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Developmental expression of IL-33 in the mouse brain
Neuroscience Letters 555 (2013) 171–176
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Developmental expression of IL-33 in the mouse brain Grzegorz Wicher a , Ena Husic a , Gunnar Nilsson b , Karin Forsberg-Nilsson a,∗ a b
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Solna 171 76, Stockholm, Sweden
h i g h l i g h t s • • • • •
IL-33 is important in inflammation, but less understood in the normal brain. We mapped expression of IL-33 in embryonic and postnatal mouse brain. IL-33 expression peaked during the first three weeks of postnatal life. Astroctyes and oligodendrocyte precursors expressed IL-33. IL-33 may thus have a role in the absence of an inflammatory response.
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Article history: Received 27 June 2013 Received in revised form 16 September 2013 Accepted 17 September 2013 Keywords: Alarmin Astrocytes Glia Development Neuroinflammation
a b s t r a c t IL-33 has important functions in inflammatory and autoimmune diseases. In the brain, models of experimental encephalomyelitis are accompanied by up-regulation of IL-33 expression, and the cytokine is seen as an amplifier of the innate immune response. Little is known, however, about IL-33 the normal brain in adult life, or during development. We have analyzed the expression of IL-33 in the mouse brain during embryonic and postnatal development. Here we report that IL-33 expression was first detected in the CNS during late embryogenesis. From postnatal day 2 (P2) until P9 the expression increased and was strongest in the cerebellum, pons and thalamus, as well as in olfactory bulbs. Expression of IL-33 then became weaker and declined until P23, and it was not present in the adult brain. Both astrocytes and oligodendrocyte precursors expressed IL-33. The vast majority of IL-33 positive cells in the brain displayed nuclear staining, and this was found to be the case also in vitro, using mixed glial cultures. Our data suggest that IL-33 expression is under tight regulation in the normal brain. Its detection during the first three weeks of postnatal life coincides with important parts of the CNS developmental programs, such as general growth and myelination. This opens the possibility that IL-33 plays a role also in the absence of an inflammatory response. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The cytokine interleukin 33 (IL-33) was described independently in 1999 as an unknown protein named DVS27 and in 2003 as a nuclear factor from high endothelial venules (NF-HEV) [1,20]. Classified in 2005 as a new member of the IL-1 cytokine family, this 30 kDa protein with high similarity to IL-18 became known as an “alarmin” with crucial roles in the innate immune response [12,21]. IL-33 binds and signals through a receptor complex consisting of the ST2 receptor and the IL-1R accessory protein [5]. Subsequent intracellular signaling events include activation of the ERK and JNK pathways (reviewed in [19]). The localization of IL-33 is nuclear, and it becomes released by necrotic cells after tissue injury. It has
∗ Corresponding author. Tel.: +46 18 471 41 58. E-mail address: [email protected] (K. Forsberg-Nilsson). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.09.046
also been shown that IL-33 is involved in transcriptional modulation [3]. IL-33 has important functions in inflammatory and autoimmune diseases (e.g. asthma, inflammatory bowel disease, autoimmune hepatitis) modulating and activating a variety of immune signaling pathways [7,9,10,21,23]. In the nervous system, high levels of IL-33 mRNA is followed by IL-33 protein expression in a subset of spinal cord astrocytes after experimental allergic encephalomyelitis [24]. Furthermore, IL-33 expression is induced in vitro in pathogenstimulated astrocytes and microglia cultures [13,24]. Additionally, IL-33 was reported to induce microglia proliferation in vitro [24]. The developmental expression of IL-33 in the central nervous system (CNS), however, has not been reported. Here we characterized the expression and anatomical location of the IL-33 protein during prenatal and postnatal mouse brain development, and in differentiating mixed glial cell culture. Our results show that IL-33 is present in the intact, non-inflamed CNS during postnatal development, and opens the possibility that it
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may have additional functions besides its role in tissue injury and trauma. 2. Materials and methods 2.1. Animals and dissection In accordance with the Swedish legislation, the local Ethics Committee for Laboratory Animals approved the animal protocol. C57Bl/6 mice were used in this study, and embryonic stage (E) was based on plug date. Embryos from three pregnant mice per stage were removed at embryonic ages E11, E13, E15, E17 and E19 and transferred for 24 h to a fixative solution consisting of 4% formaldehyde in phosphate buffered saline (PBS; 0.15 M, pH 7.4, 4 ◦ C). To analyze the postnatal (P) pattern of IL-33 expression, mice (at least 3 animals per experimental group) from ages P2, P9, P16 and P23 were euthanized with CO2 , after which brains were dissected and fixed in 4% formaldehyde in PBS. After fixation, whole embryos and brains were cryo protected overnight in 10% sucrose in phosphate buffer (4 ◦ C). Coronal and sagittal cryostat sections (14 m) were made from the whole embryo or brain (postnatal stages), and stored in −20 ◦ C before staining. 