<code lang="Ql3CJ"></code> Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or VSports app下载. mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2019 May 31;294(22):8872-8884.
doi: 10.1074/jbc.RA118.007040. Epub 2019 Apr 18.

High mobility group box 1 enables bacterial lipids to trigger receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis and apoptosis in mice

Ran Meng  1   2 , Lan Gu  1   3 , Yanyan Lu  1   3 , Kai Zhao  1   3 , Jianfeng Wu  4 , Haichao Wang  5 , Jiahuai Han  4 , Yiting Tang  6   7 , Ben Lu  8   3   9   10
Affiliations

High mobility group box 1 enables bacterial lipids to trigger receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis and apoptosis in mice

Ran Meng (V体育2025版) et al. J Biol Chem. .

Erratum in

Abstract

Receptor-interacting protein kinase 3 (RIPK3) is a key regulator of programmed cell death and inflammation during viral infection or sterile tissue injury. Whether and how bacterial infection also activates RIPK3-dependent immune responses remains poorly understood. Here we show that bacterial lipids (lipid IVa or lipid A) form a complex with high mobility group box 1 (HMGB1), released by activated immune cells or damaged tissue during bacterial infection, and that this complex triggers RIPK3- and TIR domain-containing adapter-inducing IFN-β (TRIF)-dependent immune responses. We found that these responses lead to macrophage death, interleukin (IL)-1α release, and IL-1β maturation. In an air-pouch inflammatory infiltration model, genetic deletion of Ripk3, Trif, or IL-1 receptor (Il-1R), or monoclonal antibody-mediated HMGB1 neutralization uniformly attenuated inflammatory responses induced by Gram-negative bacteria that release lipid IVa and lipid A. These findings uncover a previously unrecognized mechanism by which host factors and bacterial components work in concert to orchestrate immune responses VSports手机版. .

Keywords: HMGB1; RIPK3; apoptosis; bacteria; damage-associated molecular patterns; inflammasome; lipid A; necroptosis; tumor necrosis factor (TNF) V体育安卓版. .

