TNF gene cluster deletion abolishes lipopolysaccharide-mediated sensitization of the neonatal brain to hypoxic ischemic insult

Abstract

In the current study, we explored the role of TNF cluster cytokines on the lipopolysaccharide (LPS)-mediated, synergistic increase in brain injury after hypoxic ischemic insult in postnatal day 7 mice. Pretreatment with moderate doses of LPS (0.3 mg/g) resulted in particularly pronounced synergistic injury within 12 h. Systemic application of LPS alone resulted in a strong upregulation of inflammation-associated cytokines TNFa, LTb, interleukin (IL) 1b, IL6, chemokines, such as CXCL1, and adhesion molecules E-Selectin, P-Selectin and intercellular adhesion molecule-1 (ICAM1), as well as a trend toward increased LTa levels in day 7 mouse forebrain. In addition, it was also associated with strong activation of brain blood vessel endothelia and local microglial cells. Here, deletion of the entire TNF gene cluster, removing TNFa, LTb and LTa completely abolished endotoxin-mediated increase in the volume of cerebral infarct. Interestingly, the same deletion also prevented endothelial and microglial activation following application of LPS alone, suggesting the involvement of these cell types in bringing about the LPS-mediated sensitization to neonatal brain injury. KEYWORDS: encephalopathy; hypoxia; inflammation; ischemia; neonate; TNF Although bacteria and viruses can directly infect and injure developing brain, infections occurring outside the brain frequently will also have a damaging effect. Congenital infections appear to contribute up to 5% of cerebral palsy cases 1,2 and may sensitize the brain to perinatal hypoxic ischemic (HI) insult. [3][4][5] This synergistic effect was also reproduced in mammalian and avian animal models, combining HI insult and the lipopolysaccharide break-down product of bacteria in HI animal models.6-10 However, the molecular mediators of this endotoxin effect in vivo are currently still unknown.Both in vitro and in vivo studies show that endotoxin will upregulate numerous cytokines and chemokines, 11 upregulate signaling enzymes, such as inducible nitrogen oxide synthase (iNOS), and cyclo-oxygenase-2 (COX2) and enhance the expression of adhesion molecules on parenchymal microglia and the brain vascular endothelium. 12-15Follow on molecular studies reveal that these effects are transmitted through the classical endotoxin receptors, primarily the toll-like receptor 4 (TLR4), on blood vessel endothelia and microglia, 16,17 and involve MyD88 and NF-kappa-B components of the innate immunity cascade. 17In particular, endotoxin-induced pro-inflammatory cytokines, including TNFa and interleukin 1b (IL1b) are known to have a number of deleterious effects, including a direct toxic effect on neurones and vulnerable oligodendrocyte precursors, 18,19 astrogliosis with release of nitric oxide, and mitochondrial dysfunction, 20 as well as microglial activation with release of nitric oxide, superoxide and a panel of other inflammation-associated molecules.

Development of Cerebral Gray and White Matter Injury and Cerebral Inflammation over Time after Inflammatory Perinatal Asphyxia

“…The combined exposure to perinatal inflammation and neonatal HI results in a complex interplay of various signaling pathways of which the exact underlying mechanism has not been elucidated yet. Experimental peripheral inflammation induced by LPS is known to induce an inflammatory cerebral state leading to sensitization of the brain to HI injury [15,16,19,21,36,37]. Possible mechanisms via which peripheral LPS can sensitize the brain have been described extensively and consist of: (i) stimulation of endothelial cells of the blood-brain barrier (BBB), (ii) activation/priming of microglia via immune signals produced by endothelial and choroid plexus cells at the level of the BBB, (iii) direct transport of peripheral inflammatory molecules across the BBB, (iv) increased permeability of the BBB, which enhances the influx of peripheral immune cells into the brain, (v) stimulation of the vagal nerve by peripheral cytokines leading to signaling in the brain and (vi) the effects of inflammation on G-protein-coupled receptor signaling in the brain [17,38,39,40,41].…”

“…We showed earlier that HI-induced changes in cytokine mRNA levels were related to changes in the protein levels of these cytokines [71]. Besides the effect on oligodendrocytes, Kendall et al [16] showed that TNF-α is a key factor in the LPS-mediated sensitizing effect on HI brain damage, since deletion of the TNF gene cluster prevented the LPS-induced increase in endothelial and microglia activation and the increase in HI brain damage. Consequently, anti-TNF therapies could be promising in neonates in whom inflammation precedes HI.…”

“…The general view is that inflammation before HI sensitizes the brain and thereby aggravates HI cerebral injury. However, the exact mechanisms underlying proinflammatory sensitization are still unclear [15,16,17,18,19,20,21]. To date, the exact timing of the sensitizing effect of LPS on the intensity of cerebral gray and white matter injury and the formation of an acellular cyst after HI have not been investigated in detail.…”

Inflammation‐induced sensitization of the brain in term infants

“…Prostaglandins may also be important in LPS-induced brain damage, as injury is associated with an increase in COX-2. 66 Furthermore, both IL-1b 67 and TNFa 68 have been implicated as possible mediators of LPS-induced damage. Recent data also support an important role for microglial recruitment and activation, as well as a role for early neutrophil infiltration, in this neuroinflammatory milieu.…”

“…91 Moreover, deletion of the TNF gene cluster abolishes LPS-mediated sensitization of the neonatal brain to a hypoxic-ischaemic insult. 68 TNFa treatment is toxic to oligodendrocyte precursors 92 and potentiates IFNc-induced toxicity to these cells in vitro. 93 TNFa also stimulates proliferation, and potentiates the activation of astrocytes 94 and microglia.…”