Ana S. Falcão

Inflammatory signalling pathways involved in astroglial activation by unconjugated bilirubin

During neonatal hyperbilirubinaemia, astrocytes activated by unconjugated bilirubin (UCB) may contibute to brain toxicity through the production of cytokines. As a first step in addressing the signal transduction cascades involved in the UCB‐induced astroglial immunological response, we tested whether tumour necrosis factor (TNF)‐α receptor 1 (TNFR1), mitogen‐activated protein kinase (MAPK) and nuclear factor κB (NF‐κB) would be activated in astrocytes exposed to UCB, and examined the profile of cytokine production. Astrocyte cultures stimulated with UCB showed a rapid rise in TNFR1 protein levels, followed by activation of the MAPKs p38, Jun N‐terminal kinase1/2 and extracellular signal‐regulated kinase1/2, and NF‐κB. Interestingly, the induction of these signal effectors preceded the early up‐regulation of TNF‐α and interleukin (IL)‐1β mRNAs, and later secretion of TNF‐α, IL‐1β and IL‐6. Treatment of astrocytes with UCB also induced cell death, with levels comparable to those obtained after exposure of astrocytes to recombinant TNF‐α and IL‐1β. Moreover, loss of cell viability and cytokine secretion were reduced when the NF‐κB signal transduction pathway was inhibited, suggesting a key role for NF‐κB in the astroglial response to UCB. These results demonstrate the complexity of the molecular mechanisms involved in cell injury by UCB during hyperbilirubinaemia and provide a basis for the development of novel therapeutic strategies.

Bilirubin injury to neurons: Contribution of oxidative stress and rescue by glycoursodeoxycholic acid

It is well established that high levels of unconjugated bilirubin (UCB) can be toxic to the central nervous system, and oxidative stress is emerging as a relevant event in the mechanisms of UCB encephalopathy. In contrast, the hydrophilic bile acid, ursodeoxycholic acid (UDCA), has been reported as a cytoprotective and antioxidant molecule. In this study, we investigated if exposure of rat neurons in primary culture to clinically relevant concentrations of UCB leads to oxidative injury. The contribution of oxidative stress in UCB neurotoxicity was further investigated by examining whether the reduction of NO production by NAME, an inhibitor of nitric oxide synthase, prevents the disruption of the redox status and neuronal damage. Moreover, we evaluated the ability of glycoursodeoxycholic acid (GUDCA), the most relevant conjugated derivative in the serum of patients treated with UDCA, to abrogate the UCB-induced oxidative damage. Cultured rat neurons were incubated with 50 or 100microM UCB in the presence of 100microM human serum albumin, alone or in combination with 100microM NAME or with 50microM GUDCA, for 4h at 37 degrees C. Protein carbonyls, 4-hydroxy-2-nonenal-protein adducts, intracellular glutathione content and cell death were determined. The results obtained showed that UCB induces protein oxidation and lipid peroxidation, while diminishes the thiol antioxidant defences, events that were correlated with the extent of cell death. Moreover, these events were counteracted by NAME and abrogated in the presence of GUDCA. Collectively, this study shows that oxidative stress is one of the pathways associated with neuronal viability impairment by UCB, and that GUDCA significantly prevents such effects from occurring. These findings corroborate the antioxidant properties of the bile acid and point to a new therapeutic approach for UCB-induced neurotoxicity due to oxidative stress.

Microglia change from a reactive to an age-like phenotype with the time in culture

Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function.

MAPKs are key players in mediating cytokine release and cell death induced by unconjugated bilirubin in cultured rat cortical astrocytes.

