Theranostics 2021, Vol. 11, Issue 1 1 Ivyspring International Publisher Theranostics 2021; 11(1): 1-13. doi: 10.7150/thno.47435 Research Paper IL-37 exerts therapeutic effects in experimental autoimmune encephalomyelitis through the receptor complex IL-1R5/IL-1R8 Alba Sánchez-Fernández1,2, Stephanie Zandee3, Jesús Amo-Aparicio1,2, Marc Charabati3, Alexandre Prat3, Cecilia Garlanda4, Elan Z. Eisenmesser5, Charles A. Dinarello6,7 and Rubèn López-Vales1,2 1. Institut de Neurociencies and Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, Bellaterra, Catalonia, Spain. 2. Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED). 3. Department of Neuroscience, Faculty of Medicine, Université de Montréal, and Neuroimmunology Unit, Centre de Recherche du CHUM (CRCHUM), Montréal, Québec, Canada. 4. Humanitas Clinical and Research Center, 20089 Rozzano, Italy. 5. Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO 80238, USA. 6. Department of Medicine, University of Colorado Denver, Aurora, CO 80045, USA. 7. Department of Medicine, Radboud University Medical Center, 6500 Nijmegen, The Netherlands.  Corresponding author: Prof. Rubèn López-Vales, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. E-mail: ruben.lopez@uab.cat. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2020.04.25; Accepted: 2020.08.23; Published: 2021.01.01 Abstract Background: Interleukin 37 (IL-37), a member of IL-1 family, broadly suppresses inflammation in many pathological conditions by acting as a dual-function cytokine in that IL-37 signals via the extracellular receptor complex IL1-R5/IL-1R8, but it can also translocate to the nucleus. However, whether IL-37 exerts beneficial actions in neuroinflammatory diseases, such as multiple sclerosis, remains to be elucidated. Thus, the goals of the present study were to evaluate the therapeutic effects of IL-37 in a mouse model of multiple sclerosis, and if so, whether this is mediated via the extracellular receptor complex IL-1R5/IL-1R8. Methods: We used a murine model of MS, the experimental autoimmune encephalomyelitis (EAE). We induced EAE in three different single and double transgenic mice (hIL-37tg, IL-1R8 KO, hIL-37tg-IL-1R8 KO) and wild type littermates. We also induced EAE in C57Bl/6 mice and treated them with various forms of recombinant human IL-37 protein. Functional and histological techniques were used to assess locomotor deficits and demyelination. Luminex and flow cytometry analysis were done to assess the protein levels of pro-inflammatory cytokines and different immune cell populations, respectively. qPCRs were done to assess the expression of IL-37, IL-1R5 and IL-1R8 in the spinal cord of EAE, and in blood peripheral mononuclear cells and brain tissue samples of MS patients. Results: We demonstrate that IL-37 reduces inflammation and protects against neurological deficits and myelin loss in EAE mice by acting via IL1-R5/IL1-R8. We also reveal that administration of recombinant human IL-37 exerts therapeutic actions in EAE mice. We finally show that IL-37 transcripts are not up-regulated in peripheral blood mononuclear cells and in brain lesions of MS patients, despite the IL-1R5/IL-1R8 receptor complex is expressed. Conclusions: This study presents novel data indicating that IL-37 exerts therapeutic effects in EAE by acting through the extracellular receptor complex IL-1R5/IL-1R8, and that this protective physiological mechanism is defective in MS individuals. IL-37 may therefore represent a novel therapeutic avenue for the treatment of MS with great promising potential. Key words: multiple sclerosis, experimental autoimmune encephalomyelitis, IL-37, IL-1R8, cytokines Introduction Multiple sclerosis (MS) is the most common worldwide [1]. Although its aetiology remains demyelinating disease of the central nervous system complex, MS is thought to be autoimmune in nature (CNS) and affects more than 2.5 million individuals due to the presence of myelin-targeting T cells and http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 2 autoantibodies in the cerebrospinal fluid of MS the transgenic expression of human IL-37 mediates patients [2–5]. Therefore, blocking inflammation will broad anti-inflammatory effects in EAE and protects likely be therapeutically beneficial for treating MS mice from the functional deficits and demyelination. patients. Furthermore, we show that the beneficial effects of The IL-1 family includes prototypic cytokines IL-37 in EAE are dependent on its extracellular that promote acute and chronic inflammation in a receptor function. Relevant to the studies in mice, we broad spectrum of diseases [6, 7]. Seven of the 11 discovered that this natural anti-inflammatory members exhibit pro-inflammatory properties, mechanism is deficient in blood and brain active whereas four members reduce inflammation [8]. Of lesions of MS patients. Lastly, we provide clear these four, IL-37 broadly suppresses innate as well as evidence that the administration of recombinant adaptive immunity [8–10]. human IL-37 protein markedly reduces EAE The IL-37 gene is composed by 6 exons that give pathogenesis. Altogether, our findings reveal that rise to five different isoforms (IL-37a to IL-37e) [11, IL-37 could be employed as a new approach for the 12]. However, only the isoforms a, b and d are treatment of MS. functional since, contrary to the isoforms c and e, they have the presence of the complete sequence encoding Materials and Methods the 12β-strands structure and at least one cleavage site MS brain tissue samples required for the maturation of the cytokine [13]. Among