mir-155 is dispensable in monosodium urate-induced gouty inflammation in mice

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1 Yng et l. Arthritis Reserch & Therpy (2018) 20:144 RESEARCH ARTICLE mir-155 is dispensle in monosodium urte-induced gouty inflmmtion in mice Qiin Yng 1,3,4, Quno Zhng 2,3,4, Yufeng Qing 1, Li Zhou 3,4,5, Qingsheng Mi 3,4,5* nd Jingguo Zhou 6* Open Access Astrct Bckground: The findings of previous study y Jin et l. hve shown tht microrna (mir)-155 ws upregulted in ptients with cute gouty rthritis nd enhnced the proinflmmtory cytokines. There is no direct evidence to support tht mir-155 is indeed involved in monosodium urte (MSU)-induced inflmmtory responses in vivo. The im of this study ws to investigte the role of mir-155 knock-out (KO) or knock-in (KI) mice in MSU-induced niml models to mimic cute gout. Methods: MiR-155 expression in cultured one mrrow-derived mcrophges (BMDMs) from mir-155 KO, mir-155 KI, nd wild-type (WT) mice treted with MSU crystls in vitro ws detected y rel-time quntittive polymerse chin rection (qpcr). MiR-155 KO nd WT mice were used to induce n cute gouty inflmmtory response with MSU crystls including models of foot pd inflmmtion, nkle rthritis, ir pouch inflmmtion, nd peritonitis. Furthermore, the proinflmmtory interleukin (IL)-1β levels in lvge fluids from ir pouch nd peritonel cvity models were mesured y enzyme-linked immunosorent ssy (ELISA), nd tumor necrosis fctor (TNF)-α production from BMDMs of mir-155 KI mice treted with MSU were mesured y flow cytometry. Results: MiR-155 expression ws quickly upregulted in BMDMs from WT mice following MSU tretment in vitro.in comprison with WT mice in vivo, the swelling index of mir-155 KO mice showed no significnt difference in the murine foot pd nd nkle rthritis models for the indicted different time points. There were similr chnges in totl cell numers of lvge fluids in the ir pouch nd peritonel cvity models etween mir-155 KO nd WT mice following MSU crystl injection. Moreover, the IL-1β levels of lvge fluids in the ir pouch nd peritonitis models from mir-155 KO mice were lmostthesmesthosefromwtmice.tnf-α levels were comprle from BMDMs treted with MSU crystls in vitro etween mir-155 KI mice nd WT mice. Conclusions: MiR-155 is dispensle in MSU-induced gouty inflmmtion in mice. Deletion of mir-155 might not e n effective therpeutic pproch to relieve the inflmmtion in cute gout. Keywords: MiR-155, MSU, Gout, Inflmmtion Bckground Gout is one of the most common forms of inflmmtory rthritis disorder nd is cused y deposition of monosodium urte (MSU) crystls in nd round the joints [1]. Accumulted studies indicte tht t lest two pthwys, the Toll-like receptor (TLR)/nucler fctor (NF)- κb signling pthwy nd the NLR fmily pyrin domin * Correspondence: qmil@hfhs.org; jgzhou@cmc.edu.cn Qiin Yng nd Quno Zhng contriuted eqully to this work. 3 Henry Ford Immunology Progrm, Henry Ford Helth System, 1 Ford Plce, Detroit, MI 48202, USA 6 Deprtment of Rheumtology nd Immunology, The First Affilited Hospitl of Chengdu Medicl College, Sichun Province, Chengdu , Chin Full list of uthor informtion is ville t the end of the rticle contining 3 (NLRP3) pthwy, re involved in MSU crystl-induced inflmmtory cytokine relese from monocytes/mcrophges [2 4]. However, the moleculr mechnisms of gouty inflmmtion re still not entirely elucidted. MicroRNAs (mirnas) re n undnt clss of smll, evolutionry conserved, non-coding RNAs cting s post-trnscriptionl regultors. Our previous dt hve reported tht mirnas regulte the immune cell development nd function, nd control utoimmune disese development [5, 6]. Aerrnt mirna expression hs een oserved in numer of diseses. Recent studies suggest tht mirnas re potentilly involved in the The Author(s). 2018, corrected puliction August/2018. 