Beneficial effects of IL-37 after spinal cord injury in mice Marina Coll-Miróa, Isaac Francos-Quijornaa, Eva Santos-Nogueiraa, Abel Torres-Espina, Philip Buflerb, Charles A. Dinarelloc,d,1, and Rubèn López-Valesa,1 a Departament de Biologia Cellular, Fisiologia i Immunologia, Institut de Neurociències, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; bDepartment of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig Maximilian University of Munich, D-80337 Munich, Germany; cDivision of Infectious Diseases, University of Colorado Denver, Aurora, CO 80045; and dDepartment of Medicine, Radboud University Medical Center, 6525 HP Nijmegen, The Netherlands Contributed by Charles A. Dinarello, December 6, 2015 (sent for review August 15, 2015; reviewed by Karim Fouad and Nader Ghasemlou) IL-37, a member of the IL-1 family, broadly reduces innate in- IL-1 family are proinflammatory (9, 10), IL-37 has broad sup- flammation as well as acquired immunity. Whether the antiinflam- pressive effects on innate inflammation and acquired immunity matory properties of IL-37 extend to the central nervous system (11–14). Because a complete ORF for the mouse homolog of IL- remains unknown, however. In the present study, we subjected mice 37 has not yet been found, it was necessary to generate a strain of transgenic for human IL-37 (hIL-37tg) and wild-type (WT) mice to transgenic mice expressing human IL-37, designated hIL-37tg spinal cord contusion injury and then treated them with recombinant mice. These mice are protected against endotoxin shock, colitis, human IL-37 (rIL-37). In the hIL-37tg mice, the expression of IL-37 was hepatitis, and myocardial infarction (9, 13, 15–18); however, a barely detectable in the uninjured cords, but was strongly induced at role for IL-37 after CNS trauma remains unexplored. In the 24 h and 72 h after the spinal cord injury (SCI). Compared with WT present study, we subjected hIL-37tg mice to SCI and studied mice, hIL-37tg mice exhibited increased myelin and neuronal sparing subsequent functional impairments in comparison with wild type NEUROSCIENCE and protection against locomotor deficits, including 2.5-fold greater (WT) mice. We also administered recombinant human IL-37 speed in a forced treadmill challenge. Reduced levels of cytokines (rIL-37) to WT mice, to provide a rationale for clinical use of (e.g., an 80% reduction in IL-6) were observed in the injured cords of IL-37 as a therapeutic agent. We provide direct evidence for hIL-37tg mice, along with lower numbers of blood-borne neutrophils, the first time, to our knowledge, that IL-37 exerts marked anti- macrophages, and activated microglia. We treated WT mice with a inflammatory properties on the contused spinal cord and confers single intraspinal injection of either full-length or processed rIL-37 protection from tissue damage and functional loss. after the injury and found that the IL-37–treated mice had signifi- cantly enhanced locomotor skills in an open field using the Basso Results Mouse Scale, as well as supported faster speed on a mechanical hIL-37tg Mice Exhibit Reduced Functional Deficits and Tissue Damage treadmill. Treatment with both forms of rIL-37 led to similar benefi- After SCI. As shown in Fig. 1A, hIL-37tg mice displayed significant cial effects on locomotor recovery after SCI. This study presents novel improvement in locomotor recovery after SCI. There were signif- data indicating that IL-37 suppresses inflammation in a clinically rel- icant improvements in Basso Mouse Scale (BMS) scores starting at evant model of SCI, and suggests that rIL-37 may have therapeutic potential for the treatment of acute SCI. 7 days postinjury (dpi), and BMS scores remained significantly enhanced for the subsequent 3-wk period (Fig. 1A). At 28 dpi, all WT mice showed extensive ankle movement, but only 50% of IL-37 | spinal cord injury | inflammation | neuroprotection | cytokines these mice showed plantar paw placement but no weight-bearing T he inflammatory response plays an essential role in tissue protection after injury or invasion by microorganisms (1, 2). Regardless of the tissue, unless regulated, inflammation can Significance become chronic and result in tissue damage and loss of function Spinal cord injury (SCI) often results in severely impaired locomotor, (1, 2). This is particularly the case in spinal cord injury (SCI). sensory, and autonomic function. Although inflammation contrib- utes to the physiopathology of SCI, several clinical trials of high After spinal cord contusion or compression injury, there is a doses of dexamethasone or methylprednisolone have not resulted rapid initiation of inflammation in rodents and in humans (2). in improved recovery of function. IL-37, a member of the IL-1 family, This response is orchestrated by endogenous microglial cells and exerts broad antiinflammatory effects in several mouse models of by circulating leukocytes, especially monocytes and neutrophils, inflammatory diseases. We report here that mice expressing human which invade the lesion site during the first hours and days after IL-37 exhibit reduced