Hongquan Dong

S100A8 contributes to postoperative cognitive dysfunction in mice undergoing tibial fracture surgery by activating the TLR4/MyD88 pathway.

Neuro-inflammation plays a key role in the occurrence and development of postoperative cognitive dysfunction (POCD). Although S100A8 and Toll-like receptor 4 (TLR4) have been increasingly recognized to contribute to neuro-inflammation, little is known about the interaction between S100A8 and TLR4/MyD88 signaling in the process of systemic inflammation that leads to neuro-inflammation. Firstly, we demonstrated that C57BL/6 wide-type mice exhibit cognitive deficit 24h after the tibial fracture surgery. Subsequently, increased S100A8 and S100A9 expression was found in the peripheral blood mononuclear cells (PBMCs), spleen, and hippocampus of C57BL/6 wide-type mice within 48h after the surgery. Pre-operative administration of S100A8 antibody significantly inhibited hippocampal microgliosis and improved cognitive function 24h after the surgery. Secondly, we also observed TLR4/MyD88 activation in the PBMCs, spleen, and hippocampus after the surgery. Compared with those in their corresponding wide-type mice, TLR4(-/-) and MyD88(-/-) mice showed lower immunoreactive area of microglia in the hippocampal CA3 region after operation. TLR4 deficiency also led to reduction of CD45(hi)CD11b(+) cells in the brain and better performance in both Y maze and open field test after surgery, suggesting a new regulatory mechanism of TLR4-dependent POCD. At last, the co-location of S100A8 and TLR4 expression in spleen after operation suggested a close relationship between them. On the one hand, S100A8 could induce TLR4 activation of CD11b(+) cells in the blood and hippocampus via intraperitoneal or intracerebroventricular injection. On the other hand, TLR4 deficiency conversely alleviated S100A8 protein-induced hippocampal microgliosis. Furthermore, the increased expression of S100A8 protein in the hippocampus induced by surgery sharply decreased in both TLR4 and MyD88 genetically deficient mice. Taken together, these data suggest that S100A8 exerts pro-inflammatory effect on the occurrence and development of neuro-inflammation and POCD by activating TLR4/MyD88 signaling in the early pathological process of the postoperative stage.

Mast cells and neuroinflammation.

It has been determined that there is extensive communication between the immune system and the central nervous system (CNS). Proinflammatory cytokines play a key role in this communication. There is an emerging realization that glia and microglia, in particular, (which are the brain’s resident macrophages), are an important source of inflammatory mediators and may have fundamental roles in CNS disorders. Microglia respond also to proinflammatory signals released from other non-neuronal cells, principally those of immune origin, such as mast cells. Mast cells reside in the CNS and are capable of migrating across the blood-brain barrier (BBB) in situations where the barrier is compromised as a result of CNS pathology. Mast cells are both sensors and effectors in communication among nervous, vascular, and immune systems. In the brain, they reside on the brain side of the BBB, and interact with astrocytes, microglia, and blood vessels via their neuroactive stored and newly synthesized chemicals. They are first responders, acting as catalysts and recruiters to initiate, amplify, and prolong other immune and nervous responses upon activation. Mast cells both promote deleterious outcomes in brain function and contribute to normative behavioral functioning, particularly cognition and emotion. Mast cells may play a key role in treating systemic inflammation or blockade of signaling pathways from the periphery to the brain.

Lithium ameliorates lipopolysaccharide-induced microglial activation via inhibition of toll-like receptor 4 expression by activating the PI3K/Akt/FoxO1 pathway.

BackgroundLithium, an effective mood stabilizer for the treatment of bipolar disorders, has been recently suggested to have a role in neuroprotection during neurodegenerative diseases. The pathogenesis of neurological disorders often involves the activation of microglia and associated inflammatory processes. Thus, in this study, we aimed to understand the role of lithium in microglial activation and to elucidate the underlying mechanism(s).MethodsPrimary microglial cells were pretreated with lithium and stimulated with lipopolysaccharide (LPS). The cells were assessed regarding the responses of pro-inflammatory cytokines, and the associated signaling pathways were evaluated.ResultsLithium significantly inhibited LPS-induced microglial activation and pro-inflammatory cytokine production. Further analysis showed that lithium could activate PI3K/Akt signaling. Analyses of the associated signaling pathways demonstrated that the lithium pretreatment led to the suppression of LPS-induced toll-like receptor 4 (TLR4) expressions via the PI3K/Akt/FoxO1 pathway.ConclusionsThis study demonstrates that lithium can inhibit LPS-induced TLR4 expression and microglial activation through the PI3K/Akt/FoxO1 signaling pathway. These results suggest that lithium plays an important role in microglial activation and neuroinflammation-related diseases, which may lead to a new therapeutic strategy for the treatment of neuroinflammation-related disorders.

