Constitutive and LPS-regulated expression of interleukin-18 receptor beta variants in the mouse brain

2.1. Animals and treatments

Two month old C57BL6/J male mice were used in this study. Animals were housed in polycarbonate cages (28 × 17 × 12 cm) with ad libitum access to food and water throughout the study, maintained under a 12:12 light-dark cycle in an ambient temperature of 21 ± 3 °C with relative humidity controlled. Animals were checked for signs of discomfort as indicated by animal care and use guidelines (National Academy of Sciences. Guide for the care and use of laboratory animals, 1998, “Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research” (National Research Council 2003)). All procedures were carried out in accordance with the EC guidelines (EEC Council Directive 86/609 1987), Italian legislation on animal experimentation (Decreto Legislativo 116/92) and The Scripps Research Institute Institutional Animal Care and Use Committee.

LPS (100 μg at 1 μg/μl strain 055:B5, Sigma Aldrich L2880) were injected intraperitoneally (i.p.) (n=3) and an equal volume of vehicle (saline) was used as a control (n= 3). Tissues were harvested 3 hrs after injection and stored at −80°C until RNA extraction.

2.3. In situ hybridization

Determination of IL-18Rβ mRNA in the CNS was performed by non-isotopic in situ hybridization (ISH) using a specific DIG-labeled anti-sense riboprobe common to both isoforms, and a sense probe as negative control. cDNA [nt 759–1137 (NCBI GenBank accession number: NM_010553)] for mouse IL-18Rβ was subcloned into pDrive Cloning Vector (Quiagen^®, Hilden, Germany) in order to obtain a template for the in vitro transcription of cRNAs [antisense (using BamH I restriction site); sense (using Hind III restriction site)]. Probes (antisense –AS- and sense –S) for IL-18Rβ to be employed in in situ hybridization assay, were synthesized using the DIG-RNA labeling kit (La Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions [SP6 polymerase for IL-18Rβ AS probe – T7 polymerase for IL-18Rβ S probe].

For in situ hybridization mice were anaesthetized by isoflurane inhalation and perfused transcardially with heparinized (5.000 U.I./L) saline followed by perfusion with ice-cold 4% paraformaldehyde in PBS pH 7.4. After fixation, their brains were rapidly removed and post fixed in the same fixative solution for 4 hrs, and then cryoprotected in a 30% sucrose solution in phosphate buffer pH 7.4. In situ hybridization was carried out as previously described on 40-μm thick serial coronal sections (see Alboni et al., 2009 for further details).

The specificity of the hybridization signals in the brain regions were confirmed by comparing sections hybridized with the antisense probe to those hybridized with the sense probe. No hybridization signals were detectable in consecutive sections hybridized with the sense probe. The relative intensities of the IL-18Rβ mRNA in various brain regions were evaluated in hybridized coronal sections via transillumination microscopy by al least two independent investigators. Anatomical brain regions and nomenclature were those of the Franklin and Paxinos mouse brain atlas (Franklin and Paxinos, 1997). The staining intensities used in Table I were arbitrarily assigned to reflect the following: −, not detectable; +/−, very low signal; +, weak signal; +(++), a weak to moderate signal; ++, moderate signal; ++(+++) a moderate to strong signal; +++, strong signal. Figures were prepared using Adobe Photoshop 7.0.1 with minor adjustments to contrast and brightness.

2.4. Immunohistochemistry

Mice were sacrificed upon perfusion with 0.2% EDTA-containing PBS, under isoflorane. Brains were processed immediately after perfusion. Brains were removed and bisected in midsagittal plane, fixed in 10% formalin, embedded in paraffin, and cut into 5 μm sections. Following a 40 min 95^oC steam bath in 10mM citrate buffer (pH6.38) for antigen retrieval, representative sections of brain (plus spleen controls) were immunohistochemically stained, using an anti IL-18Rβ antibody (BAF1520, R&D Systems, Minneapolis, MN) followed by biotinylated anti-goat IgG secondary antibody (Vector Labs, Burlingame, CA) and streptavidin HRP (Invitrogen, San Diego, CA), and was developed with NovaRed (Vector Labs). Sections were counterstained with Gill’s hematoxylin (Invitrogen). Images from cells were visualized and acquired using a Zeiss (Oberkochen, Germany) Axiovert 200 inverted microscope at 20 and 32× magnification and captured by using the Zeiss Axiocam HRC associated with the Zeiss Axiovision 2.0.5 software package. Controls performed by omitting primary antibodies were negative.