inflammation in the central nervous system injury (2–4). Although these cells are required for the clearance and demonstrate significantly improved functional disabilities fol- of cellular and myelin debris, they also release cytokines and lowing SCI. The administration of recombinant forms of human IL- cytotoxic factors, which are harmful to neurons, glia, axons, and 37 enhances motor skills after SCI, suggesting that IL-37 could myelin, resulting in secondary damage to adjacent regions of the provide a new therapeutic approach to limit the harmful effects of spinal cord that had been previously unaffected by the insult inflammation in neurologic conditions. (2, 5, 6). Indeed, it is currently well accepted that inflammation is a major contributor to secondary cell death after SCI. The Author contributions: C.A.D. and R.L.-V. designed research; M.C.-M., I.F.-Q., E.S.-N., and damaging effects of inflammation are more pronounced in the R.L.-V. performed research; A.T.-E., P.B., C.A.D., and R.L.-V. contributed new reagents/ analytic tools; M.C.-M., I.F.-Q., E.S.-N., and R.L.-V. analyzed data; and M.C.-M., C.A.D., central nervous system (CNS) than in other tissues, because of and R.L.-V. wrote the paper. the limited capacity for axon regeneration and replenishment of Reviewers: K.F., University of Alberta; and N.G., Boston Children’s Hospital and Harvard damaged neurons and glial cells, which leads to irreversible Medical School. functional disabilities (7, 8). Therefore, targeting inflammation is The authors declare no conflict of interest. a valuable approach to promoting neuroprotection and limiting 1 To whom correspondence may be addressed. Email: cdinare333@aol.com or Ruben. functional deficits in SCI. Lopez@uab.cat. Cytokines are key players in the initiation, progression, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suppression of inflammation. Although several members of the 1073/pnas.1523212113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1523212113 PNAS Early Edition | 1 of 6 Fig. 1. hIL-37-tg mice show enhanced functional outcomes and reduced tissue damage after SCI. (A and B) Locomotor skill assessment using the nine-point BMS (A) and on a treadmill (B). (C) Myelin sparing at various distances rostral and caudal to the injury epicenter. (D) Representative micrographs showing myelin sparing at the injury epicenter in sections stained against LFB from WT and hIL-37tg mice. (E) Ventral horn neuron survival at various distances rostral and caudal to the injury epicenter. (F) Representative micrographs showing sparing of ventral horn neurons in WT mice (Upper) and hIL-37tg mice (Lower) in sections stained against NeuN 200 μm rostral to the injury epicenter. (Insets) Higher-magnification views of the areas outlined in the boxes. Data are expressed as mean ± SEM. n = 8 per group. *P < 0.05, two-way repeated-measures ANOVA with Bonferroni’s post hoc correction in A, C, and D and the t test in B. stepping (BMS score 2.5). In contrast, all hIL-37tg mice showed sought to evaluate whether there was enhanced axonal regen- extensive ankle movement and plantar paw placement with eration in hIL-37tg mice after complete spinal cord transection. weight support, and the majority displayed occasional stepping Both the hIL-37tg and WT mice presented complete hindlimb (BMS score 3.8) (Fig. 1A). Moreover, the hIL-37tg mice per- paralysis after the injury and lacked functional improvement at formed ∼2.5-fold faster locomotion on the treadmill compared 10 wk postinjury (BMS score 0). Histological assessment of with the WT mice (Fig. 1B), further demonstrating the protective sagittal spinal cord tissue sections revealed that transgenic ex- effect of IL-37 against functional loss in SCI. pression of IL-37 did not lead to regeneration of axons beyond We then assessed whether the improvement in motor skills of the transection site. Similarly, axonal counts at rostral distances hIL-37tg mice was associated with reduced secondary tissue to the injury did not reveal any difference between the hIL-37tg damage after SCI. Histological sections from the injury epicenter and WT mice (Fig. 2), suggesting that IL-37 does not promote stained with Luxol fast blue (LFB) revealed enhanced myelin regeneration/sprouting of corticospinal axons (Fig. 2). Overall, sparing in hIL-37tg mice compared with WT mice (Fig. 1 C and these data provide evidence that IL-37 confers protection from D). Prevention of myelin loss by transgenic expression of IL-37 functional disabilities and secondary tissue damage after spinal was evident at the lesion epicenter and at distances up to 800 μm cord contusion injury, but does not promote axonal outgrowth of caudal to the injury (Fig. 1 C and D). Assessment of neuronal the corticospinal tract. sparing in the ventral horns also demonstrated attenuated neu- ronal loss in hIL-37tg mice (Fig. 1 E and F). Spinal cord sections IL-37 Is Induced in hIL-37tg Mice After SCI. Because hIL-37tg mice stained against NeuN revealed greater numbers of neurons at were markedly protected against functional disabilities and tissue several regions