Increasing evidence suggests that IL-18 plays a pivotal role in brain injury [19, 29, 30]. However, the underlying mechanism remains unclear. The current study has demonstrated that IL-18 levels are increased and that Il-18 deficiency is neuroprotective in TBI. We observed TBI-induced expression of IL-18 in microglia and neurons post TBI. Il-18 deficiency decreased TBI-induced neuronal degeneration and apoptosis, increased neuron survival, and ultimately resulted in amelioration of neurological deficits. This protective effect is likely mediated through amelioration of neuroinflammation by attenuation of TBI-induced astrogliosis and microglial activation and reductions in inflammatory cytokine and chemokine expression. We also observed that Il-18 deficiency reduced the frequency of TBI-induced M1-like ‘proinflammatory’ microglia. The present results provide substantial evidence that IL-18 is involved in the development of secondary injury post TBI and that IL-18 is an important regulator in the process of TBI-induced inflammation.
IL-18 is a proinflammatory cytokine and an important mediator of peripheral inflammation and the host immune response. IL-18 can have autocrine and paracrine effects by binding with the IL-18R, which consists of two chains, an IL-18-binding alpha chain (IL-18Rα) and a signaling beta chain (IL-18Rβ) ; this interaction leads to activation of NF-κB signaling via a complex intracellular signaling cascade, culminating in the production and release of several cytokines, chemokines, and cellular adhesion molecules [12, 14, 32]. Several studies have demonstrated that IL-18 has important functions in the central nervous system (CNS), which initiates the inflammatory signaling cascade that contributes to neuronal injury and cell death [16, 20, 33]. Increased levels of IL-18 were observed at 3 and 7 dpi, which is consistent with previous studies [17–19]. Previous studies have shown an increase in IL-18 expression in microglia post hypoxic-ischemic brain injury, kainic acid (KA)-induced excitotoxicity), or spinal nerve ligation (SNL) [30, 34, 35]; in neurons following restraint stress ; and in astrocytes in vitro . In the present study, we observed expression of IL-18 mainly in microglia and neurons, not in astrocytes at 7 dpi in mice. The difference in cell type-specific expression of IL-18 may result from the use of different models, observation areas or animal species. Recent studies have found that the release of IL-1β and IL-18 is mediated by NLRP3 inflammasomes, which are important in the inflammatory process . High levels of IL-18 are considered to be crucial for the establishment of a chronically inflamed environment, leading to neuronal dysfunction and eventually neurodegeneration, while IL-1β is involved in the early phase of TBI [20, 39]. IL-18 is less studied post TBI. To date, researchers have demonstrated increased levels of IL-18 post TBI in mice, which result in the exacerbation of neurological deficits and apoptosis [17–19]. In the present study, Il-18 deficiency resulted in amelioration of neurological deficits, which was associated with decrease in TBI-induced neuronal degeneration and neuroinflammation, suggesting the adverse effects of IL-18 on brain injury.
Neuroinflammation is a major pathological process in the secondary response after TBI [26, 40, 41]. Post TBI, the release of the intracellular components of damaged cells activates neuroinflammatory responses, involving activation of microglia and astrocytes and production of cytokines and chemokines, which significantly contribute to substantial neural damage and behavior impairment [5, 26]. Researchers have shown that administration of an IL-18 antagonist (IL-18BP or other IL-18 neutralizing antibody) suppresses NF-κB activation and decreases the levels of inflammatory mediators including TNF-α, IL-1β, IL-4, IL-6, IL-10, and CXC chemokines following reperfusion in mouse models of ischemia/reperfusion injury [42–45]. In addition, decreased expression levels of inflammatory markers, such as TNF-α, IL-6, IL-10, IL-12p40, and CXCL1, are found in Il-18 deficient mice with pneumococcal meningitis . Here, we demonstrated that the TBI-induced secretion of pro- and anti-inflammatory cytokines, including IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL17A, G-CSF, GM-CSF, IFN-γ, and TNF-α, and chemokines, including CCL2, CCL3, CCL4, CCL5, CCL7, CCL12, CCL20, CXCL1, CXCL2, CXCL10, CXCL12, CXCL13 and CXCL16, was downregulated in Il18-KO mice, suggesting that IL-18 may participate in the regulation of cytokines and chemokines post TBI. To our knowledge, we are the first to assess the levels of TBI-induced cytokines and chemokines systematically in Il18-KO mice.