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

"VSports app下载" Figures

Figure 1.
Figure 1.
HMGB1 enables microbial lipids to trigger proinflammatory cell death. A, LDH, IL-1α, IL-1β, and TNFα measured from culture supernatants of peritoneal macrophages from wildtype (WT) and Ripk3−/− mice following stimulation with lipid IVa or lipid A (1 μg/ml) in the absence or presence of HMGB1 (0.4 μg/ml). B, Western blot for processed IL-1α and IL-1β released from WT and Ripk3−/− peritoneal macrophages stimulated with lipid IVa or lipid A (1 μg/ml) in the absence or presence of HMGB1 (0.4 μg/ml). C, flow cytometry analysis of WT or Mlkl−/− peritoneal macrophages undergoing necrosis (PI+) or apoptosis (PI−) of stimulation with lipid IVa or lipid A (1 μg/ml) in the presence of HMGB1 (0.4 μg/ml). D, the EM shows the morphology of WT and Ripk3−/− peritoneal macrophages after stimulation with HMGB1 (0.4 μg/ml) + lipid IVa or lipid A (1 μg/ml). The red arrows indicate the expansion of the cell volume, organelle swelling, and plasma membrane rupture. The blue arrows indicate intact cell membrane and condensed chromatin. Scale bars: 5 μm. E, IL-1α and IL-1β measured from culture supernatants of peritoneal macrophages from WT and Ripk3−/− upon exposure to the necrotic Hmgb1−/− or Hmgb1+/+ MEF in the presence or absence of lipid IVa or lipid A (1 μg/ml). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Graphs show the mean ± S.D. from three independent experiments.
Figure 2.
Figure 2.
HMGB1 binding is critical for microbial lipids to trigger proinflammatory cell death. A, schematic illustration of competitive binding of HMGB1 by free lipid IVa or lipid A (left). Plates coated with lipid IVa or lipid A were incubated with recombinant HMGB1 (16 μg/ml) and the indicated concentrations of free lipid IVa or lipid A. After three extensive washings, the binding capacity between plate-coated lipid IVa or lipid A and HMGB1 was measured by using a HMGB1-specific primary antibody and relevant secondary antibodies. Then the percentage of binding competition by free lipid IVa or lipid A was evaluated. B, schematic illustration of competitive binding of HMGB1 by free LPS-RS (left). Plates coated with lipid IVa or lipid A (2 μg/ml) were incubated with HMGB1 (16 μg/ml) and the indicated concentration of LPS-RS. Then the percentage of binding competition by LPS-RS was evaluated. C, IL-1α and IL-1β were measured from the supernatants of mouse peritoneal macrophages stimulated with the indicated stimuli in the absence or presence of LPS-RS (2.5 μg/ml). D, the percentage of mouse peritoneal macrophages undergoing necrosis (PI+) or apoptosis (PI−) were measured by flow cytometry after stimulation with the indicated stimuli in the absence or presence of LPS-RS (2.5 μg/ml). E, LDH, IL-1α, and IL-1β in the supernatants of WT mouse peritoneal macrophages stimulated with lipid A (1 μg/ml) + HMGB1 (400 ng/ml) in the presence of different concentrations of HPep6 for 16 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Graphs show the mean ± S.D. from three independent experiments.
Figure 3.
Figure 3.
TLR4-TRIF signaling mediates HMGB1/microbial lipid-induced proinflammatory cell death. A and D, LDH, IL-1α, IL-1β, and TNFα were measured from culture supernatants of peritoneal macrophages from WT and Tlr4−/− or TrifLps/Lps2 mice stimulated with lipid IVa or lipid A (1 μg/ml) in the absence or presence of HMGB1 (0.4 μg/ml). B and E, flow cytometry analysis of the percentage of WT and Tlr4−/− or TrifLps/Lps2 macrophages undergoing necrosis (PI+) or apoptosis (PI−) after stimulation with lipid IVa or lipid A (1 μg/ml) in the presence of HMGB1 (0.4 μg/ml). C and F, IL-1α and IL-1β measured from the supernatants of peritoneal macrophages from WT and Tlr4−/− or TrifLps/Lps2 mice upon exposure to necrotic Hmgb1−/− or Hmgb1+/+ MEF in the presence or absence of lipid IVa or lipid A (1 μg/ml). G, LDH, IL-1α, IL-1β, and TNFα measured from culture supernatants of peritoneal macrophages from mice with the indicated genotypes after stimulation with lipid IVa or lipid A (1 μg/ml) in the absence or presence of HMGB1 (0.4 μg/ml). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Graphs show the mean ± S.D. from three independent experiments.
Figure 4.
Figure 4.
TLR4-TRIF-RIPK3 signaling mediates MLKL-dependent necroptosis induced by HMGB1 and microbial lipids. A and B, Western blot analysis of phosphorylated MLKL and RIPK3 in peritoneal macrophages from WT, Ripk3−/−, and Mlkl−/− mice exposed to the indicated stimuli in the absence or presence of GSK872. C and D, Western blot analysis of phosphorylated MLKL and RIPK3 in peritoneal macrophages from WT and Tlr4−/− mice exposed to the indicated stimuli in the absence or presence of GSK872. E and F, Western blot analysis of phosphorylated MLKL and RIPK3 in peritoneal macrophages from WT and TrifLps/Lps2 mice exposed to the indicated stimuli in the absence or presence of GSK872. G, cell lysates of peritoneal macrophages from a WT mouse treated with the indicated stimuli were immunoprecipitated (IP) with RIPK3-specific antibody. The precipitated proteins were immunoblotted with RIPK3- or MLKL-specific antibodies. Whole cell lysate (Input) was used as positive control. H, flow cytometry analysis of the percentage of WT or Mlkl−/− peritoneal macrophages undergoing necrosis (PI+) or apoptosis (PI−) following stimulation with lipid IVa or lipid A (1 μg/ml) in the presence of HMGB1 (0.4 μg/ml). ***, p < 0.001; #, not significant. Graphs show the mean ± S.D. from three independent experiments.
Figure 5.
Figure 5.
TLR4-TRIF-RIPK3 signaling mediates caspase-8–dependent apoptosis induced by HMGB1 and microbial lipids. A, Western blot analysis of processed caspase-8 released from WT and Ripk3−/− mouse peritoneal macrophages stimulated with lipid IVa or lipid A (1 μg/ml) in the absence or presence of HMGB1 (0.4 μg/ml). B and C, Western blot assay for processed caspase-8 released from WT and Mlkl−/− mouse peritoneal macrophages exposed to the indicated stimuli in the absence or presence of caspase-8 inhibitor or control. D, LDH and IL-1α measured from culture supernatants of peritoneal macrophages from WT, Ripk3−/−, and Mlkl−/− mice exposed to the indicated stimuli in the absence or presence of caspase-8 inhibitor or controls. E and F, Western blot assay for processed caspase-8 released from WT and Tlr4−/− mouse peritoneal macrophages exposed to the indicated stimuli in the absence or presence of caspase-8 inhibitor or control. G and H, Western blot for processed caspase-8 released from WT and TrifLps/Lps2 mouse peritoneal macrophages exposed to the indicated stimuli in the absence or presence of caspase-8 inhibitor or control. **, p < 0.01; ****, p < 0.0001. Graphs show the mean ± S.D. from three independent experiments.
Figure 6.
Figure 6.
RIPK3 mediates the NLRP3 inflammasome-dependent IL-1β cleavage and release in response to HMGB1 and microbial lipids. A, IL-1α and IL-1β measured from culture supernatants of peritoneal macrophages from WT, Ripk3−/−, Nlrp3−/−, and Asc−/− mice stimulated with lipid IVa or lipid A (1 μg/ml) in the absence or presence of HMGB1 (0.4 μg/ml). B and C, Western blot analysis of IL-1α and IL-1β released from mouse peritoneal macrophages with the indicated genotypes stimulated with lipid IVa (1 μg/ml) + HMGB1 (0.4 μg/ml) (B) or lipid A (1 μg/ml) + HMGB1 (0.4 μg/ml) (C). D, Western blot analysis of processed IL-1α and IL-1β released from mouse peritoneal macrophages with the indicated genotypes stimulated with lipid IVa (1 μg/ml) + HMGB1 (0.4 μg/ml) or lipid A (1 μg/ml) + HMGB1 (0.4 μg/ml). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; #, not significant. Graphs show the mean ± S.D. from three independent experiments.
Figure 7.
Figure 7.
Loss of RIPK3 attenuates inflammation induced by dead E. coli and HMGB1. A, air-pouch lavage fluid was collected from WT and Ripk3−/− mice following injection with live E. coli for analysis of infiltrated white blood cell (WBC) counts by microscope (left) and neutrophils (middle) and macrophages (right) by flow cytometry. B, air-pouch lavage fluid was collected from WT and Ripk3−/− mice following injection with heat-killed E. coli for analysis of infiltrated white blood cell counts by microscope (left) and neutrophils (middle) and macrophages (right) by flow cytometry. C, WT, TrifLps/Lps2 mice and Il-1R−/− mice were selected for the same experiment as B, and leukocytes (left), neutrophils (middle), and macrophages (right) numbers are shown. D, air-pouch inflammatory infiltration was induced in the absence or presence of HMGB1-neutralizing or normal control IgGs. Then air-pouch lavage fluid was collected, and leukocytes (left), neutrophils (middle), and macrophages (right) numbers are shown. Circles represent individual mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Graphs show the mean ± S.D. from three independent experiments.
See this image and copyright information in PMC

