The role of the DAMP molecule HMGB1 in neuroinflammation and macrophage activation

High mobility group box 1 (HMGB1) is a highly conserved nuclear protein expressed in all eukaryotic cells. While its nuclear functions are vital, we know today that HMGB1 also functions as a Damage Associated Molecular Pattern (DAMP).

When HMGB1 is released from cells to extracellular space due to cell death or stress, it induces migration of immune cells and secretion of pro-inflammatory cytokines. These functions are regulated by reduction or oxidation of cysteines in the HMGB1 structure. When all cysteines are reduced, fully reduced HMGB1 (frHMGB1) induces cell migration and proliferation. HMGB1 with a disulfide bond (dsHMGB1) induces cytokine secretion. These two functions are lost when HMGB1 is terminally oxidized (oxHMGB1).

The first aim of this thesis was to determine the functions of extracellular HMGB1 as an inducer of neuroinflammation. As excessive neuroinflammation can worsen neuronal damage in diseases like stroke, HMGB1 neutralization in brain is an interesting therapeutical strategy. To develop a therapeutic against HMGB1 in neuroinflammation.

We started by determining if frHMGB1 and dsHMGB1 intracortical injections in healthy rat brains could induce neuroinflammation without any underlying neuroinflammatory state. This was to confirm the causative effect of HMGB1. Both frHMGB1 and dsHMGB1 induced local neuroinflammation and blood brain barrier (BBB) leakage. However, dsHMGB1 was more potent as it lead to activation of major histocompability complex II (MHC-II). Next, we set out to investigate neuroinflammation in a rat ischemic stroke model, M2 middle cerebral artery occlusion model (M2CAO), and simultaneously tested an Affibody molecule (ABY) as a potential therapeutic targeting HMGB1. Neuroinflammation was still present in M2CAO after four days from the induction of ischemia-reperfusion injury and an anti-HMGB1 ABY binding to HMGB1 could cross the BBB, however, no reduction of the ischemic lesion nor neuroinflammation could be detected.

Both HMGB1 injections and M2CAO induced microglia activation and macrophage infiltration and activation in the brain. Macrophages are among the immune cells recruited to brain in response to tissue injury and can obtain roles either in inducing inflammation or repairing damaged tissue. The second aim of this thesis work was to determine if and how macrophages polarize in response to HMGB1 stimulation.

To characterise the macrophage activation, frHMGB1 and dsHMGB1 were used to stimulate mouse bone marrow derived macrophages. Both dsHMGB1 and frHMGB1 activated a motile phenotype. However, only dsHMGB1 induced macrophages to obtain a pro-inflammatory phenotype including cytokine secretion whereas induction with frHMGB1 only had minor effects on the macrophage gene expression profile outside of migratory mechanisms. The results in this thesis work have expanded the understanding of the role of HMGB1 redox in inflammation and improved the understanding of HMGB1 as a therapeutic target for neuroinflammation.

List of scientific papers

I. Aucott H, Lundberg J, Salo H, Klevenvall L, Damberg P, Ottosson L, Andersson U, Holmin S, Erlandsson Harris H. Neuroinflammation in Response to Intracerebral Injections of Different HMGB1 Redox Isoforms. J Innate Immun. 2018;10(3):215-227.
https://doi.org/10.1159/000487056

II. Salo H, Little P, Linthorst N, Jussing E, Lu L, Chireh A, Wahlberg E, Bianchi E M, Andersson U, Frejd Y F, Holmin S, Erlandsson Harris H. Treatment of experimental M2CAO ischemia reperfusion stroke with an HMGB1-specific Affibody® molecule. [Manuscript]

III. Salo H, Qu H, Mitsiou D, Aucott H, Han J, Zhang XM, Aulin C, Erlandsson Harris H. Disulfide and Fully Reduced HMGB1 Induce Different Macrophage Polarization and Migration Patterns. Biomolecules. 2021 May 28;11(6):800.
https://doi.org/10.3390/biom11060800

IV. Qu H, Salo H, Heinbäck R, Ewing E, Espinosa A, Aulin C, Erlandsson Harris H. Transcriptome profile of mouse bone marrow derived macrophage upon stimulation with disulphide high mobility group box 1 (dsHMGB1) and LPS/IFN-γ reveals distinct polarization characters. [Manuscript]