Journal: International Journal of Molecular Sciences

Article Title: Drug-Tolerant Mycobacterium tuberculosis Adopt Different Survival Strategies in Alveolar Macrophages of Patients with Pulmonary Tuberculosis

doi: 10.3390/ijms241914942

Figure Lengend Snippet: M. tuberculosis ( Mtb ) survive a long-term antimicrobial chemotherapy in TB patients and resume active growth in alveolar macrophages of some patients in the prolonged ex vivo cell cultures after withdrawal of anti-TB drugs. ( a ) Mtb are acid-fast after Ziehl–Neelsen (ZN) staining and produce the virulence factors LAM, ESAT-6, and Ag38 stained with appropriate specific antibodies (green or red signals) in alveolar macrophages after ex vivo culture for 18 h and on histological sections obtained from the resected lung tuberculoma wall of patient 24 after treatment with multiple antibiotics before surgery. Black, green, and red arrows point to acid-fast, LAM- or ESAT-6-positive, and LAM- or Ag38-positive Mtb , respectively, on representative cytochemical and confocal 3D immunofluorescent images. Double arrows point to filamentous Mtb in colonies with cording morphology. Nuclei are stained by DAPI (blue signal). Collagen fibers are strongly autofluorescent (green signal) on histological immunofluorescent images. The scale bars are 10 μm each; ( b – d ) Mtb loads are estimated in the ex vivo cultures of alveolar macrophages obtained from the cavity (cav) or tuberculoma (tub) walls and the tissues distant (dist or without labeling) from the macro-TB lesions of the patients’ resected lung parts; ( b , c ) The number of alveolar macrophages with any Mtb (solitary or as colonies, including those with cording morphology), with the colonies of Mtb , including the cords, and with Mtb only in cord-colonies in them, all expressed as the percentage of the total number of alveolar macrophages stained by the ZN method, represents the Mtb load in host cells of patients after ex vivo culture for 16–18 h (16–18 h) and from two to eight days (D2–D8) without treatment with anti-TB drugs, and ( c ) after three days of ex vivo exposure to different concentrations of antibiotics, such as isoniazid (H), rifampicin (R), ofloxacin (Of), kanamycin (K), or capreomycin (Cap). Red arrows point to a trend towards increase in the number of Mtb -infected alveolar macrophages in the prolonged ex vivo cell cultures at D2–D8 versus 18 h. The numbers of patients, whose prolonged ex vivo cell cultures were characterized by this trend, are marked by red ovals; ( c ) Exposure of the patients’ alveolar macrophages to different concentrations of antibiotics does not eliminate drug-resistant or drug-susceptible Mtb in host cells in ex vivo culture during three days of treatment with anti-TB drugs; ( d ) Quantification of the alveolar macrophages with a particular number of Mtb in them expressed as the percentage of the total number of the Mtb -infected alveolar macrophages done for the patients, whose numbers are marked by red ovals in ( b , c ), after ex vivo culture for 18 h and D2–D6. Red and blue arrows point to trends towards, respectively, an increase and a decrease in the number of alveolar macrophages with a particular number of Mtb in them after ex vivo culture for various time points.

Article Snippet: Primary antibodies were against Mycobacteria LAM (Abcam, England, ab20832, 1:200 dilution), Mtb ESAT-6 (courtesy of E.V.

Techniques: Ex Vivo, Staining, Labeling, Infection

Journal: International Journal of Molecular Sciences

Article Title: Drug-Tolerant Mycobacterium tuberculosis Adopt Different Survival Strategies in Alveolar Macrophages of Patients with Pulmonary Tuberculosis

