REVIEW

High-mobility group box 1 (HMGB1) in childhood: from benchto bedside

Valeria Chirico & Antonio Lacquaniti & Vincenzo Salpietro & Caterina Munafò &

Maria Pia Calabrò & Michele Buemi & Teresa Arrigo & Carmelo Salpietro

Received: 16 January 2014 /Revised: 13 April 2014 /Accepted: 22 April 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract High-mobility group box protein 1 (HMGB1) is anonhistone nuclear protein that has a dual function. Inside thecell, HMGB1 binds DNA, regulating transcription and deter-mining chromosomal architecture. Outside the cell, HMGB1activates the innate system and mediates a wide range ofphysiological and pathological responses. HMGB1 exertsthese actions through differential engagement of multiplesurface receptors, including Toll-like receptor (TLR)2,

TLR4, and receptor for advanced glycation end products(RAGE). HMGB1 is implicated as a late mediator of sepsisand is also involved in inflammatory and autoimmune dis-eases, such as rheumatoid arthritis and systemic lupus erythe-matosus. Interestingly, HMGB1 was associated with tumorprogression, becoming a potential therapeutic target, due to itsinvolvement in the resistance to chemotherapy. Its implicationon the pathogenesis of systemic vasculitis and inflammatorybowel diseases has also been evaluated. Moreover, it regulatesneuroinflammation after traumatic brain injuries or cerebralinfectious diseases. The aim of this review is to analyze thesedifferent roles of HMGB1, both in physiological and patho-logical conditions, discussing clinical and scientific implica-tions in the field of pediatrics. Conclusion: HMGB1 plays akey role in several pediatric diseases, opening new scenariosfor diagnostic biomarkers and therapeutic strategiesdevelopment.

Keywords Autoimmune disease . Cancer . Children .

High-mobility group box 1 (HMGB1) . Neurologicaldiseases . Newborn . Preterm born . Sepsis . Vasculitis

AbbreviationsAA Acute appendicitisAGE Advanced glycation end productsAN Anorexia nervosaANCA Anti-neutrophil cytoplasmic antibodiesCSF Cerebrospinal fluidCSS Churg–Strauss syndromeCRP C-reactive proteinCD Crohn’s diseaseDAMP Damage-associated molecular patternDCs Dendritic cellsHMGB1 High-mobility group box 1KD Kawasaki diseaseJIA Juvenile idiopathic arthritis

Communicated by David Nadal

V. Chirico (*) :V. Salpietro : C. Munafò :M. P. Calabrò :T. Arrigo :C. SalpietroDepartment of Pediatric Sciences, University of Messina,98100 Messina, Italye-mail: valeriachirico@hotmail.it

V. Salpietroe-mail: salpietroenzo@yahoo.it

C. Munafòe-mail: cmunafo@unime.it

M. P. Calabròe-mail: mpcalabro@unime.it

T. Arrigoe-mail: tarrigo@unime.it

C. Salpietroe-mail: cdsalpietro@unime.it

A. Lacquaniti :M. BuemiDepartment of Internal Medicine, University of Messina, Messina,Italy

A. Lacquanitie-mail: ant.lacq@gmail.com

M. Buemie-mail: buemim@unime.it

A. LacquanitiDepartment of Internal Medicine, Mediterranean Institute forTransplantation and Advanced Specialized Therapies, ISMETT,Palermo, Italy

Eur J PediatrDOI 10.1007/s00431-014-2327-1

IC Immune complexesIBD Inflammatory bowel diseasesIFN InterferonIL InterleukinMPA Microscopic polyangiitisMAPK Mitogen-activated protein kinasesNEC Necrotizing enterocolitisNOD Nonobese diabeticNF-κB Nuclear factor-kappa Bp-ANCAs Perinuclear anti-neutrophil

cytoplasmic antibodiesRAGE Receptor for advanced glycation end productsRA Rheumatoid arthritisSLE Systemic lupus erythematosusTIM-3 T cell immunoglobulin and mucin domain-3TLR Toll-like receptorTBI Traumatic brain injuryTGF Tumor growth factorTNF Tumor necrosis factorT1D Type 1 diabetesUC Ulcerative colitisWG Wegener granulomatosis

Introduction

High-mobility group box 1 (HMGB1) is one of the mostimportant damage-associated molecular pattern (DAMP) mol-ecules, initiating and perpetuating immune response in non-infectious inflammatory responses [27]. Its role, in fact, is toact as a “danger signal”, orchestrating a homeostatic defensiveresponse in challenged tissues [46].

The recent discovery of HMGB1, as a critical mediator ofinflammation, stimulated an increasing interest in inflamma-tion research field [8]. The study of this molecule may lead tothe identification of a new diagnostic biomarker for variousdiseases, having the inflammatory process as background.Furthermore, HMGB1 could open important scenarios fornovel therapeutic strategies [77]. The aim of this review is toanalyze the role of HMGB1, reporting its physiological func-tions, its receptor interactions, and its role in pathologicalfield. In particular, pediatric diseases and data obtained fromanimal models having important clinical and scientific impli-cations in the field of pediatrics, were discussed.

Physiological role of HMGB1

HMGB1 structure

HMGB1 was first described almost 30 years ago as a nonhis-tone chromosomal protein with high electrophoretic mobility

[49]. HMGB is a family of three nuclear proteins includingHMGB1 (previously named amphoterin or HMG1), HMGB2,and HMGB3 [14, 24]. HMGB1 is a single polypeptide chainof 215-amino-acid length that contains two positively chargeddomains referred as “HMG boxes A and B” [90]. The highlyconserved N and C terminal regions of the protein areenriched with basic and acidic amino acid residues,respectively [109, 119].