2.2. Mixed-glia primary culture Mixed glial cell cultures containing astrocytes, oligodendrocytes and microglia were prepared as previously described [18]. In brief, cultures were prepared from the brain of newborn C57Bl/6 mice and placed in cold Leibovitz’s L-15 medium (Invitrogen, Stockholm, Sweden). Tissues were mechanically dissociated through a glass Pasteur pipette and passed through a 70 m nylon cell strainer (BD Falcon, US) to remove all large fragments. Dissociated cells were washed twice in cold L-15 and centrifuged for 10 min at 1000 rpm to remove debris. After centrifugation the cells were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen), 0.3% l-glutamine (Invitrogen) and 1% penicillin–streptomycin (Sigma–Aldrich, Stockholm, Sweden). Cells were then plated at a density of 2 × 105 cells/cm3 on poly-l-lysine-coated tissue culture dishes (BD, Stockholm, Sweden) or glass coverslip. Mixed-glia cultures were harvested after 1, 6 and 10 days in vitro. Samples for protein preparation were briefly washed with PBS, frozen and stored in -20. Cultures for immunocytochemistry were fixed in 4% paraformaldehyde for 10 min, cry protected in 10% sucrose in PBS, and frozen until processed for immuno-labeling. The experiment was performed three times. 2.3. Western blot analysis Frozen cell pellets were lyzed in 200 l lysis buffer (50 mM Tris–HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1% Trasylol, 2 mM NaVO4 , adjusted to pH 7.4) on ice for 30 min and mechanically homogenized and centrifuged at 16 000 × g at 4 ◦ C for 10 min. The samples were sonicated (Soniprep150, MSE, London, UK) on ice and cleared by centrifugation (16 000 × g, 4 ◦ C, 10 min). Protein concentrations were determined using BCA Protein Assay Kit and Albumin Standards (Pierce, Rockford, US). Equal amounts of protein were loaded on a 4–12% Bis-Tris polyacrylamide gradient gel (NuPAGE, Invitrogen) under reducing conditions. Proteins were blotted onto a nitrocellulose membrane (Hybond-ECL, GE Healthcare, Sweden). The membrane was blocked 1 h at room temperature (RT), with 5% bovine serum albumin in TBS-T (10 mM Tris–HCl 0.15 mM NaCl, pH 7.7, 2% Tween) followed by overnight incubation at 4 ◦ C with antibody in blocking solution. After five washes with TBS-T, the membrane was incubated for 1 h at RT with
horseradish peroxidase-labeled donkey anti-mouse immunoglobulin (1:5000; GE Healthcare) and washed in TBS-T. Bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, US) and developed on Hyperfilm-ECL (GE Healthcare). Beta-actin (monoclonal mouse beta-actin, 1:5000, Millipore, Billerica, US) was used as a loading control. WB quantitative analysis was performed in ImageJ (NIH, USA). The western blot was done twice, with different cell cultures as starting material. 2.4. Immunochemistry Defrosted tissue sections as well as cultures were first preincubated with blocking solution (1% Bovine Serum Albumin (BSA), 0.3% Triton X-100 and 0.1% NaN3 in PBS) for 1 h at RT. Samples were incubated overnight at 4 ◦ C with primary antibody (see below). After washing in 0.25% Triton X-100 in PBS, secondary antibodies (see below) were applied and incubated for 2 h at RT. Samples were washed twice and mounted with DTG mounting media (2.5% DABCO (Sigma–Aldrich), 50 mM Tris–HCl pH 8.0, 90% glycerol) with or without 0.375 ng/ml DAPI (Sigma–Aldrich). The following primary antibodies were used to label tissues and/or cell cultures: goat anti-IL-33 (1:500, #AF3626, R&D Systems, US) against recombinant mouse IL-33 (#3626-ML, R&D System), mouse anti-GFAP (1:200, #G3893, Sigma–Aldrich), rabbit anti-Olig-2 (1:500, #AB9610, Millipore), mouse anti-Map2 (1:200, #M4403, Sigma–Aldrich) and rat anti-CD11b (1:500, #CL8942AP, Cadarlane, Canada). Secondary antibodies included Cy3-coniugated donkey anti-goat (1:200, #705-165-003, Jackson ImmunoResearch, US), FITC-conjugated donkey anti-mouse (1:200, #715-095-150, Jackson ImmunoResearch), FITC-conjugated donkey anti-rat (1:200, #712-095-150, Jackson ImmunoResearch) and FITC-conjugated donkey anti-rabbit (1:200, #711-095-152, Jackson ImmunoResearch). 2.5. Specificity of IL-33 immunolabeling Immunohistochemical staining with the IL-33 antibody resulted in strong immunoreactivity (IR) in mixed glia cell cultures at 10DIV (Supplementary Fig. 1A) and in brain sections (pons) from P9 (Supplementary Fig. 1B). No IR was found in cultures (Supplementary Fig. 1C) or tissue sections (Supplementary Fig. 1D) after pre-absorption of IL-33 antibodies with an IL-33 blocking peptide or after omission of the primary antibody from the incubation procedure (data not shown). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2013.09.046. 2.6. Microscopy, image acquisition and quantification Stained slides were analyzed using a Zeiss AxioVision fluorescent microscope system. Raw images were transferred to ImageJ (NIH, USA) and semi-automated counts of cells from in vitro and in vivo samples were performed. Briefly, due to the various flouro-chromes used during immunelabeling, and the correlated variety of signal intensity, the threshold was adjust manually and images were processed for automatic counting and analysis. Each quantitative analysis was based on minimum 10 photographs from each investigated brain region or cell culture dish. Both for in vivo and in vitro analysis a minimum of 4000 cells were analyzed per each stage. For statistical analysis, oneway ANOVA was applied. Microsoft Excel was used to generate graphs. The images were processed and arranged in Adobe Photoshop CS5.
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olfactory bulbs, but not in other parts of the central nervous system. During all embryonic stages, strong IL-33 IR was found in several non-neural tissues such as bones, lung and liver, with particularly high expression in the developing liver and lungs of stages E17 and E19. Fig. 1B depicts a section through the canicular stage of lung development (E17) displaying strong expression around the developing alveoli.
3.2. Postnatal expression of IL-33 protein
Fig. 1. IL-33 expression during embryonic development. IL-33 expressing cells in neural and non-neural tissue at E11, E13, E15 and E17. (A) IL-33 positive cells in the developing retina at E19; (B) IL-33 expression in the E17 developing lung. Scale bar 100 m.
