Wang et al. Journal of Neuroinflammation (2018) 15:95 /s

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1 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 RESEARCH Open Access Centrl lockde of NLRP3 reduces lood pressure vi regulting inflmmtion microenvironment nd neurohormonl excittion in slt-induced prehypertensive rts Mo-Lin Wng 1,2, Yu-Ming Kng 1, Xio-Gung Li 3, Qing Su 1, Hong-Bo Li 1, Ki-Li Liu 1, Li-Yn Fu 1, Rolnd Osei Shene 2, Ying Li 1, Hong Tn 1,4 nd Xio-Jing Yu 1* Astrct Bckground: Inflmmtion hs een implicted in the development of crdiovsculr disese. We determined whether nod-like receptor with pyrin domin contining 3 (NLRP3) involved in the process of prehypertension, centrl lockde of NLRP3 decresed inflmmtion rection, regulted neurohormonl excittion, nd delyed the progression of prehypertension. Methods: Prehypertensive rts were induced y 8% slt diet. The rts on high-slt diet for 1 month were dministered specific NLRP3 locker in the hypothlmic prventriculr nucleus (PVN) for 4 weeks. ELISA, western lotting, immunohistochemistry, nd flow cytometry were used to mesure NLRP3 cscde proteins, pro-inflmmtion cytokines (PICs), chemokine lignd 2 (CCL2), C-X-C chemokine receptor type 3 (CXCR3), vsculr cell dhesion molecule 1 (VCAM-1), neurotrnsmitters, nd leukocytes count detection, respectively. Results: NLRP3 expression in PVN ws incresed significntly in prehypertensive rts, ccompnied y incresed numer of microgli, CD4 +,CD8 + Tcell,ndCD8 + microgli. Expressions of PICs, CCL2, CXCR3, nd VCAM-1 significntly incresed. The lnce etween 67-kD isoform of glutmte decroxylse (GAD67) nd tyrosine hydroxylse (TH) ws dmged. Plsm norepinephrine (NE) in prehypertensive rts ws incresed nd gmm-minoutyric cid (GABA) ws reduced. NLRP3 lockde significntly decresed lood pressure, reduced PICs, CCL2, VCAM-1 expression in PVN, nd restored neurotrnsmitters. Blood pressure nd inflmmtory mrkers were upregulted fter termintion of centrl lockge NLRP3. Conclusions: Slt-induced prehypertension is prtly due to the role of NLRP3 in PVN. Blockde of rin NLRP3 ttenutes prehypertensive response, possily vi downregulting the cscde rection triggered y inflmmtion nd restoring the lnce of neurotrnsmitters. Keywords: NLRP3, Hypothlmic prventriculr nucleus, Inflmmtion, Neurotrnsmitters, Microgli, Hypertension * Correspondence: yuxiojing@mil.xjtu.edu.cn Equl contriutors 1 Deprtment of Physiology nd Pthophysiology, Xi n Jiotong University School of Bsic Medicl Sciences, Key Lortory of Environment nd Genes Relted to Diseses (Xi n Jiotong University), Ministry of Eduction, Xi n , Chin Full list of uthor informtion is ville t the end of the rticle The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 2 of 12 Bckground Recent evidences hve ssocited hypertension with chronic low-grde systemic inflmmtion [1].The most susceptile mong ll orgns to incresed lood pressure is the rin [2]. The hypothlmic prventriculr nucleus (PVN) plys key role in endocrine-utonomic control y regulting roreflex function, sympthetic output, nd slt ppetite [3]. Furthermore, growing ody of evidence hve demonstrted tht incresed pro-inflmmtory cytokines (PICs) within PVN ply n importnt role in the progression of hypertension [4, 5]. Reserches descried tht hypertension is ssocited with incresed Toll-like receptor 4 (TLR4) expression in the PVN of essentil nd ngiotensin II induced hypertensive rts [6, 7]. However, the role of intrcellulr NOD like receptors, such s pyrin domin contining 3 (NLRP3) in hypertension remins unknown. Then, in this study, we nlyze the role of NLRP3, PICs, chemokine lignd 2 (CCL2), C-X-C chemokine receptor type 3 (CXCR3), vsculr cell dhesion molecule 1 (VCAM-1), nd neurohormone level in the development of prehypertension. Upon