Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation.

The epithelial-mesenchymal transition (EMT) and fibroblast activation are major events in idiopathic pulmonary fibrosis pathogenesis. Here, we investigated whether growth arrest-specific protein 6 (Gas6) plays a protective role in lung fibrosis via suppression of the EMT and fibroblast activation. rGas6 administration inhibited the EMT in isolated mouse ATII cells 14 days post-BLM treatment based on morphologic cellular alterations, changes in mRNA and protein expression profiles of EMT markers, and induction of EMT-activating transcription factors. BLM-induced increases in gene expression of fibroblast activation-related markers and the invasive capacity of primary lung fibroblasts in primary lung fibroblasts were reversed by rGas6 administration. Furthermore, the hydroxyproline content and collagen accumulation in interstitial areas with damaged alveolar structures in lung tissue were reduced by rGas6 administration. Targeting Gas6/Axl signaling events with specific inhibitors of Axl (BGB324), COX-2 (NS-398), EP1/EP2 receptor (AH-6809), or PGD2 DP2 receptor (BAY-u3405) reversed the inhibitory effects of rGas6 on EMT and fibroblast activation. Finally, we confirmed the antifibrotic effects of Gas6 using Gas6-/- mice. Therefore, Gas6/Axl signaling events play a potential role in inhibition of EMT process and fibroblast activation via COX-2-derived PGE2 and PGD2 production, ultimately preventing the development of pulmonary fibrosis.

Shrimp miR-965 transfers tumoricidal mitochondria.

BACKGROUND: Micro RNA of Marsupenaeus japonicas has been known to promote apoptosis of tumor cells. However, the detailed mechanisms are not well understood. RESULTS: Using tomographic microscope, which can detect the internal structure of cells, we observed breast tumor cells following treatment of the miRNA. Intriguingly, we found that mitochondria migrate to an adjacent tumor cells through a tunneling nanotube. To recapitulate this process, we engineered a microfluidic device through which mitochondria were transferred. We show that this mitochondrial transfer process released endonuclease G (Endo G) into tumor cells, which we referred to herein as unsealed mitochondria. Importantly, Endo G depleted mitochondria alone did not have tumoricidal effects. Moreover, unsealed mitochondria had synergistic apoptotic effects with subtoxic dose of doxorubicin thereby mitigating cardiotoxicity. CONCLUSIONS: Together, we show that the mitochondrial transfer through microfluidics can provide potential novel strategies towards tumor cell death.

Anti-Inflammatory and Neuroprotective Mechanisms of GTS-21, an α7 Nicotinic Acetylcholine Receptor Agonist, in Neuroinflammation and Parkinson's Disease Mouse Models.

Neuroinflammation is crucial in the progression of neurodegenerative diseases. Thus, controlling neuroinflammation has been proposed as an important therapeutic strategy for neurodegenerative disease. In the present study, we examined the anti-inflammatory and neuroprotective effects of GTS-21, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, in neuroinflammation and Parkinson's disease (PD) mouse models. GTS-21 inhibited the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary microglia. Further research revealed that GTS-21 has anti-inflammatory properties by inhibiting PI3K/Akt, NF-κB, and upregulating AMPK, Nrf2, CREB, and PPARγ signals. The effects of GTS-21 on these pro-/anti-inflammatory signaling molecules were reversed by treatment with an α7 nAChR antagonist, suggesting that the anti-inflammatory effects of GTS-21 are mediated through α7 nAChR activation. The anti-inflammatory and neuroprotective properties of GTS-21 were then confirmed in LPS-induced systemic inflammation and MPTP-induced PD model mice. In LPS-injected mouse brains, GTS-21 reduced microglial activation and production of proinflammatory markers. Furthermore, in the brains of MPTP-injected mice, GTS-21 restored locomotor activity and dopaminergic neuronal cell death while inhibiting microglial activation and pro-inflammatory gene expression. These findings suggest that GTS-21 has therapeutic potential in neuroinflammatory and neurodegenerative diseases such as PD.

Aging effects on the diurnal patterns of gut microbial composition in male and female mice.

