Sexually Dimorphic Role of Toll-like Receptor 4 (TLR4) in High Molecular Weight Hyaluronan (HMWH)-induced Anti-hyperalgesia

  • Ivan J.M. Bonet
    Departments of Medicine and Oral & Maxillofacial Surgery, San Francisco

    UCSF Pain and Addiction Research Center, University of California at San Francisco, San Francisco
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  • Dionéia Araldi
    Departments of Medicine and Oral & Maxillofacial Surgery, San Francisco

    UCSF Pain and Addiction Research Center, University of California at San Francisco, San Francisco
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  • Paul G. Green
    Departments of Medicine and Oral & Maxillofacial Surgery, San Francisco

    UCSF Pain and Addiction Research Center, University of California at San Francisco, San Francisco

    Departments of Preventative and Restorative Dental Sciences, University of California at San Francisco, San Francisco
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  • Jon D. Levine
    Address reprint requests to: Jon D. Levine, M.D., Ph.D., University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0440, Phone: 476-5108, Fax: 476-6305,
    Departments of Medicine and Oral & Maxillofacial Surgery, San Francisco

    UCSF Pain and Addiction Research Center, University of California at San Francisco, San Francisco

    Departments of Medicine, University of California at San Francisco, San Francisco
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      • Involvement of TLR4 in HMWH-induced anti-hyperalgesia is sexually dimorphic.
      • HMWH anti-hyperalgesia is TLR4-dependent only in male rats.
      • TLR4 dependence of HMWH anti-hyperalgesia is MyD88 mediated.


      High molecular weight hyaluronan (HMWH), a prominent component of the extracellular matrix binds to and signals via multiple receptors, including cluster of differentiation 44 (CD44) and toll-like receptor 4 (TLR4). We tested the hypothesis that, in the setting of inflammation, HMWH acts at TLR4 to attenuate hyperalgesia. We found that the attenuation of prostaglandin E2 (PGE2)-induced hyperalgesia by HMWH was attenuated by a TLR4 antagonist (NBP2-26245), but only in male and ovariectomized female rats. In this study we sought to evaluated the role of the TLR4 signaling pathway in anti-hyperalgesia induced by HMWH in male rats. Decreasing expression of TLR4 in nociceptors, by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to TLR4 mRNA, also attenuated HMWH-induced anti-hyperalgesia, in male and ovariectomized female rats. Estrogen replacement in ovariectomized females reconstituted the gonad-intact phenotype. The administration of an inhibitor of myeloid differentiation factor 88 (MyD88), a TLR4 second messenger, attenuated HMWH-induced anti-hyperalgesia, while an inhibitor of the MyD88-independent TLR4 signaling pathway did not. Since it has previously been shown that HMWH-induced anti-hyperalgesia is also mediated, in part by CD44 we evaluated the effect of the combination of ODN antisense to TLR4 and CD44 mRNA. This treatment completely reversed HMWH-induced anti-hyperalgesia in male rats. Our results demonstrate a sex hormone-dependent, sexually dimorphic involvement of TLR4 in HMWH-induced anti-hyperalgesia, that is MyD88 dependent.


      The role of TLR4 in anti-hyperalgesia induced by HMWH is a sexually dimorphic, TLR4 dependent inhibition of inflammatory hyperalgesia that provides a novel molecular target for the treatment of inflammatory pain.