The functional role of HMGB1 depends upon its location.When inside the nucleus, HMGB1 acts as an architecturalprotein that binds DNA, whereas it exerts different roles andfunctions, once outside the cell, as a proinflammatorycytokine.

Apart from its localization, structural modifications, suchas acetylation or phosphorylation, can determine differentfunctions of HMGB1 [105].

Nuclear HMGB1 actions

HMGB1 is constitutively expressed in many cell types, and alarge “pool” of preformed HMGB1 is stored in the nucleus[16, 31]. “A” and “B” HMG boxes enable HMGB1 to bindchromosomal DNA and fulfill its nuclear functions, includingnucleosomal structure and stability, and regulation of geneexpression [25]. HMGB1 is also active in DNA recombina-tion, repair, and replication [24].

HMGB1 interacts with a wide range of proteins, such astranscriptional factors [80], steroid hormone receptors [18], ma-trix metalloproteinases 2 and 9 [100], viral components [37], andseveral components of the plasminogen activation system [87].

Cytosolic HMGB1 actions

HMGB1 acts as a cell-membrane form, promoting neuriteoutgrowth, smooth muscle cell chemotaxis, and tumor cellmetastasis [89]. Cytosolic HMGB1 promotes autophagy, aconserved programmed survival pathway induced by environ-mental and intracellular stress [103].

Moreover, it represents an important biosensor of nucleicacid inside the cells. DNA or RNA derived from viruses,bacteria, or damaged cells trigger innate immune responsesthrough HMGB1, which is required for subsequent recogni-tion by specific pattern receptors [63].

Extracellular HMGB1 actions

Macrophages, monocytes, dendritic cells (DCs), and T cellsactively release HMGB1 after stimulation with exogenousbacterial products such as endotoxin, or with endogenousproinflammatory cytokines, as well as tumor necrosis factor(TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ.

As an extracellular protein, HMGB1 exerts autocrine andparacrine effects including activation of nuclear factor-kappa

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B (NF-κB), diffuse endothelial activation, systemic activationof inflammatory cells, stimulation of cell migration, facilita-tion of innate recognition of microbial products, and activa-tion of innate immune cells [71]. Moreover, HMGB1 also actson progenitor cells and may play a role in regeneration andrepair of injured tissue [85].

Bactericidal activity represents another property ofHMGB1, able to eradicate in culture more than 95 % ofbacteria within 5 min [123].

Simultaneously, the A box competes with full-lengthHMGB1 for binding sites, leading to an attenuation of theinflammatory cascade. This self-limiting action could repre-sent a negative feed-back for proinflammatory processes[116]. Table 1 summarizes the functions of HMGB1.

HMGB1 receptors

The receptor for advanced glycation end products (RAGE) andsome members of the Toll-like receptor (TLR) family, such asTLR2 and TLR4, have been involved in HMGB1 signaling [15].

After release, HMGB1 binds RAGE, activating of NF-κBand mitogen-activated protein kinases (MAPK) and, conse-quentially, several proinflammatory genes [98].

RAGE alone, however, cannot explain all observed effectsof HMGB1, suggesting the existence of additional receptorsrelevant to HMGB1 signaling.

TLR2 and TLR4 may mediate HMGB1 effects, includingchemotactic cell movement and release of proinflammatorycytokines [7, 86, 121]. HMGB1 acts also by TLR9 signaling,presenting several ligands, such as ssDNA or branched RNAstructures, to its receptor [104].

HMGB1may also suppress proinflammatory cytokine releaseinduced by HMGB1-TLR4 signaling, binding, and signalingthrough a bimolecular cell surface receptor complex formed byCD24 and Siglec-10 (in humans) or Siglec-G (in mice) [32].

T cell immunoglobulin and mucin domain-3 (TIM-3) antag-onize HMGB1 receptor, suppressing innate immune responsesthrough the inhibition of the recruitment of nucleic acids bycytosolic sensors, such as HMGB1, via a galectin-9-independent mechanism [33]. Moreover, thrombomodulin, anendothelial thrombin-binding anticoagulant cofactor, bindsHMGB1 and sequesters HMGB1 from TLRs and other recep-tors. This binding mediates proteolytic cleavage of HMGB1 bythrombin [58]. Figure 1 better explains all these interactions.

HMGB1 in childhood pathology

Sepsis

Sepsis is one of the common causes of morbidity and mortal-ity in children [111]. Early recognition and prompt initiation

of therapy are the most important measures in reducing mor-tality, but a timely diagnosis remains difficult [76].

In contrast to early inflammatory mediators, such asTNF-α and IL-1β, HMGB1 is a late mediator of sepsis [1].Pavare et al. analyzed HMGB1 in children at different age andwith different grades of infections, underlying its low diag-nostic power to detect the bacteremia [88].

Several markers were studied by Carrol et al. to diagnosebacterial infection in Malawian children, highlighting thatprocalcitonin identifies severe sepsis better, when comparedwith other biomarkers such as C-reactive protein (CRP) orHMGB1 [28].

