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. 2024 Feb 27;7(3):680–692. doi: 10.1021/acsptsci.3c00271

Modulation of Abundance and Location of High-Mobility Group Box 1 in Human Microglia and Macrophages under Oxygen–Glucose Deprivation

Patrick-Brian Bielawski †, Issan Zhang †, Clara Correa-Paz ‡, Francisco Campos ‡, Martina Migliavacca §, Ester Polo §, Pablo Del Pino §, Beatriz Pelaz §, Denis Vivien ∥,⊥, Dusica Maysinger †,*
PMCID: PMC10928888  PMID: 38481701

Abstract

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While stroke represents one of the main causes of death worldwide, available effective drug treatment options remain limited to classic thrombolysis with recombinant tissue plasminogen activator (rtPA) for arterial-clot occlusion. Following stroke, multiple pathways become engaged in producing a vicious proinflammatory cycle through the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and heat shock protein 70 kDa (HSP72). HMGB1, in particular, can activate proinflammatory cytokine production when acetylated (AcHMGB1), a form that prefers cytosolic localization and extracellular release. This study aimed at determining how HMGB1 and HSP72 are modulated and affected following treatment with the anti-inflammatory compound resveratrol and novel platelet membrane-derived nanocarriers loaded with rtPA (CSM@rtPA) recently developed by our group for ischemic artery recanalization. Under ischemic conditions of oxygen–glucose deprivation (OGD), nuclear abundance of HMGB1 and AcHMGB1 in microglia and macrophages decreased, whereas treatment with CSM@rtPA did not alter nuclear or cytosolic abundance. Resveratrol treatment markedly increased the cytosolic abundance of HSP72 in microglia. Using proximity ligation assays, we determined that HSP72 interacted with HMGB1 and with acetylated HMGB1. The interaction was differentially affected under the OGD conditions. Resveratrol treatment under the OGD further decreased HSP72-HMGB1 interactions, whereas, in contrast, treatment increased HSP72-AcHMGB1 interactions in microglia. This study points out a salient molecular interaction suited for a two-pronged nanotherapeutic intervention in stroke: enhancement of rtPA’s thrombolytic activity and modulation of cytosolic interactions between HMGB1 and HSP72 by resveratrol.

Keywords: HMGB1, HSP72, ischemic stroke, OGD, resveratrol, microglia

Stroke is a major cerebrovascular disease arising from the impaired perfusion of cerebral blood vessels, causing a transient or permanent deficit in single or multiple brain regions.1,2 With 13.7 million new cases annually and representing 10% of all deaths, strokes remain a leading cause of death and disability worldwide.3 Of the two stroke subtypes, ischemic stroke, characterized by vessel occlusion, often by blood clots, accounts for nearly 85% of all strokes.4 As cerebral blood flow ceases, the availability of both glucose and oxygen is exhausted; brain tissue degrades, and neurons begin to die. It is estimated that 1.9 million neurons die for every minute that stroke is left untreated, reinforcing the adage that “time is brain.”5

Though intensive research has been undertaken to develop new drugs for stroke treatment in recent decades, the only currently FDA-approved drug for ischemic stroke treatment remains the injection of the clot-busting recombinant tissue plasminogen activator (rtPA, commercially Alteplase).6,7 The coalescence of rtPA’s limitations—short half-life of 4.5 min, limited therapeutic time window of 4.5 h from onset, risk of hemorrhagic transformations, and contraindications for patients presenting with hemorrhagic strokes, internal bleeding, recent stroke occurrence or head trauma8—begets the fact that only 1–8% of all patients receive rtPA administration,9 though rates have been found to increase up to 20% in some primary stroke centers.10

In light of these shortcomings and in an effort to improve patient outcomes, use of adjuvant treatment with rtPA and enhanced delivery of rtPA11−13 have been considered. Incorporation of rtPA into nanocarriers has been explored to increase the efficacy of rtPA thrombolysis by increasing half-life, protecting rtPA from degradation, and enhanced targeting to clot sites.14 Some studies have already investigated the functionalization of nanoparticles formed from platelet membranes.11−13,15

A novel platelet membrane-derived biomimetic nanoparticle (cellsome, CSM) containing rtPA (CSM@rtPA) was recently developed by our team. CSM@rtPA was found to be noncytotoxic, retained the thrombolytic activity of rtPA on clots without deleterious effects on red blood cells, and decreased infarct volumes in a thromboembolic stroke mouse model compared to vehicle control without presenting immunogenic interactions.16

In ischemic stroke, several types of neural cells are adversely affected by the initial infarct and the subsequent ischemic cascade.17 Microglia, as the resident central nervous system (CNS) macrophages, are quickly activated and among the first cells to respond to injury,18 playing a key role in CNS inflammation.19 Microglia engulf and dispose of dying and dead cells at the necrotic stroke core and the surrounding penumbra.20,21 In addition to microglia, monocyte-derived macrophages infiltrate the lesion site through the damaged blood–brain barrier, assisting in the clearing of cell debris.18,22 Microglia and macrophages detect dead and dying cells via released “find-me” signals such as high-mobility group box protein 1 (HMGB1) and the presence of “eat-me” signals such as phosphatidylserine on the outer leaflet of cell membranes.23 In addition to glial cells, neurons have also been found to release HMGB1 following ischemia, helping to develop or further driving neuroinflammation.24

