Neuronal self-injury mediated by IL-1β and MMP-9 in a cerebral palsy model of severe neonatal encephalopathy induced by immune activation plus hypoxia-ischemia

Abstract

BackgroundInflammation due to remote pathogen exposure combined to hypoxia/ischemia (HI) is one of the most common causes of neonatal encephalopathy affecting at-term or near-term human newborn, which will consequently develop cerebral palsy. Within term-equivalent rat brains exposed to systemic lipopolysaccharide (LPS) plus HI, it was previously showed that neurons produce IL-1β earlier than do glial cells, and that blocking IL-1 was neuroprotective. To further define the mechanisms whereby IL-1 exerts its neurotoxic effect, we hypothesize that IL-1β plays a pivotal role in a direct and/or indirect mechanistic loop of neuronal self-injury through matrix metalloproteinase (MMP)-9.MethodsAn established preclinical rat model of LPS+HI-induced neonatal encephalopathy was used. In situ hybridization, ELISA, and immunolabeling techniques were employed. Selective blocking compounds allowed addressing the respective roles of IL-1 and MMP-9.ResultsIn LPS+HI-exposed forebrains, neuronal IL-1β was first detected in infarcted neocortical and striatal areas and later in glial cells of the adjacent white matter. Neuronal IL-1β played a key role: (i) in the early post-HI exacerbation of neuroinflammation and (ii) in generating both core and penumbral infarcted cerebral areas. Systemically administered IL-1 receptor antagonist (IL-1Ra) reached the brain and bound to the neocortical and deep gray neuronal membranes. Then, IL-1Ra down-regulated IL-1β mRNA and MMP-9 neuronal synthesis. Immediately post-HI, neuronal IL-1β up-regulated cytokine-induced neutrophil chemoattractant (CINC-1), monocyte chemoattractant protein-1 (MCP-1), and inducible nitric oxide synthase. MMP-9 would disrupt the blood–brain barrier, which, combined to CINC-1 up-regulation, would play a role in polymorphonuclear cell (PMN) infiltration into the LPS+HI-exposed brain. IL-1β blockade prevented PMN infiltration and oriented the phenotype of macrophagic/microglial cells towards anti-inflammatory and neurotrophic M2 profile. IL-1β increased the expression of activated caspase-3 and of receptor-interacting-protein (RIP)-3 within infarcted forebrain area. Such apoptotic and necroptotic pathway activations were prevented by IL-1Ra, as well as ensuing cerebral palsy-like brain damage and motor impairment.ConclusionsThis work uncovered a new paradigm of neuronal self-injury orchestrated by neuronal synthesis of IL-1β and MMP-9. In addition, it reinforced the translational neuroprotective potential of IL-1 blockers to alleviate human perinatal brain injuries.

Live imaging of the innate immune response in neonates reveals differential TLR2 dependent activation patterns in sterile inflammation and infection

“…Interestingly, Pam3CSK4 but not LPS induces IL-1β when stimulation occurs in the premature brain (Stridh et al, 2011). While IL-1β is generally considered injurious, genetic manipulations of the pathway showed rather complex effects on hypoxia-ischemia injury in neonatal brain (Doverhag et al, 2010; Girard et al, 2008; Savard et al, 2015; Stridh et al, 2011). TLR2 is regulated in hypoxia-ischemia and by LPS (Triantafilou and Triantafilou, 2002), counteracting the proliferation of neuronal progenitor cells and leading to defects in brain plasticity and behaviour (Yuan et al, 2010).…”

Perinatal Arterial Ischemic Stroke Is Associated to Materno-Fetal Immune Activation and Intracranial Arteritis

“…Labeling was assessed by conventional and confocal microscopies. We used three different techniques for the IHC and IF staining assessments, as previously described [36,41,42,43]: ( i ) for some IHC (Iba-1, ICAM-1, P-selectin, PMN, CD3, CD40, CD40L, and vWF) and IF (Iba-1, TNF-α, IL-1β PCNA, iNOS, and Arg-1) labeling, counting of labeled and unlabeled cells (data presented as ratio between labeled and total cells (%)) was performed using a positive vs. negative score; ( ii ) for the SOD-1 labeling by IF, we used a 0–3 score, in which 0 = no labeling, 1 = low-intensity, 2 = mid-intensity, and 3 = high-intensity labeling (Figure S1, data presented using this labeling score); and ( iii ) for other IHC staining assessment (IL-1β, IL1-Ra, TNF-α, and tissue factor), we used the ImageJ analysis software [44] (data presented as staining intensity and/or stained area). We first separated the immunostaining (brown) from the hematoxylin stain (purple) using the extract-brown plugin generated by the image analysis platform of the Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke (PAVI: plateforme d’analyse et de visualisation d’images).…”

“…Rats were placed on their back, four paws in the air. The time to move from paws in the air to four paws on the ground was measured and the pups were classified as having an impaired righting reflex when the delay to achieve a full righting reflex was more than 1 s. In the Elevated Body Swing Test (EBST) P16 rats were held by the base of the tail head downwards during 20 s, as previously described [42,46]. The number of effective upswings (+90°) and the total number of upswing were compared between the S + PT + H and LPS + PT + H groups.…”

Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain

“…Consequently, it would also be important in future studies to examine the effect of ischemia on in situ hybridization and zymography combined with the immunohistochemistry of tight junction proteins to understand the role of MMPs in BBB dysregulation after injury in the fetal brain. Moreover, MMP inhibitors have been shown to have neuroprotective effects in neonatal rodents after hypoxic-ischemic brain in- jury [12,13,66]. Inhibition of MMP-8 activity partially restores BBB function in a model of human brain microvascular endothelial cells [67].…”

“…MMPs are a family of neutral ECM-degrading proteases including gelatinases (MMP-2 and -9) and collagenases (MMP-8 and -13) [17][18][19][20]. The expression and activity of MMPs are altered in response to hypoxic-ischemic insults and represent potential therapeutic targets in perinatal hypoxic-ischemic brain injury [11][12][13]21]. TIMPs are a small family of 4 secreted protease inhibitors including TIMP-1, -2, -3, and -4.…”