Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice

Abstract

Sterile inflammatory insults are known to activate innate immunity and propagate organ damage through the recognition of extracellular damage-associated molecular pattern (DAMP) molecules. Although DAMPs such as endogenous DNA and nuclear high-mobility group box 1 have been shown to be critical in sterile inflammation, the role of nuclear histone proteins has not yet been investigated. We report that endogenous histones function as DAMPs after ischemic injury through the pattern recognition receptor Toll-like receptor (TLR) 9 to initiate inflammation. Using an in vivo model of hepatic ischemia/reperfusion (I/R) injury, we show that levels of circulating histones are significantly higher after I/R, and that histone neutralization significantly protects against injury. Injection of exogenous histones exacerbates I/R injury through cytotoxic effects mediated by TLR9 and MyD88. In addition, histone administration increases TLR9 activation, whereas neither TLR9 nor MyD88 mutant mice respond to exogenous histones. Furthermore, we demonstrate in vitro that extracellular histones enhance DNA-mediated TLR9 activation in immune cells through a direct interaction.

Conclusion: These novel findings reveal that histones represent a new class of DAMP molecules and serve as a crucial link between initial damage and activation of innate immunity during sterile inflammation.

Fig. 1

Pretreatment with neutralizing antibodies to histones protects against liver I/R injury. (A) Sham or I/R-treated mice were given anti-histone H3 or anti-histone H4 antibody (20 mg/kg body weight) or control antibody intravenously 30 minutes before ischemia. Data represent the mean ± SE (n = 6 mice per group). *P < 0.05. (B) Quantification of necrotic hepatocytes in hematoxylin and eosin–stained liver sections from control and anti-histone antibody-treated animals 6 hours after reperfusion. The graph is representative of liver sections from six mice per group. (C) Hepatic TNF-α and IL-6 mRNA expression after 6 hours of I/R. Results are expressed as relative increase of mRNA expression compared with sham-treated animals. Data represent the mean ± SE (n = 6 mice per group). *P < 0.05.

Fig. 2

Treatment of exogenous histones mixture exacerbate liver I/R injury. (A) Sham-treated mice and mice that underwent ischemia and 1, 3, and 6 hours of reperfusion were treated with a nonlethal dose of exogenous histone mixture (25 mg/kg body weight) or vehicle PBS immediately after ischemia. sALT levels were analyzed. Data represent the mean ± SE (n = 12 mice per group). *P < 0.05. **P < 0.01. (B) Quantification of necrotic hepatocytes in hematoxylin and eosin–stained liver tissue. The graph is representative of liver sections from six mice per group. (C) Hepatic TNF-α and IL-6 mRNA. Data represent the mean ± SE (n = 6 mice per group). *P < 0.05.

Fig. 3

Extracellular histones are released from hepatocytes after hypoxia in vitro and from the liver in vivo after hepatic I/R. (A) Systemic histones levels were assessed by way of serum enzyme-linked immunosorbent assay. Data represent the mean ± SE (n = 6 mice per group). *P < 0.05. (B) Immunofluorescent stain of histone H3 from sections of normal liver and (C) and liver 6 hours I/R (original magnification ×400). Images are representative liver sections from six mice per group. Red, histone H3; blue, nuclei; green, F-actin. (D) Cultured mouse hepatocytes were exposed to hypoxia (1% O₂) from 0 to 48 hours. Media were subjected to western blot analysis of histone H3 and H4. The blots shown are representative of three experiments with similar results. (E) Immunofluorescent stain of histone H3 and H4 from cultured mouse hepatocytes were exposed to hypoxia (1% O₂) overnight (original magnification ×600). Images are representative of three experiments with similar results. Green, histone H3 or H4; blue, nuclei; red, F-actin.

Fig. 4

Extracellular histones modulate inflammatory signaling pathways. (A) Mitogen-activated protein kinase activation was determined in sham-treated mice and mice that underwent ischemia and 1 hour of reperfusion. Animals were treated with exogenous histones mixture or vehicle PBS. (B) Mitogen-activated protein kinase activation was determined. Hepatic protein lysates from ischemic lobes were obtained; each lane represents a separate animal. The blots shown are representative of three experiments with similar results. (C) Phosphorylation at serine 536 of the p65 subunit of NF-κB after 1 hour of I/R.

Fig. 5

Extracellular histones mediate hepatic I/R injury through TLR9. (A) Serum ALT levels in TLR9 mutant and WT mice after I/R with either anti-histone antibodies or exogenous histone administration. Data represent the mean ± SE (n = 4-6 mice per group). *P < 0.05. (B) Hematoxylin and eosin–stained liver sections (original magnification ×100). Images are representative liver sections from six mice per group. The dashed line indicates the necrotic area. (C) Hepatic TNF-α and IL-6 mRNA in TLR9 mutant and WT mice after histone administration. (D) Hepatic TNF-α and IL-6 mRNA expression after histone neutralization. Results are expressed as the relative increase of mRNA expression compared with sham-treated animals. Data represent the mean ± SE (n = 4-6 mice per group). *P < 0.05. N.S., not significant.

Fig. 6

Extracellular histones-mediated hepatic I/R injury involves TLR9 signaling cascade. (A) Serum ALT levels in MyD88 KO and WT mice after 6 hours of I/R with or without histone administration. Data represent the mean ± SE (n = 4-6 mice per group). *P < 0.05. N.S., not significant. (B) Hematoxylin and eosin–stained liver sections (magnification ×100). Images are representative liver sections from six mice per group. The dashed line indicates the necrotic area. (C) sALT levels in ODN2088-treated mice after 6 hours of I/R. Data represent the mean ± SE (n = 4-6 mice per group). *P < 0.05. N.S., not significant. (D) Hematoxylin and eosin–stained liver sections from ODN2088-treated mice (magnification ×100). Images are representative liver sections from six mice per group. The dashed line indicates that necrotic area. (E) TLR9 activation by western blot analysis. The blots shown are representative of three experiments with similar results. (F) Coimmunopreciptation of histone H4 and cleaved TLR9 from liver cell lysated.

Fig. 7

Extracellular histones enhances the nucleic acid–mediated damage after hepatic I/R. (A) IL-6 mRNA expression was obtained in NPCs cocultured overnight with media from necrotic hepatocytes. NPCs were treated with vehicle PBS, Dnase I, or anti-histone H4 antibody. Results are expressed as the relative increase of mRNA expression compared with PBS treatment. Data represent the mean ± SE and are representative of three experiments with similar results. *P < 0.05. (B) IL-6 mRNA expression was observed in NPCs that were treated with vehicle PBS, CpG, exogenous histones, or both. Results are expressed as relative increase of mRNA expression compared with PBS treatment. Data represent the mean ± SE and are representative of three experiments with similar results. *P < 0.05.

Fig. 8

Hepatic I/R induces release of extracellular histones from hepatocytes. The model shows the release of extracellular histones after hepatic I/R.