doi: 10.3390/ijms241914942

Figure Lengend Snippet: M. tuberculosis ( Mtb ) with a distinct morphology re-establish in alveolar macrophages obtained from the cavity wall of patient 6 in the prolonged ex vivo cell cultures after withdrawal of anti-TB drugs. ( a ) Representative cytochemical and confocal 3D immunofluorescent images demonstrate Mtb with a distinct morphology, which are acid-fast after ZN staining and express LAM, stained with specific antibodies (green signal), in the patient’ alveolar macrophages after ex vivo culture at different time points. Single green and red arrows point to Mtb with a long filamentous and a shorter rod-shaped morphology, respectively. Double black and green arrows point to colonies with cording morphology and filamentous Mtb in them. Nuclei are stained by DAPI (blue signal). The scale bars are 10 μm each; ( b ) The number of viable and dead alveolar macrophages with different numbers of Mtb in colonies with cording morphology in them is expressed as the percentage of the total number of infected alveolar macrophages after ex vivo culture at all time points; ( c ) The number of alveolar macrophages with any short rod-shaped Mtb (solitary or as colonies) and with the colonies of Mtb with a shorter rod-shaped morphology are expressed as the percentage of the total number of the patient’ alveolar macrophages with acid-fast Mtb (filamentous or as rod-shaped) in them after ex vivo culture at all time points; ( d ) The number of viable alveolar macrophages with a particular number of filamentous Mtb (solitary or as colonies), with short rod-shaped Mtb (solitary or as colonies), and with the colonies of short rod-shaped Mtb in them expressed as the percentage of the total number of the patient’ alveolar macrophages with a particular number of acid-fast Mtb (filamentous or as rod-shaped) in them after a long-term ex vivo culture for D3 and D6.

Article Snippet: Primary antibodies were against Mycobacteria LAM (Abcam, England, ab20832, 1:200 dilution), Mtb ESAT-6 (courtesy of E.V.

Techniques: Ex Vivo, Staining, Infection

Journal: International Journal of Molecular Sciences

Article Title: Drug-Tolerant Mycobacterium tuberculosis Adopt Different Survival Strategies in Alveolar Macrophages of Patients with Pulmonary Tuberculosis

doi: 10.3390/ijms241914942

Figure Lengend Snippet: M. tuberculosis ( Mtb ) clinical isolates obtained from the lung tissues of patients 6 and 10 produce the universal stress protein Rv2623 only in stationary phase (D90), but not in exponential phase (D20 after replating onto fresh L–J medium) cultures on dense L–J medium. Representative cytochemical images of Mtb after ZN staining and the merged confocal immunofluorescent and phase contrast images of Mtb stained with appropriate specific antibodies (green and red signals) are demonstrated. White short and long arrows on the merged images and profile graphs, respectively, point to the same Rv2623-positive Mtb with no LAM production in them. Yellow arrows on the merged images point to the areas for constructing profile graphs. The scale bars are 10 μm each.

Article Snippet: Primary antibodies were against Mycobacteria LAM (Abcam, England, ab20832, 1:200 dilution), Mtb ESAT-6 (courtesy of E.V.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Drug-Tolerant Mycobacterium tuberculosis Adopt Different Survival Strategies in Alveolar Macrophages of Patients with Pulmonary Tuberculosis

doi: 10.3390/ijms241914942

Figure Lengend Snippet: Rv2623-positive Mtb not expressing the virulence factors LAM and ESAT-6 are identified in the alveolar macrophages of TB patients. ( a ) Representative confocal 3D immunofluorescent images stained simultaneously with appropriate specific antibodies demonstrate LAM- or ESAT-6-positive Mtb (green signal) not expressing the Rv2623 protein and, vice versa, Rv2623-positive Mtb (red signal) not expressing the virulence factors LAM and ESAT-6 in the patients’ alveolar macrophages on the histological sections and after ex vivo culture for 16–18 h. Close-ups of the parts of the images with Mtb , solitary and in colonies, including those with cording morphology, are shown in the upper left or right corners. Green and red arrows point to the LAM- or ESAT-6- and Rv2623-positive Mtb , respectively; ( a , c ) Mtb are characterized in alveolar macrophages obtained from the cavity (cav) and tuberculoma (tub) walls and the tissues distant (dist or without labeling) from the macro-TB lesions of the patients’ resected lung parts. To the right of the 3D immunofluorescent images and ( c ) the image after ZN re-staining: profile images of the arrow-marked Mtb are shown. Yellow arrows point to the areas for constructing profile graphs. Nuclei are stained by DAPI (blue signal). Double arrows point to Mtb in colonies with cording morphology; ( b ) The number of alveolar macrophages or caseous regions with any Rv2623-positive Mtb (single or as colonies) and with the colonies of Rv2623-positive Mtb , both expressed as the percentage of the total number of host cells or caseous regions with LAM- or ESAT-6- and Rv2623-positive Mtb in them on the histological sections. The total data of two dual-staining on the LAM/Rv2623- and ESAT-6/Rv2623-markers of Mtb for each patient are used for the construction of the graph. Mean ± SEM. Below the graph, the table presents four groups of patients with different TB lesions and characteristics, including the extent of fibrosis and activation of stress factors. Symbols (+), (++), and (+++) indicate activation of stress factors in a single, rare, and the majority of the alveolar macrophages examined; ( c ) Representative confocal 3D immunofluorescent images demonstrate the absence of intracellular lipophilic inclusions stained with Nile red dye (red signal), in the Rv2623-positive Mtb stained with specific antibodies (green signal), in the patients’ alveolar macrophages after ex vivo culture for 16–18 h. Green and black arrows point to Rv2623-positive Mtb and acid-fast Mtb in colonies with cording morphology, respectively. The scale bar is 10 μm.

