Article Dopamine Controls Systemic Inflammation
through Inhibition of NLRP3Inflammasome
Yiqing Yan,1,4Wei Jiang,1,4Lei Liu,1Xiaqiong Wang,1Chen Ding,3Zhigang Tian,1,2,*and Rongbin Zhou1,2,*
1Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease,School of Life Sciences and Medical Center, University of Science and Technology of China,Hefei230027,China
2Innovation Center for Cell Biology,Hefei National Laboratory for Physical Sciences at Microscale,Hefei230027,China
3Beijing Proteome Research Center,National Center for Protein Sciences Beijing,Beijing102206,China
4Co-first author
*Correspondence:tzg@ustc.edu(Z.T.),zrb1980@ustc.edu(R.Z.)
/10.ll.2014.11.047
SUMMARY
Inflammasomes are involved in diverse inflammatory diseases,so the activation of inflammasomes needs to be tightly controlled to prevent excessive inflam-mation.However,the endogenous regulatory mecha-nisms of inflammasome activation are still unclear. Here,we report that the neurotransmitter dopamine (DA)inhibits NLRP3inflammasome activation via dopamine D1receptor(DRD1).DRD1signaling nega-tively regulates NLRP3inflammasome via a sec-ond messenger cyclic adenosine monophosphate (cAMP),which binds to NLRP3and promotes its ubiquitination and degradation via the E3ubiquitin ligase MARCH7.Importantly,in vivo data show that DA and DRD1signaling prevent NLRP3inflamma-some-dependent inflammation,including neurotoxin-induced neuroinflammation,LPS-induced systemic inflammation,and monosodium urate crystal(MSU)-induced peritoneal inflammation.Taken together, our results reveal an endogenous mechanism of inflammasome regulation and suggest DRD1as a potential target for the treatment of NLRP3inflamma-some-driven diseases.
INTRODUCTION
The NLRP3inflammasome is a cytosolic protein complex composed of NLRP3,ASC,and caspase-1,and assembled in response to both microbial infection and endogenous‘‘danger signal’’(Davis et al.,2011;Martinon et al.,2009).The activation of NLRP3inflammasome promotes the maturation and release of several proinflammatory cytokines,such as interleukin-1b (IL-1b)and IL-18,so it plays critical roles in the initiation of inflam-mation and the development of immune responses(Lamkanfiand Dixit,2012;Schroder and Tschopp,2010).However,as excessive and persistent inflammation is quite harmful,NLRP3 inflammasome has been involved in diverse inflammatory dis-eases,including type2diabetes,atherosclerosis,and gout, thus the activation of NLRP3inflammasome should be tightly controlled(Davis et al.,2011;Lamkanfiand Dixit,2012).Several regulatory mechanisms have been identified to suppress NLRP3 inflammasome.Type I interferon has been shown to attenuate NLRP3inflammasome activation via Stat1-dependent manner, while nitric oxide has been identified as another negative regu-lator of NLRP3inflammasome activation(Guarda et al.,2011; Mishra et al.,2013).Recently,we have proposed that u-3 fatty acids can negatively regulate NLRP3inflammasome activa-tion via G protein coupled receptor120(GPR120)and GPR40 (Yan et al.,2013).Although NLRP3inflammasome has been extensively investigated,its regulatory networks,especially the endogenous mechanisms,still remain elusive.
Dopamine(DA)is a neurotransmitter,which not only can regu-late behavior,movement,endocrine,cardiovascular,renal,and gastrointestinal functions,but also functions as an important molecule bridging the nervous and immune systems(Basu and Dasgupta,2000;Beck et al.,2004;Sarkar et al.,2010). DA receptors are present in almost all immune cell subpopula-tions(Sarkar et al.,2010).Acting on its receptors,DA or agonists for DA receptors have been reported to modulate the activation, proliferation,and cytokine production in immune cells(Basu and Dasgupta,2000;Sarkar et al.,2010;Torres-Rosas et al.,2014). In addition,dopamine D2receptor(DRD2)knockout mice show remarkable inflammatory response in CNS,suggesting that DA and its downstream signaling has an antiinflammatory function (Shao et al.,2013).Consistent with this,the deficiency of DA is tightly associated with immune system abnormalities and CNS inflammation in the progression of Parkinson disease (PD)(Perry,2012;Wu¨llner and Klockgether,2003).Although the antiinflammatory effect of DA and its implication in the pa-thology of PD are emerging,the mechanisms are still poorly understood.
