ATF3 deficiency promotes alveolar macrophage pyroptosis in sepsis-induced acute lung injury.

Acute lung injury (ALI) ranks among the leading reasons for death in septic patients. As an essential transcription factor associated with stress, activating transcription factor 3 (ATF3) participates in a variety of pathophysiological processes, including immunology and inflammation. However, the specific mechanism of ATF3 in pyroptosis of sepsis-induced ALI remains elusive. A mouse model of ALI was established by administering lipopolysaccharide (LPS) in vivo and LPS combined with adenosine triphosphate (ATP) in vitro to compare differences in ATF3 expression level. The role of ATF3 in pyroptosis was then assessed by knocking down ATF3 using small interfering RNA. The levels of interleukin-6 (IL-6), tumor necrosis factor-α, IL-1β, and IL-18 in mouse serum and cell culture supernatants were measured using enzyme-linked immunosorbent assay. Moreover, immunohistochemistry, immunofluorescence, Western blotting, co-immunoprecipitation, and quantitative reverse transcription polymerase chain reaction were employed for examining pyroptosis and pyroptotic pathways. Both in vitro and in vivo ALI models were successfully established. LPS could activate the pyroptotic signaling pathway, and the expression level of ATF3 peaked 6 h after LPS and ATP stimulation in vitro. ATF3 could interact with NLRP3 and potentially influence the assembly of the inflammasome. This mechanism could involve the inhibition of the classical pyroptotic pathway, including Caspase-1 and Gasdermin D transcription and cleavage, as well as the inhibition of the non-classical pyroptotic pathway, including transcription and cleavage of Caspase-11. The results indicated that inhibition of ATF3 could exacerbate sepsis-induced ALI by regulating pyroptotic pathways. The potential of targeting ATF3 as a future treatment strategy for ALI is noteworthy.

Li D ，Ma H ，Dong X ，Fang Z ，Zhang C ，Song Q ，Wang J ，Wan X ... -  《-》

Inhibition of IRE1α/XBP1 axis alleviates LPS-induced acute lung injury by suppressing TXNIP/NLRP3 inflammasome activation and ERK/p65 signaling pathway.

Acute lung injury or acute respiratory distress syndrome (ALI/ARDS) is a devastating clinical syndrome with high incidence and mortality rates. IRE1α-XBP1 pathway is one of the three major signaling axes of endoplasmic reticulum stress that is involved in inflammation, metabolism, and immunity. The role and potential mechanisms of IRE1α-XBP1 axis in ALI/ARDS has not well understood. The ALI murine model was established by intratracheal administration of lipopolysaccharide (LPS). Hematoxylin and eosin (H&E) staining and analysis of bronchoalveolar lavage fluid (BALF) were used to evaluate degree of lung injury. Inflammatory responses were assessed by ELISA and RT-PCR. Apoptosis was evaluated using TUNEL staining and western blot. Moreover, western blot, immunohistochemistry, and immunofluorescence were applied to test expression of IRE1α, XBP1, NLRP3, TXNIP, IL-1β, ERK1/2 and NF-κB p65. The expression of IRE1α significantly increased after 24 h of LPS treatment. Inhibition of the IRE1α-XBP1 axis with 4µ8C notably improved LPS-induced lung injury and inflammatory infiltration, reduced the levels of IL-6, IL-1β, and TNF-α, and decreased cell apoptosis as well as the activation of the NLRP3 inflammasome. Besides, in LPS-stimulated Beas-2B cells, both 4µ8C and knockdown of XBP1 diminished the mRNA levels of IL-6 and IL-1B, inhibited cell apoptosis and reduced the protein levels of TXNIP, NLRP3 and secreted IL-1β. Mechanically, the phosphorylation and nuclear translocation of ERK1/2 and p65 were significantly suppressed by 4µ8C and XBP1 knockdown. In summary, our findings suggest that IRE1α-XBP1 axis is crucial in the pathogenesis of ALI/ARDS, whose suppression could mitigate the pulmonary inflammatory response and cell apoptosis in ALI through the TXNIP/NLRP3 inflammasome and ERK/p65 signaling pathway. Our study may provide new evidence that IRE1α-XBP1 may be a promising therapeutic target for ALI/ARDS.