ctivtion, NLRP3 inflmmsome is formed when NLRP3 recruits the dpter protein poptosisssocited speck-like protein (ASC) nd pro-cysteine sprtic cid protese-1 (pro-cspse-1). The NLRP3 inflmmsome is ssemled nd mtured under different exogenous nd endogenous ctivtors, resulting in the production of interleukin 1 et (IL-1β) nd interleukin 18 (IL-18) [8]. IL-1β is multifunctionl cytokine which contriutes to chronic inflmmtion nd induces strong inflmmtory response during crdiovsculr diseses [9 12]. Severl physiologicl nd endocrine djustments induced y immune ctivtion re medited nd performed y IL-1β in the rin [13]. Recent study lso suggests tht lood pressure nd serum norepinephrine level is incresed when IL-1β ws injected into the rin [14]. The stte of inflmmtion nd immune ctivtion perpetuted in hypertension my ffect severity nd progression of tissue nd orgn injury. An excessive inflmmtory response is well-recognized in determining hypertension process nd hypertensive rin dmge [15]. Activtion of monocytes correltes with hypertension in the periphery nd incresed plsm pro-inflmmtory cytokines [16 18]. The ctivtion of rin-resident strocytes, microgli, nd upregultion of dhesion molecules expression on rin endothelil cells occur s result of incresed lood pressure [19, 20]. The expression of ICAM-1 nd VCAM-1 y endothelil cells further promote inflmmtion [21]. CCL2 is lso clled monocyte-chemotctic protein (MCP-1), which ws upregulted in the kidneys of deoxycorticosterone cette (DOCA) slt-hypertensive nimls [22]. Recent studies hve shown tht chemokine CXCR3 nd its corresponding chemokine lignds, CXCL9, CXCL10, nd CXCL11, re expressed in centrl nervous system diseses [23 25]. CXCR3 is well-known tissue-homing chemokine for T cells, which is highly expressed in the circultion of hypertensive ptients. CD4 + nd CD8 + Tcellsin hypertensive ptients hve incresed renl infiltrtion thn control group [26]. Accordingly, we hypothesized tht NLRP3 ctivtion ws ssocited with hypertension, upregultion of proinflmmtory cytokines IL-1β, interleukin-6 (IL-6), tumor necrosis fctor-α (TNF-α); dhesion molecule VCAM-1, chemokine CCL2, CXCR3 in PVN, nd microglil ctivtion, contriute to the inflmmtory rection with mplifiction, which resulted in n imlnce of excittory nd inhiitory neurotrnsmitters, increse of sympthetic excitility nd elevted lood pressure. Furthermore, we postulted tht lockde of NLRP3 in PVN could hve importnt functionl consequences nd led to n ssocited decrese in hypertension y regulting inflmmtion microenvironment nd neurotrnsmitters. This reserch will revel potentil new therpeutic trget for the tretment of hypertension. Methods Experimentl nimls The experimentl nimls were mde up of mle Sprgue-Dwley (SD) rts ( g). The rts were kept nd mintined t tempertures of (20 23 C) under controlled 12 h/12 h drk/light cycle. The experimentl rules nd regultions in ccordnce with the Ntionl Institutes of Helth Guide for the Cre nd Use of Lortory Animls were duly followed. The pprovl for our study ws otined from the Xi n Jiotong University Committee for Animl Reserch. Generl experimentl protocol 0.3% NCl nd 8% NCl were dministered to normlslt (NS) group nd the high-slt (HS) group over period of 3 months, respectively. The rts were divided into 10 groups: (i) norml slt 2 months (NS2), (ii) high slt 2 months (HS2), (iii) norml slt 3 months (NS3), (iv) high slt 3 months (HS3), (v) NS + Vehicle, (vi) NS + MCC950, (vii) HS + Vehicle, (viii) HS + MCC950, (ix) NS + MCC950, (x) HS + MCC950. After high-slt diet for 4 weeks, ilterl cnnule were implnted into the PVN of (v), (vi), (vii), (viii), (ix), nd (x) groups rts for infusion of MCC950 (15 μg/h, Medchem Express), specific NLRP3 locker, or vehicle (rtificil cererospinl fluid, CSF). The dose pplied for MCC950 ws ssessed from study in rts with doses of 3, 15, nd 65 μg/h [27, 28]. The highest dose cused mortlity while the 15 μg/h produced optiml response ut the low dose recorded n incomplete inhiition. After 4 weeks of drug intervention, t the end