Here,we demonstrate that DA is an endogenous regulator of inflammasome activation and suggest the DRD1as a poten-tial target for the treatment of NLRP3inflammasome-driven diseases.
RESULTS
DA Inhibits NLRP3Inflammasome Activation
To determine the effect of DA on inflammasome activation, LPS-primed bone marrow-derived macrophages(BMDMs) were pretreated with DA before nigericin challenge.NLRP3-dependent caspase-1activation and IL-1b maturation
by
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nigericin were indeed inhibited by DA in a dose-dependent manner(Figures1A and1B and Figure S1A available online). Similarly,DA also inhibited nigericin-induced production of IL-18,another NLRP3-inflammasome-dependent cytokine(Fig-ures1C and S1B).However,the production of TNF-a,an inflam-masome-independent cytokine,was not affected by DA in this condition(Figure1D),suggesting that DA inhibited IL-1b pro-duction via affecting inflammasome activation.The physiolog-ical DA levels found in extracellularfluid surrounding neural synapses are$1m M(Basu et al.,2001;Chakrobort
y et al., 2008),but in our study,the DA concentration required for in-flammasome inhibition is over100m M,which exceeds the phys-iological concentration of DA.We speculated that the high DA concentration needed for inflammasome inhibition might be due to the instability of DA.Actually,the half-life of DA is <2min in plasma and is even much shorter in mouse brain tis-sues(Rouge-Pont et al.,2002),possibly because dopamine can be broken down into inactive metabolites by a set of enzymes, such as monoamine oxidase(MAO)and catechol-O-methyl transferase(COMT)(Youdim et al.,2006).To test this possibility, we changed the DA treatment protocol from one single treat-ment with high dose to multiple treatments with low dose.The results showed that one single treatment with45m M or90m M DA could not inhibit nigericin-induced IL-1b production,while treatment with1.5m M or3m M DA for30times(time interval is5min)inhibited nigericin-induced IL-1b production signifi-cantly,although the total doses were identical(Figure S1C). Moreover,although DA could not inhibit nigericin-induced IL-1b production at the doses under10m M,it really could suppress IL-1b production in the presence of MAO and COMT inhibitors at these doses(Figure S1D).In addition,the DA treatment had no effect on cell viability(Figure S1E).Taken together,these re-sults indicate that DA has the potential to inhibit caspase-1acti-vation and IL-1b secretion.
To test whether DA only affect nigericin-induced NLRP3in-flammasome activation,we examined other
NLRP3agonists. The results showed that DA could inhibit caspase-1cleavage and IL-1b secretion induced by all examined agonists,
including
A B C
E
D F
Figure1.DA Inhibits NLRP3Inflammasome Activation
(A)Immunoblot analysis of IL-1b and cleaved caspase-1(p20)in culture supernatants(SN)of LPS-primed BMDMs treated for3hr with various doses(above lanes)of DA and then stimulated with nigericin,and immunoblot analysis of the precursors of IL-1b(pro-IL-1b)and caspase-1(pro-caspase-1)in lysates of those cells(Input).
(B–D)ELISA of IL-1b(B),IL-18(C),and TNF-a(D)in supernatants from LPS-primed BMDMs treated for3hr with various doses(above lanes)of DA and then stimulated with nigericin.
(E)Immunoblot analysis of IL-1b and cleaved caspase-1(p20)in culture supernatants(SN)of LPS-primed BMDMs treated for3hr with DA and then stimulated with MSU,nigericin,ATP,and Alum,and immunoblot analysis of the precursors of IL-1b(pro-IL-1b)and caspase-1(pro-caspase-1)in lysates of those cells (Input).
(F)ELISA of IL-1b in supernatants from LPS-primed BMDMs treated for3hr with DA and then stimulated with MSU,nigericin,ATP,and Alum.
