Low-density lipoprotein receptor-related protein 6 ablation in macrophages differentially inhibits lung injury-mediated inflammation and metastasis.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor protein for Wnt ligands. Yet, their role in immune cell regulation remains elusive. Here we demonstrated that genetic deletion of LRP6 in macrophages using LysM-cre Lrp6fl/fl (Lrp6MKO) mice showed differential inhibition of inflammation in the bleomycin (BLM)-induced lung injury model and B16F10 melanoma lung metastasis model. Lrp6MKO mice showed normal immune cell populations in the lung and circulating blood in homeostatic conditions. In the BLM-induced lung injury model, Lrp6MKO mice showed a decreased number of monocyte-derived alveolar macrophages, reduced collagen deposition and alpha-smooth muscle actin (αSMA) protein levels in the lung. In B16F10 lung metastasis model, Lrp6MKO mice reduced lung tumor foci. Monocytic and granulocytic-derived myeloid-derived suppressor cells (M-MDSCs and G-MDSCs) were increased in the lung. In G-MDSCs, hypoxia-inducible factor 1α (HIF1α)+ PDL1+ population was markedly decreased but not in M-MDSCs. Taken together, our results show that the role of LRP6 in macrophages is differential depending on the inflammation microenvironment in the lung.

Dickkopf1 Promotes Pulmonary Fibrosis upon Bleomycin-Induced Lung Injury.

Orchestration of inflammation and tissue repair processes is critical to maintaining homeostasis upon tissue injury. Tissue fibrosis is a pathological process characterized by aberrant accumulation of extracellular matrix proteins, such as collagen, upon injury. Dickkopf1 (DKK1) is a quintessential Wnt antagonist. The role of DKK1 in bleomycin (BLM)-induced lung injury and fibrosis model remains elusive. This study shows that BLM-induced lung injury markedly elevated DKK1 protein expressions in the lungs in mice, consistent with human pulmonary fibrosis patient lung tissues. The elevated DKK1 levels coincided with immune cell infiltration and collagen deposition. Notably, the reduced expression of DKK1 in Dkk1 hypomorphic doubleridge (Dkk1d/d) mice abrogated BLM-induced lung inflammation and fibrosis. Immune cell infiltration, collagen deposition, expression of profibrotic cytokine transforming growth factor ß1 (TGF-ß1), and extracellular matrix protein-producing myofibroblast marker α-smooth muscle actin (α-SMA) were reduced in Dkk1d/d mice. Consistent with these results, local DKK1 antibody administration after BLM-induced lung injury substantially decreased lung inflammation and fibrosis phenotypes. Taken together, these results demonstrate that DKK1 is a proinflammatory and profibrotic ligand that promotes inflammation and fibrosis upon BLM-induced lung injury, placing it as an attractive molecular target for dysregulated pulmonary inflammation and tissue repair.

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia.

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S-knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

Thrombocyte-derived Dickkopf1 promotes macrophage polarization in the Bleomycin-induced lung injury model.

Immune responses are crucial to maintaining tissue homeostasis upon tissue injury. Upon various types of challenges, macrophages play a central role in regulating inflammation and tissue repair processes. While an immunomodulatory role of Wnt antagonist Dickkopf1 (DKK1) has been implicated, the role of Wnt antagonist DKK1 in regulating macrophage polarization in inflammation and the tissue repair process remains elusive. Here we found that DKK1 induces gene expression profiles to promote inflammation and tissue repair in macrophages. Importantly, DKK1 induced various genes, including inflammation and tissue repair, via JNK (c-jun N-terminal kinase) in macrophages. Furthermore, DKK1 potentiated IL-13-mediated macrophage polarization and activation. The co-inhibition of JNK and STAT6 markedly decreased gene expressions relevant to inflammation and fibrosis by DKK1 and IL-13. Interestingly, thrombocyte-specific deletion of DKK1 in mice reduced collagen deposition and decreased Arg1, CD206, HIF1α, and IL1ß protein expressions in monocyte-derived alveolar macrophages in the acute sterile bleomycin (BLM)-induced lung injury model. These data suggested that thrombocytes communicate with macrophages via DKK1 to orchestrate inflammation and repair in this model. Taken together, our study demonstrates DKK1's role as an important regulatory ligand for macrophage polarization in the injury-induced inflammation and repair process in the lung.