When activated by unconjugated bilirubin (UCB), astrocytes are important sources of inflammatory mediators such as TNF‐α, IL‐1β and IL‐6, which may contribute for the neurotoxicity observed during severe neonatal hyperbilirubinemia. In the present study, we have addressed the role of the mitogen‐activated protein kinases (MAPKs) p38, Jun N‐terminal kinase (JNK)1/2 and extracellular signal‐regulated kinase (ERK)1/2 pathways and their relation with the nuclear factor κB (NF‐κB) cascade in the signalling events involved in cytokine release and cell death caused by UCB in primary cultures of rat astrocytes. Stimulation of astrocytes with UCB in the presence of all the MAPK inhibitors prevented UCB‐induced release of TNF‐α and IL‐6, while IL‐1β secretion was only reduced by JNK1/2 and ERK1/2 inhibitors. In addition, activation of the NF‐κB transcription factor, needed for cytokine release by UCB‐stimulated astrocytes, was shown to be dependent on JNK1/2 and ERK1/2 phosphorylation. Moreover, all MAPK inhibitors prevented astroglial apoptosis triggered by UCB. Interestingly, UCB‐induced lactate dehydrogenase release was prevented by blockade of JNK1/2, ERK1/2 and NF‐κB cascades but enhanced by p38 inhibition. Taken together, our data demonstrate for the first time that MAPK transduction pathways are key players in the UCB‐induced inflammatory response and cell death in astrocytes, probably also involving NF‐κB modulation. These findings contribute to unraveling the complex mechanisms of astrocyte reactivity to UCB and may ultimately prove useful in the development of new therapeutic strategies to prevent nerve cell damage during acute bilirubin encephalopathy.

Unconjugated bilirubin activates and damages microglia.

Microglia are the resident immune cells of the brain and are the principal source of cytokines produced during central nervous system inflammation. We have previously shown that increased levels of unconjugated bilirubin (UCB), which can be detrimental to the central nervous system during neonatal life, induce the secretion of inflammatory cytokines and glutamate by astrocytes. Nevertheless, the effect of UCB on microglia has never been investigated. Hence, the main goal of the present study was to evaluate whether UCB leads to microglial activation and to the release of the cytokines tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, and IL‐6. Additionally, we investigated the effects of UCB on glutamate efflux and cell death. The results showed that UCB induces morphological changes characteristic of activated microglia and the release of high levels of TNF‐α, IL‐1β, and IL‐6 in a concentration‐dependent manner. In addition, UCB triggered extracellular accumulation of glutamate and an increased cell death by apoptosis and necrosis. These results demonstrate, for the first time, that UCB is toxic to microglial cells and point to microglia as an important target of UCB in the central nervous system. Moreover, they suggest that UCB‐induced cytokine production, by mediating cell injury, can further contribute to exacerbate neurototoxicity. Interestingly, microglia cells are much more responsive to UCB than astrocytes. Collectively, these data indicate that microglia may play an important role in the pathogenesis of encephalopathy during severe hyperbilirubinemia.

Bilirubin-induced immunostimulant effects and toxicity vary with neural cell type and maturation state.

Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. The increased vulnerability of premature infants to unconjugated bilirubin (UCB)-induced brain damage may be due to a proneness of immature nerve cells to UCB-toxic stimulus. Thus, in this study, we evaluated UCB-induced cell death, glutamate release and cytokine production, in astrocytes and neurons cultured for different days, in order to relate the differentiation state with cell vulnerability to UCB. The age-dependent activation of the nuclear factor-κB (NF-κB), an important transcription factor involved in inflammation, was also investigated. Furthermore, responsiveness of neurons and astrocytes to UCB were compared in order to identify the most susceptible to each induced effect, as an approach to what happens in vivo. The results clearly showed that immature nerve cells are more vulnerable than the most differentiated ones to UCB-induced cell death, glutamate release and tumour necrosis factor (TNF)-α secretion. Moreover, astrocytes seem to be more competent cells in releasing glutamate and in producing an inflammatory response when injured by UCB. Activation of NF-κB by UCB also presents a cell-age-dependent pattern, and values vary with neural cell type. Again, astrocytes have the highest activation levels, which are correlated with the greater amount of cytokine production observed in these cells. These results contribute to a better knowledge of the mechanisms leading to UCB encephalopathy by elucidation of age- and type-related differences in neural cell responses to UCB.