HMGB1 is a highly conserved nonhistone nuclear protein found in all cell types and is the second-most abundant protein in the nucleus,25,26 where it facilitates the binding of several regulatory partners and is continually shuttled in and out of the nucleus.27 During conditions of stress such as infection or necrotic tissue damage, HMGB1 translocates from the nucleus to the cytosol and is released extracellularly.25,28 When located extracellularly, HMGB1 acts as a damage-associated molecular pattern (DAMP), also called an alarmin, where it mediates neuroinflammatory responses in stroke.27,29 Under ischemia, the necrotic core releases HMGB1 passively via the unregulated breakdown of the cellular membrane.25,27 Once released, HMGB1 binds to the receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs), triggering the translocation and activation of nuclear factor kappa B (NF-κB) to the nucleus to induce gene expression and subsequent proinflammatory cytokine release.30,31 These proinflammatory cytokines can activate glial cells such as microglia and macrophages to promote active release of HMGB1.29 However, the redox state and post-translational modifications (PTMs) of HMGB1 affect its localization and ultimate function.32 The acetylation of HMGB1 (AcHMGB1) has been found to favor translocation out of the nucleus and prevent its nuclear reentry, advancing a proinflammatory state.28,32

In addition to HMGB1, heat shock protein 72 (HSP72) can function as a DAMP when released extracellularly by stimulating immune and inflammatory responses following states of physiological and environmental stress such as inflammation and cellular death following stroke.33,34 Intracellularly, HSP72 has been found to have a neuroprotective effect following its induction in stroke models.35 Studies have suggested that HMGB1 might interact with HSP72,36 an interesting potential therapeutic target, as it may influence the release or retention of both HMGB1 and HSP72 following stress conditions.

This study aims at revealing how ischemic conditions simulated by oxygen–glucose deprivation (OGD) modulate the subcellular localization of the alarmin HMGB1 in two of its major forms (reduced; HMGB1 and acetylated; AcHMGB1) and HSP72. Knowing that the acetylation of HMGB1 is affected by acetylases and deacetylases, we investigated the treatment of a known inhibitor of histone deacetylase (suberoylanilide hydroxamic acid; SAHA),37 an activator of the deacetylase sirtuin 1 (resveratrol)38,39 on the abundance of HMGB1 and AcHMGB1. Next, we assessed the effect of free rtPA, encapsulated rtPA in biomimetic nanocarriers (CSM@rtPA), and empty CSMs on the expression of HMGB1 and AcHMGB1 in human microglia and macrophages. This study also investigated interactions between HSP72 and HMGB1 using a proximity ligation assay and if the extent of these interactions was affected by hypoxia and resveratrol treatment.

1. Results and Discussion

1.1. Oxygen–Glucose Deprivation Decreases Nuclear Abundance of HMGB1 and AcHMGB1 in Both Human Microglia and Macrophage Cells

Clot occlusion reduces or fully interrupts cerebral blood flow, decreasing the amounts of both glucose and oxygen available to cerebral cells. This imbalance results in cells becoming stressed and, if not remedied quickly, dying. In order to simulate stroke conditions at a cellular level, an in vitro OGD model was employed in which cells are starved of glucose and oxygen. The acetylation of HMGB1 is a major post-translational modification involved in proinflammatory cytokine production and release, nucleocytosolic translocation and release from the cell, and activation of neighboring cells, resulting in further HMGB1 release and acetylation. The effect of OGD on the subcellular compartmentalization and abundance of two forms of HMGB1 (reduced or unmodified form; HMGB1, acetylated form; AcHMGB1) has not been previously studied. Therefore, we investigated this effect in human THP-1 differentiated macrophages (Figure 1B–D) and human HMC3 microglia (Figure 1E–G). Antibody for AcHMGB1 was specific to an acetylated lysine residue of HMGB1 at Lys12. While still predominantly located in cell nuclei, acetylated HMGB1 had a lower nuclear/cytosolic ratio compared to nonacetylated HMGB1 (∼20× and ∼4.5×, respectively) in microglia and macrophages, consistent with the literature, indicating that the acetylation of lysine residues of the nuclear localization sequence sites of HMGB1 shifts the balance toward cytoplasmic accumulation by the prevention of nuclear reentry.25,28 Treatment with OGD decreased the abundance of nuclear HMGB1 and AcHMGB1 in both microglia and macrophages by half while minimally affecting the cytosolic abundance of both HMGB1 forms.

Figure 1.

Figure 1

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Modulation of HMGB1 and AcHMGB1 in human macrophages and microglia under OGD. (A) Schematic of relative changes in subcellular localization of unmodified HMGB1 and acetylated HMGB1 under normoxic conditions and OGD (hypoxic) conditions. Created with ©BioRender (biorender.com). (B–G) Immunocytochemistry for (C) HMGB1 and (D) AcHMGB1 in human THP-1 differentiated macrophages and (F) HMGB1 and (G) AcHMGB1 in human HMC3 microglia. Control samples treated with RPMI or serum-free DMEM for 4 h and OGD samples treated with RPMI without glucose or HBSS media without glucose for 4 h in an Xvivo hypoxic chamber. Samples were fixed with PFA 4%, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum for 1 h. Anti-HMGB1 rabbit polyclonal (ab18256) or anti-AcHMGB1 rabbit polyclonal (MBS9404216) 1:1000. The secondary antibody, Alexa Fluor 647 (red) Goat anti-Rabbit IgG (A-21245), was diluted at 1:1000. Nuclei (blue) labeled with Hoechst 33342 and F-actin (green) labeled with Phalloidin 488. Imaged using a fluorescent microscope, analyzed, and merged using FIJI (ImageJ). (B, E) Scale bar = 20 μm. Graphs represented as scatter plots with a black bar as mean, normalized to mean nuclear control abundance. Three independent repeats. ***p < 0.001, **p < 0.01, *p < 0.05, and ns: nonsignificant.

