Leading EdgeReviewAnti-inammatory Agents:Present and FutureCharles A. Dinarello1,2,*1Department of Medicine, University of Colorado, Aurora, CO 80045, USA2Department of Medicine, Radboud University Nijmegen Medical Center, 6500 HC Nijmegen, the Netherlands*Correspondence: cdinarello@mac.comDOI 10.1016/j.cell.2010.02.043Inammation involving the innate and adaptive immune systems is a normal response to infection.However, when allowed to continue unchecked, inammation may result in autoimmune or autoin-ammatory disorders, neurodegenerative disease, or cancer. A variety of safe and effective anti-inammatoryagentsareavailable, includingaspirinandothernonsteroidal anti-inammatories,with many more drugs under development. In particular, the new era of anti-inammatory agentsincludes biologicals such as anticytokine therapies and small molecules that block the activityof kinases. Other anti-inammatories currently in use or under development include statins, histonedeacetylase inhibitors, PPAR agonists, and small RNAs. This Review discusses the current statusof anti-inammatory drug research and the development of new anti-inammatory therapeutics.IntroductionReducing pain, inammation, and fever with salicylate-contain-ing plant extracts can be traced throughout written humanhistory.Onehundredandftyyearsago,FelixHoffmanacety-lated salicylic acid and created aspirin. Aspirin inhibits thecyclooxygenase (COX) enzymes COX-1 and COX-2, whichsynthesizeinammatorymediatorscalledprostaglandinsandthromboxanes. The ability to block production of prostaglandinsand thromboxanesaccounts for aspirin being the worlds mostused therapeutic agent. Second to aspirin are nonsteroidalanti-inammatory drugs (NSAIDS), which target COX-2 andhencethesynthesisofprostaglandins,particularlyPGE2.Syn-thetic forms of natural cortisol (termed glucocorticoids) arealso widely usedtotreat many inammatory diseases, anddespitetheir sideeffects, glucocorticoidsremainamainstayfor reducing inammation. Yet, it is still the challenge of the phar-maceutical chemist todevelopmoreeffectiveandlesstoxicagentstotreatthesignsandsymptomsofacuteinammationas well as the long-term consequences of chronic inammatorydiseases.Inammation is a dynamic process with proinammatory cyto-kines such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b,andvascular endothelial growthfactor (VEGF) playingcentralroles.Anumberofbiologicalshavebeendevelopedtotreatinammation (Table 1), including agents that reduce the activityofspeciccytokinesortheirreceptors(anticytokinetherapies),blocklymphocytetrafckingintotissues, prevent thebindingof monocyte-lymphocytecostimulatorymolecules, or depleteBlymphocytes(Figure1). Current anticytokinetherapieshavefoundaplaceinthetreatment of autoimmunediseases, suchasrheumatoidarthritis,inammatorybowel disease,psoriasis,multiplesclerosis, andothers. Withoutquestion, neutralizationof specic proinammatory cytokines has canonized theircausativeroleininammationandhaschangedthelivesofmillions of patients with these diseases. One drawback of anticy-tokinetherapies is decreasedhost immunedefenseagainstinfection and possibly cancer. Nevertheless, the benets of anti-cytokinetherapies outweightherisks, andtherisks canbereduced. Comparedtotheconsequencesof long-termgluco-corticoidtreatment tosteminammation, anticytokinethera-pies are a major improvement. Indeed, organ toxicities arerarely, if ever, observed with anticytokine therapies as they oper-atealmost exclusivelyinextracellular rather thanintracellularcompartments.Kinases that act downstreamof cytokine receptors havebecomenewtargetstotameinammation, andorallyactivesmall-moleculeinhibitorsof intracellular signalingkinaseswilllikely be the newfrontier of anti-inammatory drug development.However, becausemanyintracellular signalingmoleculesareinvolved in normal cellular functions, the effective concentrationthat does not elicit organ toxicity will need to be carefully deter-mined. Statins, asafeclassof drugsusedforloweringserumcholesterol, also have anti-inammatory properties. Orally activeinhibitors of histone deacetylases, which are also safe and usedclinically, areeffectivedrugswithanti-inammatorypropertiesthat alsoblockcell proliferation. Naturallyoccurringresolvinsare also being developedas anti-inammatory agents. ThisReviewdiscussescurrent anti-inammatorydrugsaswell asthe developmentofnew orally active,safe,and effectivedrugsfor treating acute or chronic inammation.Inhibiting Prostaglandins: Targeting COX-2TheinammatorymoleculePGE2lowerspainthresholds, andtheprimarygoal of oral inhibitorsof PGE2istoreducepain.There are two pathways for synthesizing the inammatory mole-culePGE2:theconstitutiveCOX-1pathwayandtheinducibleCOX-2pathway. WhereasCOX-1accountsfor lowlevelsofPGE2 and regulates homeostatic mechanisms in health, COX-2induces at least two orders of magnitude more PGE2 comparedto COX-1 and is primarily associated with inammatory disease.Cell 140, 935950, March 19, 2010 2010 Elsevier Inc. 935Synthesis of COX-2 is absent or low in healthy individuals but isupregulatedby proinammatory cytokines suchas IL-1andTNF-a in response to infection or in inammatory disease.SpecicinhibitorsofCOX-2haveprovidedamajoradvanceinthetreatmentofpain,particularlyinpatientswithosteoarthritisor rheumatoidarthritis. For the most part, COX-2 inhibitorshave signicantly reducedgastrointestinal sideeffects com-paredtoCOX-1inhibitors. However, thechronicuseof someCOX-2-specic inhibitors has been associated with an increasein cardiovascular as well as cerebrovascular events particularlyinpatientswithanelevatedriskofthrombosis.ThisincreasedriskmaybeduetotheCOX-2-mediatedreductioninsynthe-sis of prostacyclin, which is a natural inhibitor of platelet activa-tion.Inadditiontotheirwidespreadbenetinarthritis,COX-2-specic inhibitors are used to reduce the development of coloncancer in high-risk patients as adenocarcinoma cells in the colonoverexpress COX-2. There is still a need to develop safer, moreeffective COX-2 inhibitors.ResolvinsThere are several steps in the initial inammatory cascadetriggeredbycytokines, includingrecruitment of myeloidcells(monocytes andneutrophils) intoaffectedtissues (Figure1).Inammatory products of arachidonic acid oxidation (omega-6)including inammatory prostaglandins (PGE2) and lipoxins(LTB4) arereleasedfrominltratingmyeloidcells. Incontrast,productsof eicosapentanoicacid(omega-3) oxidation, PGE3,and LTB5, have anti-inammatory activities. Products ofomega-3fattyacidoxidationincluderesolvinsof theEseries(RvE1andRvE2) (SerhanandChiang, 2008), whicharefoundnaturallyinnearlyall inammatorysitesinmammals. TheDseries of resolvins are derived fromdocosahexaenoic acid.Specicreceptorsforresolvinshavebeenidentiedandwhenactivated are functional in reducing inammation (Krishnamoor-thy et al., 2010). In general, resolvins are part of the anti-inam-matory portfolio that coexists with inammation. Synthetic formsof RvE1 are currently in clinical trials for treating ocular diseasesandother local inammatoryconditions. Inanimal modelsofsterile inammation, RvE1 suppresses the number of inltratingneutrophils and macrophages as well as decreasing expressionof the genes encoding TNF-a, IL-1b, and VEGF (Jin et al., 2009).The anti-inammatory properties of omega-3 fatty acids includesuppression of IL-1b and TNF-a production (Endres et al., 1989),and the mechanismof action of omega-3 fatty acids may includeboosting production of resolvins of the D and E series.Glucocorticoids: Suppressing Cytokine-DrivenInammationGlucocorticoids are used widely on a chronic basis to treat