2.5. RNA extraction and retro transcription

Total RNA extraction was performed using TRIzol^® reagent (Sigma^®, St. Louis, MO, USA) followed by a clean-up step on Qiagen RNeasy Spin Column and DNAse treatment to remove genomic contamination following the manufacturer’s instructions (Qiagen^®, Hilden, Germany). 1 μg of total RNA was reverse transcribed with High Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA, USA) in 20 μl of reaction mix.

2.6. Qualitative PCR analysis

Qualitative PCR analysis was carried out using GoTaq^® Flexi DNA polymerase (Promega Italia^®, Milan, Italy). Total RNA was extracted from brain areas and tissues of 4 C57BL6/J mice and pooled before RT reaction. Primers were designed based on rat short IL-18Rβ (NCBI accession number: NM_184047) to produce two specific PCR products of 101 bp and 190 bp corresponding to the canonical and short IL-18b respectively (if a mouse analogue of the rat short IL-18Rβ exists): IL-18Rβ forward: AAG GCA TGC TGC ATA TAT TGG; IL-18Rβ reverse: TCT TGA TAC AAC AGG CCA TAT CC. Cyclophilin A mRNA (NCBI GenBank accession number: NM_008907) was used for normalization; the CypA forward: AGC ATA CAG GGT CCT GGC ATC and CypA reverse: TTC ACC TTC CCA AAG ACC AC. The PCR protocol was: 95°C for 2 min, 1 cycle; 95°C for 30 sec; 60°C for 30 sec; 72°C for 1 min, 35 cycles; 72°C for 10 min, 1 cycle. To verify that the sequence of the short IL-18Rβ did not correspond to any intermediate RNA form, we used a specific primer set whose forward (GTC CTC AAA TCA TCC CAG T) and reverse primer (CGG ACT GTC CAG GAA CTC AC) matched respectively with the intron 5 and the exon 7 of the mouse IL-18Rβ gene.

2.7. Amplification and DNA sequencing

To sequence the IL-18Rβ variants, we amplified the specific PCR products (of 101 bp and 190 bp) starting from cDNA equivalent to 0,9 mg total RNA (mouse cerebral cortex or spleen) using the same forward and reverse primers used for the qualitative distribution (see section 2.6 Methods) and GoTaq^® Flexi DNA polymerase (Promega Italia^®, Milan, Italy). To increase the amount of products needed for the sequence analysis we performed a modified NESTED PCR step using the same primer pairs on 1 ml of the first PCR product. The cycling parameters were: 95°C for 2 min, 1 cycle; 95°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 25 cycles; 72°C for 10 min, 1 cycle. Amplified PCR products were analyzed by agarose gel electrophoresis and purified using Wizard® SV Gel and PCR Clean-Up System (Promega Italia^®, Milan, Italy) following the manufacturer’s instructions. 100 ng of both 101 bp and 190 bp PCR products from mouse cerebral cortex and spleen were sequenced from 5’ and 3’ ends on an ABI prism 377 DNA Sequencer using the ABI PRISM Big Dye Terminator chemistry (Perkin Elmer Biosystem^®, Milan, Italy). The DNA sequences obtained were compared with published sequences in the GenBank database, by homology search using BLAST 2.0 (basic BLAST search, nr database, accessed at http://www.ncbi.nlm.nih.gov), and all sequences with significant homology were identified.

