Background
Human newborns, especially preterm, are at high risk of brain damage [
1‐
4]. Decreased oxygen and other blood nutrient supply to the brain, remote pathogen exposure, or both combined, and the associated neuroinflammatory responses are the most important perinatal risk factors associated with brain injury and subsequent cerebral palsy and/or learning and behavioral impairments [
1‐
3]. The incidence of these forms of neonatal brain damage is inversely proportional to gestational age and thus higher in preterm than in term newborn [
4]. Type and distribution of brain lesions differ markedly between preterm and term newborns [
4,
5]. This is attributed to different levels of brain maturity and vulnerability to aggression due to regional and age-specific metabolic needs [
1,
2,
4,
5]. Post-natal bacterial infections - that are more frequent in pre-term than term newborns -, and often combined with hypoxia-ischemia (HI), are also associated with an increased risk to develop brain lesions [
6]. Our hypothesis is that developmental differences in neuroinflammatory responses contribute to the age-specific patterns of brain injury.
Pro-inflammatory cytokine expression within the brain, especially IL-1β and TNF-α, has been implicated in perinatal brain damage induced by pathogen components and/or HI both in experimental model [
7‐
12] and the human newborn brain [
13‐
23]. On the other hand, relatively little is known about anti-inflammatory and neurotrophic cytokine responses in such perinatal brain damage. Anti-inflammatory cytokines, such as IL-1 receptor antagonist (IL-1ra), IL-6, IL-10, and TGF-β1, are already known either to be constitutively expressed to support brain development, or to be induced in pathological conditions to counterbalance the pro-inflammatory response and to promote neuronal survival [
24‐
34]. Although IL-6 is often classified as a pro-inflammatory mediator in peripheral pathologies, it has not been directly implicated in perinatal brain damage and its blockage was even showed to be deleterious in some pathological circumstances [
35,
36]. During brain development, IL-6 has established neurotrophic properties [
35].
To uncover potential differences between term and preterm neuroinflammatory responses to neonatal insults, we used rat models of brain damage induced at different stages of brain development. Postnatal day 1 (P1), corresponds, in term of brain development, to the very preterm human brain (26-32 weeks of gestation), whereas P12, corresponds to the term human neonate brain [
37]. We compared the intracerebral profiles of pro- and anti-inflammatory cytokines responses, resulting chemokines responses, and related immune cell recruitments at both developmental stages.
Discussion
Using animal models of brain insults occurring at neurodevelopmental stages equivalent to the
preterm (P1) and
term (P12) human brain [
37], we showed that exposures to a bacterial endotoxin (LPS) and/or HI led to distinct patterns of neuroinflammatory responses depending on the stage of brain maturation, and on the type of insult. At P1, the neuroinflammatory reaction triggered by HI, LPS or LPS+HI was limited to IL-1β and MCP-1 - with no TNF-α - over-expression, and without any concomitant induction of classic counteracting anti-inflammatory cytokines (see Table
1). IL-1β expression at P1 was more prominent within the cerebral white matter than in the gray matter, thus correlating with predominant white matter damage occurrence previously described under our P1 experimental conditions in rodents [
40], and as also typically seen in premature human newborns [
4,
43]. At P1, anti-inflammatory cytokines' responses were absent (IL-6, IL-10), or even down-regulated (IL-1ra, TGF-β1) under HI, LPS or LPS+HI conditions. Lack of anti-inflammatory response at P1 might deprive the challenged brain of neurotrophic factors - such as TGF-β1, IL-10, IL-1ra and IL-6 - involved in neuronal survival and brain tissue repair following brain injuries [
7,
44‐
53]. Therefore, this P1 pro-inflammatory orientation (mainly intracerebral IL-1β and MPC-1 up-regulations) combined with the vulnerability of the immature neural cells might account for the brain damage induced (Table
1). Well described IL-1β neurotoxic effects in conjunction with other noxious mediators released by MCP-1 attracted macrophages, such as matrix metalloproteinases (MMPs) and reactive oxygen species, might be involved in the generation of such LPS+/-HI induced brain damage at P1 [
23,
54].
