Monoclonal antibody against Toll-like receptor 4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88- and NF-κB-dependent signaling

Abstract

The mechanisms through which mechanical ventilation causes non-infectious inflammatory diseases and lung injury are poorly understood. Animals models of this type of injury suggest that it involves signaling mediated by Toll‑like receptor (TLR)4 and 9. In this study, in order to gain further insight into the involvement of TLR4 in this type of injury, we performed in vivo and in vitro experiments to determine the mechanisms through which TLR4 triggers inflammation. We also examined whether the use of TLR4 monoclonal antibody (mAb) can alleviate this type of injury. For this purpose, rats were tracheotomized and administered intratracheal injections of anti‑TLR4 mAb or saline, and then ventilated for 4 h at a high tidal volume (HTV) of 40 ml/ kg or allowed to breathe spontaneously for the same period of time (controls). Alveolar macrophages (AMs) were isolated from the bronchoalveolar lavage fluid (BALF) of the rats and stimulated for 16 h with tumor necrosis factor (TNF)‑α in the presence or absence of anti‑TLR4 mAb. Lung injury was assessed by examining lung histopathology, lung wet/dry weight ratio, BALF total protein and cytokine levels in BALF and plasma. The mRNA and protein expression levels of TLR4, TLR9, myeloid differentiation factor 88 (Myd88) and nuclear factor (NF)‑κB were measured in cultured macrophages. Compared to the controls (spontaneous breathing), the ventilated rats exhibited greater pulmonary permeability, more severe inflammatory cell infiltration/lung edema, and higher levels of interleukin (IL)‑1β, IL‑6 and TNF‑α in BALF and plasma. The AMs from the ventilated rats expressed higher mRNA and protein levels of TLR4, TLR9, Myd88 and NF‑κB compared with the macrophages from the spontaneously breathing rats. The ventilated rats pre‑treated with anti‑TLR4 mAb exhibited markedly attenuated signs of ventilation‑induced injury, such as less lung inflammation and pulmonary edema, fewer cells in BALF, and lower levels of ILs and TNF‑α in BALF and plasma. Similarly, the TNF‑α‑dependent increases in the mRNA and protein expression of TLR4, Myd88 and NF‑κB in AMs were attenuated when TNF‑α was co‑administered with anti‑TLR4 mAb than when TNF-α was administered alone. Co‑administering anti-TLR4 mAb also reduced the TNF‑α‑dependent secretion of ILs. On the whole, our data demonstrate that TLR4 contributes significantly to ventilation‑induced lung injury by activating the Myd88/NF‑κB pathway, and pre‑treating rats with anti‑TLR4 mAb partially protects them against this type of injury by inhibiting Myd88/NF-κB signaling.

Effect of TLR4/MyD88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats, via regulation of macrophage activation and inflammatory response

“…Importantly, mAbs account for a large proportion of medicines in effective treatments for the patients with a variety of inflammatory diseases, and mAbs are created to inhibit TLR4 signaling pathway with a FcγR-binding mechanism ( 25 ). In line with our study, Huang et al also demonstrated that anti-TLR4 mAb attenuated the lung injury caused by mechanical ventilation, inflammation and edema in rats by inhibiting TLR4/MyD88 signaling pathway ( 15 ), but our study indicated that inhibition of TLR4/MyD88 signaling pathway may relieve sepsis-associated ARDS in rats through regulating macrophage activation and inflammatory response. Thus, the inhibition of TLR4/MyD88 signaling pathway might contribute to less lung injury, inflammation and edema in rats with sepsis-associated ARDS.…”

“…Castoldi et al provided evidence that the TLR4/MyD88 signaling pathway was crucial to acute kidney injury (AKI) induced by sepsis ( 14 ). The study of Huang et al exerted efforts to identify the role of monoclonal antibody against TLR4 in ventilator-induced lung injury in rats through MyD88/NF-κB signaling ( 15 ), which provides some clues for further studies in ARDS. Additionally, TLR4 is one of the key receptors associated with entire body and low-level chronic inflammatory diseases, leading to the macrophage infiltration in diabetic liver injury, through activating NF-κB and regulating pro-inflammatory genes ( 16 ).…”

High Tidal Volume Induces Mitochondria Damage and Releases Mitochondrial DNA to Aggravate the Ventilator-Induced Lung Injury

“…The animal model was established successfully based on the previous study ( 7 , 17 ). Briefly, rats were anesthetized by intraperitoneal injection of 60 mg/kg pentobarbital sodium and 80 mg/kg ketamine, and the rats were given with 15 mg/kg pentobarbital sodium every 30 min and 2 mg/kg/h pancuronium for muscle relaxation.…”

“…Alveolar macrophages were isolated and cultured according to the previous method ( 17 ). The collected BALF was centrifuged at 1,000 × g for 10 min and washed three times with pathogen-free PBS.…”

Transduced PEP-1-Heme Oxygenase-1 Fusion Protein Attenuates Lung Injury in Septic Shock Rats

“…The wide variety of genes regulated by NF- κ B includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes [ 19 ]. Accumulating evidence supports the role of the TLR4/NF- κ B pathway in lung injury caused by various factors, including ventilators [ 20 , 21 ], lipopolysaccharides (LPS) [ 22 ], and intestinal ischemia/reperfusion [ 23 ].…”