Role of the innate immune system in the regeneration of the zebrafish larvae spinal cord

Spinal cord injury leads to an increased response of innate immune cells (microglia, macrophages, neutrophils) in non-regenerating mammals and in successfully regenerating zebrafish. Here I characterize the axonal regrowth of the severed axons after complete spinal cord transection and define the role of the innate immune reaction in successful spinal cord regeneration in larval zebrafish. I found that axons start to regenerate at 18 hours post injury and by 48 hours 80% of the injured larvae show axonal growth across the injury site. The use of reporter lines to visualize the neuronal and glial elements of the spinal cord combined with immunostaining and live imaging allowed me to find that the axons and the glial processes are able to regenerate independently, following different kinetics. Inhibiting inflammation reduces and promoting it accelerates axon regrowth. Mutant analysis shows that peripheral macrophages, but not neutrophils or microglia, are crucial for axonal regeneration and functional recovery. Macrophage-deficient irf8 mutants, characterized also by increased numbers of neutrophils, fail to down-regulate pro-inflammatory cytokines TNF-α and Il-1β and to up-regulate anti-inflammatory cytokines, indicating prolonged inflammation. Because sustained inflammation is considered to be detrimental to regeneration in mammals, I determined the role of increased expression of TNF-α and IIl-1β in axonal regeneration. Inhibition of TNF-α does not rescue the mutant but impairs axonal growth in wildtype larvae. Furthermore, CRISPR/Cas9-mediated disruption of TNF-α led to increased expression of the pro-inflammatory cytokine Il-1β. Taken together these findings indicate an unexpected pro-regenerative and anti-inflammatory role of TNF-α. In contrast, inhibition of Il-1β using pharmacological and genetic approaches rescues axonal regeneration and functional recovery in irf8 mutants, indicating that Il-1β is a major determinant during successful spinal cord regeneration. In order to determine the relative importance of the neutrophils for the regenerative failure in irf8 mutants, we used morpholino injections to reduce their numbers. The treatment prevented the formation of the neutrophils and decreased the expression levels of Il-1β, indicating that neutrophils act as a source for Il-1β in the irf8 mutants. In wildtype animals, early, but not late axon regrowth is attenuated by Il-1β inhibition, indicating dynamic changes in Il-1β function during regeneration. However, the regenerative potential of adult and larval zebrafish is not restricted only to their ability to re-grow severed axons, since they can make lost neurons as well after injury. In order to assess the role of the innate immune response during neuronal regeneration, I used dexamethasone to inhibit the inflammation that occurs after injury. I found that the treatment leads to a decreased number of new-born motor neurons in the injury site, indicating the importance of the inflammatory response for axonal, as well as, neuronal regeneration of the spinal cord after injury. Furthermore, I used the macrophage deficient irf8 mutants in order to assess how lost motor neurons are regenerated in an environment with no macrophages/microglia. I found that motor neurons fail to regenerate properly after injury in the mutants, indicating the importance of macrophages/microglia, during neuronal regeneration. Taken together, these results demonstrate the complex inflammatory response that occurs after spinal cord injury and provide insight into the role of macrophages that tightly control it, by expressing TNF-α and preventing prolonged expression of Il-1β, to promote successful spinal cord regeneration in zebrafish