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 Yng et l. Arthritis Reserch & Therpy (2018) 20:144 Pge 2 of 7 development of humn cute gouty rthritis, including mir-155 [7] nd mir-146 [8]. Dleth et l. [8] found tht mir-146 expression incresed in oth humn monocytic THP-1 cells nd humn peripherl lood mononucler cells (PBMCs) using MSU crystl stimultion in vitro, ut the expression of other mirnas (including mir-155, mir-146, mir-9, nd mir-21) did not, implicting interleukin (IL)-1β regultion. Interestingly, higher levels of mir-146 were expressed in PBMCs from intercriticl gout ptients when compred with oth control groups (normouricemic control prticipnts/hyperuricemic control prticipnts) nd the cute gout group. Additionlly, mir-146 expression ws reduced during the cute flre compred with the intercriticl period in the pired smples s well s in the urte peritonitis model. Those findings reflect the more complex multicellulr in vivo response to MSU crystls. Numerous studies hve reported tht mir-155 could negtively regulte the TLR/NF-κB signling pthwy y trgeting different kinds of genes [9], nd tht mir-155 deficiency reduced inflmmtory responses in the colitis mouse model [10]. MiR-155 expression, which is closely relted to disese ctivity, is upregulted in the PBMCs nd synovil memrne of rheumtoid rthritis [11, 12]. Jin et l. [7] showed tht mir-155 ws upregulted in synovil fluid mononucler cells (SFMCs) from ptients with cute gouty rthritis nd MSU crystls strongly induced mir-155 expression in PBMCs in vitro. Furthermore, the incresed expression of mir-155 in SFMCs led to downregultion of the SH2 domin-contining inositol 5 -phosphtse 1 (SHIP1), which ctivtes the Akt/NF-kB pthwy nd enhnces the production of proinflmmtory cytokines, including IL-1β. Notly, mir-155 levels in PBMCs from gout ptients were comprle with helthy individuls. These prdoxicl dt suggest tht the sme mirna my ply different roles in different tissues/orgns nd conditions. However, there is still lck of direct evidence to further determine whether mir-155 is indeed involved in MSU-induced inflmmtion in vivo. In the present study, we took dvntge of mir-155 knock-out (KO) mice to investigte the role of mir-155 in MSU-induced gout in vivo. Four MSU-induced gout mouse models, including foot pd inflmmtion, nkle rthritis, peritonitis, nd ir pouch inflmmtion, were used. The proinflmmtory cytokine IL-1β levels in lvge fluids from the peritonel cvity nd ir pouch models nd tumor necrosis fctor (TNF)-α levels from one mrrow-derived mcrophges (BMDMs) of mir-155 knock-in (KI) mice treted with MSU crystls were mesured. Methods Animls MiR-155 / C57BL/6 knock-out (mir-155 KO) mice, Csf1r + 155Tg/Tg knock-in (mir-155 KI) mice, nd Csf1r 155Tg/ Tg wild-type (WT) mice were kindly provided y the CBR Institute for Biomedicl Reserch, Hrvrd Medicl School, Boston, USA. C57BL/6 s WT mice were purchsed from the Jckson Lortory. Colony-stimulting fctor 1 receptor (Csf1r), lso known s mcrophge colony-stimulting fctor (M-CSF) receptor, controls the production, differentition nd function of mcrophges. As we know, mcrophges ply crucil roles (such s phgocytosis nd trigging the inflmmtory response) in cute gout including in the initil phse, in development, nd in remission. The mice were housed t 24 ± 2 C under 12-h light/12-h drk cycles in pthogen-free fcility; 8- to 10-week-old mles were used to perform the experiments. The hndling of mice nd experimentl procedures in this study were performed in ccordnce with the requirements of the Institutionl Animl Cre nd Use Committee of Henry Ford Helth System. Preprtion of MSU crystls Briefly, 1.0 g uric cid (Sigm-Aldrich) ws dissolved in 200 ml oiling distilled wter contining 6.0 ml 1 M NOH. After djusting the ph of the solution to 7.2 with HCl, crystls tht formed were sterilized y heting t 180 C for 2 h [13]. The solution ws grdully cooled y stirring t room temperture nd stored overnight t 4 C. The precipitte ws filtered from the solution, dried under low het, nd suspended in phosphte-uffered sline (PBS) t concentrtion of 50 mg/ml. All regents were prepred under pyrogen-free conditions. BMDM culture nd MSU stimultion Bone mrrow cells otined from the femorl ones of C57BL/6(WT), mir-155 KO, Csf1r + 155Tg/Tg (mir-155 KI), nd Csf1r 155Tg/Tg (WT) mice, respectively, were cultured in RPMI 1640 (Gico) supplemented with 10% fetl ovine serum (FBS), penicillin (100 units/ml), nd streptomycin (100 μg/ml). To induce the prolifertion nd differentition of myeloid progenitors to mcrophges, the medium ws supplemented with 30 ng/ml M-CSF (# , Peprotech). The cells were wshed nd received fresh medium with M-CSF every 2 3 dys. After 7 dys the cells were hrvested with 0.25% trypsin. Ded cells were first gted out y propidium iodide (PI) stining. The phenotypic vlidtion of BMDMs ws performed y flow cytometry with stining using fluorescein isothiocynte (FITC)-conjugted nti-cd11 nd phycoerythrin (PE)-conjugted nti-f4/80 ntiodies (oth diluted 1:200). The BMDMs re doule-positive for CD11 nd F4/80. According to the experimentl protocol, the MSU crystl suspension (MSU 2.5 mg/ml concentrtion) ws dded to the incuted BMDMs (MSU 100 μg/ml finl concentrtion) for 4 or 8 h nd the rtio of TNF-α production from BMDMs treted with MSU for 2 or 4 h were mesured.

3 Yng et l. Arthritis Reserch & Therpy (2018) 20:144 Pge 3 of 7 Gout model Mice were plced under nesthesi (150:10 mg/kg ketmine:xylzine injected intrperitonelly) nd were injected with MSU crystls into the right foot pd (1 mg in 40 μl PBS) or nkle joint (0.5 mg in 20 μl PBS). The sme volume of sterile sline ws injected into the left foot pd or nkle joint t the sme time to serve s the control. Inflmmtion prmeters were evluted following MSU crystl injection t different time points (6, 24, nd 48 h). Pw swelling nd the size of nkle joints were mesured with n electronic cliper t the indicted time points [14 16]. For n ir pouch model, 5 ml sterile ir ws first injected sucutneously into the ck of mice to form n ir pouch, nd then 3 ml sterile ir ws injected to the ir pouch t dy 3 nd dy 5. At dy 7, MSU suspension (3 mg in 1 ml PBS) ws injected into the ir pouch [17]. For MSU-induced peritonitis, MSU crystls (3 mg in 0.5 ml PBS) were injected into the peritonel cvity s prt of the intrperitonel gouty model [7]. The totl numer of ir pouch nd peritonel cvity exudte cells were hrvested fter 4 or 8 h nd counted y hemocytometer. Rel-time quntittive polymerse chin rection The cultured BMDMs were hrvested fter 7 dys. Totl RNA of BMDMs ws extrcted using Trizol regent (Invitrogen, USA) nd reverse-trnscried into cdna using reverse trnscription regents (Invitrogen, USA) ccording to the mnufcturer s protocols. Rel-time quntittive polymerse chin rection (qpcr) ws performed using the ABI Prism 7900HT Detection System (Applied Biosystems, USA) with Power SYBR Green PCR Mster Mix (Applied Biosystems, USA). The gene primers sequences were synthesized y eurofins genomics (Louisville, USA) s follows. TNF-α: forwrd 5 - ACAAAGGTGCCGCTAACCACATGT-3, reverse 5 - ATGCTGCTGTTTCAGTCGAAGGCA-3 ; IL-1β: forwrd 5 -GGGCCTCAAAGGAAAGAATC-3, reverse 5 - CTCTGCTTGTGAGGTGCTGA-3 ; β-ctin: forwrd 5 -CAACGAGCGGTTCCGATG-3, reverse 5 -GCCA- CAGGATTCCATACCCA-3. The β-ctin s n internl control ws used to normlize the gene expression. Gene expression ws nlyzed using the 2 ΔΔCT method. The expression of mir-155 in BMDMs ws mesured using Tqmn MicroRNA Assys (Applied iosystems, Foster City, CA, USA) ccording to the mnufcturer s protocols. The Tqmn MicroRNA Assy for U6 snrna ws used to normlize the reltive undnce of mirnas. Enzyme-linked immunosorent ssy (ELISA) IL-1β protein levels in lvge fluids