rostral and caudal to the injury epicenter in hIL- loss after SCI, we studied the inflammatory response in these 37tg mice (Fig. 1 E and F). mice. We first assessed the expression profile of IL-37 in the Because infiltrating macrophages inhibit axonal outgrowth by spinal cord of hIL-37tg mice as determined by PCR. As expected, releasing soluble factors and by cell–cell interaction (19, 20), we IL-37 was not present in the intact or damaged spinal cord 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1523212113 Coll-Miró et al. observed in the colon, skin, circulating leukocytes, and cell lines transfected with IL-37 (11, 13, 16). The low levels are due to the instability sequence in human IL-37 (14). However, after con- tusion injury, induction of IL-37 in the spinal cord parenchyma was observed (Fig. 3). The IL-37 expression profile showed two peaks of expression, after 12 h postinjury (hpi) and at 3 dpi, when IL-37 levels increased by ∼17- and ∼35- fold, respectively (Fig. 3). The early peak of IL-37 coincides with the induction of cytokines in the injured spinal cord at 6–24 hpi (Figs. S1 and S2), whereas the latter peak (at 3 dpi) correlates with the infiltration of blood monocytes (2). Inhibition of Cytokine and Chemokine Expression After SCI. We next sought to examine whether the early increase in IL-37 modulates gene expression of cytokines in the contused spinal cord. We evaluated the protein levels of 32 cytokines in injured spinal cords harvested at 12 hpi and 24 hpi, periods when the protein levels of most cytokines and chemokines reach maximal con- centrations after SCI (2). At 12 hpi, cytokine levels were un- changed in the hIL-37tg mice relative to WT mice (Fig. S2); however, at 24 hpi, significant reductions in the expression of 23 of the 32 cytokines in were observed in the hIL-37tg mice (Fig. S1). Moreover, the expression of IL-3, which was detected at NEUROSCIENCE low levels in the spinal cord homogenates of WT mice (0.56 ± 0.03 pg/mg protein), was undetectable in hIL-37tg mice (<0.45 pg/mg protein). The levels of six cytokines (LIF, M-CSF, IL-2, IL-5, IL-15, and eotaxin) were unchanged in the hIL-37tg mice, whereas ex- pression of IL-4 and IL-12p40 was not detected in the contused spinal cords of both experimental groups (Fig. S1). We next evaluated the accumulation of inflammatory cells in WT mice and hIL-37tg mice following SCI. At 1 dpi, when granulocyte infiltration reaches peak levels, the spinal cords of hIL-37tg mice showed an ∼40% reduction in the number of granulocytes (Fig. 4). There were no between-group differences in cell counts of activated microglia, blood-borne macrophages, CD4 and CD8 T cells at this time point (Fig. 4). At 7 dpi, when the accumulation of activated microglia and macrophages reaches peak levels in the injured spinal cord and accounts for ∼80% of total immune cells, spinal cords from the hIL-37tg mice exhibited significantly lower numbers of these two cell subsets (Fig. 5). The number of granulocytes, which was re- duced by 90% compared with that at 1 dpi, was slightly increased Fig. 2. hIL-37tg expression does not promote axonal regeneration. (A) BDA- labeled corticospinal axons at different distances to the transection site. (B and C) Low-magnification images of complete transected spinal cords showing BDA-labeled corticospinal fibers in WT mice (B) and hIL-37tg mice (C). Lines define the transection sites. L, lesion. Data are expressed as mean ± SEM. n = 8 per group. *P < 0.05, one-way ANOVA with Bonferroni’s post hoc correction. obtained from WT mice. The hIL-37tg mice exhibited low levels Fig. 3. Expression of IL-37 after SCI. Shown is the time course of IL-37 of constitutive expression of IL-37 in the spinal cord. Low levels transcripts in the spinal cords of hIL-37tg mice after contusion injury. n = 4 of constitutive IL-37 expression in transgenic mice also has been per time point. *P < 0.05, one-way ANOVA with Bonferroni’s post hoc test. Coll-Miró et al. PNAS Early Edition | 3 of 6 3.8 in hIL-37tg; CD206: 14.1 ± 3.1 in WT vs. 15.2 ± 3.1 in hIL- 37tg) or macrophages (CD16/32: 82.2 ± 6.7 in WT vs. 79.4 ± 5.3 in hIL-37tg; CD206: 14.5 ± 1.9 in WT vs. 16.6 ± 3.2 in hIL-37tg). Intralesional Administration of rIL-37 Improves Functional Outcomes After SCI. To assess a therapeutic use of IL-37 in SCI, we ad- ministered rIL-37 to determine possible beneficial effects in SCI. We first tested the IL-37 precursor (full-length IL-37 isoform b), because previous studies have shown efficacy in vivo (21) and in vitro (22). We also administered a processed form of IL-37 with the N terminus at valine 46 (22, 23). Because the blood- brain barrier prevents the entry of most molecules into the CNS, we infused rIL-37 into the lesion site at 5 min after the contusion injury, using a glass capillary (30-μm diameter). As shown in Fig. 6A, intraspinal injection of either full-length or processed rIL-37 significantly enhanced locomotor recovery as demonstrated by the BMS score, starting at 10 dpi. At 28 dpi, mice injected with saline had a BMS score of 1.6, which corresponds to slight or