Reactive oxygen species generation required for autophagy induction during butyrate- or propionate-induced release of damage-associated molecular patterns from dying gingival epithelial Ca9-22 cells

Abstract

Purpose: Bacterial cells in mature dental plaque produce a high concentration of short-chain fatty acids (SCFAs) such as butyrate and propionate. SCFA-treatment on human gingival epithelial Ca9-22 cells induced cell death. However, the exact mechanism underlying cell death remains unclear. In this study, the relationship between reactive oxygen species (ROS) and autophagy induction during SCFA-induced cell death was examined.

Methods: Human gingival epithelial Ca9-22 cells were treated with butyrate or propionate to induce cell death and the number of dead cells were measured using SYTOX-green dye. A siRNA for ATG5 and N-acetylcysteine (NAC) were used for autophagy reduction and ROS-scavenging, respectively. Release of damage-associated molecular patterns (DAMPs) such as Sin3A-associated protein 130 (SAP130) and high-mobility group box 1 (HMGB1) were detected using western blot.

Results: Reducing autophagy significantly suppressed SCFA-induced Ca9-22 cell death. ROS generation was observed upon SCFA treatment, and scavenging ROS with NAC decreased cell death. NAC also reduced the SCFA-induced increase in microtubule-associated protein 1 light chain 3B (LC3B)-I and LC3B-II, and mitigated the release of DAMPs.

Conclusion: The findings suggest that ROS generation is necessary for autophagy, which is required for SCFA-induced cell death and accompanying DAMP release.

Introduction

Although gingivitis and periodontitis are caused by bacteria in dental plaque, the precise molecular mechanisms underlying periodontal disease onset are unclear. Bacteria in mature dental plaque produce and release high concentrations of short-chain fatty acids (SCFAs), including butyrate or propionate, as bacterial metabolites [1].

Moreover, the gingival epithelia compete with dental plaque bacteria surrounding the gingival crevicular grooves for nutrients. Therefore, gingival epithelial cells located near mature dental plaque are subjected to nutrient deprivation and SCFA exposure. These metabolites trigger a small number of apoptosis and the death of human gingival epithelial Ca9-22 cells, which is dependent on autophagy in nutrient-insufficient conditions [2,3]. Since the SCFA-induced cell death that is dependent on autophagy is necrotic [4], damage-associated molecular patterns such as high mobility group box 1 (HMGB1) that activates innate immunity are thought to be released into extracellular spaces [4,5]. Therefore, understanding SCFA-induced cell death is important for understanding the pathogenesis of periodontal disease.

Autophagy is associated with butyrate-induced Ca9-22 cell death [2,3]. Furthermore, the knockdown of microtubule-associated protein 1 light chain 3B (LC3B), which localizes to autophagosomal membranes during autophagy, reduces butyrate-induced Ca9-22 cell death [3]. In addition, Evans et al. demonstrated that AMP-dependent kinase (AMPK) activity, which induces autophagy signaling, is important for butyrate-induced Ca9-22 cell death [3]. These events consistently demonstrate the importance of autophagy induction in butyrate-induced cell death. In contrast, Uemichi et al. demonstrated that butyrate or propionate induces histone H3 acetylation by their histone deacetylase inhibitory activities, and the effects are important for SCFA-dependent death of Ca9-22 cells [6]. Although several factors associated with butyrate-induced cell death have been reported, the relationship between these factors is unclear. Because gene expressions related to autophagy induction and reactive oxygen species (ROS) production are upregulated during butyrate treatment [6], the relationship between autophagy induction and ROS generation during SCFA-induced cell death and subsequent release of damage-associated molecular patterns (DAMPs) was investigated. The potential methods for prevention of periodontal disease were additionally discussed.

Materials and Methods

Media, reagents, and antibodies

Minimum essential medium α (MEMα), Roswell Park Memorial Institute (RPMI) 1640 medium, sodium butyrate, sodium propionate, and N-acetylcysteine were purchased from Fujifilm Wako (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Biofill (Victoria, Australia). The SYTOX-Green dye was obtained from ThermoFisher Scientific (Waltham, MA, USA). Anti-ATG5 antibody was purchased from Cell Signaling Technologies (Danvers, MA, USA); an anti-LC3B antibody and anti-Sin3A associated protein 130 (SAP130) antibody were from GeneTex (Irvine, CA, USA); anti- HMGB1 antibody was from BioLegend (San Diego, CA, USA); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology (Dallas, TX, USA); and WestVision Peroxidase Polymers, anti-rabbit and mouse IgGs were from Vector Laboratories (Newark, CA, USA).

