Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: MDM-1 ( A ) or MDM-2 ( B ) primed with (bottom) or without (top) 2.5 ng/mL IFN-γ, treated with 1:1000 dilution of neutralizing antibodies (Ab) against IFN-I and IFNAR, isotype control Ab (IgG2a) or medium alone (vehicle control) for 2 h pre-infection, and infected with Mtb strain H37Rv. 4 h post infection, extracellular Mtb was washed out and medium was replaced, along with the respective Abs. Cell viability was measured 4 d post infection (left) or 2 d post infection (right). The cell viability was normalized to each donors’ value for uninfected cells not exposed to antibodies. Data Information: In ( A and B ), data are presented as mean ± standard error of mean (SEM) of the normalized cell viability. Each ● or ○ represents the average value of 3 technical replicates per donor, n = 3 (except ( A ) top right: n = 4), while n indicates biological replicates. Statistical significance was determined using a paired two-tailed, t- test (* p < 0.5; ** p < 0.01; *** p < 0.001; n.s. indicates no statistical significance). .

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Control, Infection, Two Tailed Test

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: MDM-1 and MDM-2 ( n = 3) were primed with or without 2.5 ng/mL IFN-γ for 1 day and then ( A , B ) infected with Mtb strain H37Rv or ( C , D ) treated with 3 μM di-ABZI or DMSO as a vehicle control (final DMSO, 0.06%). Supernatants were collected 1 d post infection or treatment. ( A , C ) IFN-I expression in the supernatant was measured with the IFN-I HEKBlue reporter assay and quantified by performing a sigmoidal, 4 parameter interpolation from an IFN-β standard curve. ( B , D ) TNFα expression was measured by ELISA. MDM-1 ( E ) and MDM-2 ( F ) ( n = 4) were infected with Mtb H37Rv and treated with di-ABZI (0.3 μM or 3 μM) or vehicle control (DMSO, final concentration 0.06%) after Mtb was washed out at 4 h post-infection. Supernatants were collected 1 d post infection. IFN-I expression in the supernatant was measured by the IFN-I HEKBlue reporter assay and quantified by interpolating from an IFN-β standard curve. Data Information: ( A – F ) Bar graphs report the mean ± SEM. Each point represents the average value of 2–3 technical replicates per donor, while n indicates biological replicates. ( B , D ) Values too low to be interpolated were assigned a value of 1 pg/mL, and values too high to be interpolated were assigned a value of 2000 pg/mL. ( E ) Values too high to be interpolated were assigned a value of 4800 pg/mL. ( A – F ) Statistical significance was determined using a one-tailed paired, t- test (* p < 0.5; ** p < 0.01; *** p < 0.001; n.s. indicates no significance), except for the difference between MDM-1 and MDM-2 stimulated with di-ABZI ( C ), which was determined by a two-tailed, paired t -test. .

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, Control, Expressing, Reporter Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, One-tailed Test, Two Tailed Test

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: MDM-1 and MDM-2 ( n = 2) and 10 ng/mL PMA differentiated THP-1 cells and U937 cells were infected with Mtb H37Rv. 1 d post infection, supernatants were collected. ( A ) IFN-β was measured by a high sensitivity ELISA. ( B ) TNFα was measured by ELISA. ( C , D ) MDM-1 and MDM-2 ( n = 3) were primed with or without 2.5 ng/mL IFN-γ for 1 day and then ( C ) infected with Mtb strain H37Rv ( C ) or ( D ) treated with 3 μM di-ABZI. Supernatant was collected 1 d post infection. IFN-III expression in the supernatant was measured via the IFN-III HEKBlue reporter assay and quantified by performing a sigmoidal, 4 parameter interpolation from an IFN-λ1 (IL-29) standard curve. ( E , F ) MDM-2 ( n = 3) were differentiated for only 1 week before infection with Mtb H37Rv, clinical strain 1, clinical Mtb strain 2, or treatment with 3 μM ABZI. ( E ) IFN-I expression in the supernatant was measured via the IFN-I HEKBlue reporter assay, and ( F ) TNFα expression was measured via ELISA. Data Information: ( A – F ) Bar graphs report the mean ± SEM. Each ● represents an individual donor with 2–3 technical replicates per donor. Values too low to be interpolated were assigned a value of 1 pg/mL. Statistical significance was determined using a one-tailed paired, t- test (* p < 0.5; ** p < 0.01; *** p < 0.001; n.s. indicates no significance; N/A indicates not applicable). N/A was assigned if all values between two groups being tested were below the limit of detection ( E ) or if it was not possible to compute t -test because all values per group were the same ( F ). In each panel, n indicates biological replicates.

