Following the white rabbit : release and regulation of HMGB1 in inflammation
Author: Sowinska, Agnieszka
Date: 2019-03-15
Location: CMM Lecture Hall (L8:00)
Time: 09.00
Department: Inst för medicin, Solna / Dept of Medicine, Solna
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Thesis (1.605Mb)
Abstract
Dysregulated inflammatory responses are characterized by excessive release of endogenous pro-inflammatory molecules, danger-associated molecular patterns (DAMPs). A prototypical DAMP, High Mobility Group Box 1 protein (HMGB1), is primarily bound to DNA in the nucleus of most eukaryotic cells. However, when HMGB1 exits the cell, it acquires novel functions and can trigger immune activation. The aim of this thesis was to study the release of HMGB1, follow its fate at the site of inflammation and therapeutically block its activity.
Multiple studies have reported that cells release HMGB1 in either a passive or an active manner. In order to understand the possible impact of cell death in inflammatory conditions, we performed well-controlled in vitro analyses of HMGB1 release under different cell death modes. Necrotic cells instantly released high levels of HMGB1, while apoptotic cells retained HMGB1 unless they advanced to secondary necrosis. The controlled immunogenic cell death types, such as pyroptosis and necroptosis, secreted HMGB1 along with other pro-inflammatory molecules in comparable or higher levels than cells activated with lipopolysaccharide (LPS) and interferon gamma (IFNγ).
Next, we investigated HMGB1 proteolytic regulation at the site of inflammation. Human neutrophil elastase (HNE), cathepsin G (CG) and matrix metalloproteinase 3 (MMP-3), endoproteases present in arthritis-affected joints, cleaved HMGB1 into smaller peptides in vitro. While HNE and MMP-3 truncated the acidic C-terminal tail domain of HMGB1, CG completely degraded the protein. We showed that the C-terminal tail truncation negatively regulated HMGB1 binding to Toll-like receptor 2 (TLR2). Only the protein form devoid the acidic tail domain bound TLR2 in vitro. Thus, truncation of HMGB1 by endoproteases might improve its affinity to receptors. TLR2 and HMGB1 interaction did not result in cytokine induction. However, both full-length and truncated HMGB1 formed complexes with potent TLR2 ligand peptidoglycan (PGN) to potentiate the inflammatory response in peripheral blood mononuclear cells (PBMCs). Lastly, we developed a novel tool that could enable anti-HMGB1 therapy in the clinic by engineering a chimeric, partially humanized, monoclonal antibody (h2G7). The antibody displayed anti-inflammatory effects when tested in a mouse model of paracetamol-induced liver toxicity. Experiments with mutated variants, non-Fc receptor binding and non-complement binding variants, of the novel antibody suggested HMGB1 neutralization as the mode of action.
Taken together, results presented in my thesis increased the knowledge about HMGB1 biology regarding its release during immunogenic cell death, proteolytic regulation at the site of inflammation and interaction with its receptor TLR2. In addition, the generation of a partially humanized monoclonal antibody could promote the introduction of anti-HMGB1 therapy in the clinic.
Multiple studies have reported that cells release HMGB1 in either a passive or an active manner. In order to understand the possible impact of cell death in inflammatory conditions, we performed well-controlled in vitro analyses of HMGB1 release under different cell death modes. Necrotic cells instantly released high levels of HMGB1, while apoptotic cells retained HMGB1 unless they advanced to secondary necrosis. The controlled immunogenic cell death types, such as pyroptosis and necroptosis, secreted HMGB1 along with other pro-inflammatory molecules in comparable or higher levels than cells activated with lipopolysaccharide (LPS) and interferon gamma (IFNγ).