References

    1. Takeuchi O., and Akira S. (2010) Pattern recognition receptors and inflammation. Cell 140, 805–820 10.1016/j.cell.2010.01.022 - DOI - PubMed
    1. Tang Y., Zhao X., Antoine D., Xiao X., Wang H., Andersson U., Billiar T. R., Tracey K. J., and Lu B. (2016) Regulation of posttranslational modifications of HMGB1 during immune responses. Antioxid. Redox Signal. 24, 620–634 - PMC - PubMed
    1. Lu B., Wang C., Wang M., Li W., Chen F., Tracey K. J., and Wang H. (2014) Molecular mechanism and therapeutic modulation of high mobility group box 1 release and action: an updated review. Expert Rev. Clin. Immunol. 10, 713–727 10.1586/1744666X.2014.909730 - "V体育官网" DOI - PMC - PubMed
    1. Lu B., Nakamura T., Inouye K., Li J., Tang Y., Lundbäck P., Valdes-Ferrer S. I., Olofsson P. S., Kalb T., Roth J., Zou Y., Erlandsson-Harris H., Yang H., Ting J. P., Wang H. et al. (2012) Novel role of PKR in inflammasome activation and HMGB1 release. Nature 488, 670–674 10.1038/nature11290 - DOI - PMC - PubMed
    1. Lu B., Antoine D. J., Kwan K., Lundbäck P., Wähämaa H., Schierbeck H., Robinson M., Van Zoelen M. A., Yang H., Li J., Erlandsson-Harris H., Chavan S. S., Wang H., et al. (2014) JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation. Proc. Natl. Acad. Sci. U.S.A. 111, 3068–3073 10.1073/pnas.1316925111 - DOI - PMC - PubMed

Publication types

MeSH terms (V体育ios版)