them, IL-37b (isoform 1) is the longer isoform Human brain tissue was obtained from MS and it is the one that has been used in experimental patients diagnosed with clinical and neuro- studies for its functional characterization [11]. pathological MS diagnosis according to the revised IL-37 exert anti-inflammatory actions by acting 2010 McDonald’s criteria [23]. Tissue samples were as a ligand for the extracellular receptor complex collected from MS patients with full ethical approval comprised of IL-1R5 (IL-18Rα) and the co-receptor, (BH07.001) and informed consent as approved by the IL-1R8 [14]. Similar to IL-1α and IL-33, intracellular local ethics committee. Autopsy samples were IL-37 translocates to the nucleus via caspase-1 cryopreserved and lesions classified using Luxol Fast cleavage and binding to SMAD3 [13, 15]. Blue (LFB)/Haematoxylin & Eosin staining and Oil Unlike other members of the IL-1 family, the Red O staining as previously published [24, 25]. open read frame for the IL-37 homolog is lacking in Normal appearing white matter (NAWM) and active mice. To investigate the role IL-37 in animal models of lesions representing two relapsing-remitting MS human diseases, it was thus necessary to generate a (RRMS) (one male and one female) and seven knock-in mouse expressing human IL37b (hIL-37tg) secondary-progressive MS (SPMS) (three males and 1[6]. hIL-37tg mice have demonstrated resistance four females) were used (Table S1). The median age at against many pathological conditions, such as death was 50 years (range from 26 to 65 years). systemic endotoxemia [9], colitis [17] and obesity- Regions of interest (active lesion or NAWM) for RNA induced inflammation [18]. In parallel, treatment with isolation were manually dissected from 5-6 recombinant human IL-37 protein improved cryosections per block (50 μm width). aspergillosis [19] and rheumatoid arthritis [19, 20] by MS and healthy donor PBMC samples exerting its anti-inflammatory properties. Blood samples were collected from the following Little is known about the role of IL-37 in the subjects: three from patients with secondary central nervous system. We previously reported that progressive (SPMS) (one male and two females), and IL-37 exerts marked anti-inflammatory effects after eight from patients with relapsing-remitting MS spinal cord contusion injury in mice. Notably, (RRMS) (three males and eight females) (Table S1). All hIL-37tg mice displayed significant resistance against patients were recruited at the MS Clinic at the functional deficits and demyelination after spinal cord Research Center of the University of Montreal injury (SCI) [21]. A recent report reveals that IL-37 is Hospital Center (CRCHUM). Their ages ranged from reduced in blood leukocytes of multiple sclerosis (MS) 27 to 65 years (mean age, 43 years). At time of blood [22], suggesting that this anti-inflammatory pathway sampling, only two patients were receiving treatment might be defective in MS. However, whether IL-37 with disease modifying drugs (Table S1). Seven has similar therapeutic effects in neuroinflammatory healthy volunteers were included as controls (four conditions, such as MS, has not been addressed yet. males and three females); their ages ranged from 24 to In the present study, we investigated the 42 years (mean age, 35 years). A written informed anti-inflammatory and therapeutic potential of IL-37 consent was obtained from patients and healthy in experimental autoimmune encephalomyelitis donors in accordance with the local ethics committee (EAE), a mouse model of MS. We demonstrate that (CRCHUM research ethic committee approval http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 3 number BH07.001). PBMCs were isolated from blood different experiments. samples collected in EDTA-coated vacutainer tubes EAE onset was considered on the day animals (BD Biosciences, Oakville, ON, Canada) using a Ficoll showed the first signs of disease (EAE score 0, 5 or 1; density gradient (Amersham Biosciences) as around day 9-10 post-immunization). Disease peak described previously [26, 27]. was considered on the day EAE score did not increase from the previous day (around day 15-17 post- Mice immunization). Chronic phase of the disease was All experimental procedures were approved by considered at day 21 post-immunization if EAE score the Universitat Autònoma de Barcelona Animal decreased or was maintained with respect to disease Experimentation Ethical Committee (CEEAH 2878) peak. and followed the European Communities Council Directive 2010/63/EU, and the methods were carried Drug administration out in accordance with the approved guidelines. Female C57Bl6/J mice were treated with daily Experiments were performed in adult (8-10 intraperitoneal (i.p.) injections of 1 µg of native weeks) female or male C57Bl/6 mice (Charles River), IL-3746-218 or the mutated monomeric forms IL-37D73K transgenic mice homozygote for human IL-37b (hIL- or IL-37Y85A in 200 µl of saline 28. Recombinant human 37tg), Il1r8 deficient mice (Il1r8 KO), hIL-37tg-Sigirr IL-37 protein forms were administered when mice KO mice, and their respective wildtype littermates. showed the first signs of the EAE disease, and then, Mice were randomly distributed in groups of 5-6 mice daily until the end of the follow up. Control mice were per cage. All mice were housed with food and water treated with 200 µl of saline following the same ad libitum at room temperature of 22±2ºC under 12:12 protocol. Fingolimod was dissolved in distilled water h light-dark cycle. and administered daily by oral gavage (0.3 