Downregulation of the NLRP3 inflammasome by adiponectin rescues Duchenne muscular dystrophy

17 Pages·2017·2.42 MB·English

by  Raphaël Boursereau

Boursereauetal.BMCBiology (2018) 16:33
https://doi.org/10.1186/s12915-018-0501-z
RESEARCH ARTICLE Open Access
Downregulation of the NLRP3
inflammasome by adiponectin rescues
Duchenne muscular dystrophy
Raphaël Boursereau1, Michel Abou-Samra1, Sophie Lecompte1, Laurence Noel1 and Sonia M. Brichard1,2*
Abstract
Background: The hormone adiponectin (ApN) exerts powerfulanti-inflammatory effects onskeletal muscle and can
reverse devastating myopathies, like Duchenne muscular dystrophy (DMD), where inflammation exacerbates disease
progression. The NLRP3 inflammasome plays a key role inthe inflammation process, and its aberrant activation
leads to several inflammatoryor immunediseases.Here we investigated theexpression of the NLRPinflammasome
inskeletal muscle and itscontribution to DMD.
Results: We find that NLRP3 is expressed in skeletal muscle and showthatApN downregulates NLRP3 via itsanti-
inflammatory mediator, miR-711.This repressionoccurs both invitro inC2C12 myotubes and in vivo after either
local (via muscle electrotransfer) or systemic (by using transgenic mice) ApN supplementation.To explore the role
ofthe NLRP3 inflammasome ina murine model ofDMD, we crossed mdx mice with Nlrp3-knockoutmice. In mdx
mice, all components of the inflammasome were upregulated in muscle, and the complex was overactivated. By
contrast, in mdx mice lackingNlrp3, there was a reduction incaspase-1 activation, inflammation and oxidative
stress in dystrophic muscle, and these mice showed higher global muscle force/endurance thanregular mdx mice
as well as decreased muscle damage. Toinvestigate therelevanceof NLPR3 regulation ina human disease context,
we characterizedNLRP3 expression in primary cultures ofmyotubes from DMD subjectsand found a threefold
increase compared to control subjects. This overexpression was attenuated byApN or miR-711mimic treatments.
Conclusions: The NLRP3 inflammasome plays a key pathogenic role inDMD and muscle inflammation, thereby
opening new therapeutic perspectives for theseand otherrelated disorders.
Keywords: Adiponectin,miR-711,NLRP3inflammasome,Skeletalmuscle,Inflammation,Duchennemusculardystrophy
Background structural stability and integrity to the myofibre mem-
Chronic muscle inflammation may be present as either a brane. Absence of this protein leads to membrane dam-
low-grade or a severe form. The low-grade form charac- age,allowingmassiveinfiltrationofimmunecells,chronic
terizes obesity-linked metabolic disorders [1, 2]. A more inflammation, necrosis and severe muscle degeneration
aggressiveformmayplayaroleinthedevelopmentofmy- [3].Althoughdystrophinmutationsrepresenttheprimary
opathy and in disease severity. Duchenne muscular dys- cause of DMD, it is the secondary processes involving
trophy (DMD) is the most frequently inherited human chronic and severe inflammation that likely exacerbate
myopathyandthemostdevastatingtypeofmusculardys- diseaseprogression[4,5].
trophy.DMDstemsfromX-linkedrecessivedefectsinthe Adiponectin(ApN)isahormoneabundantlysecretedby
gene encoding for dystrophin, a protein that provides adipocytes under normal conditions. Circulating ApN is
significantly decreased in obese individuals and in patients
with criteria for the metabolic syndrome [6]. Besides its
*Correspondence:[email protected]
1Endocrinology,DiabetesandNutritionUnit,InstituteofExperimentaland metabolic(mainlyinsulin-sensitizingandfat-burning)prop-
ClinicalResearch,MedicalSector,CatholicUniversityofLouvain,1200 erties,ApNhasemergedasamasterregulatorofinflamma-
Brussels,Belgium
tion/immunity in a variety of tissues including the skeletal
2IREC–Endocrinology,DiabetesandNutritionUnit,UCL/EDINB1.55.06–Av.