Composition of the gut microbiota changes with aging and plays an important role in age-associated disease such as metabolic syndrome, cancer, and neurodegeneration. The gut microbiota composition oscillates through the day, and the disruption of their diurnal rhythm results in gut dysbiosis leading to metabolic and immune dysfunctions. It is well documented that circadian rhythm changes with age in several biological functions such as sleep, body temperature, and hormone secretion. However, it is not defined whether the diurnal pattern of gut microbial composition is affected by aging. To evaluate aging effects on the diurnal pattern of the gut microbiome, we evaluated the taxa profiles of cecal contents obtained from young and aged mice of both sexes at daytime and nighttime points by 16S rRNA gene sequencing. At the phylum level, the ratio of Firmicutes to Bacteroidetes and the relative abundances of Verrucomicrobia and Cyanobacteria were increased in aged male mice at night compared with that of young male mice. Meanwhile, the relative abundances of Sutterellaceae, Alloprevotella, Lachnospiraceae UCG-001, and Parasutterella increased in aged female mice at night compared with that of young female mice. The Lachnospiraceae NK4A136 group relative abundance increased in aged mice of both sexes but at opposite time points. These results showed the changes in diurnal patterns of gut microbial composition with aging, which varied depending on the sex of the host. We suggest that disturbed diurnal patterns of the gut microbiome can be a factor for the underlying mechanism of age-associated gut dysbiosis.

The phosphodiesterase 10 inhibitor papaverine exerts anti-inflammatory and neuroprotective effects via the PKA signaling pathway in neuroinflammation and Parkinson's disease mouse models.

BACKGROUND: Neuroinflammation plays a pivotal role in the pathogenesis of Parkinson's disease (PD). Thus, the development of agents that can control neuroinflammation has been suggested as a promising therapeutic strategy for PD. In the present study, we investigated whether the phosphodiesterase (PDE) 10 inhibitor has anti-inflammatory and neuroprotective effects in neuroinflammation and PD mouse models. METHODS: Papaverine (PAP) was utilized as a selective inhibitor of PDE10. The effects of PAP on the expression of pro-inflammatory molecules were examined in lipopolysaccharide (LPS)-stimulated BV2 microglial cells by ELISA, RT-PCR, and Western blot analysis. The effects of PAP on transcription factors were analyzed by the electrophoretic mobility shift assay, the reporter gene assay, and Western blot analysis. Microglial activation and the expression of proinflammatory molecules were measured in the LPS- or MPTP-injected mouse brains by immunohistochemistry and RT-PCR analysis. The effect of PAP on dopaminergic neuronal cell death and neurotrophic factors were determined by immunohistochemistry and Western blot analysis. To assess mouse locomotor activity, rotarod and pole tests were performed in MPTP-injected mice. RESULTS: PAP inhibited the production of nitric oxide and proinflammatory cytokines in LPS-stimulated microglia by modulating various inflammatory signals. In addition, PAP elevated intracellular cAMP levels and CREB phosphorylation. Treatment with H89, a PKA inhibitor, reversed the anti-inflammatory effects of PAP, suggesting the critical role of PKA signaling in the anti-inflammatory effects of PAP. We verified the anti-inflammatory effects of PAP in the brains of mice with LPS-induced systemic inflammation. PAP suppressed microglial activation and proinflammatory gene expression in the brains of these mice, and these effects were reversed by H89 treatment. We further examined the effects of PAP on MPTP-injected PD model mice. MPTP-induced dopaminergic neuronal cell death and impaired locomotor activity were recovered by PAP. In addition, PAP suppressed microglial activation and proinflammatory mediators in the brains of MPTP-injected mice. CONCLUSIONS: PAP has strong anti-inflammatory and neuroprotective effects and thus may be a potential candidate for treating neuroinflammatory disorders such as PD.

MCP-1 and MIP-3α Secreted from Necrotic Cell-Treated Glioblastoma Cells Promote Migration/Infiltration of Microglia.

BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The defining characteristics of GBM are diffuse infiltration of tumor cells into normal brain parenchyma, rapid growth, a high degree of infiltration of microglia and macrophages, and the presence of necrosis. Microglia/macrophages are frequently found in gliomas and they extensively infiltrate GBM tissue, up to 30% of total tumor mass. However, little is known about the effect of necrotic cells (NCs) on microglia infiltration in GBM and the tumor-infiltrating microglia-induced factors in GBMs. METHODS: In this study, to address whether necrosis or necrosis-exposed GBM cells affect the degree of microglia/macrophage infiltration, migration and invasion/infiltration assays were performed. Culture supernatants and nuclear extracts of CRT-MG cells treated or untreated with necrotic cells were analyzed using a chemokine array and electrophoretic mobility shift assay, respectively. RESULTS: The presence of NCs promoted the migration/infiltration of microglia, and GBM cell line CRT-MG cells exposed to NCs further enhanced the migration and infiltration of HMO6 microglial cells. Treatment with NCs induced mRNA and protein expression of chemokines such as Monocyte Chemoattractant Protein-1 (CCL2/MCP-1) and Macrophage Inflammatory Protein-3α (CCL20/MIP-3α) in CRT-MG cells. In particular, CCL2/MCP-1 and CCL20/MIP-3α were significantly increased in NC-treated CRT-MG cells. NCs induced DNA binding of the transcription factors Nuclear Factor (NF)-κB and Activator Protein 1 (AP-1) to the CCL2/MCP-1 and CCL20/MIP-3α promoters, leading to increased CCL2/MCP-1 and CCL20/MIP-3α mRNA and protein expression in CRT-MG cells. CONCLUSION: These results provide evidence that NCs induce the expression of CCL2/MCP-1 and CCL20/MIP-3α in glioblastoma cells through activation of NF-κB and AP-1 and facilitate the infiltration of microglia into tumor tissues.

A STAT6 Inhibitor AS1517499 Reduces Preventive Effects of Apoptotic Cell Instillation on Bleomycin-Induced Lung Fibrosis by Suppressing PPARγ.

BACKGROUND/AIMS: The signal transducer and activator of transcription 6 (STAT6) transcription factor mediates PPARγ-regulated gene expression in macrophages. However, it remains largely unknown how proximal membrane signaling events initiated by apoptotic cell recognition upregulate PPARγ expression and activate the lung homeostatic program. METHODS: The STAT6 inhibitor AS1517499 was used to determine the role of STAT6 in mediating PPARγ activity, anti-inflammatory effects, and anti-fibrotic effects induced by apoptotic cell instillation after bleomycin treatment into C57BL/6 mice. Bronchoalveolar lavage fluid, alveolar macrophages and lungs were harvested at days 2, 7, and 14 and then analyzed by real-time PCR, immunoblotting, ELISA, immunocytochemistry and immunohistochemistry assays. RESULTS: Our data demonstrate that apoptotic cell instillation after bleomycin results in prolonged enhancement of STAT6 phosphorylation in alveolar macrophages and lung. Co-administration of the STAT6 inhibitor, AS1517499, reversed the enhanced PPARγ expression and activity induced by apoptotic cell instillation after bleomycin treatment. By reducing the expression of PPARγ target genes, including CD36, macrophage mannose receptor, and arginase 1, AS1517499 inhibited efferocytosis and restored pro-inflammatory cytokine expression, neutrophil recruitment, protein levels, hydroxyproline content, and expression of fibrosis markers, including type 1 collagen α2, fibronectin, and α-smooth muscle actin. STAT6 inhibition reversed the expression profile of hepatocyte growth factor and interleukin-10. CONCLUSION: These results indicate that prolonged STAT6 activation following one-time apoptotic cell instillation facilitates continuous PPARγ activation, resulting in the resolution of bleomycin-induced lung inflammation and fibrosis.

Anti-inflammatory and anti-oxidant mechanisms of an MMP-8 inhibitor in lipoteichoic acid-stimulated rat primary astrocytes: involvement of NF-κB, Nrf2, and PPAR-γ signaling pathways.

