A central nervous system-restricted isoform of the interleukin-1 receptor accessory protein modulates neuronal responses to interleukin-1

Abstract

Interleukin-1 (IL-1) has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified an isoform of the IL-1 receptor (IL-1R) accessory protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1R but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild-type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild-type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest that it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.

Figure 1. Genomic organization, sequence and TIR domain structural models of AcP isoforms

A, intron-exon map of human AcP locus and alternative splicing that leads to mRNA isoforms. Shown are the translated exons (3-12) that encode the mature AcP protein. The solid black bar indicates the extracellular exons that are deleted in the il1rap AcP knockout (Cullinan et al., 1998) and the faded bar indicates the specific exon 12b that is deleted in AcPb knockout animals (described in text). B, Alignment of the alternative C termini of AcP and AcPb isoforms. Stars indicate conserved residues between human AcP and human AcPb. Specific structural or signaling-associated motifs are indicated. C, Computational models of AcP and AcPb TIR domains. Structural folds of the ribbon structure are labeled following the convention of (Khan et al., 2004). Beta sheets are indicated in yellow, alpha helices in red and loops in blue. Shown on the bottom is the identical view with predicted surface electrostatics. Full red is equivalent to -40 kcal/mol negative charge; full blue equivalent to 40 kcal/mol positive charge and white is neutral charge. The arrow indicates the region of significant charge difference caused by the disruption of the αD helix in AcPb.

Figure 2. mRNA expression profile of AcP and AcPb

A, Relative mRNA level of AcP and AcPb isoforms in human tissues as determined by real time PCR and presented as expression relative to HPRT. Abbreviations: ‘F’, fetal, ‘sm’, small, ‘sk’, skeletal. B, Normalized relative expression of AcP and AcPb mRNAs across isolated human brain regions as compared to a neuronal- and astrocyte -specific gene (MAP2 and GFAP, respectively). Relative expression (to HPRT) for each gene in each region was normalized to its overall expression in total brain (set to 100%).C, RT-PCR amplification of AcP and AcPb mRNAs from primary hippocampal neuron and astrocyte cultures. D, In situ hybridization of adult mouse brain with RNA probes specific for AcP or AcPb isoforms. 1, whole brain hybridized with sense strand control probe. 2, whole brain hybridized with AcP antisense probe. Cerebellum is indicated with dashed box and the brain stem with a solid box. 3, 4, Bright field and dark field images of cerebellum hybridized with either AcP (3) or AcPb (4) antisense probes. Positively hybridizing Purkinje cells are indicated with an arrow for both probe sets. 5, 6, Bright field and dark field images of forebrain hybridized with either AcP (5) or AcPb (6) antisense probes. Positively hybridizing neuronal cell bodies are visible with both probes.

Figure 3. AcPb-mediated IL-1R and adapter recruitment

A, AcP surface expression on EL4.16a parental and stably-transfected cell lines utilizing an anti-AcP antibody that recognizes both forms of the receptor. B, EL4 lines were incubated with IL-1β (100 ng/ml) for 3 minutes then lysed and immunoprecipitated with a pan-AcP monoclonal antibody. Precipitations were analyzed by Western blot using either an anti-muIL-1R antibody or a pan-AcP antibody. Approximate molecular weights are indicated. C, Samples were stimulated and immunoprecipitated as in (B) and then adaptor protein recruitment was determined by Western blot with anti-MyD88 or IRAK4 antibodies, as indicated.

Figure 4. Signaling capacity of AcPb-reconstituted EL4 cells

A, EL4 lines were incubated for 24 hours in the presence of 300 ng/mL ionomycin and 100 pg/mL PMA plus or minus IL-1β (100 pg/mL), as indicated. Cytokine levels in the supernatants were quantified by Luminex (bead)-based multiplex assay and all assays were performed in triplicate. B, 2.0 × 10⁷ cells were incubated with IL-1β(10 ng/mL) or 0.5M sorbitol at 37°C for various lengths of time and lysates (approximately 0.4 × 10⁶ cell equivalents/lane) were probed by Western blot with total and phospho-specific antibodies against p38, JNK and ERK, as indicated.