mg/kg). EAE induction Real-time Quantitative PCR Assay (qPCR) Mice were sedated with intramuscular injection Total RNA was isolated from post-mortem of mixture of ketamine (22 mg/kg) (Imalgen 1000, human brain active MS lesions and normal appearing Merial) and xylazine (2.5 mg/kg) (Rompun, Bayer). white matter (NAWM), as well as, from spinal cords EAE was actively induced by subcutaneously harvested of EAE mice harvested at different stages of injection in each flank at the base of the tail of 100 µl of the disease using the RNeasy Lipid Tissue Mini Kit 3 mg/mL of myelin oligodendrocyte glycoprotein (Qiagen) following the manufacturer’s procedures. peptide 35-55 (MOG35-55) in Complete Freund’s Total RNA was also extracted from PBMCs collected Adjuvant (CFA) (Difco, Detroit, MI, USA) from MS patients and healthy donors using the supplemented with 4mg/mL of heat inactivated QIAamp RNA Blood Tissue Mini Kit (Qiagen) Mycobacterium tuberculosis (Difco, Detroit, MI, USA). according to the manufacturer’s guidelines. Immediately after induction, and on day 2, mice RNA was retrotranscribed using the cDNA received an intraperitoneal (i.p.) injection of 400 ng of Reverse Transcription Kit (Applied Biosystems). pertussis toxin (PTX) in 100 µl sterile saline (0.9% cDNA human libraries were analysed using a Bio-Rad NaCl). CFX384 (CFX Manager V3.1) and TaqMan-designed primers (ThermoFisher Scientific) for the following Functional evaluation of EAE mice human and mouse genes: IL37b (Hs00367201_m1), Mice were scored daily from day 0 to day 21 IL1R8 (Hs00222347_m1), IL1R5 (Hs00187256_m1), after induction of EAE. The researcher was blinded to GAPDH (Hs02786624_g1), Gapdh (Mm03302249_g1), experimental groups during the functional Il1r8 (Mm01275624_g1), Il1r5 (Mm00516053_m1). evaluation. A 6-point scale was used to evaluate the GAPDH/Gapdh was used as a housekeeping gene for clinical signs of EAE: 0 = normal walking; 0.5 = human and mouse samples, respectively. All data partially paralyzed tail; 1 = fully paralyzed tail; 2 = were analysed using the 2ΔΔCt method. Data from mild hind limb weakness, quick righting reflex; 3 = EAE mice were pooled from two different severe hind limb weakness, slow righting reflex, experiments. unable to bear weight; 3.5 = severe hind limb weakness and partial paralysis of hind limb; 4 = Histological analysis complete paralysis of at least one hind limb; 4.5 = Female hIL-37tg mice and WT littermates were complete paralysis of one or both hind limbs and euthanised 21 days post EAE induction with an trunk weakness; 5 = complete paralysis of one or both overdose of pentobarbital sodium (Dolethal) and hind limbs, forelimb weakness or paralysis; 6 = transcardially perfused with 4% paraformaldehyde moribund. Functional data were pooled from two (PFA) in 0.1 M of phosphate buffer (PB). Lumbar segments of spinal cord were harvested, post-fixed in http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 4 4% PFA for 2 h on ice and cryoprotected in 30% To analyse the different immune cells, the spinal sucrose in 0.1 M PB at 4ºC for at least 48 h. Next, cord, blood and lymph nodes were processed as we spinal cords were embedded in Tissue-Tek®OTC previously described in [29]. Blood samples were (Sakura, Japan), sequential transversal sections (15 incubated with red blood cell lysis buffer (BioLegend) µm-thick) between L3 and L5 segments were cut according to manufacturer’ guidelines to obtain a cell using a Leica cryostat and gelatine-coated glass slides. suspension enriched in leukocytes. Spinal cord and Samples were stored at –20ºC until further use. lymph nodes were cut in small pieces and Spinal cord slides were stained with LFB (Sigma enzymatically dissociated incubating them 30 min at Aldrich). Briefly, after graded dehydration, sections 37ºC in 1 mL of HBSS without Ca2+/Mg2+ and 0.1% were placed in 1 mg/mL of LFB solution in 96% collagenase and 0.1% DNase. Next, samples were ethanol and 0.05% acetic acid overnight at 37ºC and mechanically disintegrated by passing them with protected from light. Then, slides were washed with DMEM-10% FBS through a 70 µm cell strainer. After 96% ethanol, rehydrated with distilled water and two washes, cell suspension was split into different differentiated using a 0.5 mg/mL Li2CO3 solution in 1.5 mL microcentrifuge tubes according to the number distilled water for 3-5 min at room temperature. of antibody combinations. For surface staining, Finally, sections were washed in distilled water, samples were incubated with the following primary dehydrated again and mounted in DPX mounting antibodies from eBioscience for 1 h at 4ºC in soft medium (Sigma Aldrich). To assess the demyelinated agitation at a 1:300 concentration: CD45-PerCP, area in the spinal cords, 6 random images per spinal CD11b-PE or PE-Cy7, F4/80-PE or -APC, Ly6C-FITC, cord were captured at 10X magnification with an Ly6G-PE, CD3-FITC-APC-PerCP; CD4-APC-Cy7, Olympus DP73 Camera attached to an Olympus BX51 CD8-APC, CD49b-PE, CD24-PE and the isotype microscope. The total demyelinated area in the spinal matched-controls (1:300 eBioscience). Samples were cord was measured with Image J image analysis then fixed with 1% PFA. When needed, intracellular software. Histological data were pooled from two staining was done after extracellular staining and cell different experiments. fixation. For this purpose, cells were first permeabilizated with Permeabilization Wash Buffer Cytokine protein expression assessment (Biolegend) or FoxP3 Transcription Factor Staining Female hIL-37tg mice and WT littermates were Buffer Set (eBioscience) for 30 min in case of doing euthanised 3 days after the onset of EAE with and FoxP3 staining. Cell were then incubated for 45 min at overdose of pentobarbital sodium (Dolethal) and 4ºC with FoxP3-PE-Cy7, tBet-PerCP, RORγ-APC, transcardially perfused with 60 mL of sterile saline GATA3-PE, or the isotype matched-controls (1:300 (0.9% NaCl). Spinal cords were harvested and rapidly eBioscience) or unconjugated rabbit antibody against frozen using liquid nitrogen. To assess the protein iNOS (1:100 Abcam) and unconjugated goat levels of cytokines, spinal cord were processed as we antibodies against Arg1 (1:100 Santa Cruz) followed previously published 29. Briefly, tissue was by staining with Alexa488 or Alexa647 conjugated homogenized in HEPES and the protein concentration donkey secondary antibodies against rabbit or goat determined with a DC Protein Assay (Bio-Rad). (1:500 Molecular Probes) or isotype matched-control Protein homogenates were concentrated to 4 µg/µl for 45 min. Samples were finally analysed on a FACS and finally, protein levels of IL-4, IL-10, IL-1α, IL-1β, Canto Flow Cytometer (BD Bioscience) and all data IL-6, TNF-α, IFNγ, IL-17A, CSF-3, CCL-5, CCL-2, were processed using FlowJo® software V.10. Flow CXCL-2 and CXCL-10 were analysed using a cytometry data were obtained from a single custom-designed Mouse Cytokine Magnetic Bead experiment. Panel (Affymetrix, eBioscience) on a Luminex- MAGPIX system (Millipore). Luminex data were Haematopoiesis assay obtained from a single experiment. Bone marrow cells were obtained from femurs and tibia of WT and hIL-37tg mice after euthanasia. Flow cytometry Cells were flushed out using a 21G-needle syringe Female hIL-37tg mice and WT littermates were with 5 ml of RPMI 1640 medium (ThermoFisher euthanised at peak of EAE with an overdose of Scientific) supplemented with Penicillin/ pentobarbital sodium (Dolethal). 15 µl of blood was Streptomycin (Sigma-Aldrich). Cells were passed collected from a cardiac puncture and stored in through a 70 μm cell strainer (FisherBrand). Counts of heparinized vials at 4ºC. Then, mice were monocytes, lymphocytes, and granulocytes were transcardially perfused with 60 mL of sterile saline determined using a HemaTrue cell counter (Heska, (0.9% NaCl) and the spinal cords and lymph nodes Loveland, CO, USA). Before measuring, cell counter (cervical and inguinal) were collected. machine was calibrated using a certified blood control http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 5 authorized by Heska. At least 3 measures of the same found that IL37 transcripts were detected at very low sample were performed, and average values were levels in the spinal cord of hIL-37tg at physiological taken. conditions, but they were significantly increased (~7 fold) at the peak and chronic phase of the disease, but Experimental design and statistical analyses not at its onset (Figure 1A). All the experimental groups were gender- and Knowing that the expression of IL37 was age-matched. For all experiments, the number of mice inducible in the spinal cord upon EAE induction, we used are stated in the figure legends and text. Data are next evaluated its contribution to disease. Female shown as mean ± standard error of the mean (SEM). hIL-37tg mice were significantly protected against Statistical analysis was performed using GraphPad functional deficits compared to WT mice (Figure 1B). Prism 8. The distribution of the data was evaluated for At the end of the follow up, hIL-37tg female mice normality using D’Agostino and Pearson test. Two showed a ~1.5 point reduction in EAE clinical score. tailed Student’s t test was used for the comparison Similar beneficial effects were observed in male between two different groups (human qPCR, hIL-37tg mice suffering from EAE. However, the histological analysis, cytokine levels, accumulation of onset of disease occurred significantly earlier in male immune cells). Mean clinical score follow-up was mice expressing IL37 (Figure 1C). analysed by using two-way ANOVA repeated We then evaluated whether the enhancement in measures with post-hoc Bonferroni’s test for multiple functional outcomes of female hIL-37tg mice after comparisons. Differences were considered significant EAE induction was linked to myelin preservation. at p<0.05. Histological analysis of lumbar spinal cords harvested at the end of the follow up (21 days post induction) Results revealed that the area of demyelination was indeed Transgenic expression of IL37 reduces neuro- decreased ~50% in the hIL-37tg mice (Figure 1D-E). logical deficits and protects against de- IL-37 attenuates inflammation in mice with myelination in the spinal cord of EAE mice EAE Since the role of IL-37 in demyelinating diseases We next studied the anti-inflammatory actions of of the CNS is still poorly understood, we first sought IL-37 in EAE. For this purpose, we first measured the to explore whether this cytokine exerted beneficial protein levels of several cytokines in the spinal cord at effects in EAE mice. Since the open frame of the IL37 day 3 after disease onset. A Luminex assay revealed orthologue is not present in mice, we induced EAE in that transgenic expression of IL-37 significantly hIL-37tg mice and wildtype littermates (WT). We reduced the protein levels of 5 pro-inflammatory Figure 1. The transgenic expression of IL37 protects against functional deficits and myelin loss in the spinal cord of mice with EAE. (A) Graph showing the levels of IL37 mRNA in the spinal cord of female hIL-37tg mice at different phase of EAE disease. (B) Clinical