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Review ArticleHeat Shock Protein 72 Expressing Stress inSepsis: Unbridgeable Gap between Animal and HumanStudiesA Hypothetical Comparative Study

George Briassoulis,1 Efrossini Briassouli,2 Diana-Michaela Fitrolaki,1 Ioanna Plati,3

Kleovoulos Apostolou,4 Theonymfi Tavladaki,1 and Anna-Maria Spanaki1

Pediatric Intensive Care Unit, University Hospital, School of Health Sciences, University of Crete,Voutes Area, Heraklion, Crete, Greece

st Department of Propaedeutic Internal Medicine, Laiko, University General Hospital, University of Athens, Agiou Toma, Athens, Greece

Department of Clinical Chemistry, School of Medicine, University of Crete, Voutes Area, Heraklion, Crete, Greece National and Kapodistrian University of Athens, First Critical Care Department, Evaggelismos Hospital,

Ipsilantou , , Athens, Greece

Correspondence should be addressed to George Briassoulis; ggbriass@otenet.gr

Received April ; Accepted October ; Published January

Academic Editor: Wolgang Neuhoer

Copyright George Briassoulis et al. Tis is an open access article distributed under the Creative Commons Attribution

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Heat shock protein (Hsp) exhibits a protective roleduring timeso increasedrisk o pathogenic challengeand/or tissue damage.Te aim o the study was to ascertain Hsp protective effect differences between animal and human studies in sepsis using ahypothetical comparative study model. Forty-one in vivo (.%), in vitro (.%), or combined (.%) animal and in vivo ()or in vitro () human Hsp studies ( < 0.0001) were enrolled in the analysis. O the human studies, % showed a protectiveHsp effect compared to .% protection shown in septic animal studies ( < 0.0001). Only human studies reported Hsp-associated mortality (.%) or inection (.%) or reported results (.%) to be nonprotective ( < 0.001). In animal models,any Hsp induction method tried increased intracellular Hsp (%), compared to .% o human studies ( < 0.02), reducedproinammatory cytokines (/), and enhanced survival (/). Animal studies show a clear Hsp protective effect in sepsis.Human studies are inconclusive, showing either protection or a possible relation to mortality and inections. Tis might be dueto the act that using evermore puried target cell populations in animal models, a lot o clinical inormation regarding the netresponse that occurs in sepsis is missing.

1. Introduction

Sepsis is an inammation-induced syndrome resulting roma complex interaction between host and inectious agents. Itis considered severe when associated with acute organ dys-unction, which accounts or the main cause underlyingsepsis-induced death. Despite increasing evidence in sup-port o antioxidant [], anti-inammatory [], or immune-enhancing [] therapies in sepsis, recent studies ailed toestablish a correlation between antiseptic pathway-based

therapies and improvement o sepsis [] or septic shock []or among immune-competent patients [].

Rapid expression o the survival gene heat shock protein (Hsp) was shown to be critical or mounting cytoprotec-tion against severe cellular stress, like elevated temperature[]. Intracellular Hsps are upregulated in cells subjected tostressul stimuli, including inammation and oxidative stressexerting a protective effect against hypoxia, excess oxygenradicals, endotoxin, inections, and ever []. Recent stud-ies imply that different biological disease processes and/or

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 101023, 17 pageshttp://dx.doi.org/10.1155/2014/101023

http://dx.doi.org/10.1155/2014/101023http://dx.doi.org/10.1155/2014/101023

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simple interventions may interere with high temperaturestress, leading to different clinical outcome in patients withand without sepsis []. In septic patients, administration oantipyretics independently associated with -day mortality,without association o ever with mortality []. Importantly,ever control using external cooling was sae and decreased

vasopressor requirements and early mortality in septic shock[].Inducible Hsp is also ound extracellularly where

it exhibits a protective role by acilitating immunologicalresponses during times o increased risk o pathogenic chal-lenge and/or tissue damage []. Experimental data provideimportantinsights intothe anti-inammatory mechanismsostress proteins protection and may lead to the development oa novel strategy or treatment o inectious and inammatorydisorders []. However, although overexpression o stressproteins signals danger to inammatory cells and aids inimmune surveillance by transporting intracellular peptidesto immune cells [], it has also been linked to a deleteriousrole in some diseases []. In addition, serum Hsp levelswere shown to be modulated according to the patient oxidantstatus whereas increased serum Hsp was associated withmortality in sepsis [].

