18 TILRR Functional Mutants Selectively Inhibit Inflammatory and Anti-Apoptotic Responses

Abstract

Method Male Wistar rats at 6 months (young), 12 months (adult) and 24 months (old) (n=5 per group) were killed by a Home Office approved. The SAN region of the heart was dissected and maintained in bicarbonate-buffered saline at 37°C. Intrinsic pacemaker activity was recorded under control conditions and in the presence of 3µMM cyclopiazonic acid (CPA) to inhibit SERCA. Western blot was used to assess expression of SERCA and the SR calcium-release channel, the ryanodine receptor RYR2, both shown expressed relative to levels in the young SAN. Data are shown as mean ± SEM. Results Intrinsic SAN beating rate significantly decreased in old age (young 260±17bpm vs. old 216±15bpm; P=0.04). CPA caused slowing in both young and adult SAN by 26±9 and 49±10bpm respectively (P=0.001), but not in the old SAN. SERCA2a and RYR2 expression increased from young to adult (SERCA young 100±9.8% vs. adult 135±10.8%; P=0.002: RYR2 young 100±21.9% vs. 152±11.2%; p=0.0006), but declined substantially in old age to levels below the young (SERCA old 72±11.7%; RYR2 old 53±4.3%). Conclusion The data show a diminished SR influence on pacemaking in the old SAN. In contrast developmentally the SR may increase its pacemaker role from young to adult animals. Toll-like and IL-1 receptors control inflammatory responses. TILRR (Toll-like IL-1 Receptor Regulator), is an IL-1 co-receptor which associates with the type I IL-1 receptor (IL-1RI) to amplify activation of NF-kappaB and inflammatory responses. Earlier studies have demonstrated increased levels of TILRR in the atherosclerotic plaque. Further, that injection of a polyclonal anti-TILRR antibody, which blocks TILRR/IL-1RI association and reduces inflammatory responses, causes a 25% decrease in plaque formation in ApoE-/-mice on a high fat diet. Alanine scanning mutagenesis identified two sites within the TILRR core protein, which allow distinct control of Il-1 activities. The R425A substitution blocks enhanced cell survival, but functions as wild-type in relation to inflammatory responses. In contrast, a D448A substitution reduces MyD88-dependent inflammatory responses, but has no impact on cell survival [1]. Current studies use peptides designed to block these distinct functional sites to further analyse consequences of selective inhibition on downstream events. Results show a successive reduction in inflammatory responses by the peptide designed to block D448 dependent interactions, with no effect of the peptide targeting anti-apoptotic signals or a non-specific control. Ongoing studies are testing the effect of the peptides on IL-1-induced cell survival. TILRR FUNCTIONAL MUTANTS SELECTIVELY INHIBIT INFLAMMATORY AND ANTI-APOPTOTIC RESPONSES