of the ir pouch nd peritonel cvity models were mesured y ELISA using kits from ebioscience (ct. no ; Sn Diego, CA, USA) following the mnufcturer s instructions. The 96-well micropltes were red using VICTOR X3 plte reder. Sttisticl nlysis Dt were nlyzed with GrphPd Prism 5. Differences etween experimentl groups were tested using the unpired t test. Dt re expressed s men ± SEM. P <0.05 ws considered to denote sttisticl significnce. Results nd discussion In the present study, sed on the findings from Jin et l. [7] who found tht errnt mir-155 expression ws involved in the pthogenesis of gout, we further investigted the role of mir-155 in MSU-medited gout using mir-155 KO or KI mice in in-vitro or in-vivo experiments. We firstly nlyzed the mir-155 expression from BMDMs of mir-155 KO nd WT mice with or without MSU tretment in vitro. The purity of cultured BMDMs ws no different etween mir-155 KO nd WT mice (Fig. 1). The level of mir-155 expression in BMDMs from mir-155 KO mice ws significntly lower thn tht from WT mice (Fig. 1). The previously descried study reported tht mir-155 expression in the THP-1 cell linege experiment in vitro ws comprle with or without MSU crystl stimultion [8]. However, mir-155 expression could e induced y the MSU crystl stimultion in the PBMCs of helthy controls in vitro [7]. Our results showed tht mir-155 expression ws quickly nd strongly upregulted in BMDMs from WT mice following MSU crystl tretment, while mir-155 expression ws hrdly induced in mir-155 KO mice (Fig. 1). This is consistent with the previous report [7] of higher levels of mir-155 expression due to MSU crystl stimultion. We next tried to etter understnd whether mir-155 deficiency ffects the inflmmtory response in MSUinduced gouty inflmmtion in vivo. According to description of murine gout models [14 16], the mnifesttion of inflmmtion such s redness nd swollen joint, nd the swelling index were used to evlute the inflmmtory levels. We used MSU-induced foot pd inflmmtion nd MSU-induced nkle rthritis models. As shown in Fig. 2, lthough the swelling indexes incresed following MSU crystl tretment nd reched pek of swelling t 24 h, the swelling indexes were no different etween mir-155 KO mice nd WT mice in either the foot pd model (Fig. 2) or the nkle joint model (Fig. 2). These results indicte tht deletion of mir-155 expression does not significntly ffect the phenotype of MSU-induced gouty inflmmtion in vivo. An cute inflmmtory profile occurs in response to MSU crystls; infiltrtion of neutrophils nd monocytes/ mcrophges into the peritoneum ws oserved fter 4 h, nd proinflmmtory cytokines such s IL-1β, IL-6,

4 Yng et l. Arthritis Reserch & Therpy (2018) 20:144 Pge 4 of 7 Fig. 1 MiR-155 is upregulted in one mrrow-derived mcrophges. Bone mrrow-derived mcrophges (BMDMs) from mir-155 knock-out (KO) nd wild-type (WT) mice were cultured fter 7 dys nd the purity of BMDMs nlyzed y flow cytometry. The BMDMs were doule-positive for CD11 nd F4/80. BMDMs from mir-155ko nd WT mice were treted with MSU for 4 nd 8 h, nd mir-155 expression ws detected y Tqmn rel-time qpcr. Results re representtive of three independent experiments; n = 3 5 mice per group. **P < NS not significnt Fig. 2 Acute gouty inflmmtion ws induced in mir-155 knock-out (KO) nd wild-type (WT) mice y MSU. Swelling index of the foot pd model ws mesured y n electronic cliper t the different time points in mir-155 KO nd WT mice treted with MSU (1 mg in 40 μl PBS). Swelling index of the nkle joint model ws mesured y n electronic cliper t the different time points fter MSU tretment (0.5 mg in 20 μl PBS). Results re representtive of three independent experiments; n = 5 mice per group

