Ye-Ji Lee

Inhibiting Mer receptor tyrosine kinase suppresses STAT1, SOCS1/3, and NF-κB activation and enhances inflammatory responses in lipopolysaccharide-induced acute lung injury

Mer signaling participates in a novel inhibitory pathway in TLR activation. The purpose of the present study was to examine the role of Mer signaling in the down‐regulation of TLR4 activation‐driven immune responses in mice, i.t.‐treated with LPS, using the specific Mer‐blocking antibody. At 4 h and 24 h after LPS treatment, expression of Mer protein in alveolar macrophages and lung tissue decreased, sMer in BALF increased significantly, and Mer activation increased. Pretreatment with anti‐Mer antibody did not influence the protein levels of Mer and sMer levels. Anti‐Mer antibody significantly reduced LPS‐induced Mer activation, phosphorylation of Akt and FAK, STAT1 activation, and expression of SOCS1 and ‐3. Anti‐Mer antibody enhanced LPS‐induced inflammatory responses, including activation of the NF‐κB pathway; the production of TNF‐α, IL‐1β, and MIP‐2 and MMP‐9 activity; and accumulation of inflammatory cells and the total protein levels in BALF. These results indicate that Mer plays as an intrinsic feedback inhibitor of the TLR4‐ and inflammatory mediator‐driven immune responses during acute lung injury.

N-acetylcysteine inhibits RhoA and promotes apoptotic cell clearance during intense lung inflammation.

RATIONALEnThe resolution of pulmonary inflammation seen in various inflammatory lung conditions depends on the clearance of apoptotic cells to prevent permanent tissue damage or progressive disease. Uptake of apoptotic cells by alveolar macrophages is suppressed by oxidants through the activation of Rho signaling.nnnOBJECTIVESnWe hypothesized that antioxidant exposure would increase the ability of alveolar macrophages to clear pulmonary apoptotic cells through the inhibition of RhoA.nnnMETHODSnThe effects of the antioxidant N-acetylcysteine (NAC) on the pulmonary immune response were seen in mice treated intratracheally with LPS, LPS + NAC, or saline. Apoptotic cell clearance, RhoA activity, and changes in the lung inflammatory responses were analyzed in vivo or ex vivo.nnnMEASUREMENTS AND MAIN RESULTSnNeutrophil accumulation, apoptosis, necrosis, and oxidant production peaked at 3 days post LPS treatment. NAC enhanced the clearance of apoptotic cells and inhibited RhoA activity in alveolar macrophages at 3 days post LPS treatment. NAC suppressed LPS-induced proinflammatory mediators, enhanced the production of transforming growth factor-beta1, reduced the accumulation of inflammatory cells, and reduced levels of protein and lactate dehydrogenase in bronchoalveolar lavage fluid. In the presence of ex vivo apoptotic cells, alveolar macrophages exposed to LPS or LPS + NAC had reduced tumor necrosis factor-alpha levels and increased transforming growth factor-beta1 levels. A Rho kinase inhibitor mimicked the effects of NAC on the clearance of apoptotic cells and the inflammatory responses.nnnCONCLUSIONSnThese results indicate that NAC can expedite the resolution of LPS-induced pulmonary inflammation through the inhibition of RhoA activity and the enhancement of apoptotic cell clearance.

Upregulation of Mer Receptor Tyrosine Kinase Signaling Attenuated Lipopolysaccharide-Induced Lung Inflammation