Therefore, no exciting data about the role of HMGB1 inpediatric sepsis were obtained in these studies, but later stagesof disease, in which HMGB1 could play a more importantrole, were not assessed. In fact, HMGB1 is secreted by mac-rophages 20 hin vitro after activation with endotoxin andin vivo 20–72 h after the onset of infection [75].

HMGB1 should be evaluated as a late prognostic factor orused as a marker of response to therapy, rather than an earlymarker of sepsis. Kato et al. demonstrated that edaravone, anovel free-radical scavenger, prolonged survival in a neonatalanimal model of sepsis, preventing HMGB1 elevation [65].

Studies of sepsis have been conducted principally on ani-mal models and adult humans, with few data about pediatricpopulation and further studies are necessary to assign a role toHMGB1.

Inflammatory bowel disease

Necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is the most frequent andlethal disease affecting gastrointestinal tract of premature in-fant [45]. Although numerous risk factors including prematu-rity, hypoxia, bacterial infection, and intestinal ischemia havebeen implicated in the pathogenesis of NEC, the etiology isunclear but multifactorial [114].

Numerous inflammatory mediators have been involved inthe pathogenesis of NEC, such as fecal calpoprotectin, IL-6,TNF-α, or tumor growth factor (TGF)-β [120].

Zamora et al. examined HMGB1 in newborn rats withexperimental NEC and in inflamed intestine of human infantswith acute NEC, demonstrating its role as a mediator ofhypoxia-induced gut injury. Furthermore, the suppression ofHMGB1 with inhibitors, such as semapimod, a drug withimmunomodulatory and anti-inflammatory properties, partial-ly protected against intestinal epithelial cell death [122].

Crohn’s disease and ulcerative colitis

Crohn’s disease (CD), largely arising from a Th1 response,and ulcerative colitis (UC), mainly mediated by T cells or

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natural killer T cells, are the most important inflammatorybowel diseases (IBD) [19]. Vitali et al. demonstrated increasedHMGB1 protein levels in the stools of patients with IBD,hypothesizing an active secretion of the protein stored in thenucleus rather than a "de novo" synthesis. In addition, highHMGB1 was also revealed in patients with inactive disease,suggesting that it might be a sensitive marker of mucosalinflammation, although the disease was clinically inactive[108].

Acute appendicitis

Acute appendicitis (AA) represents a common pediatric abdom-inal surgical emergency, with high rates, around 10–30 %, ofnegative appendectomy and perforated appendicitis [12].

Diagnostic traditional methods are rather nonspecific, and itis still controversial if the use of imaging applications signifi-cantly reduces these complications [50]. Therefore, the identi-fication of novel markers of AA is clinically highly desirable.

HMGB1 was assessed in AA patients, evaluating mRNAand protein levels. In particular, Albayrak et al. demonstratedthat HMGB1 levels were high in AA patients, also in thosewith normal white blood cell counts [5]. This observation wasalso confirmed by Wu et al. who demonstrated that HMGB1,in acute simple, acute suppurative and acute gangrenous ap-pendicitis groups, were significantly high and positively cor-related with the severity of the disease [115].

These data confirm that HMGB1 plays a crucial role in thepathophysiological process of inflammatory bowel diseases,but further studies are necessary to constitute HMGB1 as aclinical biomarker.

The main data about the role of HMGB1 in inflammatoryand infectious diseases are summarized in Table 2.

Autoimmune disease

HMGB1 has been implicated in the pathogenesis of a broadspectrum of autoimmune disease, identified as an auto-antigenrecognized by perinuclear anti-neutrophil cytoplasmic anti-bodies (p-ANCAs) [26].

Table 3 reports main pediatric diseases in which a physio-pathological role of HMGB1 has been demonstrated, under-lying possible mechanisms and future clinical prospects.

Type 1 diabetes

Autoimmune response during type 1 diabetes (T1D) develop-ment is a multifactorial and complex process that requiresgenetic predisposition to synergize with unknown exogenousand/or endogenous triggers. Once triggered by those factors,early stages are characterized by insulitis and infiltration of thepancreatic islets by DCs, macrophages, and T cells [29].

Han et al. demonstrated that HMGB1, actively secreted byDCs, contributed to insulitis progression and diabetes in

Table 1 Physiological functions of HMGB1 based on its localization

Localization Functions References

Membrane Neurites growth Raucci et al. [89]

Smooth muscle cell chemotaxis Raucci et al. [89]

Tumor cell metastasis Raucci et al. [89]

Nuclear Stabilize nucleosome structure Bustin et al. [25]

Gene transcription Bustin et al. [25]

DNA repair/replication Bustin et al. [24]

Interaction with transcriptional factors Nishitani et al. [80]

Interaction with steroid hormone receptors Boonyaratanakornkit et al. [18]

Interaction with matrix metalloproteinase Taguchi et al. [100]

Interaction with viral components Costello et al. [37]

Extracellular Dendritic cell stimulation Campana et al. [27]

Th1 polarization Messmer [74]

B cell activation/proliferation Avalos [11]

Activation of immune cells with induction of proinflammatory cytokine production Lotze et al. [71]

Regeneration and repair of injured tissue Palumbo et al. [85]

Bactericidal effects Zetterstrom et al. [123]

Osteoclast formation Sakai [93]

Negative inotropic effects on cardiomyocytes Hagiwara [51]

Chemotaxis/differentiation on stem cells Chavakis [30]

HMGB1 high-mobility group box 1

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nonobese diabetic (NOD) mice. Moreover, HMGB1 signaledvia TLR4, the main receptor on β cells, to selectively damageβ cells during the development of T1D [69].