extensive movement of the ankle; however, treatment with full- length rIL-37 improved the BMS score by 1 point (Fig. 6A). The rIL-37–treated mice showed extensive movement of the ankle, and most also displayed plantar paw placement in at least one paw, but no stepping. Moreover, treatment with full-length rIL-37 increased the speed that mice were able to achieve on a treadmill by 50% (Fig. 6B), further supporting the beneficial effects of rIL-37 in preventing functional deficits after SCI. Overall, both forms of rIL- 37 led to similar beneficial effects on motor skills (Fig. 6). Discussion Although IL-37 is lacking in mice, hIL-37tg mice are protected against several inflammatory challenges. Previous studies using hIL-37tg mice have consistently demonstrated the suppressive properties of IL-37 on inflammation (11, 13, 16); however, whether IL-37 exerts a similar antiinflammatory effect in the CNS trauma has remained unknown. Here we demonstrate for the first time, to our knowledge, that IL-37 suppresses in- flammation and limits locomotor deficits and tissue damage in spinal cord contusion injury in mice. This conclusion is based on observations in hIL-37tg mice as well as in WT mice treated with either rIL-37 precursor or processed IL-37. In hIL-37tg mice, IL-37 expression is regulated by a constitutive cytomegalovirus promoter (13), and thus expression should be in- duced in all cells. However, constitutive expression of IL-37 in the spinal cord is low, owing to the presence of an instability sequence that limits the half-life of the IL-37 transcript (14), as has been shown in other models using these mice (11, 13, 16). However, in in- flammatory conditions, such as tissue trauma, activation of in- flammatory components increases the half-life of IL-37 mRNA and allows for translation of the protein IL-37. IL-37 mRNA stability is apparently regulated by instability elements present in exon 5, given that deletion of exon 5 can significantly increase the mRNA stability Fig. 4. Spinal cord immune cell counts at 1 dpi. (A–F) Representative density of both IL-37b and IL-37c. Similar mRNA stabilization on LPS plots of FACS analysis showing myeloid cells and microglia (A and B), mac- stimulation has been reported for IL-18 (14). We observed that rophages (CD45high, CD11b+, F4/80+) (C and D), and granulocytes (CD45high, mRNA levels were increased at 12 hpi, followed by a secondary in- CD11b+, Gr1high) (E and F) in the spinal cords of WT and hIL-37tg mice. crease at 3 dpi. Whether endogenous IL-37 is expressed in human (G) Quantification of the different immune cell populations in the injured spinal SCI remains unknown, however. Cytokines such as IL-1β, TLR ag- cords. Data are expressed as mean ± SEM. n = 4 per group. *P < 0.05, t test. onist, and TGF-β are known to induce IL-37 in vitro (13). Cytokine expression reaches maximal levels in the spinal cord within 6–24 hpi (2), which may account for the first peak of IL-37 expression. Al- in the hIL-37tg mice, but the difference did not reach statistical though IL-37 is expressed mainly in macrophages in hIL-37tg mice significance, whereas CD4 and CD8 lymphocyte counts were un- after several inflammatory challenges (13, 16, 24), endogenous glial changed (Fig. 3). cells (astrocytes and microglia) are likely the early source of IL-37, We also assessed whether IL-37 modulates macrophage and owing to the low amount of infiltrated leukocytes at this time microglia polarization after SCI. FACS analysis of contused spinal point. However, the later expression peak of IL-37 at 3 dpi coin- cords revealed no between-group differences in the expression of cides with the entrance of blood-borne monocytes into the spinal CD16/32 and CD206, known markers of M1 and M2 activation, cord, suggesting that macrophages are the main source of this respectively, on microglia (CD16/32: 76.2 ± 4.2 in WT vs. 74.6 ± second peak (2). 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1523212113 Coll-Miró et al. Regardless of the species or type of SCI, antiinflammatories or myeloid cell depletion consistently reveal reduced tissue damage and greater functional outcomes (2, 3). Here we show that hIL-37tg mice exhibit enhanced locomotor function associ- ated with attenuated tissue damage after SCI. Blood-borne monocytes infiltrating into the injured spinal cord mediate axo- nal retraction (19, 20), and this deleterious effect is due to products released by macrophages or by integral macrophage membrane proteins, which inhibit the growth and guidance of axons (19, 20). Other inhibitory molecules include chondroitin sulfate proteoglycans, Nogo, ephrins, and semaphorins, which are expressed at the lesion site by astrocytes, oligodendrocytes, and some precursor cells (7). In addition, adult CNS neurons have a limited ability to switch on the intrinsic regeneration machinery after axotomy (26). However, despite reduced mac- rophage numbers in the spinal cords of hIL-37tg mice, axonal outgrowth was not observed, because these other factors, which hamper the ability of axons to regenerate in the injured CNS, might not be altered in