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        • Akira S
        • Takeda K.
        Toll-like receptor signalling.
        Nat Rev Immunol. 2004; 4: 499-511
        • Alessandri-Haber N
        • Yeh JJ
        • Boyd AE
        • Parada CA
        • Chen X
        • Reichling DB
        • Levine JD.
        Hypotonicity induces TRPV4-mediated nociception in rat.
        Neuron. 2003; 39: 497-511
        • Altman RD
        • Moskowitz R.
        Intraarticular sodium hyaluronate (Hyalgan) in the treatment of patients with osteoarthritis of the knee: a randomized clinical trial. Hyalgan Study Group.
        J Rheumatol. 1998; 25: 2203-2212
        • Alvarez P
        • Green PG
        • Levine JD.
        Role for monocyte chemoattractant protein-1 in the induction of chronic muscle pain in the rat.
        Pain. 2014; 155: 1161-1167
        • Araldi D
        • Bogen O
        • Green PG
        • Levine JD.
        Role of nociceptor Toll-like Receptor 4 (TLR4) in opioid-induced hyperalgesia and hyperalgesic priming.
        J Neurosci. 2019; 39: 6414-6424
        • Araldi D
        • Ferrari LF
        • Levine JD.
        Hyperalgesic priming (type II) induced by repeated opioid exposure: maintenance mechanisms.
        Pain. 2017; 158: 1204-1216
        • Barajon I
        • Serrao G
        • Arnaboldi F
        • Opizzi E
        • Ripamonti G
        • Balsari A
        • Rumio C.
        Toll-like receptors 3, 4, and 7 are expressed in the enteric nervous system and dorsal root ganglia.
        J Histochem Cytochem. 2009; 57: 1013-1023
        • Bogen O
        • Alessandri-Haber N
        • Chu C
        • Gear RW
        • Levine JD.
        Generation of a pain memory in the primary afferent nociceptor triggered by PKCepsilon activation of CPEB.
        J Neurosci. 2012; 32: 2018-2026
        • Bonet IJM
        • Araldi D
        • Khomula EV
        • Bogen O
        • Green PG
        • Levine JD.
        Mechanisms mediating high-molecular-weight hyaluronan-induced antihyperalgesia.
        J Neurosci. 2020; 40: 6477-6488
        • Bonet IJM
        • Green PG
        • Levine JD.
        Sexual dimorphism in the nociceptive effects of hyaluronan.
        Pain. 2020;
        • Borle AB
        • Snowdowne KW.
        Measurement of intracellular free calcium in monkey kidney cells with aequorin.
        Science. 1982; 217: 252-254
        • Bourguignon LY.
        Matrix hyaluronan-activated CD44 signaling promotes keratinocyte activities and improves abnormal epidermal functions.
        Am J Pathol. 2014; 184: 1912-1919
        • Bourguignon LY
        • Shiina M
        • Li JJ.
        Hyaluronan-CD44 interaction promotes oncogenic signaling, microRNA functions, chemoresistance, and radiation resistance in cancer stem cells leading to tumor progression.
        Adv Cancer Res. 2014; 123: 255-275
        • Burch RM
        • Axelrod J.
        Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2.
        Proc Natl Acad Sci USA. 1987; 84: 6374-6378
        • Caires R
        • Luis E
        • Taberner FJ
        • Fernandez-Ballester G
        • Ferrer-Montiel A
        • Balazs EA
        • Gomis A
        • Belmonte C
        • de la Pena E.
        Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain.
        Nat Commun. 2015; 6: 8095
        • Cohen MM
        • Altman RD
        • Hollstrom R
        • Hollstrom C
        • Sun C
        • Gipson B.
        Safety and efficacy of intra-articular sodium hyaluronate (Hyalgan) in a randomized, double-blind study for osteoarthritis of the ankle.
        Foot Ankle Int. 2008; 29: 657-663
        • Cuff CA
        • Kothapalli D
        • Azonobi I
        • Chun S
        • Zhang Y
        • Belkin R
        • Yeh C
        • Secreto A
        • Assoian RK
        • Rader DJ
        • Pure E.
        The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.
        J Clin Invest. 2001; 108: 1031-1040
        • de la Pena E
        • Gomis A
        • Ferrer-Montiel A
        • Belmonte C.
        TRPV1 channel modulation by hyaluronan reduces pain.
        Channels (Austin). 2016; 10: 81-82
        • Dougados M
        • Nguyen M
        • Listrat V
        • Amor B.
        High molecular weight sodium hyaluronate (hyalectin) in osteoarthritis of the knee: a 1 year placebo-controlled trial.
        Osteoarthritis Cartilage. 1993; 1: 97-103
        • Doyle HH
        • Eidson LN
        • Sinkiewicz DM
        • Murphy AZ.