1.2. SAHA Increases AcHMGB1 Cytosolic Abundance in Human Microglia and Macrophages

The acetylation of HMGB1 is regulated by the expression and activity of histone deacetylases and histone acetyltransferases.40 Results are presented for human THP-1 macrophages (Figure 2A–C) and HMC3 microglia (Figure 2D–F). As observed above, cytosolic abundance was significantly smaller than nuclear abundance for both reduced and acetylated HMGB1, while AcHMGB1 cytosolic abundance was higher than HMGB1 cytosolic abundance under comparable treatments, further indicating that HMGB1 is a nuclear protein and that the acetylation of HMGB1 favors a cytosolic localization compared to reduced HMGB1. While resveratrol treatment only slightly increased nuclear reduced HMGB1 abundance in microglia only, SAHA markedly increased AcHMGB1 nuclear abundance in both macrophages (Figure 2C) and microglia (Figure 2F) by 3- to 5-fold compared to untreated control. Of note, however, that there was a lack of accompanying increases in cytosolic AcHMGB1 abundance under SAHA treatment. A possible explanation may be cell nucleocytosolic transport (from the nucleus to the cytosol), cytonuclear transport (from the cytosol to the nucleus), or extracellular release from the cytosol maintains relatively steady levels of cytosolic AcHMGB1.

Figure 2.

Figure 2

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Modulation of HMGB1 and AcHMGB1 following treatment with resveratrol and SAHA. (A–F) Immunocytochemistry for (B) HMGB1 and (C) AcHMGB1 in human THP-1 differentiated macrophages and (E) HMGB1 and (F) AcHMGB1 in human HMC3 microglia. Samples were treated with either 25 μM resveratrol or 10 μM SAHA for 4 h. Samples were fixed with PFA 4%, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum for 1 h. Anti-HMGB1 rabbit polyclonal (ab18256) or anti-AcHMGB1 rabbit polyclonal (MBS9404216) 1:1000. The secondary antibody, Alexa Fluor 647 (RED) Goat anti-Rabbit IgG (A-21245), was diluted at 1:1000. Nuclei (blue) labeled with Hoechst 33342 and F-actin (green) labeled with Phalloidin 488. Imaged using a fluorescent microscope, analyzed, and merged with FIJI (ImageJ). (A, D) Scale bar = 20 μm. Graphs represented as scatter plots with a black bar as mean, normalized to mean nuclear control abundance. Three independent repeats. ***p < 0.001, **p < 0.01, *p < 0.05, and ns: nonsignificant.

1.3. CSM@rtPA and CSM Do Not Alter Nuclear and Cytosolic Abundance of HMGB1 and AcHMGB1 in Human Microglia or Macrophage Cells

Having previously observed a lack of cytotoxic effects exerted by the CSMs and CSM@rtPA in human microglia and macrophages at concentrations of 0.01–20.0 μg/mL,16 it was decided to investigate if treatment with free rtPA and rtPA within our fabricated nanoparticles (Figure 3A) exerted an effect on one the contributors of neuronal death and inflammation, the stroke-associated alarmin HMGB1. Treatments were conducted under OGD conditions to more closely mimic stroke conditions experienced at a cellular level, namely, decreased oxygen and glucose supply. Treatment with rtPA, CSM@rtPA, and CSMs had no appreciable effect on HMGB1 and AcHMGB1 nuclear or cytosolic abundance in either THP-1 macrophages (Figure 3B–D) or HMC3 microglia (Figure 3E–G) compared with untreated OGD control. Once again, all treatments produced significantly higher nuclear localization compared to the cytosol, while the relative nuclear/cytosolic abundance HMGB1 ratio was observed to be consistently higher than that with AcHMGB1 in both cell types for all treatments. While it is promising that both loaded and empty CSMs produced no significant effects on alarmin abundance and localization, it is desirable in cases such as ischemic stroke to be able to modulate the progression of neuroinflammation through alarmin signaling.

Figure 3.

Figure 3

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Modulation of HMGB1 and AcHMGB1 following treatment with rtPA, CSM@rtPA, and CSMs in human macrophages and microglia. (A–F) Immunocytochemistry for (B) HMGB1 and (C) AcHMGB1 in human THP-1 differentiated macrophages and (E) HMGB1 and (F) AcHMGB1 in human HMC3 microglia. Samples were treated with 10 μg rtPA/mL of free rtPA, CSM@rtPA, or equivalent CSMs for 4 h in a hypoxic chamber. Samples were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum for 1 h. Anti-HMGB1 rabbit polyclonal (ab18256) or anti-AcHMGB1 rabbit polyclonal (MBS9404216) was diluted at 1:1000 and incubated overnight. Secondary antibody Alexa Fluor 647 (RED) Goat anti-Rabbit IgG (A-21245) was diluted at 1:1000 and incubated for 1 h. Nuclei (blue) were labeled with Hoechst 33342 and F-actin (green) was labeled with Phalloidin 488. Imaged using a fluorescent microscope, analyzed, and merged using FIJI (ImageJ). (A, D) Scale bar = 20 μm. Graphs represented as scatter plots with a black bar as mean, normalized to mean nuclear control abundance. Three independent repeats. ***p < 0.001, **p < 0.01, *p < 0.05, and ns: nonsignificant.