mostautoimmunediseases. Short-termglucocorticoidtreatment isusedingout, andintra-articular injectionsof glucocorticoidsare commonly usedtotreat painful osteoarthritic joints andtendonitis. Althoughthereareseveral mechanismsbywhichglucocorticoidsreduceinammation, amajor onemaybetoreduceexpressionofcytokine-inducedgenes.Glucocorticoidsenter all cells and bind to the cytoplasmic steroid receptor, andthen this complex translocates to the nucleus where it is recog-nized by specic DNA sequences. The major effect of binding toDNA is the suppression of transcription by opposing the activa-tionof thetranscriptionfactors AP-1andNF-kB. AP-1andNF-kBinduce expression of genes encoding nearly all proinam-matory cytokines. Glucocorticoidsalso suppress expression ofinammatory genes encodingTcell growthfactors suchasIL-2, IL-4, IL-15, and IL-17 as well as interferon-g (IFN-g). In addi-tion, glucocorticoids reduce expression of genes encodingCOX-2, induciblenitricoxidesynthase, andintracellularadhe-sionmolecule-1(ICAM-1), whicharenormallyinducedbythecytokines IL-1b and TNF-a. Glucocorticoids increase expressionof genesencodinganti-inammatorymolecules, suchasthecytokine IL-10 and the IL-1 type 2 decoy receptor.Biologicals as Anti-inammatory AgentsAnticytokine TherapiesAlthoughcytokinesarestudiedinnearlyeverybiological disci-pline, cytokine-mediatedeffectsoftendominatetheeldsofinammation, immunology, atherosclerosis, anddegenerativeTable 1. Biologicals in the Treatment of Chronic Autoimmune and Inammatory DiseasesDrugs Function Diseases TreatedAnti-CD3 (eplizumab); anti-IL-2 receptor MoAb (daclizumab) Targeting T cells Transplant rejection;Type 1 diabetesAnti-CD20 (rituximab, crelizumab, ofatumumab); anti-CD22 (epratuzumab);anti-Blys MoAb IgG1 (belimumab)Targeting B cells Type 1 diabetes; rheumatoidarthritis; multiple sclerosisAnti-TNF-a MoA (iniximab, adulimumab, golimumab); anti-TNF-a pegylatedFab (certolizumab); soluble TNF p75 receptor Fc fusion (etanercept)Reducing TNF-aactivitiesRheumatoid arthritis;Crohns disease; psoriasisAnti-IL-6 MoAb (MEDI5117); Anti-IL-6 receptor (toculizumab) Reducing IL-6activitiesRheumatoid arthritis;juvenile arthritisAnti-IL-12/23 (ustekinumab); Anti-IL-17 MoAb (AIN457/LY24398) Neutralization of IL-12,IL-23, and IL-17Rheumatoid arthritis;Crohns Disease; psoriasisIL-1 receptor antagonist (anakinra); soluble IL-1 receptor (rilonacept); anti-IL-1b(IgG1) (canakinumab); anti-IL-1b (IgG2) (Xoma 052); anti-IL-1R MoAb IgG1 (AMG 108)Reducing IL-1bactivitiesAutoinammatorydiseases (see Table S1)Anti-a4 integrins MoAb (natalizumab); anti-LFA-1 MoAb (efalizumab) Blocking cell adhesionand migrationMultiple sclerosis; CrohnsDisease; psoriasisCTLA-4 Ig fusion protein (abatacept) Blocking T cellcoreceptorsType 1 diabetes;rheumatoid arthritis936 Cell 140, 935950, March 19, 2010 2010 Elsevier Inc.processes of aging. In addition, cytokine-driven chronic inam-mation has been implicatedin cancer formation as well asmetastasis(seeReviewbyS.I.Grivennikovetal.onpage883of thisissue). Cytokinesaresecretedbyonecell andact onanothercell inordertobringaboutachangeinthefunctionofthe target cell. In a way, one can consider cytokines as the hor-monesofinammatoryresponses,butwhereasahormoneisthe primary product of a specializedcell, cytokines canbeproducedbymanydifferent cell typesincludingthoseof theimmunesystemandepithelia.Onamolarbasis,cytokinesarefar more potent than hormones. For example, the concentrationofIL-1thatinducesCOX-2is10pMandtheconcentrationofIL-12 that induces IFN-g is 20 pM. In terms of anticytokine ther-apies, the amount of a neutralizing antibody or soluble receptorthat blocks the activity of a cytokine can be relatively lowcompared to the amount of antibody needed to kill a microbe.Blockingtheactivityof theproinammatorycytokinesIL-1,TNF, IL-6, IL-12, IL-17, IL-18, or IL-23reducesinammationand suppresses specic pathways that activate T cells. BlockingIL-32 and IL-33 may also be useful for treating inammation aswell as allergy. Chemokines are also cytokines, and small-mole-culechemokinereceptorantagonistshavebeenusedtotreatCrohnsdisease, anautoimmuneinammatorybowel disease(Proudfoot et al., 2010). Chemokines drive the migration ofimmune cells butthey also affectangiogenesisandthe activityof myeloid cells.Anticytokine Therapies and Host DefenseThe conundrumin considering anticytokine therapies for treatingchronic diseases is that cytokines evolved many millions of yearsago and provided a survival benet for the host through what isnow termed the innate immune response. The innate immuneresponseislessspecicthantheadaptiveimmuneresponseand is the rst line of defense against infection or injury. It is char-acterizedbyaninammatoryresponseinvolvinginltrationofneutrophilsinresponsetocytokinesresultinginphagocytosisand intracellular killing of the pathogens and the control of infec-tion. Inmostcases, evenwithoutantibiotics, theinammationsubsidesoncetheinfectioniseliminated, andthereislittleornodamage tothe host. In fact, cytokines producedduringinammation also assist in the repair process after injury.Figure 1. Inammation and Points of Inhibi-tion by Anti-inammatory AgentsShownistheinammatorypathwayassociatedwith ischemia-induced tissue damage and theanti-inammatory drugs that can be used to blockinammation at various points in the pathway.During ischemia, mesenchymal-derived cellsundergohypoxicdamage,losemembraneinteg-rity, and release their cytoplasmic contents.This is followed by the release of biologically activeproinammatory cytokines such as IL-1 andVEGF.Thesecytokinesbindtoandactivatetheircognatereceptorsonnearbymacrophages. Theactivation of the cytokine receptors triggerstissue-resident macrophages to express an exten-sive portfolio of proinammatory genes. Somegene products such as the IL-1b and IL-18precursor proteins require processing by cas-pase-1andsecretionasactivecytokines. Thereis alsochemokine production by activatedtis-sue macrophages. A concentration gradientof secreted cytokines and chemokines formsbetween the macrophage and the endothelium ofthemicrovasculature. Activationof cytokineandchemokinereceptorsontheendotheliumtakesplace resulting in the opening of endothelialjunctions. Thisleadstoacapillaryleakandthepassage of plasma proteins into the ischemicarea. During the ischemic event, there is activationof complement withinductionof cytokinesandchemokines,aswell asadirecteffectofC5aonchemoattraction. As part of the cytokine activationof the endothelium, there is expression of theadhesionmoleculeVCAM-1,whichfacilitatestherolling of innate immune cells such as monocytesand neutrophils (myeloid cells) and their adherence to the endothelium. These inammatory cells then leave the circulation and enter the tissue space by diape-desis.Withincreasingnumbersofinltratingmyeloidcells,thehealthytissuesurroundingtheischemicarea(calledthepenumbra)issubjectedtodamagefromthe cellular inltrate due to the release of cytokines and chemokines into the penumbral tissue. Release of damaging proteases fromneutrophils contributesto the loss of tissue in the penumbra. Adherence of platelets to the endothelium causes further endothelial activation by increasing expression of receptors forplatelets. During the development of an inammatory inltrate in the ischemic tissue, a cascade of clotting factors is initiated by cytokine-induced endothelialtissue factor. In addition, the cytokine-activated endotheliumreleases inhibitors of brinolysis. With increased coagulation in the microvasculature and decreasedbrinolysis, tissue perfusion slows and hypoxia and tissue damage worsen.Cell 