BACKGROUND: Recent evidence suggests that reactive astrocytes play an important role in neuroinflammation and neurodegenerative diseases. Thus, controlling astrocyte reactivity has been suggested as a promising strategy for treating neurodegenerative diseases. In the present study, we investigated whether a matrix metalloproteinase (MMP)-8 inhibitor, M8I, could control neuroinflammation in lipoteichoic acid (LTA)-stimulated rat primary astrocytes. METHODS: The effects of M8I on the expression of inducible nitric oxide synthase, cytokines, and MMPs were examined in LTA-stimulated rat primary astrocytes by ELISA, RT-PCR, and Western blot analysis. The effects of M8I on reactive oxygen species (ROS) generation and phase II antioxidant enzyme expression were examined by the DCF-DA assay, RT-PCR, and Western blot analysis. The detailed molecular mechanisms underlying the anti-inflammatory and antioxidant effects of M8I were analyzed by the electrophoretic mobility shift assay, the reporter gene assay, Western blot, and RT-PCR analysis. RESULTS: Treatment with LTA, a major cell wall component of Gram-positive bacteria, led to astrocyte activation and induced the expression of inflammatory molecules such as iNOS, COX-2, and pro-inflammatory cytokines. In addition, LTA induced the expression of MMPs such as MMP-1, MMP-3, MMP-8, MMP-9, and MMP-13 in rat primary astrocytes. Based on previous reports showing that MMP-8 plays a role as a proinflammatory mediator in microglia, we investigated whether MMP-8 is also involved in inflammatory reactions of reactive astrocytes. We found that treatment of astrocytes with M8I significantly inhibited LTA-induced expression of iNOS, TNF-α, IL-1ß, IL-6, and TLR-2. In addition, M8I inhibited LTA-induced NF-κB, MAP kinase, and Akt activities, while it increased the anti-inflammatory PPAR-γ activities. Moreover, M8I showed antioxidant effects by suppressing ROS production in LTA- or H2O2-stimulated astrocytes. Interestingly, M8I increased the expression of phase II antioxidant enzymes such as hemeoxygenase-1, NQO1, catalase, and MnSOD by modulating the Nrf2/ARE signaling pathway. CONCLUSIONS: The data collectively suggest the therapeutic potential of an MMP-8 inhibitor in neuroinflammatory disorders that are associated with astrocyte reactivity.

Exogenous Hydrogen Peroxide Induces Lipid Raft-Mediated STAT-6 Activation in T Cells.

BACKGROUND/AIMS: CD4+ T cells are a critical component of the adaptive immune response. While the mechanisms controlling the differentiation of the Th1, Th17, and regulatory T cell subsets from naïve CD4+ T cells are well described, the factors that induce Th2 differentiation are still largely unknown. METHODS: The effects of treatment with exogenous H2O2 on STAT-6 phosphorylation and activation in T cells were examined by immunoblotting, immunofluorescence and gel shift assay. Anti-CD3 antibody and methyl-ß-cyclodextrin were utilized to induce lipid raft assembly and to investigate the involvement of lipid rafts, respectively. RESULTS: Jurkat and EL-4 T cells that were exposed to H2O2 showed rapid and strong STAT-6 phosphorylation, and the extent of STAT-6 phosphorylation was enhanced by co-treatment with anti-CD3 antibody. The effect of H2O2 on STAT-6 phosphorylation and translocation was inhibited by disruption of lipid rafts. STAT-6 activation in response to H2O2 treatment regulated IL-4 gene expression, and this response was strengthened by treatment with anti-CD3. CONCLUSION: Our results indicate that reactive oxygen species such as H2O2 can act on upstream and initiating factors for activation of STAT-6 in T cells and contribute to formation of a positive feedback loop between STAT-6 and IL-4 in the Th2 differentiation process.

Anti-inflammatory mechanism of lonchocarpine in LPS- or poly(I:C)-induced neuroinflammation.

