Journal: Hepatology Communications

Article Title: Post-translational modifications drive the effects of HMGB1 in alcohol-associated liver disease

doi: 10.1097/HC9.0000000000000549

Figure Lengend Snippet: Hmgb1 ablation in hepatocytes or myeloid cells partially protects, while ablation in both prevents steatosis, inflammation, IL1B production, and alcohol-induced liver injury. WT, Hmgb1 ΔHep , Hmgb1 ΔMye , and Hmgb1 ΔHepΔMye mice were fed the LDC diets for 6 weeks. H&E staining showing steatosis (black arrows) and inflammation (yellow arrows) (A). The liver-to-body weight ratio, serum ALT activity (U/L), liver TG (µg/mg), and the histopathological scores (steatosis, hepatocyte ballooning degeneration, inflammation) (B). IHC for HMGB1, where the orange arrows indicate ablation of HMGB1 in either hepatocytes, myeloid cells, or both (insets: ×630). IHC shows NASDCA + (green arrows), F4/80 + (blue arrows), or IL1B + (pink arrows) cells (C). Quantitative HMGB1 morphometry analysis (left). NASDCA, F4/80, and IL1B indexes (number of positive cells in 10 fields at ×200, right) (D). Quantification of liver IL1B (pg/mg) by ELISA (E). Heatmaps of IL1R and NFκB pathways (F). Results are expressed as mean ± SEM. n = 8/group. * p < 0.05, ** p < 0.01, and *** p < 0.001 for Hmgb1 ΔHep , Hmgb1 ΔMye , and Hmgb1 ΔHepΔMye versus WT; • p < 0.05 and •• p < 0.01 for ethanol-fed versus control diet-fed. Abbreviations: H&E, hematoxylin and eosin; HMGB1, high-mobility group box-1; Hmgb1 ΔHep , conditional Hmgb1 knockout mice in hepatocytes; Hmgb1 ΔMye , conditional Hmgb1 knockout mice in myeloid cells; Hmgb1 ΔHepΔMye , conditional Hmgb1 knockout mice in hepatocytes and myeloid cells; IHC, immunohistochemistry; LDC, Lieber-DeCarli; NASDCA, naphthol AS-D chloroacetate; TG, triglyceride.

Article Snippet: To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and Rage ΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGBiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.

Techniques: Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Knock-Out, Immunohistochemistry

Journal: Hepatology Communications

Article Title: Post-translational modifications drive the effects of HMGB1 in alcohol-associated liver disease

doi: 10.1097/HC9.0000000000000549

Figure Lengend Snippet: Neutralization of HMGB1 prevents, whereas injection of [H] or [O] HMGB1 worsens AALD. Schematic representation of the HMGB1 protein structure where cysteines (C) can undergo oxidation are written in yellow, and lysines (K) that can undergo acetylation are written in blue. These residues were mutated in the constructs to engineer the AAV8 vectors or in the conditional knockin mice (A). WT mice were injected with a pan-HMGB1 neutralizing Ab, [H] HMGB1, or [O] HMGB1 throughout the ethanol feeding regimen of 6 weeks. IHC shows NASDCA + (green arrows), F4/80 + (blue arrows), or IL1β + (pink arrows) cells in these mice (B). NASDCA, F4/80, and IL1β indexes (number of positive cells in 10 fields at ×200) (left) and concentration of HMGB1 in serum (ng/dL) (right) (C). Results are expressed as mean ± SEM. n = 8/group. * p < 0.05, ** p < 0.01 and *** p < 0.001 for cotreated versus ethanol + BSA. Abbreviations: AAV8, adeno-associated viruses serotype-8; BSA, bovine serum albumin; HMGB1, high-mobility group box-1; [H] HMGB1, native or fully-reduced HMGB1; [O] HMGB1, disulfide HMGB1; NASDCA, naphthol AS-D chloroacetate; WT, wild-type.

Article Snippet: To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and Rage ΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGBiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.