140, 935950, March 19, 2010 2010 Elsevier Inc. 937However,the same cytokines that orchestratethe inltrationofneutrophilsacutelyinorder toght infectionareresponsiblefor tissue remodeling and organ damage when produced chron-ically. Chronic inammation destroys the joints, the ability of lungtissuetoexchangegases, thepatencyof bloodvessels, theintestinal barrier, andthemyelinsheaththat insulates nervebersinthebrainandspinal cord. Hence, eventhoughtheirprimaryfunctionistoprotectthehostwhenchallengedandtorepair tissuewheninjured, thesecytokinesaremediators ofdiseaseandthusaretargetsforanticytokinetherapy. AnotherparadoxisprovidedbyIFN-g, whichisessential for defenseagainst intracellular microorganisms suchas Mycobacteriumtuberculosis, whichcausestuberculosis. IFN-gisalsoamajorcytokineinthepathogenesisof several autoimmunediseasesincluding multiple sclerosis, psoriasis, and lupus.Blocking cytokines may reduce inammation but also rendersthehost susceptibleto infectionandmaybe even cancer. Anti-TNF-atreatment increasesopportunisticinfections(reviewedin Dinarello, 2005). On the other hand, anticytokine therapy haslittleor noorgantoxicityor gastrointestinal disturbancesandso is well tolerated.Biologicals for Treating Autoimmune DiseasesAutoimmune versus Autoinammatory DiseasesSome chronic inammatory diseases are autoimmune, whereasothers areautoinammatory. Inautoimmunediseases, theT celldominatesas the primarydysfunctionalcell orinitiator ofthediseaseprocess. Acluster of cytokines suchas TNF-a,IFN-g, IL-2, IL-12, IL-23, andIL-17participateinmaintainingautoreactiveTcells. Rheumatoidarthritis, inammatoryboweldisease, type 1 diabetes, psoriasis, lupus, and multiple sclerosisareexamplesofautoimmunediseasesinwhichtheinamma-tion is secondary to a disease process that is driven by autoreac-tiveTcells(SeeEssaybyL.A. Zenewiczet al., inthisissue).Incontrast, autoinammatory diseases are not mediatedbytheadaptiveimmunesystemanddonot involveTcellsbutrather arecausedbydysfunctional macrophages(seeEssayby D.L. Kastner et al. in this issue). The mechanismof autoinam-matorydiseaseappearstobeduetoincreasedsecretionofIL-1b,andtreatmentisuniquelybasedonreducingtheactivityof IL-1b.Most autoimmunediseasescanbetreatedwithanyoneofa number of biologicals (Table 1). The best example is rheuma-toidarthritiswhere neutralizingTNF-a, blockingIL-6receptors,depleting B lymphocytes, or preventing T cell costimulation areall effectivetherapeuticapproaches.Even blockingIL-1 recep-tors or neutralizingIL-1bis effectivefor treatingrheumatoidarthritis, but this effect is due to protection of bone and cartilageratherthanreductionofTcell activation. Thus, preventingtheactivity of only one cytokine or reducing the effect of onepathway can be effective in treating autoimmune diseases.OtherexamplesincludepsoriasisandCrohnsdisease, whichcanbe effectivelytreatedbyblocking IL-12,IL-23, or TNF-a orby using a humanized monoclonal antibody to block a4 integrins.For example, a monoclonal antibody directed against a4 integ-rins(natalizumab) preventsthemigrationof immunecellsintotissues. Onegoal intreatingautoimmunediseaseistoreducetheinltrationof autoreactiveTcellsintotheaffectedtissue.Another is the depletion of B cells using anti-CD20 monoclonalantibodies such as rituximab, ocrelizumab, or ofatumumab.Biologicals offer several options for treating autoimmunediseases. Althoughtheeldof biologicalsbeganwiththeuseofanti-TNF-amonoclonal antibodiestotreatCrohnsDisease,it has expanded considerably. Antibodies to IL-12, IL-17, IL-23,IL-1b, and the IL-6 receptor have been approved, and antibodiesto IL-23 and IL-17 are in clinical trials. Each biological can beeffective in the treatment of more than one autoimmune disease,suggestingthatthereisconsiderableoverlapincytokinefunc-tions ininammation. Thesuccess of abiological is bestobservedinpatientswithpoorlycontrolleddiseasewhoarereceivingthestandardoftherapyforthatdisease,forexamplerheumatoidarthritis. Addingabiological tostandardtherapyoften decreases disease severity. The physician has an increas-inglist of biologicalsfromwhichtochoose: anti-TNF-aanti-bodiesorsolubleTNFreceptorstoneutralizeTNF-a,anti-IL-6receptor antibodies to block IL-6, CTLA-4-Ig to block the bindingof costimulatory molecules, rituximab (an anti-CD20 monoclonalantibody) todepleteBcells, neutralizingantibodiestoblockIL-12andIL-23activity, ornatalizumabtoreducelymphocytetrafcking.Althoughthedegreeofbenetwill differdependingon the patient, most experience improved control of theirdisease.Why Just One?Despite the multiple cellular andcytokine-mediated mecha-nismsfor sustainingautoimmunediseases, blockingjust onecytokinecanbesufcient tobringthediseaseunder control.Why is this? There is evidence that cytokines exist in cascadesand that interrupting one cytokine interrupts the cascade.For example, blockingTNF-areducestheactivityof IL-6andIL-1b(Dinarelloetal., 1986; Fonget al., 1989), blockingIL-1breducesIL-6(Fitzgeraldet al., 2005; Goldbach-Manskyet al.,2006;Hoffmanetal.,2004;Pascual etal.,2005),andblockingIL-12andIL-23reducesIFN-g. BlymphocytesmakeTNF-aandIL-1b, andthismayaccount for thebenecial effectsofdepletingBcellswithmonoclonal antibodies. IL-12andIL-23sustainproductionofIL-17,whichmayexplainthesuccessofusing IL-12 and IL-23 inhibitors to treat autoimmune diseases.There is also ample evidence that cytokines act in a synergisticratherthanadditivefashion.Forexample,synergisticcytokinepairsincludeIL-1andTNF-a,TNF-aandIFN-g,andIL-1bandIL-6(reviewedinDinarello, 1996, 2009a). Thus, blockingonecytokineinterruptsthissynergyandreducesdiseaseseverity.PartofthesuccessofTNF-ainhibitorsfortreatingrheumatoidarthritis, Crohnsdisease, andpsoriasisisthelossof TNF-a-bearing T cells. TNF-a is located on the T cell surface and anti-TNF-a monoclonal antibodies of the IgG1 class crosslink mem-brane TNF-a and induce death of T cells (reviewed in Dinarello,2005).Autoimmune Disease and B Cell DepletionRituximab andother monoclonal antibodies that target anddepleteCD20-positiveBcellshaveanunexpectedbenet intreating rheumatoid arthritis, psoriasis, Crohns disease, multiplesclerosis,andtype1diabetes(Table1).AlthoughdepletionofB cells reduces immunoglobulin levels, this is unlikely to explaintheefcacyof Bcell depletionfor treatingtheseautoimmunediseases. Bcellscontributetoantigenpresentationandthus938 Cell 140, 935950, March 19, 2010 2010 Elsevier Inc.help in T cell activation. Also, increasing evidence links the path-ogenesisofmostautoimmunediseaseswithTcellsproducingIL-17. IL-17 is a proinammatory cytokine that induces chemo-kine production andpromotes inltration of neutrophils andmacrophages. There was a marked reduction in IL-17 producedbysynovialcellsfromthejointsofrheumatoidarthritispatientstreated with rituximab (van de Veerdonk et al., 2009b). In periph-eral blood mononuclear cells, the presence of rituximab reducedthe levels of IL-17 as well as the number of T cells producing thecytokine.Side Effects of BiologicalsThe major side effect of biologicals is a reduction in host defenseagainst infections. When detected early, these infections can beeffectivelytreatedwithantibiotics. However, threebiologicalsusedtotreat autoimmunediseaseshaveresultedincasesofprogressive multifocal leukoencephalopathy (PML). PML isa rapidly demyelinating and potentially fatal disease that iscausedbyavirusandisoftenobservedinpatientstreatedwithimmunosuppressivedrugsorinpatientswithAIDS. PMLhasbeenassociatedwithpatientswithmultiplesclerosis orCrohns disease treated with the monoclonal antibody natalizu-mab(Major,2009).PMLalsodevelopsinpatientstreatedwiththe