3 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 3 of 12 of 8 weeks rts of (v), (vi), (vii), nd (viii) groups were dministered n nesthesi of ketmine (80 mg/kg) nd xylzine (10 mg/kg) mixture (ip); following this, the rins, herts, orts, nd peripherl lood were removed nd immeditely frozen on dry ice. But (ix) nd (x) groups were kept, nd the rts were fed with highslt diet for nother 1 month without tretment until the end of the experiment. The intr-prventriculr nucleus cnnul ppliction for chronic infusion A 28-dy mini osmotic pump (infusion rte 0.25 μl/h; Alzet, model 2004, Durect Corportion, Cupertino, CA, USA) ws through ctheter tue connected to the infusion cnnul to deliver MCC950 or vehicle in the PVN, s descried previously [29]. Blood pressure mesurements The noninvsive computerized til-cuff system (NIBP, AD Instruments, Austrli) ws pplied for mesuring lood pressure of the til rtery in conscious rts, s descried previously in our report [30].The men lood pressure of ll rts were determined nd recorded per week. Hemtoxylin nd eosin stining 5 μm pthology slices from the rin, ort, nd hert were prepred to discern morphology y stining with hemtoxylin nd eosin (H&E). Immunohistochemistry stining The following ntiodies were immunohistochemiclly determined: NLRP3 (1:200, Snt Cruz, CA, USA), IL-1β (1:200, Snt Cruz, CA, USA), ASC (1:100, Snt Cruz, CA, USA), pro-csp-1 (1:100, Snt Cruz, CA, USA), CD8 + (1:200, Snt Cruz, CA, USA), I-1 (1:200, Snt Cruz, CA, USA), TH (1:200, Snt Cruz, CA, USA), nd GAD67 (1:200, Snt Cruz, CA, USA). The detiled immunostining protocol performed ws the sme s in our previous reserch [29, 31]. The Imge-Pro Plus softwre ws pplied in the nlysis of the integrl opticl density nd fluorescence intensity. Isoltion of peripherl lood mononucler cells (PBMCs) 4 ml of Ficoll-Hypque were dded to 50 U/ml heprinized venous loods nd centrifuged t 400 g for 30 min t room temperture. PBMC interfce lyer ws wshed in phosphte uffer sline (PBS) twice nd ws centrifuged for 10 min t 300 g. RPMI medium supplemented with fetl clf serum ws used to resuspend cells to finl concentrtion of monocytes/ml [32]. Western lotting The rin ws sectioned serilly in 300 μm increments from the regm to lmd, oth sides of the PVN tissues were isolted y the use of punch-out technique with cryostt [33, 34], nd the PVN tissue ws stored t 80 C until use. Western lotting nlysis ws performed in the sme mnner s previously descried [6]. The protein levels were determined from tissue homogente otined from the PVN for the following ntiodies: NLRP3 (1:2000, Snt Cruz, CA, USA), ASC (1:500, Snt Cruz, CA, USA), pro-cspse-1 (1:2000, Acm, MA, USA), IL-1β (1:500, Snt Cruz, CA, USA), CXCR3 (1:2000, Acm, MA, USA), VCAM- 1, ICAM-1 (1:2000, Acm, MA, USA), nd CCL2 (1: 2000, Snt Cruz, CA, USA), I-1 (1:500, Snt Cruz, CA, USA). The β-ctin ntiody ws used s n internl stndrd, nd nd densities were nlyzed with NIH ImgeJ softwre. Flow cytometric nlysis of leukocyte in PBMCs PBMCs were trnsferred to tue nd stined with n ntiody cocktil consisting of CD3, CD4, nd CD8 (BD Bioscience, USA) diluted in PBS. Smples were then nlyzed y flow cytometry using FACS Cliur TM cytometer (BD Biosciences, Sn Jose, CA, USA). Cytokines mesure Commercilly ville rt ELISA kits were used to quntify tissue TNF-α, IL-6, nd plsm norepinephrine (Invitrogen Corportion, CA, USA) following the mnufcturer s instructionl mnul. Dt nlysis The dt were recorded s men±sem. Dt were nlyzed etween two groups