Background
Hypoxia-ischemia (HI) combined to inflammation is an important pathophysiological component of neonatal encephalopathy (NE) affecting up to 1 % of newborns. Indeed, combined pathogen-induced inflammation complicated by HI was recognized as one of the most common causes of human perinatal brain damage [
1‐
8]. Moderate and severe forms of NE lead either to death or lifelong neurodisabilities such as cerebral palsy (CP) or other neurobehavioral disabilities including learning difficulties [
1,
4]. Combination of inflammation/infection plus HI has been reproduced experimentally in rodents at neurodevelopmental stages equivalent to early and late preterm development of human neonates—i.e., in rat pups between P1 and P7 [
5,
9‐
13]. Inflammation/infection and HI have also recently been modeled in rats at a neurodevelopmental stage (P12) corresponding to full-term human neonate development. Profound differences in the neuropathological impacts from lipopolysaccharide (LPS) plus HI-induced patterns of innate immune responses have been shown between P1 and P12 rats [
13,
14]. Importantly, these neuropathological differences reproduce those observed between preterm and term human newborns [
14]. Thus, LPS+HI-induced brain injury of P12 rat pups is a relevant model for the study of term human NE [
15].
We previously showed that, within term-matched rat brains exposed to systemic LPS+HI, neurons produce IL-1β earlier than do glial cells, and that IL-1 receptor antagonist (IL-1Ra) was neuroprotective [
15]. To further define the neurotoxic mechanisms of IL-1 in the immature brain, we hypothesize that IL-1 induces directly and/or indirectly neuronal self-injury involving matrix metalloproteinase (MMP)-9, blood–brain barrier (BBB) disruption, and polymorphonuclear cell (PMN) infiltration.
Discussion
Our key findings are that IL-1β and MMP-9 from neuronal origin play a central role in LPS+HI-induced injury of the newborn brain. Neuronal IL-1β might be up-regulated both via mRNA synthesis and NLRP1 inflammasome activation. IL-1Ra (peripherally administered) reaches the brain and interacts directly with neuronal cells to interfere with IL-1β mRNA and protein and MMP-9 synthesis. Neuronal IL-1β up-regulates chemokines (CINC-1, MCP-1), as well as other pro-inflammatory and neurotoxic mediators, namely iNOS and MMP-9. MMP-9—likely in combination with iNOS—might participate to disrupt the BBB [
23,
24]. BBB leaks, combined to CINC-1 up-regulation, would facilitate PMN infiltration within the brain. IL-1β modulates macrophages’ polarization since its blockade maintains the M1/M2 balance towards an M2 anti-inflammatory and neuroprotective phenotype. Finally, IL-1β activates neural cell death pathways, namely RIP-3-associated necroptosis and caspase-3-mediated apoptosis. Altogether, these results show that IL-1β and MMP-9 contribute to neuronal self-injury and that their blockade curbs this effect.
Pro-IL-1β is cleaved by a macromolecular complex called the inflammasome for IL-1β secretion to occur [
25]. Stimuli triggering inflammasome assembly and activation, within LPS+HI-exposed neurons, could be damage-associated molecular patterns (DAMPs) release due to the release of endogenous molecules following cellular damage such as that induced by LPS+HI [
20]. Inflammasome-dependent IL-1β processing relies on distinct NLRs that respond to the intracellular milieu and trigger inflammasome assembly [
20]. We showed that NLR expression within neural cells is developmentally regulated, NLRP1 being expressed at P12 (but not P1) within neurons. NLRP3 however was undetected in the P1 or P12 immature brain and only expressed later. Thus, exposure to LPS+HI might release DAMPs within the cytoplasm of neurons leading to exacerbated activation of IL-1β, through NLRP1 inflammasome activation. In addition, LPS might interact with TLR-4 to increase the synthesis of IL-1β via NF-κB activation. Such ability of neurons to produce IL-1β mRNA and protein has also been reported by others in rodents subjected to either HI, traumatic or excitotoxic brain injury [
26‐
28]. Thus, exposure to LPS+HI at a specific term-like stage of brain development may lead to an autocrine/paracrine loop of neuronal self-injury, mediated by IL-1β and subsequent neurotoxic mechanisms such as IL-1β-induced MMP-9 and NO production and/or IL-1β exacerbation of excitotoxic damage [
25,
29]. Consistent with such mechanisms, IL-1Ra blockade of the IL-1β signaling pathway decreases the extent of LPS+HI-induced brain injury. Another mechanism whereby IL-1Ra limits LPS+HI-induced injury appears to be by preventing the IL-1β-induced M1 polarization of macrophages [
30].
MMP-9 competitive inhibitor shrunk the size of LPS+HI-induced brain lesions to a same extent as IL-1Ra treatment. MMP-9 induces cell death via anoikis, an integrin-mediated form of caspase-3-induced apoptosis [
31]. When the cell loses its tether to the extracellular matrix, anoikis is induced via caspase-9 and the apoptosome [
31]. Such a cell death mechanism is relevant to our findings showing LPS+HI-induction of activated caspase-3 and subsequent neuronal cell death—that is preventable by MMP-9 inhibitor treatment. MMP-9 also alters the BBB by degrading the lamina of intra-cerebral blood vessels. Such disruption would enable pro-inflammatory and/or neurotoxic mediators to leak through the BBB and thus increase the extent of intra-cerebral lesions. NO is also known for its deleterious effects on the BBB [
32]. Causing a rise of expression of iNOS in LPS+HI brains, together NO and MMP-9 could alter the BBB permeability.
We also observed cerebral expression of TNFα following LPS+HI exposure (data not shown). It is known that TNFα—the synthesis of which is induced by IL-1β—mediates receptor-interacting-protein (RIP) kinase-dependent necroptosis [
33]. RIP-1 and -3 associate with TNF receptor-associated death domain (TRADD) and migrate to the mitochondria [
33]. This complex releases reactive oxygen species (ROS) and DNAses from the mitochondria, inducing necroptosis [
33]. Accordingly, our findings show that RIP-3 expression is increased after LPS+HI. IL-1β-induced MMP-9 (see Fig.
8) also activates necroptosis via FAS-FAS ligand (FASL) interaction through FAS-associated death domain (FADD) [
31,
33]. RIP kinases associated with FADD, as well as TRADD, activate necroptosis [
33]. Either of these cell death mechanisms might be involved in LPS+HI-induced brain damage in term NE. In human NE, the only current treatment known to be efficient is hypothermia [
34,
35]. Hypothermia acts on penumbra but not core lesions in animal models of neonatal HI encephalopathy [
34]. In human NE, hypothermia imparts only partial neuroprotection. IL-1Ra, which acts on both, may one day afford additional neuroprotective benefits to hypothermia [
34,
35]. However, this needs to be tested in future clinical trials.
Acknowledgements
We thank Karine Belleville and Jean Lainé for their assistance. We thank Jasna Kriz for the in situ hybridization protocol. This work was supported by: Canadian Institutes for Health Research (CIHR), Fonds de la Recherche du Québec—Santé (FRQ-S), Foundation of Stars, Heart and Stroke Foundation Canada, Centre Mère-Enfant de l’Université de Sherbrooke, and Centre des Neurosciences de l’Université de Sherbrooke, Canada. GS is a member of the FRQ-S-funded Centre de Recherche Clinique du CHU de Sherbrooke (CHUS)—.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AS, CG, and MC carried out the experiments. AS performed the statistical analyses. AS and MEB drafted the manuscript. GS conceived the study, coordinated the project, and further edited the manuscript. CG and DG helped to draft the manuscript. All authors read and approved the final manuscript.