Neuroinflammation plays an important role in the progression of various neurodegenerative diseases. In this study, we investigated the anti-inflammatory effects of lonchocarpine, a natural compound isolated from Abrus precatorius, under in vitro and in vivo neuroinflammatory conditions induced by challenge with lipopolysaccharide (LPS)- or polyinosinic-polycytidylic acid (poly(I:C)). Lonchocarpine suppressed the expression of iNOS and proinflammatory cytokines in LPS or poly(I:C)-stimulated BV2 microglial cells. These anti-inflammatory effects were verified in brains of mice with systemic inflammation induced by administration of LPS or poly(I:C). Lonchocarpine reduced the number of Iba-1-positive activated microglia, and suppressed the mRNA expression of various proinflammatory markers in the cortex of LPS- or poly(I:C)-injected mice. Molecular mechanistic experiments showed that lonchocarpine inhibited NF-κB activity by reducing the phosphorylation and degradation of IκBα in LPS- or poly(I:C)-stimulated BV2 cells. Analysis of further upstream signaling pathways in LPS-stimulated microglia showed that lonchocarpine inhibited the phosphorylation of IκB kinase and TGFß-activated kinase 1 (TAK1). Moreover, lonchocarpine suppressed the interaction of myeloid differentiation factor 88 (MyD88) and intereleukin-1 receptor-associated kinase 4 (IRAK4). These data suggest that toll-like receptor 4 downstream signals such as MyD88/IRAK4-TAK1-NF-κB are at least partly involved in the anti-inflammatory mechanism of lonchocarpine in LPS-stimulated microglia. Its strong anti-inflammatory effects may make lonchocarpine an effective preventative drug for neuroinflammatory disorders that are associated with systemic inflammation.

Matrix metalloproteinase-8 plays a pivotal role in neuroinflammation by modulating TNF-α activation.

Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood-brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A(74)/Q(75), A(76)/V(77) residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain.

RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages.

Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

Noggin improves ischemic brain tissue repair and promotes alternative activation of microglia in mice.

We previously reported that bone morphogenetic proteins (BMPs) and their endogenous antagonist noggin are expressed in the brain weeks after an ischemic insult. Here, to define their roles in ischemic brain tissue repair and remodeling, we infused recombinant BMP7 or noggin into the ipsilateral ventricle of mice for 2weeks starting 2weeks after transient middle cerebral artery occlusion (MCAO). Four weeks after MCAO, we measured ischemic brain volume, functional recovery, and molecules related to neurogenesis and angiogenesis such as synaptophysin, GAP-43, and VEGF. Noggin-treated mice but not BMP7-treated mice showed preserved ipsilateral brain volume and reduced neurological deficits compared with artificial cerebrospinal fluids (aCSF)-treated mice. Noggin treatment also decreased glial scar thickness, increased levels of GAP-43 and VEGF protein, and increased the number of Iba1-positive activated microglia in the ipsilateral brain. Furthermore, noggin treatment decreased M1 markers (IL-1ß, TNF-α, IL-12, CCL2 and CD86) and increased M2 markers (IL-1ra, IL-10, arginase 1, CD206 and Ym1) of activated microglia, suggesting a shift from M1 to M2 phenotypes. These results suggest that noggin improves functional recovery from ischemic stroke and enhances alternatively activated microglia, thereby promoting tissue repair and remodeling.

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.

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-1ß, 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.

IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1ß by keratinocytes via the ROS-NLRP3-caspase-1 pathway.

BACKGROUND: The pathogenesis of inflammatory skin disease involves the release of cytokines from keratinocytes, and one of these, IL-1ß, has been previously implicated in inflammatory skin disease. T(h)17 cells, a subset of T(h) cells involved in autoimmunity and inflammation, possess IL-1ß receptors and secrete cytokines such as IL-17 and IL-22 in response to IL-1ß stimulation. A mutation in the inflammasome protein NLRP3 (NACHT, LRR and PYD domains-containing protein 3) causes excess production of IL-1ß, resulting in an augmentation of T(h)17-dominant pathology. METHODS: To determine the feedback effect, if any, of IL-17 and/or IL-22 on the secretion of IL-1ß from keratinocytes, we stimulated the human keratinocyte cell line HaCaT, as well as caspase-1-deficient mice, with IL-17 or IL-22. RESULTS: We found that treatment with IL-17 and IL-22 causes an increase in IL-1ß via the activation of NLRP3 by a process that involves the generation of reactive oxygen species. Moreover, skin inflammation induced by IL-17 and IL-22 was lower in caspase-1 knockout (KO) mice relative to that induced by IL-1ß treatment. Additionally, skin inflammation induced by the drug imiquimod was lower in caspase-1 KO mice than in wild-type mice. CONCLUSION: These results indicate that cytokines from T(h)17 cells may potentiate IL-1ß-mediated skin inflammation and result in phenotypic alterations of keratinocytes via a feedback mechanism.

