Journal: bioRxiv

Article Title: Glutamine is required for M1-like polarization in response to Mycobacterium tuberculosis infection

doi: 10.1101/2022.01.11.475775

Figure Lengend Snippet: BMDMs were infected with Mtb , and cells and supernatants were collected at different times for single-cell mRNA analysis by sm-RNA-FISH and for measurement of glutamine uptake/utilization, respectively. Glutaminase (GLS) inhibitor BPTES was added to sets of cultures for GLS inhibition experiments at a final concentration of 10 μM. For rescue experiments, 1.5 mM dimethyl α-ketoglutarate (DMKG) was added to sets of cultures treated with the inhibitor. High rate of glutamine uptake/utilization by Mtb -infected macrophages corresponding to the M1-like polarization ( A ). Cell culture supernatants collected at the indicated times were subjected to glutamine determination using the Glutamine/Glutamate-Glo (TM) Assay kit (Promega, Madison, WI). Kinetics of glutamine uptake/utilization (μm per hour per well) were calculated based on its changes in the culture medium. Data are shown as means ± SDs from three independent experiments. Increased mRNA molecules for glutaminase gene Gls in M1-like macrophages ( B ). Gls mRNA molecules in infected BMDMs were detected and analyzed by sm-RNA-FISH as in . Diminished M1-like polarization by GLS inhibition with BPTES, and alleviation of the inhibition by treatment with DMKG ( C ). mRNA molecules in infected BMDMs, with or without 10 μM BPTES and/or 1.5 mM of DMKG at 8 hrs p.i were analyzed for Il1b, Nos2, Glut1, Mct4 , and Pfkfb3 by sm-RNA-FISH as in . Enhanced Mtb growth by GLS inhibition with BPTES ( D ). CFU of Mtb was determined by plating assay of cell lysates of infected BMDMs with indicated treatments at day 3 p.i. Dampened M1 polarization in THP-1 GLS KO cells ( E - F ). Wild-type THP1 cells, and GLS KO cells, generated by the Synthego Corporation (Menlo Park, CA), were subjected to differentiation and Mtb infection. mRNA expression of IL1B and GLUT1 were analyzed by sm-RNA-FISH ( E ) as in . FISH data are shown as means ± 95% CI and represent three independent experiments. Mitochondrial mass was evaluated using MitoView Fix 640 (Biotium) by flow cytometry (left) and quantified (right) ( F ). Data are shown as means ± SDs from three independent experiments. Statistical significance at * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 was based on two-tailed Student’s T-test.

Article Snippet: Glutamine in culture supernatant was determined using the Glutamine/Glutamate-Glo (TM) Assay kit (Promega, Madison, WI) and used for calculating the kinetics of glutamine uptake/utilization, following manufacturer’s instructions.

Techniques: Infection, Inhibition, Concentration Assay, Cell Culture, Generated, Expressing, Flow Cytometry, Two Tailed Test