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Reporter Assay, One-tailed Test

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: ( A , B ) MDM-1 and MDM-2 ( n = 3) were infected with Mtb H37Rv, treated with 3 μM di-ABZI or vehicle controls (medium or DMSO, final concentration 0.06%). Lysates were collected 1 d post-treatment or post-infection for RNA sequencing. MDM were collected from 3 donors (MDM differentiation and Mtb infection were done with cells from 1–2 donors at a time). ( A ) Heatmap of log 2 fold changes in gene expression of stimulated/infected MDMs compared to unstimulated MDMs. ( B ) Ingenuity Pathway Analysis (QIAGEN) of the top 6 canonical pathways upregulated in Mtb-infected MDM-1 (top) and Mtb-infected MDM-2 (bottom) as determined by –log(adjusted p -value) and absolute z-scores >3. Numbers above bars display z-scores. ( C ) Mtb-infected MDM-1 ( n = 2 or 3) were treated with a 1:1000 dilution anti-IFN-I/anti-IFNAR neutralizing Abs or IgG2a isotype Ab for 2 h pre-infection, infected with Mtb H37Rv, again treated with the respective Ab after Mtb was removed by washing the monolayers 4 h post infection, and lysates were collected 1 d post infection. IFITM3 expression was measured by RT-qPCR. Data Information: Bar graphs report the mean ± SEM. Each ● represents the average value of 3–4 technical replicates per donor, while n indicates biological replicates. Statistical significance was determined using QIAGEN’s IPA analysis, which employs Fisher’s exact test and the Benjamini–Hochberg method to correct the FDR (Krämer et al, ) ( B ) and using a one-tailed paired, t- test (* p < 0.5; n.s. indicates no significance) ( C ). .

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, Concentration Assay, RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR, One-tailed Test

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: ( A ) MDM-1 and MDM-2 ( n = 3) were infected with Mtb H37Rv or treated with 3 μM di-ABZI or vehicle controls (equal volume of DMSO as in 3 μM di-ABZI). Lysates were collected 1 day post-treatment or post-infection for RNA sequencing. Volcano plots show log 2 fold changes of ISGs determined by Interferome between stimulated/infected MDM-1 compared to unstimulated MDM-1 and the corresponding −log 10 adjusted p- values. ( B ) Ingenuity Pathway Analysis (QIAGEN) of the top 6 canonical pathways upregulated in di-ABZI treated MDM-1 (left) and di-ABZI treated MDM-2 (right) determined by –log(adjusted p -value) and absolute z-scores >3. ( C ) Mtb-infected MDM-1 from 2 donors (left) and 1 donor (right) were treated with anti-IFN-I neutralizing Abs or IgG2a isotype Ab for 2 h pre-infection, infected with Mtb H37Rv and again treated with the respective Ab after Mtb was washed out 4 h post infection. Lysates were collected 1 d post infection. ISG15 expression was measured by RT-qPCR. Data Information: ( A ) Each ● represents 1 gene. Statistical significance was determined using the DESeq2 package, which uses the Wald test and Benjamini–Hochberg method to correct the FDR ( A ), and using QIAGEN’s IPA analysis, which employs Fisher’s exact test and the Benjamini–Hochberg method to control the false discovery rate (Krämer et al, ) ( B ). ( C ) Each ● represents the average value of 4 technical replicates per donor, while n indicates biological replicates.