Next, we investigated HMGB1 proteolytic regulation at the site of inflammation. Human neutrophil elastase (HNE), cathepsin G (CG) and matrix metalloproteinase 3 (MMP-3), endoproteases present in arthritis-affected joints, cleaved HMGB1 into smaller peptides in vitro. While HNE and MMP-3 truncated the acidic C-terminal tail domain of HMGB1, CG completely degraded the protein. We showed that the C-terminal tail truncation negatively regulated HMGB1 binding to Toll-like receptor 2 (TLR2). Only the protein form devoid the acidic tail domain bound TLR2 in vitro. Thus, truncation of HMGB1 by endoproteases might improve its affinity to receptors. TLR2 and HMGB1 interaction did not result in cytokine induction. However, both full-length and truncated HMGB1 formed complexes with potent TLR2 ligand peptidoglycan (PGN) to potentiate the inflammatory response in peripheral blood mononuclear cells (PBMCs). Lastly, we developed a novel tool that could enable anti-HMGB1 therapy in the clinic by engineering a chimeric, partially humanized, monoclonal antibody (h2G7). The antibody displayed anti-inflammatory effects when tested in a mouse model of paracetamol-induced liver toxicity. Experiments with mutated variants, non-Fc receptor binding and non-complement binding variants, of the novel antibody suggested HMGB1 neutralization as the mode of action.
Taken together, results presented in my thesis increased the knowledge about HMGB1 biology regarding its release during immunogenic cell death, proteolytic regulation at the site of inflammation and interaction with its receptor TLR2. In addition, the generation of a partially humanized monoclonal antibody could promote the introduction of anti-HMGB1 therapy in the clinic.
List of papers:
I. Mode of cell death influences amounts of released HMGB1. Agnieszka Sowinska, Peter Lundbäck, Daniel Applegren, Lena Klevenvall, Manoj Neog, Ulf Andersson, Helena Erlandssoon Harris. [Manuscript]
II. Proteolytic cleavage of the alarmin HMGB1 and its potential role in regulating HMGB1 activity in inflammatory disease. Agnieszka Sowinska, Merlin Rensing, Lena Klevenvall, Manoj Neog, Peter Lundbäck, Helena Erlandsson Harris. [Submitted]
III. Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain. Hannah Aucott, Agnieszka Sowinska, Helena Erlandsson Harris, Peter Lundbäck. Mol Med. 2018 May 4;24(1):19.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and post-injury inflammation in mice. Peter Lundbäck, Jonathan D. Lea, Agnieszka Sowinska, Lars Ottosson, Camilla Melin Fürst, Johanna Steen, Cecilia Aulin, Joanna I. Clarke, Anja Kipar, Lena Klevenvall, Huan Yang, Karin Palmblad, B. Kevin Park, Kevin J. Tracey, Anna M. Blom, Ulf Andersson, Daniel J. Antoine, Helena Erlandsson Harris. Hepatology. 2016 Nov;64(5):1699-171.
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Mode of cell death influences amounts of released HMGB1. Agnieszka Sowinska, Peter Lundbäck, Daniel Applegren, Lena Klevenvall, Manoj Neog, Ulf Andersson, Helena Erlandssoon Harris. [Manuscript]
II. Proteolytic cleavage of the alarmin HMGB1 and its potential role in regulating HMGB1 activity in inflammatory disease. Agnieszka Sowinska, Merlin Rensing, Lena Klevenvall, Manoj Neog, Peter Lundbäck, Helena Erlandsson Harris. [Submitted]
III. Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain. Hannah Aucott, Agnieszka Sowinska, Helena Erlandsson Harris, Peter Lundbäck. Mol Med. 2018 May 4;24(1):19.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and post-injury inflammation in mice. Peter Lundbäck, Jonathan D. Lea, Agnieszka Sowinska, Lars Ottosson, Camilla Melin Fürst, Johanna Steen, Cecilia Aulin, Joanna I. Clarke, Anja Kipar, Lena Klevenvall, Huan Yang, Karin Palmblad, B. Kevin Park, Kevin J. Tracey, Anna M. Blom, Ulf Andersson, Daniel J. Antoine, Helena Erlandsson Harris. Hepatology. 2016 Nov;64(5):1699-171.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Erlandsson Harris, Helena
Co-supervisor: Andresson, Ulf; Lundbäck, Peter
Issue date: 2019-02-21
Rights:
Publication year: 2019
ISBN: 978-91-7831-314-3
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