Techniques: Neutralization, Injection, Construct, Knock-In, Concentration Assay

Journal: Hepatology Communications

Article Title: Post-translational modifications drive the effects of HMGB1 in alcohol-associated liver disease

doi: 10.1097/HC9.0000000000000549

Figure Lengend Snippet: [O] HMGB1 induces liver injury with higher inflammation, whereas [Ac] HMGB1 protects from AALD. Hmgb1 ΔHepΔMye and Hmgb1 ΔHep mice were transduced with AAV8 vectors containing the cDNA encoding for WT or for mutations of HMGB1 to prevent oxidation (Δ[O] HMGB1), acetylation (Δ[Ac] HMGB1), or both (Δ[O + Ac] HMGB1) of the protein. Two weeks later, mice were fed with the ethanol LDC diet for 6 weeks. H&E staining shows steatosis (black arrows), hepatocyte ballooning degeneration (open black arrow), and inflammation (yellow arrows) (A). The liver-to-body weight ratio, serum ALT activity (U/L), liver TG (µg/mg), and the histopathological scores (steatosis, hepatocyte ballooning degeneration, inflammation) (B). IHC shows HMGB1 expression (orange arrows), NASDCA + (green arrows), F4/80 + (blue arrows), or IL1B + (pink arrows) cells in these mice (C, D). Quantitative HMGB1 morphometric analysis (left). NASDCA, F4/80, and IL1β indexes (number of positive cells in 10 fields at ×200, right) (E). Results are expressed as mean ± SEM. n = 8/group. * p < 0.05, ** p < 0.01, and *** p < 0.001 for Δ[O], Δ[Ac], or Δ[O + Ac] HMGB1 versus WT HMGB1; • p < 0.05 for Hmgb1 ΔHep versus Hmgb1 ΔHepΔMye . Abbreviations: AALD, alcohol-associated liver disease; AAV8, adeno-associated viruses serotype-8; H&E, hematoxylin and eosin; HMGB1, high-mobility group box-1; [Ac] HMGB1, acetylated HMGB1; [O] HMGB1, disulfide HMGB1; Hmgb1 ΔHep , conditional Hmgb1 knockout mice in hepatocytes; Hmgb1 ΔMye , conditional Hmgb1 knockout mice in myeloid cells; Hmgb1 ΔHepΔMye , conditional Hmgb1 knockout mice in hepatocytes and myeloid cells; IHC, immunohistochemistry; NASDCA, naphthol AS-D chloroacetate; TG, triglyceride.

Article Snippet: To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and Rage ΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGBiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.

Techniques: Transduction, Staining, Activity Assay, Expressing, Knock-Out, Immunohistochemistry

Journal: Hepatology Communications

Article Title: Post-translational modifications drive the effects of HMGB1 in alcohol-associated liver disease

doi: 10.1097/HC9.0000000000000549

Figure Lengend Snippet: [O] HMGB1 induces liver injury, whereas [Ac] HMGB1 protects from AALD. [WT] Hmgb1 KI Hep and mice with conditional overexpression of different HMGB1 isoforms (Δ[O] Hmgb1 KI Hep , Δ[Ac] Hmgb1 KI Hep , Δ[O + Ac] Hmgb1 KI Hep ) were fed the ethanol LDC diet for 6 weeks. H&E staining shows steatosis (black arrows) and inflammation (yellow arrows) (A). The liver-to-body weight ratio, serum ALT activity (U/L), liver TG (µg/mg), and histopathological scores (steatosis, hepatocyte ballooning degeneration, and inflammation) are shown (B). Results are expressed as mean ± SEM. n = 8/group. * p < 0.05 for Δ[O], Δ[Ac], or Δ[O + Ac] HMGB1 versus [WT] HMGB1; • p <0.05 and •• p <0.01 for ethanol versus control. Abbreviations: AALD, alcohol-associated liver disease; H&E, hematoxylin and eosin; HMGB1, high-mobility group box-1; [Ac] HMGB1, acetylated HMGB1; [O] HMGB1, disulfide HMGB1; [WT] Hmgb1 KI Hep , conditional WT Hmgb1 knockin mice in hepatocytes; Δ[Ac] Hmgb1 KI Hep , conditional Δ[Ac] Hmgb1 knockin mice in hepatocytes; Δ[O] Hmgb1 KI Hep , conditional Δ[O] Hmgb1 knockin mice in hepatocytes; Δ[O + Ac] Hmgb1 KI Hep , conditional Δ[O + Ac] Hmgb1 knockin mice in hepatocytes; LDC, Lieber-DeCarli; TG, triglyceride.