Article Snippet: Primary antibodies were against Mycobacteria LAM (Abcam, England, ab20832, 1:200 dilution), Mtb ESAT-6 (courtesy of E.V.

Techniques: Expressing, Staining, Ex Vivo, Labeling, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Drug-Tolerant Mycobacterium tuberculosis Adopt Different Survival Strategies in Alveolar Macrophages of Patients with Pulmonary Tuberculosis

doi: 10.3390/ijms241914942

Figure Lengend Snippet: Rv2623-positive M. tuberculosis ( Mtb ) reveal an acid-fast-negative phenotype in alveolar macrophages of patients and guinea pig with TB disease. Representative confocal 3D or single immunofluorescent images of host cells stained with antibodies reacting with the Mtb LAM or ESAT-6 (green signal) and Mtb Rv2623 (red signal) markers, and images of the same host cells after ZN re-staining (ZN) demonstrate Rv2623-positive Mtb , solitary and as colonies, with an acid-fast-negative phenotype in the patients’ and guinea pig’s alveolar macrophages after ex vivo culture for 18–20 h. Nuclei are stained by DAPI (blue signal). Close-ups of the parts of the images with Mtb , solitary and in colonies, including those with cording morphology, are shown in the upper left or right corners. Green and red arrows point to the LAM- or ESAT-6- and Rv2623-positive Mtb , respectively. Brown short arrows point to the host cells with the Rv2623-positive Mtb in them. Black arrows point to the acid-fast Mtb on the ZN images. Double arrows point to Mtb in colonies with cording morphology Mtb are characterized in the ex vivo cultures of alveolar macrophages and, in parallel, on the histological sections obtained from the cavity (cav) and tuberculoma (tub) walls and the tissues distant (dist or without labeling) from the macro-TB lesions of the patients’ resected lung parts. The scale bars are 10 μm each.

Article Snippet: Primary antibodies were against Mycobacteria LAM (Abcam, England, ab20832, 1:200 dilution), Mtb ESAT-6 (courtesy of E.V.

Techniques: Staining, Ex Vivo, Labeling

Journal: Nanoscale Advances

Article Title: Thymoquinone-loaded lipid nanocapsules with promising anticancer activity for colorectal cancer †

doi: 10.1039/d3na00445g

Figure Lengend Snippet: Effect of TQ and TQ-loaded LNCs on immune system response. Levels of (A) GM-CSF (ng mL −1 ), and (B) TFN-α (pg mL −1 ) for the different treated groups (a: P < 0.05 versus control group, b: P < 0.05 versus TQ group).

Article Snippet: Tumor cytokine levels were measured in a homogenate of 50 mg of tumor mass in 1 mL of PBS using a commercial ELISA kit for GM-CSF (Abcam, ab174448) and TNF-α (Abcam, ab208348).

Techniques:

Journal: Acta Neuropathologica

Article Title: Inflammation and the pathological progression of Alzheimer’s disease are associated with low circulating choline levels

doi: 10.1007/s00401-023-02616-7

Figure Lengend Snippet: A choline deficient diet (Ch-) results in both elevated amyloidosis and hyperphosphorylated tau in 3xTg-AD mice. Correlations between choline, ACh, TNFα and the various forms of amyloid and pathological tau epitopes in 3xTg-AD mice. a – d In the cortex of 3xTg-AD mice, the Ch− diet elevated soluble Aβ 42 , insoluble Aβ 40 , insoluble Aβ 42 , soluble pTau Thr 181, insoluble pTau Thr 181, soluble pTau S 396, and insoluble pTau S 396. e – r Correlations between plasma choline, cortex choline, and soluble and insoluble Aβ 40 and Aβ 42 , soluble and insoluble pTau Thr 181, and soluble and insoluble pTau S 396. s – y Correlations between plasma TNFα and soluble and insoluble Aβ 40 and Aβ 42 , soluble and insoluble pTau Thr 181, and soluble and insoluble pTau S 396. Data are reported as means ± SEM. * P < 0.05, **** P < 0.0001

Article Snippet: We used commercially available ELISA kits (Invitrogen-ThermoFisher Scientific) to quantify levels of soluble and insoluble Aβ 40 and Aβ 42 , and levels of phosphorylated Tau (pTau) at threonine (Thr) 181 and serine (S) 396, and TNFα (Abcam, ab208348) as previously described [ , ].

