Toll-like receptor 4 mediates acute lung injury induced by high mobility group box-1

Abstract

Background: Acute lung injury (ALI) is considered to be the major cause of respiratory failure in critically ill patients. Clinical studies have found that in patients with sepsis and after hemorrhage, the elevated level of high mobility group box-1(HMGB-1) in their circulation is highly associated with ALI, but the underlying mechanism remains unclear. Extracellular HMGB-1 has cytokine-like properties and can bind to Toll-like Receptor-4 (TLR4), which was reported to play an important role in the pathogenesis of ALI. The aim of this study was to determine whether HMGB-1 directly contributes to ALI and whether TLR4 signaling pathway is involved in this process.

Methods: Recombinant human HMGB-1 (rhHMGB-1) was used to induce ALI in male Sprague-Dawley rats. Lung specimens were collected 2 h after HMGB-1 treatment. The levels of TNF-α, IL-1β, TLR4 protein, and TLR4 mRNA in lungs as well as pathological changes of lung tissue were assessed. In cell studies, the alveolar macrophage cell line, NR8383, was collected 24 h after rhHMGB-1 treatment and the levels of TNF-α and IL-1β in cultured medium as well as TLR4 protein and mRNA levels in the cell were examined. TLR4-shRNA-lentivirus was used to inhibit TLR4 expression, and a neutralizing anti-HMGB1 antibody was used to neutralize rhHMGB-1 both in vitro and in vivo.

Results: Features of lung injury and significant elevation of IL-1β and TNF-α levels were found in lungs of rhHMGB-1-treated animals. Cultured NR8383 cells were activated by rhHMGB-1 treatment and resulted in the release of IL-1β and TNF-α. TLR4 expression was greatly up-regulated by rhHMGB-1. Inhibition of TLR4 or neutralization of HMGB1 with a specific antibody also attenuated the inflammatory response induced by HMGB-1 both in vivo and in vitro.

Conclusion: HMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce ALI through a mechanism that relies on TLR-4.

Figure 1. Lung inflammatory response after HMGB-1 treatment.

Normal lung architecture was observed in the control group and 0 µg HMGB-1 group (A, B). Intense interstitial accumulation of neutrophils and edema was observed in 50 µg and 100 µg HMGB-1 groups (C, D). Rats exposed to 0 µg HMGB1 showed low lung histological scores. Intratracheal exposure of 50 µg and 100 µg of HMGB1 increased lung histological scores significantly (E). IL-1β and TNF-α levels in lungs were evaluated by ELISA. Compared with the control group, inflammatory mediators in the 0 µg HMGB-1 group were not significantly different, and marked increases were observed in 50 µg and 100 µg HMGB-1 groups (F, G). The increases in the 100 µg HMGB-1 group were higher than those in the 50 µg HMGB-1 group (F, G). Data are shown as the mean± SD, n = 6, *P<0.05, **P<0.01,scale bar 50 µm, original magnification 200×.

Figure 2. IL-1β and TNF-α levels in culture media change after HMGB-1 stimulation in NR8383.

NR8383 cells were stimulated with rhHMGB-1at different concentrations and then the IL-1β and TNF-α levels in the culture media were evaluated by ELISA assay at 24 h post-treatment. After treatment, the IL-1β levels increased slightly in the 10 ng/ml HMGB-1 group. In the 50 and 100 ng/ml HMGB-1 groups, the levels of IL-1β increased dramatically (A). Compared with the control group, TNF-α levels did not change in the 10 ng/ml HMGB-1 group, but clearly increased in the 50 and 100 ng/ml HMGB-1 groups (B). Data are shown as the mean±SD, n = 6, *P<0.05, **P<0.01.

Figure 3. Expression of TLR4 after HMGB-1 treatment in vitro and in vivo.

The expression of TLR4 was assessed by western blot analysis as well as real-time quantitative PCR analysis. For the in vitro studies, the expression of TLR4 protein and TLR4 mRNA levels were elevated after HMGB-1 stimulation, and every group displayed significant elevation (A, C). For the in vivo studies, the TLR4 protein and TLR4 mRNA levels in the 0 µg HMGB-1 treated group were comparatively equal to that of control. In addition, after HMGB-1 stimulation, the protein and mRNA levels were both clearly increased in a dose-dependent manner (B, D). Data are shown as the mean±SD, n = 6, **P<0.01.

Figure 4. The effect of TLR4 inhibition on HMGB-1-induced ALI.

RNAi was used to inhibit the expression of TLR4, and the animals received 100 µg of rhHMGB-1. Lung histological observation was performed after transfection and 24 h after HMGB-1 treatment. The negative and shTLR4 groups displayed normal lung structure similar to the control (A, B, and D) Stronger ALI effects were seen in the HMGB-1 group (C), but weaker ALI effects were observed in the shTLR4+HMGB-1 group (F).The anti-HMGB-1 and HMGB-1+anti-HMGB-1 groups also showed normal lung structure(E, G). Lung histopathological scores showed the degree of interstitial accumulation of neutrophils and edema: the HMGB-1 group was the highest, and the shTLR4+HMGB-1 was clearly lower than the shNT+HMGB-1 group, but still higher than the other three groups (H). The levels of TNF-α and IL-1β in lung were analyzed by ELISA after transfection and 24 h after HMGB-1 administration. The levels of IL-1β in lung were not significantly different between the control, negative, shTLR4, and anti-HMGB-1 groups. After treatment with HMGB-1, the levels of IL-1β and TNF-α increased dramatically in the HMGB-1 group and increased slightly in the shTLR4+HMGB-1 group, but did not change in the HMGB-1+anti-HMGB-1 group (J, K). TLR4 protein expression was measured by western blot analysis and TLR4 mRNA expression was measured by real-time quantitative PCR after transfection and 24 h after HMGB-1 administration. The TLR4 protein and mRNA levels were very low in the shTLR4 group, but dramatically increased in the HMGB-1 group. The levels of TLR4 expression in the other five groups were equal to each other (I, L). Data are shown as the mean±standard deviation (SD), n = 6, *P<0.05, **P<0.01 vs. control, ++ P<0.01 vs. HMGB-1.

Figure 5. The effect of TLR4 inhibition on the inflammatory response by HMGB-1-treated NR8383 cells.

RNAi was used to inhibit TLR4 expression in NR8383 cells. The levels of IL-1β and TNF-α in the culture supernatants were determined by ELISA after transfection and 24 h after HMGB-1 administration. The levels of IL-1β and TNF-α in negative, shTLR4, and anti-HMGB-1 groups were comparable to the control group. After treatment with HMGB-1, the IL-1β and TNF-α concentrations in the HMGB-1 group were higher than those in the shTLR4+HMGB-1 group, while there were no changes in these concentrations in the HMGB-1+anti-HMGB-1 group (A, B). The expression of TLR4 was assessed by western lot and real-time quantitative PCR after transfection and 24 h after HMGB-1 administration. After transfection, The TLR4 protein and mRNA levels were very low in the shTLR4 group, decreasing by approximately 60%. After treatment with HMGB-1, the levels of TLR4 protein and mRNA in the shTLR4+HMGB-1 group returned to the levels of the control. In addition, the levels of TLR4 protein and mRNA remained the same in the anti-HMGB-1 and HMGB-1+anti-HMGB-1 groups (C, D). Data are shown as the mean±SD, n = 6, **P<0.01 vs. control, ++ P<0.01 vs. shNT+HMGB-1.