Figure 5. IL-1-induced responses in primary cortical neurons

A, C57BL/6 embryonic cortical neuron cultures were stained by immmunohistochemistry after 9 days of culture using antibodies that recognize either the neuronal-specific protein MAP-2 or the astrocyte-specific protein GFAP, as indicated. B, RT-PCR demonstrating specific expression of specific AcP isoforms in lentiviral vector-transduced neuronal cultures but not in non-transduced AcP^−/− culture 2 days post transduction. C, RT-PCR detection of CCL2 transcript in transduced neuronal cultures stimulated for 4 or 18 hours with IL-1β (10 ng/mL), as indicated. D, Gene intensity profiles of AcP-deficient and AcP isoform-reconstituted neuronal cultures (as indicated) stimulated with IL-1β (10 ng/mL) for 4 hours. RNA samples from triplicate stimulations for each cell population were used to hybridize a 45,000 probe set microarray as described in the methods.

Figure 6. Generation of AcPb knockout mouse

AcPb-deficient animals were generated as described in the methods and as indicated in Figure 1. A, RT-PCR primers specific for AcP, AcPb or HPRT (as a control) were used to amplify cDNA generated from whole brain tissue from wild type, AcP^−/− or AcPb^−/− mice. Plasmid DNA for each cDNA was used as positive PCR controls and H₂O as a negative PCR control. B, immunoprecipitations of AcP- and AcPb-EL4 cell lysates or whole brain lysates from wild typeor AcPb^−/− mice were performed with a pan-AcP antibody and analyzed by Western blot using a polyclonal anti-AcP antibody, as described in the methods. C, Brains were collected from 10 week old mice and coronal plane sections were stained with Thionine- Nissl to reveal cell bodies and general morphology. Images are indicative of observations across the interval of sections from 3 age-matched mice per genotype. D, Splenocytes were isolated from wild type, AcP^−/− or AcPb^−/− mice and stimulated with IL-1β at the indicated concentrations. Supernatants were collected for determination of IL-6 levels after 48 hours. Results were generated using splenocytes from 3 individual mice per genotype and average fold induction is indicated (N.S. = not significant, ** p<0.001).

Figure 7. Effect of AcP and AcPb deletion in vivo: EAE (A-D) and acute LPS challenge in CNS (E-H)

Female C57Bl/6 wild type, AcP^−/− or AcPb^−/− mice (approximately 10 weeks old) were immunized s.c. with MOG_35-55 in CFA on day 0 to induce experimental autoimmune encephalomyelitis. Pertussis toxin (350 ng/mouse) in PBS was injected i.v. 48 hr after immunization. Animals were monitored, weighed, and scored for clinical disease every other day until disease was observed then daily thereafter. A, AcPb^−/− mice (n=14) and wild type controls(n=15) had 100% disease incidence and a median day of onset of day 14 post immunization. AcP^−/− mice (n=15) had significantly reduced disease incidence (5%, 1/18) compared to wild type controls or AcPb^−/− mice (p<0.0001, Logrank test). B, At day 11 post-immunization, the AcPb^−/− mice had a statistically significant increase in weight loss compared to wild type controls (p<0.05, One way ANOVA, Tukey's post hoc test). C, AcPb^−/− mice had a similar disease progression to that of wild type controls. AcP^−/− mice had a significant reduction in mean clinical score on days 14-20 post-immunization (p<0.05, One way ANOVA, Tukey's post hoc test) and also suffered significant weight loss compared to wild type controls. D, RNA was extracted on day 20 post-immunization from spinal cords of 5 mice per strain, as indicated, and gene expression was profiled by real-time PCR. Average gene expression relative to HPRT is plotted for each gene. E, Mice (approximately 10 weeks old) were anesthetized and LPS (1.0 mg/mL) was injected intracerebrally over a one minute period. 3 days later, mice were deeply anesthetized and perfused, fixed brains were collected and processed as described in the methods. IL-1β mRNA was quantified following LPS administration by in situ hybridization. OD =optical density of IL-1β signal on radiographic film (not shown). Error bars represent variation among replicate animals per group. F, Proteolipid protein (PLP) in situ hybridization (black and white image) and Fluoro-Jade B (FJB) staining (cell bodies of degenerating neurons stain green) of brains 3 days post LPS administration. White dashed lines indicate location of injection tract and red dashed boxes indicate regions of localized PLP loss and FJB-positive neurons (also shown at 200× magnification from AcPb brain). The relative area of lost PLP expression (G) or the number of FJB-positive degenerating neurons (H) were quantified by ImageJ software. Results are expressed as mean ± SEM (* P < 0.05, *** P < 0.001), Data are representative of brains from either 3 or 4 individual mice per treatment group.