score of females and (C) male hIL-37tg mice and WT littermates. (D) Graph showing the quantification of the area of demyelination in the spinal cord of female hIL-37tg and WT littermates at 21 days post induction. (E) Representative images of lumbar spinal cord from female hIL-37tg mice and WT littermates at day 21 after EAE induction. *p<0.05; **p<0.01; ***p<0.001 vs. control. One-way ANOVA with Bonferroni post hoc test in A (n=7 per time point); Two-way repeated measures ANOVA with Bonferroni’s post hoc test in B (n=15 WT; n=14 hIL-37tg) and C (n=9 per group); Unpaired t-test in D (n=15 WT; n=14 hIL-37tg. Data were pooled from two different experiments. Data shown as mean±sem. http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 6 cytokines (IL-1α, IL-1β, IL-6, TNFα and IFNγ), each of and neutrophils (CD45+, CD11b+, F4/80–, Ly6G+) them linked to development of EAE pathophysiology (Figure 2B, Figure S2B, F). Of note, transgenic (Figure 2A). Since cytokines coordinate the expression of IL37 significantly reduced the recruitment and activation of immune cells into the proportion of pathogenic Th1 cells (CD3+, CD4+, CNS parenchyma, we next evaluated whether tBet+) and increased the proportion of classical transgenic expression of IL-37 reduced the regulatory CD4 cells (CD3+, CD4+, FoxP3+) in the accumulation of immune cells in the spinal cord at the spinal cord (Figure 2C, Figure S3A). Moreover, IL-37 peak of the disease. Flow cytometry analysis showed reduced the expression of CD16/32+ and iNOS+ in that hIL-37tg mice had significant lower numbers of T macrophages by 20% (Figure 2D, Figure S3B), but did cells (CD3+), including CD4 and CD8 T cells, not alter the expression of Ly6C (Figure 2E, Figure macrophages (CD45high, CD11b+, F4/80+), total S3C). These data indicate that IL-37 not only reduced microglial cells (CD45low, CD11b+) and activated the accumulation of immune cells in the spinal cord, microglial cells (CD45low, CD11b+, F4/80+) (Figure but also modulated their phenotype towards a more 2B, Figure S1-S2). IL-37, however, did not attenuate anti-inflammatory state. the number of B cells (CD45+, CD11b–, CD3–, CD24+) Figure 2. IL-37 reduces the inflammatory response in the spinal cord of mice at the peak of EAE. (A) Protein level profile of cytokines (IL-4, IL-10, IL-1α, IL-1β, IL-6, IL-17A, IFNγ, TNFα and chemokines (CXCL-2, CXCL-10, CCL-2, CCL-5, CSF-3) in the spinal cord of female hIL-37tg relative to female WT at the peak EAE. (B) Number of infiltrated immune cells and (C) percentage of CD4+ T cells expressing the transcription factors CD49b, FoxP3, tBet, GATA3 or RORγ in the spinal cord of female WT and female hIL-37tg mice at the peak of EAE. (D) Percentage of macrophages expressing the pro-inflammatory markers CD16/32 and iNOS or (E) Ly6C in the spinal cord of female WT and female hIL-37tg mice at the peak of EAE. *p<0.05; **p<0.01; ***p<0.001 vs. WT. Unpaired t-test (n=5 per group in A; n=4 per group in B-E). Data were obtained from a single experiment. Data shown as mean±SEM. http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 7 Figure 3. Effects of IL-37 on immune cell counts in the lymph nodes and blood in EAE mice. (A) Number of immune cells in the lymph nodes and (E) blood of female WT and female hIL-37tg mice at the peak of EAE. (B) Number of dendritic cells in the lymph nodes. (C) Percent of CD4+ T cells expressing CD49b, FoxP3, tBet, GATA3 or RORγ in lymph nodes and (F) blood. (D) Percentage of monocytes expressing Ly6C in lymph nodes and (G) blood. *p<0.05; **p<0.01; ***p<0.001 vs. WT. Unpaired t-test (n=4 per group). Data were obtained from a single experiment. Data shown as mean±SEM. We then explored whether the reduced unaltered in lymph nodes (Figure 3C). In the blood accumulation of immune cells in the spinal cord of samples, we found that IL-37 did not decrease the hIL-37tg mice in EAE was due to a central or numbers of the different leukocyte populations peripheral effect of this cytokine. Consequently, we (Figure 3D) but increased the proportion of non- first assessed the immune cell counts in the lymph classical regulatory CD4 T cells (Figure 3E) as nodes and blood at EAE disease peak. Similar to the reflected by increased CD49b expression. The spinal cord, flow cytometry analysis demonstrated proportion of monocytes expressing Ly6C remained that hIL-37tg mice featured a significantly reduced unaltered in blood (Figure 3F). number of CD4 T cells (Figure 3A) but no changes in Finally, we performed a haematopoiesis assay to their phenotype (Figure 3B) in the lymph nodes, discard that the reduction in the number of immune suggesting that IL-37 attenuated the expansion but cells in the CNS and the periphery was due to a lower not polarization of this T cell subset, respectively. basal progenitor number in the bone marrow of IL-37 also reduced the number of macrophages and hIL-37tg. This assay revealed that transgenic dendritic cells (CD45+, CD11b+, CD11c+) (Figure 3A), expression of IL-37 did not alter lymphocytes, which is compatible with reduced T cell priming monocytes and granulocytes formation in the bone observed in the spinal cord (Figure 2B). The marrow (Figure S4). proportion of monocytes expressing Ly6C remained http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 8 Figure 4. The protective role of IL-37 expression against EAE is mediated by the IL-1R8. A-B, Graphs showing the relative expression the Il1r5 (A) and Il1r8 (B) in the spinal cord of female EAE and naïve mice. (C-D), Clinical score of female WT, hIL-37tg, Il1r8 KO and hIL-37tg x Il1r8 