Cell culture condition

Ca9-22 human gingival epithelial cells, which are frequently used as counterparts to human gingival epithelial cells [7], were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan). The cells were maintained in 10% FBS MEMα containing 1% penicillin/streptomycin (Fujifilm Wako) at 37°C in a 5% CO₂ atmosphere.

ATG5 knockdown and SYTOX-Green cell death assay

ATG5, which is required for LC3-recruiting to autophagosomal membranes, is important for autophagy flux [8]. siRNA chimera duplex for ATG5 knockdown (5′ –AACAAGUUGGAAUUCGUCCTT– 3′) was synthesized by Hokkaido System Science (Sapporo, Japan). The siPerfect negative control chimera duplex was purchased from RNAi (Tokyo, Japan). siRNA transfection was performed using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instruction. After transfection for 24 h, cells were seeded at 3.0 × 10³ cells/well density onto wells of a 96-well black optical-bottom plate and incubated overnight at 37°C. Residual cells were collected and lysed in lysis buffer, and the lysates were used for western blot to confirm the ATG5 reduction. Transfected cells were treated with 5 mM sodium butyrate or 10 mM sodium propionate in 1% FBS RPMI1640 for 48 h. Cell death assays using SYTOX-Green dye were performed as previously described [3,6].

ROS assay

ROS generated from Ca9-22 cells during SCFA-treatment were detected using the ROS-Assay Kit with photo-oxidation resistant DCFH-DA (Dojindo, Mashiki, Japan) according to the manufacturer’s instructions. In brief, Ca9-22 cells were seeded onto wells of a 24-well plate (2 × 10⁵ cells/well) and incubated overnight at 37°C. Then, cells were treated for 30 min with DCFH-DA working solution at 37°C. After two washes with phenol red-free RPMI1640, the cells were treated with or without 5 mM sodium butyrate or 10 mM sodium propionate for 40 min (four wells for each condition). After two washes with RPMI1640, fluorescence signal imaging was performed using a Keyence BZ-X810 fluorescence microscope (Keyence, Osaka, Japan). Eight fields of view from four wells are photographed under the same conditions. The mean fluorescence intensities of each photo were measured using Image J software, and the averages and standard deviations were calculated, and presented as the average of intensities ± standard deviation.

ROS quenching experiments

Ca9-22 cells were pretreated for 1 h with 30 mM N-acetylcysteine (NAC), a reducing agent that scavenges ROS, and then treated for 48 h with sodium butyrate (5 mM) or sodium propionate (10 mM) in the presence of the same concentration of NAC. The number of dead cells was measured using the SYTOX-Geen dye cell death assay as previously described. To examine the effect of ROS quenching on autophagy, cells were pretreated with 0, 20, or 30 mM NAC for 1 h and then treated for 6 h with sodium butyrate (5 mM) or sodium propionate (10 mM) in the presence of the same concentration of NAC. Cell lysates were then prepared, and 5 µG of total protein from each sample was subsequently applied to the western blot. An anti-LC3B was used as the primary antibody to detect the conversion of LC3B-I to LC3B-II. During autophagosome maturation, the C-terminal of LC3B is cleaved by the activated ATG4 (the N-terminal fragment is referred to as LC3B-I). LC3B-I then binds to phosphatidylethanolamine on the phagosomal membrane. The lipid-binding form of LC3B is known as LC3B-II. Because LC3B-II is much more hydrophobic than LC3B-I, the LC3B-II band was detected at a lower molecular weight than LC3B-I.

Damage-associated molecular patterns (DAMPs) detection

Ca9-22 cells (2 × 10⁵ cells/well in 24-well plates) were pretreated for 1 h with or without NAC (30 mM) in 10% FBS MEMα media and subsequently treated for 48 h with or without sodium butyrate or sodium propionate in the presence of NAC (30 mM) in 1% FBS RPMI1640 media. Cell culture supernatants were collected and cell debris in it was precipitated by centrifugation. Aliquots (10 µL) of the resulting supernatants were subjected to western blot analysis.

Statistical analysis

All statistical analyses were performed using the EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [9]. Shapiro-Wilk test was conducted to determine whether the data distribution was normal. For normally distributed data, Bartlett’s test was conducted to test for homogeneity of variances. Since the data were not homoscedastic, Welch’s one-way analysis of variance (ANOVA) followed by Bonferroni’s test was used to examine the differences. If the data were not normally distributed, the Kruskal-Wallis test and subsequently the Steel test was used to determine differences. Results with P < 0.01 were considered statistically significant.