B cell-depleting antibody rituximab and in psoriasis patientstreatedwiththemonoclonal antibodyefalizumab.Natalizumabandefalizumabprevent themigrationof Tcellsintotissues,whereasrituximablysesCD20-bearingBcellsanddoesnotaffect T cell migration. It is not clear why natalizumab, rituximab,or efalizumab cause PML.Biologicals for Treating Autoinammatory DiseasesAutoinammatory diseases are chronic inammatory conditionscharacterizedbymacrophagedysfunctionandlocal aswell assystemicinammation(seeEssaybyD.L.Kastneretal.inthisissue; TableS1availableonline). Autoinammatory diseasesarenoninfectiousbutcanbeexacerbatedbyinfection.Recur-rent feversarecommon, withpainful jointsandmusclesandelevated blood neutrophil counts. By inhibiting only IL-1b, thesediseases rapidly are brought under control; neutralizationofTNF-a has little or no effect. Some of these debilitating diseasesare due to gain-of-function mutations that affect the activation ofcaspase-1, which leads to increased processing of the inactiveIL-1b precursor and release of active IL-1b (reviewed in Masterset al., 2009). Caspase-1 activation is initiated by the inamma-some, a multiprotein intracellular complex (see ReviewbyK. Schroder andJ. Tschopponpage821of thisissue). TherstmutationaffectingtheprocessingofIL-1bwasdiscoveredinthenucleotide-bindingdomainandleucine-richrepeatcon-tainingprotein3(NLRP3), acomponent of theinammasome(Hoffmanet al., 2001). Mutationsassociatedwiththeclassicautoinammatory disease familial Mediterranean fever alsoincreasethesecretionof IL-1b(Masterset al., 2009). Anotherclassicautoinammatorydiseaseishyper-IgD(Drenthet al.,1999). Mutations in the gene encoding mevalonate kinase causehyper-IgD and also periodic fever syndrome (InternationalHyper-IgDStudyGroup).Ineachofthesediseases, IL-1bcanstimulate its own production, which plays a pivotal role inautoinammatorydiseasepathogenesis(Dinarelloetal., 1987;Gattorno et al., 2007).Is Type 2 Diabetes an Autoinammatory Disease?Type 1 diabetes is a classic autoimmune disease in which autor-eactive T cells attack the insulin-producing b cells of thepancreas. Ontheother hand, intype2diabetes, thereisnoTcell involvement but rather achronicstateof inammationinwhichIL-1bisproducedandkillsthebcells. Thus, type2diabetesfallsintotheautoinammatoryclassofdiseases,andthebenecial effectsofIL-1bblockadeinpatientswithtype2diabetes supports that concept. IL-1b is also cytotoxic forbisletcellsinautoimmunetype1diabetes(Mandrup-Poulsenetal., 1986),butintype2diabeteshighlevelsofglucosecanstimulate IL-1bproductionby the bislet cell itself (Maedleretal., 2002). IL-1bisalsoproducedbyfatcells, anadditionalsourceofIL-1bintype2 diabetes.Ingeneral,theloss ofb cellmass inthe pancreas progresses over several years duringwhichtimepatientsareinapre-diabeticstate.It isthereforepossibletosalvage bisletcellsbyreducingIL-1b-mediatedinammation.Proof of a specic role for IL-1b in the pathogenesis of type 2diabetes was demonstrated in patients in a 13 week randomized,placebo-controlled study using the IL-1 receptor antagonist(IL-1Ra), also called anakinra, to block IL-1b. There was a statis-tically and clinically signicant improvement in insulin productionand glycemic control (Larsen et al., 2007a). The clinical responsetoanakinrawaslinkedtovariantsinthegeneencodingIL-1Rathat result in lowcirculatinglevels of IL-1Ra (Larsen et al.,2009), raisingthe possibility that insufcientIL-1Ra contributesto inammation in type 2 diabetes. In the 39 weeks after anakinratreatment,patientswhorespondedtoanakinraused66%lessinsulin to obtain the same level of glycemic control. This obser-vationsuggests that blockingIL-1bevenfor ashort periodrestores the function of pancreatic b cells or possibly allows forsomeregenerationof bcells. Several trialsblockingTNF-aintype 2 diabetes have succeeded in reducing C-reactive proteinbut did not improve glycemic control. The anakinra trial observa-tionshavebeenconrmedusingaspecicneutralizingmono-clonal antibodytoIL-1b(Donathet al., 2009). Thesestudiesprovide clinical evidence that type 2 diabetes is a chronic auto-inammatory disease in which IL-1b-mediated inammationprogressively destroys the insulin-producing pancreatic b cells.In diabetic mice, administration of a caspase-1 inhibitorreduces insulin resistance; in mice decient in caspase-1, thereis improved sensitivity to insulin (Stienstra et al., 2009). In addi-tion, the thioredoxin-interacting protein (TXNIP) binds to NLRP3,which activates caspase-1 and the subsequent processing andsecretion of active IL-1b (Zhou et al., 2010). TXNIP-decient miceexhibit improvedglucosetoleranceandreducedinsulinresis-tance (Zhou et al., 2010). There are several studies linking TXNIPexpressiontotype2diabetesandglucoseregulation. Thus,itappearsthat ininsulin-secretingpancreaticbcells, glucose-stimulatedIL-1bactivityiscaspase-1dependent; productionofIL-1bbyadipocytesisalsocaspase-1dependent(Stienstraet al., 2009).IL-1 Blockade in Stroke and Heart DiseaseIL-1 receptor blockade in stroke patients results in a decrease incirculating neutrophils and IL-6 levels, which is often associatedwith a reduction in IL-1b activity. Moreover, the severity of neu-rological impairment appears todecrease instroke patientsCell 140, 935950, March 19, 2010 2010 Elsevier Inc. 939treated with IL-1 receptor blockade compared to placebo-treatedcontrols(Emsleyet al., 2005). DoesIL-1contributetoheart failureafter myocardial infarction?Animal studiesshowthat reducingIL-1activitypreventspost-infarctionmyocardialremodeling (Abbate et al., 2008). In a placebo-controlled trial ofanakinrainpatientswithmyocardial infarction, dailyanakinratreatmentwasaddedtostandardtherapythedayafterangio-plastyandcontinuedfor 14days. Threemonthslater, therewasastatisticallysignicant differenceintheleft ventricularend-systolic volume index (a surrogate marker of damageseverity in myocardial infarction) in patients treated with anakinracompared to patients given a placebo (Abbate et al., 2010). Themechanismfor thebenecial effect of the14daycourseoftherapy is likely to be due to a reduction in the myocardial remod-eling that takes place after loss of viable heart muscle, and thatresults in heart failure. The heart seems to be exquisitely sensi-tivetoIL-1blockadeastheamount of anakinrausedtotreatthe myocardial infarction patients was the same as that used inrheumatoidarthritis,thatis,100mgsubcutaneouslyeachday.Thisdoseresultsinabrief (4hr) riseinbloodlevelsnotmorethan 1.0 mg/ml.Agents for Blocking IL-1 ActivitySpecic monoclonal antibodies, such as canakinumab andXoma 052, are the best agents for blocking IL-1b activity. Cana-kinumab is approved for the treatment of a group of autoinam-matory diseases nowtermed cryopyrin-associated periodicsyndromes(CAPS)that includes familialcold autoinammatorysyndrome, Mucle-Wellssyndrome, andNeonatal Onset Multi-inammatoryDisease(seeEssaybyD.L. Kastneretal. inthisissue; Lachmannet al., 2009). BothcanakinumabandXoma052 are in clinical trials for treating type 2 (and type 1) diabetes,gout, Behcetsdisease, andotherautoinammatorydiseases.Rilonacept isaconstruct of thetwoextracellular domainsoftheIL-1receptorcomplexandbindstobothIL-1aandIL-1b.Rilonacept has been approved for the treatment of familial coldautoinammatory syndrome (Hoffman et al., 2008) and hasbeen used to treat gout. The extracellular domains of IL-1receptor type II preferentially bind to IL-1b. However, for optimalneutralization,theextracellulardomainofIL-1receptoracces-sory protein is required and, in clinical testing, the type II receptorwas not effective. The