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, RNA Sequencing, Expressing, Quantitative RT-PCR, Control

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: ( A ) MDMs ( n = 3) were differentiated under method 2, except that they were cultured without cytokines, with 10 ng/mL M-CSF, or with 0.5 ng/mL GM-CSF and TNFα for 1 or 2 weeks. MDMs were stimulated with 3 μM di-ABZI and supernatants were collected 1 d post infection. IFN-I expression in the supernatant was measured via the IFN-I HEKBlue reporter assay and quantified by performing a sigmoidal, 4 parameter interpolation from an IFN-β standard curve. Right figure displays all 3 donors. Left image displays the MDMs of 2 donors without the outlier. ( B , C ) MDMs ( n = 3) were differentiated under method 1, except that they were cultured with 10% FBS or 40% autologous human plasma and infected with Mtb H37Rv. Lysates were collected 1 d post infection. Values too low to be interpolated were assigned a value of 1 pg/mL. ( B ) IFITM3 expression was measured by RT-qPCR. ( C ) Representative images of MDM-1 (upper, left) ( n = 3) 9 days post differentiation, or MDM-2 (upper, right) ( n = 3) 16 days post differentiation isolated from different donors. Representative images ( n = 3) of MDM-1 from the same donor differentiated under method 1 (bottom, left) or MDM-1 differentiated under method 1 except in 40% autologous human plasma instead of FBS (bottom, right) 7 days after differentiation. Scale bars = 100 μm. Images were taken at 20X using phase contrast. Data Information: Bar graphs report the mean ± SEM. Each ● represents the average value of ( A ) 3 technical replicates per donor or ( B ) 3–4 technical replicates per donor. ( A , B ) Statistical significance was determined using a two-tailed paired, t- test (** p < 0.01; *** p < 0.001; n.s. indicates no significance). In each panel, n indicates biological replicates. .

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Cell Culture, Infection, Expressing, Reporter Assay, Clinical Proteomics, Quantitative RT-PCR, Isolation, Two Tailed Test

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: MDM-1 were infected with Mtb H37Rv and supernatants and lysates were collected at indicated timepoints. For the timepoint 4 h post infection, extracellular Mtb was not washed out. ( A ) IFN-I expression was measured via the IFN-I HEKBlue reporter assay and quantified by performing a sigmoidal, 4 parameter interpolation from an IFN-β standard curve. Values too low to be interpolated were assigned a value of 1 pg/mL. ( B ) IFITM3 expression was measured by RT-qPCR. ( C ) MDM-1 ( n = 1–3) were stimulated with 0.1–1000 pg/mL IFN-β, lysates were collected at indicated timepoints, and IFITM3 expression was measured by RT-qPCR. Data Information: Bar graph reports the mean ± standard error of mean (SEM) ( n = 3). Each ● represents the average value of 2 technical replicates ( A ) or 3–4 technical replicates ( B and C ) per donor, n indicates biological replicates. ( A ) Statistical significance was determined using a two-tailed paired, t- test (n.s. indicates no significance). ( B ) Correlation between time post infection and log2fold change of IFITM3 from uninfected was determined by simple linear regression of the means of the donors per timepoint (R squared = 0.8282). .

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, Expressing, Reporter Assay, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: ( A ) MDM-1 ( n = 2) were infected with Mtb H37Rv and lysates were collected at indicated timepoints. ISG15 expression was measured by RT-qPCR. ( B ) MDM-1 ( n = 1) were stimulated with 1–100 pg/mL IFN-β, supernatants were collected at indicated timepoints, and IFN-I expression was measured via the IFN-I HEKBlue reporter assay. IFN-I was quantified by performing a sigmoidal, 4 parameter interpolation from an IFN-β standard curve. ( C ) MDM-1 ( n = 1–3) were treated with IFN-β and lysates were collected at indicated timepoints. ISG15 expression was measured by RT-qPCR. Each ● represents the average value of 3–4 technical replicates per donor, while n indicates biological replicates. Data Information: Each ● represents the average value of 3–4 technical replicates per donor ( A ), the average value of 2 technical replicates per donor ( B ), and the average value of 3–4 technical replicates per donor ( C ), n indicates biological replicates.