Results

Sodium butyrate or sodium propionate treatment-induced cell death of human gingival epithelial cells is autophagy-dependent

As previously reported, butyrate treatment induces the autophagy-dependent death of human gingival epithelial Ca9-22 cells [2,3]. Since both butyrate and propionate are found in high concentration in mature dental plaque [1], the role of autophagy in propionate-induced cell death was examined. Autophagy signaling induces the binding of ATG5 to ATG12, which is necessary for autophagosome formation. Therefore, the effects of ATG5 knockdown on butyrate- and propionate-induced death of human gingival epithelial Ca9-22 cells were examined. As shown in Fig. 1A, the band from ATG5-siRNA-treated cells was weaker than that from the negative control siRNA-treated cells. In addition, ATG5 knockdown significantly decreased both sodium butyrate- and sodium propionate-induced cell death (n = 8, P < 0.01, Fig. 1B).

Fig. 1 Reduction of autophagy by ATG5 knockdown reduces cell death induced by butyrate or propionate treatment on human gingival epithelial Ca9-22 cells

(A) siRNAs for ATG5 or for the negative control were transfected into Ca9-22 cells. After 24 h incubation, the knockdown level was confirmed using western blot. (B) Transfected cells were treated for 48 h with butyrate or propionate. The number of dead cells was measured using SYTOX-green dye (n = 8; *P < 0.01).

Sodium butyrate or sodium propionate-treated Ca9-22 cells generate ROS, and ROS quenching results in reduction of butyrate or propionate-induced Ca9-22 cell death

It has been demonstrated that sodium butyrate treatment of Ca9-22 cells altered the expression of many autophagy- and ROS generation-related genes [6]. Therefore, whether Ca9-22 cells release ROS by treatment with sodium butyrate or sodium propionate was examined. Sodium butyrate (5 mM) or sodium propionate (10 mM) treatment of Ca9-22 cells generated significantly more ROS than the non-treated cells (Fig. 2A, B). In addition, NAC pretreatment resulted in a significant reduction in sodium butyrate- or sodium propionate-induced cell death (Fig. 2C).

Fig. 2 Ca9-22 cell death induced by treatment with butyrate or propionate requires reactive oxygen species (ROS) production

(A) ROS generated by Ca9-22 cells during 40 min treatment with butyrate or propionate were detected using the ROS Assay Kit-Photo-oxidation Resistant DCFH-DA. Green fluorescence images were visualized by Keyence BZ-X810. (B) Eight fields of view from four wells of the same condition were photographed, and the mean green-fluorescent intensities of each photo were measured using Image J software. Fluorescent intensities were indicated as the average of mean intensities ± standard deviation (n = 8; *P < 0.01). (C) Ca9-22 cells were pretreated for 1 h with 30 mM N-acetylcysteine (NAC), followed by butyrate or propionate treatment (for 48 h). The number of dead cells was determined using the SYTOX-Green cell death assay (n = 8; *P < 0.01).

Pretreatment of Ca9-22 cells with NAC, a reducing agent, suppresses sodium butyrate or sodium propionate-induced autophagy

In the present study, it was demonstrated that butyrate- and propionate-induced Ca9-22 cell death is dependent on autophagy and ROS-generation. The effect of ROS quench on autophagy was examined to determine the relationship between autophagy and ROS generation. Sodium butyrate (5 mM) and sodium propionate (10 mM) induced LC3B and the conversion of LC3B-I to LC3B-II (Fig. 3). Pretreatment of Ca9-22 cells with 30 mM NAC reduced the butyrate- or propionate-induced upregulation of the LC3B-Ⅰ and LC3B-II bands (Fig. 3).

Fig. 3 Butyrate or propionate-induced autophagy requires reactive oxygen species (ROS) production

Ca9-22 cells were pretreated for 1h with 0, 20, or 30 mM N-acetylcysteine (NAC) and treated for 6 h with butyrate or propionate in the presence of NAC. Cell lysates were collected, and 10 µG of total proteins were subjected to western blot using an anti-microtubule-associated protein 1 light chain 3B (anti-LC3B) antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for internal control.