extracellular domains of IL-1 receptor typeI, which preferentially bind to IL-1Ra and IL-1a, were not effectivein patients.Is it possible to specically inhibit IL-1 activity with orally activeagents? Currently, there are no orally active agents that preventthebindingof IL-1toitsreceptor. Nevertheless, orallyactiveagentsthat target theprocessingandsecretionof IL-1bhavebeendeveloped. ForIL-1btobereleasedfromthecell asanactive cytokine, several steps are necessary (Figure 2). The puri-nergic receptor P2X7 is activated by extracellular ATPandappearstobeacriticalearlystepinthereleaseofactiveIL-1b(Netea et al., 2009; Perregaux et al., 2000). Once the purinergicreceptorP2X7isactivated,thereisarapidefuxofpotassiumions from the cell, subsequent oligomerization of the inamma-some, activation of caspase-1, cleavage of the IL-1b precursor,and release of active IL-1b via secretory lysosomes. Orallyactive,specicinhibitorsoftheP2X7receptorreducediseasein several models of inammation and are being tested inhumanswithrheumatoidarthritis.However,theP2X7receptorservesothercellularfunctions,anditisuncleartowhatextentFigure 2. Production and Release of IL-1b(Top left) Primary blood monocytes, tissue macro-phages, or dendritic cells are activatedby thecytokine IL-1b (blue oval) resulting in the formationof the IL-1 receptor complex (composed of IL-1RIandIL-1RAcP). Theintracellular TIRdomainsofthe IL-1 receptor complex recruit MyD88 andothersignalingcomponentsleadingtotransloca-tion of the transcription factor NF-kBinto thenucleus. Thisresultsintranscriptionof thegeneencoding the precursor of IL-1b and its translationinto protein (blue dumb-bell) (Bocker et al., 2001).ATPreleasedfromactivatedmonocytes(orfromdyingcells) accumulatesoutsidethecell. Astheextracellular levels of ATP increase, the P2X7receptor is activated, triggering the efux of potas-siumionsfromthecell. Lowintracellular potas-sium ion levels enable the components of the cas-pase-1 inammasome to assemble. Assemblyresults in the conversion of procaspase-1 to activecaspase-1. There is evidence that the compo-nentsof theinammasomelocalizetosecretorylysosomes together with the IL-1b precursorprotein and lysosomal enzymes (Andrei et al.,2004). In the secretory lysosome, active cas-pase-1 cleaves the IL-1b precursor, generating active mature IL-1b. Mature IL-1b is released along with the IL-1b precursor and the contents of the secretorylysosomes (Andrei et al., 2004). There is evidence that secretion of active IL-1b requires an increase in intracellular calcium ion levels (Kahlenberg and Dubyak,2004).Processingof the IL-1b precursorcan also takeplacein the cytosolindependently of caspase-1 andthe inammasome(Brough andRothwell,2007).Studies have alsoshownthatRab39a, amemberoftheGTPasefamily,contributesto thesecretionofmatureIL-1bbyhelpingIL-1btrafcfromthecytosolintothevesicularcompartment(Beckeretal.,2009)fromwhereitissecretedbyexocytosis(Quetal.,2007)(not shown).MatureIL-1bcanalsoexitthecellthrough a loss in membrane integrity (Laliberte et al., 1999) or exocytosis via small vesicles (MacKenzie et al., 2001).940 Cell 140, 935950, March 19, 2010 2010 Elsevier Inc.inhibition of this receptor contributes to the amelioration ofinammation. Moreover, sites for caspase-1 activity are presentinfour members of theIL-1family: IL-1b, IL-18, IL-33, andIL-1F7.InthecaseofIL-33,inhibitionofcaspase-1decreasesthe intracellular destruction of the IL-33 precursor. Orally activecaspase-1inhibitorsalsoreduceinammationinseveral ani-mal models of disease and are effective in humans with autoin-ammatorydiseases(Stacket al., 2005). However, similar toinhibitionoftheP2X7receptor,specicinhibitionofcaspase-1will also impact processing of other IL-1 family members. More-over, there are animal models of IL-1b-mediated disease that areindependent of caspase-1 processing of the IL-1b precursor.Althoughtheoligomerizationof inammasomecomponentsinitiates the conversion of pro-caspase-1 into the active enzymeforprocessingtheIL-1bprecursor, thereareampleexamplesof caspase-1-independent activities of IL-1b. For example,theinammationinducedbymuscleinjuryresultsinalargeinuxofneutrophils, tissuedamage,andhighIL-6levels.Thisresponse is absent in mice decient in IL-1b but fully present inmice decient in caspase-1 (Fantuzzi et al., 1997). Intra-articularinjection of cell wall fragments derived from Streptococcus pyo-genes into mice resulted in joint inammation, cartilage destruc-tion, and a robust inltration of neutrophils. However, caspase-1-decient miceexhibitedthesameresponse(Joostenet al.,2009).Similarly,injectionofuratecrystalsintraperitoneallyintomiceinducedabriskperitonitisinmicewithor without cas-pase-1 (Guma et al., 2009). These ndings are important for thepathogenesisofautoinammatorydiseasesasnon-caspase-1processingofIL-1bintheextracellularcompartmentaccountsfor IL-1b-mediated inammation under conditions involvingelevated neutrophil activation. Neutrophil enzymes that canprocess the IL-1b precursor include elastase, proteinase-3,chymases, granzyme A, and cathepsin G (Fantuzzi et al., 1997;Gumaetal., 2009; Joostenetal.,2009). Proteinase-3cleavestheIL-1bprecursor intoanactivemolecule(Coeshott et al.,1999; Joosten et al., 2009) (see Figure 1). Alpha-1 antitrypsin isan endogenous inhibitor of elastase and proteinase-3 andexhibitsabroadrangeof anti-inammatoryproperties(Lewiset al., 2008).Natural Inhibition of CytokinesSomecytokinesfunctionasnatural inhibitorsof inammation,but are they useful therapeutically? Rabbits passively immunizedagainst their own endogenous IL-1 receptor antagonist (IL-1Ra)experience severe colitis (Ferretti et al., 1994), and mice decientinIL-1Radevelopdiseasessuchasdestructivearthritis(Horaietal., 2000), arteritis(Nicklinetal., 2000), andapsoriasis-likeskin disorder. Furthermore, mice decient in endogenous IL-1Radevelop aggressive tumors after exposure to carcinogens (Krelinet al., 2007). These data support the concept that relativeamountsofIL-1versusIL-1Raaffecttheseverityofsomedis-eases. Asingle-nucleotidepolymorphism(SNP) isassociatedwith lower circulating levels of IL-1Ra (Larsen et al., 2009; Raqet al., 2007) andaconcomitant increaseintype2diabetes(Larsenet al., 2009). Thesamepolymorphismisassociatedwithreducedsurvival inpatientswithcoloncarcinomacom-pared to those with the wild-type allele (Graziano et al., 2009).Thenotionof animbalancebetweenIL-1bandIL-1Rahasgained considerable legitimacy with reports of infants bornwithageneticabnormalitythatresultsinnonfunctional IL-1Ra(Aksentijevevich et al., 2009; Reddy et al., 2009). Soon after birth,the affected infants exhibit impressive local and systemic inam-mation.UnlesstreatedwithexogenousIL-1Ra,theinfantsdie.The infants suffer from multiple neutrophil-laden skin eruptions,vasculitis, bone abnormalities with large numbers of osteoclasts,osteolyticlesions, andsterileosteomyelitis. Theinammationresemblesaninfectionwithsepsis-likemultiorganfailure, butall culturesweresterilefor microbes. WhentreatedwithdailyIL-1Ra (anakinra), the inammation abated and the bone lesionswerereversed. Theinfantsareanextremeexampleof whathappenswithoutfunctional IL-1Ra:theactivityofendogenousIL-1 is unopposed and IL-1-driven inammation runs rampant.Fromthese studies, one can propose that the outcome of inam-mation is the balance of proinammatory versus anti-inamma-tory cytokines (Dinarello, 2009b).Systemic InammationSepsis is an example of severe systemic inammation and fallsintotheautoinammatoryclassof diseasesasmacrophagesbut not T cells play a dominant role. The rst studies that blockedcytokine activity in humans were in patients with sepsis. The clin-ical trials of IL-1or TNF-ablockade werebasedonanimalmodels of systemic inammation caused by lethal endotoxin orlive infection. The data from the animal studies were impressive:blocking either IL-1 or TNF-a reduced mortality. Hence, itseemedplausiblethatpatientswithsepsiscouldbe treatedbyreducing IL-1 or TNF-a activity. Despite sophisticated intensivecare,thereare morethan 500,000deathsfromsepsisannuallyin the US. Consequently, millions of dollars have been investedin the development of IL-1 and TNF-a blocking agents, and thesehavebeentestedinplacebo-controlledclinical trials inover12,000patients. Onlymarginal reductionsin28daymortalitywere achieved, insufcient to gain regulatory approval. A meta-analysis of the clinical trial data concluded that a survival benetofblockingIL-1orTNF-awasonlyobservedinthosepatientsmost likely to die (Eichacker et al., 2002).OtheranticytokinetherapiessuchasblockingIL-4orIL-5inthe treatment of asthma were based on well-established animalmodels of airway antigenchallenge, yet thedata inseveralplacebo-controlled trials did not show sufcient efcacy to war-rantmovingforwardwiththesecytokineblockers.However,inautoimmune diseases and autoinammatory diseases, blockingcytokines has provenconsistently benecial. ThesameIL-1receptor antagonist andthesamemonoclonal antibodies toTNF-aorsolubleTNFreceptorsthat failedinclinical trialsforsepsishavebeenapprovedfor thetreatment of rheumatoidarthritis,Crohnsdisease,plaquepsoriasis,andaspectrumofautoinammatory diseases.Protease InhibitorsProteases play an important role in initiating as well as sustaininginammation. Manystudieshavefocusedonblockingmatrixmetalloproteasesinordertostabilizefragileplaquesinarteriesor reduce the loss of cartilage in arthritic conditions. Posttransla-tional cleavage of the N-terminal amino acids of certain chemo-kines (CCL3, CCL5, andCCL23, for example) by proteasesincreasestheir potency(somebyover 100-fold). Inhibitionofproteinase-activatedreceptor2(PAR-2) reducesinammationCell 140, 935950, March 19, 2010 2010 Elsevier Inc. 941in mouse models of rheumatoid arthritis. Inhibition of PAR-2 alsoreducesthedestructionofchondrocytesincartilagebyIL-1b.Antibodiestocomplement factor 5(C5) prevent itsprotease-mediatedconversiontoC5a, theterminal componentincom-plement-mediatedinammation. Thus, inhibitionof proteasesis a valid approach to controlling inammation.There are several serum protease inhibitors to combatprotease-mediatedinammation. Theserumproteina-1anti-trypsininhibitsserineproteasesincludingneutrophil elastaseand also proteinase-3. Activation of complement is also inhibitedby a-1 antitrypsin. Humans born with a genetic deciency in a-1antitrypsindeveloppulmonaryemphysemaearlyinlife, possi-bly due to progressive destruction of lung alveoli due to insuf-cient inhibition of neutrophil-derived elastase by a-1 antitrypsin.Although a-1 antitrypsin inhibits neutrophil-derived elastaseinvitro, it alsoblocksendotoxin-stimulatedIL-1bandTNF-aproduction by human blood monocytes and reduces transloca-tion of the transcription factor NF-kB to the nucleus in responseto cytokine stimulation. Whole-blood cytokine production inpatientslackinga-1antitrypsiniselevatedbut decreasesfol-lowinganinfusionof exogenous a-1antitrypsin(Pott et al.,2009). Administration of exogenous a-1 antitrypsin to miceprotects against allograft rejection (Lewis et al., 2008) and blocksIL-1b-induced death of insulin-producing pancreatic b cells(Lewiset al., 2008); a-1antitrypsinalsopreventsHIV-1entryinto host cells in vitro (Shapiro et al., 2001; Munch et al., 2007).Orally Active Anti-inammatoriesSeveral orally active, disease-modifying drugs usedtotreatautoimmune diseasesmethotrexate, cyclosporine, tacrolimus,and rapamycinalso possess anti-inammatory properties. Themost widely used of these is methotrexate, which is primarily anantiproliferativeagent duetoitsabilitytoinhibit dihydrofolatereductase. However, methotrexatealsodecreasesproductionof TNF-a and chemokines in vivo. The antiangiogenic propertiesof methotrexatemayalsocontributetoits anti-inammatoryprole. Cyclosporine, tacrolimus, andrapamycinareimmuno-suppressive agents, which are used broadly in many clinical situ-ationsfrompreventingallograft rejectiontotreatingpsoriasisand controlling vasculitis. Their primary mechanism of action isas inhibitors of IL-2 andIFN-gproduction, which results inlymphocytesproducinglessTNF-a. Reducinginammationispart of their effectiveness but is likely to be an indirect effect.Colchicine, whichbindstotubulinandblocksmicrotubuleformation, remains the standard of therapy for preventingattacks of urate crystal arthritis (gout) and for treating the autoin-ammatorydiseasefamilial Mediterraneanfever(seeEssaybyD.L. Kastner et al. in this issue). Colchicine is also used to treatavarietyof chronicinammatorydiseasesincludingautoim-mune biliary cirrhosis. The mechanism of action of colchicine istoprevent theinltrationof neutrophilsintoinamedtissues.Colchicinealsoinhibitsthemobilityofmonocytesandmacro-phages, and colchicine therapy in patients with familial Mediter-raneanfever hasdramaticallyreducedthedevelopmentoflife-threateningamyloidosis.Bothfamilial MediterraneanfeverandgoutyarthritisareIL-1b-mediateddiseases. For example, theIL-1receptorantagonist(anakinra)aswell asmonoclonal anti-bodiestoIL-1b(canakinumab)will reducethepainandinam-mation of gouty arthritis. Similarly, in familial Mediterranean feverand Behcets disease, blocking IL-1b results in a rapid and sus-tained remission of inammation.In 1991, Gilla Kaplan reported that clinically achievable levelsof thalidomide inhibited endotoxin-induced TNF-a in humanbloodmonocytes (Sampaioet al., 1991). Thalidomide, orallyactiveandstructurally relatedtoglutamicacid, hadcausedlimbmalformationsinchildrenbornof motherswhousedthedrugtocombat nauseaduringpregnancy. Thalidomidewasapproved to treat patients with leprosy and later, due to its anti-angiogenicproperties, wasusedwidelytotreat patientswithmultiplemyeloma. Itsuseintreatingvariousautoimmunedis-eases isbased on its immunosuppressiveeffects and abilitytoaffect the production as well as the activities of several cytokinesincludingIL-1, IL-2, IL-6, IL-12, IL-23, TNF-a, andIFN-g. Forexample, abenecial effectof thalidomideinCrohnsdiseasepatients is thought to be due to reducedTNF-a andIL-23production. However, peripheral neuropathy and other adverseeffectsareamajorproblemwiththalidomide. Newanalogsofthis drug such as a-uoro-4-aminothalidomide exhibit increasedpotency in blocking cytokine production. Analogs increasecAMP levels in macrophages by inhibiting phosphodiesterase-4,which results in reduced TNF-a production. The anti-inamma-tory cytokine IL-10 is elevated in response to thalidomideanalogs(Thieleet al., 2002). Thethalidomideanaloglenalido-midehasbeenapprovedintheUSandEuropeincombinationwith dexamethasone as maintenance therapy for multiple mye-loma(Falcoet al., 2008). Althoughlenalidomidehasreducedneurotoxicity, ahypercoagulationstatehasbeenobservedinclinical trials. In controlled studies, lenalidomide failed to induceremission or be effective for treating Crohns disease. Neverthe-less, lenalidomides clinical benet in multiple myeloma is due, inpart, toitsanticytokineandantiangiogenicproperties, anditcontinuestobeevaluatedinthetreatment of several chronicautoimmune and autoinammatory diseases.Small-Molecule Inhibitors of Signal TransductionIt has been estimated that there are over 300 kinases that phos-phorylate intracellular proteins, including autophosphorylation ofkinases