Dickkopf1: An Immunomodulator in Tissue Injury, Inflammation, and Repair.

Upon injury, inflammation and repair processes are orchestrated to maintain tissue homeostasis. The Wnt ligands play essential roles in cell differentiation and proliferation for tissue repair and regeneration. It is increasingly clear that Wnt ligands play crucial immune-modulatory roles in inflammatory diseases. It is predicted that comprehensive research regarding the cross-talk between nonimmune and immune cells in tissue injury and repair will flourish. The Wnt system and immune system interaction will be critical to understanding tissue injury, inflammation, and repair. In this study, we will first introduce the Wnt system and review the role of the Wnt system in tissue regeneration and repair. We will review the previous literature regarding how the Wnt ligands regulate the immune system. Next, we will discuss the current and future perspectives of Wnt ligands to target cancer and other immunological diseases. Finally, we will discuss the quintessential Wnt antagonist Dickkopf1 as an immunomodulatory ligand.

Establishing a 3D In Vitro Hepatic Model Mimicking Physiologically Relevant to In Vivo State.

Three-dimensional (3D) bioprinting is a promising technology to establish a 3D in vitro hepatic model that holds great potential in toxicological evaluation. However, in current hepatic models, the central area suffers from hypoxic conditions, resulting in slow and weak metabolism of drugs and toxins. It remains challenging to predict accurate drug effects in current bioprinted hepatic models. Here, we constructed a hexagonal bioprinted hepatic construct and incorporated a spinning condition with continuous media stimuli. Under spinning conditions, HepG2 cells in the bioprinted hepatic construct exhibited enhanced proliferation capacity and functionality compared to those under static conditions. Additionally, the number of spheroids that play a role in boosting drug-induced signals and responses increased in the bioprinted hepatic constructs cultured under spinning conditions. Moreover, HepG2 cells under spinning conditions exhibited intensive TGFß-induced epithelial-to-mesenchymal transition (EMT) and increased susceptibility to acetaminophen (APAP)-induced hepatotoxicity as well as hepatotoxicity prevention by administration of N-acetylcysteine (NAC). Taken together, the results of our study demonstrate that the spinning condition employed during the generation of bioprinted hepatic constructs enables the recapitulation of liver injury and repair phenomena in particular. This simple but effective culture strategy facilitates bioprinted hepatic constructs to improve in vitro modeling for drug effect evaluation.

Stem cell-secreted 14,15- epoxyeicosatrienoic acid rescues cholesterol homeostasis and autophagic flux in Niemann-Pick-type C disease.

We previously demonstrated that the direct transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) into the dentate gyrus ameliorated the neurological symptoms of Niemann-Pick type C1 (NPC1)-mutant mice. However, the clinical presentation of NPC1-mutant mice was not fully understood with a molecular mechanism. Here, we found 14,15-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P450 (CYP) metabolite, from hUCB-MSCs and the cerebella of NPC1-mutant mice and investigated the functional consequence of this metabolite. Our screening of the CYP2J family indicated a dysregulation in the CYP system in a cerebellar-specific manner. Moreover, in Purkinje cells, CYP2J6 showed an elevated expression level compared to that of astrocytes, granule cells, and microglia. In this regard, we found that one CYP metabolite, 14,15-EET, acts as a key mediator in ameliorating cholesterol accumulation. In confirming this hypothesis, 14,15-EET treatment reduced the accumulation of cholesterol in human NPC1 patient-derived fibroblasts in vitro by suppressing cholesterol synthesis and ameliorating the impaired autophagic flux. We show that the reduced activity within the CYP system in the cerebellum could cause the neurological symptoms of NPC1 patients, as 14,15-EET treatment significantly rescued cholesterol accumulation and impaired autophagy. We also provide evidence that the intranasal administration of hUCB-MSCs is a highly promising alternative to traumatic surgical transplantation for NPC1 patients.