Hippocrate55,Harvey55,B-1200Brussels,Belgium muscle[7,8].ApNturnedouttobesufficientlypowerfulto
©Brichardetal.2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Boursereauetal.BMCBiology (2018) 16:33 Page2of17
offset severe inflammation/oxidative stress and muscle stimulation. Fas-associated protein with death domain
damage in dystrophic muscles of mdx mice (a model of (FADD) is indeed one of the target genes of miR-711, and
DMD). This was demonstrated in our very own mouse this protein serves as an apical mediator of both priming
model (mdx-ApN), where mdx animals were crossed with andactivationofNLRP3inflammasomethroughtheactiva-
transgenic mice overexpressing ApN [9]. Conversely, ApN tionofcaspase-8[12,13].
deficiencyworsenedthemdxphenotype,whilemuscleelec- Theinflammasomeisinvolvedintheinitiationorprogres-
trotransfer of the ApN gene reversed inflammation/oxida- sionofdiseaseswithahighimpactonpublichealth,suchas
tive stress and disease progression, thereby suggesting a metabolic disorders and neurodegenerative diseases [14].
therapeuticpotentialforApNinDMD[10]. The best characterized inflammasome is NLRP3, so named
We have also recently shown that the anti-inflammatory because the NLRP3 protein belongs to the family of
actionofApNonskeletalmusclewasatleastinpartmedi- nucleotide-binding and oligomerization domain-like recep-
ated by a micro RNA (miRNA), miR-711. Thus, ApN up- tors (NLRs). NLRP3 must be primed before activation.
regulatedmiR-711inmuscle,whichinturnrepressedgenes NLRP3 priming results from activation of NF-κB, which in
belonging to inflammation/immunity signalling cascades turn upregulates transcription of inflammasome compo-
[11](thesegenesareindicatedinboldinFig.1).Thesecas- nents/targets, including inactive NLRP3, pro-interleukin
cades split into two branches. The first branch, which has (IL)-1β and pro-IL-18 [14, 15] (see Fig. 1). Next, activation
been described recently, leads to nuclear factor kappa B involves NLRP3 binding to apoptosis-associated speck-like
(NF-κB) activation [11]. The second branch, still unde- protein(ASC)andpro-caspase-1,formingacomplextermed
scribed, could potentially lead to NLRP3 inflammasome the inflammasome. This triggers pro-caspase-1 self-cleavage
Fig.1Inflammation/immunesignallingpathwaysrepressedbymiR-711inmuscle.ThetargetgenesofmiR-711areindicatedinboldandoutlinedinblack.
Theyareinvolvedininflammatory/immunesignallingpathwaysthatsplitintotwobranches.Thefirstone,depictedingrey,hasbeenrecentlydescribed[11]
andresultsinNF-κBactivation.Briefly,bindingofLPSorIL-1βtotheirrespectivereceptorTLR4orIL-1Rtriggerstheassemblyofacomplex(composedof
MyD88andTOLLIP),whileactivationofTNFRsignallingbyTNFαtriggerstheassemblyofanothercomplexincludingTRADD.Theseintracellularscaffolds
stimulatetheIKKcomplexthroughactivationofTAB1orPI3K.Next,theIKKcomplexreleasesNF-κBfromitsrepressorIκB,therebypermittingitstranslocation
intothenucleus.Thisinturnupregulatestranscriptionofpro-inflammatoryfactorssuchasTNFαandinflammasometargets/componentslikepro-IL-1β/-18
andNLRP3(i.e.primingofNLRP3).ThesecondbranchinfuchsialeadsthroughFADDandcaspase-8activationtoNLRP3activation.FADDcanberecruited
bytheTRADDorMyD88complex.InflammasomeactivationinvolvesassemblyofNLRP3withASCandpro-caspase-1.Thiscomplextriggerspro-caspase-1
self-cleavageintoactivecaspase-1,whichthencleavespro-IL-1βandpro-IL-18intotheirbiologicallyactivecytokines.Abbreviations:AKTproteinkinaseB,
ASCapoptosis-associatedspeck-likeprotein,FADDFas-associatedproteinwithdeathdomain,IκBinhibitorofkappaB,IKKIκBkinase,IL-1Rinterleukin-1
receptor,IRAK1/4interleukin-1receptor-associatedkinase1/4,LPSlipopolysaccharide,MyD88myeloiddifferentiationprimaryresponse88,NF-κBnuclearfactor
kappaB,PI3Kphosphatidylinositol-4,5-bisphosphate3-kinase,RIP1receptorinteractingprotein1,TAB1transforminggrowthfactorbeta(TGF-β)activated