using the Student s t test. For experiments tht involved multiple groups, dt nlyses were conducted y either one-wy or two-wy ANOVA followed y post hoc Bonferroni test. A proility vlue of P <0.05 ws inferred to e sttisticlly significnt. Results The role of NLRP3 on lood pressure in slt-induced prehypertensive rts The initil lood pressure seline levels were similr. By the fourth week of high-slt diet, the lood pressure of high-slt diet rts hd grdully incresed. The MAP of high-slt group incresed significntly fter 8 weeks compred with NS groups (Fig. 1). Chronic PVN infusion of MCC950, specific NLRP3 ntgonist for 4 weeks of high-slt diet, lood pressure of HS + MCC950 group ws no longer incresed; however, the MAP of HS + MCC950 group ws incresed compred with the control group, fter the centrl occlusion of

4 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 4 of 12 Fig. 1 Effects of NLRP3 on MAP in slt-induced prehypertension rts. The lood pressure of the high-slt group incresed grdully fter 2 months of high-slt diet in comprison with control group. Compred with HS + vehicle rts, chronic PVN infusion of MCC950 specificity NLRP3 ntgonist for 4 weeks ttenuted hypertension. Blood pressure continued to rose fter 4 weeks of discontinution tretment. Vlues re expressed s mens ± SEM, n = 6 8. *P < 0.05, NS vs HS, or NS + vehicle vs HS + vehicle, P < 0.05, HS + MCC950 vs HS + vehicle,#p < 0.05, HS + MCC950 vs NS + MCC950 NLRP3 ws stopped nd the high slt feeding for nother 4 weeks (Fig. 1). The expression of NLRP3/ASC/IL-1β in the PVN of high-slt diet rts y the second month In this study, we found tht NLRP3, ASC, nd IL-1β expressions in the PVN of high-slt diet incresed significntly y the second month, with corresponding increse in the lood pressure. Most interestingly, the NLRP3 pthwy proteins expression hd no difference etween the high-slt diet for 2 nd 3 months (Fig. 2). The expression of NLRP3 in the hert, ort, nd plsm pro-inflmmtory cytokines in HS group y the second month Our study demonstrted tht HS group hd shown significntly incresed NLRP3 in the hert nd ort since the second month, s shown in Fig. 3 c. ELISA results showed tht plsm TNF-α nd IL-6 in the HS groups y the second month were higher thn those in the NS rts (Fig. 3e, f). Morphologiclly, we oserved tht the crdic myocytes nucleus enlrged in the HS model since the third month, which indicted erly myocrdil hypertrophy (Fig. 3d). The numer of microgli ws incresed in the cortex nd hippocmpus of rts with high slt for 2 nd 3 months Immunohistochemistry nd western lotting showed tht the numer of microgli in hippocmpus nd cortex of the HS group ws higher thn tht of the control group (Fig. 4). CD4 + nd CD8 + T cells in PBMCs nd CD8 + microglis/ mcrophges in the rin were incresed in of high-slt diet rts since the third month We performed flow cytometric nlysis to determine leukocyte numers in PBMCs. Compred to NS rts, the numer of T cell in PBMCs ws elevted in HS rts. Further nlysis of T cell susets showed tht the popultions of CD4 + nd CD8 + T cells incresed significntly (Fig. 5). In peripherl circultion, the expression of CD4 + nd CD8 + T

5 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 5 of 12 c Fig. 2 The expression of NLRP3 pthwy protein in PVN. A representtive immunohistochemistry imge of NLRP3, scle r 100 μm, immunofluorescence imge of IL-1β, scle r 50 μm, showing the chnges of NLRP3, IL-1β in the high-slt diet rts from 2 to 3 months. A representtive immunolot of NLRP3/ASC/IL-1β. c Anlysis of NLRP3 pthwy expression in vrious groups; n = 3. Vlues re expressed s mens ± SEM. *P < 0.05, **P < 0.001, vs control (NS), 3 V, third ventricle c d e f Fig. 3 A representtive immunohistochemistry imge of NLRP3 in the hert nd ort, scle r 50 nd 20 μm, showing the chnges of NLRP3 in the high-slt diet rts from 2 to 3 months. Anlysis integrted OD of NLRP3 in vrious hert tissue groups, n =6 8. c Anlysis integrted OD of NLRP3 in vrious ort tissue groups, n =6 8. d The tissue sections of the left ventricles were stined y HE stining, scle r 50 μm. e ELISA detection plsm TNF-α expression in the vrious groups, n = 6 8. f ELISA detection plsm IL-6 expression in the vrious groups, n = 6 8.Vlues re expressed s mens ± SEM. **P < 0.001, ***P < vs control (NS)