3. Results 3.1. Embryonic expression of IL-33 protein We analyzed the embryonic mouse brain for IL-33 expression by immunohistochemistry on coronal and sagittal sections. IL-33 protein was detected from embryonic day 19 (E19). At this developmental stage, nuclear IL-33 immunoreactivty (IR) was found in the developing retina (Fig. 1A) and in the glomerular layer of the
In contrast to its scant expression in the embryonic brain, intense IL-33 IR was readily detected during postnatal brain development. In Figs. 2B and S2, photomicrographs depict the change in expression pattern from early postnatal (P2) to weaning-stage (P23) mice. This is accompanied by graphical representations of the photographed slides (Fig. 2A) giving an overview of IL-33 IR detected on sagittal sections, as well as quantitative analysis of IL33 IR cells in the different areas investigated (Fig. 2C). At P2, IL-33 IR cells were mostly detected in pons while at P9 IL-33 IR cells were spread over large areas of the brain. At this time, the strongest IL33 IR was found in the hindbrain, with distinct labeling of cells in the cerebellum and pons. Additionally, numerous IL-33 labeled cells were found in the midbrain, thalamic structures and olfactory bulbs. In the frontal cerebral cortex, IL-33 IR cells were present in low numbers, without any specificity to specific cortical layers. No IL-33 IR cells were found in the striatum of P9 mouse brain. At P16 there were fewer IL-33 IR cells than at P9, and the numbers had further decreased by P23. We still found IL-33 IR cells in pons, as well as in the olfactory bulb, but the number of IL-33 IR cells in olfactory bulb at P23 was much lower then that observed at P16.
Fig. 2. Postnatal expression of IL-33 in the mouse brain. (A) Graphical representation of IL-33 expression in postnatal stages (P2, P9, P16 and P23). (B) At P2, IL-33 IR was not seen in thalamus and cerebellum but found in hindbrain, including pons. At P9, robust IL-33 expression was found in thalamus, midbrain and hindbrain regions including pons and cerebellum. At P16, IL-33 expression was detectable in thalamus, pons and the hindbrain except for cerebellum At P23, IL-33 expression had almost completely vanished from thalamus (J) and midbrain where only single positive cells persisted. In pons and hindbrain except for cerebellum, the expression of IL-33 remained strong. Scale bar 100 m. (C) Quantification of the average number of IL-33 IR cells at different postnatal developmental stages, and in various brain regions (OB, olfactory bulb, FC, frontal cortex, TH, thalamus, PO, pons, CB, cerebellum).
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Fig. 3. Identification of IL-33 expressing cells in vivo. (A) GFAP/IL-33 and (B) Olig2/IL-33 (B) double positive cells at P9 in the frontal cerebral cortex (arrows). (C) Map2-positive neurons were negative for IL-33, also at P9. Scale bar 100 m. (D) Quantitative analysis of glia type-cell contributions to the IL-33 positive cell population.
IL-33 IR cells were present in thalamus and midbrain structures of P16 but were further diminished by P23. No IL-33 IR cells were observed in the cerebellum at P16 or P23, and striatum remained negative with regard to immunostaining for IL-33 also at these stages. Taken together, we report that IL-33 was absent from the mouse brain until late embryogenesis, and then increased to reach its peak expression around P9, after which it decreased progressively until weaning, when levels were similar to those of P2. IL-33 expression could not be detected in the adult mouse brain (not shown). In all developmental stages examined, the expression of IL-33 was found to be predominantly located in the nuclei. However, a low level of immunoreactivity for IL-33 could also be found in the cytoplasm (Fig. S3A). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j. neulet.2013.09.046. 3.3. Identification of IL-33 immunoreactive cells in brain To determine the phenotype of the IL-33 IR cells we analyzed co-expression of IL-33 and markers for astrocytes (GFAP), oligodendrocyte progenitors (Olig2) or neurons (Map2) in postnatal stage P9 (Fig. 3). We detected GFAP-positive astrocytes (Fig. 3A) and Olig-2-positive oligodendrocyte progenitors (Fig. 3B)
expressing IL-33 while most Map2-positive neurons in frontal cortex were negative for IL-33 (Fig. 3C). Quantitative analysis of double-labeled cells showed that 30% of the IL-33 positive cells were oligodendrocyte progenitors and 20% were astrocytes. Thus, with the near absence of neurons co-staining with IL-33 we conclude that glia is the main neural cell population expressing IL-33 during postnatal brain development (Fig. 3D). 3.4. Expression of IL-33 in mixed glial cell cultures We next examined the expression pattern of IL-33 in mixed-glial cultures obtained from the perinatal mouse brain. Astrocytes, oligodendrocytes and microglia sequentially differentiate and increase in number in mixed glia cultures as they do in vivo. At 10 DIV our mixed glial cultures contained approximately 50–60% astrocytes, 20–30% oligodendrocytes/oligodendrocyte precursors, 10–20% microglia and very few neurons (<5%, data not shown). Cultures were kept for 1, 6 or 10 days in vitro (DIV), during which differentiation proceeded, and IL-33 expression was examined with immunocytochemistry (Fig. 4A–C) and quantified (Fig. 4D). At the first day of mixed-glia cells in culture, we found occasional IL-33 IR cells (Fig. 4A), and the number of positive cells increased with time in culture (Fig. 4B and C) as can be seen from quantification of the number of cells expressing IL-33 (Fig. 4D). IL-33 protein expression was analyzed by western blot (Fig. 4E) and quantification (Fig. 4F)
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Fig. 4. IL-33 expression in primary mixed-glia cultures. At 1 day (A) in vitro (DIV) single IL-33 cells were seen while at day 6 (B) numerous positive cells were present and the numbers had further increased by 10 DIV (C). Scale bar 100 m. Quantitative representation of temporal pattern of IL-33 IR in cultures (D) showing statistically significant increasing number of IL-33 IR cells over time DIV (one way ANOVA, n = 4, ***p < 0.001; **p < 0.01). Immunoblot of IL-33 protein (E) with quantification (F) at the corresponding time points, showing expression of the 30 kDa form of the IL-33 protein (n = 2).