Astrocytes and microglia, which can secret and response to various cytokines and chemokines, are crucial in inflammatory cascades post TBI . After TBI, astrocytes are activated, showing a hypertrophic morphology involving swelling of cell bodies and extension of processes, and the number of astrocytes increases significantly and peaks at 7 dpi . Recent investigations have shown that astrocytes can produce numerous inflammatory mediators, including IL-1β, IL-6, IL-10, IL-17, TNF-α, CCL2, CCL5, CCL7, CCL8, CCL12, CXCL1, CXCL10, CXCL12 and CXCL16 . Multiple reports provide evidence that IL-18 is involved in neuroinflammation in a variety of experimental models, including brain injury [12, 15, 16]. Previous studies have shown that treatment with an anti-IL-18 antibody blocks the activation of astrocytes in an SNL model , while Il18-deficient mice exhibit enhanced KA-induced astrogliosis . In the present study, Il-18 deficiency attenuated TBI-induced astrogliosis, with astrocytes showing relatively small cell bodies with slender, relatively short and branched processes, which may contribute to the reductions in inflammatory cytokine and chemokine levels observed post TBI in Il18-KO mice in our study.
Microglia are considered a key player in initiating inflammatory responses post injury . During neuroinflammation, IL-18 may have an important role in the activation of microglia. IL-18-mediated microglial activation potentiates caspase-1 expression, metalloproteinase expression, and proinflammatory cytokine production, which augment inflammatory responses in the brain [30, 49]. After TBI, microglia become activated, and the cells number peaks at 7 dpi . Microglia release proinflammatory cytokines, including IL-1β, IL-6, IL-12, and TNF-α, and chemokines, including CCL2, CCL3, CCL5, CXCL10, and CXCL12 . Activated microglia are described by the nomenclature ‘M1’ and ‘M2’ [7, 9]. Microglia polarize from a resting phenotype into the M1-like phenotype after exposure to danger-associated molecular patterns (DAMPs), free radicals, or proinflammatory cytokines such as IFN-γ, TNF-α and IL-6 . M1-like cells are characterized by the production of proinflammatory cytokines (e.g. IL-1β, IL-6, IL-12p40, IL-18, and TNF-α) and chemokines (e.g. CCL2, CCL3, CCL4, and CXCL9), reactive oxygen species (ROS) generation, and reduced phagocytic activity [7, 9, 50, 51]. M1-like ‘pro-inflammatory’ cells are considered neurotoxic following brain injury [7, 52]. In response to anti-inflammatory cytokines (e.g. IL-4 adn IL-13), microglia polarize towards the M2-like phenotype, which is considered neuroprotective [7, 9, 52]. M2-like microglia are associated with the production of anti-inflammatory cytokines (e.g. IL4, IL-10, and IL-13), chemokines (e.g. CCL22) and trophic factors, and increased phagocytic activity [50, 53, 54]. Microglia can synthesize and respond to IL-18 . Previous studies have shown that Il-18 deficiency reduces stress and KA-induced microglial activation, possibly by reducing IFN-γ expression [56, 57]. Furthermore, recent studies show a reduction in the IL-18 level may be related to inhibition of the activation of M1 microglia  and may induce microglial polarization towards the M2 profile . Previous studies have shown that suppressing M1 phenotype activation and promoting M2 phenotype activation of microglia can rescue damaged neurons, reduce lesion volume, and improve neurological functional recovery following TBI [60–62]. In this study, although the levels of the cytokines IL-4 and IL-13, which can induce microglial polarization towards the M2-like phenotype, were downregulated, M2-like polarization was still upregulated post TBI in Il18-KO mice, suggesting that TBI-induced M2-like polarization in Il18-KO mice may be induced by other more important mechanisms . Furthermore, Il-18 deficiency reduced the frequency of TBI-induced M1-like ‘proinflammatory’ microglia, which may be the result of Il-18 deficiency-induced decrease in the levels of IFN-γ, TNF-α and IL6, which can induce microglial polarization towards the M1-like phenotype. In conclusion, IL-18 promotes microglial activation and polarization towards the M1-like phenotype post TBI, which enhances microglial caspase-1 and IL-18 expression and forms a feed-forward loop to subsequently promote the inflammatory response .
Although we found an important role for IL-18 in glial activation and numerous cytokines and chemokines, there are still some topics that need to be addressed in future work. First, in the present study, we used only global Il18-KO mice to illustrate the role of IL-18 in TBI-induced neuroinflammation. Thus, the effects of Il-18 ablation on other organs or tissues in the pathogenesis of secondary injury after TBI cannot be neglected. It would be better to illustrate the role of IL-18 with glia-specific conditional knockout mice. Furthermore, we explored the role of IL-18 in neuroinflammation only through assessing glial activation and the expression of cytokines and chemokines post TBI. Further studies are needed to explore the relationship between IL-18 and the NLRP3 inflammasome, because NLRP3-mediated inflammation is regarded as a key pathway in the process of pyroptosis [20, 38]. We are conducting an experiment to evaluate the effects of IL-18 on pyroptosis in the late phase of TBI. Finally, more animals in each group are needed to better control for variation.