kinase1-bindingprotein1,TAK1TGF-βactivatedkinase1,TIRAPToll-interleukin1receptordomain-containingadapterprotein,TLR4Toll-likereceptor4,TNFR
tumournecrosisfactorreceptor,TOLLIPTollinteractingprotein,TRADDTNFRtype1-associateddeathdomainprotein,TRAF2/6TNFRassociatedfactor2/6
Boursereauetal.BMCBiology (2018) 16:33 Page3of17
intoactivecaspase-1,whichthencleavespro-IL-1βandpro- ofFADDactuallyrepressedNlrp3mRNAandproteinex-
IL-18 to their active forms [14, 15]. So far, the NLRP3 pressionininflamedmyotubes(Fig.3b),therebyextending
inflammasomehasbeenpoorlystudiedinmuscle[16,17]. dataobtainedinFADD-deficientmacrophages[12].
The aims of this work were first to study whether
NLRP3 was present in skeletal muscle, more specifically MuscleelectrotransferofeitherApNcomplementaryDNA
within myofibres, and second to test whether it was reg- (cDNA)orpre-miR-711preventsLPSupregulationof
ulated by ApN through the miR-711. Third, we investi- NLRP3inmice
gated whether NLRP3 may play a crucial role in the We next tested whether thisregulationwasalsoeffective
pathogenesis of DMD. For this matter, we crossed mdx in vivo. To this end, we took advantage of our previous
mice with Nlrp3-knockout (Nlrp3-KO) mice to generate model, inwhich local administration of ApN or miR-711
mdx mice with NLRP3 depletion (mdx/Nlrp3-KO). Fi- was able to protect muscles of ApN-KO mice against
nally, wetranslatedourdata tohumans. LPS-induced inflammation [11]. Briefly, one tibialis an-
terior muscle was injected with a plasmid containing the
Results ApN sequence (p-ApN) or the pre-miR-711 (p-miR-
NLRP3inflammasomeispresentwithinmyofibres 711), while the contralateral one received an empty plas-
We first provided evidence for NLRP3 expression in mid; the muscles were then electroporated. Nine days
myofibres. To this end, wild-type (WT) and Nlrp3-KO later, mice were challenged by ip injection of LPS. Mus-
mice were challenged by intraperitoneal (ip) injection of cleswere sampled 24hlater.
lipopolysaccharide (LPS) to induce inflammation or re- Herein, we studied the expression of NLRP3 by im-
ceived vehicle only. In basal conditions (no LPS), NLRP3 munochemistry. Muscle electrotransfer of the ApN gene
staining was faint in WT mice and undetectable in or pre-miR-711 reduced NLRP3 staining by ~25–30%
Nlrp3-KO ones (Fig. 2). After LPS, NLRP3 was detected (Fig. 4a, b). These data indicate that in inflamed muscle
as stained clusters in the sarcoplasm of WT mice, while NLRP3isattenuatedbyApNormiR-711invivo.
there was still no labelling in Nlrp3-KO muscle (Fig. 2).
These results were confirmed by Western blot analysis. NLRP3isupregulatedinmuscleofdystrophicmicebut
Taken together, our data showed that NLRP3 is pro- partiallycorrectedbyApN
duced by mouse myofibres and could play an important We next explored whether NLRP3 may play a role in the
role inmuscleinflammation. pathogenesis of DMD, where inflammation markedly ex-
acerbates the disease. Nlrp3 mRNA and protein levels
NLRP3expressionisregulatedbyApNandmiR-711as were four- and threefold higher in muscles of mdx mice
wellasbyFADDinmurinemyotubes thaninWTones,respectively.However,thisupregulation
We next examined whether NLRP3 is regulated by the wasattenuatedintransgenicmdxmicewithchronicover-
anti-inflammatory hormone ApN and its mediator miR- expression of ApN [9] (Fig. 5a, b). Interestingly, gene ex-
711. This hypothesis was first tested in vitro in inflamed pression of miR-711, the mediator of ApN, was strongly
myotubes(Fig.3a).Nlrp3messengerRNAs(mRNAs)were decreased in mdx mice, while it was increased in mdx
increased in C2C12 cells challenged by LPS (compare the mice overexpressing ApN (Fig. 5c). These results suggest
firstwhitecolumnwithbasalconditionsrepresentedbythe that NLRP3, which is inversely related to miR-711 and
dotted line (i.e. no LPS and any other treatments)). Both ApN,couldbeinvolvedinthepathogenesisofDMD.
