. int., small intestine; LTA, lipoteichoic acid; IEC, intestinal epithelial cell; Veh, vehicle; S . Tm, Salmonella Typhimurium; Uninf., uninfected; WT, wild-type. MFI, median fluorescence intensity. Data shown in panels A , B , D , and F are representative of 2 to 3 independent experiments. Each bar graph data point represents one mouse. Significance was determined by one-way ANOVA.

Article Snippet: For i.p. administration, mice received 40 ng/g body weight of ultrapure flagellin (InvivoGen, cat. #tlrl-epstfla), LPS (InvivoGen, cat. #tlrl-pb5lps), or LTA (InvivoGen, cat. #tlrl-pslta).

Techniques: Injection, In Vivo, Fluorescence, Flow Cytometry, Mutagenesis, Infection, Mass Spectrometry, Real-time Polymerase Chain Reaction