Data are means±SEM,*p<0.05,**p<0.01,***p<0.001.
See also Figure S1.
Cell160,62–73,January15,2015ª2015Elsevier Inc.63
MSU,Alum,and ATP,similar to nigericin (Figures 1E and 1F),suggesting that DA is a potent and broad inhibitor for NLRP3inflammasome activation.Moreover,DA pretreatment had minimal effect on poly (dA:dT)transfection-induced AIM2in-flammasome activation or Salmonella typhimurium (Salmonella )infection-induced NLRC4inflammasome activation (Figures S1F–S1I).Taken together,these results demonstrate that DA specifically inhibits NLRP3inflammasome activation and subse-quent IL-1b production.
DA Inhibits NLRP3Inflammasome Activation via DRD1Signaling
Next,we investigated the mechanisms underlying the inhibitory activity of DA on NLRP3inflammasome activation.DA exerts its effects by binding to the activating receptors located on the surface of cells.There are at least five subtypes of dopamine re-ceptors identified,termed DRD1–DRD5,and all of them can be detected in immune cells,including macrophages and dendritic cells (McKenna et al.,2002;Meredith et al.,2005;Ricci et al.,1999).To determine which receptor was involved in DA-induced
NLRP3inflammasome inhibition,these receptors were silenced by small interfering RNA (siRNA)in BMDMs,respectively (Fig-ures S2A and S2B).Knockdown of Drd1in BMDMs significantly suppressed the inhibitory effect of DA on inflammasome activa-tion,while knockdown of Drd2,Drd3,or Drd4in BMDMs had no effect (Figure 2A).In addition,knockdown of Drd5in BMDMs had a little bit of an effect on DA-induced inflammasome inhibition (Figure 2A ).These results suggest that DA inhibits NLRP3inflam-masome activation primary through DRD1.To further confirm this,we tested the role of DA in Drd1À/Àcells.The results showed that the inhibitory effect of DA on nigericin-induced IL-1b secretion in BMDMs was inhibited completely or partially when Drd1was absent,depending on the doses of DA (Figures 2B and 2C).We also examined whether the agonist of DRD1could inhibit inflam
masome activation.First,we found that the agonist of DRD1A-68930inhibited nigericin-induced IL-1b secretion significantly,while it had no effect on cell viability,simi-larly to DA (Figures 2D and S2C),and the agonists of DRD2,DRD3,or DRD4had mild or no effect on NLRP3inflammasome activation (Figure S2D).In addition,A-68930-induced
NLRP3
A B
C D E
Figure 2.DA Inhibits NLRP3Inflammasome Activation via DRD1
(A)ELISA of IL-1b in supernatants from LPS-primed BMDMs transfected with control siRNA with a scrambled sequence or Drd1-Drd5-specific siRNA as indi-cated,treated for 3hr with DA and stimulated with nigericin.
(B)ELISA of IL-1b in supernatants from LPS-primed BMDMs of Drd1À/Àand Drd2À/Àmice treated for 3hr with DA and stimulated with nigericin.
(C)Immunoblot analysis of IL-1b and cleaved caspase-1(p20)in culture supernatants (SN)of LPS-pri
med BMDMs from Drd1À/Àmice treated for 3hr with DA and then stimulated with nigericin,and immunoblot analysis of the precursors of IL-1b (pro-IL-1b ),caspase-1(pro-caspase-1)and b -actin in lysates of those cells (Input).
(D)Immunoblot analysis of IL-1b and cleaved caspase-1(p20)in culture supernatants (SN)of LPS-primed BMDMs treated for 3hr with various doses (above lanes)of A-68930and then stimulated with nigericin,and immunoblot analysis of the precursors of IL-1b (pro-IL-1b )and caspase-1(pro-caspase-1)in lysates of those cells (Input).
(E)ELISA of IL-1b in supernatants from LPS-primed BMDMs of Drd1À/Àand Drd2À/Àmice treated for 3hr with A-68930and then stimulated with nigericin.Data are means ±SEM,*p <0.05,**p <0.01,***p <0.001.See also Figure S2.
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inflammasome inhibition was not affected by DRD1deficiency (Figure 2E).