5 Yng et l. Arthritis Reserch & Therpy (2018) 20:144 Pge 5 of 7 nd TNF-α were elevted within 2 h nd peked t 4 h [18]. Given tht elevted levels of mir-155 re involved in MSU-induced gouty inflmmtion, we hypothesized tht loss of mir-155 my reduce the inflmmtory meditor of MSU-induced gout. To test this hypothesis, MSU crystls were injected into the peritonel cvity of mir- 155 KO nd WT mice, referring to Jin et l. s protocol [7] who used the synovil cvity, to induce n cute inflmmtory response. Mice were scrificed t 0, 4, or 8 h following MSU crystl injection. The totl cell numer of lvge fluids ws counted in the peritonitis model, nd there were similr levels of totl cell numers etween mir-155 KO nd WT mice regrdless of drmticlly incresing tendency oserved in oth of these s time goes on (Fig. 3). We lso used n MSUinduced ir pouch model, model system chrcterized y the genertion of synovium-like lining cell lyer [19]. There were lso no remrkle chnges in totl cell numers in lvge fluids in the ir pouch (Fig. 3c) etween mir-155 KO nd WT mice following MSU tretment. Additionlly, TNF-α (Fig. 3d) nd IL-1β (Fig. 3e) mrna expression from the ir pouch lvge fluids cells were not significntly different etween mir-155 KO nd WT mice. Finlly, we lso found tht the proinflmmtory cytokine IL-1β levels in lvge fluids from the peritonel cvity (Fig. 3) nd the ir pouch (Fig. 3f) from mir-155 KO mice were comprle with those from WT mice. Thus, our findings indicte tht the loss of mir-155 is not n effective relief strtegy for MSUinduced ir pouch nd peritonitis in vivo. It hs een found tht overexpression of mir-155 promotes TNF-α nd IL-1β levels in the superntnt of c d e f Fig. 3 Acute gouty inflmmtion ws induced in the peritonel cvity nd ir pouch models from mir-155 knock-out (KO) nd wild-type (WT) mice treted with MSU., c Totl cell numer in lvge fluids from the peritonel cvity () nd ir pouch (c) models were counted y hemtocytometer. d, e The mrna expression of tumor necrosis fctor (TNF)-α (d) nd interleukin (IL)-1β (e) were mesured y rel-time qpcr in the totl cells from ir pouch lvge fluids of mir-155 KO nd WT mice treted with MSU crystls for 0, 4, or 8 h., f ThecytokineIL-1β levels in lvge fluids from peritonel cvity () nd ir pouch (f) models were mesured y ELISA. Results re representtive of three independent experiments; n =3 5 mice per group

6 Yng et l. Arthritis Reserch & Therpy (2018) 20:144 Pge 6 of 7 THP-1 cells treted with MSU in vitro [7]. Thus, we further ssessed the effects of mir-155 overexpression on the MSU-induced inflmmtory response. The BMDMs from Csf1r + 155Tg/Tg (mir-155 KI) nd Csf1r 155Tg/Tg (WT) mice with MSU stimultion were used in the present study. In comprison with WT mice, oth purity nd numer of cultured BMDMs were comprle in mir-155 KI mice (Fig. 4). We vlidted the mir-155 expression in BMDMs from mir-155 KI mice. As shown in Fig. 4, drmticlly incresed expression of mir- 155 ws oserved in BMDMs from mir-155 KI mice compred with those from WT mice. The cytokine TNF-α levels from BMDMs following MSU tretment for 2 or 4 h were ssyed y flow cytometry. We found tht the percentge of BMDMs producing TNF-α in mir-155 KI mice ws similr to tht of WT mice lthough overexpression of mir-155 ws likely to promote more TNF-α production (Fig. 4c). Our results further support tht mir-155 does not directly ffect MSU-induced gouty inflmmtion. This is in disgreement with the previous result of high mir- 155 expression promoting TNF-α production [7]. This discrepncy might result from overexpression of mir- 155 in vitro or in vivo. More reserch is required to untngle the confusing role of mir-155 in gouty inflmmtion. Overll, our dt suggest tht mir-155 is dispensle in MSU-induced gouty inflmmtion in mice. Given tht mny mirnas re regulted in monocytes/mcrophges upon MSU stimultion (our unpulished dt), mir-155 is more likely redundnt with other regulted mirnas during gout development. c Fig. 4 Cytokine tumor necrosis fctor (TNF)-α ws produced from BMDMs of Csf1r + 155Tg/Tg (KI) nd Csf1r 155Tg/Tg (WT) mice with MSU stimultion. Bone mrrow-derived mcrophges (BMDMs) from mir-155 KI nd WT mice were cultured for 7 dys, nd the purity nd numer of BMDMs were nlyzed y flow cytometry. The BMDMs were doule-positive for CD11 nd F4/80. MiR-155 expression ws detected y Tqmn rel-time qpcr in BMDMs from mir-155 KI nd WT mice. c BMDMs from mir-155ki nd WT mice were treted with MSU for 0, 2, or 4 h, nd the rtio of cytokine TNF-α production from BMDMs following MSU tretment ws ssyed y flow cytometry. Results re representtive of three independent experiments; n =3 mice per group. **P <0.01.NS not significnt

7 Yng et l. Arthritis Reserch & Therpy (2018) 20:144 Pge 7 of 7 Conclusion The mir-155 expression ws quickly upregulted in BMDMs from WT mice with MSU-induced cute gouty inflmmtion in vitro. MiR-155 deficiency did not significntly ffect the phenotype in diverse murine MSUinduced gouty inflmmtion in vivo. Therefore, deletion of mir-155 might not e n effective therpeutic pproch to relieve the inflmmtion in cute gout. Arevitions BMDM: Bone mrrow-derived mcrophge; Csf1r: Colony-stimulting fctor 1 receptor; ELISA: Enzyme-linked immunosorent ssy; IL: Interleukin; KI: Knock-in; KO: Knock-out; M-CSF: Mcrophge colony-stimulting fctor; mirna: MicroRNA; MSU: Monosodium urte; NF: Nucler fctor; NLRP3: NLR fmily pyrin domin contining 3; PBMC: Peripherl lood mononucler cell; PBS: Phosphte-uffered sline; qpcr: Quntittive polymerse chin rection; SFMC: Synovil fluid mononucler cell; SHIP1: SH2 domin-contining inositol 5 -phosphtse 1; TNF: tumor necrosis fctor; TLR: Toll-like receptor; WT: Wild-type Acknowledgements We thnk ll lortory memers for their help nd encourgement. Funding This study ws supported in prt y the Henry Ford Helth System Reserch Grnts for the Immunology Progrm (T71016 to QM nd T71016 to LZ), the Ntionl Nturl Science Foundtion of Chin ( to JZ), the Eduction Agency of Sichun Province Grnts (15ZB0202 to QY), the Sichun Medicl Assocition Grnts (S16027 to QY), nd the Ntionl Key Reserch Progrm of Chin (2016YFC to YQ). The funders hd no role in the study design, dt collection, nlysis, or interprettion of the dt, or in writing the mnuscript or the decision to sumit the mnuscript. Avilility of dt nd mterils The dtsets used nd/or nlyzed during the current study re ville from the corresponding uthor on resonle request. Authors contriutions QY nd QZ performed the experiment. QY drfted the mnuscript. YQ, LZ, QM, nd JZ designed experiments nd criticlly revised the mnuscript. All uthors red nd pproved the finl mnuscript. Ethics pprovl All niml experimentl procedures in the current study were pproved y the Institutionl Animl Cre nd Use Committee of Henry Ford Helth System (Detroit, USA). Consent for puliction Not pplicle. 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 Rheumtology nd Immunology, Affilited Hospitl of North Sichun Medicl College, Sichun Province, Nnchong , Chin. 2 Deprtment of Gerontology, Affilited Hospitl of North Sichun Medicl College, Sichun Province, Nnchong , Chin. 3 Henry Ford Immunology Progrm, Henry Ford Helth System, 1 Ford Plce, Detroit, MI 48202, USA. 4 Deprtment of Dermtology, Henry Ford Helth System, 1 Ford Plce, Detroit, MI 48202, USA. 5 Deprtment of Internl Medicine, Henry Ford Helth System, 1 Ford Plce, Detroit, MI 48202, USA. 6 Deprtment of Rheumtology nd Immunology, The First Affilited Hospitl of Chengdu Medicl College, Sichun Province, Chengdu , Chin. Received: 31 Octoer 2017 Accepted: 21 Ferury 2018 References 1. Mrtinon F, Glimcher LH. Gout: new insights into n old disese. J Clin Invest. 2006;116(8): Busso N, So A. Mechnisms of inflmmtion in gout. Arthritis Res Ther. 2010; 12(2): Qing YF, Zhng QB, Zhou JG, Jing L. 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