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Journal: Frontiers in Immunology

Article Title: Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani

doi: 10.3389/fimmu.2021.632512

Figure Lengend Snippet: Glycolysis stress test profile of L. donovani -infected neutrophils. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C and 5% CO 2 . Uninfected cells served as control. The infection rate was determined by Giemsa staining of cytocentrifuged samples. Subsequently, free, non-ingested parasites were removed by washing. After 6 h post infection the glycolysis stress test profile was determined by using the Seahorse extracellular flux analyzer (A) . Extracellular acidification rate (ECAR) measurements following the sequential injection of 5 mM glucose, 1 μM oligomycin, and 10 mM 2-DG (dotted lines indicate injection time) were used to calculate key parameters of glycolytic function. Glycolysis (B) was calculated by subtraction of 2-DG-mediated ECAR from glucose-mediated ECAR. Glycolytic capacity (C) was calculated by subtraction of 2-DG-mediated ECAR from oligomycin-mediated ECAR. Bar graphs show mean ± SD ( n = 8), ** p ≤ 0.01.

Article Snippet: The preparations contained ≥99% granulocytes, of which >95% were neutrophils and 1–4 % were eosinophils, as determined by Giemsa staining (Diff Quick Fix, Medion Diagnostics, Berlin, Germany) of cytocentrifuged (Shandon) samples.

Techniques: Infection, Control, Staining, Injection

Journal: Frontiers in Immunology

Article Title: Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani

doi: 10.3389/fimmu.2021.632512

Figure Lengend Snippet: Mitochondrial stress test profile of L. donovani -infected neutrophils. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C and 5% CO 2 . Uninfected cells served as control. Successful infection was determined by Giemsa staining of cytocentrifuged samples. Subsequently, free, non-ingested parasites were removed by washing. After 6 h post infection the mitochondrial stress test profile was determined by using the Seahorse extracellular flux analyzer (A) . The measurement of basal oxygen consumption rate (OCR) was followed by sequential injections of 1 μM oligomycin, 1.5 μM FCCP, and 1 μM rotenone/antimycin A (dotted lines indicate injection time). OCR measurements were used to calculate key parameters of mitochondrial function. Non-mitochondrial respiration (B) was calculated as OCR after rotenone/antimycin A injection. Basal respiration (C) was calculated by subtraction of rotenone/antimycin A-mediated OCR from basal OCR. Maximal respiration (D) was calculated by subtraction of rotenone/antimycin A-mediated OCR from FCCP-mediated OCR. Proton leak (E) was calculated by subtraction of non-mitochondrial respiration from oligomycin-mediated OCR. ATP production (F) was calculated by subtraction of oligomycin-mediated OCR from basal OCR. Bar graphs show mean ± SD ( n = 3), ** p ≤ 0.01, ns, not significant.

Article Snippet: The preparations contained ≥99% granulocytes, of which >95% were neutrophils and 1–4 % were eosinophils, as determined by Giemsa staining (Diff Quick Fix, Medion Diagnostics, Berlin, Germany) of cytocentrifuged (Shandon) samples.

Techniques: Infection, Control, Staining, Injection

Journal: Frontiers in Immunology

Article Title: Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani

doi: 10.3389/fimmu.2021.632512

Figure Lengend Snippet: 2-NBDG uptake of L. donovani -infected neutrophils. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C and 5 % CO 2 . Uninfected cells served as control. The infection rate was determined by Giemsa staining of cytocentrifuged samples. Subsequently, free, non-ingested parasites were removed by washing. After 6 h post infection the fluorescent glucose analog 2-NBDG was added to the cells in glucose-free medium for the last 10 min of incubation time and 2-NBDG uptake was analyzed by flow cytometry. The bar diagram shows the autofluorescence corrected mean fluorescence intensity (MFI) of 2-NBDG uptake ± SD ( n = 3), *** p ≤ 0.001.

Article Snippet: The preparations contained ≥99% granulocytes, of which >95% were neutrophils and 1–4 % were eosinophils, as determined by Giemsa staining (Diff Quick Fix, Medion Diagnostics, Berlin, Germany) of cytocentrifuged (Shandon) samples.