1.4. CSM@rtPA and Resveratrol Increase Cytosolic HSP72 Abundance in Microglia

While the role of HMGB1 as a DAMP is critical in the progression and resolution of neuroinflammation in ischemic stroke, it is not the only DAMP involved. HSP72 functions as a DAMP when released extracellularly by binding to Toll-like Receptor 4 (TLR4) and activating nuclear translocation of NF-κB following stressful stimuli such as stroke. In contrast, HSP72 has a protective effect when it is induced intracellularly. The next aim was to investigate whether CSMs or rtPA had an effect on the cytosolic abundance of HSP72. Samples were treated with DMEM with glucose, hydrogen peroxide (as a positive control), HBSS, rtPA, empty CSM, or resveratrol (as a sirtuin 1 activator) for 4 h. Results indicate that while hydrogen peroxide slightly increased cytosolic abundance of HSP72, resveratrol treatment markedly increased cytosolic abundance (Figure 4A,B). Resveratrol has been observed to induce HSP72 levels.41 Resveratrol acts an anti-inflammatory by preventing NF-κB nuclear translocation through its activation of SIRT1 and induction HSP72 abundance and through the inhibition of AcHMGB1 release into the cytosol (Figure 4C)

Figure 4.

Figure 4

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Modulation of the cytosolic HSP72 abundance in human microglia. (A–B) Immunocytochemistry for HSP72 in human HMC3 microglia. Samples were treated with serum-free DMEM with glucose as the control, 200 μM H2O2, 10 μg rtPA/mL of free rtPA, CSM@rtPA or CSMs, 50 μM resveratrol, or HBSS without glucose (glucose-deprived). Samples were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum for 1 h. Anti-HSP70 mouse monoclonal primary antibody (ab2787) was diluted at 1:1000 and incubated overnight. Secondary antibody Alexa Fluor 647 (RED) Goat anti-Rabbit IgG (A-21245) was diluted at 1:1000 and incubated for 1 h. Nuclei (blue) were labeled with Hoechst 33342, and F-actin (green) was labeled with Alexa Fluor phalloidin 488. Imaged using a fluorescent microscope, analyzed, and merged using FIJI (ImageJ). (F) Scale bar = 20 μm. Graphs represented as scatter plots with a black bar as mean, normalized to mean cytosolic control abundance. ***p < 0.001, **p < 0.01, *p < 0.05, and ns: nonsignificant. (C) Schematic of the action of resveratrol on HSP72. Induction of intracellular HSP72 can decrease nucleocytoplasmic translocation and release of AcHMGB1 and HMGB1.

1.5. HSP72 Interacts Differentially with HMGB1 and AcHMGB1 under OGD and Resveratrol Treatment in Microglia

Previous papers have shown that HSP72 overexpression inhibited HMGB1 release from macrophages following LPS or hydrogen peroxide treatment by inhibiting exportin 1 translocation.36,42 They also found that overexpression of HSP72 inhibited HMGB1-induced cytokine expression and release, correlating closely with NF-κB pathway inhibition. However, these papers did not distinguish between the various possible redox states and PTMs of HMGB1, including acetylated HMGB1, which play a vital role in determining the role and effect of HMGB1 extracellularly. A proximity ligation assay was employed to investigate if (1) HSP72 interacted with either form of HMGB1 and if (2) HSP72 differentially interacted with HMGB1 and AcHMGB1 under the OGD conditions and treatment with resveratrol, which was observed to increase HSP72 abundance. It has been suggested that HSP72 inhibits the translocation and activation of NF-κB.43,44 The prevention of AcHMGB1 release and maintenance of its intracellular localization may prevent subsequent proinflammatory events, mitigate neuronal cell death, and improve functional outcomes. Initial proximity ligation assay finding (Figure S1) demonstrated considerable differences in the number of interactions between HSP72–HMGB1 and HSP72–AcHMGB1 (235.8 dots/cell vs 11.8 dots/cell) under normoxic control and glucose-deprived conditions, suggesting that the form of HMGB1 plays a key role in potential binding or interaction with HSP72. The effects of oxygen–glucose deprivation and/or treatment with resveratrol on interactions between HSP72 and HMGB1 were studied. OGD treatment decreased the number of interactions for both HSP72–HMGB1 and HSP72–AcHMGB1 compared to the control; however, resveratrol treatment in the OGD conditions produced conflicting results. HSP72–AcHMGB1 interactions returned to similar levels as the control, while HSP72–HMGB1 interactions markedly decreased, further suggesting that the form of HMGB1 plays a critical role in how it interacts with HSP72 and how these interactions are affected by pharmacological stimuli (Figure 5).

Figure 5.