Fig. 5. Identification of IL-33 expressing cells in vitro. At 10 DIV we detected GFAPpositive astrocytes (A, arrows) and Olig-2-positive oligodendrocytes (B, arrows) double positive for IL-33. Scale bar 100 m.
showed an increase in total amount of IL-33 during the first six days in culture. We detected the unprocessed 30 kDa form of IL-33 while the processed, 18 kDa form was not seen. Furthermore, subcellular localization of IL-33 protein revealed mainly nuclear staining, but also some cytoplasmic localization in mixed-glial cultures (Supplementary Fig. 3B). 3.5. Identification of IL-33-immunoreactive cells in vitro To determine the phenotype of the IL-33 IR cells in mixed glial cell cultures we analyzed co-expression of IL-33 with GFAP and Olig2 at 10 DIV (Fig. 5). We detected co-labeling of IL-33 with GFAPpositive astrocytes (Fig. 5A) and Olig-2-positive oligodendrocyte progenitors (Fig. 5B). 4. Discussion In the present study, we describe how the IL-33 expression pattern changes with time in the CNS, from early embryonic stages until the mouse brain has matured. Our data do not suggest
a crucial role for IL-33 in normal development of the first two trimesters of prenatal life since it could not be detected until E19. In contrast, IL-33 was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased, and was then absent from the normal adult brain. Our study provides evidence that IL-33 expression during development does not demand activation of immune response pathways inducing inflammation. Instead, the temporal and spatial pattern of IL-33 expression during development suggests that IL-33 is correlated to naturally occurring processes of growth and maturation. From postnatal day 2 to shortly after weaning, there is a rapid growth of the average mouse brain size [2,11,17]. The rodent brain then continues to expand in total mass over the first three months, but the fastest growth occurs until P25 [2,11,17]. It is interesting to note that the IL-33 IR pattern observed in our study correlates to this period of rapid growth of the brain. This is also a time when substantial mechanical tension occurs, due to the major remodeling processes and compaction of brain tissue. Recent studies have shown that mechanical stress can induce expression of IL-33 [15] and one possible cause of IL-33 up-regulation could be mechano sensing. IL-33 expression in both the developing and maturing CNS was found almost exclusively in the nucleus, both in vivo and in vitro, with only a small fraction of IL-33 staining detected in the cytoplasm. This is in line with previous observations in vitro, where IL-33 was nuclear and associated with heterochromatin [2,11,17]. Our expression data and the recent finding that nuclear IL-33 can act as a transcription factor [6] in endothelial cells opens interesting possibilities for transcriptional regulation by IL-33 also in the normal nervous system. The IL-33 detected was of the intracellular unprocessed form. This is in line with a previous study [24], and suggests that under regular culture conditions, the appropriate proteases to digest the 30 kDa IL-33 into the 18 kDa form are not present. Co-localization of IL-33 with GFAP expression in the postnatal stages suggests a role for IL-33 in mature astrocytes. Astrocytes are of great importance in many postnatal developmental processes such as myelination, synaptogenesis, synapse pruning and elimination [16,22], and further investigations are needed to explore the precise role of IL-33 in astrocyte maturation. The characterization of IL-33 as an alarmin suggests this cytokine as an essential player
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in pro-inflammatory events [4,12]. Recent studies demonstrate an encephalomyelitis virus-induced IL-33-ST2 signaling in astrocytes, which may modulate innate immune responses [13,24], but no role has so far been ascribed to IL-33 in astrocytes that are not subject to injury. Oligodendrocyte precursors were also positive for IL-33. This is to our knowledge the first report of IL-33 expression in immature oligodendrocytes. The sequence of IL-33-IR cells appearance in the developing mouse brain correlates with maturation of cortico-spinal tracts (CST), involving nerve sprouting, and early synaptogenesis [8]. Formation and functional selection of corticospinal projection occurs during the first two weeks of rodent life, which is when most fibers reach their target structures [14]. IL-33 may have a role in this process. IL-33 expression in oligodendrocyte precursors also correlated with fiber myelination in the brain, which occurs after birth and is completed by the third postnatal week. Taken together, our data suggest that the inflammatory cytokine IL-33 has a function in the brain, also in the absence of an inflammatory Conflict of interest The authors declare no conflict of interests Acknowledgement This study was supported by grants from the Swedish Research Council. References [1] E.S. Baekkevold, M. Roussigne, T. Yamanaka, F.E. Johansen, F.L. Jahnsen, F. Amalric, P. Brandtzaeg, M. Erard, G. Haraldsen, J.P. Girard, Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules, Am. J. Pathol. 163 (2003) 69–79. [2] F. Bandeira, R. Lent, S. Herculano-Houzel, Changing numbers of neuronal and non-neuronal cells underlie postnatal brain growth in the rat, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 14108–14113. [3] V. Carriere, L. Roussel, N. Ortega, D.A. Lacorre, L. Americh, L. Aguilar, G. Bouche, J.P. Girard, IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 282–287. [4] C. Cayrol, J.P. Girard, The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 9021–9026. [5] A.A. Chackerian, E.R. Oldham, E.E. Murphy, J. Schmitz, S. Pflanz, R.A. Kastelein, IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex, J. Immunol. 179 (2007) 2551–2555. [6] Y.S. Choi, J.A. Park, J. Kim, S.S. Rho, H. Park, Y.M. Kim, Y.G. Kwon, Nuclear IL-33 is a transcriptional regulator of NF-kappaB p65 and induces endothelial cell activation, Biochem. Biophys. Res. Commun. 421 (2012) 305–311.