KO mice showed as the mean clinical score (C), maximum and cumulative score (D). (E), Graph showing the quantification of myelin loss in the spinal cord of female WT, hIL-37tg, Il1r8 KO and hIL-37tg x Il1r8 KO mice at 21 days post-induction. (F) Representative images of the spinal cords from each of the experimental groups. *p<0.05; **p<0.01; ***p<0.001 vs. WT. One-way ANOVA in A and B (n=6 in onset and n=7 in the other groups). Two-way ANOVA with repeated measures, Bonferroni’s post hoc test in C (n=21 in WT, n=16 in hIL-37tg, n=14, n=14 in Il1r8 KO and n=14 in hIL-37tg x Il1r8 KO. One-way ANOVA in D (n=21 in WT, n=16 in hIL-37tg, n=14 in Il1r8 KO and n=14 in hIL-37tg x Il1r8 KO. and E (n=20 in WT, n=16 in hIL-37tg, n=13 in Il1r8 KO and n=13 in hIL-37tg x Il1r8 KO. Data were pooled from two different experiments. Data shown as mean±SEM. purpose, we crossed hIL-37tg mice with Il1r8 KO IL-1R8 is critical for IL-37 to mediate beneficial mice. We selected IL-1R8 for gene deletion to assess effects in EAE the importance of the extracellular IL-37 signalling As reported previously, IL-37 is a dual-function instead of IL-1R5 because IL-1R8 is a unique cytokine that can mediate its biological effects by co-receptor for IL-37. In contrast, IL-1R5 is also used acting as an extracellular cytokine [30] or by by IL-18, and consequently, gene deletion of IL-1R5 translocating to the nucleus [15, 31]. Here, we the also alters IL-18 signalling. This double transgenic sought to demonstrate the importance of the mouse (hIL-37tg x Il1r8ko) expresses the human form extracellular function of IL-37 in EAE. of IL-37b in the homozygous, but this cytokine has For this purpose, we first characterized the only nuclear function, since IL-1R8, which is crucial expression of IL-37 receptor complex in the spinal for the extracellular function of IL-37, has been cord in different phases of EAE. We found that both genetically deleted. components Il1r5 and Il1r8, were expressed in the EAE induction in Il1r8 ko mice revealed that the spinal cord at physiological conditions and during lack of this receptor does not alter EAE pathogenesis. EAE disease progression (Figure 4A and 4B), although However, the beneficial effects of IL-37 were Il1r5 transcripts significantly increased at peak and in completely absent in hIL-37tg mice lacking Il1r8 chronic phase of EAE. (Figure 4C-D). Accordingly, histological analysis Knowing that the components of the IL-37 revealed that the protective actions of IL-37 against receptor complex were expressed in the spinal cord, demyelination were abrogated in the absence of Il1r8 we aimed at evaluating to what degree the (Figure 4E-F). These experiments provide clear extracellular IL-37 was important to mediate the evidence that the extracellular function of IL-37 is beneficial effects of this cytokine in EAE. For this crucial in conferring protection against EAE. http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 9 Figure 5. The administration of recombinant human IL-37 protein in EAE mice. (A) Clinical score of female WT EAE mice treated with IL-3746-218, IL-37D73K, IL-37Y85A or vehicle showed as mean clinical score and as (B) maximum and cumulative score. (C) Graph showing the quantification of demyelinating areas in the spinal cord of female WT mice treated with native IL-3746-218, IL-37D73K, IL-37Y85A or vehicle and (D) representative images of these spinal cords. *p<0.05; **p<0.01; ***p<0.001 vs. vehicle. Two-way ANOVA with repeated measures, Bonferroni’s post hoc test in A (n= 9 per group in vehicle, IL-37D73K and IL-37Y85A and n=10 in native IL-3746-218). One-way ANOVA in B and C (n= 9 per group in vehicle, IL-37D73K and IL-37Y85A and n=10 in native IL-3746-218). Data were pooled from two different experiments Data shown as mean±SEM. assessed the expression of IL37 as well as IL-1R5 and Administration of recombinant human IL-37 IL-1R8 in PBMCs of MS patients and healthy controls. protein reduces neurological deficits and de- In addition, we examined brains samples from MS myelination in EAE mice patients. qPCR analysis revealed that IL37 transcripts Since mice expressing IL-37 have less disease in were barely detectable in PBMC from healthy the EAE model, we administered recombinant human controls, and that the levels of this anti-inflammatory IL-37 protein in EAE mice starting at disease onset. cytokine were not significantly increased in MS Since IL-37 form spontaneous dimers, which have patients (Figure 6A), although its receptor reduced anti-inflammatory actions 28, 32, we components were expressed (Figure 6B-C). However, compared the efficacy of the native IL-37 (IL-3746-218) IL37 was detectable albeit at very low levels in to two mutant monomeric forms (IL-37D73K and NAWM of MS brains. Strikingly, IL37 levels tended to IL-37Y85A), which do not dimerize. We found that decrease in MS active lesions, except in two of the daily administration of 1 µg of native IL-3746-218, patients who showed a marked increase in the IL-37D73K or IL-37Y85A from the onset of EAE enhanced transcripts for this cytokine (Figure 6D). In line with functional outcomes and protected against the PBMC data, IL1R5 and IL1R8 were also expressed demyelination (Figure 5A-D). Among the three in the NAWM of brain MS samples, and the level of recombinant IL-37 proteins, the mutant monomeric IL1R5 tended to increase in active lesions (Figure form IL-37D73K showed the greatest efficacy at both 6E-F). These data reveal that while the components of functional and histological levels, although not the IL-37 receptors are present in