6 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 6 of 12 c Fig. 4 The expression of I-1 in the microgli ws incresed. A representtive immunohistochemistry imge of I-1 in cortex nd hippocmpus, scle r 10 nd 50 μm. A representtive immunolot of I-1. c Anlysis of I-1 expression in vrious groups, n = 3. Vlues re expressed s mens ±SEM.*P <0.05,**P < 0.001, vs control (NS) Fig. 5 Flow cytometric nlysis T cells in peripherl circultion. CD4 + T cell (CD3 + CD4 + ). CD8 + T cell (CD3 + CD8 + ). Dt is represented s the percentge of CD3 + CD4 +, CD3 + CD8 +

7 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 7 of 12 cells incresed, nd expression of CXCR3 in PVN ws upregulted. Therefore, we sought to find infiltrting T cells in the rin prenchym. We did not find infiltrting T cells in the rin, ut we found microglis/mcrophges expressing CD8 molecules in the cortex, corpus cllosum, hippocmpus, choroid plexus, s well s neurons expressing CD8 molecules in hippocmpus nd PVN. Jnder hs previously reported the expression of CD8 + mcrophges in the centrl nervous system [35, 36](Fig.6). Blockde NLRP3 suppression of ASC/pro-cspse-1/IL-1β expression in PVN of high-slt diet rts To determine whether suppression of NLRP3 in PVN ttenutes inflmmtion response, we exmined the protein level of NLRP3 pthwy in PVN. Chronic PVN infusion of MCC950 significntly decresed ASC, pro-cspse-1, nd IL-1β expression in high-slt diet rts (Fig. 7, ). However, whenthedruginterventionwsstoppedndcontinued with high-slt feeding for month, we oserved tht the ASC nd IL-1β of HS + MCC950 group ws significntly incresed in PVN thn those of control group (Fig. 7c e). Centrl lockde NLRP3 inhiited VCAM-1, CXCR3, CCL2, nd PICs expression in the PVN of high-slt diet rts Western lotting reveled tht severl inflmmtory mrkers dhesion molecule VCAM-1, chemokine CXCR3, nd CCL2 were incresed in prehypertensive rts compred to NS rts. Centrl lockde NLRP3 ttenuted VCAM-1, CXCR3, nd CCL2 expression (Fig. 8, ). Termintion of intervention nd continued high-slt feeding for month, led to significnt upregultion VCAM-1,CXCR3, nd CCL2 in the MCC950 tretment group, s shown in Fig. 8c, d. ELISA nlysis showed tht HS + vehicle group hd significnt increse in IL-6 nd TNF-α expression in the PVN compred with NS + vehicle group. The upregultion of TNF-α nd IL-6 were significntly ttenuted y centrl lockde NLRP3 (Fig. 8e, f). Centrl lockde NLRP3 regultes PVN excittory nd inhiitory neurotrnsmitters nd plsm norepinephrine nd GABA Compred with control groups, hypertensive rts hd higher expression of TH nd lower expression of GAD67 in the PVN. Chronic infusion of MCC950 into the PVN for 4 weeks decresed the expression of TH nd incresed GAD67 expression in prehypertensive rts induced y slt. Plsm NE in prehypertensive rts ws incresed nd GABA ws reduced; interestingly, centrl lockde of NLRP3 significntly decresed plsm NE nd upregulted GABA levels in PVN (Fig. 9). Discussion In this study, the role of NLRP3 expression in the PVN of slt-induced prehypertension ws defined. The novel Fig. 6 Incresed ppering of CD8 + microglis/mcrophges in the rin of high-slt diet rts. A representtive immunohistochemistry imge of CD8 molecule microglis/mcrophges nd neurons in cortex (ctx), corpus cllosum (cc), hippocmpus (hipp), choroid plexus (CP), nd hypothlmic prventriculr nucleus (PVN), scle r 50 μm, showing the chnges of CD8 + cells in the high-slt diet rts for 3 months, n =6 8, 3 V, third ventricle