themselves. A key kinase of interest is the p38 mitogen-activated protein kinase (MAPK), which regulates several funda-mental mediatorsof inammationincludingcytokines, chemo-kines, COX-2, andnitricoxidesynthase. Thediscoveryof p38MAPKbeganwithascreenfor small moleculesthat inhibitedendotoxin-inducedIL-1bproductionaspart of aprogramtodeveloporallyactivedrugsfor treatingrheumatoidarthritis. Scien-tistsat SmithKlineBeechamfoundthat oneof thecompounds(SB20358) reducedIL-1bproductionandalsoreducedarthri-tisinmice. Inorder toascertainthemechanismof actionofSB20358, they radiolabeledthe compound, addedit tocelllysates, andisolatedanintracellular proteinthat wascloselyrelatedtoHOG-1(highosmoticglucose-1), whichisfoundinyeastandenablesgrowthinhighsugarconcentrations(Kumaretal.,1995). Theinductionof cytokinesandchemokinesbyimmunecellstreatedwith hyperosmoticconcentrations ofsodiumchlo-ride is dependent on p38 MAPK (Shapiro and Dinarello, 1995).After the discovery of p38 MAPK in the drug screen, the nextstep was to develop small-molecule inhibitors against the942 Cell 140, 935950, March 19, 2010 2010 Elsevier Inc.several isoforms of p38 MAPK. In general, specic inhibitors ofp38MAPKdonotaffectthedownstreamsignalingcomponentc-JunNH2-terminal kinase(JNK). Inhibitorsof thep38MAPKaisoformreducetheproductionof IL-1bandTNF-ainendo-toxin-stimulated human monocytes, whereas the b, g, and d iso-form inhibitors are less effective. Most animal models for sepsis,type1diabetes, rheumatoidarthritis,lupus,multiplesclerosis,andinammatorybowel diseaseexhibit markedimprovementwhentreatedwithpan-p38MAPKinhibitors.Themoststudiedinhibitor of p38 MAPK is SB20358,which inhibits phosphoryla-tion of its substrate ATF-2.Orally active p38 MAPK inhibitors are well tolerated in phase Iclinical trials in healthy subjects, but hepatic toxicity is observedwith increasing doses. Clinical targets for orally active inhibitorsof p38 MAPK havefocusedon rheumatoidarthritiswith limitedsuccess. Althoughthereisimprovement inclinical scoresinrheumatoid arthritis patients treated with most p38 MAPK inhib-itors, the windowof efcacy and unwanted toxicity appears to benarrow and some clinicaltrialshave beenhalted. The develop-mentofnewp38MAPKinhibitorsforclinical testingcontinuesbecause the kinase plays such a central role in many inamma-tory processes (Marin et al., 2001).TyrosinekinasessuchastheJanuskinases(Jak)playakeyroleinmanyproliferativediseasesincludingleukemiasandarepart of theangiogenicprocessinsolidtumors. Inrheumatoidarthritis, thesynovial membrane, usuallyasingle-cell layer inhealthysubjects, proliferatesandbecomesvascularized. Likea tumor, the proliferating synovium(pannus) invades anddestroysthesurroundingjoint. Jak3isatyrosinekinasethatdrives the proliferation and differentiation of lymphocytes.Frominvitrostudies, inhibitionof theJaksignalingpathwayappearstobeastrategyintreatingrheumatoidarthritisandpossibly other chronic inammatory conditions. Clinical trials oftheorallyactiveJakinhibitorCP-690550inpatientswithrheu-matoid arthritis, renal allografts, or psoriasis showed a reductionininammationmarkersandimprovedqualityof lifeforthesepatients. Mechanisms of action includea reduction in dendriticcell activation and proinammatory cytokine production as wellas increased production of the anti-inammatory cytokineIL-10. Arelated Jak inhibitor reduced IL-17 and IFN-g productionbyperipheral bloodmononuclear cells. At higher doses, Jakinhibitors are associated with increased serum creatinine levels,indicating possible renal toxicity. An increase in infections,nausea, abdominal pain, diarrhea, anddyspepsiahavebeenreported as the dose increases.Spleentyrosinekinase(Syk) participatesinintracellularsig-nalinginBcellsaswell asincellsexpressingFc-greceptors.Fc-greceptorsareactivatedbyIgG1andareexpressedbymonocytes,macrophages,neutrophils,andmastcells.Aspe-cicinhibitorofSykreducedproductionofIL-1b,TNF-a,IL-6,and IL-18. There are controversial data on whether Syk inhibitsIL-1b via the inammasome (Gross et al., 2009; van de Veerdonket al., 2009a). In a mouse model of rheumatoid arthritis (collagen-inducedarthritis),Sykinhibitionreducedboneerosion,pannusformation, and synovitis. In a blinded, placebo-controlled clinicalstudy in patients with rheumatoid arthritis, who were unrespon-sive to methotrexate, there was a rapid and signicant improve-ment in all parameters of disease severity. Matrix metalloprotei-nase3andserumIL-6levelsdecreasedwithintherstweek.However, side effects includedgastrointestinal disturbancesandneutropenia(Weinblattetal., 2008). Inhibitionof Sykmaybeavalidstrategyfor reducinginammationinautoimmunediseases.Imatinib(Gleevec) isatyrosinekinaseinhibitor that isusedclinically to block the kinase activity of the BCR-ABL oncoproteinin patients with chronic myeloid leukemia. Imatinib is also effec-tiveinamelioratingdiseaseinamousemodel of rheumatoidarthritis andin reducing TNF-a production by synovial uidmononuclear cells frompatients with rheumatoid arthritis (Pania-gua et al., 2006). This kinase inhibitor reduced TNF-a productionbyhumanbloodmonocytesinresponsetolipopolysaccharidebutdidnotsuppressproductionoftheanti-inammatorycyto-kineIL-10. Inmurinemodelsof acutehepatitis, imatinibpre-vented TNF-a-dependent inammatory damage to the liverinducedbyinjectingconcanavalinA(Wolf etal., 2005). Thesendings suggest that imatinib is able to act as an anti-inamma-torybecauseit reducesTNF-aproduction. It remainsunclearwhether these observations will be of therapeutic value for treat-ing TNF-a-mediated diseases.Statins as Anti-inammatory AgentsMevalonateisthesubstrateforthesynthesisofcholesterol aswell as several biologically active sterols. Thegenerationofmevalonaterequiresthereductionof theenzymehydroxyme-thylglutaryl-coenzymeA(HMG-CoA) byHMG-CoAreductase.Syntheticsmall-moleculeinhibitorsofthereductasearecalledstatins. Statins were developedto lower the endogenoussynthesisof cholesterol inpatientsat highriskformyocardialinfarctioninwhomelevatedlevelsof low-densitylipoproteins(LDL) were difcult tocontrol. Initial epidemiological studiesshowedahighlysignicantreductionincardiovasculareventswith statin treatment, and the number of high-risk patients beingtreated with statins increased rapidly. Several analogs of statinsthat alsoinhibit HMG-CoAreductaseenteredclinical trials; itbecameclear that all statinsreducedLDLlevelstothesameextent but that the decrease in cardiovascular events wasgreater for some statins than for others. An analysis of the Fra-minghamcohort (along-termprospectivestudyof thehealthof residents of Framingham, MA, USA) for serumlevels ofC-reactive protein (CRP) provided a value for predicting cardio-vascular risk, althoughtheassociationof elevatedCRPwithinammationincoronaryarterydiseasewasnot new(Liuzzoet al., 1994).A large, placebo-controlled trial of nearly 18,000 patients eval-uatedtheeffect of rosuvastatinfor cardiovascular events inpatients with normal LDL-cholesterol levels (less that 130mg/dL). After 1.9 years, CRP decreased by 37%in the treatmentgroupandthereweresignicantlyfewer(rangeof 48%55%)cardiovasculareventsincludingstroke. Deathfromanycausewas20%lowercomparedtotheplacebogroup(Ridkeretal.,2008).ThestudyknownastheJUPITERtrial raisedtheepide-miological issueof thebenet of statinsinapparentlyhealthysubjects. CRPlevelsabove2.0mg/l, abiomarker for occultinammation including coronary artery inammation, wereused as an entry criterion for the JUPITER trial (4% of the USpopulation have CRP levels above 2.0 mg/l). The overallCell 140, 935950, March 19, 2010 2010 Elsevier Inc. 