Te purpose o this basic research-related review incritical care is to document the available evidence on therole o Hsp in sepsis, reporting both the state o theart and the uture research directions. It might be thatpotential therapeutic use o stress proteins in prevention ocommon stress-related diseases involves achieving optimalbalance between protective and immunogenic effects o thesemolecules []. In this review, we will attempt to classiyexperimental and clinical studies on Hsp in sepsis and tocompare their results on inammation, organ unction, andoutcome; we will also briey discuss the mechanisms on howstress proteins might exert their protective or negative rolein the disease development and highlight the potential clinictranslation in the research eld.

2. Materials and Methods

Human or animal in vivo or in vitro studies examining thebenecial effect o intra- or extracellular Hsp expression insepsis were included in this study. Te PRISMA [] searchmethod or identication o studies consisted o searches oPubMed database ( to September ) and a manual

review o reerence lists using the search term: Hsp or .Te search output was limited with the search lter or anyo: sepsis; severe sepsis; bacterial lipopolysaccharide (LPS);endotoxin. Reerences in selected studies were examinedalso. Te title and abstract o all studies identied by theabove search strategy were screened, and the ull text orall potentially relevant studies published in English wasobtained. Te ull text o any potentially relevant studies wasassessed by ve authors (DMF, EB, IP, AK, and ). Te sameauthors extracted data rom the published studies.

.. Statistical Analysis. Proportions o methods used and

results ndings were compared by the 2 test. A two-sided

alpha o . was used or statistical signicance. Te resultswere analyzed using SPSS sofware (version ., SPSS,Chicago, IL, USA).

3. Results

Our search identied PubMed titles and abstracts.Afer excluding duplicates, studies with no original data, ordata insufficient to evaluate or those whose outcome wasischemia/reperusion injury or others, articles were nallyincluded or analysis. Te aim o this minireview was notto examine the quality o studies, but to describe inductionmethods and to compare in vivo and in vitro methods andresults regarding a potential protective role or Hsp inhuman and animal sepsis.

.. Animals. Forty-one in vivo (, .%), in vitro (,.%), or combined (, .%) animal studies ullling theresearch criteria regarding the role o Hsp in sepsis were

enrolled in analysis (ables (a), (b), and (c)). In only studies transgenic animals (Hsp/ (.%), overexpressingthe human Hspab gene (.%)) were used (.%), all inmice ( < 0.03). Hsp induction methods used in ratsdiffered rom those used in mice ( < 0.0001). Hspinduction was attempted most ofen using heat shock (rats, .%; mice , .%), glutamine (Gln) (rats , .%; mice, %; sheep , %), or combined Gln with additionalinducer (rats , .%; mice , .%). In rats Hsp wasinduced through adenoviral vector Hsp (AdHSP) (, .%o studies in rats) or various recombinant Hsp (rHsp)preparations (, .%) compared to mice studies whereAdHSP, bovine rHsp preconditioning, or overexpressed

Hsp within the intestinal epithelium was used (.%).Hsp gene-transected models (, .%) or cecal ligationand puncture (CLP) with LPS or injection o microorganisms(, .%) were used only in mice studies.

In more than hal o the studies induction was attemptedin a pretreatment mode (, .% or mice; , .% orrats induction afer LPS injection or CLP), ollowed by aconcomitant mode in rats (, %) or a posttreatment onein mice (, %). Te different time intervals used beore orafer experimental sepsis, most ofen - hours, did not differamong groups. Preventive effect was achieved by most induc-tion methods used in mice or rats (/, .%), irrespectiveo the challengeperiod or timing used (Figures(a)and(b)).

wo studies, one carried out in sheep and one in rats,were inconclusive. In all septic animal models, any Hspinduction method tried increased intracellular Hsp (/,%), reduced proinammatory cytokines (/ studiesinvolving cytokine measurements), organ damage (/),clinical deterioration (/), and enhanced survival (/).

.. Patients. Only human in vivo () and in vitro ()Hsp studies were identied (ables(a) and(b)): humanperipheral blood mononuclear cells (hPBMC) studies,.%; polymorphonuclear leukocytes (hPMNL) studies,.%; lymphocytes (hPBLC) study, .%; in vivo (childrenor adults serum levels) studies, .%. O those, hPBMC

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T:(a)Resultsoanimalinvivostudiesexaminingthepreventiveroleointra-

orextracellularHsp(Hsp)expression

andHsp(Hsp)expressioninexperim

entalsepsisorsepsis-

relatedpathophysiology.(

b)Resultsoan

imalinvitrostudiesexaminingthepreventiveroleointracellularHsp(Hsp)expressioninexperimentalsepsisorsepsis-relatedpathophysiology.

