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Effects of Endotoxin Tolerance Induced by Porphyromonas gingivalis Lipopolysaccharide on Inflammatory Responses in Neutrophils

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Abstract

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.

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Author notes

  1. Jian-yu Gu and Yu-jie Liu contributed equally to this work.

Authors and Affiliations

  1. Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, China

    Jian-yu Gu, Yu-jie Liu, Xiang-qing Zhu, Jia-ying Qiu & Ying Sun

  2. Department of Periodontology, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, China

    Jian-yu Gu, Yu-jie Liu, Xiang-qing Zhu, Jia-ying Qiu & Ying Sun

  3. Department of Stomatology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China

    Xiang-qing Zhu

  4. Department of Periodontology, Suzhou Stomatology Hospital, Suzhou, China

    Jia-ying Qiu

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  1. Jian-yu Gu
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Contributions

The experiments were designed by YS; performed by JG, YL, XZ, and JQ; and analyzed by JG and YL. The manuscript was written by JG and YL, and reviewed and edited by YS. All authors read and approved the final manuscript.

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Correspondence to Ying Sun.

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This study was approved by the Ethical Committee of Nanjing Medical University in accordance with the principles of the Declaration of Helsinki (Permit Number: 20130204) and written informed consents were obtained from all recruits.

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Gu, Jy., Liu, Yj., Zhu, Xq. et al. Effects of Endotoxin Tolerance Induced by Porphyromonas gingivalis Lipopolysaccharide on Inflammatory Responses in Neutrophils. Inflammation 43, 1692–1706 (2020). https://doi.org/10.1007/s10753-020-01243-8

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