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Developmental expression of IL-33 in the mouse brain Grzegorz Wicher a , Ena Husic a , Gunnar Nilsson b , Karin Forsberg-Nilsson a,∗ a b
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Solna 171 76, Stockholm, Sweden
h i g h l i g h t s • • • • •
IL-33 is important in inflammation, but less understood in the normal brain. We mapped expression of IL-33 in embryonic and postnatal mouse brain. IL-33 expression peaked during the first three weeks of postnatal life. Astroctyes and oligodendrocyte precursors expressed IL-33. IL-33 may thus have a role in the absence of an inflammatory response.
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 16 September 2013 Accepted 17 September 2013 Keywords: Alarmin Astrocytes Glia Development Neuroinflammation
a b s t r a c t IL-33 has important functions in inflammatory and autoimmune diseases. In the brain, models of experimental encephalomyelitis are accompanied by up-regulation of IL-33 expression, and the cytokine is seen as an amplifier of the innate immune response. Little is known, however, about IL-33 the normal brain in adult life, or during development. We have analyzed the expression of IL-33 in the mouse brain during embryonic and postnatal development. Here we report that IL-33 expression was first detected in the CNS during late embryogenesis. From postnatal day 2 (P2) until P9 the expression increased and was strongest in the cerebellum, pons and thalamus, as well as in olfactory bulbs. Expression of IL-33 then became weaker and declined until P23, and it was not present in the adult brain. Both astrocytes and oligodendrocyte precursors expressed IL-33. The vast majority of IL-33 positive cells in the brain displayed nuclear staining, and this was found to be the case also in vitro, using mixed glial cultures. Our data suggest that IL-33 expression is under tight regulation in the normal brain. Its detection during the first three weeks of postnatal life coincides with important parts of the CNS developmental programs, such as general growth and myelination. This opens the possibility that IL-33 plays a role also in the absence of an inflammatory response. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The cytokine interleukin 33 (IL-33) was described independently in 1999 as an unknown protein named DVS27 and in 2003 as a nuclear factor from high endothelial venules (NF-HEV) [1,20]. Classified in 2005 as a new member of the IL-1 cytokine family, this 30 kDa protein with high similarity to IL-18 became known as an “alarmin” with crucial roles in the innate immune response [12,21]. IL-33 binds and signals through a receptor complex consisting of the ST2 receptor and the IL-1R accessory protein [5]. Subsequent intracellular signaling events include activation of the ERK and JNK pathways (reviewed in [19]). The localization of IL-33 is nuclear, and it becomes released by necrotic cells after tissue injury. It has
∗ Corresponding author. Tel.: +46 18 471 41 58. E-mail address: [email protected] (K. Forsberg-Nilsson). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.09.046
also been shown that IL-33 is involved in transcriptional modulation [3]. IL-33 has important functions in inflammatory and autoimmune diseases (e.g. asthma, inflammatory bowel disease, autoimmune hepatitis) modulating and activating a variety of immune signaling pathways [7,9,10,21,23]. In the nervous system, high levels of IL-33 mRNA is followed by IL-33 protein expression in a subset of spinal cord astrocytes after experimental allergic encephalomyelitis [24]. Furthermore, IL-33 expression is induced in vitro in pathogenstimulated astrocytes and microglia cultures [13,24]. Additionally, IL-33 was reported to induce microglia proliferation in vitro [24]. The developmental expression of IL-33 in the central nervous system (CNS), however, has not been reported. Here we characterized the expression and anatomical location of the IL-33 protein during prenatal and postnatal mouse brain development, and in differentiating mixed glial cell culture. Our results show that IL-33 is present in the intact, non-inflamed CNS during postnatal development, and opens the possibility that it
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may have additional functions besides its role in tissue injury and trauma. 2. Materials and methods 2.1. Animals and dissection In accordance with the Swedish legislation, the local Ethics Committee for Laboratory Animals approved the animal protocol. C57Bl/6 mice were used in this study, and embryonic stage (E) was based on plug date. Embryos from three pregnant mice per stage were removed at embryonic ages E11, E13, E15, E17 and E19 and transferred for 24 h to a fixative solution consisting of 4% formaldehyde in phosphate buffered saline (PBS; 0.15 M, pH 7.4, 4 ◦ C). To analyze the postnatal (P) pattern of IL-33 expression, mice (at least 3 animals per experimental group) from ages P2, P9, P16 and P23 were euthanized with CO2 , after which brains were dissected and fixed in 4% formaldehyde in PBS. After fixation, whole embryos and brains were cryo protected overnight in 10% sucrose in phosphate buffer (4 ◦ C). Coronal and sagittal cryostat sections (14 m) were made from the whole embryo or brain (postnatal stages), and stored in −20 ◦ C before staining. 