        Sex differences in microglia activity within the periaqueductal gray of the rat: A potential mechanism driving the dimorphic effects of morphine.
        J Neurosci. 2017; 37: 3202-3214
        • Ferrari LF
        • Araldi D
        • Bogen O
        • Levine JD.
        Extracellular matrix hyaluronan signals via its CD44 receptor in the increased responsiveness to mechanical stimulation.
        Neuroscience. 2016; 324: 390-398
        • Ferrari LF
        • Khomula EV
        • Araldi D
        • Levine JD.
        Marked sexual dimorphism in the role of the ryanodine receptor in a model of pain chronification in the rat.
        Sci Rep. 2016; 6: 31221
        • Ferrari LF
        • Khomula EV
        • Araldi D
        • Levine JD.
        CD44 signaling mediates high molecular weight hyaluronan-induced antihyperalgesia.
        J Neurosci. 2018; 38: 308-321
        • Fields CT
        • Chassaing B
        • Castillo-Ruiz A
        • Osan R
        • Gewirtz AT
        • de Vries GJ.
        Effects of gut-derived endotoxin on anxiety-like and repetitive behaviors in male and female mice.
        Biol Sex Differ. 2018; 9: 7
        • Furuta J
        • Ariyoshi W
        • Okinaga T
        • Takeuchi J
        • Mitsugi S
        • Tominaga K
        • Nishihara T.
        High molecular weight hyaluronic acid regulates MMP13 expression in chondrocytes via DUSP10/MKP5.
        J Orthop Res. 2017; 35: 331-339
        • Gomis A
        • Miralles A
        • Schmidt RF
        • Belmonte C.
        Nociceptive nerve activity in an experimental model of knee joint osteoarthritis of the guinea pig: effect of intra-articular hyaluronan application.
        Pain. 2007; 130: 126-136
        • Green PG
        • Dahlqvist SR
        • Isenberg WM
        • Miao FJ
        • Levine JD.
        Role of adrenal medulla in development of sexual dimorphism in inflammation.
        Eur J Neurosci. 2001; 14: 1436-1444
        • Green PG
        • Dahlqvist SR
        • Isenberg WM
        • Strausbaugh HJ
        • Miao FJ
        • Levine JD.
        Sex steroid regulation of the inflammatory response: sympathoadrenal dependence in the female rat.
        J Neurosci. 1999; 19: 4082-4089
        • Huang TL
        • Chang CC
        • Lee CH
        • Chen SC
        • Lai CH
        • Tsai CL.
        Intra-articular injections of sodium hyaluronate (Hyalgan(R)) in osteoarthritis of the knee. a randomized, controlled, double-blind, multicenter trial in the Asian population.
        BMC Musculoskelet Disord. 2011; 12: 221
        • Kataoka Y
        • Ariyoshi W
        • Okinaga T
        • Kaneuji T
        • Mitsugi S
        • Takahashi T
        • Nishihara T.
        Mechanisms involved in suppression of ADAMTS4 expression in synoviocytes by high molecular weight hyaluronic acid.
        Biochem Biophys Res Commun. 2013; 432: 580-585
        • Kawai T
        • Sato S
        • Ishii KJ
        • Coban C
        • Hemmi H
        • Yamamoto M
        • Terai K
        • Matsuda M
        • Inoue J
        • Uematsu S
        • Takeuchi O
        • Akira S.
        Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6.
        Nat Immunol. 2004; 5: 1061-1068
        • Lennon FE
        • Singleton PA.
        Role of hyaluronan and hyaluronan-binding proteins in lung pathobiology.
        Am J Physiol Lung Cell Mol Physiol. 2011; 301: L137-L147
        • Mestre C
        • Pelissier T
        • Fialip J
        • Wilcox G
        • Eschalier A.
        A method to perform direct transcutaneous intrathecal injection in rats.
        J Pharmacol Toxicol Methods. 1994; 32: 197-200
        • Mizrahy S
        • Raz SR
        • Hasgaard M
        • Liu H
        • Soffer-Tsur N
        • Cohen K
        • Dvash R
        • Landsman-Milo D
        • Bremer MG
        • Moghimi SM
        • Peer D.
        Hyaluronan-coated nanoparticles: the influence of the molecular weight on CD44-hyaluronan interactions and on the immune response.
        J Control Release. 2011; 156: 231-238
        • Oliveira-Fusaro MCG
        • Zanoni CIS
        • Dos Santos GG
        • Manzo LP
        • Araldi D
        • Bonet IJM
        • Tambeli CH
        • Dias EV
        • Parada CA
        Antihyperalgesic effect of CB1 receptor activation involves the modulation of P2×3 receptor in the primary afferent neuron.
        Eur J Pharmacol. 2017; 798: 113-121
        • Parada CA
        • Yeh JJ
        • Reichling DB