2.8. Real Time PCR

Real Time PCR was performed in ABI PRISM 7900 HT (Life Technologies Corporation, Carlsbad, CA, USA) using Power SYBR Green mix (Life Technologies Corporation, Carlsbad, CA, USA). To distinguish between the two splice variants of IL-18Rβ we designed a common forward primer complementary to nt 673–692 of the coding exon 5 and two specific reverse primers: full-length IL-18Rβ : GGG GGC TCC TAA TTC TGG G and short IL-18Rβ: GTC CTG TGA GCA CTT GTC TA. IL-18 (NCBI GenBank accession number: NM_008360) IL-1β (NCBI GenBank accession number: NM_008361), IL-18Rα type I (NCBI GenBank accession number: NM_008365), IL-18Rα type II (NCBI GenBank accession number: BC023240), and IL-18Rβ levels were normalized for each well to endogenous control cyclophilin A (CypA). The following forward and reverse sequences were used at the final concentration of 150 nM: IL-18 forward: TGA AGA AAA TGG AGA CCT GGA, IL-18 reverse: GGC TGT CTT TTG TCA ACG AAG; IL-1β forward: TGA AAG CTC TCC ACC TCA ATG, IL-1β reverse: CCA AGG CCA CAG GTA TTT TG; IL-18Rα type I forward: GAG TAA CTG TGC TTG TTC TCG CCT CTG T, IL-18Rα type I reverse: GGG TAA CGT CTC CAC ACG AAA AGT AT; IL-18Rα type II forward: GGC ACC CTA GCT CAT GTT TT; IL-18Rα type II reverse: AAC GAG GCT CAG AGA TCA TTA GT. The cycling parameters were: 95°C 10 min and 95 °C 15 s, 60° 1 min for 40 cycles. Single PCR products were subjected to a heat dissociation protocol (gradual increase of temperature from 60°C to 95 °C) and agarose gel separation to verify the absence of artifacts, such as primer-dimers or non-specific products. Direct detection of PCR products was monitored by measuring an increase in fluorescence intensity caused by binding of SYBR GREEN I dye to neo-formed double strand DNA during the amplification phase. Ct (cycle threshold) value was determined by the SDS software 2.2.2 (Life Technologies Corporation, Carlsbad, CA, USA) and was utilized to calculate mRNA fold changes using the delta delta ct (ΔΔCt) method. The equation used was $2^{\mathrm{\Delta }\mathrm{\Delta }\text{ct}}=2^{{({\text{Ct}}_{\text{X}}-{\text{Ct}}_{\text{R}})}_{\text{reference}}-{\left({\text{Ct}}_{\text{X}}-{\text{Ct}}_{\text{R}}\right)}_{\text{target}}}$, where Ct_X was the threshold cycle of the gene of interest (that were: IL-18 and the IL-18R chains) and Ct_R was the threshold cycle of the house-keeping gene (that was: cyclophilin A).

For an appropriate application of comparative ΔΔCt method, it was demonstrated that amplification efficiency of the target gene and endogenous control gene was approximately equal. Each cDNA sample was run in triplicate and the mean values were used to calculate the gene expression levels.

2.9. Statistical analysis

The relative quantity of short and full-length IL-18Rβ variants gene expression was analyzed by the ΔΔCt method using as calibrator average of the cerebral cortex of control (saline injected) animals. The mRNA levels of the targets genes were normalized on the intensity of the house-keeping gene, cyclophilin A. Cv values were obtained by an interpolate study. Regional brain differences in the expression of the canonical and the short IL-18Rβ variants were analyzed with a one factor analysis of variance (ONE-WAY ANOVA) followed by multiple post-hoc comparisons (Tukey HSD). Differences were considered significant if p< .05. The effect of LPS on the expression pattern of the full- and short-length IL-18Rβ mRNA isoforms in the various brain areas were analyzed using a two-factor ANOVA. Individual planned post-hoc comparisons were performed (t-test) between LPS and saline treated animals for each individual brain area (p values below .05 were considered significant). The relative expression of full- and short-length IL-18Rβ in animals injected with LPS or vehicle (ratio) was analyzed using a two-factor ANOVA followed by planned post-hoc pair-wise comparisons. Differences were considered significant if p < .05. Finally the analysis of the effects of a peripheral LPS injection on the expression of the cytokine IL-18 and the two isoforms of the IL-18Rα (type I and II) was performed with a Student’s t-test (comparative ΔΔCt method was performed using as calibrator average of saline control animals for each brain area; p≤.01 was considered significant).