Techniques:

Journal: Frontiers in Immunology

Article Title: Suppression of microglial Ccl2 reduces neuropathic pain associated with chronic spinal compression

doi: 10.3389/fimmu.2023.1191188

Figure Lengend Snippet: Depletion of Ccl2 in microglia alters pain-associated cytokines and factors in spine ELISA for known cytokines and factors related to pain. (A) Il1β. (B) TNFα. (C) IFNɣ. (D) Il6. (E) Il17. (F) ARG1. (G) CD163. (H) TGFβ1. (I) Il10. *p<0.05. ns, non-significant.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of mouse Ccl2 (ab208979; Abcam, Hangzhou, China), mouse Il1β (ab197742; Abcam), mouse tumor necrosis factor alpha (TNFα, ab208348; Abcam), mouse interferon gamma (IFNɣ, ab282874; Abcam), mouse Il6 (ab100712, Abcam), mouse Il17 (ab100702; Abcam), mouse arginase 1 (ARG1, ab269541; Abcam), mouse CD163 (ab272204, Abcam), mouse transforming growth factor β1 (TGFβ1, ab119557, Abcam) and mouse Il10 (ab108870, Abcam) in cell lysis.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: HDAC3 promotes macrophage pyroptosis via regulating histone deacetylation in acute lung injury

doi: 10.1016/j.isci.2023.107158

Figure Lengend Snippet: HDAC3 contributed to LPS-induced inflammatory response in macrophages (A) Western blot analysis of HDAC3 in differentiated THP-1 cells transfected with three pairs of si- Hdac3 and si- Gapdh (n = 6). (B–E) Relative mRNA levels of Tnf-α, IL-6, Ccl-2, and IL-1β in Hdac3-knockdown THP-1 cells following LPS/nigericin induction (n = 6). (F) Cell viability in Hdac3-knockdown THP-1 cells following LPS/nigericin induction (n = 6). (G) Western blot analysis of HDAC3 in differentiated THP-1 cells transduced with Ad- Hdac3 . (H–K) Relative mRNA levels of Tnf-α, IL-6, Ccl-2, and IL-1β in Ad- Hdac3 -transduced THP-1 cells following LPS/nigericin induction (n = 6). (L) Cell viability in Ad- Hdac3 -transduced THP-1 cells following LPS/nigericin induction (n = 6). Values represent the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the matched groups, n.s. represents no significance.

Article Snippet: TNF-α ELISA kits , Abcam , Cat# ab285312, ab208348.

Techniques: Western Blot, Transfection, Transduction

Journal: iScience

Article Title: HDAC3 promotes macrophage pyroptosis via regulating histone deacetylation in acute lung injury

doi: 10.1016/j.isci.2023.107158

Figure Lengend Snippet: Comparison of the severity of lung pathological and inflammation between HDAC3-C and HDAC3-CKO mice after LPS induction (A) HDAC3 flox/flox mice were generated by inserting two LoxP sequences in the same direction into the introns flanked with the exon 4, 5, 6, and 7 of HDAC3 using CRISPR-Cas9 system, producing a nonfunctional HDAC3 protein. Lyz2-CreERT2 transgenic mice were then crossed with HDAC3 flox/flox mice to generate the macrophage-specific HDAC3-knockout mice, named as Lyz2-CreERT2 + -HDAC3 flox/flox . (B and C) Western blot analysis of HDAC3 in lung tissues and primary macrophages from HDAC3-C and HDAC3-CKO mice (n = 6). (D and E) Histological analyses of the H&E staining and lung injury score of HDAC3-C and HDAC3-CKO mice after LPS induction (n = 6). (F) Lung wet/dry weight (n = 6). (G–I) Relative mRNA levels of IL-6, IL-1β, and Tnf-α in HDAC3-C and HDAC3-CKO mice after LPS induction (n = 6). (J) Immumohistochemical staining of IL-6 in lung tissues. (K) The protein levels of IL-18, IL-1β, and TNF-α in the obtained bronchial lavage fluid detected by ELISA (n = 6). Values represent the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the matched groups, n.s. represents no significance.

