Monocyte-Derived Macrophages Induce Alveolar Macrophages Death via TNF-α in Acute Lung Injury.

Acute lung injury (ALI) and its subsequent progression to acute respiratory distress syndrome (ARDS) are severe respiratory conditions. They are marked by rapid lung function deterioration and extensive pulmonary inflammation, often resulting in critical patient outcomes. Alveolar macrophages (AMs) and monocyte-derived macrophages (MDMs) are two distinct subsets of lung macrophages present in the alveoli during ALI. Both are critical mediators of pulmonary inflammation. Our study examined the interplay between AMs and MDMs in the inflammatory environment of ALI/ARDS. Mice were treated with lipopolysaccharide (LPS) to establish ALI models. The lung tissues of mice were subjected to hematoxylin-eosin staining to observe the degree of tissue damage. In vivo, CCR2-deficient mice or depleting peripheral blood mononuclear cells by clodronate liposomes were used to reduce MDMs recruitment. The bronchoalveolar lavage fluid (BALF) supernatants were used for cytokine and total protein analyses. AMs and MDMs in the BALF were analyzed by flow cytometry. The levels of AMs death were determined through propidium iodide staining and measured by flow cytometry. In vitro, primary AMs were exposed to MDM-conditioned medium or TNF-α, and their death levels were assessed under a fluorescence microscope with propidium iodide staining. AMs significantly decrease in number and undergo extensive cell death during ALI. The reduced MDMs recruitment can increase the number of AMs, reduce AMs death, and alleviate lung injury. In vitro, MDM-conditioned medium can induce AMs death and TNF-α is one of the major secretions. It indicates that TNF-α stimulation in vitro promotes AMs death. In vivo, MDMs are identified as the primary cells secreting TNF-α. Additionally, the treatment with TNF-α antagonists can reduce AMs death and the severity of lung injury. Our study demonstrates that MDMs contribute to AMs death during ALI through TNF-α. Targeting TNF-α may offer a therapeutic strategy to mitigate AMs death and lung injury in ALI/ARDS.

Xiao J ，Hou F ，Wang H ，Wang R ，Liu Y ，Wu X ，Xie L ... -  《Immunity Inflammation and Disease》

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》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

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 ... -  《-》

Pharmacological inhibition of P300 with C646 ameliorates LPS-induced acute lung injury by modulating CXCL1 in M1 alveolar macrophages.

Acute lung injury (ALI) is an excessive inflammatory condition with the involvement of M1 alveolar macrophage (AM) polarization. Given the high mortality rate of ALI, elucidating its underlying mechanisms is crucial for identifying therapeutic targets. Inhibition of P300, a lysine acetyltransferase, has illustrated the potential to alleviate inflammatory diseases through the regulation of immune cell activation. However, little is known whether P300 inhibition could ameliorate ALI through regulating the polarization of M1 AMs. We established an LPS-induced ALI model and evaluated the effects of the P300 inhibitor C646 on pulmonary pathology, inflammation and M1 AM polarization via H&E staining, ELISA and flow cytometry. Additionally, the specific inflammatory mediators regulated by P300 in M1 AMs affecting ALI were analyzed by RNA sequencing and validated by intratracheal instillation experiment. Intratracheal instillation of LPS resulted in neutrophil accumulation within the pulmonary alveoli and interstitial areas, along with increased levels of total inflammatory cells and IL-1β in the lung. However, administration of C646 ameliorated these pulmonary pathology and inflammation, accompanied by a diminished proportion and quantity of M1 AMs in BALF. Furthermore, by taking the intersection of P300-targeted genes in macrophages from the Cistrome, genes upregulated after M1 polarization of AMs, and genes downregulated following C646 treatment in M1 AMs, we identified 'Cxcl1' among the intersecting genes. Also, intratracheal instillation of CXCL1 aggravated pulmonary pathology and inflammation in C646 treated-ALI models. Our study suggested that pharmacological inhibition of P300 with C646 ameliorated LPS-induced ALI by modulating CXCL1 in M1 AMs.

Deng Y ，Wen G ，Yin Y ，Chen D ，Li D ，Chen R ... -  《-》