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, Expressing, Quantitative RT-PCR, Reporter Assay

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: ( A ) IFN-β or ( B ) IFN-α were added to Mtb H37Rv infected MDM-1 ( n = 3) at 4 h post infection after extracellular Mtb was washed out. Cell viability was determined by CellTiter Glo assay at 2 d post infection. MDM-1 were primed with ( C ) IFN-β or ( D ) IFN-α; 1 day later MDM-1 ( n = 3) were infected with Mtb H37Rv. Cell viability was measured by CellTiter Glo assay at 2 d post infection. Cell viability was normalized to values for the respective uninfected, unstimulated or non-primed MDMs. The fold change of the normalized cell viability was calculated by dividing the normalized cell viability of IFN-I stimulated MDMs by that of unstimulated MDMs within the same group (uninfected or infected) per donor. ( E ) Images of MDM-1 ( n = 3) primed with IFN-β 1 day pre-infection, infected with Mtb H37Rv, and imaged at 20x at 2 d post infection. Scale bars = 20 μm. Data Information: ( A – D ) Bar graphs report the mean ± SEM of the fold change. Each ● represents the average value of 3 technical replicates per donor ( n = 3), n indicates biological replicates. Cells from each donor were tested in independent experiments. Statistical significance was determined using a paired, two-tailed t -test (* p < 0.5; ** p < 0.01; n.s. indicates no significance). .

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, Glo Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: ( A ) Representative images of MDM-1 ( n = 1) that were primed with IFN-α 1 day pre-infection, infected with Mtb H37Rv, stained with 7-aminoactinomycin D (7-AAD) and imaged at 10×2 d post infection. Scale bars = 100 μm. ( B ) MDM-2 ( n = 1) were primed with IFN-β 1 day pre-infection, infected with Mtb H37Rv, and cell viability was measured via CellTiter Glo assay. Data Information: ( B ) Dot plot reports mean value of 3 technical replicates per 1 donor, n indicates biological replicates.

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Infection, Staining, Glo Assay

Journal: EMBO Reports

Article Title: Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

doi: 10.1038/s44319-024-00171-0

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mycobacterium tuberculosis strain H37Rv , ATCC , Cat # 25618.

Techniques: Recombinant, Sequencing, Modification, Saline, Enzyme-linked Immunosorbent Assay, Cell Viability Assay, Membrane, Sterility, Pore Size, Western Blot, Stripping, Software, Spectrophotometry, Microscopy, Microplate Reader Luminescence Measurement, Autoradiography, Imaging

Journal: Journal of Clinical Microbiology

Article Title: Point-of-Care System for Detection of Mycobacterium tuberculosis and Rifampin Resistance in Sputum Samples

doi: 10.1128/JCM.02209-13

Figure Lengend Snippet: Representative Highlighter Probe melt curve data from 5 copies of genomic DNA from cultured strain H37Rv (ATCC 25618, USA) amplified using the Genedrive system. The data show first-order derivative melt peaks for M. tuberculosis at 62°C (blue), internal positive control at 54°C (green), and RIF wild type (WT) at 69°C (red).

Article Snippet: The analytical limit of detection (LOD) for the Genedrive system was determined using an M. tuberculosis strain H37Rv genomic DNA standard (ATCC 25618).

Techniques: Cell Culture, Amplification, Positive Control

Journal: Clinical Proteomics

Article Title: Antigen 85B peptidomic analysis allows species-specific mycobacterial identification

doi: 10.1186/s12014-017-9177-6

Figure Lengend Snippet: Sequence homology among reviewed Ag85B UniProt protein entries

Article Snippet: M. tuberculosis ATCC 25618/H37Rv , P9WQP1 , 100.00.

Techniques: Sequencing