Article Snippet: TNF-α ELISA kits , Abcam , Cat# ab285312, ab208348.

Techniques: Generated, CRISPR, Transgenic Assay, Knock-Out, Western Blot, Staining, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: HDAC3 promotes macrophage pyroptosis via regulating histone deacetylation in acute lung injury

doi: 10.1016/j.isci.2023.107158

Figure Lengend Snippet: cGAS overexpression abolished the inhibition of LPS-induced inflammation and pyroptosis by HDAC3 knockdown in macrophage (A–C) Relative mRNA levels of Tnf-α, IL-6, and Ccl-2 in the indicated groups (n = 6). (D–E) IL-1β and IL-18 content in the indicated groups (n = 6). (F) Western blot analysis of cGAS, P-STING, T-STING, NLRP3, and GSDMD-N in the indicated groups (n = 6). Values represent the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the matched groups, n.s. represents no significance.

Article Snippet: TNF-α ELISA kits , Abcam , Cat# ab285312, ab208348.

Techniques: Over Expression, Inhibition, Western Blot

Journal: iScience

Article Title: HDAC3 promotes macrophage pyroptosis via regulating histone deacetylation in acute lung injury

doi: 10.1016/j.isci.2023.107158

Figure Lengend Snippet: miR-4767 antagomir abolished the protective roles of HDAC3 deficiency in macrophage in LPS-treated mice (A and B) Histological analyses of the H&E staining and lung injury score of LPS-treated HDAC3-CKO mice injected with antagomir NC or miR-4767 antagomir (n = 6). (C) Lung wet/dry weight (n = 6). (D) The level of miR-4767 in lung tissues (n = 6). (E and F) Relative mRNA levels of Tnf-α and IL-6 in LPS-treated HDAC3-CKO mice injected with antagomir NC or miR-4767 antagomir (n = 6). (G) Western blot analysis of cGAS, P-STING, T-STING, NLRP3, and GSDMD-N in LPS-treated HDAC3-CKO mice injected with antagomir NC or miR-4767 antagomir (n = 6). Values represent the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the matched groups, n.s. represents no significance.

Article Snippet: TNF-α ELISA kits , Abcam , Cat# ab285312, ab208348.

Techniques: Staining, Injection, Western Blot

Journal: iScience

Article Title: HDAC3 promotes macrophage pyroptosis via regulating histone deacetylation in acute lung injury

doi: 10.1016/j.isci.2023.107158

Figure Lengend Snippet: Intratracheal administration of Hdac3 siRNA-loaded liposomes prevented LPS-induced ALI in mice (A) A schematic diagram showing the preparation of Hdac3 siRNA-loaded liposomes. (B) Representative IVIS images of the mouse treated R-labeled liposomes as well as ex vivo fluorescence images of heart, liver, spleen, lung, and kidney from mice. (C) Temporal Hdac3 mRNA level alterations in the lung tissues from mice treated with Hdac3 siRNA-loaded or scrambled siRNA-loaded liposomes (n = 6). (D) A schematic diagram showing experimental design of LPS-treated mice administered with either Hdac3 siRNA-loaded or Scrambled siRNA-loaded liposomes (n = 6). (E and F) Histological analyses of the H&E staining and lung injury score of LPS-treated WT mice administered with either Hdac3 siRNA-loaded or Scrambled siRNA-loaded liposomes. (n = 6). (G) Lung wet/dry weight (n = 6). (H and I) Relative mRNA levels of Tnf-α and IL-6 in LPS-treated WT mice administered with either Hdac3 siRNA-loaded or Scrambled siRNA-loaded liposomes. (n = 6). (J) The level of miR-4767 in lung tissues (n = 6). (K) Western blot analysis of cGAS, P-STING, T-STING, NLRP3, and GSDMD-N in LPS-treated mice administered with either Hdac3 siRNA-loaded or Scrambled siRNA-loaded liposomes (n = 6). Values represent the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the matched groups, n.s. represents no significance.

Article Snippet: TNF-α ELISA kits , Abcam , Cat# ab285312, ab208348.

Techniques: Labeling, Ex Vivo, Fluorescence, Staining, Western Blot

Journal: iScience

Article Title: HDAC3 promotes macrophage pyroptosis via regulating histone deacetylation in acute lung injury

doi: 10.1016/j.isci.2023.107158

Figure Lengend Snippet:

Article Snippet: TNF-α ELISA kits , Abcam , Cat# ab285312, ab208348.

Techniques: Recombinant, CCK-8 Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay