Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages is necessary for functional spinal cord regeneration in zebrafish

Abstract

Spinal cord injury leads to a massive response of innate immune cells (microglia, macrophages, neutrophils) both, in non-regenerating mammals and in successfully regenerating zebrafish, but the role of these immune cells in functional spinal cord regeneration in zebrafish has not been addressed. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in larval zebrafish. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary and sufficient for full regeneration.Macrophage-less irf8 mutants show prolonged inflammation with elevated levels of Il-1β and Tnf-α. Decreasing Il-1β levels or number of Il-1β + neutrophils rescues functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1β function impairs regeneration in irf8 and wildtype animals.Inhibiting Tnf-α does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-α. Hence, inflammation is tightly and dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.

Macrophages: The Potent Immunoregulatory Innate Immune Cells

“…In addition macrophages are also shown to involve in the process of regeneration of heart/cardiomyocytes in different animals (Zebra fish, Salamander, and the laboratory mouse) [165][166][167]. Even studies have also shown the involvement of macrophages in the regeneration of spinal cord and tail fin of Zebra fish [168,169]. Wnt signaling in macrophages plays a critical role in driving parenchymal regeneration in animal models of liver injury [170].…”

Microglia Stimulate Zebrafish Brain Repair Via a Tumor Necrosis Factor-α-Initiated Inflammatory Cascade

“…Macrophages are also critical for regeneration of fin, retina, and heart in zebrafish (Huang et al, 2012; Petrie et al, 2014; Sanz-Morejon et al, 2019). In contrast, several studies showed that microglia are not necessary for murine axonal regeneration (Hilla et al, 2017) and spinal cord repair in zebrafish (T. M. Tsarouchas et al, 2018). Though many studies have shown that the microglial response after injury, whether microglia are required for successful repair after acute brain injury in zebrafish is unknown.…”

“…In contrast, acute inflammation appears necessary for promoting regeneration after injury in zebrafish. Inflammatory mediators implicated in the zebrafish CNS pro-regenerative response include leukotrienes in brain (Kyritsis et al, 2012), interleukin (il) 6, il11, and leptin in retina (Wan et al, 2014; Zhao et al, 2014), and tnf α and Il1 β in spinal cord (T. M. Tsarouchas et al, 2018).…”

Microglial response patterns following damage to the zebrafish olfactory bulb

“…The decrease in amoeboid profiles that occurs between 4 h and 12 h after cautery deafferentation may be due to a delayed recruitment phase. A recent study reported anti-inflammatory cytokines, such as tgf-β1a and tgf-β3, being expressed at low levels during initial regeneration, and upregulated during late regeneration in zebrafish, suggesting a biphasic immune response within the 48 h after injury (Tsarouchas et al, 2018). Other studies have suggested that adult microglia have a low proliferation level and slow turn-over rate in mice (McCarthy and Leblond, 1988; Lawson et al, 1992; Inman and Downs, 2007), suggesting proliferation and reconstitution of microglia to be the limiting factor, perhaps exacerbated by the severity of the damage.…”

“…Sections were rinsed in PBS and placed in blocking solution (3% normal goat serum, 0.4% Triton X-100 in PBS) for 1 h at room temperature and in 4C4 mouse monoclonal antibody (1:100, a kind gift of Drs. Pamela Raymond and Peter Hitchcock, University of Michigan) to label microglia (Becker and Becker, 2001; Raymond et al, 2006; Craig et al, 2008; Baumgart et al, 2012; Tsarouchas et al, 2018). Sections were rinsed in PBS and then incubated in biotinylated goat anti-mouse secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature.…”