Pediatric T1D patients have high circulating levels ofHMGB1, which could trigger TLR2 and TLR4 activationand leading to a proinflammatory state [38].

The challenge for future studies would be the developmentof HMGB1 blockade drugs to evaluate the natural history ofT1D and its complications.

Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex, chronicmultisystem autoimmune inflammatory disease [66]. Up to

20 % of cases are diagnosed during childhood with a lessfavorable prognosis [21]. Environmental triggers, such asinfectious agents, operate in the context of both susceptibilitygenes and epigenetic modifications, resulting in alterations inantigen presentation, lymphoid signaling, apoptosis, andantigen/immune complexes (IC) clearance [48].

HMGB1 is significantly elevated in lupus sera and hasbeen regarded as one of the components in DNA-containingIC that enhances cytokine production through TLR9 orRAGE ligation [70, 104].

These observations derived from adult studies, whereasthere are no data about HMGB1 in children with SLE, exceptGarcia-Romo’s evaluation of neutrophil proteins, includingHMGB1, in pediatric SLE patients [47].

Fig. 1 Release modalities, receptors, pathways, and actions of HMGB1.The full-length HMGB1 contains two homogenous domains (boxes Aand B) and an acidic C-terminal tail. HMGB1 is actively secreted byinnate immune cells in response to exogenous bacterial products (e.g.,endotoxin or CpG-DNA) or endogenous inflammatory stimuli (e.g., TNFand IFN-γ) and passively released by damaged or virus-infected cells.HMGB1 initiates cellular responses by interacting with several differentcell surface receptors. HMGB1 can associate with LPS and activateTLR4, or, when associated with the nucleosome, released during second-ary necrosis, TLR2 is preferentially engaged. HMGB1 also bind to IL-1βand signal through the IL-1R. HMGB1 can bind to DNA and RNA andsignal through RAGE. Engagement of these receptors results in receptor-dependent signaling via the MyD88-dependent pathway involvingNF-κB activation. Activated NF-κB migrates to the nucleus, where itdirectly upregulates the transcriptions of proinflammatory products,

including HMGB1. HMGB1 binds CpG-DNAs, ssRNAs, and dsRNAspromoting their recognition. These complex are endocytosed intoendosomal compartments where they engage the intracellular TLR9,TLR7, and TLR3 resulting in MyD88 and TRIF recruitment and thesubsequent immune response via NF-κB. TIM-3 and CD24/Siglec-10represent two inhibitory HMGB1 receptors. TIM-3, binding HMGB1,inhibits the recruitment of nucleic acids into endosomes and subsequentnucleic acid-mediated immune responses. The complex HMGB1-CD24/Siglec-10 determines a suppression of proinflammatory cytokine release.Abbreviations: HMGB1 high-mobility group protein B1, RAGE receptorfor advanced glycation end products, TLR toll-like receptor, IL interleu-kin, LPS lipopolysaccharide, MyD88 myeloid differentiation primaryresponse gene 88, TRIF TIR-domain-containing adapter-inducinginterferon-β, NF-kB nuclear factor-kappa B, TIM-3T cell immunoglobu-lin and mucin domain-3

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Furthermore, recently it has been demonstrated thatHMGB1, significantly increased in juvenile SLE pa-tients with or without active nephritis, was positivelycorrelated with the disease activity, sustaining the in-flammatory action of interferon-α [62].

Studies assessing HMGB1, anti-HMGB1 and their com-plexes in the pathogenesis of SLE are needed in childhoodpopulation, to further elucidate the role of this novel inflam-matory protein. Therapeutic targeting of HMGB1 might opennew therapeutic opportunities.

Rheumatoid arthritis

HMGB1 is clearly involved in the pathogenesis of rheumatoidarthritis (RA), enhancing the expression of cytokines andother proinflammatory factors [68].

Juvenile idiopathic arthritis (JIA) is a group of chronicchildhood arthritides of unknown origin. While there areseveral observations that confirm the role of HMGB1 in adultarthritis, few studies are conducted in children, in which it wasdemonstrated that patients with JIA were characterized byantibodies direct against HMGB1 or HMGB-2 [92, 113].

It is plausible that HMGB1-binding antibodies might serveas a limiting overwhelming cytokine release, after tissue

damage. Furthermore, high levels of HMGB1 in inflamedjoints were demonstrated in children with JIA, with thehighest levels assessed in patients with a disease onset in earlyage (before age 10) [95].

These data confirm that HMGB1 represents a mediator ofinflammatory arthritis and may serve as a potential therapeutictarget. Further studies should be conducted in children withJIA to confirm the role of HMGB1 and to evaluate if phar-macological interference with this pathway can be an excel-lent strategy for pediatric arthritis.

Vasculitis

Kawasaki disease

Kawasaki disease (KD) is an acute, self-limited vasculitis,with potential cardiovascular complications, developing in∼15 to 25 % of untreated children [79].