(c)ResultsogeneticanimalstudiesexaminingthepreventiveroleointracellularHsp(Hsp)expressioninexperimenta

lsepsisorsepsis-relatedpathophysiology.

(a)

Invivo

Induction

Organsst

udied

Expressionin

cells/Hsp

challenge

Extracellular

Hsplevels

Inhibitors

Functional

Pathways

Interleukins

Organ

damage

Survival

CLPsepsis

rats[,]

LPS-treated

rats[]

LPS-treated

mice[]

Heatstress

Lungs()

Heart()

Splenocytes()

Rostral

Ventrolateral

medulla(

)

Mitochon

drial

unction()

Brain()

Induced()

Hsp

inhibitors

(KNKor

pithrin-m)

abrogatedthe

abilityothe

thermal

treatmentto

enhanceTNF-

()

Alleviated

hypotension,

bradycardia,

sympathetic

vasomotor

activity()EEG

andepileptic

spikes

attenuated()

Suppre

ssediNOS

mRNA

NF-B

activation,I

Bkinase

activation,I

B

degrad

ation()

Preven

ted

downregulationo

Grp,preserved

cytoch

romec

oxidase()enhanced

phosphorylationo

IKK,IkB,N

F-B

nuclear

translo

cation,

bindingtothe

TNF-promoter()

Cytokines

declined()

HMGB

inhibited()

enhanced

LPS-induced

TNF-

production()

Reduced

()

Prevented

sepsis-

associated

encepha

lopa-

thy()

Enhanced()

LPS-treated

mice[,]

rats[]

sheep[]

CLPsepsis

rats[,]

CPLsepsis

mice[,]

Glutamine

Heart()

Lungs()

Liver()

Aorta()

Kidneys(

)

Brain()

Blood()

Multipleorgans

()

Induced()

Bloodsamples:

increasedHsp

onlyafercoad-

ministrationo

Glnand

ciprooxacin

[]

Quercetin

blocked

Gln-mediated

enhancemento

Hspand

HSF--p

expressionsand

survivalbenet

()

LDdoseoP.

aeruginosaand

ciprooxacinin

combinations

()

Prevented

ARDS()

arterial

pressure,

cardiac

contractility

restoredinthe

Glnthaninthe

LPSshock()

Quercetin

preventedGln

protection()

Nodifferencein

hemodynamic

parameters()

Inhibitedactivation,

translo

cationo

NF-B

tothe

nucleu

sdegradation

oIKBalpha,

phosphorylationo

pMAPK,E

RK,

increasedMKP-()

lungH

MGB-

expressionNF-B

DNA-binding

activitysuppressed

()Red

uced

peroxide

biosynthesis()

Attenuated

TNF-alpha(),

IL-.I

L-,

MDA,H

MGB-,

apoptosis()

increasedIL-

()

Reduced

()

Enhanced()

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(a)Continued.

Invivo

Induction

Organsst

udied

Expressionin

cells/Hsp

challenge

Extracellular

Hsplevels

Inhibitors

Functional

Pathways

Interleukins

Organ

damage

Survival

LPS-treated

ratsbovineor

Advirusor

rHsp[]

CLPsepsis

ratsand

tracheas

AdHSP[]

Exogenous

rHsp

AdTrackor

Advirus

Liver()

Peritonea

l

macrophages

()

MLE-c

ells()

Myocardium()

Lungs()

Induced()

Normalized

hemostasis()

hemodynamics

()

Biochemical

parameters()

Inhibited

LPS-in

duced

decreaseNO

expressionin

macrophages,

norma

lized

neutro

philapoptosis

()inhibitedIB

degrad

ationand

NF-B

,pnuclear

translo

cation()

apopto

ticcellular

pathwayscaspases,

,()

Modied

myeloidcells

responsetoLPS

()prevented

LPS-induced

increasein

TNF-andIL-

()Reduced

ICAM-,

attenuated

cardiac

dysunction()

Attenuated

cardiac

dysunction

()reduced

alveolar

cell

apoptosis()

Enhanced()

(b)

Invitro

Induction

Organsstudied

Intracellular

Hspexpression

Inhibitors

Pathways

Interleukins

Organdamage

Survival

Murine

macrophage-

likeRAW

.cells

[]

Heatshocked

Macrophages()

CellsromHS

overexpressed

Hsp()

Inhibited

phosphorylationo

p,J

NK,

ERK/MAPK,IB

degradation,

NF-Bpnuclear

translocation()

HSinhibited

HMGB-

induced

cytokines

TNF-and

IL-

()

Enhanced()

CLP-treated

murine

peritoneal

macrophage

cellline

RAW.

