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The development of microglial endotoxin tolerance (ET) is a critical event in protecting neurons against excessive immune responses when microglia are administered two consecutive lipopolysaccharide (LPS) challenges. However, the intrinsic mechanisms of microglia shape ET programs and protect neurons remain unclear. This study aimed to determine whether extracellular autocrine cascades or intracellular signaling pathways are involved in ET microglia-mediated tumor necrosis factor-alpha (TNF-α) reduction and neuroprotection. Neuron-glia cultures composed of astroglia, neurons, and microglia were performed in different conditions: with or without serum or LPS-binding proteins (LBP), along with an induction approach of ET. Enzyme-linked immunosorbent assay results revealed that LPS induced TNF-α tolerance of microglia in an LBP-dependent manner. Furthermore, we determined whether the early pro-inflammatory cytokines induced by LPS might contribute to the development of microglial ET. Our data showed that the neutralization of TNF-α using an anti-TNF-α antibody had no change in the TNF-α tolerance of microglia during the ET challenge. Furthermore, pre-incubation of TNF-α, interleukin-1 beta, and prostaglandin E2 failed to induce any TNF-α tolerance in microglia after LPS treatment. Moreover, using three specific chemical inhibitors that respectively blocked the activities of the mitogen-activated protein kinases (MAPKs) namely p38, c-Jun N-terminal kinase and extracellular signal-related kinases revealed that inhibition of p38 MAPK by SB203580 disrupted the tolerated microglia-mediated TNF-α reduction and neuroprotection. In summary, our findings demonstrated that the LPS pre-treatment immediately programmed the microglial ET to prevent endotoxin-induced TNF-α production and neuronal damage through the intracellular p38 MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Periodontitis is a dental plaque–induced chronic inflammatory disease. Long-term exposure of the host to periodontal pathogens leads to a hyporesponsive state to the following stimulations, which is described as endotoxin tolerance. Neutrophils are the most abundant innate immune cells in the body. To clarify the roles of endotoxin tolerance in periodontitis, inflammatory responses in Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS)–tolerized neutrophils were explored in this study. Here, apoptosis and respiratory burst in neutrophils upon single or repeated P. gingivalis LPS stimulations were explored by flow cytometry. Cytokine production (TNF-α, IL-8, and IL-10) in tolerized neutrophils or neutrophils co-cultured with peripheral blood mononuclear cells was determined by ELISA. Phagocytosis of P. gingivalis by tolerized neutrophils was also assayed by flow cytometry. In addition, quality and quantitation of neutrophil extracellular trap (NET) formation were detected using immunofluorescence microscope and microplate reader, respectively. The protein expressions of extracellular signal–regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) were examined to identify possible mechanisms for the abovementioned changes. Tolerance induced by P. gingivalis LPS significantly suppressed apoptosis, reactive oxygen species (ROS) generation, and phagocytosis in neutrophils (p < 0.05). In both neutrophils alone and co-culture system, repeated P. gingivalis LPS stimulations significantly decreased TNF-α production, but increased IL-10 secretion (p < 0.05). Moreover, in tolerized neutrophils, NET formations were strengthened and there were more released extracellular DNA (p < 0.05). In P. gingivalis LPS-tolerized neutrophils, phosphorylation of ERK1/2 was suppressed compared with that in non-tolerized cells. Taken together, immune responses in neutrophils were reprogrammed by P. gingivalis LPS-induced tolerance, which might be related with the development of inflammation in periodontal tissues. Moreover, ERK1/2 might play important roles in endotoxin tolerance triggered by P. gingivalis LPS. [ABSTRACT FROM AUTHOR]