ApN treatment and miR-711 transfection reversed this
stimulation of Nlrp3 expression (first two pairs of histo- EffectsofNLRP3depletiononphysicalperformanceand
grams: compare grey/black vs white column). Likewise, as muscleinjuryofmdxmice
shownonWesternblot,NLRP3proteinlevels,whichwere To further investigate the pathogenic role of the inflam-
expressedrelativetobasalcondition(noLPS),tendedtobe masome in DMD, we generated mdx mice which were
decreased by both ApN and miR mimic. Conversely, miR- Nlrp3-deficient (mdx/Nlrp3-KO). Thesemice were com-
711 silencing further augmented Nlrp3 expression (histo- pared to their mdx littermates (mdx). Nlrp3-KO mice
grams,3rdpairofcolumns).Moreover,theinhibitoryeffect andWTmice werealso usedforcomparison.
ofApNonNlrp3mRNAwasreversedbytheanti-miR(last Thesefourgroupsofmiceweresubmittedtothreediffer-
pairof histograms).Takentogether,thesedata suggestthat ent functional tests in order to evaluate the effects of
NLRP3isimplicatedinmuscleinflammationandisdown- NLRP3depletiononmuscleforceandendurance.Thegrip
regulatedbyApNthroughmiR-711. test quantitatively measures the global force in limb mus-
To search for mechanisms by which miR-711 may cles.Thestrengthofcombinedfore-andhindlimbmuscles
downregulate NLRP3, we studied one of its target genes, was ~44% lower in mdx than in WT mice, while mdx/
FADD, which is known to promote NLRP3 priming and Nlrp3-KOmiceshowedasignificantimprovement(Fig.6a).
activation[12].Weshowedthatsilencinggeneexpression The wire test evaluates muscle force and resistance to
Boursereauetal.BMCBiology (2018) 16:33 Page4of17
a
b
Fig.2EvidenceforNLRP3expressionwithinmousemyofibres.Wild-typemice(WT)andNlrp3-KOmicewerechallengedbyintraperitoneal(ip)LPSor
receivedvehicleonly(noLPS),andtibialisanteriormusclesweresampled24hlater.aImmunochemistrywasperformedwithaspecificantibodydirected
againstNLRP3.Clustersofstaining,shownbyarrowsinthemagnificationinset,weredetectedinmyofibresofWTmiceafterLPS,whiletherewasno
labellinginNlrp3-KOmice.Micereceivingvehicleonlyareshownforcomparison.Scalebar=50μmforallimagesincludingtheinset.Representative
sectionsforsixmicepergrouparepresented.bWesternblotanalysiswasalsoperformedinthedifferentgroupsofmice;datawerenormalizedtoactin
levelsandpresentedaspercentagesofWTwithoutLPS.Valuesaremeans±standarderrorofthemean(SEM)forthreetosixmicepergroup.**P<0.01
fortheeffectofLPS
fatigue.Inthistest,thetimeduringwhichthemouseissus- Eventually, endurance was evaluated by an eccentric exer-
pendedonahorizontalwireisrecorded.Themdxmicefell cise. On the 3rd and last day of the exercise, the distance
down much faster than WT mice, while the mdx/Nlrp3- (metres) covered by each mouse was measured. WT mice
KO mice stayed on the wire twice as long (Fig. 6b). covered the maximum distance(100m), while the running
Boursereauetal.BMCBiology (2018) 16:33 Page5of17
Fig.3(Seelegendonnextpage.)