[]Neonatal

ratcardiomy-

ocytes[]

IEC-rat

intestinal

epithelial

cells[]

Glutamine

Peritoneal

ma

crophages()

Cardiomyocytes

()Int

estinal

epithelialcells()

IncreasedHsp

expression()

Gln

protection

mimickedby

PUGNA,

banishedby

alloxan()

DFMO

ornithine

decarboxy-

laseinhibitor

()

ReducedLDH,

increased

O-ClcNAc,HSF-,

transcription

activity()

increasedHSF

bindingtoHSE()

Invitro

TNF-dose-

time-Gln.

DependentIn

vivolower

intracellular

TNF-level

Attenuated

LPS-induced

cardiomyocyte

damage()

Enhanced()

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(b)Continued.

Invitro

Induction

Organsstudied

Intracellular

Hspexpression

Inhibitors

Pathways

Interleukins

Organdamage

Survival

LPS-treated

rats[]

LPS

stimulation-

mouse

macrophage-

likecellline

(RAW.

cells)[,]

Transected

withHsp

plasmidor

HS

Myocardium()

Macrophages()

Hspplasmidor

HSinduced

Hsp()

iNOSmRNA

completely

abolishedbyHS-

Hsptransected

cells()

HSinhibited

LPS-induced

NF-BandHSF-

activity()

increasesboth

cellularSKmRNA

andproteinlevels

()

Enhanced()

CLPrats,

murinelung

epithelial-

cellsin

culture[]

Murine

macrophage-

likeRAW

.cells

[]

Exogenous

Hsp

Lungs()

Macrophages()

Overexpression

oHspin

RAW/Hspcells

()

Limitednuclear

translocationo

NF-B,

phosphorylationo

IkappaBalpha()

Inhibitionothe

MAPkinases(p,

JNK,andERK)()

Inhibitiono

theNF-B-

HMGB-

induced

releaseo

TNF-,I

L-

()

LimitedNF-B

activation()

Enhanced()

CLP-treated

mice[]

Arsenite

(Positive

control)

Lungs()

Induced-

Inhibitors

blockedHsp

expression,(

)

Anti-human

Hsp()

Pretreatmentwith

neutralizing

antibodiesto

Hspdiminished

neutrophilkilling

()

Survivorshigher

oTcells()

Enhanced()

(c)

KOanimals

Induction

Organsstudied

IntracellularHsp

expression

Pathways

Interleukins

Organdamage

Survival

CLPsepsis

Hsp.

//KO

mice[]

Glutamine

Lungs()

Hsp.

//mice

didnotincrease

Hsp()

Hsp.

/((/))

miceincreasedNF-B

binding/activation()

IncreasedTN

F-,

IL-inKO()

Increasedlunginjury

inKO()

DecreasedinKO()

CLPsepsis,

injectiono

microorganisms

Hsp/KO

mice[]

Imipenem/

cilastatin

Gut()

Lungs()

Hsp/micedid

notincreaseHsp

()

Increasedapoptosis

andinammation

Hsp/increased

TNF-,I

L-,I

L-,

IL-b

KO-increasedgut

epithelialapoptosis,

pulmonary

inammation()

D

ecreasedinKOage

dependent()

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(c)Continued.