Induction of Microglial Activation by Mediators Released from Mast Cells

Background/Aims: Microglia are the resident immune cells in the brain and play a pivotal role in immune surveillance in the central nervous system (CNS). Brain mast cells are activated in CNS disorders and induce the release of several mediators. Thus, brain mast cells, rather than microglia, are the “first responders” due to injury. However, the functional aspects of mast cell-microglia interactions remain uninvestigated. Methods: Conditioned medium from activated HMC-1 cells induces microglial activation similar to co-culture of microglia with HMC-1 cells. Primary cultured microglia were examined by flow cytometry analysis and confocal microscopy. TNF- alpha and IL-6 were measured with commercial ELISA kits. Cell signalling was analysed by Western blotting. Results: In the present study, we found that the conditioned medium from activated HMC-1 cells stimulated microglial activation and the subsequent production of the pro-inflammatory factors TNF-α and IL-6. Co-culture of microglia and HMC-1 cells with corticotropin-releasing hormone (CRH) for 24, 48 and 72 hours increased TNF-α and IL-6 production. Antagonists of histamine receptor 1 (H1R), H4R, proteinase-activated receptor 2 (PAR2) or Toll-like receptor 4 (TLR4) reduced HMC-1-induced pro-inflammatory factor production and MAPK and PI3K/AKT pathway activation. Conclusions: These results imply that activated mast cells trigger microglial activation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS inflammation-related diseases.

Enhancement of LPS-Induced Microglial Inflammation Response via TLR4 Under High Glucose Conditions

Background: Microglia activation mediated by toll-like receptor 4 (TLR4) plays an important role in neuroinflammation and postoperative cognitive dysfunction (POCD). Diabetes mellitus (DM) has been recently suggested as an independent risk factor for POCD. In this study, we investigate the potential exacerbation of the inflammatory response in primary microglia due to high glucose conditions. Methods: Primary microglial cells were exposed to normal glucose (25 mmol/L) and high glucose (35 mmol/L) levels alone or with lipopolyscaccharide (LPS 0, 2, 5, 10 ng/mL). The pro-inflammatory response of the cells was assessed by measuring changes in cytokine levels and the evaluation of associated signaling pathways. Results: Neither high glucose nor low LPS (≤5ng/ml) alone had an effect on TNF-a and IL-6 levels, but the combination of low LPS and high glucose stimulated the inflammatory response. Analyses of the associated signaling pathways demonstrated that high glucose enhanced the LPS-induced microglial activation via the TLR4/JAK2/STAT3 pathway. Conclusion: This study demonstrates that high glucose, one of the key abnormalities characteristic of DM, can augment LPS-induced microglial activation and inflammatory cytokine levels through the TLR4/JAK2/STAT3 pathway, offering new insight into the pathophysiological relationship between DM and POCD.

Suppression of Brain Mast Cells Degranulation Inhibits Microglial Activation and Central Nervous System Inflammation

Brain inflammation has a critical role in the pathophysiology of brain diseases. Microglia, the resident immune cells in the brain, play an important role in brain inflammation, while brain mast cells are the “first responder” in the injury rather than microglia. Functional aspects of mast cell-microglia interactions remain poorly understood. Our results demonstrated that site-directed injection of the “mast cell degranulator” compound 48/80 (C48/80) in the hypothalamus induced mast cell degranulation, microglial activation, and inflammatory factor production, which initiated the acute brain inflammatory response. “Mast cell stabilizer” disodium cromoglycate (cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced microglial activation, inhibition of MAPK and AKT pathways, and repression of protein expression of histamine receptor 1 (H1R), histamine receptor 4 (H4R), protease-activated receptor 2 (PAR2), and toll-like receptor 4 (TLR4) in microglia. We also demonstrated that C48/80 had no effect on microglial activation in mast cell-deficient KitW-sh/W-sh mice. These results implicate that activated brain mast cells trigger microglial activation and stabilization of mast cell inhibits microglial activation-induced central nervous system (CNS) inflammation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.