2.2. Mixed-glia primary culture Mixed glial cell cultures containing astrocytes, oligodendrocytes and microglia were prepared as previously described [18]. In brief, cultures were prepared from the brain of newborn C57Bl/6 mice and placed in cold Leibovitz’s L-15 medium (Invitrogen, Stockholm, Sweden). Tissues were mechanically dissociated through a glass Pasteur pipette and passed through a 70 m nylon cell strainer (BD Falcon, US) to remove all large fragments. Dissociated cells were washed twice in cold L-15 and centrifuged for 10 min at 1000 rpm to remove debris. After centrifugation the cells were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen), 0.3% l-glutamine (Invitrogen) and 1% penicillin–streptomycin (Sigma–Aldrich, Stockholm, Sweden). Cells were then plated at a density of 2 × 105 cells/cm3 on poly-l-lysine-coated tissue culture dishes (BD, Stockholm, Sweden) or glass coverslip. Mixed-glia cultures were harvested after 1, 6 and 10 days in vitro. Samples for protein preparation were briefly washed with PBS, frozen and stored in -20. Cultures for immunocytochemistry were fixed in 4% paraformaldehyde for 10 min, cry protected in 10% sucrose in PBS, and frozen until processed for immuno-labeling. The experiment was performed three times. 2.3. Western blot analysis Frozen cell pellets were lyzed in 200 l lysis buffer (50 mM Tris–HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1% Trasylol, 2 mM NaVO4 , adjusted to pH 7.4) on ice for 30 min and mechanically homogenized and centrifuged at 16 000 × g at 4 ◦ C for 10 min. The samples were sonicated (Soniprep150, MSE, London, UK) on ice and cleared by centrifugation (16 000 × g, 4 ◦ C, 10 min). Protein concentrations were determined using BCA Protein Assay Kit and Albumin Standards (Pierce, Rockford, US). Equal amounts of protein were loaded on a 4–12% Bis-Tris polyacrylamide gradient gel (NuPAGE, Invitrogen) under reducing conditions. Proteins were blotted onto a nitrocellulose membrane (Hybond-ECL, GE Healthcare, Sweden). The membrane was blocked 1 h at room temperature (RT), with 5% bovine serum albumin in TBS-T (10 mM Tris–HCl 0.15 mM NaCl, pH 7.7, 2% Tween) followed by overnight incubation at 4 ◦ C with antibody in blocking solution. After five washes with TBS-T, the membrane was incubated for 1 h at RT with
horseradish peroxidase-labeled donkey anti-mouse immunoglobulin (1:5000; GE Healthcare) and washed in TBS-T. Bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, US) and developed on Hyperfilm-ECL (GE Healthcare). Beta-actin (monoclonal mouse beta-actin, 1:5000, Millipore, Billerica, US) was used as a loading control. WB quantitative analysis was performed in ImageJ (NIH, USA). The western blot was done twice, with different cell cultures as starting material. 2.4. Immunochemistry Defrosted tissue sections as well as cultures were first preincubated with blocking solution (1% Bovine Serum Albumin (BSA), 0.3% Triton X-100 and 0.1% NaN3 in PBS) for 1 h at RT. Samples were incubated overnight at 4 ◦ C with primary antibody (see below). After washing in 0.25% Triton X-100 in PBS, secondary antibodies (see below) were applied and incubated for 2 h at RT. Samples were washed twice and mounted with DTG mounting media (2.5% DABCO (Sigma–Aldrich), 50 mM Tris–HCl pH 8.0, 90% glycerol) with or without 0.375 ng/ml DAPI (Sigma–Aldrich). The following primary antibodies were used to label tissues and/or cell cultures: goat anti-IL-33 (1:500, #AF3626, R&D Systems, US) against recombinant mouse IL-33 (#3626-ML, R&D System), mouse anti-GFAP (1:200, #G3893, Sigma–Aldrich), rabbit anti-Olig-2 (1:500, #AB9610, Millipore), mouse anti-Map2 (1:200, #M4403, Sigma–Aldrich) and rat anti-CD11b (1:500, #CL8942AP, Cadarlane, Canada). Secondary antibodies included Cy3-coniugated donkey anti-goat (1:200, #705-165-003, Jackson ImmunoResearch, US), FITC-conjugated donkey anti-mouse (1:200, #715-095-150, Jackson ImmunoResearch), FITC-conjugated donkey anti-rat (1:200, #712-095-150, Jackson ImmunoResearch) and FITC-conjugated donkey anti-rabbit (1:200, #711-095-152, Jackson ImmunoResearch). 2.5. Specificity of IL-33 immunolabeling Immunohistochemical staining with the IL-33 antibody resulted in strong immunoreactivity (IR) in mixed glia cell cultures at 10DIV (Supplementary Fig. 1A) and in brain sections (pons) from P9 (Supplementary Fig. 1B). No IR was found in cultures (Supplementary Fig. 1C) or tissue sections (Supplementary Fig. 1D) after pre-absorption of IL-33 antibodies with an IL-33 blocking peptide or after omission of the primary antibody from the incubation procedure (data not shown). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2013.09.046. 2.6. Microscopy, image acquisition and quantification Stained slides were analyzed using a Zeiss AxioVision fluorescent microscope system. Raw images were transferred to ImageJ (NIH, USA) and semi-automated counts of cells from in vitro and in vivo samples were performed. Briefly, due to the various flouro-chromes used during immunelabeling, and the correlated variety of signal intensity, the threshold was adjust manually and images were processed for automatic counting and analysis. Each quantitative analysis was based on minimum 10 photographs from each investigated brain region or cell culture dish. Both for in vivo and in vitro analysis a minimum of 4000 cells were analyzed per each stage. For statistical analysis, oneway ANOVA was applied. Microsoft Excel was used to generate graphs. The images were processed and arranged in Adobe Photoshop CS5.
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olfactory bulbs, but not in other parts of the central nervous system. During all embryonic stages, strong IL-33 IR was found in several non-neural tissues such as bones, lung and liver, with particularly high expression in the developing liver and lungs of stages E17 and E19. Fig. 1B depicts a section through the canicular stage of lung development (E17) displaying strong expression around the developing alveoli.
3.2. Postnatal expression of IL-33 protein
Fig. 1. IL-33 expression during embryonic development. IL-33 expressing cells in neural and non-neural tissue at E11, E13, E15 and E17. (A) IL-33 positive cells in the developing retina at E19; (B) IL-33 expression in the E17 developing lung. Scale bar 100 m.