943conclusionofthestudywasthatstatinshadanti-inammatorypropertiesindependentoftheirabilitytolowercholesterol andthat reducing occult inammation with statins couldreducedegenerative processes of aging.The statin-mediated reduction in mevalonate also reduces thesynthesis of cholesterol via the isoprenoid pathway. The functionof several intracellular signalingmoleculesisalsoaffectedbystatinsthroughareductioninproductsoftheisoprenoidpath-way that are important for the formation of signaling complexes.Thereisnodearthofstudiesontheanti-inammatoryproper-ties of statins invitroandinanimal models of autoimmuneandinammatory diseases. In vitro, statins reduce cytokineproductionandexpressionofendothelial adhesionmolecules.Statinsdecreaseexpressionof major histocompatibilitycom-plex(MHC) classII moleculesbut donot affect MHCclassI.They also downregulate the maturation of dendritic cells. Statinsbindtoa unique allostericsite inb2 integrin known as lympho-cytefunction-associatedantigen-1(LFA-1), whichparticipatesinmanyinammatoryprocesses. However, theanti-inamma-toryeffectofbindingtoLFA-1isindependentoftheinhibitionof HMG-CoA reductase. Fluvastatin induced apoptosis ofsynovial cellsfrompatientswithrheumatoidarthritisthrougha caspase-3 mechanism. Both atorvastatin and simvastatinsuppressedexpressionof theRANKligandbybroblast-likesynovial cells, and atorvastatin inhibited osteoclast forma-tioninacocultureofperipheral bloodmononuclearcells,sug-gestingthatstatinsmayinhibitosteoclastformationandbonedestruction.However,studiesontheanti-inammatoryeffectsof statinsinvitrohavealsoyieldedinconsistent data: somestudies report areductionincytokineproductionby humanblood monocytes, whereas others report an increase in cytokineproduction (Kuijk et al., 2008). It seems that animal models revealconsistentndingsfortheanti-inammatoryeffectsof statins.It is important to note that although the anti-inammatoryproperties of statins arehighly consistent inanimal models,statinsdonotlowercholesterol inmice.Manystudiesdemon-strate reduced disease severity in animals treated with clinicallyachievable levels of statins and suffering froma variety of inam-matoryconditionsincludingcolitis, uveitis,myocarditisexperi-mental allergic encephalitis, lethal sepsis, allograft rejection,and asthma (Abeles and Pillinger, 2006).Statins have been used to reduce inammation, tame immunecell activation, or arrest degenerativeprocesses. Becauseoftheir widespread use and long-term safety record, some physi-cians prescribe statin therapy for nonapproved indications.A number of case reports describe the dramatic effects of statinsaddedtostandardtherapies,butbenecial effectsneedtobeconrmedincontrolledstudies.Forexample,inthreepatientswith lupus glomerulonephritis who were not responding tothestandardof therapy(cyclophosphamideandprednisone),addinghighdoses of simvastatinbrought about adramaticreductionindiseaseactivity. Therearerandomized, placebo-controlled studies, but two issues cloud the results: the particularstatin used and the dose. Clearly the anti-inammatory proper-ties of statins vary, whereas the cholesterol-lowering propertiesare similar. Six clinically used statins were examined in vitro fortheir ability to affect NF-kB, phosphorylation of IkB, and activa-tionof tissuefactor, therst stepincoagulation. Therewasa distinct difference between the statins: cerivastatin, atorvasta-tin, and simvastatin were more effective in reducing theseparametersthanuvastatin, lovastatin, or pravastatin(Hilgen-dorff et al., 2003).Nevertheless, there seems to be no dearth of case reports andsmall uncontrolledtrialsthat showthebenetsof statinsforreducing inammation. In the case of rheumatoid arthritis wheretheanti-inammatoryeffectsof blockingcytokineshavebeenshown repeatedly, there have been more than ten trials ofstatinsresultinginamoderatereductioninjoint inammationassociatedwithafall inCRPandredcell sedimentationrateanddecreasedcytokineproductionbycirculatingmonocytes.Asmanypatientswithlong-standingrheumatoidarthritisarealso at high risk for cardiovascular disease, adding statin therapytothestandardof care(methotrexate) ishighlycosteffective.Addingstatins totheregimen ofcyclosporineandsirolimusforkidneytransplant patientsloweredtherateof organrejection.In patients with relapsing-remitting multiple sclerosis, statintherapyfor6monthssignicantlyloweredthenumberofbrainlesions detected by gadolinium, an established marker ofdisease activity. Examination of peripheralblood cells revealednosuppressioninTcell responsesbutdidreveal anincreasein production of IL-10, an anti-inammatory cytokine (Paulet al., 2008).Histone Deacetylase InhibitorsInordertocompactchromatin,DNAistightlywrappedaroundnuclearhistones,whicharemaintainedinastateofdeacetyla-tionbyhistonedeacetylases(HDACs). Histoneacetylases, ontheother hand, hyperacetylatehistones, unravelingDNAandthuspermittingtranscriptionfactorstobindtogenepromotersandinitiategeneexpression. Inhumans, thereare18HDACsdivided into classes based on their dependence on zinc for enzy-maticactivity(Khanet al., 2008). InadditiontotheirabilitytodeacetylatethehighlyconservedN-terminal lysinesofnuclearhistones, HDACs also target many non-histone-containing cyto-plasmic proteins affecting ribosomal function, cytoskeletal poly-merization, and signaling pathways.Synthetic inhibitors of HDACs are orally active and used widelyinmedicine.Forexample,valproicacid,thedrugofchoicefortreating epilepsy as well as obsessive disorders (Gerstneret al., 2008), isanHDACinhibitor (Renet al., 2004). ValproicacidhasalsobeenusedinpatientswithHIV-1topurgethelatentlyinfectedpool of memoryTcells(Archinet al., 2008).TheHDACinhibitor sodiumbutyrateisusedtotreat patientswith sickle cell anemia and b-thalassemia (Atweh and Schechter,2001). The development of newer, small-molecule syntheticinhibitors of HDACs began with the work of Paul Marks, a cancerresearcher(Marksetal.,2001).Thegoal wastoinhibitHDACsin order to increase the expression of several proapoptoticgenesthat areoftensilencedinmalignant cells. Therst ofthisclasswastheHDACinhibitor suberoylanilidehydroxamicacid(SAHA).Atmicromolarplasmaconcentrations,(OConnoret al., 2006) SAHA has been approved for treating patients withcutaneousTcell leukemia(Santini etal.,2007)andhasshownbenet in the treatment of patients with acute myeloid leukemia(Garcia-Manero et al., 2008). Many hematopoietic malignanciesappear toberesponsivetoHDACinhibitors, possiblyrelated944 Cell 140, 935950, March 19, 2010 2010 Elsevier Inc.totheabilityofthesecompoundstoreducetheproductionofcytokines, many of which are growth factors for leukemiasandmyeloproliferative neoplasms (Guerini et al., 2008). Indeed,HDACinhibitors such as SAHA exhibit immunosuppressiveand anti-inammatorypropertiesbyreducing cytokineproduc-tion (Leoni et al., 2002, 2005). Importantly, the anti-inammatoryproperties of HDAC inhibitors are observed in the lownanomolarrangecomparedtothemicromolarrangeneededtotreatcan-cer where the mechanism of action is to increase apoptosis byupregulating expression of proapoptotic genes. The recentexpandinginterest inHDACinhibitors as orally active, safe,anti-inammatory agents has been fueled by the ability of theseinhibitors to reduce disease severity in several animal models ofinammatory and autoimmune diseases (Table S2).ITF2357 is a hydroxamic acid-containing, orally active inhibitortargeting class I and II HDACs. In a phase II clinicaltrial in chil-dren with active systemic onset juvenile idiopathic arthritis,a daily oral dose of ITF2357 at 1.5 mg/kg for 12 weeks resultedinnoorgantoxicityandasignicant (p