these mice. Therefore, our data indicate that attenuation of inflammation in the hIL-37tg mice did not overcome the extrinsic and intrinsic factors that curtail the rel- atively limited regenerative potential of injured corticospinal axons. It is possible that increasing the dosage of rIL-37 could NEUROSCIENCE enhance the axonal outgrowth of other motor or sensory tracts with better regeneration capabilities. Once synthesized after activation, up to 30% of the IL-37 precursor is cleaved intracellularly by activated caspase-1 and translocates to the nucleus, most likely in association with Smad3 (13, 17). Indeed, the antiinflammatory effects of IL-37 Fig. 5. Recruitment of immune cells within the spinal cord at 7 dpi. (A–F) Representative density plots of FACS analysis showing myeloid cells and microglia (A and B), macrophages (CD45high, CD11b+, F4/80+) (C and D), and activated microglia (CD45low, CD11b+, F4/80+) (E and F) in the spinal cords of WT and hIL-37tg mice. (G) Quantification of the different immune cell populations in the injured spinal cords. Data are expressed as mean ± SEM. n = 4 per group. *P < 0.05; *P < 0.001; #P = 0.001, t test. IL-37 reduces the expression of several proinflammatory cyto- kines in cell cultures and in various inflammatory disorders (13, 15, 16, 18, 25). We also found that IL-37 attenuates the protein levels of most proinflammatory cytokines evaluated in the contused spinal Fig. 6. Infusion of rIL-37 promotes functional recovery after SCI. Locomotor cord, with IL-6 the most markedly effected. The capacity of IL-37 to performance of C57BL/6 mice treated with intraspinal injections of saline, reduce cytokine production after SCI affects the infiltration and full-length IL-37b (IL-371–218), or the processed form of IL-37b (IL-37V46–218) activation of immune cells. Indeed, we found that IL-37 reduced the after SCI using the BMS (A) and treadmill (B). Data are shown as mean ± recruitment of neutrophils and macrophages into the injured spinal SEM. n = 14 for saline; n = 9 for rIL-371–218 and rIL-37V46–218. *P < 0.05, two- cord, as well as the activation state of the microglia; however, it did way repeated-measures ANOVA with Bonferroni’s post hoc correction in A not affect the infiltration of T cells. and the t test in B. Coll-Miró et al. PNAS Early Edition | 5 of 6 are abolished in LPS-stimulated macrophages when incubated with In summary, rIL-37 prevents functional deficits and secondary caspase-1 inhibitors, or when the caspase-1 site (D20) is mutated tissue damage in SCI, suggesting a potential clinical application. and cannot translocate to the nucleus (27). Similarly, in vivo studies To our knowledge, this is the first report suggesting that IL-37 also have revealed that the antiinflammatory actions of IL-37 are may be an effective therapy during the acute phase after SCI, for lost when Smad3 is silenced after LPS challenge (13). which there is currently no effective treatment. On the other hand, the precursor form of IL-37 is released from human monocytes, or cell lines transfected with IL-37 after LPS Materials and Methods stimulation (13). The release of IL-37 is independent of caspase-1 Animals. All experimental procedures were approved by the Universitat cleavage at D20, however (27). The administration of neutralizing Autònoma de Barcelona Animal Experimentation Ethical Committee (CEEAH antibodies against IL-37 enhances cytokine production in in vitro 1188R3-DMAH 6131) and followed the European Communities Council Di- rective 2010/63/EU, and the methods for each procedure were carried out in and in vivo, highlighting the extracellular antiinflammatory function accordance with the approved guidelines. Experiments were performed in of IL-37 (22, 27). These observations are further supported by adult (8–10 wk) female mice expressing the human form of IL-37 (hIL-37tg) studies showing the ability of rIL-37 protein to reduce cytokine and C57BL/6 (WT) mice (Charles River Laboratory). After SCI, motor skills production (22). Extracellular IL-37 carries out its antiinflammatory were assessed in hIL-37tg mice, WT mice, and WT mice treated with rIL-37. actions by recruiting the decoy IL-1R8 to the IL-18Rα/IL-37 com- Detailed information on SCI models, rIL-37 delivery, and functional assess- plex (28–30). Administration of rIL-37 improves functional recovery ment is provided in SI Materials and Methods. after SCI, indicating that extracellular IL-37 is sufficient for its beneficial effects in the CNS. Nevertheless, we cannot rule out the Tissue Processing. Spinal cords were cut on a cryostat and stained for as- possibility that the translocation of IL-37 to the nucleus also plays a sessment of neuron survival, myelin sparing, and axonal regeneration. Spinal cords were also processed for RNA, cytokine protein level, and FACS analyses. role in the transgenic mice, as well as in humans. Details are provided in SI Materials and Methods. Processing of the IL-37 precursor appears to occur extracellularly. Edman degradation of supernatants from cells lines transfected with ACKNOWLEDGMENTS. This work was supported by grants from the Fundació IL-37b revealed a processed form starting at valine 46 (IL-37V46–218) La Marató, Spanish Ministry of Economy and Competitiveness (SAF2013-48431- (23). Both full-length and processed IL-37 exhibit antiinflammatory R), International Foundation for Research in Paraplegia, and Fondo de Inves- effects (22). We found that both IL-37 forms exerted similar bene- tigación Sanitaria of Spain [Red de Terapia Celular (TERCEL) and Centro de Investigación Biomédica en Red, Enfermedades Neurodegenerativas ficial effects on functional outcomes after SCI, suggesting that the (CIBERNED)], all to R.L.