TLR-3 receptor activation protects the very immature brain from ischemic injury

Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors that play a key role in host defense against pathogen infection. These receptors recognize a variety of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide, peptidoglycan, bacterial DNA, and double-stranded RNA [1]. There are 13 mammalian TLRs with TLRs 1 to 9 being conserved between humans and mice. The expression of TLRs and their role in inflammation and ischemic injury in the adult brain is well documented. TLR-4 expression has been observed in the meninges, choroid plexus, and circumventricular organs of the adult rat brain [2]. In the human CNS, microglia express TLRs 1 to 9, astrocytes express robust TLR-3 and low-level TLRs 1,4,5,9 and oligodendrocytes express TLR-3 and TLR-2 [3, 4]. Cerebral ischemia results in increased TLR-4 and TLR-2 expression in the brains of adult mice [5]. Furthermore, mice deficient in TLR-4 and TLR-2 display reduced infarct size after ischemic injury compared to wild-type mice [5]. Taken together, these results indicate the TLRs play an important role in ischemia-induced injury in the adult brain.

While there is accumulating knowledge on the expression and function of TLRs in the adult CNS, little is known about TLRs in the developing brain. TLR-8 and TLR-3 are expressed in neurons of embryonic and neonatal mouse brains where they regulate neuronal growth [6, 7]. We have shown that TLR-4 is expressed in postnatal day 7, 9, and 14 rat brains [8]. More recent studies have shown that TLRs 1 to 9 are expressed in the P9 mouse brain [9]. Cerebral ischemia has been shown to increase the expression of a number of TLRs in neonatal mice [9]. However, the role of TLRs in ischemic injury of the developing brain is yet to be determined.

Ischemic tolerance or preconditioning is a phenomenon by which a sub-injurious stimulus is applied to a tissue such as the brain. After a certain delay, the brain develops tolerance to ischemic injury caused by the injurious stimulus. Ischemic preconditioning, therefore, protects against subsequent ischemic injury. The delay to protection may be minutes to few hours (rapid or early preconditioning) or days (delayed preconditioning) requiring protein synthesis [10‐12]. Since Kitagawa and colleagues first reported on delayed preconditioning in 1991 [13], this phenomenon has been well documented in the brain. Although brief cerebral ischemia or hypoxia is the typical ischemic preconditioning stimulus [14], ischemic preconditioning may also be induced by exposing the brain to a variety of stimuli such as inflammation, oxidative stress, hyperthermia, and spreading depression [11, 12]. Activation of TLR2 and TLR9 by their highly specific ligands (Pam3CSK4 and unmethylated CpG ODN, respectively) has been shown to induce ischemic preconditioning in adult stroke models [15, 16]. We have recently shown a robust delayed preconditioning against ischemic injury in the neonatal rat [8] and piglet [17] brains induced by lipopolysaccharide (LPS), a TLR-4 specific agonist.

We reported that LPS-induced neuroprotection against cerebral ischemic injury was offered to P7, P9, and P14 rat pups. LPS neuroprotection was ineffective in P3 and P5 rat pups, and the brains of these pups expressed significantly less TLR-4 compared to P7, P9, and P14 rats [8]. In light of these findings, we sought in this study to investigate the effect of brain maturity on TLRs expression and to examine whether TLRs other than TLR-4 offer neuroprotection to the developing brain against cerebral ischemic injury. We chose TLR-2 and TLR-9 because of their potential capability of mediating preconditioning in the rat immature brain given their neuroprotective effect in adult brain and heart [15, 16, 18]. We also examined the expression of TLR3 because it is the only receptor that share MyD88-indpendent signaling pathway with TLR4. It is plausible that TLR3 has a neuroprotective function specific to the developing brain independent of TLR4.

Many studies showed that low doses of LPS provided neuroprotection when administered 24 h prior to ischemic injury in adult stroke models [26‐29]. Fewer studies demonstrated the same LPS-induced neuroprotection in neonatal stroke models [17, 30]. Recently, we found that LPS administered 48 h prior to HI injury reduced brain damage and infarct volume by 90% at 1 week post injury [8]. This robust LPS-induced neuroprotection was observed in P7, P9, and P14 rat pups. However, LPS was ineffective in protecting P3 and P5 rats from HI injury [8].

The mechanism of how an inflammatory stimulus, such as LPS, induces preconditioning against ischemic injury is still being investigated. The requirement of de novo protein synthesis and the induction of pro-inflammatory cytokines such as TNFα, IL-1β, and IL-6 are believed to be essential for achieving preconditioning [31‐33]. The dependency of this neuroprotective phenomenon on pro-inflammatory cytokines raises the possibility that a preconditioning dose of LPS may activate TLR-4 causing a mild inflammatory response that will trigger the expression of negative feedback inflammatory inhibitors including the TLR-4 signaling inhibitors (for example, phosphatidylinositol 3-kinase). These endogenous inhibitors are only induced in response to TLRs or cytokine receptors activation (de novo protein synthesis) and will remain upregulated until the subsequent ischemic insult occurs. At this state of suppressed innate immunity, the ischemic injury will be unable to elicit an inflammatory response, resulting in reduced brain damage [34‐36]. Recent studies have shown that LPS-TLR4 are not the only mediators of preconditioning against ischemic injury. Neuroprotection can also be achieved by activating TLR-2 and TLR-9 in the adult ischemic mouse brain [16, 37].

