The IL-1RI Co-Receptor TILRR (FREM1 Isoform 2) Controls Aberrant Inflammatory Responses and Development of Vascular Disease

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SUMMARY
Expression of the interleukin-1 receptor type I (IL-1RI) co-receptor Toll-like and interleukin-1 receptor regulator (TILRR) is significantly increased in blood monocytes following myocardial infarction and in the atherosclerotic plaque, whereas levels in healthy tissue are low. TILRR association with IL-1RI at these sites causes aberrant activation of inflammatory genes, which underlie progression of cardiovascular disease. The authors show that genetic deletion of TILRR or antibody blocking of TILRR function reduces development of atherosclerotic plaques. Lesions exhibit decreased levels of monocytes, with increases in collagen and smooth muscle cells, characteristic features of stable plaques. The results suggest that TILRR may constitute a rational target for site-and signal-specific inhibition of  (1)(2)(3)(4). Activation is induced by ligand binding and by association of systemspecific co-receptors, which regulate signal amplification and transcriptional activity (1,4,5). Changes in co-receptor expression and causative mutations impact responses to infection, tissue damage and stress, and affect development of disease (5).
Proteoglycans and glycosylated proteins act as co-receptors in a number of regulatory systems.
Recruited to the receptor complex, they control receptor function, ligand binding, and extracellular interactions associated with aberrant signal activation and disease (6)(7)(8). The IL-1 receptor type I (IL-1RI), and its ligand, the cytokine IL-1, are potent activators of nuclear factor-kappa B (NF-kB) and intrinsically linked with acute and chronic inflammation (9). Dysregulation of NF-kB and IL-1-induced gene activity underlie development and progression of conditions such as atherosclerosis (10,11). Our earlier studies identified Toll-like and IL-1 receptor regulator (TILRR) (FREM1 isoform 2) (12), a cell surface proteoglycan, as an IL-1RI co-receptor (13,14). We These targeted embryonic stem clones were expanded and analyzed by long-range PCR for confirmation before using them for embryonic stem cell-morula aggregations (KSOM embryo culture medium, overnight, 37 C) and development of blastocysts for generation of chimeric animals.
Chimeric animals were bred with ROSA26-Flpe mice (Jax #003946, Jackson Laboratory, Bar Harbor, Maine) to remove the PGKneo cassette to generate the conditional knock-in mice, or Hprt-Cre mice (Jax #004302) to generate the global KO mice. C57BL/6J wild-type littermates were used as control mice.
Apolipoprotein E (ApoE)-deficient (ApoE -/-) mice (Jax #2052) were obtained from the Jackson Laboratory. P C R g e n o t y p i n g . Ear clippings were lysed in 50-ml alkaline lysis reagent (95 C, 2 h) before addition of neutralization reagent (50 ml), and 1 ml used for each PCR reaction. Each reaction used 12.5 ml BioMixRed (2Â, Bioline), 0.6 ml of each primer (10 mM), and 1 ml of DNA in a total volume of 25 ml (Tables 1 to 4 R e s p o n s e t o i n j u r y . This was assessed using carotid ligation, as previously (17). The right carotid artery of wild-type and TILRR KO mice was exposed and permanently ligated with a 6-0 suture just below the bifurcation. The contralateral artery received a sham ligation. Arteries were harvested from animals after 28 days following perfusion fixation.
Arteries were embedded in paraffin wax, sectioned and stained for morphometric analysis and immunohistochemistry.  Smith et al. days. Cells were seeded at 5 Â 10 6 in 10 cm dish with  Abbreviations as in Tables 1 and 2. En face staining of the whole aorta was carried out as described (21). In brief, the aortas were perfused MICROSCOPY. Microscopy was carried out using a Nikon E600 microscope (Nikon UK Limited) and quantitation made using NIS-Elements software.    [NIAID], NIH) (25,26), and data expressed as log2-fold change of activity in TILRR -/relative to wild-type.    Figure 1), which demonstrated that TILRR deletion caused a reduction in activated blood monocytes from 28.13 AE 5.04% to 11.34 AE 1.81% ( Figure 1C).   (Figures 2D and 2E).  Figure 3D). Further, plaque levels in the descending aorta were reduced from 8.3 AE 2.5% to 1.9 AE 0.5% of the cross-sectional area of the vessel ( Figure 3E).   Figure 2C). Levels, expressed relative to activities in cultures incubated with a nonspecific IgG control, were reduced from 1.00 AE 0.07 to Abbreviations as in Figure 1.   Figures 5I and 5J).

DISCUSSION
These studies establish a central role for the IL-1RI  complex following blocking of the 448 residue, which is located within the predicted region of proteinprotein association (13,14,27). A mechanism involving alterations in TILRR binding to the receptor complex is consistent with results from our previous studies, which show that TILRR function is dependent on its interaction with the signaling receptor (13). It is also in agreement with effects of inhibiting co-receptor