Bilirubin-induced inflammatory response, glutamate release, and cell death in rat cortical astrocytes are enhanced in younger cells.

Unconjugated bilirubin (UCB) encephalopathy is a predominantly early life condition resulting from the impairment of several cellular functions in the brain of severely jaundiced infants. However, only few data exist on the age-dependent effects of UCB and their association with increased vulnerability of premature newborns, particularly in a sepsis condition. We investigated cell death, glutamate efflux, and inflammatory cytokine dynamics after exposure of astrocytes at different stages of differentiation to clinically relevant concentrations of UCB and/or lipopolysaccharide (LPS). Younger astrocytes were more prone to UCB-induced cell death, glutamate efflux, and inflammatory response than older ones. Furthermore, in immature cells, LPS exacerbated UCB effects, such as cell death by necrosis. These findings provide a basis for the increased susceptibility of premature newborns to UCB deleterious effects, namely when associated with sepsis, and underline how crucial the course of cell maturation can be to UCB encephalopathy during moderate to severe neonatal jaundice.

Features of bilirubin-induced reactive microglia: From phagocytosis to inflammation

Microglia constitute the brains immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.

Unconjugated bilirubin differentially affects the redox status of neuronal and astroglial cells.

We investigated whether nerve cell damage by unconjugated bilirubin (UCB) is mediated by oxidative stress and ascertained the neuronal and astroglial susceptibility to injury. Several oxidative stress biomarkers and cell death were determined following incubation of neurons and astrocytes isolated from rat cortical cerebrum with UCB (0.01-1.0 microM). We show that UCB induces a dose-dependent increase in neuronal death in parallel with the oxidation of cell components and a decrease in the intracellular glutathione content. Comparison of the results obtained in both cell types demonstrates that neurons are more vulnerable than astrocytes to oxidative injury by UCB, for which accounts the lower glutathione stores in neuronal cells. Moreover, neuronal oxidative injury is prevented by supplementation with N-acetylcysteine, a glutathione precursor, whereas astroglial sensitivity to UCB is enhanced by inhibition of glutathione synthesis, using buthionine sulfoximine. Collectively, we demonstrate that oxidative stress is involved in UCB neurotoxicity and depict a new therapeutic approach for UCB-induced oxidative damage.

Apoptosis and impairment of neurite network by short exposure of immature rat cortical neurons to unconjugated bilirubin increase with cell differentiation and are additionally enhanced by an inflammatory stimulus.

Nerve cell injury induced by unconjugated bilirubin (UCB) has been implicated in brain damage during severe neonatal hyperbilirubinemia, although the molecular mechanisms underlying UCB neurotoxicity are still not clarified. It has been suggested recently that there is an association between hyperbilirubinemia and long‐term neurologic dysfunctions. We incubated immature neurons with UCB to evaluate the short‐ and long‐term effects of UCB on apoptotic death and on neuritic outgrowth and ramification. We also evaluated whether mature neurons, exposed previously to UCB in an early stage of differentiation, are more sensitive to apoptosis or to neuritic breakdown when treated with inflammatory agents, such as lipopolysaccharide and tumor necrosis factor‐α. Results show that exposure of immature neurons to UCB increased apoptosis and provoked a reduction of both neurite extension and number of nodes. These injurious effects observed in immature cells treated with UCB were increasingly perpetuated along cell differentiation, as compared to neurons incubated in the absence of UCB. In addition, neurons that were exposed to UCB when immature showed an increased susceptibility to death by apoptosis, as well as an additional decrease in neurite outgrowth when incubated with an inflammatory agent afterward. This work shows, for the first time, that UCB induces neurite changes consistent with neurodevelopment abnormalities. Furthermore, pre‐exposure to UCB followed by an inflammatory stimulus leads to an enhanced susceptibility to long‐term apoptosis, as well as a greater neuritic breakdown. These data support the association between neonatal hyperbilirubinemia and the later development of mental illness, such as schizophrenia.