Wang S ，Hu L ，Fu Y ，Xu F ，Shen Y ，Liu H ，Zhu L ... -  《RESPIRATORY RESEARCH》

Mitoxantrone attenuates lipopolysaccharide-induced acute lung injury via inhibition of NEDD8 activating enzyme.

Lipopolysaccharide (LPS) triggers the activation of nuclear factor kappa B (NF-κB) by interacting with Toll-like receptor 4 (TLR4), leading to the production of various proinflammatory enzymes and cytokines that are crucial in the development of acute lung injury (ALI). Mitoxantrone (MTX) has been demonstrated to mitigate the inflammatory response caused by LPS; however, its precise function in the context of ALI is not fully comprehended. This study aimed to investigate the inhibitory effects and underlying mechanisms of MTX against LPS-induced ALI. ALI was induced in C57BL/6 mice via a single intratracheal administration of LPS (5 mg/kg), followed by an intraperitoneal injection of MTX to evaluate its therapeutic potential. The effects of MTX on lung injury and the progression of inflammation in ALI mice were assessed using a comprehensive range of techniques, including hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), myeloperoxidase activity measurement, cell enumeration in bronchoalveolar lavage fluid (BALF), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Additionally, IHC, Western blotting, and co-immunoprecipitation were used to elucidate the specific signaling pathways and molecular mechanisms by which MTX exerted its anti-inflammatory effects in ALI mice. Surface plasmon resonance (SPR) and molecular docking were used to examine the target to which MTX binds directly to reduce inflammation. We also established a lung epithelial cell injury model using LPS-treated A549 cells. The polyubiquitination of IκBα and TRAF6 in LPS-induced A549 cells was detected through Western blotting following immunoprecipitation. In mice with LPS-induced ALI, MTX exhibits anti-inflammatory effects by ameliorating histopathological abnormalities caused by LPS, reducing inflammatory cell infiltration, and decreasing the production of proinflammatory enzymes and cytokines. It has been observed that MTX directly binds to the NEDD8 activating enzyme (NAE), thereby inhibiting the transfer of NEDD8 to the substrates UBC12, Cul1, and Cul5. Consequently, the polyubiquitination of IκBα and TRAF6 is disrupted, leading to the suppression of TAK1 activation by TRAF6. This suppression of TAK1 activity hindered the phosphorylation of IKK and MAPK. By stabilizing IκBα through dephosphorylation via IKK inhibition and preventing polyubiquitination, NF-κB activation is reduced. This cascade of events ultimately leads to a reduction in the production of proinflammatory enzymes and cytokines, effectively mitigating the inflammatory response in ALI. In A549 cells, MTX reduces the LPS-induced K48-linked polyubiquitination of IκBα and K63-linked polyubiquitination of TRAF6. This process can be reversed by the overexpression of NEDD8. Additionally, treatment with MG-132, a proteasome inhibitor, can restore the polyubiquitination of IκBα that was inhibited by MTX. These findings confirm the essential role of Cul1/5 neddylation in ALI and suggest that MTX could be a promising therapeutic agent for ALI.

Liu H ，Liu Y ，Lin X ，Fan J ，Huang Z ，Li A ... -  《-》

Eclipta prostrata improves alveolar development of bronchopulmonary dysplasia via suppressing the NLRP3 inflammasome in a DLD-dependent manner.

