Oxidation of HMGB1 Is a Dynamically Regulated Process in Physiological and Pathological Conditions

Abstract

Acute inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens or cell damage, and is essential for immune defense and proper healing. However, unresolved inflammation can lead to chronic disorders, including cancer and fibrosis. The High Mobility Group Box 1 (HMGB1) protein is a Damage-Associated Molecular Pattern (DAMP) molecule that orchestrates key events in inflammation by switching among mutually exclusive redox states. Fully reduced HMGB1 (frHMGB1) supports immune cell recruitment and tissue regeneration, while the isoform containing a disulphide bond (dsHMGB1) promotes secretion of inflammatory mediators by immune cells. Although it has been suggested that the tissue itself determines the redox state of the extracellular space and of released HMGB1, the dynamics of HMGB1 oxidation in health and disease are unknown. In the present work, we analyzed the expression of HMGB1 redox isoforms in different inflammatory conditions in skeletal muscle, from acute injury to muscle wasting, in tumor microenvironment, in spleen, and in liver after drug intoxication. Our results reveal that the redox modulation of HMGB1 is tissue-specific, with high expression of dsHMGB1 in normal spleen and liver and very low in muscle, where it appears after acute damage. Similarly, dsHMGB1 is highly expressed in the tumor microenvironment while it is absent in cachectic muscles from the same tumor-bearing mice. These findings emphasize the accurate and dynamic regulation of HMGB1 redox state, with the presence of dsHMGB1 tightly associated with leukocyte infiltration. Accordingly, we identified circulating, infiltrating, and resident leukocytes as reservoirs and transporters of dsHMGB1 in tissue and tumor microenvironment, demonstrating that the redox state of HMGB1 is controlled at both tissue and cell levels. Overall, our data point out that HMGB1 oxidation is a timely and spatially regulated process in physiological and pathological conditions. This precise modulation might play key roles to finetune inflammatory and regenerative processes.

Keywords: cancer cachexia; inflammation; injury; leukocyte; liver; muscle; regeneration; tumor.

Figure 1

HMGB1 redox isoforms expression and leukocyte infiltration during acute muscle injury. (A) Representative images of immunohistochemical staining for CD45 (upper panel) and HMGB1 (lower panel) on tibialis anterior (TA) muscle sections at indicated time points after cardiotoxin (CTX) injection. Scale bars, 50 μm. Ctrl, uninjured control muscles. (B–E) Western blot probed with anti-CD45 (upper panel) and anti-HMGB1 (middle panel) antibodies in reducing conditions or with anti-HMGB1 antibody in non-reducing conditions (lower panel) on muscle lysates at indicated time points after CTX injection. The upper and lower bands in non-reducing conditions correspond to the fully reduced-HMGB1 (frHMGB1) and the disulphide-HMGB1 (dsHMGB1) isoforms, respectively. (C) Quantification of total HMGB1 protein expression levels, relative to control (Ctrl) and normalized on Ponceau staining, at indicated time points after CTX injection. A.U. = arbitrary unit (n ≥ 10 muscles, 3 mice/time point). (D) Quantification of CD45 and HMGB1 redox isoforms expression (frHMGB1 and dsHMGB1), normalized on Ponceau staining, at indicated time points. (E) Distribution of HMGB1 redox isoforms expression in muscle lysates in absence (controls and at 1 h post-injury) or presence of CD45-positive cells (from 6 h to day 7 post-injury). (F–I) Western blot probed with anti-HMGB1 antibody in reducing (upper panel) and non-reducing conditions (lower panel) on supernatant of muscles isolated at indicated time points after CTX injection (F). Total HMGB1 protein expression at indicated time points after CTX injection (G). A.U. = arbitrary unit (n = 6 muscle supernatants, 3 mice/time point). (H) Quantification of HMGB1 redox isoforms expression (frHMGB1 and dsHMGB1) in muscle supernatants, from Western blot assays in non-reducing conditions, at indicated time points after CTX injection and compared with CD45 expression as in (D). (I) Distribution of HMGB1 redox isoforms expression in supernatants of muscle in absence (controls and at 1 h post-injury) or presence of CD45-positive cells (from 6 h to day 7 post-injury). Data represent the means ± SEM and statistical significance was calculated by One-way (C,D,G,H) and Two-way ANOVA (E,I). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2

Disulphide-HMGB1 derives from non-myogenic cells in injured muscle. (A) Western blot probed with anti-CD45 (upper panel) and anti-HMGB1 (lower panel) antibodies in reducing conditions on tibialis anterior (TA) muscle lysates from WT or HMGB1 mKO mice at indicated time points after cardiotoxin (CTX) injection. Ctrl, control uninjured muscles. (B,C) Quantification of CD45 (B) and HMGB1 (C) protein expression, normalized on Ponceau staining, before (Ctrl) and after CTX injection (CTX at 1, 2, and 7 d) in TA and triceps muscle lysates (n ≥ 4 muscles/time point, n = 3 mice/genotype). A.U. = arbitrary unit. (D,E) Western blot probed with anti-HMGB1 antibody in non-reducing conditions (D) on TA muscle lysates from WT or HMGB1 mKO mice at indicated time points after CTX injection. The upper band corresponds to the fully reduced-HMGB1 (frHMGB1) and the lower band to the disulphide-HMGB1 (dsHMGB1). (E) Percentage of HMGB1 redox isoforms expression from WT or HMGB1 mKO mice before (Ctrl) and after CTX injection (CTX at 1, 2, 5, and 7 d) in TA and triceps muscle lysates (n ≥ 3 muscles/time point; n ≥ 4 mice/genotype). Data represent the means ± SEM and statistical significance was calculated by Student T-test (B,C) and Two-way ANOVA (E). **P < 0.01; ***P < 0.001; ^##P < 0.01 (Ctrl vs. CTX).

