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: Vaccines

Article Title: Prophylactic Sublingual Immunization with Mycobacterium tuberculosis Subunit Vaccine Incorporating the Natural Killer T Cell Agonist Alpha-Galactosylceramide Enhances Protective Immunity to Limit Pulmonary and Extra-Pulmonary Bacterial Burden in Mice

doi: 10.3390/vaccines5040047

Figure Lengend Snippet: Sublingual immunization with Mycobacterium tuberculosis (Mtb) proteins Ag85B and ESAT-6 in the presence of alpha-galactosylceramide (α-GalCer) adjuvant induces systemic and mucosal T cell responses. Separate groups of mice ( n = 4–6) were immunized by sublingual route with one or two doses of mixture of Mtb proteins Ag85B and ESAT-6 in the presence or absence of α-GalCer adjuvant (w/aGC and w/o aGC, respectively) at 7 day intervals and sacrificed 7 days after each immunization to determine immune responses. Single cell suspensions from spleen and lung tissues were analyzed for IFN-γ producing cells specific for ESAT-6 (Panels ( A , B )) and Ag85B (Panels ( C , D )) using a standard IFN-γ ELISpot assay. Responses specific to the Mtb proteins were determined by subtracting the background values of medium stimulation from that of the individual protein stimulation and the mean ± S.D values were expressed as spot forming cells (SFC) per 10 5 input cells. Significant responses ( p < 0.05) between groups vaccinated in the presence and absence of α-GalCer (*) and between one and two doses (#) of vaccine delivered were noted. The experiment was performed using 5 mice/group and repeated 2 times.

Article Snippet: Three weeks after final immunization, the animals were infected with Mycobacterium tuberculosis Erdman (ATCC 35801) by the aerosol route (100 CFU/mouse through Glas-Col apparatus) as described previously [ ].

Techniques: Adjuvant, Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: A Dual Marker for Monitoring MDR-TB Treatment: Host-Derived miRNAs and M. tuberculosis -Derived RNA Sequences in Serum

doi: 10.3389/fimmu.2021.760468

Figure Lengend Snippet:

Article Snippet: The membranes were incubated with anti-CD63 (ab216130, Abcam, UK), -CD9, -CD81, -hsp70 ExoAb Antibody Kit (cat. EXOAB kit, System Biosciences, USA), ESAT-6 (ab26246, Abcam, U.K), -CFP-10 (PA1-19445, Thermo Fisher, USA), -Ag-38kDa (NB100-62769, Novus Biological, USA), -Ag-85B (ab43019, Abcam, U.K), and -MPT64 (CSB-PA14947A0Rb, Cusabio, USA), and then with anti-IgG-HRP (cat. EXOAB kit) or anti-IgG-HRP (65-6120, Thermo Fisher, USA).

Techniques:

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 1. Comparison of the percentage of apop- tosis and necrosis of U937 macrophages infected with M. tuberculosis H37Rv and H37Ra. The MOI used was 10.

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Comparison, Infection

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 3. Alveolar epithelial cell apoptosis after M. tuberculosis H37Rv infection. A549 cells were either uninfected (A and B) or infected (C and D) with M. tuberculosis (MOI 10) and treated with staurosporine as described in Experimental procedures. Microscopic examination of staurosporine- treated cells indicated that: (A) staurosporine-treated uninfected A549 cells underwent nuclear morphological changes typical of apoptosis as demonstrated by the TUNEL assay (B); M. tuberculosis infection before adding staurosporine suppressed staurosporine-induced apoptosis in A549 cells (C and D).

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Infection, TUNEL Assay

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 6. A and B. Western blot analysis of bad and bcl-2 proteins in macrophage and alveolar epithelial cells. U937 and A549 cells were infected with M. tuberculosis H37Rv at an MOI 1:10 or cultured with medium alone for 48 h. Concentrated cell lysate-free supernatants were subjected to Western blotting (see Experimental procedures). Line 1, A549 uninfected control; line 2, infected A549; line 3, U937 uninfected control; line 4, infected U937.

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Western Blot, Infection, Cell Culture, Control

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 7. Effect of bacterial growth inhibition by rifampicin (protein syn- thesis inhibitor) on apoptosis levels of macrophages (A) and alveolar epithelial cells (B) after M. tuberculosis H37Rv infection. U937 mac- rophages as well as A549 alveolar epithelial cells were infected with M. tuberculosis virulent strain with an MOI of 10. To one part of the cell cultures, rifampicin was added 24 h after infection at a concen- tration 0.1 mg ml-1. Apoptosis response was assessed after 3 and 5 days by Cell Death Detection ELISA. Percentage of apoptosis was calculated in infected; infected/rifampicin-treated macrophages and alveolar epithelial cell populations in comparison with uninfected; uninfected/rifampicin-treated monolayers. *Statistically significant dif- ference between groups (P < 0.05).

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Inhibition, Infection, Enzyme-linked Immunosorbent Assay, Comparison