Bronchopulmonary dysplasia (BPD), the most common late morbidity in preterm infants, is characterized by impaired alveolar development caused by persistent lung inflammation. Studies have shown that NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome-mediated inflammation is critically involved in the development of BPD. As a traditional Chinese medicinal herb, Eclipta prostrata (EAP) exhibits potent anti-inflammatory properties. Our study aims to investigate whether EAP could improve the lung development of BPD by suppressing the lung inflammatory response. The BPD rat model was established by intra-amniotic injection of lipopolysaccharide (LPS) and postnatal exposure to hyperoxia. Changes in the NLRP3 inflammasome and pyroptosis were assessed by treatment with EAP. The effect of EAP on the NLRP3 inflammasome was tested in vitro using the THP-1 cell line and primary alveolar macrophages. Proteomics analysis was used to elucidate the mechanism of action of EAP. Histopathological and immunofluorescence results of lung tissues revealed that LPS and hyperoxia induced lung injury and triggered NLRP3 inflammasome activation and pyroptosis in alveolar macrophages. EAP ameliorated BPD lung injury, inhibited NLRP3 inflammasome activation and reduced gasdermin D (GSDMD) expression in alveolar macrophages. EAP downregulated the expression of NLRP3 inflammasome pathway molecules (NLRP3, caspase-1, and IL-1β) and GSDMD in LPS-stimulated THP-1 macrophages and primary alveolar macrophages. In addition, proteomics analysis identified that dihydrolipoamide dehydrogenase (DLD) interacted with EAP. Inhibition of DLD activity abolished the protective effects of EAP. Our study suggested that EAP could attenuate arrest of alveolar development via inhibiting NLRP3 inflammasome in a DLD-dependent way, and could be a potential therapeutic method for BPD.

Zheng X ，Tan Z ，Zhu D ，Zhao D ，Liu C ，Wang S ，Wang X ，Zhang Y ... -  《-》

Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

The role of the JAK2-STAT1/PD-L1 pathway in the phagocytic activity of alveolar macrophages (AMs) during LPS-induced acute lung injury in mice remains poorly understood. This study aims to explore whether the JAK2-STAT1/PD-L1 pathway is upregulated on AMs in LPS-induced mice acute lung injury and to further explore the impact of the JAK2-specific inhibitor CEP-33779 on the LPS-induced impairment of AMs phagocytic activity and lung injury. ALI was induced in mice via intratracheal administration of LPS, followed by intragastric administration of JAK2 inhibitor CEP-33779 suspension. Immunohistochemistry was conducted to assess PD-L1 expression in lung tissue, as well as p-JAK2, p-STAT1, and PD-L1 expression on AMs in bronchoalveolar lavage fluid (BALF) using immunofluorescence. Levels of TNF-α and IL-6, as well as protein concentration in BALF, were measured using enzyme-linked immunosorbent assay and Bicinchoninic acid assays, respectively. Hematoxylin-eosin staining and lung injury score were employed to evaluate pathological changes in mouse lungs. Total cell count in BALF was determined using a cell counter. Furthermore, western blot and immunofluorescence was conducted to assess the effect of JAK2 and STAT1 inhibitor on JAK2-STAT1 pathway activation and PD-L1 expression, while confocal microscopy with latex beads rabbit IgG FITC complex was used to observe MH-S cells phagocytic ability. The study revealed that LPS stimulation triggered the activation of the JAK2-STAT1 pathway and an upregulation of PD-L1 on AMs in both LPS-induced acute lung injury mice and MH-S cell lines. Moreover, treatment with the JAK2 and STAT1 inhibitor effectively reduced the activation of JAK2-STAT1 signaling, downregulated PD-L1 expression on AMs in BALF from LPS-induced ALI mice and LPS-stimulated MH-S cells, and significantly improved the LPS-induced reduction in phagocytic activity in MH-S cells. Most notably, CEP-33779 treatment significantly mitigated the pulmonary inflammatory response and lung injury in mice with LPS-induced ALI. Collectively, these findings imply that the JAK2-STAT1 pathway plays a role in the upregulation of PD-L1, which in turn is associated with the diminished phagocytic activity in LPS-induced AMs as well as lung injury. Furthermore, our study highlights that CEP-33779 treatment can effectively improve the reduced phagocytic activity of AMs and relieve lung injury induced by LPS through suppression of the JAK2-STAT1/PD-L1 pathway.

Wang YH ，Li AG ，Wang HY ，Tu YS ... -  《Frontiers in Immunology》