Hoshina et al. measured HMGB1 levels in 27 childrenaffected by KD, demonstrating higher HMGB1 values thanhealthy controls. Furthermore, the highest values were detectedin early acute phase with a gradually decrement after deferves-cence [55]. In addition, analyzing KD patients after intravenousimmunoglobulin treatment, Eguchi et al. demonstrated that

Table 2 HMGB1 role in inflammatory and infective diseases

Disease HMGB1 Mechanisms Pediatric data (reference) Future prospects

Sepsis Serum ↑or→

Innate immune cells actively release HMGB1which can be released passively fromdamaged cells or cells infected by viruses.HMGB1 triggers an inflammatoryresponse

2 studies; Pavare [88] andCarrol [28] studied childrenwith different grades ofinfections, including sepsis

Late prognostic factor

Marker of therapy response

HMGB1 inhibition as therapy

IBD Stoolsand H↑↑

Intracellular localization of HMGB1with its active secretion storedin the nucleus rather than a"de novo" synthesis

1 study; Vitali [108] analyzedthe stools and bioptic tissuesof 19 children with CDand 21 with UC

Early fecal diagnostic marker

Marker of mucosal inflammation

Marker of therapy response

NEC H ↑↑ Over expression of HMGB1 andRAGE is related to the enterocyteapoptosis/necrosis cell death andhypoxia-induced gut barrier failureassociated with NEC

1 study; Zamora [122] studiedbiopsies in 7 infants andin newborn rats

HMGB1 inhibition as therapy

AA Serumand H↑↑

Bacteria invade and infect the wallof the appendix, causing releasingof bacterial endotoxin andproinflammatory cytokines, which activatemonocytes/macrophages to release HMGB1. HMGB1is also passively released from injuredor necrotic cells

2 studies; few adolescentswere enrolled with adultsin these studies [5, 108]

Diagnostic marker of appendicitis

Newborn infectionPRM,hypoxemia

AMF andUCB↑↑

In response to inflammatory or infectivestimuli, HMGB1 is secreted by fetalimmune cells, which triggerinflammatory responses

6 studies; HMGB1 wasevaluated in several newborndiseases [2, 23, 39, 77, 82, 94]

Diagnostic marker of inflammatoryor infective diseases in newborns

HMGB1 high-mobility group box 1,H histological data, CSF cerebrospinal fluid, IBD inflammatory bowel disease, CD Cronh’s disease,UC ulcerativecolitis,NEC necrotizing enterocolitis, AA acute appendicitis, AMF amniotic fluid,UCB umbilical cord blood,PRM prolonged rupture of the membranes,TLR toll-like receptor

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poor-responder group was characterized by high HMGB1,representing a potential marker of therapy resistance [40].

Anti-neutrophilic cytoplasmatic antibodies–associatedvasculitis

The primary small-vessel systemic vasculitic disorders asso-ciated with anti-neutrophic cytoplasmic antibodies (ANCA)

include Wegener granulomatosis (WG), microscopic polyan-giitis (MPA), and Churg–Strauss syndrome (CSS) [60].

CSS is a vasculitis with a propensity for lung involvement,accompanied by asthma and an eosinophil-rich inflammatoryinfiltrate with granuloma formation [81, 125]. It is generallyconsidered a disease of adults, and its occurrence in childrenhas been reported infrequently [20].

Taira et al. revealed an involvement of HMGB1 in the path-ogenesis of CSSwith HMGB1 levels strictly related with ANCA

Table 3 HMGB1 involvement in autoimmune disorders and vasculitis

Disease HMGB1 Mechanisms Pediatric data (reference) Future prospects

T1D Serum ↑ Dendritic cells contributesto insulitis progression,secreting HMGB1 that,signaling via TLR4,selectively damages β cells

1 study; Devaraj [38]evaluated HMGB1levels in pediatric patients

HMGB1inhibitionas therapy

SLE Serum and H ↑↑ HMGB1 enhances cytokineproduction through TLR9or RAGE ligation. It is alsoinvolved in the abnormalhyperactivation of JAK-STAT1signaling pathway. HMGB1sustains the inflammatoryaction of interferon-α

2 studies; 1 in vitro studyconducted on neutrophilsisolated from the blood ofpediatric SLE patients [47] and1 in vivo study in juvenileSLE patients [62]

Therapeutictargeting ofHMGB1pathway

JIA Serum and SF ↑↑ Innate immune cells activelyrelease HMGB1 which canbe released passively fromdamaged cells or cells infectedby viruses. HMGB1 triggersan inflammatory response

2 studies; antibodies directagainst HMGB1 weredetected in ANA positivechildren. High HMGB1were recorded in synovialfluid [92, 115]

Diagnosticmarker of JIA

HMGB1inhibition astherapy

Vasculitis

KD Serum and H activephase (↑) andremission phase(↓)

HMGB1 is actively secretedby immune cells and passivelyreleased by injured and necroticcells. HMGB1 serum levelsreflect remaining low-gradeinflammatory activity ortissue damage

2 studies; different levelsof HMGB1 in acute orchronic vasculitis [38],before and after treatment [52]

Marker ofinflammationin vasculitisdisease

HMGB1inhibitionas therapy

Marker oftherapyresponse

ANCA-associated VASCULITIS

CSS Serum and H ↑ HMGB1 is a eosinophilchemotaxis inductor.Eosinophil are able torelease HMGB1

1 study; Taira [95] demonstratedthat eosinophils stainedpositive for HMGB1 positivelycorrelated with peripheraleosinophil counts

Diagnosticmarkerof CSS

WS Serum and H activephase (↑) andremission phase(↓)

HMGB1 is passively released bynecrotic and apoptotic cells.Furthermore, its high levels,especially during the active phase,are also due to immune cell activeproduction

2 studies; HMGB1 levels wereelevated in active Wegenercases [106] and WG withpredominant granulomatousmanifestations [51]

