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Modulation of the Inflammatory Response After Spinal Cord Injury

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Modulation of the Inflammatory Response After Spinal Cord Injury ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en MODULATION OF THE INFLAMMATORY RESPONSE AFTER SPINAL CORD INJURY Presented by Jesús Amo Aparicio ACADEMIC DISSERTATION To obtain the degree of PhD in Neuroscience by the Universitat Autònoma de Barcelona 2019 Directed by Dr. Rubèn López Vales Tutorized by Dr. Xavier Navarro Acebes INDEX SUMMARY Page 7 INTRODUCTION Page 13 - Spinal cord Page 15 - Spinal cord injury Page 17 - Incidence and causes Page 18 - Types of SCI Page 18 - Biological events after SCI Page 20 - Studying SCI Page 24 - Animal models Page 24 - Lesion models Page 24 - Current therapies for SCI Page 25 - Basic principles of the immune system Page 27 - Innate immune response Page 27 - Adaptive immune response Page 28 - Inflammatory response Page 29 - Inflammatory response after SCI Page 30 - Modulation of injury environment Page 36 - Interleukin 1 Page 36 - Interleukin 37 Page 40 - Interleukin 13 Page 44 OBJECTIVES Page 47 MATERIALS AND METHODS Page 51 - Approvals Page 53 - Mice Page 53 - Genotyping Page 54 - Spinal cord injury surgery Page 55 - Treatment injections Page 55 - Flow cytometry Page 55 - Gene expression (qPCR) Page 56 - Luminex (bead-based multiplex assay) Page 57 - Functional assessment Page 57 - Histology Page 58 - Cell sorter and RNA extraction Page 59 - Low-input RNA-seq Page 59 - RNA-seq processing and analysis Page 60 - Cell culture Page 60 - ELISA Page 60 - Gene expression (cytokine array) Page 60 - Cell metabolism Page 61 - Statistics Page 61 PRODUCT’S REFERENCES Page 63 RESULTS Page 67 - Chapter 1: Protocols Page 69 - Abstract Page 69 - Introduction Page 69 - Protocols Page 70 - Commentary Page 81 - References Page 85 - Chapter 2: MABp1 and OLT1177 Page 87 - Abstract Page 87 - Introduction Page 88 - Materials and methods Page 89 - Results Page 91 - Discussion Page 94 - References Page 97 - Chapter 3: IL-37 Page 101 - Abstract Page 101 - Introduction Page 102 - Materials and methods Page 103 - Results Page 106 - Discussion Page 113 - References Page 116 - Chapter 4: IL-13 Page 119 - Abstract Page 119 - Introduction Page 120 - Materials and methods Page 121 - Results Page 124 - Discussion Page 133 - Supplementary figures Page 137 - References Page 142 GENERAL DISCUSSION Page 145 FINAL CONCLUSIONS Page 157 COMPLETE REFERENCES Page 161 ABREVIATIONS Page 179 SUMMARY - 7 - - 8 - MODULATION OF INFLAMMATORY RESPONSE AFTER SPINAL CORD INJURY Spinal cord injury (SCI) consists in the disruption of the ascending and/or descending information pathways due to the death of nervous tissue. Since axons from the central nervous system do not regenerate, injury results in a permanent lack of communication between the brain and the regions of the body below the lesion site. This lack of communication translates into loss of movement, loss of sensation, autonomic deficits, and pain. The final magnitude of these dysfunctions is defined by the level, the severity, and the type of injury. However, there are many secondary degenerative processes that take place in the spinal cord after primary injury and that contribute to the final dysfunctions. Among these processes, inflammatory response is one of the most important. Spinal cord injury elicits an inflammatory response produced mainly by macrophages and microglia. All the process is orchestrated by cytokines. These cells and mediators are fundamental for the clearance of cellular debris and the healing of damaged tissue. However, in contrast to other tissues, inflammatory response in the central nervous system is not properly resolved. Exacerbated and uncontrolled inflammatory response produces damage to healthy neighboring tissue, increasing the lesion size and worsening the final deficits. The main objective of this thesis is the modulation of the inflammatory response after SCI, maintaining the essential benefits of an immune response but avoiding dangers of exacerbated immunity. We hypothesize that this modulation will result in improvement of tissue preservation and functional recovery in a murine model of SCI. First two strategies of this thesis are based on the suppression of pro-inflammatory environment targeting IL-1a and IL-1b, main cytokines in the first stages of the inflammatory response. Targeting of IL-1a was performed by MABp1, a monoclonal antibody recently approved for clinical use. This antibody neutralizes the soluble form of IL-1a and inhibits its binding