Although our results showed that DA did not affect LPS-induced TNF-a production when macrophages were treated with DA after LPS (Figure 1D),previous results have shown that DA can i
nhibit some inflammasome-independent cytokine production,including TNF-a (Basu and Dasgupta,2000).To examine the role of DRD1signaling in inflammasome-indepen-dent cytokine production,we treated BMDMs with DA before LPS and found that DA could suppress LPS-induced TNF-a production,but the dose was higher than that needed for inflam-masome inhibition (Figure S2E).In addition,the effect of DA on LPS-induced TNF-a production were significantly impaired in Drd2À/Àcells,while slightly reduced in Drd1À/Àcells,suggesting that DA inhibits LPS-induced TNF-a production primarily via DRD2(Figure S2E).These results were consistent with a new study in which they reported that Drd2À/Àmice showed sponta-neous inflammation in the brain and DRD2can suppress the transcription and production of proinflammatory cytokines,including TNF-a (Shao et al.,2013).These results suggest that DA signals through DRD1to inhibit inflammasome activation and signals through DRD2to suppress the transcription proin-flammatory cytokines.
DA and DRD1Signaling Promote NLRP3Ubiquitination and Autophagy-Mediated Degradation
Next,we asked how DA and DRD1signaling inhibit NLRP3inflammasome activation.Interestingly,we found that DA treatment induced NLRP3degradation in a dose-and time-dependent manner (Figures 3A and 3B).DA-induced protein degradation was specific to NLRP3,because the expression of AIM2,NLRC4,ASC,caspase-1,and pro-IL-1b were stable in DA-treated BMDMs (Figures 3A and 3B).
Similar to DA,treat-ment with the DRD1agonist A-68930also induced NLRP3degradation in BMDMs (Figures S3A and S3B).Further study showed that DA-induced NLRP3degradation was inhibited in Drd1À/Àmacrophages (Figure 3C),suggesting that DA promotes NLRP3degradation via DRD1signaling.Consistent with the degradation of NLRP3,treatment with DA induced a K48-linked polyubiquitination of NLRP3in macrophages (Figure 3D).Impor-tantly,DA-induced NLRP3polyubiquitination was also impaired in Drd1À/Àmacrophages (Figure 3E).
We then asked whether proteasome or autophagy mediated the degradation of ubiquitinated NLRP3protein.Our results showed that autophagy inhibitor 3-Methyladenine (3-MA)could suppress DA-induced NLRP3degradation,while proteasome in-hibitor MG132could not (Figures 3F and 3G),suggesting that ubiquitinated NLRP3is degraded via autophagy.Indeed,DA treatment induced EGFP-NLRP3to form big aggregates in HEK293T cells (Figure S3C),which might explain why ubiquiti-nated NLRP3is degraded by autophagy,not by proteasome.Consistent with this,DA treatment could induce autophagy in BMDMs via DRD1signaling (Figures 3H and 3I).Importantly,DA-induced inflammasome inhibition could be rescued by auto-phagy inhibitor 3-MA (Figure S3D).These results suggest that autophagy mediates DA-induced NLRP3
degradation.
A B C
D E F G H I Figure    3.DRD1Signaling Promotes NLRP3Ubiquitination and Degradation to Inhibit NLRP3Inflammasome
(A)Immunoblot analysis of NLRP3,AIM2,NLRC4,ASC,Pro-caspase-1,and b -actin from LPS-primed BMDMs treated for 3hr with various doses of DA.
(B)Immunoblot analysis of NLRP3,ASC,Pro-caspase-1,and b -actin from LPS-primed BMDMs treated for different time points of DA.
(C)Immunoblot analysis of NLRP3,ASC,Pro-caspase-1,and b -actin from LPS-primed BMDMs
of Drd1À/Àmice treated for 3hr of DA.
(D)LPS primed-BMDMs were treated with DA
modulate
(0.2mM).Immunoblot analysis of K48-Ub and K63-Ub proteins in cell lysates immunoprecipi-tated with NLRP3antibody.