Figure 5

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Resveratrol treatment differentially alters interactions of HSP72 with reduced and acetylated HMGB1 under OGD. (A) Schematic of resveratrol’s effects on HSP72 interactions with HMGB1 and AcHMGB1. Created with ©BioRender (biorender.com). (B–E) Proximity ligation assay in HMC3 microglia for HSP72 and unmodified HMGB1 (B–C) or acetylated HMGB1 (D–E). Cells treated with either serum-free DMEM with glucose, HBSS without glucose under hypoxia (OGD), or resveratrol (50 μM) for 4 h. Antibodies—Anti-HMGB1 rabbit polyclonal 1:1000 (ab18256, Abcam) or Anti-AcHMGB1 rabbit polyclonal 1:2500 (MBS9404216, MyBioSource) and Anti-HSP70 mouse monoclonal 1:1000 (ab2787, Abcam). Red spots indicate interactions of either (B) HSP72–HMGB1 or (D) HSP72–AcHMGB1. Nuclei stained with Hoechst 33342 (blue), F-actin (green) labeled with Alexa Fluor 488 phalloidin. Quantification of HSP72–HMGB1 (C) and HSP72–AcHMGB1 (E) interactions by # of spots per cell represented by bar graphs with individual cell counts mean ± SD. Scale bar of whole cells at 20 μM, scale bar of selected subsection at 10 μM. ***p < 0.001, **p < 0.01, *p < 0.05, and ns: nonsignificant.

We investigated the effects of OGD and treatment with resveratrol, SAHA, and biomimetic platelet membrane-derived nanocarriers functionalized with the current stroke treatment drug, rtPA, on the abundance and subcellular localization of nonmodified HMGB1, acetylated HMGB1, and HSP72. OGD significantly decreased the nuclear abundance of both forms of HMGB1 compared to normoxic controls in both human HMC3 microglia and THP-1 macrophage cell lines. The decrease in nuclear abundance observed under the OGD for both HMGB1 and AcHMGB1 may be explained by release from functionally impaired or dying cells and/or stress due to depletion of nutrients and energy sources. A previous report demonstrated that hypoxia induced an increase in extracellular HMGB1 levels.45

Treatment under the OGD with empty cellsomes (CSMs), loaded cellsomes (CSM@rtPA), or rtPA alone displayed no effect on HMGB1 and AcHMGB1 subcellular distribution compared with the effect of the OGD alone. These findings suggest that the platelet membrane-derived nanoformulation both with and without rtPA did not cause additional adverse effects from OGD alone, a favorable outcome as any exacerbation of stress incurred by cells during acute ischemic stroke may negate any potential benefit of improved treatment delivery. Resveratrol treatment alone did not substantively alter the HMGB1 or AcHMGB1 abundance and localization. In contrast, SAHA treatment, which is a deacetylase inhibitor, markedly increased AcHMGB1 abundance in both cell lines. Rabadi et al. found that 7-day resveratrol pretreatment in a mouse renal ischemia-reperfusion injury model decreased HMGB1 acetylation through activation of SIRT1 and blunted nucleocytoplasmic translocation.46 Multiple mechanisms have been suggested to be involved in the neuroprotective function of resveratrol, such as inhibiting neuroinflammation, oxidative stress, apoptosis, lipid peroxidation, glutathione depletion, and mitochondrial dysfunction.47 However, it is not yet clear how impacting each mechanism with resveratrol treatment affects other molecular mechanisms involved in stroke pathophysiology.

Apart from serving as a molecular chaperone, HSP72 overexpression has been associated with improved outcomes in a stroke model, attenuation of NF-κB activation in mouse microglia, and involvement in multiple pathways of neuroprotection.35,44 We observed a 3-fold increase in cytosolic HSP72 abundance in microglia following 24 h of treatment with resveratrol, indicating a promising role of resveratrol as a candidate neuroprotectant. Resveratrol treatment 2 h following ischemia has been found to downregulate the TLR4 pathway in a rat ischemia-reperfusion model, reducing the ischemia-induced increases in TLR4, NF-κB p65, and IL-1β expression levels.48 As both HSP72 and HMGB1 serve as extracellular DAMPs but confer protective roles intracellularly, we wanted to investigate whether HMGB1 and HSP72 interacted intracellularly. Using PLA, the use of OGD was seen to decrease the number of interactions of both reduced and acetylated HMGB1 with HSP72, while treatment with resveratrol under the use of the OGD increased HSP72-AcHMGB1 interactions to similar levels as control conditions but further decreased HSP72-HMGB1 interactions.

This differential effect following resveratrol treatment may point to a unique binding pattern between the two HMGB1 forms with HSP72. This is a promising new axis for targeting neuroinflammation in stroke, as AcHMGB1 but not HMGB1 produces proinflammatory cytokines when released, and HSP72 itself has a cytoprotective effect when maintained intracellularly. Modulating this interaction would be beneficial in the context of stroke recovery by decreasing the proinflammatory response of alarmins in the stroke penumbra while increasing or maintaining the chaperoning function of both reduced HMGB1 and HSP72 within the cell. Some previously described inducers of HSP72 such as geranylgeranylacetone,49 dulaglutide,50 and other polyphenols similar to resveratrol should be studied to determine if similar effects on AcHMGB1-HSP72 and HMGB1-HSP72 interactions are observed, or if resveratrol uniquely affects interactions differentially. Future studies will need to determine if the increase in the level of HSP72-AcHMGB1 interactions can lead to a decrease in proinflammatory cytokine production and maintenance of cellular functions.

Within seconds to minutes following initial cerebral blood flow reduction, ischemic stroke initiates a plethora of pathophysiological changes from excitotoxicity, neuroinflammation, oxidative stress, and ultimately, cell death, with effects lasting from hours to months poststroke.1,17,51,52 After initial damage or neuronal cell death, multiple constituents of the neurovascular unit, such as microglia, macroglia (astrocytes), macrophages, and even endothelial cells, orchestrate an interconnected ballet of inflammatory responses to mitigate and repair damage.53,54 Use of transcriptomics with single-cell (sc-) or single-nucleus (sn-) RNA sequencing is one method currently being used to uncover the spatial and temporal responses of various cell types and subpopulations.55−59 A better understanding of cellular responses to stroke by cell subtype, interactions with neighboring cell types, and how and when these interactions are affected during and after a stroke is crucial for the development of more effective therapeutic strategies.

