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Anti-oxidant inhibition of hyaluronan fragment-induced inflammatory ...

Anti-oxidant inhibition of hyaluronan fragment-induced inflammatory ...

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Journal of Inflammation 2008, 5:20http://www.journal-inflammation.com/content/5/1/20cytokines [39]. The exact molecular source of ROS uponLPS challenge was recently discovered by Park et al whoshowed that LPS-induced ROS generation is mediated bydirect interaction of TLR-4 with NADPH oxidase 4 (Nox4)[40]. LPS-signaling via TLR 4 shares many similarities toHA fragment signaling via TLR-2 [19]. Thus it was importantto exclude LPS signaling effects via LPS contaminationof LMW HA fragment preparations. To this end, allexperiments were performed in the presence of polymyxinB and the primary macrophages were derived from LPShyporesponsive CeH/HeJ mice. It has been shown in thepast that under these conditions LPS fails to induceinflammatory gene up-regulation [21,22,25].Our data add to the growing body of literature highlightingthe role of ROS as important signaling molecules thatare able to modulate various gene transcription via activationof redox-sensitive protein kinases and transcriptionfactors [41]. ROS have been identified as second messengersin cells, and play a role in receptor signaling and posttranslationalmodification of signaling molecule activity[41,42]. Many kinases involved in direct or indirect activationof NF-κB are affected by oxidants and therefore, havethe potential to alter NF-κB activity [41,42]. The transcriptionfactor NF-κB plays a major role in coordinatinginnate and adaptive immunity, cellular proliferation,apoptosis and development and is a key transcription factorfor many HA fragment induced inflammatory geneexpression. In macrophages and alveolar epithelial cells,HA fragments can induce chemokines via activation of theNF-κB pathway [33,43]. We have shown that the anti-oxidantsNAC and DMSO inhibit the HA fragment inducedinflammatory gene expression by interfering with HAfragment-induced activation of NF-κB. This finding is inline with other investigations showing the importance ofROS in the activation of NF-κB [44]. Schreck et al. werethe first to demonstrate that direct addition of ROS, specificallyH 2 O 2 to the culture medium of a subclone of Jurkatcells could activate NF-κB. Now several lines ofevidence support a model suggesting that ROS mainlyactivate NF-κB via IKK activation and IκBa degradation[2,44].Pulmonary fibrosis is associated with chronic inflammation,increased ROS, and accumulation and turnover ofextracellular matrix. During lung inflammation in bothhuman diseases and bleomycin injured animal models,activated phagocytes release large amounts of reactiveoxygen species (ROS) that have been demonstrated to beinvolved in tissue injury and to impede tissue repair, thusleading to pulmonary fibrosis [3,27]. Anti-oxidant treatmentprotects against bleomycin-induced lung damage inrodents [27-30]. Additionally, mice deficient in extracellularSuperoxide Dismutase (SOD) develop an exaggeratedfibrosis in response to bleomycin [45]. Inghilleri et al.compared the in situ oxidative burden and anti-oxidantenzyme activity in bleomycin-injured rat lungs and normalcontrols and found after treatment with bleomycin,ROS production was enhanced in both phagocytes and intype II alveolar epithelial cells [46]. Interestingly Manouryet al. have demonstrated that mice deficient in thep47phox subunit of the NADPH oxidase complex areunable to produce ROS via the NADPH oxidase pathwayand do not develop pulmonary fibrosis after intranasaladministration of bleomycin [47]. In addition, recentclinical trials show favorable effects on lung functiondecline in patients with idiopathic pulmonary fibrosistreated with high doses of N-acetylcysteine [6].ConclusionIn conclusion we have shown that the anti-oxidant NACinhibits HA fragment-induced cytokine expression via NFκBinhibition in macrophages and epithelial cells. Takentogether our data provides further insight into the basicmechanisms of beneficial effects anti-oxidants have demonstratedin animal models of pulmonary fibrosis andpossibly in patients with Idiopathic Pulmonary Fibrosis.The findings in this investigation point towards a centralrole of ROS in the pathophysiologic "vicious cycle" ofinflammation: tissue injury generates ROS, which generatefragments of the extracellular matrix HA, which in turnsynergize with the ROS to activate the innate immune systemvia TLR-2. Activation of the immune system leads tofurther production of ROS by activated macrophages, activationof NF-κB and induction inflammatory cytokinesand chemokines that promote further inflammation andcontinued fragmentation of the extracellular matrix HA,generation of ROS, more injury, more inflammation andultimately fibrosis (Figure 6). Thus, multi-targeted therapeuticinterventions addressing this self perpetuating spiralof tissue injury, ROS production, matrix degradationthat leads to further matrix-induced inflammation mayhold a promise of improving clinical outcomes in patientswith inflammatory diseases in the future.Competing interestsThe authors declare that they have competing interests.Authors' contributionsME co-conceived the study, carried out the NAC andDMSO cellular studies, participated in the ELISAs andtransfections and drafted the manuscript. KAS participatedin the ELISAs and transfections. KEB participated inthe ELISAs and transfections. SLC preformed the FACSexperiments. YC participated in the ELISAs, transfectionsand ROS cellular experiments. JDP participated in thedesign of the study and helped to draft the manuscript.MRH co-conceived of the study, participated in its designand coordination, performed the statistical analysis andPage 8 of 10(page number not for citation purposes)