Techniques: Infection, Control, Staining, Incubation, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani

doi: 10.3389/fimmu.2021.632512

Figure Lengend Snippet: Lactate secretion and pyruvate content of L. donovani -infected neutrophils. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C and 5% CO 2 . Uninfected cells served as control. The infection rate was determined by Giemsa staining of cytocentrifuged samples. Subsequently, free, non-ingested parasites were removed by washing. Whole cell lysates were generated after 6 h post infection by boiling cells at 90°C for 5 min. Cell-free supernatants were collected after 6 h post infection. Lactate was detected in culture supernatants by using a lactate assay kit. Pyruvate was detected in whole cell lysates by using a pyruvate assay kit. Bar diagrams show mean concentration of lactate (A) after subtraction of the medium blanks and pyruvate (B) calculated by interpolation from standard curve ± SD ( n = 5), * p ≤ 0.05, **** p ≤ 0.0001.

Article Snippet: The preparations contained ≥99% granulocytes, of which >95% were neutrophils and 1–4 % were eosinophils, as determined by Giemsa staining (Diff Quick Fix, Medion Diagnostics, Berlin, Germany) of cytocentrifuged (Shandon) samples.

Techniques: Infection, Control, Staining, Generated, Lactate Assay, Pyruvate Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani

doi: 10.3389/fimmu.2021.632512

Figure Lengend Snippet: ATP concentration in L. donovani -infected neutrophils as response to metabolic inhibitors. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C and 5% CO 2 . Uninfected cells served as control. The infection rate was determined by Giemsa staining of cytocentrifuged samples. Subsequently, free, non-ingested parasites were removed by washing. After 6 h post infection cells were treated with 10 mM 2-DG for 3 h at 37°C and 5% CO 2 . PBS served as solvent control. Whole cell lysates were prepared and the ATP concentration was determined by using the ATP determination kit. Bar diagrams show the mean ATP concentration ± SD ( n = 3) in uninfected (A) and L. donovani -infected neutrophils (B) , * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The preparations contained ≥99% granulocytes, of which >95% were neutrophils and 1–4 % were eosinophils, as determined by Giemsa staining (Diff Quick Fix, Medion Diagnostics, Berlin, Germany) of cytocentrifuged (Shandon) samples.

Techniques: Concentration Assay, Infection, Control, Staining, Solvent

Journal: Frontiers in Immunology

Article Title: Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani

doi: 10.3389/fimmu.2021.632512

Figure Lengend Snippet: Survival of L. donovani promastigotes in 2-DG-treated neutrophils. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C, 5% CO 2 . The infection rate was determined by Giemsa staining of cytocentrifuged samples. After removing the free, non-ingested parasites the infected cells were treated with PBS, 5 mM, 50 mM, or 100 mM 2-DG. Survival of parasites was assessed after 24 h post infection by using the limiting dilution assay. The bar diagram shows the mean survival rates (%) normalized to PBS-treated control cells ± SD ( n = 3), **** p ≤ 0.0001.

Article Snippet: The preparations contained ≥99% granulocytes, of which >95% were neutrophils and 1–4 % were eosinophils, as determined by Giemsa staining (Diff Quick Fix, Medion Diagnostics, Berlin, Germany) of cytocentrifuged (Shandon) samples.

Techniques: Infection, Staining, Limiting Dilution Assay, Control

Journal: Frontiers in Immunology

Article Title: Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani

doi: 10.3389/fimmu.2021.632512

Figure Lengend Snippet: ROS production of 2-DG-treated L. donovani -infected neutrophils. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C, 5% CO 2 . The infection rate was determined by Giemsa staining of cytocentrifuged samples. After removing the free, non-ingested parasites the infected and uninfected cells were treated with PBS, 5 mM, 50 mM, or 100 mM 2-DG. After 24 h post infection the MPO-derived ROS production was measured for 1 h at 37°C and 5% CO 2 after the stimulation with 20 nM PMA by using the luminol-based chemiluminescence assay. A representative curve of luminol chemiluminescence is shown in panel (A) . The bar diagram (B) shows the mean area under the curve (AUC) values ± SD ( n = 3), * p ≤ 0.05.

Article Snippet: The preparations contained ≥99% granulocytes, of which >95% were neutrophils and 1–4 % were eosinophils, as determined by Giemsa staining (Diff Quick Fix, Medion Diagnostics, Berlin, Germany) of cytocentrifuged (Shandon) samples.

Techniques: Infection, Staining, Derivative Assay, Chemiluminescence Immunoassay