8 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 8 of 12 c d e Fig. 7 Centrl lockde NLRP3 decreses ASC/Pro-cps-1/IL-1β expression of prehypertensive rts. A representtive western lot of NLRP3 pthwy. Densitometric nlysis expression of NLRP3 pthwy in the PVN of different groups, n =3.c A representtive immunofluorescence imge of ASC, scle r 100 μm, nd immunohistochemistry imge of IL-1β, scle r 50 μm, showing the chnges of ASC nd IL-1β expression when centrl lockde NLRP3 ws stopped. d Densitometric nlysis of immunofluorescent intensity of ASC in vrious groups. e Densitometric nlysis of integrted OD of IL-1β in vrious groups, n =6 8. Vlues re expressed s mens ± SEM. *P < 0.05, **P < 0.001, ***P < , 3 V, third ventricle outcomes of this present reserch re the following: (1) NLRP3 expression in the PVN of high slt ws incresed with corresponding elevted lood pressure, (2) NLRP3 ctivtion in the PVN ws ccompnied y high expression of PICs, dhesion molecule VCAM-1, chemokine CCL2 nd CXCR3, s well s reking the lnces of neurotrnsmitters in the PVN, (3) microglil ctivtion, CD8 + microglis/ mcrophges were incresed in rin prenchym of prehypertensive rts, (4) centrl lockde of NLRP3 in the PVN ttenuted prehypertension resulting from regulting the inflmmtion microenvironment nd restoring the lnces of neurotrnsmitters in PVN, (5) NLRP3 ws not only upregulted in the PVN, ut lso highly expressed in the peripherl tissue, hert nd ort. PVN in the rin is notle s n importnt endocrineutonomic control re which contriutes to sympthetic regultion of lood pressure nd ody fluid homeostsis [37]. The sympthetic outflow from the PVN depends on the lnce of different kinds of neurotrnsmitter ctivities. Incresingly evidences demonstrted tht the incresed sympthetic ctivtion is due to n increse in excittory drenergic nd glutmtergic ctivities nd decrese in GABAergic ctivity in the PVN [38]. In chronic sterile inflmmtion, inflmmtory responses re cused nd mintined y the NLRP3 inflmmsome [39], which ctivtion of pro-cspse-1 nd IL-1β, IL-1β is recognized s the min ctivtor of inflmmtion, which serves s meditor to trigger the cscde relese of other cytokines. Reports hve demonstrted tht vrious PICs such s TNF-α, IL-1β, nd IL-6 ply vitl role in the development of hypertension [31, 40 42]. Tissue cells synthesize inflmmtory meditors nd chemokine in response to injury nd stress, to recruit inflmmtory cells. Positive feedck loops result s inflmmtory cells perpetute the relese of cytokines nd express dhesion molecules. Studies hve shown tht dhesion molecules ICAM-1, VCAM- 1, immune cells, chemokine CCL2, nd CXCR3 re key fctors in humn hypertension [21, 22, 26]; these series of events results in the progression of hypertension. In this study, we provide the evidence tht NLRP3 plys key role in prehypertension induced y slt. Our study demonstrted tht HS rts recorded significnt increse

9 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 9 of 12 c d e f Fig. 8 Centrl lockde NLRP3 suppresses of VCAM-1, CXCR3, nd CCL2 expression in the PVN of prehypertensive rts. A representtive immunolot of VCAM-1, CXCR3, nd CCL2. Densitometric expression nlysis of VCAM-1, CXCR3, nd CCL2 in vrious groups, n =3.c Terminted tretment of nti-nlrp3, representtive immunolot of VCAM-1, CXCR3, nd CCL2. d Densitometric protein nlysis of VCAM-1, CXCR3, nd CCL2 in vrious groups; n =3.e ELISA detection IL-6 expression in the PVN of the vrious groups, n =6 8. f ELISA detection TNF-α expression in the PVN, n =6 8. Vlues re expressed s mens ± SEM. *P <0.05,**P <0.001.