Inflammation plays a critical role in all stages of atheroscleroticplaque development, particularly so in inducing an unstable plaquephenotype [1]. Cytokines modulate this inflammatory process, withemergingdatashowingthatbothpro-andanti-inflammatorycytokinesare present in atherosclerotic plaques [2]. The cytokines IL-31, IL-32,IL-33 and IL-34 have recently been characterised with regard tovarious inflammatory conditions, with IL-31, -32 and -34 having pro-inflammatory effects whereas IL-33 has been shown to be potentiallyathero-protective both in vitro and in animal studies of experimentalatherosclerosis [3–6]. Our recently published data described the distri-butionofthesecytokinesinpost-mortemcoronarysamples[7];howev-er their expression in ex-vivo human carotid plaques, particularly theircorrelationwithstableversusunstableplaquephenotypesandrelation-ship with patient characteristics, has not yet been explored.Forty-five consecutive patients with haemodynamically significantcarotid artery disease and in whomcarotid endarterectomy was deemedappropriate were selected to participate in the study [8]. Patients wereclassified as symptomatic (n = 23) or asymptomatic (n = 22) accordingto the presence or absence of cerebrovascular symptoms, respectively.Aspreviouslydescribed,themajorityofplaquesfromasymptomaticsub-jects were Type V lesions, whilst plaques from symptomatic subjectswere predominantly Type VI lesions, as defined by the American HeartAssociation Vascular Lesion Classi fication [9]. Cardiovascular risk factors,lipidprofileandmedicationusewererecordedforeachpatient.Informedconsent was obtained from each patient and the study was approvedby the South Western Sydney Area Health Ethics Committee, NSW,Australia.Excised plaques were immediately fixed in formalin and transverse5mmparaffin blocks were prepared from each plaque, from which5 μM serial sections were obtained. Sections were stained withhaematoxylin–eosin and Movat's pentachrome, and further adjacentsections were used for immunohistochemistry (IL-31–IL-34). Each ofthe polyclonal rabbit anti-human primary antibodies (Abcam, Sydney,Australia)was followed bypolymer HRP goat anti-rabbit immunoglob-ulin (Dako, Sydney, Australia). The positive staining was photographedandquantifiedusingImage-Pro®Plus7.Quantitativereal-timePCRwasperformed on cDNA prepared from total RNA isolated from snap frozencarotid plaques(10asymptomatic, 8symptomatic). Gene expression ofIL-31–IL-34wasquantifiedaccordingtothemanufacturer'sinstructions(Qiagen, Chadstone, Australia) using relevant primers. The statisticalanalyses were performed using GraphPad Prism® Version 5.0 by oneway ANOVA. p-Value b 0.05 was considered significant.Asymptomatic and symptomatic groups were well matched interms of baseline patient characteristics (Table 1). IL-31, -32, -33and -34 were detected in plaques from both asymptomatic andsymptomatic patients. Immunostaining for IL-31, -32 and -34 wassignificantly greater in plaques from symptomatic compared withasymptomatic subjects (2.4-, 3.5- and 2.3-fold higher, respectively,p b 0.05 for all). However, IL-33 immunostaining was 1.9-fold greaterin stable versus unstable plaques (p b 0.05) (Fig. 1). Similarly mRNAtranscriptlevelsofIL-31,-32and-34weresignificantlyhigherinunsta-ble v stable plaques (1.5-, 1.8-, 1.9-fold, respectively, p b 0.05 for all)whereasfor IL-33there wasa significant2.1-foldincreaseinmRNAex-pression in stableversus unstableplaques (Fig. 2).When statin-treatedand statin-naive patients were compared, IL-33 immunostaining washigher in treated versus untreated (1.6-fold, p b 0.05), with thesegroups otherwise being well matched in terms of the presence orabsence of symptoms or other cardiovascular risk factors. There was anon-significant increase in IL-33 mRNA levels in statin-treated com-paredwithstatinnaivepatients.Cardiovascularriskfactorswereother-wise not significantly associated with the presence of any of thesecytokines.IL-31, -32, and -34 are potent inflammatory cytokines, whosepresencehasbeendemonstratedininflammatorydiseasessuchascoli-tis and psoriasis. We have previously shown the presence of these