Studies on TLRs expression in the developing brain are scarce. Protein expression of TLR-8 and TLR-3 has been shown during embryonic development [6, 7]. More recently mRNA expression has been detected for all TLR1-9 and regulated by HI injury in neonatal (P9) mouse brain [9]. We have shown high expression of TLR-4 in P7, P9, and P14 but low expression levels in P3 and P5 rat pups [8]. To identify TLRs that may play a role in preconditioning the very immature brain (rat pups aged < P7), we investigated the effect of brain maturity on TLR-2, TLR-3, and TLR-9 expression because of their potential role in neuroprotection in the adult brain. TLR-2 and TLR-3 were highly expressed in P3 and P5 compared to P7 rat pups. These results, taken together, indicate that TLRs expression is developmentally determined.

TLRs are expressed in a variety of cell types including brain cells. Using in vitro studies, several laboratories have shown that human microglia and astrocytes express TLR mRNAs [3, 4, 38]. Microglia of corpus callosum and cerebellum in neonatal rats express TLR-4 and this expression has been shown to be upregulated after hypoxia [39]. This is similar to what we reported here on the increase of TLR-3 expression in microglia after HI injury. Recent studies have also shown that cultured rodent [5] and human [40] neurons express TLR-2, TLR-3, and TLR-4. TLR-2 is also expressed in neurons of neonatal mice and its activation seems to contribute to the HI injury [9]. We have shown here in vivo that TLR-3 is expressed in neurons of P5 rat brain. These results indicate that neurons have the capacity to contribute to the ischemia-induced inflammatory response in the developing brain.

The highest expression of TLR-3 is in the P5 neonatal rat brain making it the most likely candidate to induce preconditioning against ischemic injury in this age group. Indeed, pre-treating P5 pups with poly I:C, TLR-3 specific agonist, resulted in a significant reduction in infarct volume. This reduction in brain damage was not observed in P7 pre-treated pups indicating that the neuroprotective effect of TLR-3 receptor activation is age specific. TLR-3 activation has been shown to reduce proliferation of adult human cultured astrocytes and to promote neuronal survival in cultured human brain slices by inducing the expression of neuroprotective mediators and modulating the inflammatory response [41]. There is emerging evidence that TLR-3 is expressed in embryonic brain cells where it plays a role in regulating neurogenesis in the developing mouse brain [7]. To our knowledge, this is the first evidence of a neuroprotective role of TLR-3 against ischemic brain injury.

Stimulation of TLR-3 by poly I:C recruits TRIF, the key adaptor protein in TLR-3 signaling pathways. Recruitment of TRIF leads to the activation of several transcription factors including IRF3 and NF-κB (for review 42). Our data showed that exposing P5 pups to HI injury increased NF-κB expression compared to normal rats. This increase was reversed in P5 rats pre-treated with poly I:C. HI injury alone, on the other hand, did not modulate IRF3 expression. An increase in IRF3 expression was only seen when P5 pups were pre-treated with poly I:C prior to HI injury. Activation of NF-κB and IRF3 results in subsequent production of IL-12 and IFN-β, respectively [42]. IL-12 is a known pro-inflammatory cytokine whereas IFN-β is shown to have anti-inflammatory and neuroprotective effects in adult stroke model [43, 44]. We hypothesize, therefore, that TLR-3-induced preconditioning is mediated by upregulation of IRF3 anti-inflammatory pathway and concurrent downregulation of pro-inflammatory NF-κB pathway. We are currently investigating this potential mechanism of TLR-3-induced preconditioning using NF-κB and IRF3 knockout mice.

Preconditioning is not only observed in animals and in vitro studies; this phenomenon may occur in the human brain. Several studies have reported that stroke patients with previous transient ischemic attacks had milder neurological deficit at presentation and better outcome [45, 46]. The challenge remains in determining how to utilize this phenomenon in a new paradigm that will provide prophylactic therapy for patient populations at high risk of brain ischemic injury, such as children with congenital heart disease. From these children, 1:185 will have a stroke within 72 h of their cardiac procedure that will leave 72% of them with neurological deficit [47]. Preconditioning has the potential to protect patients at high risk of brain ischemic injury from devastating neurological outcome and improve their quality of life. However, we still need to understand the pathways leading to preconditioning to achieve this goal.

TLRs expression and function are developmentally determined. TLR-3 activation induces preconditioning against ischemic injury in the very premature brain. This neuroprotection is mediated by modulation of TLR-3 signaling pathways. The results of this study will be utilized to design neuroprotective therapies for premature infants who are at known high risk of hypoxic-ischemic brain injury.