Figure 3

High expression of disulphide-HMGB1 in human leukocytes. (A) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions (upper panels) or probed with anti-HMGB1 antibody in non-reducing conditions (lower panel) on peripheral blood mononuclear cells (PBMCs) isolated from four healthy human donors. The upper band corresponds to the fully reduced-HMGB1 (frHMGB1) and the lower band to the disulphide-HMGB1 (dsHMGB1) in the lower panel. (B) Quantification of HMGB1 redox isoforms expression normalized on Ponceau staining. A.U. = arbitrary unit (n = 4 healthy donors). (C,D) Western blot probed with anti-HMGB1 antibody in non-reducing conditions on PBMCs stimulated with anti-CD3/anti-CD28 antibodies or lipopolysaccharide (LPS) for 24 or 72 h (C). Percentage of HMGB1 redox isoforms expression (D). Ctrl, control unstimulated cells (n = 2 healthy donors). Data represent the means ± SEM and statistical significance was calculated by Two-way ANOVA (D).

Figure 4

Leukocytes operate as transporter of dsHMGB1 in tumor microenvironment. (A) Representative images of immunohistochemical staining for CD45 (upper panel) and HMGB1 (lower panel) on tibialis anterior (TA) muscle sections from control (Ctrl) vs. Lewis lung carcinoma (LLC)-bearing mice. Scale bars, 50 μm. (B,C) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions (B), and quantification of total CD45 and HMGB1 protein levels normalized on GAPDH (C) (n = 4 mice). In (B), spleen lysate (5 μg) was added as positive control for CD45 expression. (D) Western blot probed with anti-HMGB1 antibody in non-reducing conditions on tibialis anterior (TA) lysates from control or LLC-bearing mice. The upper and lower bands in non-reducing conditions correspond to the fully reduced-HMGB1 (frHMGB1) and the disulphide-HMGB1 (dsHMGB1) isoforms, respectively. (E) Percentage of HMGB1 redox isoforms expression. A.U. = arbitrary unit (n = 4 mice/group). (F) Immunohistochemical staining for CD45 (upper panel) and HMGB1 (lower panel) on tumoral sections from LLC-bearing mice. Scale bars, 50 μm. (G–J) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions on LLC cells and tumoral masses isolated from mice injected with LLC cells (G), and quantification of total CD45 and HMGB1 protein levels normalized on GAPDH (H). (I) Western blot probed with anti-HMGB1 antibody in non-reducing conditions on LLC cells and tumoral masses isolated from mice injected with LLC cells. (J) Percentage of HMGB1 redox isoforms expression in LLC cultured cells and tumoral masses from LLC-injected mice (J). A.U. = arbitrary unit (n = 5 cell replicates and n = 4 mice for tumoral masses). Data represent the means ± SEM and statistical significance was calculated by Student T-test (C,H) and Two-way ANOVA (E,J). *P < 0.05; ****P < 0.0001.

Figure 5

Redox modulation of HMGB1 in spleen and in drug-intoxicated liver. (A) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions (upper panels) or probed with anti-HMGB1 antibody in non-reducing conditions (lower panel) on lysates of spleen and liver isolated from control WT mice. In the lower panel, the upper band corresponds to the fully reduced-HMGB1 (frHMGB1) and the lower band to the disulphide-HMGB1 (dsHMGB1). (B,C) Quantification of total CD45 and HMGB1 protein levels normalized on GAPDH (B), and HMGB1 redox isoforms percentage (C) in spleen and liver lysates. A.U. = arbitrary unit (n = 4 mice/group). (D–G) Drug-induced liver injury (DILI) was induced by i.p. injection of acetaminophen (APAP), 300 mg/kg (body weight). Serum collection and necroscopy were performed at the indicated time points. (D) Representative images of DAPI and Evans Blue (EB) staining, Haematoxylin & Eosin (H&E) staining, and CD45 and HMGB1 immunostaining in liver sections from control mice (Ctrl) and at days 1, 2, 3, and 7 after DILI. Scale bars, 50 μm. (E) Alanine aminotransferase (sALT) and HMGB1 levels in serum before and after APAP injection in mice (n ≥ 5 mice/group). (F) Quantification of total number of intrahepatic leukocytes (IHLs) in control mice and at days 1 and 2 post-APAP injection (n = 4 mice/group). (G) Quantification of HMGB1 redox isoforms percentage, from Western blot assays performed in non-reducing conditions with anti-HMGB1 antibody, in IHLs isolated from control mice and at days 1 and 2 post-APAP injection (n = 4 mice/group). Data represent the means ± SEM and statistical significance was calculated by Student T-test (B), One-way (E,F) and Two-way ANOVA (C,G). ***P < 0.001; ****P < 0.0001; ns, not significant.