Diagnosticmarkerof WS

Prognosticmarker

HMGB1 high-mobility group box 1, T1D type 1 diabetes, TLR toll-like receptor, SLE systemic lupus erythematosus,H histological data,RAGE advancedglycation end products, JIA juvenile idiopathic arthritis, SF synovial fluid, IF immunofluorescence, ANA antinuclear antibodies,MGmyasthenia gravis,HSP Henoch–Schönlein purpura, BD Behcet’s disease, KD Kawasaki disease, CSS Churg–Strauss syndrome, WS Wegener’s syndrome, ANCA anti-neutrophilic cytoplasmatic antibodies

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antibodies and higher values than those observed in asthmaticpatients.Moreover, eosinophils stained positive for HMGB1, oneof themost important eosinophil chemotaxis inductor, which wasalso positively correlated with peripheral eosinophil counts [101].

WG typically occurs in early adolescence, and glomerulone-phritis and pulmonary disease are common at the diagnosis [4].

Wibisono et al. reported increased serum HMGB1 levelsonly in active WG, suggesting that HMGB1 could vary withthe amount of necrotic tissue [112].

These data were confirmed by Henes et al. who demon-strated that HMGB1 serum levels were significantly high inWG with predominant granulomatous manifestations, corre-lating with the volumes of pulmonary “granuloma” [54].

HMGB1 may be used as a marker of inflammation invasculitis disease, but further studies are necessary to betterunderstand its physiopathological functions and to confirm itsdiagnostic and prognostic role. Furthermore, if HMGB1 isproven to be an inflammatory mediator in vasculitis (in par-ticular, if anti-HMGB1 antibodies will diminish the diseaseactivity in ANCA-associated vasculitis models), reducingHMGB1 expression may be an interesting new drug target.

Cancer

The involvement of HMGB1 in cancer is complex, with itsimplication in tumor formation, progression and in the re-sponse to chemotherapeutics [41] (Fig. 2).

Expression of HMGB1 is upregulated in nearly everytumor type and involved in the regulation of invasive tumorcell migration [44].

Autophagy has recently attracted increasing attention for itsrole in conferring resistance to various commonly used anti-cancer therapies [59].

The balance between apoptosis ("programmed cell death")and autophagy ("programmed cell survival") is important intumor development and response to therapy. HMGB1 and p53form a complex that regulates the balance between tumor celldeath and survival, with a pivotal role of HMGB1 in autoph-agy regulation [56].

Anticancer agents, including doxorubicin, cisplatin, andmeth-otrexate, induce HMGB1 upregulation in human osteosarcomacells, determining an increased drug resistance mediated byautophagy. Therefore, HMGB1 represents a critical factor forthe development of chemoresistance, as a regulator of autophagy,offering a novel target for improving osteosarcoma therapy [57].

In addition, Yang et al. found that HMGB1 was expressedabundantly in various kinds of both leukemia and nonbloodcancer cell lines, and its expression was positively correlatedwith clinical status in childhood leukemia. In leukemia cells,autophagy was induced by HMGB1, enhancing leukemia cellchemoresistance [117].

Another implication of HMGB1 in childhood oncologywas provided by Kang et al. who explored the clinical

significance of HMGB1alterations in acute lymphocytic leu-kemia, one of the most common malignancies in childhood,demonstrating its prognostic role [64].

Further studies are needed to verify if HMGB1 blockagecan be used to improve cancer treatments.

Neurological diseases

Autistic disorders

Autism is a neurodevelopmental disability characterized byimpairments in verbal communications, reciprocal social in-teractions, and restricted repetitive stereotyped behaviors [84].

Dysregulated immune function is a recurrent finding, includ-ing evidence of brain reactive antibodies, altered cytokine levelsin the brain, and altered function of innate immune cells [96].

HMGB1 receptors are involved in the pathophysiologicalmechanisms of autism. Enstrom et al. described abnormal sen-sitivity of peripheral blood monocytes, isolated from childrenwith and without autism, to TLR ligands, suggesting a dysfunc-tion in monocyte pathogen recognition and/or TLR signalingpathways [43]. Junaid et al. showed high incidence of A-allelehomozygosis in theGLO1 gene, with reduction in Glo1 activity[61]. This condition determines an accumulation in the brain ofmethylglyoxal, leading to the formation of advanced glycosyl-ated end products (AGE), which ultimately induces the RAGE-mediated downstream signaling cascade [13]. Autistic childrenare also characterized by abnormal serum levels of HMGB1when compared with healthy controls [42].

High serum levels of HMGB1 may be a biomarker of theimpai red rec ip roca l soc ia l in te rac t ions in th i sneurodevelopmental disorder.

Anorexia nervosa

Anorexia nervosa (AN) is a serious disorder affecting adoles-cents and young adults, decreasing quality of life over longperiod [97]. Several findings from clinical and animal studiesindicate that proinflammatory cytokines are involved in path-ophysiological mechanisms of eating disorders [36].

Agnello et al. demonstrated that administration of HMGB1into cerebral ventricles in rats reduces food intake; whereas, ina model of endotoxemia, passive immunization with anti-HMGB1 antibodies attenuated the development ofhypophagia [3]. In addition, HMGB1 was evaluated in pa-tients with AN at baseline and after nutritional rehabilitationand cognitive behavior therapy, with high HMGB1 valuesobserved in AN patients who did not respond to therapy [118].

