A novel mycobacterial <i>In Vitro</i> infection assay identifies differences of induced macrophage apoptosis between CD4⁺ and CD8⁺ T cells

<div><p>Macrophages are natural host cells for pathogenic mycobacteria, like <i>Mycobacterium tuberculosis (M</i>.<i>tb)</i>. Immune surveillance by T cells and interaction with <i>M</i>.<i>tb</i> infected macrophages is crucial for protection against <i>M</i>.<i>tb</i> reactivation and development of active tuberculosis. Several factors play a role in the control of <i>M</i>.<i>tb</i> infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional <i>in vitro</i> assays which allow concomitant determination of i) mycobacterial eradication; ii) cytotoxic effects on host macrophages; and iii) effector T-cell functions. We established a novel functional <i>in vitro</i> assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a <i>Mycobacterium bovis</i> BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated <i>in vitro</i> and infected with defined LD-BCG numbers. After short-term MDM/LD-BCG co-incubation with autologous effector T cells or in the presence of antibiotics, proportions of MDM containing live or dead LD-BCG were determined by flow cytometry. Concomitant measure of defined numbers of added beads allowed comparison of absolute MDM numbers between samples. Differential effects of T-cell subpopulations on anti-mycobacterial cytotoxicity and on MDM apoptosis were determined. Flow cytometry measure of MDM/LD-BCG treated with rifampicin correlated well with mycobacterial colony forming units and fluorescence microscopy results. Co-culture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. <i>M</i>.<i>tb</i> protein specific CD4⁺ and CD8⁺ T-cells contributed similarly to anti-mycobacterial cytotoxicity but CD4⁺ T cells induced higher levels of apoptosis in infected MDMs. This novel assay enables rapid quantification of anti-mycobacterial cytotoxicity and characterization of effector functions. Our functional <i>in vitro</i> assay has the potential to contribute to the identification of biomarkers for protective T-cell responses against tuberculosis.</p></div

<i>In vitro</i> generation of effector T cells (E) and co-culture with LD-BCG infected MDM (M) at different E/M ratios.

<p>(A) Workflow depiction for the generation of effector T cells, LD-BCG infected MDM, E/M co-culture, flow cytometry analyses of infected MDM and effector T cells. (B) Analyses of MDM infected with LD-BCG after co-culture with effector T cells stimulated with <i>Staphylococcus</i> Enterotoxin B (SEB), <i>M</i>. <i>tuberculosis</i> Purified Protein Derivative (PPD), and without stimulation (w/o). Different E/M ratios are shown on the x-axes. Proportions of infected MDM with live (grey) or dead LD-BCG (open) are shown as stacked boxes (left graph). Absolute numbers of infected MDM are shown as symbols (middle graph) and numbers of MDM infected with live LD-BCG are shown as boxes (right graph). The dotted line in the middle graph indicates MDM numbers infected with LD-BCG w/o effector T-cell co-culture. Median with range of triplicates from a representative experiment are depicted.</p

Proportional and absolute differences of LD-BCG infected MDM and comparison with Colony Forming Units (CFU) and fluorescence microscopy.

<p>Rifampicin treatment of MDM infected with LD-BCG at different indicated concentrations is shown. (A) Flow cytometry (FACS) analysis of MDM proportions infected with live (grey) or dead (open) LD-BCG are shown as stacked boxes. (B) Comparison of absolute MDM numbers infected with LD-BCG measured by bead corrected flow cytometry. Bead-based standardization of measured sample volume is shown in the upper graphs. Absolute numbers of all infected MDM (lower left graph) as well as MDM containing live and dead LD-BCG (lower right graph) at different rifampicin concentrations are shown. Median with range of triplicates are depicted. (C) Comparison of live LD-BCG infected MDM numbers measured by FACS and mycobacterial culture (CFU). Circles indicate FACS values adjusted for multiple infections as determined by fluorescence microscopy. Median values with range of triplicates are depicted. (D) Fluorescence microscopy analyses of MDMs infected with LD-BCG with or w/o ATC or non-infected MDM are shown. Blue color indicates MDM nuclei; red color indicates mCherry expressing LD-BCG; green color indicates GFP-expressing live LD-BCG. A representative experiment of three is shown.</p

Cytotoxic effects of total effector T cells and T-cell subpopulations against LD-BCG infected MDM and markers of induced cell death on MDM.

<p>Combined analyses of autologues E/M samples from healthy donors (n = 8) for (A), (n = 6) for (B), and (n = 5) for (C) are shown. Total effector T cells (A) or enriched CD4⁺/CD8⁺ T-cell subpopulations (B, C) were applied. (A, B) Cytotoxicity of differentially stimulated effector T cells against MDM infected with LD-BCG was assessed by flow cytometry. Different E/M ratios were applied (x-axes) and absolute numbers of MDM infected with live LD-BCG are indicated (y-axes). Mean and standard deviations are shown. (C) Apoptosis marker expression on MDM with or w/o LD-BCG infection after coculture with differentially stimulated CD4⁺ or CD8⁺ effector T cells. E:M ratios of 1:3 are shown. The proportions of non-apoptotic (open), early apoptotic (bright grey), and late apoptotic (dark grey) MDM are depicted as pie charts. Non-infected MDM (left panel) as well as MDM infected with live (middle) and dead (left) LD-BCG are shown. Median values of five independent experiments are depicted. The Mann-Whitney U-test were applied. Asterisks indicate significant differences (***: p<0.001; **: p<0.01; *: p<0.05).</p

Comparison of expression of apoptotic markers induced by differentially activated CD4⁺ and CD8⁺ T cells in MDM with live BCG (E:M 1:1)

<p>Comparison of expression of apoptotic markers induced by differentially activated CD4⁺ and CD8⁺ T cells in MDM with live BCG (E:M 1:1)</p

Antibacterial and Cytotoxic Phenolic Metabolites from the Fruits of Amopha fruticosa

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Antibacterial and Cytotoxic Phenolic Metabolites from the Fruits of Amorpha fruticosa

Fourteen new natural products, namely, 2-[(Z)-styryl]-5-geranylresorcin-1-carboxylic acid (1), amorfrutin D (2), 4-O-demethylamorfrutin D (3), 8-geranyl-3,5,7-trihydroxyflavanone (4), 8-geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone (5), 6-geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone (6), 8-geranyl-7,3′-dihydroxy-4′-methoxyisoflavone (7), 3-O-demethyldalbinol (8), 6a,12a-dehydro-3-O-demethylamorphigenin (9), (6aR,12aR,5′R)-amorphigenin (10), amorphispironones B and C (11 and 12), resokaempferol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside-7-O-α-l-rhamnopyranoside (13), and daidzein 7-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (14), together with 40 known compounds, were isolated from the fruits of Amorpha fruticosa. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic analysis as well as from the mass spectrometry data. ECD calculations were performed to determine the absolute configurations of 11 and 15. Compounds 1, 4–6, and 16–23 showed potent to moderate antibacterial activities against several Gram-positive bacteria with MIC values ranging from 3.1 to 100 μM. In addition, compounds 11 and 24–33 were significantly cytotoxic against the L5178Y mouse lymphoma cell line and exhibited IC50 values from 0.2 to 10.2 μM

Expanding the Metabolic Profile of the Fungus Chaetomium sp. through Co-culture with Autoclaved Pseudomonas aeruginosa

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