Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia

The developing brain is highly vulnerable to oxygen deprivation or hypoxia [1, 2]. Risk factors including placental insufficiency, decreased utero-placental blood flow, as well as neonatal pulmonary and/or cardiac dysfunction can compromise neonatal oxygenation, thus affecting the development and growth of the brain [3, 4]. Neuroinflammation, characterized by microglial activation, has been reported to play an important role in the hypoxic injuries in the neonatal brain [5, 6]. A large number of a nascent form of microglia, known as the amoeboid microglial cells (AMCs), preponderate in the corpus callosum as well as the cerebellum of the developing brain [1]. Hypoxia-induced activation of AMCs is known to result in the production of excessive amounts of inflammatory cytokines, such as, TNF-α and IL-1β, along with nitric oxide (NO) and reactive oxygen species (ROS). Collectively, they cause oligodendrocyte death and axonal degeneration, as well as disruption of the immature blood–brain-barrier (BBB) in the periventricular white matter (PWM), leading to neonatal mortality and long-term neurodevelopmental deficits [1, 6‐8]. A similar phenomenon is observed in the hypoxic developing cerebellum in which activated AMCs have been shown to induce Purkinje neuronal death through production of TNF-α and IL-1β [9]. However, the mechanism via which hypoxia induces microglial activation remains to be fully explored. Hence, determination of the various mechanisms controlling microglial activation will play an important part in the suppression of neuroinflammation.

Toll-like receptors (TLRs) are first-line molecules for initiating innate immune responses. Among more than ten mammalian TLRs identified [5], TLR4 has been shown to be expressed on microglia and mediates neuroinflammatory diseases [10]. Numerous studies have demonstrated TLR4-dependent activation of microglia in neurodegenerative diseases and trauma in the central nervous system (CNS), such as Alzheimer’s disease (AD) and Parkinson’ s disease (PD) [11, 12], as well as brain injury induced by ethanol [13]. Besides the above, TLR4 is also reported to be involved in hypoxia-related diseases. It has been reported recently that TLR4 is involved in brain damage and inflammation after stroke and spinal cord injury in adult mice or rats [14, 15]. In fact, increased expression of TLR4 after hypoxic treatment in microglia has also been reported in vitro[16]; however, the expression and putative roles of TLR4 in microglia of neonatal rats following hypoxic injury have remained elusive.

In light of the critical role of TLR4 in neuroinflammation and hypoxic-ischemic-related diseases, the current study was undertaken to determine the expression, putative roles and mechanism of TLR4 in the microglia of hypoxic neonatal rats both in vivo and in vitro. Considering the involvement of hypoxia-inducible factor-1 alpha (HIF-1α) in the induction of TLR4 expression in tissue macrophages exposed to hypoxic stress [17], we sought to determine its role in TLR4 expression in hypoxic microglia. We report here that TLR4 participated in microglial activation in the hypoxic developing brain and microglia. TLR4 expression was constitutively expressed in microglia distributed in the corpus callosum and cerebellum and was noticeably increased in the brain of hypoxic pup rats. The increase in TLR4 expression in hypoxic microglia was dependent on HIF-1α, and TLR4 was found to mediate the release of pro-inflammatory mediators through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. All these could collectively contribute to neonatal brain damage resulting from hypoxic exposure. Hence, regulation of TLR4 expression in microglia may therefore present as a novel therapeutic target for the treatment of various pathological states that involve hypoxia in the CNS.

Microglial cells are the primary immune effector cells in the brain and play a pivotal role in the neuroinflammatory processes associated with a variety of neurological and pathological disorders [23]. We have reported that microglia are involved in hypoxic injuries in the developing brain, affecting both neurons and oligodendrocytes [1, 6, 24]. Hence, a fuller understanding of the underlying molecular mechanisms of microglial activation under such conditions would be desirable for the design of a novel therapeutic strategy for the management of hypoxic damage.

It is well documented that TLR4 in microglia is a key player of neuroinflammation in several neurodegenerative and CNS trauma diseases [12‐14, 25]. The data presented herein provide the first evidence of the role of microglial TLR4 in a neonatal rat model of hypoxic injury. It is unequivocal from the present results that TLR4 signaling participates in the microglial activation in the hypoxic developing brain. TLR4 immunoexpression was localized in microglia in the neonatal rat corpus callosum and cerebellum and was increased in hypoxia. In vitro studies further demonstrated that TLR4 expression in hypoxic microglia was dependent on HIF-1α and more importantly, that TLR4 mediates the production of inflammatory mediators through the NF-κB pathway. The effect of TLR4 as demonstrated in vitro was further confirmed in vivo by TLR4 inhibition with its specific inhibitor, namely, CLI-095. It is therefore suggested that this TLR4 pathway activation contributes to the neonatal brain damage resulting from hypoxic exposure.