3.1. Identification of a mouse homologue of the short rat brain IL-18Rβ isoform

We demonstrated the existence of a short isoform of the mouse IL-18Rβ (sIL-18Rβ) which is supposedly similar to that described by Andre and coworkers (Andre et al., 2003). We used a forward primer matching the exon 5 and a reverse primer matching the exon 6 of mouse IL-18Rβ mRNA. In the presence of both beta chain isoforms these primers were expected to produce two specific products by RT-PCR. Qualitative PCR performed on mouse cDNA obtained from different brain areas as well as from spleen amplified two products of 101 bp and 190 bp for the canonical and the small IL-18Rβ isoform, respectively (Fig. 1). As demonstrated by sequencing, the latter PCR product includes a 89 bp fragment, downstream of the exon 5, previously known to be intronic (according to the homology with the published mouse sequence NCBI GenBank accession number: NM_010553).

The relative expression (abundance) of the canonical (full-length) and the short IL-18Rβ variants in the CNS were then evaluated using Real Time PCR. One-Way ANOVA revealed a statistically significative difference [F (7,16) = 4.68; p = .005] among the mean of the ΔΔct values (see Methods section) for the full-length 18Rβ mRNA expression in the different brain areas evaluated. Transcript levels of the full-length IL-18Rβ were highest in the olfactory bulb and in the frontal cortex followed by the striatum, the hypothalamus, the thalamus, the cerebellum and the cerebral cortex. The lowest relative levels of IL-18Rβ mRNA expression were found in the hippocampus (Fig. 2A). Post-hoc tests revealed a significant difference in full-length 18Rβ mRNA expression between the frontal cortex and the thalamus (p= .007). Moreover, the expression of the canonical IL-18Rβ mRNA was significantly higher in the olfactory bulb with respect to the striatum (p= .034), the hypothalamus (p= .050), the thalamus (p= .000), the hippocampus (p= .005), the cerebellum (p= .034) and the cerebral cortex (p= .014). Furthermore, the levels of IL-18Rβ mRNA were relatively lower in all these brain regions when compared to the spleen (mean ± S.E.M.; 1480.08 ± 201.02) (data not shown).

With respect to the IL-18Rβ short isoform, a one-Way ANOVA revealed that the expression of this isoform doffers significantly among the brain areas considered [F (7,16) = 6.16; p = .001]. Similar to the full-length IL-18Rβ variant, the short IL-18Rβ mRNA variant was expressed at high levels in the olfactory bulb and in the frontal cortex and in the thalamus, with somewhat lower levels expressed in the cerebellum, the hippocampus, the hypothalamus, the striatum and the cerebral cortex (Fig. 2B). Post-hoc comparisons between areas revealed that the levels of the sIL-18Rβ mRNAs where higher in the olfactory bulbs compared to that of the striatum (p= .001), the hypothalamus (p= .002), the thalamus (p= .032), the hippocampus (p= .013), the cerebellum (p= .025) and the cerebral cortex (p= .001). Moreover, mRNA levels of the sIL-18Rβ were relatively lower in all these brain regions when compared to the spleen (2489.84 ± 756.81) (data not shown).

3.2. Basal expression and distribution of IL-18Rβ in the mouse brain

We next determined the anatomical distribution and localization of IL-18Rβ mRNA in the mouse brain. This was performed by in situ hybridization with digoxigenin-labeled cRNA anti-sense probes directed against a portion of the CDS (using a sense probe as a control) designed to detect the transcript for the canonical IL-18Rβ. An overall view of the distribution of the IL-18Rβ mRNA is presented in figure 3 and supplementary figure 2, while a comprehensive summary of its expression is presented in Table I.