Article Snippet: To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and Rage ΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGBiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.

Techniques: Over Expression, Staining, Activity Assay, Control, Knock-In

Journal: Hepatology Communications

Article Title: Post-translational modifications drive the effects of HMGB1 in alcohol-associated liver disease

doi: 10.1097/HC9.0000000000000549

Figure Lengend Snippet: [O] HMGB1 stimulates MF migration and activation. RAW 264.7 cells were plated on cell culture inserts, each HMGB1 isoform or BSA (control) was added at a concentration of 1 nM, and cells were cultured for 24 hours. Hematoxylin staining of cells that migrated to the bottom side of the cell culture insert membrane (A). Western blot analysis of intracellular and secreted IL1B under the same culture conditions as in panel (A, B). The experiment was repeated 3 times. *** p < 0.001 for [H] or [O] HMGB1 versus BSA. Abbreviations: BSA, bovine serum albumin; [H] HMGB1, native or fully-reduced HMGB1; [O] HMGB1, disulfide HMGB1; MF, macrophage.

Article Snippet: To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and Rage ΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGBiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.

Techniques: Migration, Activation Assay, Cell Culture, Control, Concentration Assay, Staining, Membrane, Western Blot

Journal: Hepatology Communications

Article Title: Post-translational modifications drive the effects of HMGB1 in alcohol-associated liver disease

doi: 10.1097/HC9.0000000000000549

Figure Lengend Snippet: Ethanol-fed Rage ΔMye but not Tlr4 ΔMye , Rage ΔHep , or Tlr4 ΔHep mice are protected from AALD. WT, Rage ΔHep , Tlr4 ΔHep , Rage ΔMye , and Tlr4 ΔMye mice were fed the ethanol LDC diet for 6 weeks. H&E staining shows steatosis (black arrows) and inflammation (yellow arrows) (A). The liver-to-body weight ratio, serum ALT activity (U/L), liver TG (µg/mg), and the histopathological scores (steatosis, hepatocyte ballooning degeneration, and inflammation) (B). IHC shows HMGB1 expression (orange arrows), NASDCA + (green arrows), F4/80 + (blue arrows), or IL1B + (pink arrows) cells in these mice (C). Quantitative HMGB1 morphometry analysis and concentration of HMGB1 in serum (ng/dL) (left). NASDCA, F4/80, and IL1β indexes (number of positive cells in 10 fields at ×200, right) (D). In the HMGB1 morphometry, the quantification was expressed as fold-change of the WT, which was assigned a value of 1; the rest of the results are given as mean ± SEM. n = 8/group. * p < 0.05, ** p < 0.01, and *** p < 0.001 for ethanol-fed Rage ΔHep , Tlr4 ΔHep , Rage ΔMye , and Tlr4 ΔMye versus WT. Abbreviations: AALD, alcohol-associated liver disease; H&E, hematoxylin and eosin; HMGB1, high-mobility group box-1; IHC, immunohistochemistry; LDC, Lieber-DeCarli; NASDCA, naphthol AS-D chloroacetate; Rage ΔHep , conditional Rage knockout mice in hepatocytes; Rage ΔMye , conditional Rage knockout mice in myeloid cells; WT, wild-type; Tlr4 ΔHep , conditional Tlr4 knockout mice in hepatocytes; Tlr4 ΔMye , conditional Tlr4 knockout mice in myeloid cells; TG, triglyceride.

Article Snippet: To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and Rage ΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGBiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.

Techniques: Staining, Activity Assay, Expressing, Concentration Assay, Immunohistochemistry, Knock-Out

Journal: Hepatology Communications

Article Title: Post-translational modifications drive the effects of HMGB1 in alcohol-associated liver disease