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a hyporesponsive state to subsequent challenge, which is termed endotoxin tolerance. In this experiment, we studied the cytokine production in THP-1 cells upon single or repeated Porphyromonas gingivalis ( P. gingivalis) lipopolysaccharide (LPS) or Escherichia coli ( E. coli) LPS stimulation by ELISA. In addition, the protein expression profiles of Toll-like receptor 2 (TLR2), TLR4, IL-1 receptor-associated kinase 4 (IRAK4) and IRAK-M and the gene expression changes of Toll-interacting protein (Tollip) and suppressor of cytokine-signaling-1 (SOCS1) were explored to identify possible mechanisms for changes in cytokine secretion. After repeated stimulation with P. gingivalis LPS or E. coli LPS, secretions of TNF-α and IL-1β were decreased significantly compared with those following single challenge, while the levels of IL-10 were increased ( p < 0.05). Only comparable levels of IL-8 were confirmed in P. gingivalis LPS-tolerized cells ( p > 0.05). In addition, severe downregulation of TLR2 was detected in THP-1 cells retreated with P. gingivalis LPS, and the reduction of TLR4 expression was observed in cells restimulated with E. coli LPS ( p < 0.05). Precondition with P. gingivalis LPS or E. coli LPS also led to an enhancement of IRAK-M and SOCS1, while maintaining the expressions of IRAK4 and Tollip. This pattern of cytokine production indicates the different effects of endotoxin tolerance triggered by P. gingivalis LPS and E. coli LPS, which might contribute to limiting inflammatory damage. Moreover, TLR2, TLR4, IRAK-M, and SOCS1 might play important roles in developing tolerance. [ABSTRACT FROM AUTHOR]

Endotoxin tolerance is an important mechanism for preventing uncontrolled inflammatory cytokine production in bacterial sepsis. However, its molecular mechanisms remain largely unknown. It was reported that Twist-2 protein was a negative regulator for cytokine signaling by repressing the nuclear factor (NF)-κB-dependent cytokine pathway. However, the relationship between Twist-2 and endotoxin tolerance is unclear. Endotoxin tolerance models of BABL/c mice and isolated Kupffer cells (KCs) were established to observe the changes of Twist-2 during endotoxin tolerance. Then, Twist-2 shRNA was used to specifically inhibit Twist-2 gene in KCs to further explore the role of Twist-2 in endotoxin tolerance. The expression of Twist-2 was analyzed by immunohistochemistry, reverse transcription polymerase chain reaction, and Western blotting, respectively. The responses to lipopolysaccharide were assessed by the activation of nuclear factor-κB and the production of tumor necrosis factor-α. The histopathologic changes in the liver of the non-endotoxin tolerance group were more serious than those of the endotoxin tolerance group. Endotoxin tolerance also led to less activation of nuclear factor-κB, lower expression levels of tumor necrosis factor-α mRNA, and more expression of Twist-2 than those of non-endotoxin tolerance group in liver and KCs. Moreover, the inhibitive effects partly weaken in KCs transfected with Twist-2 shRNA. Twist-2 was involved in endotoxin tolerance through inhibiting NF-κB trans-activation and cytokines transcriptional activities. It may be a new target for the clinical treatment of sepsis and other inflammatory diseases. [ABSTRACT FROM AUTHOR]

Prior exposure of innate immune cells to lipopolysaccharide (LPS) has caused them to be refractory to further endotoxin stimulation, also termed endotoxin tolerance (ET). Bacterial LPS signals through Toll-like receptor (TLR) 4, which was thought to enable the innate immune system to deal with invasive pathogens and to restrain systemic inflammation efficiently. We established a robust model of ET and determined the level of TNF-α and IL-6 in cultured human monocytes. Then, microarray assay was applied to assess gene expression in this model. The results showed that 356 non-tolerizable genes were differentially expressed at a high level in tolerant monocytes. The genes selected were classified into several categories based on gene ontology (GO) and KEGG pathway database. And then literature annotations, protein-protein interaction (PPI) network, and functional consistency were applied to analyze the non-tolerizable genes. Finally, the microarray results were verified by quantitative real-time PCR of seven representative genes, including the two candidate genes, Spry2 and Smurf2, which were supposed to play a critical role in TLRs-induced inflammation based on literature retrieval. Our results would provide useful information for further analysis of regulating TLRs-induced inflammation, and would facilitate the study of associated mechanisms. [ABSTRACT FROM AUTHOR]