Hundreds of clinical trials with drugs targeting one or multiple of these mechanisms affected following stroke, termed neuroprotectants, have been completed to date with little to no success.60 Combination therapy with neuroprotectants in addition to a thrombolytic agent such as rtPA and mechanical thrombectomy would be of particular benefit in the context of ischemic stroke13 where early intervention during clot occlusion and subsequent cerebral tissue repair and maintenance require therapeutic agents with varying treatment profiles. One nexus of inflammation that has been targeted is the release and receptor pathway of extracellular HMGB1,61−63 with agents such as resveratrol.64 While combination therapy can entail the use of drugs administered concurrently but individually, a vesicular nanocarrier such as presented here can be used to deliver both agents together. An agent for clot degradation can be encapsulated within the nanocarrier core, while a lipophilic agent such as resveratrol65 could be noncovalently incorporated within the lipid bilayer, allowing for dual-agent delivery of a thrombolytic and neuroprotectant. The usefulness of combination therapy lies in the ability to target the multiple pathways of pathogenesis of diseases, with recent strides in the field of cancer.66 In fact, nanocarriers sensitive to hypoxic microenvironments have been developed for treating drug-resistant tumor cells,67 and could be reasonably expanded to one day include controlled release of thrombolytics in an ischemic stroke setting. While the effectiveness of some nanomaterials can be hindered by their rapid uptake and clearance by macrophages,68 the proinflammatory state of macrophages in ischemic stroke means that rapid uptake of neuroprotectants would actually be beneficial.

Ischemic stroke remains a major disabling event that has multifaceted and often overlapping and interconnected molecular mechanisms, and as such, the employment of multipronged interventions is necessary to improve neurological outcomes and reduce mortality. Use of functionalized nanocarriers, such as the one presented in this study, could be utilized to deliver multiple agents at a target site, improving recanalization and reperfusion rates with thrombolytics while reducing neurological impairments and preventing further deterioration with delivery of localized neuroprotectants such as resveratrol.

2. Conclusions

This study aimed at revealing the modulation and subcellular localization of the alarmin HMGB1 in both its reduced and acetylated (AcHMGB1) forms under the conditions of the OGD, an in vitro stroke model mimicking the lack of oxygen and glucose supply to cerebral cells. OGD itself decreased the nuclear abundance of HMGB1 and AcHMGB1 without affecting cytosolic abundance in both cell types. Treatment with SAHA, a histone deacetylase inhibitor, significantly increased the nuclear abundance of AcHMGB1 but not of reduced HMGB1, while resveratrol, a histone deacetylase activator, did not produce marked effects on either HMGB1 form. Treatment with the thrombolytic agent rtPA loaded in a biomimetic platelet membrane-derived nanocarrier (CSM@rtPA), free rtPA, or empty CSMs did not produce observable effects on either the abundance or relative localization of HMGB1 and AcHMGB1. The modulation of a second but nonetheless important alarmin, HSP72, was investigated. HSP72, while acting as a DAMP extracellularly, plays cytoprotective roles intracellularly. Treatment with resveratrol increased cytosolic abundance of HSP72 in microglia, while glucose deprivation itself did not affect abundance. Using proximity ligation assays, HSP72 was found to interact with both forms of HMGB1, but this interaction was differentially affected by resveratrol treatment under OGD conditions. Resveratrol treatment decreased the number of HSP72-HMGB1 interactions; it increased the number of HSP72-AcHMGB1 interactions back to control levels. This study reveals a promising axis for targeting the neuroinflammatory cycle under stroke that has not been previously discussed and a new mechanism of neuroprotection with resveratrol-preventing extracellular release of alarmins AcHMGB1 and HSP72 while maintaining their localization intracellularly, thus maintaining their protective functions. Further studies are needed to elucidate the exact manner in which AcHMGB1 and HMGB1 interact with HSP72 and how resveratrol treatment exactly affects this interaction. Modulating these interactions may provide for improved stroke recovery as constraining extracellular AcHMGB1 release can prevent proinflammatory cytokine production and inducing HSP72 intracellular can provide additional cytoprotection following injury and damage.