KOanimals

Induction

Organsstudied

IntracellularHsp

expression

Pathways

Interleukins

Organdamage

Survival

LPS-treatedmice

Hsp/or

overexpressed

Hsp[]

LPS

Intestinal

epithelium()

Pharmacologic

Hsp

upregulation

HspreducedTLR

signalingin

enterocytes()

Hspreversed

TLR-cytok

ines,

enterocyteapoptosis

()

Preventedandtreated

experimentalNEC()

LPS-treatedmice

overexpressing

thehuman

Hspabgene[]

LPS

Heart()

Overexpressiono

HSPAB

Preventeddecrement

intheactivationo

PIK/proteinkinaseB

signalingin

myocardium()

Decreasedth

e

expressiono

VCAM-/ICAM-()

Decreasedleucocyte

inltrationin

myocardium()

Attenuatedcardiac

dysunction()

:numberostudies;PBMC:peripheralbloo

dmononuclearcells;LPS:bacteriallipopolysaccharide;CLP:cecalligationandpuncture;TNF

-:tumornecrosisactor-alpha;AdHSP:adenoviralvectorHsp;Gln:

glutamine;HS:heatstress;Hspgene:Hspgene-transectedmodels;HSF:HSactor;HSE

:heatshockelement;IKK:IBkinase;IkB:IkappaBalpha.

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Treatment time related to LPS/CLP

Both Con Post Pre

AdVhsp

CLP+

ExofHsp

Gln

Gln+

HS

Hspgene

Animal iHsp72induction studies

InconclusiveProtective

(a)

LPS/CLP iHsp72 interval (min)

0 10 30 60 120

AdVhsp

CLP+

ExofHsp

Gln

Gln+

HS

Hspgene

Animal iHsp72induction studies

InconclusiveProtective

(b)

F : (a) Preventive effect was achieved by all induction methods used irrespective o the challenge period or (b) time lapse betweenthe sepsis insult and the Hsp induction: LPS, bacterial lipopolysaccharide; CLP, caecal ligation and puncture; iHsp, inducible heat shockprotein ; Pre, pre-treatment; Post, posttreatment; both, trials with pre- and postexperiments; Con, concomitant; AdHSP, adenoviral vectorHsp; exogHsp, exogenous Hsp preparations; Gln, glutamine; +, additional challenge; HS, heat stress; Hspgene, Hsp gene-transectedmodels.

were used in only studies with septic patients but in withhealthy volunteers. Heat stress (HS) or acclimation was usedin studies (.%), Gln administration in in associationwith LPS (.%), recombinant human Hsp in (.%),and either inhibitor or agonist in (.%). In studies nochallenge or only LPS (.%) was used. In only out o (.%) studies in septic patients induction Hsp methodswere attempted compared to % in the studies with healthy() or ARDS () patients ( < 0.006). Protection markersstudied were apoptosis ( studies, .%), HS ( studies,.%), oxidative damage, hospital inections, hemodynamicinstability, and ARDS ( study each, .%).

Intracellular Hsp was induced in in vitro studies(.%, in healthy, in septic) and inhibited in (.%, in

septic, in ARDS patients). O the studies in septic patients,intracellular Hsp was increased in (%), inhibited in (%), and not measured in . With the exception o sodiumarsenite, neither Gln nor HS were tested in these studies.Extracellular Hsp, measured in in vitro and in in vivostudies, was shown to increase in sepsis, especially in septicshock or in those who died (.% o human studies).

Increased intracellular Hsp was protective in hal othe human studies (%); regarding the positive (HS, Gln,exogenous Hsp) in vitro induction Hsp human studies (.%) were protective (Figure (a)) and inconclusive(.%) or nonprotective (.%). O the induction methodsused, protection offered HS (/, %), glutamine (/, %),

rHsp and sodium arsenite (/, % each) (Figure (b)).Incontrast, o the in vivo (serum Hsp measurements), in

vitro endotoxin induced (LPS or CLP), and Hsp inhibitorhuman studies, none was shown to be associated with a betteroutcome ( < 0.02); studies were associated with mortality(%) and with inection (%) or were inconclusive (%).Septic patients studies were positive or protection in only out o (.%) compared to out o (.%) in healthy and% in ARDS patients ( < 0.06).

.. Human Compared to Animal Studies. Out o a total o enrolled studies, only in vivo human studies (.%) havebeen reported on the role o Hsp in sepsis compared to

mice (.%) and rat (.%) in vivo studies ( < 0.0001);in contrast human (.%) studies have been reportedin vitro compared to only in rats (.%) and in mice(.%); mice (.%) and rat (.%) combined in vitro-in vivo studies have also been reported. O the humanstudies, % showed a protective Hsp effect comparedto .% protection shown in animal studies (Figure (a)).When restricted to the septic patients studies, however, only out o (.%) demonstrated an Hsp protective effectcompared to .% protection shown in animal studies (