In accordance with the PCR results (Fig. 2), high hybridization signal was observed in the telencephalon, in the orbitofrontal cortex (e.g. frontal association cortex-FrA and prelimbic cortex-PrL) (supplementary Fig. 2) and in the olfactory system (e.g. piriform cortex-Pir and anterior olfactory nuclei-AOD, AOL and AOV) (supplementary Fig. 2). Furthermore, a moderate IL-18Rβ mRNA signal was found in each layer (I to VI) of the cerebral cortex (Fig. 3) with a similar distribution pattern in the different cortical areas (data not shown). In the hippocampal formation the expression of the IL-18Rβ mRNA was mainly neuronal-like, with heavy IL-18Rβ mRNA staining in the pyramidal cell layer of the Ammon’s horn (CA1-CA3) (Fig. 3). In the cerebral cortex and in the hippocampus, as observed also for other areas or nuclei, the staining was mainly confined to the cell body of the pyramidal neurons and was not found in their processes (Fig. 3). No, or very weak, staining was present in the stratum radiatum (Rad) and in the layers molecular (mol), lacunosum molecular (Lmol) and oriens (Or) of the hippocampus (Fig. 3). Moreover, a weak staining was observed in the granule cell layer (GrDG) and in the polymorph layer (PoDG) of the dentate gyrus (DG) (Fig. 3). Finally a moderate hybridization signal was observed in the indusium griseum (IG) and in the subiculum (S, including presubiculum - PrS and parasubiculum - PaS) of the hippocampal formation. While the IL-18Rβ mRNA staining in the basal ganglia was weak, a moderate to strong IL-18Rβ mRNA signal was detected in the amygdala (Fig. 3). Especially high levels of IL-18Rβ mRNA expression were found in the basolateral amygdaloid nucleus (anterior - BLA, and posterior - BLP, parts), in the posterior part of the basomedial amygdaloid nucleus (BMP), and in the posteromedial cortical amygdaloid nucleus (PMCo). Strong IL-18Rβ mRNA staining was also observed in the septum especially in the nucleus of the vertical limb of the diagonal band (VDB). In the diencephalon high levels of IL-18Rβ mRNA expression were observed in the medial habenular nucleus (MhB), in the thalamus (particularly in the anterior thalamic nuclei such as the anteroventral thalamic nucleus - AV, the parataenial thalamic nucleus-PT and the anterior part of the paraventricular thalamic nucleus - PVA) and in the hypothalamus (with especially high levels in the paraventricular hypothalamic lateral magnocellular part - PaLM, in the paraventricular hypothalamic dorsal cap - PaDC and in the ventromedial hypothalamic nucleus dorsomedial part - VMHDM) (Fig. 3). Finally in the cerebellar cortex the cell body of the Purkinje cells showed a very dense staining for IL-18Rβ mRNA whereas the staining was very low in the molecular layer, confined only to a few cell bodies in the granular layer and almost absent in the white matter (Fig. 3).

3.3. Peripheral LPS treatment strongly elevated IL-18Rβ mRNA levels in the mouse brain

The LPS-induced regulation of IL-18Rβ mRNA expression in the mouse brain was assessed three hours after intraperitoneal injection of 100 μg (1μg/μl) of LPS or vehicle (saline-100 μl) followed by Real Time PCR on the same brain regions investigated for the basal level.

A two-factor ANOVA (LPS or saline injection × brain area) demonstrated an overall main effect of treatment [F (15,32) = 331.17; p < .0001] and brain area [F (15,32) = 5.46; p < .0001]. Moreover, an interaction was observed between the two terms [F (15,32) = 3.10, p = .01]. Three hours after LPS injection (100 μg/mouse), a marked increase in the expression of full-length IL-18Rβ was observed in all brain regions with respect to control levels detected in each distinct brain area (Fig. 4A) (in detail: frontal cortex: t = 5.25, p = .006; olfactory bulb: t = 5.17, p = .007; striatum: t = 6.89, p = .002; hypothalamus: t = 8.01, p = .001; thalamus: t = 5.39, p = .006; hippocampus: t = 8.85, p = 9.01 ×10⁻⁴; cerebral cortex: t = 7.48, p = .002; cerebellum: t = 16.02, p = 8.86×10⁻⁵).