doi: 10.1097/HC9.0000000000000549

Figure Lengend Snippet: Hepatocyte-derived [O] HMGB1 signals through RAGE in myeloid cells to drive AALD. Hmgb1&Rage ΔHepΔMye mice were transduced with AAV8 vectors containing the cDNA encoding for WT or mutations of HMGB1 to prevent oxidation (Δ[O] HMGB1), acetylation (Δ[Ac] HMGB1), or both (Δ[O+Ac] HMGB1) of the protein. Two weeks later, mice were fed the ethanol LDC diet for 6 weeks. H&E staining showing steatosis (black arrows) and inflammation (yellow arrows) (A). The liver-to-body weight ratio, serum ALT activity (U/L), liver TG (µg/mg), and histopathological scores (steatosis, hepatocyte ballooning degeneration, inflammation) are shown (B), n = 16/group. Abbreviations: AALD, alcohol-associated liver disease; AAV8, adeno-associated virus serotype-8; H&E, hematoxylin and eosin; HMGB1, high-mobility group box-1; Δ[Ac] HMGB1, acetylation-incompetent HMGB1 mutant; Δ[O] HMGB1, oxidation-incompetent HMGB1 mutant; Hmgb1&Rage ΔHepΔMye , conditional Hmgb1 and Rage knockout mice in hepatocytes and myeloid cells; [O] HMGB1, disulfide HMGB1; LDC, Lieber-DeCarli; RAGE, receptor for advanced-glycation end-products; WT, wild-type; TG, triglyceride.

Article Snippet: To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and Rage ΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGBiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.

Techniques: Derivative Assay, Transduction, Staining, Activity Assay, Virus, Mutagenesis, Knock-Out

Journal: Journal of Toxicologic Pathology

Article Title: High mobility group box1 as a danger signal inducing the infiltration of neutrophils and macrophages in thioacetamide-induced rat liver injury

doi: 10.1293/tox.2024-0055

Figure Lengend Snippet: ( a–d ) The liver samples from rats treated with TAA and IgY (TAA+IgY) and ( e–h ) with TAA and HMGB1 neutralizing antibody (TAA+αHMHG1). Treatment with IgY or αHMHG1 were administered at 6 h after TAA injection via the tail vein. ( a, e ) At 0 h no histological changes were observed in the livers of the TAA+IgY group and TAA+αHMHG1 group. ( b, f ) At 12 h after TAA administration, slight hepatocellular necrosis with presence of some inflammatory cells was observed in the centrilobular areas. ( c, g ) At 18 h, a smaller number of injured hepatocytes and inflammatory cells were observed in the TAA+αHMHG1 group compared with the TAA+IgY group. ( d, h ) At 24 h, more extensive hepatocellular necrosis and infiltration of inflammatory cells were observed; however, the inflammation and hepatocyte injury were less severe in the TAA+αHMHG1 group compared with that in the TAA+IgY group. HMGB1: high-mobility group box-1; TAA: thioacetamide; αHMGB1: HMGB1 neutralizing antibody. CV: central vein; H&E: hematoxylin and eosin.

Article Snippet: At 6 h after TAA injection, the rats were injected with an HMGB1 neutralizing antibody derived from chickens (αHMGB1; 300 μg/rat; Shino-Test, Tokyo, Japan) via the tail vein.

Techniques: Injection

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Molecular targets in bone cancer pain: a systematic review of inflammatory cytokines

doi: 10.1007/s00109-024-02464-2

Figure Lengend Snippet: Comprehensive overview of the key characteristics of the included studies

Article Snippet: [ ] , Wistar rats (adult, female, 180–200 g) , Walker 256 TIBIA , HMGB1 , Intrathecal administration • polyclonal neutralizing antibodies against HMGB1 , ▪ 50% PWT , Spinal cord lumbar SDH HMGB1, IL-1β (WB) , After TCI ↑ HMGB1 ↑ IL-1β ↑ mechanical allodynia After blocking HMGB1 ↓IL-1β ↓ mechanical allodynia.

Techniques: Expressing, Activation Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Injection, Plasmid Preparation, Small Interfering RNA, Over Expression, Translocation Assay, Clinical Proteomics, Staining

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Three experimental designs were used to study the impact of HMGB1 on mitochondria during ODE exposure. For acute exposure, primary NHBE cells were transfected with HMGB1 targeted siRNA to induce knockdown followed by exposure to either media (control) or ODE (1%) for 24 h (a). For chronic exposure, NHBE cells were treated with media alone (control) or ODE (1%) followed by media (control) or HMGB1 neutralization antibody (10 μg/mL) for 8 h per day for five days (b). Air-liquid interface (ALI) culture model was developed by seeing NHBE cells onto a semi-permeable membrane to develop and the differentiated cells were exposed to ODE, with or without HMGB1 neutralization for 1 h per day for 5 days (c).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Transfection, Knockdown, Control, Neutralization, Membrane