Hypoxia results in changes in the signaling pathways and gene expression related to physiological as well as pathological responses. We have shown that TLR4 in microglia was overexpressed in the cerebrum and cerebellum of neonatal rats after hypoxic treatment. Very interestingly, many in vivo experimental and clinical studies have similarly reported that TLR4 expression is elevated in cells and tissues in hypoxia-related disease. The levels of TLR4 mRNA and protein have been found to be increased in murine hearts after myocardial ischemic injury, and in human hearts derived from patients with dilated cardiomyopathy and myocarditis [26, 27]. In addition, TLR4 expression is significantly increased in Kupffer cells in rat liver grafts, and in tubular epithelial cells and infiltrating leucocytes within the kidney following ischemia [28, 29]. Here, we have shown that TLR4 protein expression in primary microglia subjected to hypoxic exposure for 24 h was increased. In BV-2 cells, TLR4 protein overexpression was sustained with long-term hypoxic exposure from 4 to 12 h compared with the significant increase in mRNA expression from 2 to 6 h hypoxia exposure (data not shown). Consistent with our result, the increase in TLR4 protein expression in BV-2 microglial cells or primary microglial cultures is also reported with exposure to an extremely low concentration of oxygen (O₂ tension <0.2%) for 8 or 24 h [17]. Hypoxic stress induced either by a low oxygen tension or CoCl₂ can also enhance TLR4 expression in macrophages [16]. However, some studies report that relatively long-term hypoxia for 48 to 72 h diminishes TLR4 expression as a result of mitochondrial generation of ROS in human umbilical vein endothelial cells [30]. It would appear, therefore, from the above studies that differences in duration of hypoxic exposure and the type of cells investigated may account for the discrepancy in TLR4 expression over time.

It is widely demonstrated that exogenous ligands to TLR4 such as lipopolysaccharide (LPS) and endogenous ligands to TLR4, such as members of the heat shock protein family and proteoglycans, can lead to the induction of proinflammatory cytokines [20, 31]. Intracerebral injection of LPS into the developing periventricular white matter of immature rodents results in the loss of oligodendrocytes, hypomyelination and formation of periventricular cysts through the action of TLR4 [32]. Hence, a hypothesis that TLR4 mediates the production of proinflammatory cytokines, which in turn, contributes to damage in the developing brain after hypoxia, was formed. To test this hypothesis, we adopted siRNA transfection and antibody neutralization in BV-2 cells and primary cultured microglia, separately, in the present study. We show here that inhibition of TLR4 expression in microglia effectively suppressed the overexpression of inflammatory factors elicited by hypoxic stress. This suggests that increased microglial TLR4 activation correlates with the production of inflammatory factors after hypoxic stress, even in the absence of exogenous TLR4 ligands such as LPS. A similar function of TLR4 has also been reported in microglia stimulated with other factors. For example, we have reported previously that TLR4 signaling mediated the inflammation in microglia that was stimulated with saturated fatty acid [33]. It has also been reported that TLR4 is upregulated in ethanol-induced microglial activation and response, suggesting that the activation of microglial TLR4 signaling could trigger the release of inflammatory mediators, which in turn could induce white matter abnormalities and neuronal dysfunctions [13]. Accumulating evidence has shown a general participation of TLR4 in the neuroinflammatory process and brain damage of different pathological factors, suggesting that TLR4 exacerbates brain damage in neuroinflammatory diseases. In contrast, studies with TLR4 mutation mice indicate that microglia are activated via TLR4 signaling to reduce β-amyloid deposits and preserve cognitive functions from β-amyloid-mediated neurotoxicity [12]. In this case, activation of microglia through TLR4 appears to be neuroprotective in Alzheimer’s disease. Therefore, whether TLR4 is neuroprotective or neurotoxic in neuroinflammatory disorders may vary with different pathological conditions and the different stages of these diseases. TLR4 activation appears to be tightly controlled, in order to regulate the switch between its different roles in immune surveillance or neuroinflammatory propagation. The present results tend to support the theory that TLR4 promotes the production of major inflammatory mediators such as TNF-α, IL-1β, iNOS, ROS and NO, which have been implicated in oligodendrocyte and neuronal death, thus, suggesting its detrimental role in hypoxic neonatal brain injury.