Boursereauetal.BMCBiology (2018) 16:33 Page6of17
(Seefigureonpreviouspage.)
Fig.3EffectsofApN,miR-711(a)andFADD(b)onNLRP3expressioninvitro.aC2C12myotubeswereeithertreatedornotwithApNortrans-
fectedwithmiR-711mimicoritscontrol(Ctrl+)for24h.Cellswerealsotransfectedwithanti-miR-711oritsrelativecontrol(Ctrl–)for28h,while
ApNwasaddedduringthelast24h.AllconditionspresentedhereinwereobtainedinC2C12challengedbyLPSexceptforthebasalcondition
(noLPS,notransfectionoranyothertreatments)representedbythedottedline.mRNAlevelswerenormalizedtocyclophilin,andthesubsequent
ratioswerepresentedasrelativeexpressioncomparedtobasalcondition.Valuesaremeans±SEMforsevenrepeatedexperiments.AWestern
blotwithfiveexperimentalconditions(basal,LPSalone,ApN+LPS,controlmiR+LPSandmiRmimic+LPS)isalsoshownabovethehistograms.
NLRP3proteinexpressionwasnormalizedtoactinlevelsandthesubsequentratiospresentedasrelativeexpressioncomparedtobasalcondition.Thisblot
isrepresentativeoftwodifferentexperiments;themeanNLRP3valueisindicatedatthetopofeachcondition.bEffectsofFADDsilencingonNLRP3expres-
sioninC2C12cellschallengedbyLPS.Myotubesweretransfectedfor24hwithsmallinterferingRNAs(siRNAs)againstFADDoranegative(non-targeting,
NT)control.Next,cellswerechallengedbyLPSduringthelast20h.mRNAlevelswerenormalizedandpresentedasrelativeexpressioncomparedtothe
basalcondition(noLPS,notransfection)representedbythedottedline.NLRP3proteinexpressionwasmeasuredbyWesternblot,normalizedtoactinlevels,
andalsopresentedaspercentagesofthebasalcondition.Valuesaremeans±SEMforfive(mRNA)andthree(protein)repeatedexperiments.**P<0.01,
***P<0.001forindicatedconditions(a)orvsNTsiRNA(b)
distance of mdx mice fell drastically (~38 m); that of the confirmed the ablation of the Nlrp3 gene in KO
mdx/Nlrp3-KO mice was intermediate (~73 m) (Fig. 6c). mice (mdx or not) and the upregulation in regular
For each exercise, there were no differences between WT mdx animals (Fig. 7). NLRP3 protein showed a com-
andNlrp3-KOmice. parable pattern of expression when quantified by
Wenextmeasuredinbasalconditionscreatinekinaseac- Western blotting (Fig. 7) or measurement of 3,3’-dia-
tivity,aplasmamarkerofmuscledamage.Thismarkerwas minobenzadine (DAB) staining areas on immuno-
~2.6foldhigherinmdxthaninWTmice,whileitdeclined chemistry sections (like those shown on Fig. 8), the
bymorethan20%inmdx/Nlrp3-KOmice(Fig.6d). correlation between both techniques being excellent
(Additional file 1: Figure S1). Besides Nlrp3, mRNA
EffectsofNLRP3depletiononmuscleinflammationand levels of the adapter protein ASC were also upregu-
oxidativestressinmdxmice lated in mdx mice and partially corrected in mdx/
We first examined the components of the inflamma- Nlrp3-KO mice (Fig. 7). Gene expression and total
some complex in the four groups of mice. We protein levels of the last component of this complex,
a
b
Fig.4EffectsofApNcDNAandpre-miR-711electrotransferonNLRP3inskeletalmusclesofApN-KOmice.OnetibialisanteriormuscleofApN-KO
micewasinjectedandelectroporatedwithApNcDNA-containingplasmid(p-ApN)orwithpre-miR-711-containingplasmid(p-miR-711),whereas
thecontralateralmusclewasinjectedandelectroporatedwiththerespectivecontrolplasmid(p-ctrl).Ninedayslater,micewerechallengedby
LPSandthetibialisanteriormusclesweresampled24hlater.aImmunochemistrywasperformedwithaspecificantibodydirectedagainstNLRP3.