NAC-pretreatment of Ca9-22 cells suppresses the sodium butyrate or sodium propionate-induced damage-associated molecular patterns (DAMPs) release

Ca9-22 cell death induced by butyrate- or propionate-treatment was reported to be necrotic [4]. Therefore, death is followed by the release of DAMPs such as HMGB1 and SAP130. HMGB1 has a role in proinflammatory cytokines that activate receptor for advanced glycation end products (RAGE) and Toll-like receptor (TLR) 4 signaling that activate innate immunity [4]. In addition, SAP130 binds to macrophage-inducible C-type lectin (MINCLE) and induce phosphorylation of spleen tyrosine kinase (SYK) that promote osteoclast differentiation [10,11,12]. To examine whether NAC pretreatment reduces butyrate- or propionate-induced DAMP-release, the number of DAMPs in the cell culture supernatants after Ca9-22 cells were pretreated with NAC in the presence of butyrate or propionate was determined. As shown in Fig. 4, butyrate or propionate induced the release of HMGB1 and SAP130, whereas NAC pretreatment reduced DAMP release.

Fig. 4 Quenching reactive oxygen species by N-acetylcysteine reduces high-mobility group box 1 (HMGB1) and Sin3A-associated protein 130 (SAP130)-release from short-chain fatty acid (SCFA)-treated Ca9-22 cells

After pretreatment for 1h with 0 or 30 mM N-acetylcysteine (NAC), Ca9-22 cells were treated for 48 h with 5 mM sodium butyrate or 10 mM sodium propionate in the presence of NAC. The same volume of cell culture supernatants was subjected to western blot.

Discussion

It was shown in this study that human gingival epithelial Ca9-22 cell death induced by butyrate or propionate treatment is dependent on both autophagy (Figs. 1, 5) and ROS generation (Figs. 2, 5). In addition, induction of autophagy by butyrate or propionate treatment was ROS-dependent (Figs. 3, 5). Therefore, these findings indicate that treatment of Ca9-22 cells with butyrate or propionate induces ROS generation, which causes an increase in autophagy, leading to cell death and following release of DAMPs (Figs. 4, 5). The current is indicated by the blue characters with arrows in Fig. 5.

Gingival epithelial cells in patients with gingivitis are associated with mature dental plaque. In such situations, gingival cells compete with plaque bacteria for nutrients and experience energy deprivation. Therefore, a low FBS (1%) condition has been used instead of nutrient-insufficient conditions in this and previous studies. Low FBS conditions activate AMPK, which can turn on autophagy induction signals. Butyrate treatment in the presence of low FBS concentrations increase AMPK activity, resulting in enhanced autophagy and subsequent cell death [3]. ROS can enhance AMPK activation [13,14], and ROS production by Ca9-22 cells may further enhance AMPK activation and subsequent enhancement of autophagy when cells are treated with butyrate or propionate. Notably, findings in this study revealed that ROS are important for the induction of autophagy (Fig. 3) and cell death induced by butyrate or propionate (Fig. 2). In addition, cell death was dose-dependently inhibited in the presence of a compound C (dorsomorphin), an AMPK inhibitor [3]. Furthermore, Uemichi et al. showed that butyrate or propionate treatment increased histone H3 acetylation levels through their histone deacetylase (HDAC) inhibitory activity, and pretreatment of cells with an inhibitor of histone acetyltransferase (HAT) reduces histone H3 acetylation and butyrate- or propionate-induced cell death [6]. Increased histone acetylation levels of histones epigenetically control gene expression. Because histone lysine residues are cationic, negatively charged DNA is strongly associated with non-acetylated histones. This makes it difficult for the molecules required for transcription to bind to promoters and enhancers, leading to an inactive transcriptional status (closed chromatin). In contrast, since the acetylation of histones weakens their ability to bind to DNA, the transcriptional machinery can easily bind to the DNA regions required for gene expression. Therefore, butyrate- or propionate-induced histone acetylation drastically alters a wide range of gene expressions [6]. In addition, pre-inhibition of HAT activities by C646, which results in reduced histone acetylation levels, reverses the change (749 upregulated genes by butyrate treatment were restored) and reduces cell death induced by treatment with HDAC inhibitors, including butyrate and propionate [6]. Therefore, the butyrate- or propionate-upregulated genes that were also restored by HAT pre-inhibition were related to the induction of cell death. Since many genes related to autophagy and ROS were found among the 749 genes, these genes may be related to butyrate- or propionate-induced cell death [6].