The identification of factors responsible for the induction of TLR4 expression in microglia under hypoxia has remained elusive. HIF-1 has been reported to mediate the TLR4 expression in macrophages under hypoxic conditions [16]. This has prompted us to determine the role of HIF-1 in TLR4 expression in hypoxic microglia. HIF-1 is a major transcription factor activated during hypoxia in pathological conditions and it plays critical roles in inducing hypoxia-related gene expression and cellular responses. HIF-1 is a heterodimeric protein that consists of HIF-1β and HIF-1α, among which HIF-1β is constitutively expressed while HIF-1α expression is tightly regulated by oxygen [34, 35]. Therefore, the overall activity of HIF-1 is dictated by the intracellular HIF-1α level. In this study, a hypoxia model was first achieved in BV-2 cells by placing the cells in a chamber filled with a gas mixture of 3% O₂, 5% CO₂ and 92% N₂ for 0.5 h or more followed by assessing the HIF-1α protein expression. Remarkably, there was no significant difference in HIF-1α mRNA expression level between the hypoxic and control cells. On the other hand, HIF-1α protein expression was significantly increased in hypoxia. It has been reported that HIF-1α is degraded rapidly by the hydroxylation of prolyl hydroxylase, and the enzymatic activity of prolyl hydroxylase is oxygen-dependent. Under hypoxic conditions, the enzymatic activity of prolyl hydroxylase is significantly reduced, resulting in the increase in HIF-1α [36, 37]. Our findings of increased HIF-1α protein expression in hypoxic BV-2 cells is in accordance with the mechanism of HIF-1α increase in hypoxic conditions. A major finding in this study was that TLR4 expression was regulated by HIF-1α in hypoxic microglia. This is evidenced by the fact that TLR4 expression decreased on neutralization of HIF-1α with its antibody. Indeed, several recent studies support the notion of the link between HIF-1α and TLR4. Studies in macrophages found that TLR4 expression is upregulated via HIF-1α in response to hypoxic stress. Interestingly, some studies also found that LPS, a ligand of TLR4 can induce accumulation and DNA-binding activity of HIF-1α protein in murine microglial cells [38], macrophage-differentiated cells [39] and macrophages [40]. In other words, LPS can raise the level of HIF-1α in a TLR4-dependent fashion, suggesting that TLR4 may act conversely to regulate HIF-1α expression. Positive correlation between TLR4 and HIF-1α has also been found in pancreatic ductal adenocarcinoma cells indicating that there may exist a crosstalk between the TLR4 signal pathway and the HIF-1 signal pathway, which may act synergistically to promote the progression of pancreatic ductal adenocarcinoma [41]. We have demonstrated the role of HIF-1α in TLR4 expression in hypoxic microglia; but it remains to be elucidated whether the two act synergistically.

NF-κB is a transcription factor known to regulate genes of a spectrum of processes such as inflammation, stress responses, innate, and acquired immunity [42, 43]. Under normal physiological conditions NF-κB is localized in the cytoplasm in an inactive state bound to Inhibitor of nuclear factor-κB (IκB). Under pathological conditions IκB is phosphorylated and degraded, resulting in active NF-κB [44]. Activated NF-κB is in turn phosphorylated and subsequently translocated to the nucleus where it regulates target gene expression. It has been reported that hypoxia induces rapid degradation of phosphorylated IκB and an increase in phosphorylated NF-κB in hypoxic microglia of neonatal rats [45]. Activation of the NF-κB pathway through the TLR4 pathway has been reported not only in LPS-treated but also in non-TLR4 ligands, such as fatty acid-treated microglia [17, 33]. The present results showed nuclear translocation of NF-κB in hypoxic BV-2 microglial cells, and more importantly, the translocation was prevented by TLR4 knockdown. Moreover, the increase in phosphorylated NF-κB expression, which was increased after hypoxic exposure, was inhibited after TLR4 knockdown. Results with primary cultured microglia are in accordance with the observations made in BV-2 cells. The present results suggest that TLR4 mediates the activation/nuclear translocation of NF-κB in microglia after hypoxia stress. In contrast, it has been reported that hypoxia suppressed LPS-induced NF-kB activation in microglia [17]. It is suggested that the mechanism involved in cells treated with hypoxia alone may be different from LPS stimulation along with hypoxic exposure. Although our results have shown that NF-κB signaling represents one of the downstream pathways of TLR4-induced production of inflammatory factors, the possibility of involvement of other pathways such as mitogen-activated protein kinase (MAPK) [46] and Notch pathways in hypoxia-induced inflammation (unpublished data for Notch pathway) should also be considered.

CLI-095, a specific inhibitor of TLR4, also called TAK 242, has been reported to specifically and effectively inhibit TLR4 signaling in different animal models [47‐50]. We have demonstrated the effect of TLR4 inhibition on microglial activation in vivo and this has further confirmed the role of TLR4 in neonatal brain damage following hypoxia. Very interestingly, the protective effect observed with microglia in both the cerebrum and cerebellum indicates that TLR4 may mediate in damage in both oligodendrocytes and Purkinje neurons. Indeed, obvious reduction in apoptosis of oligodendrocytes and Purkinje neurons was found after TLR4 blockade in hypoxic rats (data not shown), although direct evidence for the contribution of the inhibition of the activation of microglia awaits further investigation. Notwithstanding, it can be confidently concluded from the present results that TLR4 inhibition can effectively inhibit release of inflammatory mediators by microglia in hypoxic neonatal rats.

The present findings have highlighted that TLR4 signaling regulates the expression of hypoxia-induced inflammatory mediators via the NF-κB pathway and is linked to the inflammatory process in neonatal hypoxic brain injury. Thus, inhibiting the activation of microglia via TLR4 in neonatal hypoxic injury may be neuroprotective, for which the TLR4 signaling pathway represents a potential therapeutic target.