Circulating histone H4 increased in Cl2 induced ARDS in mice
Different concentrations of Cl2 (10, 20, 50, 100, 200, 400, 600, 800 ppm) were used to induce ARDS in mice (n = 12, exposure time 30 min). The effect of Cl2 on the severity of acute lung injury was concentration dependent. Cl2 in low concentrations (≤ 100 ppm) merely caused brief tachypnea, and the mice (12/12) all survived. When the concentration increased to 200 ppm, Cl2 caused dyspnea and eight mice (8/12) survived for 72 h. The concentration of 400 ppm caused evident dyspnea, and five mice (5/12) survived for 72 h. The concentration of 600 ppm caused serious dyspnea and only three mice (3/12) survived for 72 h. Almost, all mice (11/12) died in 72 h under the concentration of 800 ppm. Pulmonary interstitial edema, inflammatory cells infiltration, hemorrhage, atelectasis, microthrombus formation, and epithelium necrosis were obvious. The pathological changes in lung tissue 24 h after Cl2 exposure were shown in Figure S1 (supplemental data).
The circulating histone H4 was very low in normal state. After Cl2 exposure, circulating histone H4 increased, particularly when the concentration of Cl2 reached to 400 ppm. As shown in Fig. 1A, there was a significant positive correlation between the concentrations of Cl2 (from 10 to 800 ppm) and histone H4 in plasma (r = 0.8017, p = 0.0289). As shown in Fig. 1B, the change of histone H4 in plasma was proved by means of Western blot.
Effect Of Histone H4 And Tlrs On Survival Rate
In order to study the role of histone H4, mice were injected via tail vein with histone H4 or anti-H4 antibody 30 min prior to Cl2 exposure. As shown in Fig. 2A, nine mice (9/12) died within 72 h after Cl2 exposure of lethal concentration (600 ppm, 30 min). Pretreated with intravenous histone H4 (10 mg/kg), nearly all mice (11/12) died within 72 h after Cl2 exposure. Instead, only three mice (3/12) died when pretreated with intravenous anti-H4 antibody (20 mg/kg). The lethality rate between the mice merely challenged with Cl2 exposure and those pretreated with anti-H4 antibody was statistically different (p = 0.0223).
Tlr2-KO and Tlr4-KO mice were used to investigate the role TLRs. As shown in Fig. 2B, five WT mice (5/6) died within 72 h after Cl2 exposure of lethal concentration (600 ppm, 30 min). Compared with the WT mice, four Tlr2-KO mice (4/6) and only one Tlr4-KO mice (1/6) died within 72 h after Cl2 exposure. The lethality rate between the WT mice and the Tlr4-KO mice was statistically different (p = 0.0201). Thus, the Tlr2-KO and Tlr4-KO mice were resistant to Cl2 exposure of lethal concentration to some extent.
Role of histone H4 and TLRs in Cl
2 induced acute lung injury
As shown in Fig. 3A, Cl2 exposure caused obvious hypoxemia in the WT mice. Pretreated with intravenous histone H4, the PaO2 decreased much more seriously. On the contrary, the values of PaO2 were improved when mice were pretreated with intravenous anti-H4 antibody. Compared with the WT mice, the PaO2 only decreased slightly 24 h after Cl2 exposure in the Tlr4-KO mice. More importantly, the effect of histone H4 or anti-H4 antibody observed in the WT mice was diminished greatly in the Tlr4-KO mice. Different from the Tlr4-KO mice, the change of PaO2 and the effect of histone H4 or anti-H4 antibody in the Tlr2-KO mice were similar with those in the WT mice.
As shown in Fig. 3B, the pulmonary edema was serious after Cl2 exposure in the WT mice. Pretreatment with histone H4 further increased the lung wet/dry mass ratio. On the other side, pretreatment with anti-H4 antibody attenuated the degree of pulmonary edema. Compared with the WT mice, the gene deletion of Tlr4 provided obvious protection against challenge of Cl2 exposure and intravenous histone H4, while Tlr2 deficiency only showed slightly protective effect.
Cl2 exposure caused evident endothelial activation which was shown as P-selectin translocation and release of vWF from endothelial Weibel-Palade bodies (WPBs) (Fig. 3C, 3D). Pretreatment with intravenous histone H4 aggravated P-selectin translocation and release of vWF further. However, pretreatment with the anti-H4 antibody produced some protective effects. To a great extent, the deficiency of Tlr4 prevented the endothelial activation, while the impact of Tlr2 deficiency was not significant.
Pulmonary neutrophil infiltration and activation were prominent after Cl2 exposure, which were measured by neutrophil specific Ly6G marker and MPO activity (Fig. 3E, 3F). Pretreatment with intravenous histone H4 increased neutrophil infiltration and MPO activity further while pretreatment with the anti-H4 antibody showed some antagonistic effects. In comparison with the WT mice, the Tlr4-KO mice but not Tlr2-KO mice were shown to be resistant to challenge of Cl2 exposure and intravenous histone H4.
TLRs involved in histone H4 mediated endothelial inflammation in vitro
Blocking antibodies against TLR1, TLR2, TLR4, and TLR6 were used to investigate the role of TLRs in histone H4 mediated endothelial inflammation. Histone H4 (10 mg/L) treatment triggered the expression of inflammatory cytokines in the pulmonary vascular endothelial cells, which included TNF-α and IL-1β. As shown in Fig. 4A, a blocking antibody against TLR4 distinctly reduced the transcription of TNF-α (38% decrease versus H4 group, p < 0.05). Additionally, a blocking antibody against TLR2 slightly reduced the transcription (p > 0.05). However, the blocking antibody against TLR1 or TLR6 showed little effect. The effect of the blocking antibody against TLR1, TLR2, TLR4 or TLR6 on the protein expression of TNF-α was similar with the transcription (Fig. 4C).
As shown in Fig. 4B, the blocking antibody against TLR4 obviously reduced the transcription of IL-1β (41% decrease versus H4 group, p < 0.05), while the effect of the blocking antibody against TLR2 on the transcription was much weaker (p > 0.05). The blocking antibody against TLR1 or TLR6 nearly did not affect the transcription. The effect of the blocking antibodies on IL-1β was also validated by protein expression (Fig. 4D).