Following butyrate- or propionate-induced cell death, DAMPs, such as HMGB1 and SAP130, were released into the extracellular milieu (Fig. 4). HMGB1, one of the best-known DAMPs, induces neutrophil migration, which is dependent on its concentration, and inflammatory cytokine production through its binding to TLR2, TLR4, and receptor for advanced glycation end products (RAGE), resulting in the activation of innate immunity [15,16,17]. Binding of SAP130 to C-type lectin receptors that are expressed on dendritic cell surfaces, which activate nuclear factor-κB (NF-κB) through RAF1and SYK pathways, also induces activation of innate immunity [17,18]. Therefore, DAMPs released following SCFA-induced cell death may play an important role in the onset of gingival and periodontal inflammation. This finding supports the importance of uncovering the mechanisms of SCFA-induced cell death to elucidate the mechanisms underlying oral bacteria-induced gingival inflammation.

Figure 5 illustrates the proposed mechanism of gingivitis. When the mature dental plaque comes into contact with the gingival epithelium, gingival epithelial cells are subjected to both nutrient deprivation and exposure to highly concentrated SCFAs. Low-nutrient conditions can activate the AMPK signaling pathway, which in turn activates the autophagy signaling pathway. High levels of SCFA generate ROS in the gingival epithelial cells, which can increase autophagy. In addition, SCFA-induced histone acetylation upregulates ROS and autophagy-related gene expression. This may potentiate excessive autophagy and induce gingival epithelial cell death associated with the release of DAMPs. The released DAMPs then bind to specific receptors and might induce gingival inflammation. In this scheme, scavenging ROS could be the simplest and best way to prevent gingivitis. This is because obtaining reducing agents that can scavenge ROS from safe drugs and food ingredients, such as NAC and ascorbic acid, is easier. These compounds may help maintain healthy periodontal tissues when added to toothpaste and mouthwashes.

Fig. 5 Schematic diagram of putative gingivitis onset mechanisms

Gingival epithelial cells adjacent to mature dental plaque must deal with both low nutrient availability and high levels of short-chain fatty acids (SCFAs) such as butyrate and propionate. AMP-activated protein kinase (AMPK) signaling is triggered by low-nutrient conditions and this activation leads to autophagy. High concentration of SCFAs elicit reactive oxygen species (ROS) production from gingival epithelial cells, which can also enhance autophagy. Moreover, histone acetylation by SCFA increased ROS and autophagy-related gene expression. These may cause excessive autophagy and lead to gingival epithelial cell death and subsequent damage-associated molecular patterns (DAMP) releases. Released DAMPs bind to their specific receptors and may trigger gingival inflammation. Findings in this study are indicated with a sequential word line in blue characters.

Abbreviations

AMPK: AMP-activated protein kinase; DAMP: damage-associated molecular pattern; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAT: histone acetyltransferase; HDAC: histone deacetylase; HMGB1: high-mobility group box Ⅰ; LC3B: microtubule-associated protein 1 light chain 3B; MEMα: minimum essential medium α; MINCLE: macrophage-inducible C-type lectin; NAC: N-acetylcysteine; RAGE: receptor for advanced glycation end products; ROS: reactive oxygen species; RPMI1640: Roswell Park Memorial Institute 1640; SAP130: sin3A-associated protein 130; SCFS: short-chain fatty acid; SYK: spleen tyrosine kinase; TLR: Toll-like receptor

Conflicts of Interest

All authors declare no potential conflicts of interest with respect to the research, authorship, and publication of the article.

Funding

This research was funded by KAKENHI (20K09913 and 23K09222) and the Sato Fund (H.T., 2023) of Nihon University School of Dentistry.

Author contributions

KM: investigation, data acquisition, and writing; YM: conceptualization and methodology, review; TA: data acquisition; TT: review; KS: review; MT: review; YY: review; HT: conceptualization, methodology, writing, review, editing, and supervision. All authors read and approved the final version of the manuscript.

ORCID iD

^1,2)KM: [e-mail]

³⁾YM: [e-mail], NA

^1,4)TA: [e-mail], NA

⁵⁾TT: [e-mail], https://orcid.org/0009-0004-0275-425X

²⁾KS: [e-mail], https://orcid.org/0000-0002-0291-5779

²⁾MT: [e-mail], NA

⁴⁾YY: [e-mail], https://orcid.org/0000-0002-3626-9023

^6,7)HT*: [e-mail], https://orcid.org/0000-0002-2047-2262

Acknowledgments

The authors would like to thank Editage (www.editage.com) for the English language editing.

Data Availability Statements

Data generated during the current study are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article.