3. Results 3.1. Embryonic expression of IL-33 protein We analyzed the embryonic mouse brain for IL-33 expression by immunohistochemistry on coronal and sagittal sections. IL-33 protein was detected from embryonic day 19 (E19). At this developmental stage, nuclear IL-33 immunoreactivty (IR) was found in the developing retina (Fig. 1A) and in the glomerular layer of the
In contrast to its scant expression in the embryonic brain, intense IL-33 IR was readily detected during postnatal brain development. In Figs. 2B and S2, photomicrographs depict the change in expression pattern from early postnatal (P2) to weaning-stage (P23) mice. This is accompanied by graphical representations of the photographed slides (Fig. 2A) giving an overview of IL-33 IR detected on sagittal sections, as well as quantitative analysis of IL33 IR cells in the different areas investigated (Fig. 2C). At P2, IL-33 IR cells were mostly detected in pons while at P9 IL-33 IR cells were spread over large areas of the brain. At this time, the strongest IL33 IR was found in the hindbrain, with distinct labeling of cells in the cerebellum and pons. Additionally, numerous IL-33 labeled cells were found in the midbrain, thalamic structures and olfactory bulbs. In the frontal cerebral cortex, IL-33 IR cells were present in low numbers, without any specificity to specific cortical layers. No IL-33 IR cells were found in the striatum of P9 mouse brain. At P16 there were fewer IL-33 IR cells than at P9, and the numbers had further decreased by P23. We still found IL-33 IR cells in pons, as well as in the olfactory bulb, but the number of IL-33 IR cells in olfactory bulb at P23 was much lower then that observed at P16.
Fig. 2. Postnatal expression of IL-33 in the mouse brain. (A) Graphical representation of IL-33 expression in postnatal stages (P2, P9, P16 and P23). (B) At P2, IL-33 IR was not seen in thalamus and cerebellum but found in hindbrain, including pons. At P9, robust IL-33 expression was found in thalamus, midbrain and hindbrain regions including pons and cerebellum. At P16, IL-33 expression was detectable in thalamus, pons and the hindbrain except for cerebellum At P23, IL-33 expression had almost completely vanished from thalamus (J) and midbrain where only single positive cells persisted. In pons and hindbrain except for cerebellum, the expression of IL-33 remained strong. Scale bar 100 m. (C) Quantification of the average number of IL-33 IR cells at different postnatal developmental stages, and in various brain regions (OB, olfactory bulb, FC, frontal cortex, TH, thalamus, PO, pons, CB, cerebellum).
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Fig. 3. Identification of IL-33 expressing cells in vivo. (A) GFAP/IL-33 and (B) Olig2/IL-33 (B) double positive cells at P9 in the frontal cerebral cortex (arrows). (C) Map2-positive neurons were negative for IL-33, also at P9. Scale bar 100 m. (D) Quantitative analysis of glia type-cell contributions to the IL-33 positive cell population.
IL-33 IR cells were present in thalamus and midbrain structures of P16 but were further diminished by P23. No IL-33 IR cells were observed in the cerebellum at P16 or P23, and striatum remained negative with regard to immunostaining for IL-33 also at these stages. Taken together, we report that IL-33 was absent from the mouse brain until late embryogenesis, and then increased to reach its peak expression around P9, after which it decreased progressively until weaning, when levels were similar to those of P2. IL-33 expression could not be detected in the adult mouse brain (not shown). In all developmental stages examined, the expression of IL-33 was found to be predominantly located in the nuclei. However, a low level of immunoreactivity for IL-33 could also be found in the cytoplasm (Fig. S3A). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j. neulet.2013.09.046. 3.3. Identification of IL-33 immunoreactive cells in brain To determine the phenotype of the IL-33 IR cells we analyzed co-expression of IL-33 and markers for astrocytes (GFAP), oligodendrocyte progenitors (Olig2) or neurons (Map2) in postnatal stage P9 (Fig. 3). We detected GFAP-positive astrocytes (Fig. 3A) and Olig-2-positive oligodendrocyte progenitors (Fig. 3B)
expressing IL-33 while most Map2-positive neurons in frontal cortex were negative for IL-33 (Fig. 3C). Quantitative analysis of double-labeled cells showed that 30% of the IL-33 positive cells were oligodendrocyte progenitors and 20% were astrocytes. Thus, with the near absence of neurons co-staining with IL-33 we conclude that glia is the main neural cell population expressing IL-33 during postnatal brain development (Fig. 3D). 3.4. Expression of IL-33 in mixed glial cell cultures We next examined the expression pattern of IL-33 in mixed-glial cultures obtained from the perinatal mouse brain. Astrocytes, oligodendrocytes and microglia sequentially differentiate and increase in number in mixed glia cultures as they do in vivo. At 10 DIV our mixed glial cultures contained approximately 50–60% astrocytes, 20–30% oligodendrocytes/oligodendrocyte precursors, 10–20% microglia and very few neurons (<5%, data not shown). Cultures were kept for 1, 6 or 10 days in vitro (DIV), during which differentiation proceeded, and IL-33 expression was examined with immunocytochemistry (Fig. 4A–C) and quantified (Fig. 4D). At the first day of mixed-glia cells in culture, we found occasional IL-33 IR cells (Fig. 4A), and the number of positive cells increased with time in culture (Fig. 4B and C) as can be seen from quantification of the number of cells expressing IL-33 (Fig. 4D). IL-33 protein expression was analyzed by western blot (Fig. 4E) and quantification (Fig. 4F)
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Fig. 4. IL-33 expression in primary mixed-glia cultures. At 1 day (A) in vitro (DIV) single IL-33 cells were seen while at day 6 (B) numerous positive cells were present and the numbers had further increased by 10 DIV (C). Scale bar 100 m. Quantitative representation of temporal pattern of IL-33 IR in cultures (D) showing statistically significant increasing number of IL-33 IR cells over time DIV (one way ANOVA, n = 4, ***p < 0.001; **p < 0.01). Immunoblot of IL-33 protein (E) with quantification (F) at the corresponding time points, showing expression of the 30 kDa form of the IL-33 protein (n = 2).