Journal of Inflammation 2008, 5:20http://www.journal-inflammation.com/content/5/1/20The central role of ROS in a pathophysiologic "vicious cycle"Figure 6The central role of ROS in a pathophysiologic "vicious cycle": A) Tissue injury generates ROS, which mediate fragmentationof the extracellular matrix HA. B) Fragmented HA and ROS synergize to activate the innate immune system via TLR-2,followed by further production of ROS, activation of NF-κB and expression of inflammatory cytokines and chemokines, promotingfurther inflammation. This cycle perpetuates continued fragmentation of the extracellular matrix HA and generation ofROS, thus leading to further injury, inflammation and ultimately fibrosis. Anti-oxidants have the potential to ameliorate thisvicious cycle.helped draft the manuscript. All authors read andapproved the final manuscript.AcknowledgementsThe authors would like to thank Dr Roy C. Ziegelstein for helpful suggestions.NIH R01 HL073855 (MRH) and FAMRI (MRH) funded this work.References1. Gutteridge JM, M J: Redox imbalance in the critically ill. Br MedBull 1999, 55:49-75.2. Gloire GL-PS, Piette J: NF-kappaB activation by reactive oxygenspecies: fifteen years later. Biochem Pharmacol 2006,72:1493-1505.3. Cross CE, vd VA, O'Neill CA, Eiserich JP: Reactive oxygen speciesand the lung. Lancet 1994, 344:930-933.4. Day BJ: Antioxidants as potential therapeutics for lung fibrosis.Antioxid Redox Signal 2008, 10:355-370.5. Gao F, Kinnula VL, Myllarniemi M, Oury TD: Extracellular superoxidedismutase in pulmonary fibrosis. Antioxid Redox Signal2008, 10:343-354.6. Demedts M, Behr J, Buhl R, Costabel U, Dekhuijzen R, Jansen HM,MacNee W, Thomeer M, Wallaert B, Laurent F, et al.: High-doseacetylcysteine in idiopathic pulmonary fibrosis. N Engl J Med2005, 353:2229-2242.7. Liu RM: Oxidative stress, plasminogen activator inhibitor 1,and lung fibrosis. Antioxid Redox Signal 2008, 10:303-319.8. Saari H: Oxygen derived free radicals and synovial fluidhyaluronate. Ann Rheum Dis 1991, 50:389-396.9. Saari H, Konttinen YT, Friman C, Sorsa T: Differential effects ofreactive oxygen species on native synovial fluid and purifiedhuman umbilical cord hyaluronate. Inflammation 1993,17:403-415.10. Laurent T, Fraser J: Hyaluronan. FASEB J 1992, 6:2397-2404.11. Laurent TC: Functions of Hyaluronan. A Rheum Dis 1995, 54:429.12. Hance AJ, Crystal RG: The connective tissue of lung. Am Rev RespDis 1975, 112:657-668.Page 9 of 10(page number not for citation purposes)

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