Further studies with a larger sample size are needed toinvestigate the relationship between serum HMGB1 and path-ogenic mechanisms of anorexia.

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Traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of morbidityand mortality in children [14].

HMGB1 in part reflects the degree of necrosis and apopto-sis present after TBI, contributing to cell death and neurolog-ical morbidity [99].

Anti-HMGB1 monoclonal antibodies may provide a noveland effective therapy for TBI by protecting against blood–brainbarrier disruption and reducing the inflammatory responses in-duced by HMGB1 [83]. Moreover, adult observations highlight-ed that HMGB1 levels and Glasgow Coma Scale score providedsimilar prognostic information, in patients with severe TBI [110].

Only one pediatric study was performed to evaluateHMGB1 levels in ventricular cerebrospinal fluid (CSF) afterTBI, demonstrating that increased CSF levels of HMGB1were associated with poor outcome [10].

Targeting HMGB1 signaling may be a promising therapeu-tic approach for the treatment of TBI in pediatric and adultpopulation, but further studies are needed to better understandthe physiopathological role of this molecule in TBI.

Neurological infectious disease

Inflammation plays an important role in the neuropathologicalchanges induced by bacterial meningitis [17]. Moreover,

Fig. 2 Different roles of HMGB1 in the tumor microenvironment. Stim-ulated secretion of HMGB1 from tumor cells and passive release bynecrotic, dying tumor cells into the extracellular milieu has both pro-and anti-tumorigenic effects. The anti-tumorigenic effects are as follows:(1) Acute release of HMGB1 after antitumor treatments stimulates mat-uration of DCs through interactionwith TLR4 and RAGE. Activated DCsdetermine a clonal expansion of tumor antigen-specific T cells withimmune antitumor responses. (2) HMGB1 enhances chemotaxis andpromotes migration of monocytes, DCs, and Tcells. The pro-tumorigeniceffects are as follows: (1) Angiogenesis—persistent hypoxia in growingtumors leads to necrosis and apoptosis, causing a chronic release ofHMGB1, which activates protumor responses promoting angiogenesisand tumor growth through the recruitment of endothelial precursor cells(EPCs) and activation of local endothelial cells through RAGE signaling.Furthermore, activated macrophages synthetizes pro-angiogenetic fac-tors, such as VEGF. (2) Autophagy induction—during low oxygen,

HIF-1α initiates TLR4 transcription, which can then be engaged byHMGB1. The engagement of TLR4 results in signaling through adaptormolecules MyD88 and TRIF which both inhibit the interaction of Bcl-2with Beclin 1. Cytosolic HMGB1 disrupts the interaction betweenBeclin1 and Bcl-2 by competitively binding to Beclin1, inducing autoph-agy. HMGB1 induces autophagy by its direct binding to the RAGE andactivating ROS and NF-kB pathways. Autophagy improves the tumorgrowth inducing chemotherapy resistance, inhibiting apoptosis, and en-hancing cell survival. Abbreviations: HMGB1 high-mobility group pro-tein B1, RAGE receptor for advanced glycation end products, TLR toll-like receptor, IL interleukin, TNF tumor necrosis factor, EPCs endothelialprogenitor cells, VEGF vascular endothelial growth factor,HIF1 hypoxiainducible factor-1,MyD88myeloid differentiation primary response gene88, TRIF TIR-domain-containing adapter-inducing interferon-β, Bcl-2 Bcell lymphoma 2, ROS reactive oxygen species, NF-kB nuclear factor-kappa B

Eur J Pediatr

tissue damage secondary to meningeal inflammation is in-duced by TLR signalling activation [53, 67, 94].

Several studies demonstrated a pivotal role of HMGB1 inmeningitis. In particular, CSF levels of HMGB1were elevatedin children with bacterial and aseptic meningitis, with thehighest values revealed in bacterial infection. Furthermore, asignificant correlation between white blood cell counts, glu-cose levels, and HMGB1 was underlined, confirming a diag-nostic role of this protein in meningitis [102].

These observations were confirmed by Asano et al., whoanalyzed HMGB1 in different infectious neurological disease.A marked elevation of HMGB1was seen only in bacterial andaseptic meningitis, concluding that HMGB1 was a poor dis-ease marker for acute encephalopathy [9]. In addition, Allevaet al. revealed a pathogenetic role of HMGB1 in childrenshowing cerebral symptoms as a consequence of severefalciparum malaria. Malarial patients were characterized byhigher HMGB1 levels than controls, and strictly related withpatient’s survival [6].

However, further studies about HMGB1 and its receptorare needed to reveal its exact role in pediatric meningitis.

Epilepsy

Febrile seizures are themost common form of childhood seizures[52]. Experimental studies provide evidence that induction ofspecific proinflammatory pathways in forebrain mediatesproconvulsive effects, and their pharmacological modulation rep-resents a potential strategy to reduce seizure activity [106, 107].