***P < inmapincomprisonwithnsgroupythe2monthsof high-slt diet, nd the chnges were prlleled with NLRP3 incresed expression in HS group. HS group feed with high slt for 2 months hd significnt increse in NLRP3 not only in the PVN ut lso in the hert nd ort peripherl tissues, s well s upregulted plsm IL-6 nd TNF-α.We lso found tht VCAM-1, chemokine CCL2, nd CXCR3 in the PVN were incresed since the second month of highslt diet. During inflmmtory responses, chemokine clssiclly medite, migrte, nd trffic cells to sites of inflmmtion [43]. Arteril hypertension ffects one mrrow hemtopoietic niche directly y incresed levels of monocytes nd neutrophils in the circultion [44]. In fct, previous studies showed tht the lood nd rin of nive SHR expressed incresed monocyte nd neutrophil counts [45 47]. Our dt illustrted tht the numer of microgli in the cortex nd hippocmpus nd totl T cells in the peripherl circultion in HS rts were incresed compred to NS rts, nd further nlysis of the immune cell susets reveled tht the popultions of CD4 + nd CD8 + Tcellswere incresed significntly. We try to find the infiltrtion of T cells in the rin prenchym without success, ut we found the numer of CD8 + microglis/mcrophges in the rin incresed. Both CD4 nd CD8 ntigens were originlly descried s ntigen coreceptors on helper nd cytotoxic/suppressor T lymphocytes, respectively. However, Jnder et l. descried popultion of ctivted centrl nervous system (CNS) mcrophges chrcterized y the expression of the CD8 molecule [35, 36]. Our next step sought to determine whether the nti- NLRP3 therpy could decrese cscde rection of inflmmtion, regulte neurotrnsmitters, nd counterct hypertension. We found tht lockde NLRP3 therpy mrkedly reversed hypertension, in consistent with the results reported y Krishnn [48], s well s significntly

10 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 10 of 12 c d e Fig. 9 Centrl lockde NLRP3 regultes PVN excittory nd inhiitory neurotrnsmitters. A representtive immunohistochemistry imge of TH nd GAD67, scle r 10 nd 20 μm, showing the chnges of TH nd GAD67 in the high-slt diet rts fter centrl lockde NLRP3. Expression of TH in vrious groups; n =6 8. c Expression of GAD67 in vrious groups, n =6 8. d ELISA detection plsm NE expression in vrious groups, n =6 8. e ELISA detection GABA expression in vrious groups, n =6 8.Vlues re expressed s mens ± SEM. *P < 0.05, ***P < , 3 V, third ventricle reduced NLRP3 pthwy protein expression, decresed PICs, CCL2, CXCR3, nd VCAM-1, djusted TH, GAD67, nd GABA in PVN nd plsm NE. However, when we stopped the drug intervention nd continued feeding with the high-slt diet for month, we oserved tht HS + MCC950 group hd significnt ctivtion of ASC, IL-1β, incresed VCAM-1, CCL2, nd CXCR3 expression gin in the PVN compred to the control group. Conclusions In summry, our results show tht NLRP3 plys key role in prehypertensive rts induced y high-slt diet, vi n inflmmtory mechnism tht increses IL-1β, IL-6, nd TNF-α; chemokine CCL2, CXCR3; dhesion molecule VCAM-1 nd djusts excittory neurotrnsmitters nd inhiitory neurotrnsmitters, s shown in Additionl file 1: Figure S1. These findings shed new lights into the moleculr mechnism tht controls hypertension; however, whether NLRP3 could e used s therpeutic trget for hypertension remins to e clrified. Limittions Our study hs limittions. We oserved tht dhesion molecule VCAM-1 nd chemokine CCL2, CXCR3 re highly expressed in the PVN; however, it is uncler which cells express these molecules, endothelil cells, glil cells or infiltrting leukocytes? Then, wht ctivtes the NLRP3? Wht re the first steps tht trigger the inflmmtion response in the PVN? All these questions needs to e further explored nd discussed. Additionl