-V.; the National Institutes of Health (Grant AI- IL-37 precursor may be processed when administered in the injured 15614, to C.A.D.); and the Deutsche Forschungsgemeinschaft (Grant BU spinal cord. 1222/3-3, to P.B.). 1. Serhan CN (2014) Pro-resolving lipid mediators are leads for resolution physiology. 19. Evans TA, et al. (2014) High-resolution intravital imaging reveals that blood-derived Nature 510(7503):92–101. macrophages but not resident microglia facilitate secondary axonal dieback in trau- 2. David S, López-Vales R, Wee Yong V (2012) Harmful and beneficial effects of in- matic spinal cord injury. Exp Neurol 254:109–120. flammation after spinal cord injury: Potential therapeutic implications. Handb Clin 20. Horn KP, Busch SA, Hawthorne AL, van Rooijen N, Silver J (2008) Another barrier to Neurol 109:485–502. regeneration in the CNS: Activated macrophages induce extensive retraction of dys- 3. Popovich PG (2014) Neuroimmunology of traumatic spinal cord injury: A brief history trophic axons through direct physical interactions. J Neurosci 28(38):9330–9341. and overview. Exp Neurol 258:1–4. 21. Moretti S, et al. (2014) IL-37 inhibits inflammasome activation and disease severity in 4. David S, Greenhalgh AD, López-Vales R (2012) Role of phospholipase A2s and lipid murine aspergillosis. PLoS Pathog 10(11):e1004462. mediators in secondary damage after spinal cord injury. Cell Tissue Res 349(1): 22. Li S, et al. (2015) Extracellular forms of IL-37 inhibit innate inflammation in vitro and 249–267. in vivo but require the IL-1 family decoy receptor IL-1R8. Proc Natl Acad Sci USA 5. López-Vales R, et al. (2011) Phospholipase A2 superfamily members play divergent 112(8):2497–2502. roles after spinal cord injury. FASEB J 25(12):4240–4252. 23. Pan G, et al. (2001) IL-1H, an interleukin 1-related protein that binds IL-18 receptor/IL- 6. Alexander JK, Popovich PG (2009) Neuroinflammation in spinal cord injury: Thera- 1Rrp. Cytokine 13(1):1–7. peutic targets for neuroprotection and regeneration. Prog Brain Res 175:125–137. 24. Quirk S, Agrawal DK (2014) Immunobiology of IL-37: Mechanism of action and clinical 7. Silver J, Schwab ME, Popovich PG (2015) Central nervous system regenerative failure: perspectives. Expert Rev Clin Immunol 10(12):1703–1709. Role of oligodendrocytes, astrocytes, and microglia. Cold Spring Harb Perspect Biol 25. Ye L, et al. (2014) IL-37 inhibits the production of inflammatory cytokines in periph- 7(3):a020602. eral blood mononuclear cells of patients with systemic lupus erythematosus: Its cor- 8. Rowland JW, Hawryluk GW, Kwon B, Fehlings MG (2008) Current status of acute relation with disease activity. J Transl Med 12:69. spinal cord injury pathophysiology and emerging therapies: Promise on the horizon. 26. Liu K, et al. (2010) PTEN deletion enhances the regenerative ability of adult cortico- Neurosurg Focus 25(5):E2. spinal neurons. Nat Neurosci 13(9):1075–1081. 9. Garlanda C, Dinarello CA, Mantovani A (2013) The interleukin-1 family: Back to the 27. Bulau AM, et al. (2014) Role of caspase-1 in nuclear translocation of IL-37, release of future. Immunity 39(6):1003–1018. the cytokine, and IL-37 inhibition of innate immune responses. Proc Natl Acad Sci USA 10. Dinarello CA, Simon A, van der Meer JW (2012) Treating inflammation by blocking 111(7):2650–2655. interleukin-1 in a broad spectrum of diseases. Nat Rev Drug Discov 11(8):633–652. 28. Yang Y, et al. (2015) IL-37 inhibits IL-18–induced tubular epithelial cell expression of 11. Luo Y, et al. (2014) Suppression of antigen-specific adaptive immunity by IL-37 via pro-inflammatory cytokines and renal ischemia-reperfusion injury. Kidney Int 87(2): induction of tolerogenic dendritic cells. Proc Natl Acad Sci USA 111(42):15178–15183. 396–408. 12. Dinarello CA, Bufler P (2013) Interleukin-37. Semin Immunol 25(6):466–468. 29. Nold-Petry CA, et al. (2015) IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to 13. Nold MF, et al. (2010) IL-37 is a fundamental inhibitor of innate immunity. Nat carry out its multifaceted anti-inflammatory program upon innate signal trans- Immunol 11(11):1014–1022. duction. Nat Immunol 16(4):354–365. 14. Bufler P, Gamboni-Robertson F, Azam T, Kim SH, Dinarello CA (2004) Interleukin-1 30. Lunding L, et al. (2015) IL-37 requires IL-18Rα and SIGIRR/IL-1R8 to diminish allergic homologues IL-1F7b and IL-18 contain functional mRNA instability elements within airway inflammation in mice. Allergy 70(4):366–373. the coding region responsive to lipopolysaccharide. Biochem J 381(Pt 2):503–510. 