At P12, in sharp contrast to P1, both pro- (IL-1β peaking 8-fold higher than TNF-α at 4 h post LPS+HI) and anti-inflammatory cytokines (namely IL-1ra, IL-6, IL-10 and TGF-β1) were over-expressed within brains exposed to HI or LPS+HI (Table
1). Despite the combined induction of pro- and anti-inflammatory cytokines at P12, the balance of pro/anti effects remained strongly oriented towards inflammation, at a neurodevelopmental stage equivalent to the term human brain. HI-, LPS- or LPS+HI-induced chemokine responses (MCP-1 and/or CINC-1), known to derive in part from the activation of the IL-1β pathway [
55,
56], were much more prominent at P12 than at P1, particularly under LPS+HI conditions compared to HI alone (Table
1). Combined, and likely interacting, IL-1β, TNF-α and chemokine inductions were also associated at P12 (but not at P1) with BBB leakage and massive neutrophil infiltration within HI and LPS+HI damaged brain areas. Known TNF-α myelinotoxicity and IL-1β neurotoxicity, joined to other neurotoxic mediators released by neutrophils, such as MMPs and reactive oxygen species, might well play a central role in the induction of such brain damage (Table
1), as also suggested by neonatal human brain studies [
7,
18].
After identical endotoxin and/or HI exposures, premature human newborn commonly present patchy areas of white matter damage whereas term newborns display major cortico-subcortical infarcts associated with BBB disruption and leukocyte infiltration [
4]. The distinct neuroinflammatory responses to similar aggressions were documented in both preterm- (P1) and term-like (P12) brains, and their mechanistic features might contribute to the strikingly different age-dependent neuropathological differences of perinatal brain damage observed in rodents [
38,
40,
57] as well as between preterm and term human newborns [
4].
Systemic adaptive and innate immune responses of the human neonates are known to differ from the adult's one in several ways, such as a bias towards Th2 response, and peripheral blood monocyte stimulation from pathogens leading to higher IL-6, and IL-10 responses, but lower TNF-α expression than in the adults [
58,
59]. Certain developmental comparisons have already been performed between neonatal and adult neuroinflammatory responses and across neonatal period [
60,
61]. For instance, a "window of susceptibility" to intracerebral IL-1β exposure was detected in juvenile (2-6 week-old) as opposed to neonatal (P1) or adult (P90) rats, with juvenile rats displaying a peculiar age-dependent hyper-response in term of neutrophil infiltration and neural cell damage [
62‐
64]. Our experimental data further delineate that over a short time frame, the P1 brain reacts differently than the P12 brain following exposure to systemic endotoxin and/or HI aggressions (Table
1). Importantly, the recent demonstration of much lower expression of TLR-4, within P1 rat brain compared to slightly more mature rat brains, might provide a mechanistic explanation to our results [
65]. The neuroinflammatory response, even when induced at both developmental stages, was weaker in P1 than in P12 brains in its neurotoxic, as well as neuroprotective components. In agreement with these experimental results, both IL-1ra, IL-1β, and subsequent MMP-9 expressions were shown
in situ, to be more weakly expressed in preterm than in term damaged white matter from
human brains [
7]. In addition, studies testing the transmigration across barriers of peripheral immune cells showed only modest capacities for circulating leucocytes, such as neutrophils, to migrate into the brain of preterm human newborns as compared to term newborns [
59]. This is known to be linked to immature adhesion molecule expression by blood or BBB cells. In addition, the reduced P1 (preterm-like) expressions of MCP-1 and CINC-1 we showed at the most immature stage of postnatal brain development might also contribute to the weak preterm recruitment of systemic cells (neutrophils and CD68+ cells) within the brain in our model, as also observed by others [
57,
62]. In agreement with the age-dependant level of CINC-1 production we showed, neutrophils have been already shown to be implicated - but only after P7 - in rat ischemic brain damage, with a peak of deleterious effects at P12-P30 [
37,
62,
64]. The fact that we did not detect any immune cell recruitment at P1 as compared to P12 (and P7 as described by others [
64,
66‐
68]), shows that the stage of brain and immune system development is crucial in the modulation of neuroinflammatory responses. Recent data disclosed that the neonatal BBB is more mature than previously thought, and that after an inflammatory challenge at P1, the BBB was less permeable than later on [
62,
69]. This is in keeping with our observations showing that albumin extravasation and leucocytes infiltrations across the BBB were abundant in rat brains exposed to HI, or LPS+HI, at P12, but absent following the same insults at P1. This might also be due to the weaker P1, than P12, neuroinflammatory response avoiding BBB leakage.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MEB and SG are co-first authors with equivalent contributions. MEB, SG and KL carried out the experiments and performed statistical analyses. MEB and SG drafted the manuscript. GS conceived the study. GS and SG designed and coordinated the project. GS helped to draft the manuscript. All authors read and approved the final manuscript.