Scalebar=50μm.bQuantificationof3,3’-diaminobenzadine(DAB)stainingareaswithinmuscles.Resultsaremeans±SEMfortwogroupsoffive
mice(thecontralateralmusclebeingusedascontrol).*P<0.05fortheeffectofApNormiR-711
Boursereauetal.BMCBiology (2018) 16:33 Page7of17
a b
c
Fig.5Effectsoflong-termApNoverexpressiononNLRP3andmiR-711levelsinmusclesofdystrophicmice.Threegroupsofmicewereused:
mdxmiceoverexpressingApN,whichweregeneratedbytransgenesis(mdx-ApN[9]),theirmdxlittermates(mdx)andWTmice.Nlrp3mRNA(a)
andprotein(b)aswellasmiR-711levels(c)weremeasuredintibialisanteriormuscles,normalizedtocyclophilin(a,c)ortoactin(b),andthe
subsequentratioswerepresentedasrelativeexpressioncomparedtoWTvalues.Resultsaremeans±SEMforthreetosevenmicepergroup.#P
<0.05,##P<0.01,###P<0.001vsWT;*P<0.05,**P<0.01formdx-ApNvsmdxmice
caspase-1, were increased in both regular mdx and NLRP3expressionandregulationinhumanDMD
Nlrp3-deficient mdx mice. However, caspase-1 activ- myotubes
ity (percentage cleaved 20 kDa subunits) was upreg- We measured NLRP3 mRNA levels in primary cultured
ulated in regular mdx mice only and was actually myotubesobtainedfromhealthycontrols(C)orfromDMD
even downregulated in mdx/Nlrp3-KO mice. subjects studied in basal conditions (Fig. 10a). NLRP3
mRNA and protein levels of caspase-1 targets, IL-1β mRNAlevelswerethreefoldhigherinDMDthanincontrol
and IL-18 were upregulated in mdx mice and partially myotubes. This increase was partly attenuated by ApN
or completely corrected by NLRP3 depletion in dys- treatment. miR-711 levels showed a reverse pattern of ex-
trophic mice. IL-1β was detected with a specific anti- pression: a 40% decrease in DMD myotubes compared to
body for its bioactive form and IL-18 with an antibody controlones,whileApNtreatmentupregulatedtheselevels,
that recognized its total form (Figs. 8 and 9). Other therebyleadingtotheirnormalizationinDMD(Fig.10a).
markers of inflammation (tumour necrosis factor alpha We next tested the hypothesis that, as in C2C12 cells,
(TNFα)) or oxidative stress (peroxiredoxin 3 (PRDX3)) miR-711maybeamediatoroftheanti-inflammatoryaction
behaved similarly with a complete or partial correction ofApNinCandDMDmyotubes.Tomimictheinflamma-
by Nlrp3 ablation of the otherwise upregulated levels tory microenvironment which prevails in DMD, we chal-
found in mdx mice. Conversely, gene expression of the lenged the myotubes by an inflammatory stimulus (TNFα/
anti-inflammatory cytokine, IL-10, which was increased interferon gamma (IFNγ)) [18], as already described [19]
in mdx mice likely to compensate inflammation, was (Fig. 10b, c). These cells were then transfected with miR-
furtherupregulatedbyNlrp3ablation(Fig. 9). 711 mimic or anti-miR-711 (and their respective controls:
Taken together, these data suggest that abrogation Ctrl+and–)andtreatedornotwithApN.InCmyotubes,
of the inflammasome protects the dystrophic muscle NLRP3 expression was markedly upregulated by the in-
against excessive inflammatory reactions and oxidative flammatory challenge (first white column vs dotted line,
stress. whichrepresentsbasalconditions,i.e.noinflammationand
Boursereauetal.BMCBiology (2018) 16:33 Page8of17
a b
c d
Fig.6EffectsofNLRP3depletiononglobalforce,resistanceandmuscleinjuryinmdxmice.Fourgroupsofmicewereused:mdxmicethatwere
alsodeficientforNlrp3(mdx/Nlrp3-KO),regularmdx(mdx),Nlrp3-KOmiceandWTones.Severalfunctionalinvivostudieswerecarriedout.aThe
animalswereloweredonagridconnectedtoasensortomeasurethemuscleforceofbothfore-andhindlimbs;datawerethenexpressedin
