Journal: mBio

Article Title: Glutamine Is Required for M1-like Polarization of Macrophages in Response to Mycobacterium tuberculosis Infection

doi: 10.1128/mbio.01274-22

Figure Lengend Snippet: Requirement of glutamine for M1-like polarization. BMDMs were infected with M. tuberculosis , and cells and supernatants were collected at various times for single-cell mRNA analysis by sm-RNA-FISH and for measurement of glutamine uptake/utilization. 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. (A) High rate of glutamine uptake/utilization by M. tuberculosis -infected macrophages corresponding to the M1-like polarization. Cell culture supernatants collected at the indicated times were subjected to glutamine determination using the glutamine/glutamate-glo assay kit. 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 ± S.D. from three independent experiments. (B) Increased mRNA molecules for glutaminase gene Gls in M1-like macrophages. Gls mRNA molecules in infected BMDMs were detected and analyzed by sm-RNA-FISH as described in Fig. 1 . (C) Diminished M1-like polarization by GLS inhibition with BPTES and alleviation of the inhibition by treatment with DMKG. mRNA molecules in infected BMDMs, with or without 10 μM BPTES and/or 1.5 mM DMKG at 8 hpi, were analyzed for Il1b , Nos2 , Glut1 , Pfkfb3 , and Mct4 by sm-RNA-FISH as described in Fig. 1 . (D) Enhanced M. tuberculosis growth by GLS inhibition with BPTES. CFU of M. tuberculosis was determined by plating assay of cell lysates of infected BMDMs with indicated treatments at day 3 p.i. (E) Increased expression of Arg1 and decreased Il1b in infected and BPTES-treated BMDMs. (F and G) Expression of Il1b and Arg1 was determined by RT-PCR and normalized to the expression level of Actb . Dampened M1 polarization in THP-1 Gls KO macrophages. Wild-type THP1 cells and Gls KO cells, generated by the Synthego Corporation, were subjected to differentiation and M. tuberculosis infection. mRNA expression of IL1B and GLUT1 was analyzed by sm-RNA-FISH (F) as described in Fig. 1 . FISH data are shown as means ±95% CI and represent three independent experiments. (G) Mitochondrial mass was evaluated using MitoView Fix 640 by flow cytometry and quantified (left: gating strategy; right: quantification). Data are shown as means ± S.D. 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: For GLS inhibition experiments, inhibitors CB-839 or BPTES (MedChemExpress LLC, Monmouth Junction, NJ) were added to sets of cultures 12 h prior to infection at a final concentration of 10 μM, according to literature ( , ).

Techniques: Infection, Single Cell, Inhibition, Concentration Assay, Cell Culture, Glo Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Flow Cytometry, Two Tailed Test