Mer receptor tyrosine kinase (Mer) signaling plays a central role in the intrinsic inhibition of the inflammatory response to Toll-like receptor activation. Previously, we found that lung Mer protein expression decreased after lipopolysaccharide (LPS) treatment due to enhanced Mer cleavage. The purpose of the present study was to examine whether pharmacologically restored membrane-bound Mer expression upregulates the Mer signaling pathways and suppresses lung inflammatory responses. Pretreatment with the ADAM17 (a disintegrin and metalloproteinase-17) inhibitor TAPI-0 (tumor necrosis factor alpha protease inhibitor-0) reduced LPS-induced production of soluble Mer protein in bronchoalveolar lavage (BAL) fluid, restored membrane-bound Mer expression, and increased Mer activation in alveolar macrophages and lungs after LPS treatment. TAPI-0 also enhanced Mer downstream signaling, including phosphorylation of protein kinase b, focal adhesion kinase, and signal transducer and activator of transcription 1. As expected from enhanced Mer signaling, TAPI-0 also augmented suppressor of cytokine signaling-1 and -3 mRNA and protein levels and inhibited nuclear factor κB activation at 4 and 24 hours after LPS treatment. TAPI-0 suppressed LPS-induced inflammatory cell accumulation, total protein level elevation in BAL fluid, and production of inflammatory mediators, including tumor necrosis factor-α, interleukin-1β, and macrophage inflammatory protein-2. Additionally, the effects of TAPI-0 on the activation of Mer signaling and the production of inflammatory responses could be reversed by cotreatment with specific Mer-neutralizing antibody. Restored Mer protein expression by treatment with TAPI-0 efficiently prevents the inflammatory cascade during acute lung injury.

Apoptotic cell instillation after bleomycin attenuates lung injury through hepatocyte growth factor induction

Apoptotic cell clearance by macrophages and neighbouring tissue cells induces hepatocyte growth factor (HGF) secretion. HGF plays a key role in alveolar epithelial regeneration and reconstruction after lung injury. Direct in vivo exposure to apoptotic cells enhances HGF production, resulting in attenuation of pulmonary injury. We investigated the direct effect of in vivo exposure to apoptotic cells in bleomycin-stimulated lungs (2 days old) on HGF induction. Furthermore, sequential changes of bleomycin-induced HGF production following apoptotic cell instillation related to the changes in inflammatory and fibrotic responses were assessed. At 2 h after apoptotic cell instillation into bleomycin-stimulated lungs, the levels of HGF mRNA and protein production, and apoptotic cell clearance by alveolar macrophages were enhanced. Furthermore, HGF induction persistently increased following apoptotic cell instillation up to 21 days after bleomycin treatment. Apoptotic cell instillation attenuated bleomycin-induced pro-inflammatory mediator production, inflammatory cell recruitment and total protein levels. Apoptotic cell instillation also induced antiapoptotic and antifibrotic effects. These anti-inflammatory and antiapoptotic effects could be reversed by co-administration of HGF-neutralising antibody. These findings indicate that in vivo exposure to apoptotic cells enhances transcriptional HGF production in bleomycin-stimulated lungs, resulting in attenuation of lung injury and fibrosis.

Coordinated induction of cyclooxygenase-2/prostaglandin E2 and hepatocyte growth factor by apoptotic cells prevents lung fibrosis

Apoptotic cell instillation after bleomycin induces persistent HGF production and protects from pulmonary fibrosis, but the underlying mechanism remains unclear. We investigated immediate and prolonged effects of in vivo instillation of apoptotic cells into bleomycin‐stimulated mouse lungs (2 days old) on COX‐2 expression in lung tissue and alveolar macrophages and PGE2 production in BALF. Furthermore, functional interaction between these molecules and HGF, following apoptotic cell instillation in a bleomycin‐induced lung fibrosis model, was assessed. Apoptotic cell instillation results in enhanced immediate and prolonged expression of COX‐2 and PGE2 when compared with those from bleomycin‐only‐treated mice. Coadministration of the COX‐2‐selective inhibitor NS398 or the selective PGE2R EP2 inhibitor AH6809 inhibited the increase in HGF production. Inhibition of HGF signaling using PHA‐665752 inhibited increases in COX‐2 and PGE2. Long‐term inhibition of COX‐2, PGE2, or HGF reversed the reduction of TGF‐β, apoptotic and MPO activities, protein levels, and hydroxyproline contents. Up‐regulation of COX‐2/PGE2 and HGF through a positive‐feedback loop may be an important mechanism whereby apoptotic cell instillation exerts the net results of anti‐inflammatory, antiapoptotic, and antifibrotic action.

Preventing cleavage of Mer promotes efferocytosis and suppresses acute lung injury in bleomycin treated mice.