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Stock and working concentrations of treatments

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Concentration Assay, Neutralization

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Transmission electron microscopy (TEM) of NHBE cells treated with medium or ODE (1%) followed by medium or HMGB1 neutralization antibody (10 μg/mL) for 8 h per day for 5 days show changes in the mitochondrial morphology. A number of mitochondria displayed fused and elongated mitochondrial morphology (a-a’’; scale bar, 0.5–1 μm) on treatment with ODE. Cells treated with ODE followed by HMGB1 neutralization showed noticeably healthier mitochondria with a few morphological changes. Mitochondrial mass was measured (c) and the morphology was visualized by staining with Mito-tracker dye (b-b’’; Scale bar = 100 μm). MTT assay was performed to measure cell viability on treatment (d). Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 6/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Transmission Assay, Electron Microscopy, Neutralization, Staining, MTT Assay, Comparison, Control

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Markers of mitochondrial biogenesis in NHBE cells with or without siRNA mediated HMGB1 knockdown and treated with ODE (1%) for 24 h was measured. Immunoblotting of whole cell lysates of NHBE cells was performed to measure PGC1α (a, b) and NRF2 expression (c, d). TFAM expression was compared in the mitochondrial (e, f) and cytosolic (e, g) fractions of the NHBE cells. mRNA fold change of gene targets downstream of NRF2, gstp1 (h), hmox1 (i), and nqo1 (j), was measured by qPCR. All the protein bands were normalized over β-actin (37 kD) and percentage intensity relative to control was analyzed. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Knockdown, Western Blot, Expressing, Control, Comparison

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Changes in mitochondrial morphology in NHBE cells with or without siRNA mediated HMGB1 knockdown and treated with ODE (1%) for 24 h was assessed. Immunoblotting was performed to measure the mitochondrial fusion proteins MFN1 (a, b), MFN2 (c, d) and OPA1 (e, f) and fission protein DRP1 (g, h) and compared. Mitochondrial mass was measured (i) and the morphology was visualized by staining with Mito-tracker dye (j-j’’’; Scale bar = 100 μm). All the protein bands were normalized over β-actin (37 kD) and percentage intensity relative to control was analyzed. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3–6/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Knockdown, Western Blot, Staining, Control, Comparison

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Immunoblotting of NHBE cells, with or without siRNA mediated HMGB1 knockdown and treated with ODE (1%) for 24 h, was performed to measure expression of mitophagy markers, PINK1 (a, b) and Parkin (c, d), and BNIP3 (e, f). All the protein bands were normalized over β-actin (37 kD) and percentage intensity relative to control was analyzed. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Western Blot, Knockdown, Expressing, Control, Comparison

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Mitochondrial DNA (mtDNA) leakage into the cytosol (a) and extracellularly (b) in NHBE cells, with or without siRNA mediated HMGB1 knockdown and treated with ODE (1%) for 24 h was analyzed via qPCR. Immunoblotting was performed to measure expression of TLR9 (c, d), cGAS (e, f), IFI16 (g, h) and IRF3 (i, j). mRNA fold change of gene targets of mtDNA release, ifna1 (k), ifna4 (l), and ifnb (m), was measured by qPCR. All the protein bands were normalized over β-actin (37 kD) and percentage intensity relative to control was analyzed. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Knockdown, Western Blot, Expressing, Control, Comparison

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: NHBE cells differentiated on air-liquid interface (ALI) culture treated with medium or ODE (1%) followed by medium or HMGB1 neutralization antibody (10 μg/mL) for 1 h per day for 5 days. mRNA fold change of markers of motile cilia, foxj1 (a) and cfap157 (b), and tight junction, ocln (c) and cldn 1 (d), was measured by qPCR. Tight junction integrity was assessed by trans-epithelial electrical resistance (TEER) measurement (e). Samples for all assays were derived from the same experiment and were processed in parallel. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Neutralization, Derivative Assay, Comparison, Control

