HMGB1A powerful biomarkerfor a range of disease states .

What is HMGB1?

HMGB1 is an abundant, highly conserved nuclear protein that was named after its rapid migration in the polyacrylamide gel electrophoresis system, as well as its group box structure.2 HMGB1 contains two consecutive DNA binding domains – the HMG A box domain and HMG B box domain – followed by a C-terminal acidic tail and a short, but functionally significant, N-terminal region.3 In a healthy cell, HMGB1 plays a significant role as a DNA chaperone within the nucleus. It is essential for maintaining the structure and function of chromosomes and for regulating replication, transcription and translation processes.4 However, in response to cellular stress, inflammation or injury, it can shuttle from the nucleus to the cytoplasm for release into the extracellular space.

What is the role of this protein outside of the nucleus?

HMGB1 has multiple roles outside of the nucleus, where it can induce innate immune responses and autophagy in the cytosol, or act as a damage-associated molecular pattern (DAMP) that stimulates immunoreceptors and promotes inflammation in the extracellular environment.5, 6, 7 It is also secreted by immune cells, acting as an important mediator in a range of pathological conditions, including infectious diseases, ischemia-reperfusion injury, autoimmune conditions, cardiovascular diseases, neurodegeneration and cancer.5 In fact, it was the first protein thought to fulfill the criteria described by Polly Matzinger as ‘danger theory’, which stipulates that the immune system responds to signals of danger, rather than solely to the presence of foreign pathogens.6

However, HMGB1 does not simply act as a danger signal. The protein is a broad sensor of cellular stress, and plays a role in balancing cell death and survival responses that are essential for cellular homeostasis and tissue maintenance.5 The position and activity of HMGB1 – and its ability to induce inflammatory or survival pathways – are affected by posttranslational modifications and redox reactions.

How does HMGB1 orchestrate an immune response?

HMGB1 translocation results from two main processes that commonly occur together in response to inflammation or autoimmune disease: activation of inflammatory cells – including macrophages, monocytes and dendritic cells – and cell death. The particular mechanism of cell death is important; HMGB1 is usually released when cells die, but is retained by cells undergoing apoptosis. Where many forms of cell death – such as necrosis – are considered to be proinflammatory, apoptosis is considered to be a noninflammatory mechanism.7 These cellular events therefore have contrasting effects on the protein, leading to varying posttranslational modifications and changes to the redox states of three cysteine residues within its structure.

HMGB1 can exist in three primary redox states:

• fully reduced HMGB1 – containing cysteine residues in the thiol form – is typically found within the nucleus of healthy cells.7 However, it can be released by stressed, injured or dying cells, as well as by immune cells, particularly dendritic cells and macrophages.8 Outside of the cell, reduced HMGB1 acts as a chemoattractant, binding to the chemokine CXCL12 to induce chemotaxis of inflammatory cells via CXCR4;8, 9
• disulfide HMGB1 – which results from some cysteine residues forming disulfide bonds – can be found in extracellular spaces and in circulation under conditions of mild oxidative stress.7 Disulfide HMGB1 can induce cytokine production, by binding to toll-like receptor 4 (TLR4) and receptors for advanced glycation end-products (RAGE), activating an innate immune response;8
• fully oxidized HMGB1 is inactive.3

The ability of the protein to switch between different forms – depending on factors such as oxidative stress, cytokine activity and the molecular machinery of cell death – suggests that it plays a dynamic role in the course of inflammatory disease.

How does HMGB1 promote tissue healing?

Aside from inducing an immune response, HMGB1 has also been found to be involved in the anti-inflammatory processes required for tissue healing.9, 6 While its initial role is to initiate inflammation and to recruit immune cells, such as macrophages and neutrophils, to the site of injury – processes that are crucial to clear debris, dead cells and potential pathogens – HMGB1 also plays an important role in shifting macrophages from a pro-inflammatory M1 phenotype to an M2 phenotype, promoting tissue remodeling.10 The protein can also encourage the release of pro-regenerative growth factors by various cell types, which help to recruit stem cells and aid their proliferation and fusion to repair the damaged tissue. Finally, HMGB1 can promote reconstruction of the capillary bed, enhancing perfusion.10 These processes are all vital for tissue repair and regeneration.

What is the role of HMGB1 in tumor biology?

Like any condition that causes necrotic cell death, a tumor will result in HMGB1 release, and the recruitment of inflammatory cells into the tumor environment, which can help to kill tumor cells. However, HMGB1 has also been shown to have pro-tumor activities.10, 11 Cancer cells can take advantage of the reparative actions of HMGB1, manipulating the protein – and its ability to recruit anti-inflammatory macrophages and neutrophils to the tumor environment – to promote their survival and proliferation, and to help them evade the immune system.10 When this occurs, HMGB1 can promote tumor cell growth, survival, and resistance to apoptosis, and can functionally impair immune cells in close proximity to the tumor. Some evidence also suggests that the protein can aid metastasis by interacting with various receptors and signaling pathways.11

Considering the seemingly contradictory actions of HMGB1, what is its value as a biomarker and therapeutic target?

It does have multiple – and seemingly conflicting – roles in healthy and diseased tissues and, for this reason, further research is needed to fully understand the context-dependent actions of HMGB1 and to harness it as a therapeutic target.12 However, it is currently a useful biomarker for identifying a wide range of disease states. Raised levels of HMGB1 were originally recognized in acute medical settings – such as in the context of sepsis – but the protein was soon found to be elevated in various inflammatory and autoimmune conditions.7 As a result, HMGB1 lacks specificity as a biomarker, but it is a valuable indicator of inflammation and stress on the body.

HMGB1 is present in the blood of healthy patients at low levels – approximately 2 ng/ml13 – but this number is elevated significantly in disease states such as cancer, rheumatoid arthritis and respiratory illness. The extent of this increase is often related to the severity of a patient’s condition, so monitoring levels of HMGB1 can help clinicians to keep an eye on disease activity and responses to treatment. It is also becoming a useful biomarker for a range of neurological conditions, as it can cross the blood brain barrier in both directions.14 As a result, high levels of HMGB1 have been detected in the blood of elderly patients experiencing post-surgical cognitive decline, as well as in various cases of epilepsy, stroke, Parkinson’s disease, Alzheimer’s disease, and depression. 14, 15

Dr Marco E. Bianchi

How do we measure levels of this protein?

The key role that HMGB1 plays in many diseases has led many researchers and clinicians on a hunt for reliable methods to measure it – and its various isoforms – in a broad range of sample types.16 Previously, the accurate measurement of the protein using ELISA was fraught with difficulty, mainly due to non-specific activity or cross-reactivity associated with HMGB1 antibodies.17 However, innovations in diagnostics have led to the development of reproducible assays that enable accurate measurement of the protein in serum, plasma. Furthermore, these assays can be used to research HMGB1 content in synovial fluid, cerebrospinal fluid and cell culture extracts.17 Many thanks to Dr Marco Bianchi for discussing this fascinating molecule and for outlining its potential as a disease biomarker. With its outstanding accuracy and reproducibility, the HMGB1 Express kit*, along with its predecessor, has earned the trust of key opinion leaders, establishing itself as the gold standard1 for quantitative HMGB1 analysis and has been featured in over 1400 publications to date.

To find out more or to see our full immunology portfolio please visit:

www.ibl-international.com/cytokines-adhesion-molecules

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* In the US: For research use only. Not for use in diagnostic procedures.