Lethal effects of mitochondria via microfluidics.

Tumor cells can respond to therapeutic agents by morphologic alternations including formation of tunneling nanotubes. Using tomographic microscope, which can detect the internal structure of cells, we found that mitochondria within breast tumor cells migrate to an adjacent tumor cell through a tunneling nanotube. To investigate the relationship between mitochondria and tunneling nanotubes, mitochondria were passed through a microfluidic device that mimick tunneling nanotubes. Mitochondria, through the microfluidic device, released endonuclease G (Endo G) into adjacent tumor cells, which we referred to herein as unsealed mitochondria. Although unsealed mitochondria did not induce cell death by themselves, they induced apoptosis of tumor cells in response to caspase-3. Importantly, Endo G-depleted mitochondria were ineffective as lethal agents. Moreover, unsealed mitochondria had synergistic apoptotic effects with doxorubicin in further increasing tumor cell death. Thus, we show that the mitochondria of microfluidics can provide novel strategies toward tumor cell death.

RIPK1 Regulates Microglial Activation in Lipopolysaccharide-Induced Neuroinflammation and MPTP-Induced Parkinson's Disease Mouse Models.

Increasing evidence suggests a pivotal role of receptor-interacting protein kinase 1 (RIPK1), an initiator of necroptosis, in neuroinflammation. However, the precise role of RIPK1 in microglial activation remains unclear. In the present study, we explored the role of RIPK1 in lipopolysaccharide (LPS)-induced neuroinflammation and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice by using RIPK1-specific inhibitors necrostatin-1 (Nec-1) and necrostatin-1 stable (Nec-1s). Nec-1/Nec-1s or RIPK1 siRNA inhibited the production of proinflammatory molecules and the phosphorylation of RIPK1-RIPK3-MLKL and cell death in LPS-induced inflammatory or LPS/QVD/BV6-induced necroptotic conditions of BV2 microglial cells. Detailed mechanistic studies showed that Nec-1/Nec-1s exerted anti-inflammatory effects by modulating AMPK, PI3K/Akt, MAPKs, and NF-κB signaling pathways in LPS-stimulated BV2 cells. Subsequent in vivo studies showed that Nec-1/Nec-1s inhibited microglial activation and proinflammatory gene expression by inhibiting the RIPK1 phosphorylation in the brains of LPS-injected mice. Furthermore, Nec-1/Nec-1s exert neuroprotective and anti-inflammatory effects in MPTP-induced PD mice. We found that p-RIPK1 is mainly expressed in microglia, and thus RIPK1 may contribute to neuroinflammation and subsequent cell death of dopaminergic neurons in MPTP-induced PD model mice. These data suggest that RIPK1 is a key regulator of microglial activation in LPS-induced neuroinflammation and MPTP-induced PD mice.

IL-33 induces Th17-mediated airway inflammation via mast cells in ovalbumin-challenged mice.

Allergic asthma is characterized by infiltration of eosinophils, elevated Th2 cytokine levels, airway hyperresponsiveness, and IgE. In addition to eosinophils, mast cells, and basophils, a variety of cytokines are also involved in the development of allergic asthma. The pivotal role of eosinophils in the progression of the disease has been a subject of controversy. To determine the role of eosinophils in the progression of airway inflammation, we sensitized and challenged BALB/c wild-type (WT) mice and eosinophil-deficient ΔdblGATA mice with ovalbumin (OVA) and analyzed different aspects of inflammation. We observed increased eosinophil levels and a Th2-dominant response in OVA-challenged WT mice. In contrast, eosinophil-deficient ΔdblGATA mice displayed an increased proportion of mast cells and a Th17-biased response following OVA inhalation. Notably, the levels of IL-33, an important cytokine responsible for Th2 immune deviation, were not different between WT and eosinophil-deficient mice. We also demonstrated that mast cells induced Th17-differentiation via IL-33/ST2 stimulation in vitro. These results indicate that eosinophils are not essential for the development of allergic asthma and that mast cells can skew the immune reaction predominantly toward Th17 responses via IL-33 stimulation.

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.