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: mRNA expression profiles of genes specific to mitochondrial respiration in NHBE cells treated with medium or ODE (1%) followed by medium or HMGB1 neutralization antibody (10 μg/mL) for 8 h per day for 5 days was measured using a targeted gene array. Heatmap of mRNA transcripts of genes that had a 10-fold change in expression were selected (a). Selected upregulated (b, d) and downregulated (c, e) pathways from reactome pathway analysis of significantly altered mRNA transcripts with FDR corrected p-value cut-off <0.05 were selected and represented by gene counts. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 2/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Expressing, Neutralization, Comparison, Control

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Immunoblotting of mitochondrial and mitochondria-free cytosolic fractions of NHBE cells with or without siRNA mediated HMGB1 knockdown and treated with ODE (1%) for 24 h, to detect the presence of Cytochrome C. Cytochrome C expression was compared between the mitochondrial (a, b) and cytosolic (a, c) fractions of the cells. Concentration of secreted nitrites was measured using griess assay (d) and mRNA levels of nos2 (e) was measured by qPCR. Using CM-H2DCFDA and Mito-SOX dyes, the levels of superoxide anions (SOX) intracellularly (f) and within the mitochondria (g) was measured, respectively. Intra-mitochondrial calcium levels in mitochondria isolated from the treated cells was quantified (h) and visualized (i-i’’’; Scale bar = 100 μm) by Rhod-2AM staining. For western blot, all the protein bands were normalized over β-actin (37 kD) and percentage intensity relative to control analyzed. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3–6/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Western Blot, Knockdown, Expressing, Concentration Assay, Griess Assay, Isolation, Staining, Control, Comparison

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Markers of hypoxia response in NHBE cells with or without siRNA mediated HMGB1 knockdown and treated with ODE (1%) for 24 h was measured. Immunoblotting of whole-cell lysates of NHBE cells were performed to measure HIF1α (a, b). mRNA fold change of ubiquitin ligase, vhl (c), and slc2a6 (d) and eno1 (e), involved in glucose uptake and glycolysis, were measured by qPCR. 2-NBDG uptake to measure cellular glucose uptake was performed (f). For western blot, all the protein bands were normalized over β-actin (37 kD) and percentage intensity relative to control analyzed. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3–6/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Knockdown, Western Blot, Ubiquitin Proteomics, Control, Comparison

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: Markers of hypoxia response in NHBE cells treated with medium or ODE (1%) followed by medium or HMGB1 neutralization antibody (10 μg/mL) for 8 h per day for 5 days was measured. mRNA fold change of hypoxia response factor, hif1a (a) and ubiquitin ligase, vhl (b), and Slc2a6 (c) and Eno1 (d), involved in glucose uptake and glycolysis were measured by qPCR. 2-NBDG uptake to measure cellular glucose uptake was performed (e). Samples for all assays were derived from the same experiment and were processed in parallel. Data was analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and represented as mean ± SEM with n = 3–6/treatment (* indicates significant difference from control).

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Neutralization, Ubiquitin Proteomics, Derivative Assay, Comparison, Control

Journal: Cell and tissue research

Article Title: Transcriptomic and Ultrastructural Evidence Indicate That Anti-HMGB1 Antibodies Rescue Organic Dust Induced Mitochondrial Dysfunction

doi: 10.1007/s00441-022-03602-3

Figure Lengend Snippet: HMGB1 increases autophagy, inhibits apoptosis, and regulates mitochondria functions. ODE exposure promoted mitochondrial fusion and mtDNA release promoting a pro-inflammatory response through TLR9, NFκB, and IRF3 signaling pathways resulting in pro-inflammatory cytokine release. On the other hand, HMGB1 knockdown or use of Anti-HMGB1 antibody rescues ODE-induced mtDNA release and promotes mitochondrial fission. ODE exposure also induces a decrease in OXPHOS and glucose uptake while increasing ROS generation and mitochondrial membrane permeability. HMGB1 knockdown or use of Anti-HMGB1 antibody treatment decreases ROS generation, promotes mitochondrial biogenesis, and upregulates markers associated with OXPHOS.

Article Snippet: We would like to thank Dr. Kevin Tracey (Feinstein Institutes for Medical Research, Northwell Health, NY) for providing us with the anti-HMGB1 neutralization antibody and Dr. Y.S.

Techniques: Protein-Protein interactions, Knockdown, Membrane, Permeability