The expression of the short IL-18Rβ variant was also strongly induced three hours after the LPS injection (100 mg/mouse) (Fig. 4B) but in a more pronounced way than observed for the full-length isoform. A two-factor ANOVA (type of injection × brain area) demonstrated an overall main effect of type of injection [F (15,32) = 266.51; p < .0001] and area [F (15,32) = 3.48; p =.007] whereas an interaction between the two terms was not observed. Significant LPS-induced up-regulation of the sIL-18b mRNA expression was observed in all areas evaluated (in detail: frontal cortex: t = 6.64, p = .002; olfactory bulb: t = 6.27, p = .02; striatum: t = 102.08, p = 9.59×10⁻⁵; hypothalamus: t = 8.97, p = .012; thalamus: t = 4.56, p = .01; hippocampus: t = 5.08, p = .007; cerebral cortex: t = 12.31, p = 2.50×10⁻⁴; cerebellum: t = 9.00, p = 8.51×10⁻⁴). As observed for the canonical beta chain, also the expression of the short variant of the accessory chain was not significantly changed following LPS injection in the spleen (data not shown).

In the spleen (used for comparison and for monitoring treatment efficacy), LPS treatment induced a tenfold increase in IL-1β mRNA expression (t = 8.57, p = .001) and a four hundred fold elevation of IL-6 mRNA (t = 5.74, p = .004). however, no effect was found with respect to the expression levels of IL-18Rβ mRNA or, as previously reported by Abu Elhija and co-workers (2008), on IL-18Rα transcript (data not shown).

We also analyzed the relative expression of full- and short-length IL-18Rβ in animals injected with LPS or vehicle (supplementary Fig. 1). A two-factor ANOVA (LPS or saline injection × brain area) demonstrated an overall main effect of type of injection (LPS or vehicle) [F (15,32) = 24.77; p < .0001] and an interaction between the two terms [F (15,32) = 2.47; p = .038]. Planned post-hoc analyses revealed that relative differences in the expression of the full-length/short IL-18Rβ ratio in mice exposed to LPS or exposed to saline differed significantly only in the frontal cortex (t = −3.33, p = .03), in the olfactory bulb (t = −3.521, p = .02), and in the striatum (t = −23.27, p < .001). Ratios did not differ significantly in hypothalamus and cortex. This is possibly due to the high variance observed in these areas, which can in part be explained by the fact that in basal conditions the short IL-18Rβ variant was expressed at very low levels in these two areas (ct values were higher than 35).

Finally we evaluated the central effect of the same LPS challenge on other components of the IL-18 system including the cytokines IL-18 and the two isoforms of the alpha chain of the IL-18 receptor (supplementary Tab. 2).

Compared to saline injected animals, the peripheral administration of LPS significantly increased the levels of IL-18Rα type II specifically in the cerebral cortex (t = 4.70; p = .009) and in the hippocampus (t = 4.48; p = .01) whereas no significant (p ≥ .01) effect was observed in the expression of IL-18Rα type I and IL-18 mRNAs in all brain areas evaluated (supplementary Tab. 2).

3.4. CNS basal and LPS-induced IL-18Rβ protein

Using immunohistochemistry, on paraffin-embedded brain sections, we have identified regions with endogenous basal expression of IL-18Rβ. We have highlighted the amigdala (Fig. 5A and 5B), cerebellum (Fig. 5C and 5D), hippocampus (Fig. 5E and 5F) and POA (Fig. 5G and 5H), In these regions as well as in others, LPS increased the expression of IL-18Rβ protein (Fig. 5B, D, F and H) in comparison to PBS-injected controls (Fig. 5A, C, E and G). Particularly in the cerebellum, we have noticed that the IL-18Rβ up-regulation occurred in neuronal cell bodies (Fig. 5C and 5D).