gram-forcerelativetobodyweight.bMicewerenextsubjectedtoawiretestwheretheyweresuspendedbytheirforelimbsandthetimeuntil
theycompletelyreleasedthewireandfelldownwasrecorded.cMicewerealsosubmittedtoadownhilltreadmillexercisefor10min;the
covereddistance(metres)wasmeasuredforeachmouse,100mbeingthemaximaldistance.dMuscleinjurywasassessedbyplasmacreatine
kinase(CK)activitymeasuredinthebasalstateandexpressedasIU/L.Resultsaremeans±SEMforeighttotwelvemicepergroup.##P<0.01,
###P<0.001vsWT;*P<0.05,**P<0.01fortheindicatedconditions
anyothertreatments;Fig.10b).OverexpressionofmiR-711 as skeletal muscle [16, 17]. NLRP3 has been reported to
downregulated NLRP3 mRNAs (compare the first pair of be upregulated in human skeletal muscle biopsies from
columns;Fig.10b),whiletheanti-miRtendedtoinducean dysferlin-deficient patients [17] and from subjects sub-
opposite effect (second pair of columns; Fig.10b). Like the mitted to a high palmitic acid diet [20], suggesting that
miR-711 mimic, ApN downregulated gene expression of skeletal muscle can actively participate in inflammasome
NLRP3(columns5 vs3), an inhibitionwhich wasreversed upregulation. However, localization of NLRP3 has not
by miR-711 silencing (last pair of columns). Changes of yet been investigated within skeletal muscle. We unam-
TNFα wererather similar to thoseofNLRP3: downregula- biguously showed that NLRP3 was produced in vivo
tion by miR-711 and ApN, upregulation by miR-711 within myofibres under inflammatory conditions. After
blockade and attenuation of ApN anti-inflammatory effect acute ip LPS injection, NLRP3 was detected as stained
by this blockade (Fig. 10b). Data obtained in DMD myo- clusters in myocytes, suggesting activation and forma-
tubeswerequalitativelysimilartothoseinthecontrols(Fig. tion of the inflammasome complex, which may reach up
10c). Eventually, as in C2C12 cells, we confirmed that to3μmindiameterinvitro[21].Duringchronicmuscle
TOLLIPandFADDwerealsotwotargetgenesofmiR-711 inflammation, like that seen in mdx mice, additional
in both human C and DMD cells (Additional file 2: Figure NLRP3 staining was detected to some extent in inflam-
S2) and could thus be early steps leading to activation of matory infiltrates(datanot shown).
theinflammasomecomplex. Since ApN exerts anti-inflammatory effects on muscle
via miR-711 and because miR-711 inhibits target genes
Discussion that could lead to the inflammasome pathway [11], we
The inflammasome complex has been well characterized explored whether NLRP3 could be regulated by ApN
in cells that participate in innate immunity; however, and its miRNA mediator in muscle. Both ApN and miR-
there is still little information about its expression, acti- 711 mimic actually inhibited NLRP3 expression in
vationandpotentialroleinnon-hematopoieticcellssuch C2C12 myocytes, while miR-711 blockade had opposite
Boursereauetal.BMCBiology (2018) 16:33 Page9of17
Fig.7EffectsofNLRP3depletiononcomponentsoftheinflammasomecomplexinmdxmuscles.TibialisanteriormusclesweresampledfromWTmice,
Nlrp3-KOmice,mdxmiceandmdx/Nlrp3-KOmice.Membersoftheinflammasomecomplex(NLRP3,ASCandcaspase-1)weremeasuredatthemRNA
and/orproteinlevels.mRNAlevelswerenormalizedtocyclophilinandpresentedasrelativeexpressioncomparedtoWTvalues.Proteinquantificationwas
performedbyWesternblot.NLRP3andtotalcaspase-1levels(45kDaproenzymeanditsactivecleavedsubunits)werenormalizedtoactinlevelsand
presentedaspercentagesofWTvalues.Caspaseactivity(activated20kDasubunit)wasexpressedaspercentageoftotalforms.Resultsaremeans±SEM