the PBMC and reaching statistical significance when compared to brains of MS individuals, there appears to be a IL-3746-218 and IL-37Y85A (Figure 5A-D). Interestingly, paucity of IL-37 transcripts, indicative of a relative we found that human recombinant IL-3746-218 protein deficiency state. led to similar therapeutic actions than Fingolimod, one of the most effective drugs to treat relapsing MS Discussion (Figure S5). In the present study, we investigated whether Overall, these data support the therapeutic the anti-inflammatory cytokine IL-37 protects hosts actions of exogenous administration of recombinant from EAE pathogenesis. We observed amelioration of human IL-37 protein in EAE. the clinical signs of EAE and reduced demyelination IL37 is aberrantly induced in MS patients in mice expressing human IL-37. The suppression of the inflammatory response correlates with a decrease Since IL-37 plays a beneficial role in EAE, we in the protein levels of several pro-inflammatory finally evaluated IL-37 expression in MS patients. We http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 10 cytokines in the spinal cord. Accordingly, IL-37 similar to previous findings [21], we found that IL37 reduces the number of CD4 T cells, macrophages and mRNA is present at very low levels in the spinal cord dendritic cells in the lymph nodes, and attenuates the of hIL-37tg mouse at steady-state conditions but is accumulation of most of the immune cell populations markedly upregulated during EAE. Indeed, IL37 into the CNS. Moreover, IL-37 switches the phenotype mRNA reached the maximum expression at peak of of CD4 T cells and macrophages in the CNS resulting EAE, a disease phase when leukocytes are highly in more regulatory and less inflammatory accumulated in the spinal cord, which may suggest phenotypes. We also observed that the beneficial that leukocytes are the main source of this cytokine actions of IL-37 are mediated by signalling via its [39]. It is important to also note that IL37 expression surface receptor complex IL-1R5/IL-1R8. Lastly, we was associated with reduced neurological provided a proof of concept that the administration of impairments and demyelination of EAE mice, recombinant human IL-37 protein in EAE mice supporting the beneficial action of IL-37 in neuro- alleviates disease symptomatology and protects inflammatory conditions, as we previously against demyelination. Thus, we hypothesise that this demonstrated in spinal cord injury [21]. natural mechanism to attenuate inflammation might It is well established that the cytokines produced be aberrant in MS patients. by the initial wave of CNS-infiltrating leukocytes are IL-37 is a member of the IL-1 family that exerts the dominant force that orchestrates the ensuing potent anti-inflammatory action [33]. Although this inflammatory cascade in MS and EAE [40, 41]. These cytokine is not present in mice, the generation of a cytokines play a key role in the recruitment and transgenic mouse that expresses the human form of activation of more immune cells from the circulation, IL-37, and the use of recombinant human IL-37 as well as in the triggering of glial reactivity [41]; they protein, have demonstrated its protective properties are thus key contributors to MS pathology. In support from a myriad of pathological challenges, such as of this concept, cytokines appear among the most colitis [34], metabolic syndrome and type 2 diabetes associated risk factor genes for MS in genome-wide [18], lung inflammation [19], cancer [35], arthritis [36], association study [42]. calcific valve disease [37] and spinal cord injury [21]. Here, we report that transgenic expression of In hIL-37tg mice, IL37 transcription is artificially IL37 silenced several pro-inflammatory cytokines in regulated by a cytomegalovirus promoter, which is the spinal cord that have a key contribution to EAE constitutively expressed in all the cells [9]. However, and MS pathology [43–47]. The effects of IL-37 on IL-37 is not constitutively expressed as expected in the cytokine levels were associated with reduced hIL-37tg mouse due to the presence of an instability infiltration of immune cells into the spinal cord at the sequence in the mRNA that limits its half-life. peak severity of the disease. This is quite relevant Nonetheless, under inflammatory conditions, IL37 since leukocytes accumulate in demyelinating areas mRNA is stabilized and, consequently, the translation and their numbers correlate to tissue damage in MS of the protein is allowed [38]. Along this line and patients and EAE mice [48, 49]. Figure 6. Characterization of the expression of IL37, IL1R5 and IL1R8 in PBMC and brain from healthy and MS patients. (A-F) Graphs showing the expression of IL-37 and the components of its extracellular receptor in PBMCs (A-C) and brain (D-F) from healthy and MS patients. Unpaired t-test in A, B and C (n=7 in healthy controls, n=11 in MS). Paired t-test in D, E and F (n=9 per group). Data shown as mean±SEM. http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 11 Cytokines are also responsible for the influence MS, as we recently observed in endotoxemia polarization of immune cells, including T cells [41]. in mice [15]. The increased proportion of T regulatory cells has Although IL-37 is up-regulated after been defined as a hallmark of the recovery phase in inflammatory challenges there is accumulating EAE mice [50] as well as, of the remission phase in evidence corroborating that IL-37 is deficient in some RRMS individuals [51]. In this line, transgenic pathological conditions [37, 