3. Materials and Methods

3.1. Preparation of Biomimetic Nanoparticles

Biomimetic platelet cell-derived nanoparticles (cellsomes, CSM) were prepared following the protocol we previously described to obtain monodisperse cellsomes of size around 200 nm.16,69 To create the CSMs, human platelet concentrate (#SER-PCEX) was purchased from Zen-bio©. Platelets were aliquoted at 5 mL (109–1011 cells) and stored at 4 °C until use. The collected cells (10 × 106 cells) were washed with precooled phosphate-buffered saline (PBS, pH 7.4, Thermo Fisher No. 14190169) and centrifuged at 600g for 5 min. The cell pellet was resuspended in 10 mL of hypotonic buffer (0.25× PBS) containing 1x protease inhibitor cocktail (PIC, Sigma-Aldrich #P2714-BTL) and incubated in an ice bath for 10 min. Then, cell lysis was carried out using a freeze–thaw method consisting of 4 cycles of freezing in liquid nitrogen for 1 min followed by thawing at 37 °C for 10 min. Finally, the solution was placed in a bath sonicator for 5 min. To purify the cell membrane fragments, the solution was subjected to several centrifugation steps. First, the solution was centrifuged at 700g for 10 min at 4 °C. Then, the cell membrane fragments remaining in the supernatant were precipitated by centrifugation at 15,000g for 30 min at 4 °C. To allow for self-assembly of the membrane fragments into CSMs, a mechanical extrusion process was applied. The pellet was dispersed in 1 mL of 1× PBS and subjected to 10 cycles of extrusion by using an Avanti Mini extruder with an 800 nm polycarbonate membrane. Fluorescently labeled CSMs were produced using fluorescent phospholipids that can be intercalated in the lipidic bilayer. In particular, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine labeled with Atto 647N (DOPE Atto 647N, Sigma-Aldrich #42247) was added to the CSM. One mL of the obtained CSM solution dispersed in 1x PBS was mixed with 5 μL of 1 mg·mL–1 of DOPE Atto 647N (dissolved in dichloromethane) and sonicated for 10 min. The resulting fluorescent CSMs were extruded 10 times using an Avanti Mini extruder. Then, recombinant tissue plasminogen activator (rtPA) was encapsulated in the platelet-derived CSM (termed CSM@rtPA). Five mg of commercially available rtPA (Actilyse) was labeled with fluorescein isothiocyanate isomer I (FITC) by mixing 25 FITC/rtPA (mol/mol) in 1x PBS with l-arginine (3.5 mg·mL–1). The reaction was left overnight at 4 °C and then purified from the excess of FITC by size exclusion chromatography using a PD-10 desalting column. 0.4 mL of rtPA-FITC (fraction 2, 1 mg·mL–1) was added to 0.5 mL of platelet-derived CSM fluorescently labeled with DOPE Atto 647N. The solution was sonicated for 15 min, extruded 10 times using an Avanti Mini extruder with an 800 nm polycarbonate membrane, and purified from the excess of rtPA-FITC and DOPE Atto 647N by centrifugation (1 h, 70,000g, 4 °C). CSM@rtPA was finally resuspended in 1 mL of 1× PBS (see Supporting Information Figure S3A). To ensure long-term storage and cross-laboratory accuracy, samples containing rtPA (CSM@rtPA) and empty CSM were lyophilized. These lyophilized CSM@rtPA samples were found to retain similar physicochemical characteristics as nonlyophilized samples.16 The physicochemical features of CSM and CSM@rtPA were characterized by dynamic light scattering (DLS) for hydrodynamic size and colloidal stability, nanoparticle tracking analysis (NTA) for concentration determination and size distribution analysis, and fluorescence spectroscopy for quantification of rtPA-FITC loading (see Supporting Information Figure S3B). In addition, the amidolytic activity of encapsulated rtPA was determined by a chromogenic peptide substrate (Sigma-Aldrich #T2943) that is permeable to the CMS surface (see Supporting Information Figure S3C) (summarized in Table S1). To ensure equivalent treatment concentrations in all experiments, the relative concentration of CSM@rtPA required was calculated to match the desired final rtPA concentration. Once calculated, the concentration of empty CSM required for treatment can be determined and matched to the number of nanoparticles in the CSM@rtPA treatment.

3.2. Cell Cultures

Human microglia clone 3 (HMC3) cells were obtained from the American Tissue Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, #11995-065), supplemented with 5% fetal bovine serum (FBS; Wisent #080-450) and 1% Penicillin–Streptomycin (P–S; Invitrogen, #15140–122). THP-1 human monocyte cells were obtained from ATCC. THP-1 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640; Gibco, #11835-030) containing 10% (FBS), 1% penicillin–streptomycin (P/S), and 50 μM of β-mercaptoethanol (Bio-Rad, #161-0710) in a 75 cm2 vented cell culture flask (Sarstedt, 83.3911.002). Differentiation of THP-1 monocytes into macrophages was induced over a period of 5 days with phorbal-12-myristate-13-acetate (PMA; Abcam, ab120297) at a concentration of 75 ng/mL. Differentiating THP-1 cells were refreshed with media containing PMA on the third day. HMC3 cells were kept at passages below 35, while THP-1 passages were limited to 12. Both cell types were incubated at 37 °C, with 5% CO2 and atmospheric O2.

3.3. Oxygen–Glucose Deprivation

A BioSpherix Xvivo System model X3 Hypoxia Hood with a glovebox was employed for experiments requiring hypoxic conditions. To achieve hypoxic conditions, the chamber temperature was raised, and nitrogen gas was used to flush oxygen at least 2 h prior to use to achieve hypoxic conditions of 37 °C, 5% CO2, and between 1.0–2.0% O2. All treatments under hypoxic conditions lasted 4 h. For immunocytochemistry experiments, cells were fixed with 4% paraformaldehyde (BDH #29447) for 10 min while still in the hypoxic chamber to mitigate the effects of reoxygenation. Remaining immunocytochemistry protocol steps were conducted outside of the hypoxic chamber. For treatments requiring a lack of glucose, Hanks’ balanced salt solution (HBSS; Gibco, #14025-092) was used for HMC3 microglia cells, and RPMI 1640 without glucose (Gibco, #11879-020) was used for THP-1 differentiated macrophages (not used during the differentiation protocol).