31. Klopstein A, et al. (2012) Beneficial effects of alphaB-crystallin in spinal cord contu- 15. Ballak DB, et al. (2014) IL-37 protects against obesity-induced inflammation and in- sion injury. J Neurosci 32(42):14478–14488. sulin resistance. Nat Commun 5:4711. 32. Fry EJ, Chagnon MJ, Lopez-Vales R, Tremblay ML, David S (2010) Corticospinal tract 16. McNamee EN, et al. (2011) Interleukin 37 expression protects mice from colitis. Proc regeneration after spinal cord injury in receptor protein tyrosine phosphatase sigma Natl Acad Sci USA 108(40):16711–16716. deficient mice. Glia 58(4):423–433. 17. Bulau AM, et al. (2011) In vivo expression of interleukin-37 reduces local and 33. Stirling DP, Yong VW (2008) Dynamics of the inflammatory response after murine systemic inflammation in concanavalin A-induced hepatitis. ScientificWorldJournal spinal cord injury revealed by flow cytometry. J Neurosci Res 86(9):1944–1958. 11:2480–2490. 34. Basso DM, et al. (2006) Basso Mouse Scale for locomotion detects differences in re- 18. Teng X, et al. (2014) IL-37 ameliorates the inflammatory process in psoriasis by sup- covery after spinal cord injury in five common mouse strains. J Neurotrauma 23(5): pressing proinflammatory cytokine production. J Immunol 192(4):1815–1823. 635–659. 6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1523212113 Coll-Miró et al. Supporting Information Coll-Miró et al. 10.1073/pnas.1523212113 SI Materials and Methods Various combinations of markers were used to identify activated Surgical Procedure. All surgical procedures were approved by the microglia (CD45low, CD11b+, F4/80+), granulocytes (CD45high, Universitat Autònoma de Barcelona Animal Care Committee were CD11b+, F4/80−, Gr-1high), macrophages (CD45high, CD11b+, F4/ performed in accordance with the guidelines of the European 80+), CD4 T cells (CD45+, CD11b−, CD3+, CD4+), and CD8 T Commission on Animal Care. Adult (8–10 wk old) female C57BL/6 cells (CD45+, CD11b, CD3+, CD8+) (31). In addition, microglia mice (Charles River Laboratory) and hIL-37tg mice (13) were an- and macrophages were further differentiated based on CD16/32 esthetized with ketamine (90 mg/kg, i.m.) and xylazine (10 mg/kg, and CD206 and to assess M1 and M2 polarization, respectively. i.m.). After laminectomy at the 11th thoracic vertebrae, the exposed Cells were analyzed using FlowJo software on a FACSCanto flow spinal cord was contused with a force of 60 kdyn using the Infinite cytometer (BD Biosciences). Horizon Impactor device (Precision Scientific Instrumentation) (31) or was completely transected using a microscalpel. At 56 d after Functional Assessment. Locomotor recovery was evaluated at 1, 3, 5, complete spinal cord transection, the corticospinal tract was labeled 7, 10, 14, 21, and 28 dpi in an open-field test using the nine-point by injecting a 10% (wt/vol) solution of the axonal tracer biotinylated BMS (34), which was developed specifically for locomotor testing dextran amine (BDA) into the sensorimotor cortex (32). after contusion injuries in mice. The BMS analysis of hindlimb Administration of rIL-37 protein (b isoform) was performed movements and coordination was performed by two independent intraspinally by means of a glass capillary (30 μm internal diameter; and blinded assessors, and a consensus score was derived. In ad- Eppendorf) coupled to a 10-mL syringe (Hamilton #701). Then, at dition, at the end of the follow-up period (28 dpi), the highest 5 min postinjury, 1 μL of saline, saline containing 100 ng of full- locomotion speed of the mice was evaluated on a belt of a motorized length (rIL-371–218), or rIL-37V46–218 was injected into the injured treadmill. In brief, each mouse was allowed to explore the treadmill spinal cord at the lesion site. Injections were made at a perfusion compartment for 5 min, with the motor speed set to zero. Then the speed of 2 μL/min, controlled by an automatic injector (KDS 310 speed was increased by 2 cm/s every 5 s until the animal was not able Plus; KD Scientific), and the tip of the glass capillary was left in the to maintain the speed. The maximum speed at which each mouse cord tissue for 3 min after each injection to avoid liquid reflux. was able to achieve for 5 s was recorded. RNA Isolation, Reverse Transcription, and Real-Time PCR. The mice Histology. Mice were perfused with 4% paraformaldehyde in 0.1 M were perfused with sterile saline, and a 5-mm-long section of un- phosphate buffer (PB) at 12 hpi, 3 dpi, and 28 dpi. A 5-mm length of injured spinal cord was removed. Tissue was homogenized with spinal cord containing the lesion site was removed, cryoprotected with QIAzol lysis reagent (Qiagen), and RNA was extracted using the 30% sucrose in 0.1 M PB at 4 °C, and six series of 10-μm-thick RNeasy Lipid Tissue Kit (Qiagen), according to the manufacturer’s sections were picked up on glass slides. Thus, adjacent sections on the protocol. RNA thus obtained was treated with DNase I (Qiagen) same slide were 100 μm apart. For demyelination analyses, sections to eliminate genomic DNA contamination. Then 1 μg of RNA was were stained with LFB (Sigma-Aldrich). After graded dehydration, primed with random hexamers (Promega) and reverse-transcribed sections were placed in a 1 mg/mL LFB solution in 95% ethanol and using the Omniscript RT Kit (Qiagen). RNase inhibitor (Roche) 0.05% acetic acid overnight at 37 °C. Sections were then washed in was added (1 U/μL final concentration) to avoid RNA degrada- 95% ethanol and distilled water before being placed into a solution tion. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served of 0.5 mg/mL Li2CO3 in distilled water for 1 min at room temper- as a housekeeping gene. ature. After washing in distilled water, sections were dehydrated and mounted in DPX mounting medium (Sigma-Aldrich). For neuronal Cytokine Protein Expression. The mice were perfused with sterile assessment, sections were incubated overnight at 4 °C with bio- saline, and 5-mm lengths of spinal cord centered on the lesions tinylated antibodies against NeuN. After several washes in PBS, were collected at 12 hpi and 24 hpi and then snap-frozen. The sections were stained using the ABC kit (Vector Laboratories), and a spinal cords were then homogenized, and protein concentration coverslip was applied in DPX mounting medium (Sigma-Aldrich). was determined using the DC Protein Assay (Bio-Rad). Samples The epicenter of the injection or contusion injury impact was were concentrated to 4 μg/μL using MicroCon centrifugation determined for each mouse spinal cord by localizing the tissue filters (Millipore) to ensure equal amounts of protein. The section with the greatest damage using LFB-stained sections. protein levels of 32 cytokines and chemokines were then ana- lyzed using the Milliplex MAP Mouse Cytokine/Chemokine Myelin sparing after SCI was calculated by delineating the spared Magnetic Bead Panel on a Luminex system (EMD Millipore) in LFB- stained tissue area from images taken at 40× magnification accordance with the manufacturer’s protocol. at the injury epicenter and every 200 μm rostral and caudal to the lesion site. Neuronal survival was assessed by manually counting Flow Cytometry. Immune cells from the injured spinal cords were the number of NeuN+ cells in high-magnification (400×) images analyzed by flow cytometry at 1 dpi and 7 dpi as described previously obtained at both ventral horns at the injury epicenter and every (33). In brief, spinal cords were cut into small pieces and passed 200 μm rostral and caudal to the lesion site. ImageJ software was through a 70-μm cell strainer (BD Falcon) using saline. The cell used to quantify all of the histological parameters. suspension was centrifuged at 300 × g for 10 min at 4 °C. The pellet At 10 d after the BDA tracer injections (66 dpi), mice were was then resuspended and centrifuged a second time. The samples perfused, and the transected spinal cords were harvested and cut were divided, and cells alone and isotype-matched control samples into 20-μm-thick sagittal sections as described above. Visualization were generated to control for nonspecific binding of antibodies and of corticospinal fibers was done by incubating the spinal cord for autofluorescence. The following antibodies were purchased sections with Alexa Fluor 594-conjugated streptavidin. Pictures from eBioscience: CD45-PerCP, CD11b-PE-Cy7, Gr1-FITC, F4/ from the spinal cord covering the transection lesion and up 3,000 μm 80-APC, F4/80-PE, CD3-FITC, CD4-APC, CD8-APC, and CD19- rostral were obtained at 200× magnification and then merged PE. After 30 min of incubation with combinations of antibodies at into collages using Adobe Photoshop software. Only sections that 4 °C, the samples were washed and fixed in 1% paraformaldehyde. contained BDA-labeled fibers were acquired. The number of pixels Coll-Miró et al. www.pnas.org/cgi/content/short/1523212113 1 of 3 of BDA-labeled fibers with a dorsoventral line was counted every and subscores, along with histological analyses of myelin and 150 μm over the rostrocaudal axis using ImageJ. neuronal sparing as well as BDA+ fibers, were done using two-way repeated-measures ANOVA. Post hoc comparisons were carried Statistical Analysis. All analyses were conducted with SPSS version out only when a main effect showed statistical significance. P values 19. The two-tailed Student’s t test was used for single comparisons for multiple comparisons were adjusted using Bonferroni’s post between two groups. Maximal speed on a treadmill was analyzed hoc correction. Results are expressed as mean ± SEM. Differences using the Mantel–Cox test. Functional follow-up for BMS scores were considered significant at P < 0.05. Fig. S1. Multiplex analysis of cytokine protein profiles from spinal cords of WT and hIL-37tg mice at 24 hpi. Data are expressed as fold change vs. WT mice (mean ± SEM). Coll-Miró et al. www.pnas.org/cgi/content/short/1523212113 2 of 3 Fig. S2. Multiplex analysis of cytokine protein profiles from spinal cords of WT and hIL-37tg mice at 12 hpi. Data are expressed as fold change vs. WT mice (mean ± SEM). Coll-Miró et al. www.pnas.org/cgi/content/short/1523212113 3 of 3