Toll-like receptor 4 and high-mobility group box 1 are critical mediators of tissue injury and survival in a mouse model for heatstroke

Abstract

The molecular mechanisms that initiate the inflammatory response in heatstroke and their relation with tissue injury and lethality are not fully elucidated. We examined whether endogenous ligands released by damaged/stressed cells such as high-mobility group box 1 (HMGB1) signaling through Toll-like receptor 4 (TLR4) may play a pathogenic role in heatstroke. Mutant TLR4-defective (C3H/HeJ) and wild type (C3H/HeOuJ) mice were subjected to heat stress in an environmental chamber pre-warmed at 43.5 °C until their core temperature reached 42.7°C, which was taken as the onset of heatstroke. The animals were then allowed to recover passively at ambient temperature. A sham-heated group served as a control. Mutant mice displayed more histological liver damage and higher mortality compared with wild type mice (73% vs. 27%, respectively, P<0.001). Compared to wild type mice, mutant mice exhibited earlier plasma release of markers of systemic inflammation such as HMGB1 (206 ± 105 vs. 63 ± 21 ng/ml; P = 0.0018 and 209 ± 100 vs. 46 ± 32 ng/ml; P<0.0001), IL-6 (144 ± 40 vs. 46 ± 20 pg/ml; P<0.001 and 184 ± 21 vs. 84 ± 54 pg/ml; P = 0.04), and IL-1β (27 ± 4 vs. 1.7 ± 2.3 pg/ml; P<0.0001 at 1 hour). Both strains of mice displayed early release of HMGB1 into the circulation upstream of IL-1β and IL-6 responses which remained elevated up to 24 h. Specific inhibition of HMGB1 activity with DNA-binding A Box (600 µg/mouse) protected the mutant mice against the lethal effect of heat stress (60% A Box vs. 18% GST protein, P = 0.04). These findings suggest a protective role for the TLR4 in the host response to severe heat stress. They also suggest that HMGB1 is an early mediator of inflammation, tissue injury and lethality in heatstroke in the presence of defective TLR4 signaling.

Figure 1. Core body temperature response of mutant and wild mice type to environmental heat stress.

Mice implanted intraperitoneally with radiotelemetric transmitters (Data Science International were allowed to recover for 2 weeks after surgery before subjected to heat stress or sham heat stress. (A) Core body temperature recorded in mutant and wild type mice at baseline (BL), and during sham heat exposure. (B) Core body temperature recorded in mutant and wild type mice at baseline, during cooling and recovery after heatstroke. Values represent median and IQR. *P<0.05 at T0+4 hours between the two groups tested by the generalized estimating equations model.

Figure 2. Comparison of heatstroke-induced lethality in mutant and wild type mice.

Survival rates (%) of wild type and mutant mice after heatstroke are shown over 72 hours from the onset of heatstroke. Heatstroke was induced by passive exposure to environmental heat until the core temperature reached 42.7°C. **P<0.001 between the two groups analyzed by the Kaplan-Meier log-rank test.

Figure 3. Histopathological comparison of heatstroke-induced liver injury in mutant and wild type mice.

Heatstroke was induced in mice by passive exposure to environmental heat until the core temperature reached 42.7°C. Sham-heated mice were used as control. Liver sections from mutant (A–D) and wild type (E–H) mice either sham-heated (A and E) or after induction of heatstroke (B–D and F–H, respectively) were stained with Hematoxylin & Eosin. In both mice strains, no morbid changes were observed after sham-heat stress (A and E). Heatstroke-induced in mutant mice resulted in hepato-cellular necrosis (arrows) and inflammatory cell infiltration (broken arrow) at onset (B), and massive perilobular necrosis with severe sinusoidal congestion (*) were observed at follow-up (C, D) time points. Minimal histopathological changes were noted in wild type subjected to heatstroke (F–H).

Figure 4. Comparison of plasma IL-6, IL-1β and HMGB1 levels in mutant and wild type mice.

(A) Plasma levels (mean ± SD) of IL-6, (B) IL-1β and (C) HMGB1 are compared with mutant and wild type mice at the onset of heatstroke (T0) and at T0+0.5, +1, +4 and +24 hours post heatstroke. Plasma levels from sham-heated animals’ represent baseline. *P<0.05; **P<0.001; and ***P<0.0001 statistical significance at given time points between the two groups tested by the generalized estimating equations model.

Figure 5. Inhibition of HMGB1 activity protects mutant mice against the lethal effects of heatstroke.

(A) SDS-PAGE analysis of the recombinant A-Box and GST control proteins expressed and purified from E. coli is shown. Increasing amounts of purified proteins were resolved by SDS-PAGE and stained with Coomassie blue; (B) Mutant mice were pretreated (i.p. injection) with A Box 600 µg/mouse; or GST control and heatstroke was induced by passive exposure to environmental heat until the core temperature reached 42.7°C. Pretreatment with A Box before heat stress protected the mutant mice against the lethal effects of heatstroke. *P = 0.04 Kaplan-Meier log-rank testing between the two groups.