Mer receptor tyrosine kinase (Mer) regulates macrophage activation and promotes apoptotic cell clearance. Mer activation is regulated through proteolytic cleavage of the extracellular domain. To determine if membrane-bound Mer is cleaved during bleomycin-induced lung injury, and, if so, how preventing the cleavage of Mer enhances apoptotic cell uptake and down-regulates pulmonary immune responses. During bleomycin-induced acute lung injury in mice, membrane-bound Mer expression decreased, but production of soluble Mer and activity as well as expression of disintegrin and metalloproteinase 17 (ADAM17) were enhanced . Treatment with the ADAM inhibitor TAPI-0 restored Mer expression and diminished soluble Mer production. Furthermore, TAPI-0 increased Mer activation in alveolar macrophages and lung tissue resulting in enhanced apoptotic cell clearance in vivo and ex vivo by alveolar macrophages. Suppression of bleomycin-induced pro-inflammatory mediators, but enhancement of hepatocyte growth factor induction were seen after TAPI-0 treatment. Additional bleomycin-induced inflammatory responses reduced by TAPI-0 treatment included inflammatory cell recruitment into the lungs, levels of total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, as well as caspase-3 and caspase-9 activity and alveolar epithelial cell apoptosis in lung tissue. Importantly, the effects of TAPI-0 on bleomycin-induced inflammation and apoptosis were reversed by coadministration of specific Mer-neutralizing antibodies. These findings suggest that restored membrane-bound Mer expression by TAPI-0 treatment may help resolve lung inflammation and apoptosis after bleomycin treatment.

The TAM-family receptor Mer mediates production of HGF through the RhoA-dependent pathway in response to apoptotic cells

The receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. Study of the induction of HGF during the interaction of macrophages with apoptotic cells shows that only Mer is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.

Mer signaling increases the abundance of the transcription factor LXR to promote the resolution of acute sterile inflammation.

The receptor tyrosine kinase Mer resolves local and systemic inflammation by increasing the abundance of liver X receptors. Resolving inflammation with Mer Unchecked inflammatory responses in the body can do more harm than good; thus, the body has mechanisms for resolving inflammation and restoring normal tissue function. Choi et al. found that mice with impaired signaling by the receptor tyrosine kinase Mer had exacerbated inflammation in response to acute sterile tissue damage, as well as decreased abundance of liver X receptor (LXR) transcription factors (which are implicated in inhibiting macrophage responses) in their macrophages, spleens, and lungs. Conversely, treating macrophages in culture with an agonist of Mer increased LXR abundance and activity. Furthermore, an LXR agonist reduced inflammation in mice even in the context of reduced Mer signaling. The finding that Mer-dependent increases in LXR abundance lead to the resolution of inflammation might be exploited therapeutically. The receptor tyrosine kinase Mer plays a central role in inhibiting the inflammatory response of immune cells to pathogens. We aimed to understand the function of Mer signaling in the resolution of sterile inflammation in experiments with a Mer-neutralizing antibody or with Mer-deficient (Mer−/−) mice in a model of sterile, zymosan-induced acute inflammation. We found that inhibition or deficiency of Mer enhanced local and systemic inflammatory responses. The exacerbated inflammatory responses induced by the lack of Mer signaling were associated with reduced abundance of the transcription factors liver X receptor α (LXRα) and LXRβ and decreased expression of their target genes in peritoneal macrophages, spleens, and lungs. Similarly, treatment of mice with a Mer/Fc fusion protein, which prevents the Mer ligand Gas6 (growth arrest–specific protein 6) from binding to Mer, exacerbated the inflammatory response and decreased the abundance of LXR. Coadministration of the LXR agonist T0901317 with the Mer-neutralizing antibody inhibited the aggravating effects of the antibody on inflammation in mice. In vitro exposure of RAW264.7 cells or primary peritoneal macrophages to Gas6 increased LXR abundance in an Akt-dependent manner. Thus, we have elucidated a previously uncharacterized pathway involved in the resolution of acute sterile inflammation: Enhanced Mer signaling during the recovery phase increases the abundance and activity of LXR to inactivate the inflammatory response in macrophages.

Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF.

Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors.

Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2 and HGF Expression via a Positive Feedback Loop

Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.