(E)LPS primed-BMDMs from Drd1À/Àmice were
treated with DA (0.2mM)for 1hr.Immunoblot analysis of K48-Ub protein in cell lysates immu-noprecipitated with NLRP3antibody.
(F)Immunoblot analysis of NLRP3and b -actin in cell lysates from LPS-primed BMDMs treated with
different doses of 3-MA for 30min and then stim-ulated with DA (0.2mM)for 3hr.
(G)Immunoblot analysis of NLRP3and b -actin in cell lysates from LPS-primed BMDMs treated with different doses of MG132for 30min and then stimulated with DA (0.2mM)for 3hr.
(H)Immunoblot analysis of LC3and b -actin in cell lysates from LPS-primed BMDMs treated for different time points of DA (0.2mM).(I)Immunoblot analysis of LC3and b -actin in cell lysates from LPS-primed BMDMs of Drd1À/Àmice treated with DA (0.2mM)for 3hr.See also Figure S3.
Cell 160,62–73,January 15,2015ª2015Elsevier Inc.65
DRD1Signaling Induces NLRP3Ubiquitination via Cyclic AMP-Dependent Manner
We further investigated how DRD1signaling promotes NLRP3ubiquitination.DRD1signaling can stimulate the activity of ad-enylate cyclase and the production of cyclic AMP (cAMP),which is a second messenger and is important in many biological pro-cesses (Beaulieu et al.,2004;Nishi et al.,2011).Recently,cAMP has been proposed to negatively regulate NLRP3inflammasome
activation (Lee et al.,2012),so we examined the role of cAMP in DA-induced inflammasome inhibition.We found that the in-crease of the cAMP levels with adenylate cyclase (ADCY)acti-vator forskolin inhibited NLRP3inflammasome activation with a dose-dependent manner (Figures 4A and 4B),consistent with a previous report (Lee et al.,2012).Furthermore,forskolin treat-ment promoted NLRP3degradation and ubiquitination in mac-rophages (Figures 4C and S4A),similar with DA
treatment.
A B C
D E F
G H I
Figure 4.DRD1Signaling Promotes NLRP3Ubiquitination and Degradation via cAMP
(A)ELISA of IL-1b in supernatants from LPS-primed BMDMs treated for 3hr with forskolin and then stimulated with nigericin.
(B)Immunoblot analysis of IL-1b and cleaved caspase-1(p20)in culture supernatants (SN)of LPS-primed BMDMs treated for 3hr with various doses (above lanes)of forskolin and then stimulated with nigericin,and immunoblot analysis of the precursors of IL-1b (pro-IL-1b )and caspase-1(pro-caspase-1)in lysates of those cells (Input).
(C)Immunoblot analysis of NLRP3and b -actin in cell lysates from LPS-primed BMDMs treated for different time points of forskolin (100m M).
(D)ELISA of IL-1b in supernatants from LPS primed-BMDMs treated with different doses of KH7for 30min before 3hr DA treatment and then stimulated with nigericin.
(E)Immunoblot analysis of IL-1b and cleaved caspase-1(p20)in culture supernatants (SN)of LPS primed-BMDMs treated with KH7(5m M)for 30min before 3hr DA treatment and then stimulated with
nigericin.
(F)Immunoblot analysis of NLRP3and b -actin in cell lysates from LPS primed-BMDMs treated with different doses of KH7for 30min before 3hr DA treatment.(G)LPS primed-BMDMs were treated with KH7(5m M)for 30min before 1hr DA treatment.Immunoblot analysis of K48-Ub protein from the cell lysates immunoprecipitated with anti-NLRP3antibody.
(H)Lysates from LPS primed-BMDMs were treated with different doses of cAMP for 30min.Immunoblot analysis of K48-Ub protein from the cell lysates immunoprecipitated with anti-NLRP3antibody.
(I)LPS primed-BMDMs were treated with DA (0.2mM)for 1hr.Immunoblot analysis of cAMP and NLRP3proteins from the cell lysates immunoprecipitated with anti-NLRP3antibody.
Data are means ±SEM,*p <0.05,**p <0.01,***p <0.001.See also Figure S4.
66Cell 160,62–73,January 15,2015ª2015Elsevier Inc.

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