foreighttoten(mRNA)orsix(protein)micepergroup.#P<0.05,###P<0.001vsWT;**P<0.01forindicatedconditions
effects and reversed the anti-inflammatory action of WenextexaminedwhetherApNwasalsoabletodown-
ApN. Qualitatively similar results were obtained in vivo regulate the inflammasome in a context of severe and
by using muscle electrotransfer of ApN cDNA or pre- chronicinflammationinmuscle.Tothisend,wechosethe
miR-711. Taken together, our data indicate that miR-711 mdxmouse,amodelofDMD,wheremarkedinflammation
mediates the ApN-induced inhibition of NLRP3 expres- exacerbatesdiseaseprogression[4,5].CirculatingApNwas
sion and thus NLRP3 transcriptional priming. Although markedly diminished in mdx mice [9, 24]. Replenishment
Nlrp3 is not a target gene of miR-711, miR-711 indir- of ApN due to transgenesis strikingly reduced muscle in-
ectly represses NLRP3 through two mechanisms: (1) by flammation,oxidativestressaswellasmuscledamagewhile
inhibiting NF-κb (see Fig. 1), which is involved in increasing global force and endurance in mdx-ApN mice
NLRP3 priming as shown in macrophages [22, 23], and [9]. Herein, we further showed that ApN also reduced the
(2) by inhibiting its target gene FADD ([11] and this marked(fourfold)riseofNlrp3mRNAandproteininmdx
paper), which is also known to promote priming and ac- mice, thereby contributing to its anti-inflammatory action.
tivation ofNLRP3([12]andourown data). This upregulation of Nlrp3 expression in DMD is at
Boursereauetal.BMCBiology (2018) 16:33 Page10of17
Fig.8ImmunodetectionofinflammationandoxidativestressmarkersinmdxmuscleswithNlrp3abrogation.Tibialisanteriormuscleswere
sampledfromWTmice,Nlrp3-KOmice,mdxmiceandmdxmicethatwerealsoNlrp3-deficient(mdx/Nlrp3-KO).Immunodetectionwasper-
formedwithspecificantibodiesdirectedagainstNLRP3,threepro-inflammatorycytokines(IL-1β,IL-18andTNFα)andoneoxidativestressmarker
(peroxiredoxin3,PRDX3).IL-1βantibodywasspecificforthematureformoftheprotein,whileIL-18antibodyrecognizeditstotalform.Represen-
tativesectionsforsixmicepergroupareshown.Scalebar=50μm
variance with another study conducted in a small number ligation and puncture surgery. The polymicrobial sepsis
ofanimals[17].WhencomparedtoNLRP3,miR-711levels was systemic [25]. To get a better insight into the role of
exhibitedareversepatternofexpression:adownregulation NLRP3inmuscleinflammation,weinvestigatedtheimpact
in mdx mice, in line with the decrease in circulating ApN, ofNLRP3deletioninmdxmice,inwhichtheinflammation
while an upregulation was observed in mdx-ApN animals. originates from the muscle. We thus generated mdx mice
Thus, ApN downregulates NLRP3 expression likely via with NLRP3 deletion. Each component of the NLRP3
miR-711 in dystrophic mice. Moreover, as this hormone inflammasome complex was overexpressed in regular mdx
was also found to reduce mature IL-1β protein levels in mice whencompared to WT ones. Moreover, activationof
muscle of these mice [9], this reinforces the concept men- theinflammasomewasalsoenhancedindystrophicmuscle,
tioned above that ApN inhibits both NLRP3 priming and as shown by higher caspase-1 activity and larger levels of
activationinmuscle. matureIL-1βandIL-18.Inmdx/Nlrp3-KOmice,caspase-1
The role of NLRP3 in muscle needs to be unravelled. A activityandabundanceofIL-1β/IL-18werecorrected.This
veryrecentstudyhasshownthatmuscleatrophywasatten- contributed to normalize another major pro-inflammatory
uated in Nlrp3-KO mice made acutely septic by cecal cytokine (TNFα) and an oxidative stress marker (PRDX3).