file Additionl file 1: Figure S1. Slt-induced prehypertension is prtly due to the role of NLRP3 in PVN, vi n inflmmtory mechnism. Blockde of rin NLRP3 ttenutes prehypertensive response, possily vi downregulting the cscde rection triggered y inflmmtion nd restoring the lnce of neurotrnsmitters (PPTX 203 k) Arevitions ASC: Adpter protein poptosis-ssocited speck like protein; BP: Blood pressure; CCL2: Chemokine lignd 2; CXCR3: C-X-C chemokine receptor type 3; DAMP: Dnger-ssocited moleculr pttern; DOCA: Deoxycorticosterone cette; GABA: Gmm-minoutyric cid; GAD67: 67-kD isoform of glutmte decroxylse; I-1: Ionized clcium-inding dptor

11 Wng et l. Journl of Neuroinflmmtion (2018) 15:95 Pge 11 of 12 molecule-1; ICAM-1: Intercellulr dhesion molecule 1; IL-18: Interleukin 18; IL- 1β: Interleukin 1β; IL-6: Interleukin 6; MAP: Men rteril pressure; mmhg: Millimeter of mercury; NF-κB: Nucler fctor-kpp B; NLRP3: Nod-like receptor with pyrin domin contining 3; PBMC: Peripherl lood mononucler cell; PICs: Pro-inflmmtory molecules; pro-cspse-1: Pro-cysteine sprtic cid protese-1; PVN: Hypothlmic prventriculr nucleus; SD rts: Sprgue-Dwley rts; TH: Tyrosine hydroxylse; TLR4: Toll-like receptor 4; TNF-α: Tumor necrosis fctor-α; VCAM-1: Vsculr cell dhesion molecule 1 Funding This study ws supported y Ntionl Nturl Science Foundtion of Chin (nos , ), Chin Postdoctorl Science Foundtion (nos M602835, 2016 M592802), Shnxi Postdoctorl Science Foundtion (nos. 2016BSHEDZZ91, 2016BSHEDZZ88), nd Jimusi University project (no. JMSUJCMS ). The funders hd no role in the study design, dt collection nd nlysis, decision to pulish, or preprtion of the mnuscript. Avilility of dt nd mterils Dt shring is not pplicle to this rticle s no dtsets were generted or nlyzed during the current study. Authors contriutions YK designed the study. MW, XY, QS, XL, KL, LF, YL, nd HL performed the dt nlysis nd drfted the mnuscript. MW, QS, RS, HT, nd HL performed ll the experiments nd prticipted in the dt nlysis. All uthors reviewed the mnuscript. All uthors red nd pproved the finl mnuscript. Ethics pprovl nd consent to prticipte The experimentl rules nd regultions in ccordnce with the Ntionl Institutes of Helth Guide for the Cre nd Use of Lortory Animls were duly followed. The pprovl for our study ws otined from the Xi n Jiotong University Committee for Animl Reserch. Competing interests The uthors declre tht they hve no competing interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Author detils 1 Deprtment of Physiology nd Pthophysiology, Xi n Jiotong University School of Bsic Medicl Sciences, Key Lortory of Environment nd Genes Relted to Diseses (Xi n Jiotong University), Ministry of Eduction, Xi n , Chin. 2 Deprtment of Immunology, School of Bsic Medicl Sciences, Jimusi University, Jimusi , Chin. 3 Deprtment of Rehilittion Medicine, People s Hospitl of Bon District, Shenzhen , Chin. 4 Deprtment of Pthology, Xi n Jiotong University School of Bsic Medicl Sciences, Xi n Jiotong University Helth Science Center, Xi n , Chin. Received: 12 Octoer 2017 Accepted: 15 Mrch 2018 References 1. Puletto P, Rttzzi M. Inflmmtion nd hypertension: the serch for link. Nephrol Dil Trnsplnt. 2006;21: Mnci G, Grssi G. The utonomic nervous system nd hypertension. Circ Res. 2014;114: Srirmul S, Hque M, Mjid DS, Frncis J. 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Inflmmsome ctivity is essentil for one kidney/deoxycorticosterone cette/slt-induced hypertension in mice. Br J Phrmcol. 2016;173: Sumit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-sumission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online sumission Thorough peer review Inclusion in PuMed nd ll mjor indexing services Mximum visiility for your reserch Sumit your mnuscript t