Fig. 5. Identification of IL-33 expressing cells in vitro. At 10 DIV we detected GFAPpositive astrocytes (A, arrows) and Olig-2-positive oligodendrocytes (B, arrows) double positive for IL-33. Scale bar 100 m.
showed an increase in total amount of IL-33 during the first six days in culture. We detected the unprocessed 30 kDa form of IL-33 while the processed, 18 kDa form was not seen. Furthermore, subcellular localization of IL-33 protein revealed mainly nuclear staining, but also some cytoplasmic localization in mixed-glial cultures (Supplementary Fig. 3B). 3.5. Identification of IL-33-immunoreactive cells in vitro To determine the phenotype of the IL-33 IR cells in mixed glial cell cultures we analyzed co-expression of IL-33 with GFAP and Olig2 at 10 DIV (Fig. 5). We detected co-labeling of IL-33 with GFAPpositive astrocytes (Fig. 5A) and Olig-2-positive oligodendrocyte progenitors (Fig. 5B). 4. Discussion In the present study, we describe how the IL-33 expression pattern changes with time in the CNS, from early embryonic stages until the mouse brain has matured. Our data do not suggest
a crucial role for IL-33 in normal development of the first two trimesters of prenatal life since it could not be detected until E19. In contrast, IL-33 was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased, and was then absent from the normal adult brain. Our study provides evidence that IL-33 expression during development does not demand activation of immune response pathways inducing inflammation. Instead, the temporal and spatial pattern of IL-33 expression during development suggests that IL-33 is correlated to naturally occurring processes of growth and maturation. From postnatal day 2 to shortly after weaning, there is a rapid growth of the average mouse brain size [2,11,17]. The rodent brain then continues to expand in total mass over the first three months, but the fastest growth occurs until P25 [2,11,17]. It is interesting to note that the IL-33 IR pattern observed in our study correlates to this period of rapid growth of the brain. This is also a time when substantial mechanical tension occurs, due to the major remodeling processes and compaction of brain tissue. Recent studies have shown that mechanical stress can induce expression of IL-33 [15] and one possible cause of IL-33 up-regulation could be mechano sensing. IL-33 expression in both the developing and maturing CNS was found almost exclusively in the nucleus, both in vivo and in vitro, with only a small fraction of IL-33 staining detected in the cytoplasm. This is in line with previous observations in vitro, where IL-33 was nuclear and associated with heterochromatin [2,11,17]. Our expression data and the recent finding that nuclear IL-33 can act as a transcription factor [6] in endothelial cells opens interesting possibilities for transcriptional regulation by IL-33 also in the normal nervous system. The IL-33 detected was of the intracellular unprocessed form. This is in line with a previous study [24], and suggests that under regular culture conditions, the appropriate proteases to digest the 30 kDa IL-33 into the 18 kDa form are not present. Co-localization of IL-33 with GFAP expression in the postnatal stages suggests a role for IL-33 in mature astrocytes. Astrocytes are of great importance in many postnatal developmental processes such as myelination, synaptogenesis, synapse pruning and elimination [16,22], and further investigations are needed to explore the precise role of IL-33 in astrocyte maturation. The characterization of IL-33 as an alarmin suggests this cytokine as an essential player
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in pro-inflammatory events [4,12]. Recent studies demonstrate an encephalomyelitis virus-induced IL-33-ST2 signaling in astrocytes, which may modulate innate immune responses [13,24], but no role has so far been ascribed to IL-33 in astrocytes that are not subject to injury. Oligodendrocyte precursors were also positive for IL-33. This is to our knowledge the first report of IL-33 expression in immature oligodendrocytes. The sequence of IL-33-IR cells appearance in the developing mouse brain correlates with maturation of cortico-spinal tracts (CST), involving nerve sprouting, and early synaptogenesis [8]. Formation and functional selection of corticospinal projection occurs during the first two weeks of rodent life, which is when most fibers reach their target structures [14]. IL-33 may have a role in this process. IL-33 expression in oligodendrocyte precursors also correlated with fiber myelination in the brain, which occurs after birth and is completed by the third postnatal week. Taken together, our data suggest that the inflammatory cytokine IL-33 has a function in the brain, also in the absence of an inflammatory Conflict of interest The authors declare no conflict of interests Acknowledgement This study was supported by grants from the Swedish Research Council. References [1] E.S. Baekkevold, M. Roussigne, T. Yamanaka, F.E. Johansen, F.L. Jahnsen, F. Amalric, P. Brandtzaeg, M. Erard, G. Haraldsen, J.P. Girard, Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules, Am. J. Pathol. 163 (2003) 69–79. [2] F. Bandeira, R. Lent, S. Herculano-Houzel, Changing numbers of neuronal and non-neuronal cells underlie postnatal brain growth in the rat, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 14108–14113. [3] V. Carriere, L. Roussel, N. Ortega, D.A. Lacorre, L. Americh, L. Aguilar, G. Bouche, J.P. Girard, IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 282–287. [4] C. Cayrol, J.P. Girard, The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 9021–9026. [5] A.A. Chackerian, E.R. Oldham, E.E. Murphy, J. Schmitz, S. Pflanz, R.A. Kastelein, IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex, J. Immunol. 179 (2007) 2551–2555. [6] Y.S. Choi, J.A. Park, J. Kim, S.S. Rho, H. Park, Y.M. Kim, Y.G. Kwon, Nuclear IL-33 is a transcriptional regulator of NF-kappaB p65 and induces endothelial cell activation, Biochem. Biophys. Res. Commun. 421 (2012) 305–311.
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