Effects Of Hsp72 And Quercetin On Release And Pro-inflammatory Function Of HMGB1

High mobility group box 1 (HMGB 1), a nuclear protein, has recentlybeen identified as an important mediator of local and systemicinflammatory diseases when released into the extracellular milieu. Heatshock response (HSR) is an effective auto-protective mechanism whenthe host encountered harmful stimuli. HSR not only has anti-injury andanti-apoptosis roles, but also has an anti-inflammatory role.Anti-inflammatory regulation by HSR is associated with the induciblesynthesis of heat shock proteins (HSPs), but the related mechanismremains incompletely delineated. Heat shock protein 72 (Hsp72) is thebest characterized and stress inducible HSPs. Quercetin, one of flavanoids,has wide pharmacologic actions with little adverse reactions. However,the potential role of Hsp72 and quercetin in the regulation of HMGB1release and function has not been investigated previously.Firstly, the investigators explored that the clinical significance ofHMGB1 alteration in childhood infectious diseases. (1) Detection ofHMGB1 in serum of pediatric patients with sepsis. The serumconcentrations of HMGB1 were significantly higher in sepsis cases onadmission to Pediatric Intensive Care Unit (PICU) as compared withthose in normal control group. There were lower levels of HMGB1 onimproved group, but higher levels of HMGB1 on aggravated group ascompared with those on admission to PICU. (2) Detection of HMGB1and heat shock proteins in cerebrospinal fluid of pediatric patients withmeningitis. The cerebrospinal fluid (CSF) levels of HMGB1 in thepurulent meningitis (PM) group were significantly higher than those inthe control, viral meningitis (VM) or tubercular meningitis (TM) group.However, there was no significant difference in CSF HMGB1 levelsbetween control and TM group, and between VM and TM group. TheCSF levels of Hsp72 were significantly higher in the PM, VM, and TMgroup as compared with those in the control group. Moreover, CSF levelsof Hsp72 were significantly higher in the PM and TM group as comparedwith those in the VM group. No significant difference was found betweenPM and TM group. The CSF levels ofαB-crystallin were significantly higher in the PM, VM, and TM groups as compared with those in thecontrol group. However, there was no significant difference between thePM, VM and TM group. Notably, heat shock protein 90 (Hsp90) was notdetectable in CSF samples of all patients. It suggests that themeasurement of serum HMGB 1 is helpful to evaluate the prognosis of thechildhood sepsis. HMGB1 or Hsp72 levels may be valuable indistinguishing patients with purulent bacterial meningitis from viralmeningitis.Secondly, the investigators explored the roles of Hsp72 in theregulation of HMGB1 translocation, release and pro-inflammatoryfunction. Although residing predominantly in the nucleus of quiescentmacophages, HMGB 1 can be actively secreted in response to exogenousand endogenous inflammatory stimuli such as lipopolysaccharide (LPS),tumor necrosis factor (TNF)-αand hydrogen peroxide (H₂O₂). Thesestimuli also increased the cytoplasmic translocation and expression ofchromosome region maintenance 1 (CRM1), a nuclear export factor ofHMGB1, and increased the interaction of CRM1 with HMGB1. Inaddition, H₂O₂ induced histone-3 (H3) acetylation in macophages.However, mild HSR or enhanced expression of Hsp72 (by genetransfection) similarly rendered macrophages resistant to thesestimuli-induced HMGB1 cytoplasmic translocation and release. Hsp72inhibited the interaction between HMGB1 and CRM1 by competitivelybinding to HMGB1 in macrophages after inflammatory stimuli treatment.Genetic deletion of the nuclear localization sequence (NLS) and peptidebinding domain (PBD) from Hsp72 prevented H₂O₂-inducedHMGB1-Hsp72 interaction in the nucleus and completely abolishedHsp72-mediated suppression of HMGB1 cytoplasmic translocation andrelease. In addition to HMGB1, nuclear Hsp72 also interacted withhistoric deacetylase 1 (HDAC1) and inhibited H3 acetylation, whichpotentially prevented HMGB1 acetylation and cytoplasmic translocation.Overexpression Hsp72 also inhibited H₂O₂-induced CRM1 expression.These findings suggest that Hsp72 inhibits HMGB1 release mainlythrough interfering with cytoplasmic translocation of HMGB1. Theresults also showed that oxidative stress such as H₂O₂ induced active and/or passive HMGB1 release from macrophage and monocyte culturesin a time- and dose-dependent fashion. H₂O₂ stimulated macrophages andmonocytes to actively release HMGB1 in JNK-or ERK-dependent, butp38-independent mechanisms. On the other hand, overexpression ofHsp72 strongly inhibited HMGB 1-induced cytokine (e.g. TNF-α, IL-1β)expression and release which correlated closely with (1) inhibition of theMAPKs (p38, JNK, and ERK) and (2) inhibition of the NF-κB pathway(IκBαdegradation, NF-κB p65 nuclear translocation, and NF-κB DNAbinding activity) by elevated Hsp72.Eventually, the investigators observed that roles of quercetin inpreventing lethality in mice with endotoxemia and inhibiting HMGB1release and translocation. Quercetin pretreatment prevented endotoxinlethality and inhibited TNF-αand HMGB1 release in vivo. Delayedadministration of quercetin also attenuated the lethality of endotoxemiaand the release of HMGB1 in vivo. In addition, quercetin not onlyinhibited LPS- and TNF-α-induced HMGB 1 relese and translocation, butalso inhibited LPS- and HMGBl-induced expression and release ofTNF-αand IL-1βin vitro. Furtermore, quercetin inhibited LPS-andTNF-α-induced activation of the MAPKs and the NF-κB pathway. Itsuggests that quercetin prevents endotoxemia by attenuating the releaseand pro-inflammatory function of HMGB1.Taken together, these experiments suggest that HMGB1 playimportant roles in onset of sepsis and central nervous system infections.The anti-inflammatory activity of Hsp72 and quercetin is achieved partlyby interfering both with the release and pro-inflammatory function ofHMGB1. Our experimental data provide important insights into theanti-inflammatory mechanisms of Hsp72 and quercetin, and may lead tothe development of novel therapies of human infectious diseases.