55–57]. A recent study expression of IL37 reduced the percentage of revealed that the production of IL-37 by T cell is pathogenic Th1 effector cells while increasing the reduced in MS patients. In addition, in the same study percent of T regulatory cells in the spinal cord at the the authors demonstrate that those patients who had peak of the EAE disease. Importantly, we found that higher levels of IL-37 in PBMCs were more protected the polarizing effects of IL-37 were not restricted to against to the exacerbation of the disease than those CD4 T cells, since hIL-37tg mice also showed lower with lower levels [22]. Although we did not observed proportion of macrophages expressing CD16/32 and reduction of IL-37 in PBMCs of MS patients, this iNOS, which have potent pro-inflammatory effects cytokine was not up-regulated as compared to heathy and contribute to EAE pathology [52]. We also found volunteers. In this line, we also observed that IL-37 decreased numbers of various immune cell was not upregulated in brain active lesions of most populations in the lymph nodes of EAE mice with MS patients. Nonetheless, the components of the transgenic IL37 expression. For example, there was a extracellular receptors for IL-37 were expressed in ~50% reduction in the number of dendritic cells, PBMCs and brain samples of healthy donors and MS which are potent antigen-presenting cells endowed patients. These data therefore suggest that although with the ability to prime T-cell responses. Both the MS individuals have the machinery to activate this number of MS plaques and the severity of EAE have beneficial mechanism to contain inflammation, IL-37 been found to correlate with the presence and signalling is not properly induced due to low functional status of dendritic cells [53, 54]. The production of IL-37. Importantly, we showcase that number of CD4 T cells was also significantly reduced the exogenous administration of recombinant human in the lymph nodes of hIL-37tg mice, suggesting that IL-37 protein in EAE mice ameliorated the clinical and the expansion of this T cell subset was attenuated by histopathological signs of the disease, suggesting the IL-37. However, this cytokine did not alter the therapeutic potential of IL-37 in MS. Highlight that phenotype of CD4 T cells in the lymph nodes. treatment with recombinant IL-37 protein was Similarly, IL-37 did not impede the efflux of immune initiated once mice showed the first clinical signs of cells into the circulation since the number of the EAE, which makes these findings clinically relevant. different immune cells analysed did not change after Recent studies have reported that IL-37 forms its transgenic expression. However, IL-37 may have dimers with nanomolar affinity, resulting in limited favoured the mobilization of regulatory CD4 T cells bioactivity [32]. They uncovered that mutations in the into the circulation since this immune cell subset was dimer interface, such as IL-37D73K and IL-37Y85A, increased in the hIL-37tg mice after EAE. These data specifically disrupt dimer formation, resulting in therefore suggest that the reduced accumulation of stable monomers that provide greater suppression of the immune cells in the spinal cord of EAE mice with inflammation compared with native IL-37 transgenic expression of IL37 was likely due to the (Eisenmesser et al., 2019). However, we did not find ability of this cytokine to reduce their infiltration into any significant enhanced effect of the two mutant CNS rather than suppressing their mobilization into monomers IL-37D73K and IL-37Y85A compared to the the circulation. native IL-37 form, although the monomer IL-37D73K IL-37 is a dual-function cytokine that can exert its resulted in slight greater efficacy in EAE. This may anti-inflammatory activity through two different indicate that the amount of IL-37 we administered pathways: (i) the activation of the extracellular was not high enough to favour the formation of receptor complex IL-1R5/IL-1R8 or (ii) its dimers. Importantly, we found that human translocation into the nucleus [14]. We here recombinant IL-37 protein exerted similar therapeutic demonstrate that the interaction of IL-37 with its potential than fingolimod, one of the most effective extracellular receptor complex is required to mediate treatment for relapsing-remitting forms of MS. its protective effects in EAE, by showing that the Interestingly, a recent study reported that serum genetic deletion of IL-1R8, the co-receptor for IL-37, levels of IL-37 were higher in MS individuals treated abolished its beneficial actions in EAE. It is important with fingolimod as compared to patients receiving no to note that this does not rule out the possibility that treatment, suggesting that this drug could exert its anti-inflammatory effects linked to the nuclear beneficial actions in MS, at least in part, by increasing function of IL-37 likely take place in humans and may the levels of this anti-inflammatory cytokine [22]. http://www.thno.org Theranostics 2021, Vol. 11, Issue 1 12 In conclusion, in the present study we provide 12. C. A. Dinarello and P. Bufler. Interleukin-37. Semin Immunol. 2013; 25: 466– 468. clear evidence that IL-37 wields beneficial effects in 13. Bulau AM, Nold MF, Li S, Nold-Petry CA, Fink M, Mansell A, et al. Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 EAE by signalling through the surface receptor inhibition of innate immune responses. Proc Natl Acad Sci. 2014; 111: 2650– complex IL-1R5/IL-1R8. Although further studies are 2655. 14. 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