3.4. Immunocytochemistry

Cells were seeded at either 7000 cells/coverslips for HMC3 microglia cells or 10,000 cells/coverslip for THP-1 monocytes on 12 mm circular glass coverslips (Karl Hecht, 41001112). THP-1 cells were differentiated over 5 days as per the protocol mentioned above, and HMC3 cells were cultured for 24 h prior to treatment. Following treatment, cells were fixed in 4% PFA for 10 min, permeabilized for 10 min with 0.1% Triton X-100, and blocked with 10% goat serum in PBS. Cells were then incubated with primary antibodies overnight at 4 °C [rabbit anti-HMGB1 (1:1000, Abcam, ab18256)], rabbit anti-acetylated-HMGB1 (1:1000, MyBioSource, MBS9404216) or mouse anti-HSP70 (1:1000, Invitrogen, A-21245). Cells were then washed three times with PBS prior to incubation with secondary antibodies for 1 h at room temperature [goat anti-rabbit Alexa Fluor 647 (1:1000, Invitrogen, A-21245)]. Cells were washed twice with PBS and stained for nuclei with Hoechst 33342 (10 μM, Molecular Probes #H-1399) and F-actin with Alexa Fluor 488 (1:400, Invitrogen, A-12379) for 20 min. Cells were washed twice for a final time with PBS, and coverslips were mounted onto microscope slides (Fisherbrand 4951602811T) using Aqua-Poly/Mount (Polysciences, #18606-20). Samples were then imaged with a Leica fluorescent microscope (Leica DMI4000 B), and fluorescence was analyzed using FIJI (ImageJ). The nuclear and cytoplasmic fluorescence of HMGB1 or acetylated-HMGB1 (AcHMGB1) in each cell was measured from the mean fluorescence and subtracted from the mean background fluorescence value.

3.5. Proximity Ligation Assay

A Millipore Duolink Proximity Ligation Assay kit was used following a protocol previously described.70 HMC3 microglia were seeded at 7,000 cells/coverslip on 12 mm circular glass coverslips (Karl Hecht, 41001112) 24 h prior to treatment. Following treatment, cells were fixed with 4% PFA for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked using the 1X Duolink blocking solution (DUO82007-4ML) for 1 h at 4 °C. Primary antibody pairs [rabbit anti-HMGB1 (1:1000, Abcam, ab18256) and mouse anti-HSP70 (1:1000, Abcam, ab2787)] and [rabbit anti-acetylated-HMGB1 (1:1000, MyBioSource, MBS9404216)] and mouse anti-HSP70 (1:1000, Abcam, ab2787) were diluted in 1X Duolink antibody diluent (DUO82008-2.5ML) and incubated overnight at 4 °C. The following day, samples were washed thrice with Duolink wash buffer A (DUO82046) and incubated with PLA probes A (Duolink Probe anti-rabbit MINUS 5X, DUO82002) and B (Duolink Probe antimouse PLUS 5X, DUO82004) diluted 1:5 in Duolink diluent for 1 h at 37 °C. Samples were washed thrice with wash buffer A and incubated with a ligase solution [Duolink ligase (DUO82029), ligation buffer 5X (DUO82009), and purified distilled water] for 30 min at 37 °C. Following ligation, samples were incubated with the amplification polymerase solution [Duolink polymerase (DUO82030), Duolink Amplification Far Red (DUO82012), and purified distilled water] for 100 min at 37 °C. Samples were washed twice and were stained for F-Actin with Alexa Fluor 488 phalloidin (1:400, Invitrogen, A-12379) for 20 min. Cell nuclei were stained, samples were mounted with Duolink in situ mounting medium with DAPI (DUO82040) and imaged with a Leica DMI4000 B fluorescent microscope, and PLA interaction dots were counted using the Cell Counter plugin in FIJI (ImageJ) (Figure 6).

Figure 6.

Figure 6

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Schematic of proximity ligation assay. Cells are first seeded on glass coverslips in a 6-well plate, treated with respective treatments, fixed, and permeabilized. A pair of primary antibodies of different species origins are used to bind to two proteins of interest. A second pair of PLA probes recognizing the species’ origin binds to the primary antibodies attached to the proteins of interest. The addition of PLA ligase produces a circular DNA template that can be amplified by using a DNA polymerase. Fluorescently labeled oligonucleotide Duolink probes bind to amplified DNA, which can be imaged and analyzed with a fluorescent microscope, appearing as a distinct “spot.” Created with ©BioRender (biorender.com).

Acknowledgments

This project was partially supported by the ISCII (AC20/00031 and AC20/0041), ANR under the framework of EuroNanoMed III_2020 (PLATMED_project), the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN2020-07011), and the Fonds de Recherche du Québec–Santé (EuroNanoMed III - PLATMED) (FRQ-S #294233).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00271.

  • Proximity ligation assay between HSP72 with HMGB1 and AcHMGB1 in microglia; modulation of HSP72 and HMGB1 by resveratrol based on in silico models of binding; fabrication steps and characterization of rtPA-loaded cellsomes; and a table of physicochemical characterization of cellsomes (PDF)

Author Contributions

Experiments were designed by P.-B.B., I.Z., and D.M. and conducted by P.-.B.B. and I.Z. The preparation and characterization of nanocarriers were performed by C.C.-P., M.M., P.D.P., and B.P. Data was analyzed by P.-B.B. and I.Z. The manuscript was prepared and reviewed by all authors and edited by D.M., F.C., E.P., and D.V. All authors read and approved the final manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt3c00271_si_001.pdf (496.7KB, pdf)

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