Lithium Ameliorates LPS-Induced Astrocytes Activation Partly via Inhibition of Toll-Like Receptor 4 Expression

Background/Aims: Astrocytes are critical for the development of postoperative cognitive dysfunction (POCD). In addition, astrocytes express toll-like receptors 4 (TLR4) and build up responses to innate immune triggers by releasing pro-inflammatory molecules. The pathogenesis of neurological disorders often involves the activation of astrocytes and associated inflammatory processes. Lithium, a primary drug for the treatment of bipolar disorder, has recently been suggested to have a role in neuroprotection during neurodegenerative diseases. In this study, we aimed to investigate whether lithium can ameliorate LPS-induced astrocytes activation via inhibition of TLR4 expression. Methods: Primary astrocytes cells were pretreated with lithium and stimulated with lipopolysaccharide (LPS). Cellular activation, cytokine production, and TLR4 expression, were assessed. Results: Lithium significantly inhibited LPS-induced astrocytes activation and pro-inflammatory cytokine production, as well as LPS-induced TLR4 expression. Conclusions: Lithium can inhibit LPS-induced TLR4 expression and astrocytes activation. These results indicate that lithium plays an important role in astrocytes activation and neuroinflammation-related diseases, which may open new avenues for neuroscience and biomedical research, and also offers new insight into the treatment of POCD.

Bidirectional relationship of mast cells-neurovascular unit communication in neuroinflammation and its involvement in POCD

HighlightsNeuroinflammation is involved in the development of postoperative cognitive dysfunction.Activated Mast cells contribute to the progress of neuroinflammation.Mast cells‐neurovascular unit communication promotes the pathological process of neuroinflammation and cognitive dysfunction. Abstract Postoperative cognitive dysfunction (POCD) has been hypothesized to be mediated by surgery‐induced neuroinflammation, which is also a key element in the pathobiology of neurodegenerative diseases, stroke, and neuropsychiatric disorders. There is extensive communication between the immune system and the central nervous system (CNS). Inflammation resulting from activation of the innate immune system cells in the periphery can impact central nervous system behaviors, such as cognitive performance. Mast cells (MCs), as the“first responders” in the CNS, can initiate, amplify, and prolong other immune and nervous responses upon activation. In addition, MCs and their secreted mediators modulate inflammatory processes in multiple CNS pathologies and can thereby either contribute to neurological damage or confer neuroprotection. Neuroinflammation has been considered to be linked to neurovascular dysfunction in several neurological disorders. This review will provide a brief overview of the bidirectional relationship of MCs‐neurovascular unit communication in neuroinflammation and its involvement in POCD, providing a new and unique therapeutic target for the adjuvant treatment of POCD.

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells.

Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

Prophylactic lithium alleviates splenectomy-induced cognitive dysfunction possibly by inhibiting hippocampal TLR4 activation in aged rats

Though the pathogenesis of postoperative cognitive dysfunction (POCD) remains unclear, evidence is accumulating for a pivotal role of neuroinflammation in the disease process. Advanced age and severe surgical trauma are two main risk factors for POCD. Lithium, a neuroprotective agent, can alleviate peripheral surgery-induced memory impairment in aged rats. The results of in vivo and in vitro experiments also showed that toll like receptor 4 (TLR4) was associated with the occurrence and development of neuroinflammation and POCD. So we hypothesized that inhibition of TLR4 signaling in the hippocampus maybe involved in the protective effects of prophylactic lithium on the occurrence of inflammation and POCD. In the present study, we incubated BV-2 microglia with 1μg/ml lipopolysaccharide (LPS) to mimic neuroinflammation in vitro. We found that pretreatment with 10mM of lithium or 100nM of TLR4 siRNA could inhibit the tumor necrosis factor (TNF)-α and TLR4 mRNA expression induced by LPS in BV-2 microglia. Furthermore, combination of prophylactic lithium and TLR4 siRNA even decreased their mRNA expression to the baseline levels, which showed that TLR4 signaling may be vital in protective effects of prophylactic lithium on neuroinflammation. So we further undergone the in vivo experiment. Then, we firstly demonstrated that prophylactic 2mM/kg of lithium alleviated splenectomy-induced cognitive impairments, decreased splenectomy-associated systemic, central, and hippocampal TNF-α and interleukin (IL)-1β expression and reduced the increase of CD11b(+) area in hippocampal CA1 region caused by the surgery. Then, we also found that splenectomy merely increased hippocampal TLR2 and TLR4 mRNA levels in aged rats. At last, we confirmed that prophylactic lithium reduced the increased levels of hippocampal TLR4/NF-κB induced by splenectomy. Taken together, these results demonstrate that TLR4 signaling inactivation may contribute to the protective effects of prophylactic lithium on the occurrence of POCD by inhibiting systemic inflammation and especially neuroinflammation.