References

• 1.  Pöllänen MT, Salonen JI, Uitto VJ (2003) Structure and function of the tooth-epithelial interface in health and disease. Periodontol 2000 31, 12-31. doi.org/10.1034/j.1600-0757.2003.03102.x
• 2.  Tsuda H, Ochiai K, Suzuki N, Otsuka K (2010) Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells. J Periodontal Res 45, 626-634. doi.org/10.1111/j.1600-0765.2010.01277.x
• 3.  Evans M, Murofushi T, Tsuda H, Mikami Y, Zhao N, Ochiai K et al. (2017) Combined effects of starvation and butyrate on autophagy-dependent gingival epithelial cell death. J Periodontal Res 52, 522-531. doi.org/10.1111/jre.12418
• 4.  Ebe N, Hara-Yokoyama M, Iwasaki K, Iseki S, Okuhara S, Podyma-Inoue KA et al. (2011) Pocket epithelium in the pathological setting for HMGB1 release. J Dent Res 90, 235-240. doi.org/10.1177/0022034510385688
• 5.  Castiglioni A, Canti V, Rovere-Querini P, Manfredi AA (2011) High-mobility group box 1 (HMGB1) as a master regulator of innate immunity. Cell Tissue Res 343, 189-199. doi.org/10.1007/s00441-010-1033-1
• 6.  Uemichi K, Mikami Y, Watanabe T, Shinozuka K, Tonogi M, Tsuda H (2023) Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death. Odontology 111, 658-667. doi.org/10.1007/s10266-022-00775-9
• 7.  Ohshima M, Noguchi Y, Ito M, Maeno M, Otsuka K (2001) Hepatocyte growth factor secreted by periodontal ligament and gingival fibroblasts is a major chemoattractant for gingival epithelial cells. J Periodontal Res 36, 377-383. doi.org/10.1034/j.1600-0765.2001.360605.x
• 8.  Dikic I, Elazar Z (2018) Mechanism and medical implications of mammalian autophagy. Nat Rev Mol Cell Biol 19, 349-364. doi.org/10.1038/s41580-018-0003-4
• 9.  Kanda Y (2013) Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant 48, 452-458. doi.org/10.1038/bmt.2012.244
• 10.  GuezGuez A, Prod'homme V, Mouska X, Baudot A, Blin-Wakkach C, Rottapel R et al. (2010) 3BP2 adapter protein is required for receptor activator of NFkappaB ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells. J Biol Chem 285, 20952-20963. doi.org/10.1074/jbc.m109.091124
• 11.  Andreev D, Liu M, Weidner D, Kachler K, Faas M, Gruneboom A et al. (2020) Osteocyte necrosis triggers osteoclast-mediated bone loss through macrophage-inducible C-type lectin. J Clin Invest 130, 4811-4830. doi.org/10.1172/jci134214
• 12.  Gong T, Liu L, Jiang W, Zhou R (2020) DAMP-sensing receptors in sterile inflammation and inflammatory diseases. Nat Rev Immunol 20, 95-112. doi.org/10.1038/s41577-019-0215-7
• 13.  Hinchy EC, Gruszczyk AV, Willows R, Navaratnam N, Hall AR, Bates G et al. (2018) Mitochondria-derived ROS activate AMP-activated protein kinase (AMPK) indirectly. J Biol Chem 293, 17208-17217. doi.org/10.1074/jbc.ra118.002579
• 14.  Marino A, Hausenloy DJ, Andreadou I, Horman S, Bertrand L, Beauloye C (2021) AMP-activated protein kinase: a remarkable contributor to preserve a healthy heart against ROS injury. Free Radic Biol Med 166, 238-254. doi.org/10.1016/j.freeradbiomed.2021.02.047
• 15.  Huang W, Tang Y, Li L (2010) HMGB1, a potent proinflammatory cytokine in sepsis. Cytokine 51, 119-126. doi.org/10.1016/j.cyto.2010.02.021
• 16.  Berthelot F, Fattoum L, Casulli S, Gozlan J, Marechal V, Elbim C (2012) The effect of HMGB1, a damage-associated molecular pattern molecule, on polymorphonuclear neutrophil migration depends on its concentration. J Innate Immun 4, 41-58. doi.org/10.1159/000328798
• 17.  Roh JS, Sohn DH (2018) Damage-associated molecular patterns in inflammatory diseases. Immune Netw 18, e27. doi.org/10.4110/in.2018.18.e27
• 18.  Geijtenbeek TB, Gringhuis SI (2009) Signalling through C-type lectin receptors: shaping immune responses. Nat Rev Immunol 9, 465-479. doi.org/10.1038/nri2569