In particular, recent data, obtained from different experi-mental models of acute and chronic seizures, identified thecrucial role played by the activation of HMGB1-TLR4 sig-naling in the hippocampus in the generation and recurrence ofseizures [73]. Choi et al. demonstrated that active inflamma-tion, including HMGB1 and proinflammatory cytokines, oc-curred in children with febrile seizures and pediatric epilepsy[34]. Zurolo et al. evaluated the role of HMGB1-TLR4 axis inpediatric, adolescent, and adult patients who underwent

Table 4 Neurological diseases and HMGB1 involvement

Disease HMGB1 Mechanisms Pediatric data (reference) Future prospects

Autism Serum ↑ Dysregulated immune functionis a recurrent finding in autisticchildren. In addition, the TLRand RAGE signaling pathwaysare altered with a dysfunctionin monocyte pathogen recognition

4 studies; atudies [13, 43, 61]investigated TLR and RAGEpathway dysfunction in children.HMGB1 levels were assessed inautistic children [42]

Diagnostic marker of autism

AN Serum→or ↑

Proinflammatory cytokines playroles in AN. administration ofHMGB1 into the cerebral ventriclesin rats decreases food intake, whilea passive immunization withanti-HMGB1 antibodies attenuatedthe development of hypophagia

No evidence in children. Only 1study evaluated HMGB1 in youngadults affected by AN. It analyzed basalHMGB1 values and after treatment [118]

Diagnostic marker of AN

Marker of therapy resistance

TBI Serumand H↑

HMGB1 in part reflect the degreeof necrosis and apoptosis presentafter TBI. RAGE and HMGB1expression increased in microgliain the region surrounding thecontused area

1 study; HMGB1 was evaluated in CSFof children with TBI, highlighting itsprognostic role [10]

HMGB1 inhibition as therapy

Prognostic marker

Meningitis CSF ↑↑ HMGB1, massively released intothe cerebrospinal fluid, acts as aninflammatory cytokine throughTLR pathway, mediating meningealinflammation. MeningococcalCpG-DNA-HMGB1 enters inthe cells by endocytosis and thenbinds to TLR9, inducing activation ofinflammatory cytokines

4 studies; in pediatric populations,the physiopathological role ofHMGB1 has been studied inmeningitis and other neurologicalinfectious diseases

Diagnostic marker of meningitisbut not for acute enchephalothy

Prognostic marker

Marker of therapy response

Epilepsy Serumand H↑

Active neuroinflammation and markedcellular injury occur in pediatricepilepsy.The activation of HMGB1-TLR-RAGEsignaling plays a pivotal role in thegeneration and recurrence of seizures

2 studies; children with epilepsypresented an active inflammationstate and high HMGB1 levels [34].Histologically, it assessed anoverexpression of HMGB1 and itsreceptors in cerebral bioptic tissue [124]

HMGB1 inhibition as therapy

Prognostic marker

HMGB1 high-mobility group box 1, TLR toll-like receptor, H histological data, RAGE advanced glycation end products, AN anorexia nervosa, TBItraumatic brain injury, CSF cerebrospinal fluid

Eur J Pediatr

therapeutic surgical resection for refractory epilepsy, demon-strating an intralesional overexpression of HMGB1, TLR2,TLR4, and RAGE [124].

A pharmacological modulation of HMGB1-TLR/RAGEaxis with receptor antagonists or inactivating antibodies mayrepresent a potential novel antiepileptic strategy.

All information concerning HMGB1 involvement in pedi-atric neurological disorders has been reported in Table 4.

Prematurity born

The poor outcome observed in many preterm children is notentirely dependent on their gestational age at birth, but severalrisk factors are significantly associated with an increased riskof cerebral palsy, such as hypoxia, intra-amniotic infection,and inflammation [23].

It is well known that the host’s response to microbial path-ogens involves a series of carefully orchestrated mechanisms,including DAMP peptides, such as HMGB1, and their receptorpathways. A significant change in expression of RAGE andHMGB1 was demonstrated in the setting of inflammation-induced preterm birth, indicating that these peptides may beimportant mediators of cellular injury in fetus delivered [22].

Moreover, Romero et al. demonstrated high amniotic fluidHMGB1 concentrations in newborn with intra-amniotic infec-tion and preterm prelabor rupture of membranes [39, 91].HMGB1 levels were also high in infants born before 32 weeksof gestation and requiring mechanical ventilation, with a strictlycorrelation with the development of bronchopulmonary dyspla-sia or death [2]. HMGB1 was also investigated in neonates,with higher levels in unscheduled than scheduled caesariandelivery and associated with ischemic reperfusion injury [78].

Moreover, in neonates with asphyxia, it has been suggestedthat the elevation of HMGB1 might be associated with abnor-mal inflammatory responses, involving the excessive produc-tion of proinflammatory cytokines [82].

The important role of HMGB1 in fetal immune system hasbeen corroborated by data demonstrating that HMGB1 trig-gers inflammatory responses through upregulation of adhe-sion molecules and release of soluble proinflammatory medi-ators from endothelial cells [72]. Furthermore, HMGB1 exertsan essential role of neonatal immune responses with importantbiological functions, as a bridge between innate and adaptiveimmune responses [35].

Conclusion and future prospects

HMGB1 represents a common causal agent for various typesof diseases and may serve as a future target molecule poten-tially useful for understanding disease processes. Future stud-ies will define the pathways for HMGB1 release, elucidate themost effective strategies for blocking the effect of this

mediator and determine the nature of any side effects devel-oped when HMGB1 levels are reduced. Certain substances,able to inhibit the release or inactivate HMGB1 protein, areunder investigation. The study of HMGB1 should open newscenarios for diagnostic biomarkers and therapeutic strategies,both in childhood and in adult patients.

Funding source No external funding was secured for this study.

Financial disclosure The authors have no financial relationships rele-vant to this article to disclose.

Conflict of interest The authors have no conflicts of interest todisclose.

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