to receptors. Besides promising findings in other inflammatory-mediated diseases, MABp1 failed to promote protection after SCI. Targeting of IL-1b was performed by OLT1177, an inhibitor of the NLRP3 inflammasome needed for the processing and release of IL-1b and IL-18. OLT1177 successfully promoted tissue protection and locomotor improvement when injected intraperitoneally after SCI. Since OLT1177 is safe in humans, it is a promising candidate for the treatment of SCI. Last two strategies of this thesis are based on the potentiation of the anti-inflammatory environment by IL-37 and IL-13. Previous result from our group showed that increasing IL-37 levels promoted a protective effect after SCI. Here we studied the contribution of the nuclear and extracellular pathways to the role of IL-37. We demonstrated that, although both pathways promote protective effects, benefits after SCI were attributed mainly to extracellular signaling. Increasing levels of IL-13 promoted anti-inflammatory environment after SCI, as previously reported by our group with IL-4. However, in contrast to IL-4, IL-13 failed to confer functional - 9 - improvement. To determine differences between these two cytokines, we performed the first cell-specific RNA sequencing of macrophages and microglia after SCI. We observed that, although both cytokines have the same effect on the modulation of the injury environment, they induce differences in the metabolism of macrophages and microglia. Metabolism induced by IL-4 was more oxidative whereas metabolism induced by IL-13 was more glycolytic. Since inflammatory response is a very demanding process, how cell obtain energy may be crucial for the development of the immune response and may explain differences observed at functional level. Future of SCI is multidisciplinary. Through this thesis we provide evidences to support that trophic modulation of the injury environment is a fundamental component of novel therapeutic approaches to treat SCI. - 10 - MODULACIÓN DE LA RESPUESTA INFLAMATORIA DESPUÉS DE UNA LESIÓN DE MÉDULA ESPINAL La lesión de médula espinal (LME) consiste en la disrupción de la vías ascendentes y/o descendentes de información debido a un daño del tejido nervioso. Puesto que los axones del sistema nervioso central no regeneran, el daño conduce a una falta de comunicación permanente entre el cerebro y las regiones del cuerpo que están por debajo de la zona de lesión. Esta falta de comunicación se traduce en pérdida de movimiento, pérdida de sensación, déficits autonómicos y dolor. La magnitud final de estos déficits se define en base al nivel, la severidad y el tipo de lesión. Sin embargo, hay muchos procesos secundarios degenerativos que ocurren en la médula espinal después del daño primario y que contribuyen a los déficits finales. Entre estos procesos, la respuesta inflamatoria es uno de los más importantes. La LME activa una respuesta inflamatoria producida principalmente por macrófagos y microglía. Todo el proceso está dirigido por citoquinas. Estas células y mediadores son fundamentales para la limpieza de los restos celulares y la recuperación del tejido dañado. Sin embargo, a diferencia de otros tejidos, la respuesta inflamatoria del sistema nervioso central no se resuelve adecuadamente. Una respuesta inflamatoria exacerbada puede producir daños en el tejido vecino sano, incrementando el tamaño de la lesión y empeorando los déficits finales. El objetivo principal de esta tesis es la modulación de la respuesta inflamatoria después de la LME, manteniendo los beneficios esenciales de la respuesta inflamatoria pero evitando los daños atribuidos a una respuesta exacerbada. Según nuestra hipótesis, esta modulación resultará en una mejora de la preservación del tejido y de la recuperación funcional en un modelo murino de LME. Las primeras dos estrategias de esta tesis están basadas en la supresión del ambiente proinflamatorio actuando sobre IL-1a e IL-1b, citoquinas principales en las primeras fases de la respuesta inflamatoria. La actuación sobre IL-1a se realizó mediante MABp1, un anticuerpo monoclonal recientemente aprobado para su uso clínico. Este anticuerpo neutraliza la forma soluble de la IL-1a e inhibe su unión a los receptores. A pesar de los hallazgos prometedores en otras enfermedades mediadas por la respuesta inflamatoria, MABp1 no promovió protección después de una LME. La actuación sobre IL-1b se realizó mediante OLT1177, un inhibidor del inflamasoma NLRP3 necesario para el procesamiento y la liberación de IL-1b e IL- 18. OLT1177 promovió de manera exitosa la preservación del tejido
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