        • Levine JD.
        Transient attenuation of protein kinase Cepsilon can terminate a chronic hyperalgesic state in the rat.
        Neuroscience. 2003; 120: 219-226
        • Quanhong Z
        • Ying X
        • Moxi C
        • Tao X
        • Jing W
        • Xin Z
        • Li W
        • Derong C
        • Xiaoli Z
        • Wei J.
        Intrathecal PLC(beta3) oligodeoxynucleotides antisense potentiates acute morphine efficacy and attenuates chronic morphine tolerance.
        Brain Res. 2012; 1472: 38-44
        • Randall LO
        • Selitto JJ.
        A method for measurement of analgesic activity on inflamed tissue.
        Arch Int Pharmacodyn Ther. 1957; 111: 409-419
        • Song MJ
        • Wang YQ
        • Wu GC.
        Additive anti-hyperalgesia of electroacupuncture and intrathecal antisense oligodeoxynucleotide to interleukin-1 receptor type I on carrageenan-induced inflammatory pain in rats.
        Brain Res Bull. 2009; 78: 335-341
        • Su L
        • Wang C
        • Yu YH
        • Ren YY
        • Xie KL
        • Wang GL.
        Role of TRPM8 in dorsal root ganglion in nerve injury-induced chronic pain.
        BMC Neurosci. 2011; 12: 120
        • Sun JL
        • Xiao C
        • Lu B
        • Zhang J
        • Yuan XZ
        • Chen W
        • Yu LN
        • Zhang FJ
        • Chen G
        • Yan M.
        CX3CL1/CX3CR1 regulates nerve injury-induced pain hypersensitivity through the ERK5 signaling pathway.
        J Neurosci Res. 2013; 91: 545-553
        • Taiwo YO
        • Coderre TJ
        • Levine JD.
        The contribution of training to sensitivity in the nociceptive paw-withdrawal test.
        Brain Res. 1989; 487: 148-151
        • Taiwo YO
        • Levine JD.
        Prostaglandin effects after elimination of indirect hyperalgesic mechanisms in the skin of the rat.
        Brain Res. 1989; 492: 397-399
        • Tavianatou AG
        • Caon I
        • Franchi M
        • Piperigkou Z
        • Galesso D
        • Karamanos NK.
        Hyaluronan: molecular size-dependent signaling and biological functions in inflammation and cancer.
        FEBS J. 2019; 286: 2883-2908
        • Taylor KR
        • Yamasaki K
        • Radek KA
        • Di Nardo A
        • Goodarzi H
        • Golenbock D
        • Beutler B
        • Gallo RL.
        Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.
        J Biol Chem. 2007; 282: 18265-18275
        • Toole BP.
        Hyaluronan-CD44 interactions in cancer: Paradoxes and possibilities.
        Clin Cancer Res. 2009; 15: 7462-7468
        • Triantaffilidou K
        • Venetis G
        • Bika O.
        Efficacy of hyaluronic acid injections in patients with osteoarthritis of the temporomandibular joint. A comparative study.
        J Craniofac Surg. 2013; 24: 2006-2009
        • Vigetti D
        • Karousou E
        • Viola M
        • Deleonibus S
        • De Luca G
        • Passi A.
        Hyaluronan: biosynthesis and signaling.
        Biochim Biophys Acta. 2014; 1840: 2452-2459
        • Vigetti D
        • Viola M
        • Karousou E
        • De Luca G
        • Passi A.
        Metabolic control of hyaluronan synthases.
        Matrix Biol. 2014; 35: 8-13
        • Wang Y
        • Abarbanell AM
        • Herrmann JL
        • Weil BR
        • Manukyan MC
        • Poynter JA
        • Meldrum DR.
        TLR4 inhibits mesenchymal stem cell (MSC) STAT3 activation and thereby exerts deleterious effects on MSC-mediated cardioprotection.
        PLoS One. 2010; 5: e14206
        • Wu PT
        • Kuo LC
        • Su FC
        • Chen SY
        • Hsu TI
        • Li CY
        • Tsai KJ
        • Jou IM.
        High-molecular-weight hyaluronic acid attenuated matrix metalloproteinase-1 and -3 expression via CD44 in tendinopathy.
        Sci Rep. 2017; 7: 40840
        • Yamamoto M
        • Sato S
        • Hemmi H
        • Uematsu S
        • Hoshino K
        • Kaisho T
        • Takeuchi O
        • Takeda K
        • Akira S.
        TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway.
        Nat Immunol. 2003; 4: 1144-1150
        • Zhang Z
        • Qin P
        • Deng Y
        • Ma Z
        • Guo H
        • Guo H
        • Hou Y
        • Wang S
        • Zou W
        • Sun Y
        • Ma Y
        • Hou W.
        The novel estrogenic receptor GPR30 alleviates ischemic injury by inhibiting TLR4-mediated microglial inflammation.
        J Neuroinflammation. 2018; 15: 206