Eun Young Choi

Innate immune training involves myelopoiesis, dynamic gene modulation, and functional reprogramming of myeloid cells in response to secondary heterologous challenges. The present study evaluates whether systemic innate immune training can protect tissues from local injury. Systemic pretreatment of mice with β‐glucan, a trained immunity agonist, reduces the mortality rate of mice with bleomycin‐induced lung injury and fibrosis, as well as decreasing collagen deposition in the lungs. β‐Glucan pretreatment induces neutrophil accumulation in the lungs and enhances efferocytosis. Training of mice with β‐glucan results in histone modification in both alveolar macrophages (AMs) and neighboring lung epithelial cells. Training also increases the production of RvD1 and soluble mediators by AMs and efferocytes. Efferocytosis increases trained immunity in AMs by stimulating RvD1 release, thus inducing SIRT1 expression in neighboring lung epithelial cells. Elevated epithelial SIRT1 expression is associated with decreased epithelial cell apoptosis after lung injury, attenuating tissue damage. Further, neutrophil depletion dampens the effects of β‐glucan on macrophage accumulation, epigenetic modification in lung macrophages, epithelial SIRT1 expression, and injury‐mediated fibrosis in the lung. These findings provide mechanistic insights into innate immune training and clues to the potential ability of centrally trained immunity to protect peripheral organs against injury‐mediated disorders.

Pancreatic cancer is characterized by late detection, frequent drug resistance, and a highly metastatic nature, leading to poor prognosis. Antibody-based immunotherapy showed limited success for pancreatic cancer, partly owing to the low delivery rate of the drug into the tumor. Herein, we describe a poly(lactic-co-glycolic acid;PLGA)-based siRNA nanoparticle targeting PD-L1 (siPD-L1@PLGA). The siPD-L1@PLGA exhibited efficient knockdown of PD-L1 in cancer cells, without affecting the cell viability up to 6 mg/mL. Further, 99.2% of PDAC cells uptake the nanoparticle and successfully blocked the IFN-gamma-mediated PD-L1 induction. Consistently, the siPD-L1@PLGA sensitized cancer cells to antigen-specific immune cells, as exemplified by Ovalbumin-targeting T cells. To evaluate its efficacy in vivo, we adopted a pancreatic PDX model in humanized mice, generated by grafting CD34⁺ hematopoeitic stem cells onto NSG mice. The siPD-L1@PLGA significantly suppressed pancreatic tumor growth in this model with upregulated IFN-gamma positive CD8 T cells, leading to more apoptotic tumor cells. Multiplex immunofluorescence analysis exhibited comparable immune cell compositions in control and siPD-L1@PLGA-treated tumors. However, we found higher Granzyme B expression in the siPD-L1@PLGA-treated tumors, suggesting higher activity of NK or cytotoxic T cells. Based on these results, we propose the application of siPD-L1@PLGA as an immunotherapeutic agent for pancreatic cancer.

Distant metastasis represents the primary cause of cancer-associated death. Pulmonary metastasis is most frequently seen in many cancers, largely driven by lung inflammation. Components from primary tumor or recruited leukocytes are known to facilitate metastasis formation. However, contribution of target site–specific host factor to metastasis is poorly understood. Here, we show that developmental endothelial locus–1 (DEL-1), an anti-inflammatory factor abundant in the lung and down-regulated by inflammatory insults, protects from melanoma lung metastasis independently of primary tumor development and systemic immunosurveillance. DEL-1 deficiency is associated with gene profiles that favor metastatic progression with inflammation and defective immunosurveillance. Mechanistically, DEL-1 deficiency primarily influences Ly6G⁺ neutrophil accumulation in lung metastatic niche, leading to IL-17A up-regulation from γδ T cells and reduced antimetastatic NK cells. In support, neutrophil depletion or recombinant DEL-1 treatment profoundly reverses these effects. Thus, our results identify DEL-1 as a previously unrecognized link between tumor-induced inflammation and pulmonary metastasis.

Ependymal cells (ECs) are multiciliated neuroepithelial cells that line the ventricles of the brain and the central canal of the spinal cord (SC). How ependymal motile cilia are maintained remains largely unexplored. Here we show that zebrafish embryos deficient in Wnt signaling have defective motile cilia, yet harbor intact basal bodies. With respect to maintenance of ependymal motile cilia, plcδ3a is a target gene of Wnt signaling. Lack of Connexin43 (Cx43), especially its channel function, decreases motile cilia and intercellular Ca²⁺ wave (ICW) propagation. Genetic ablation of cx43 in zebrafish and mice diminished motile cilia. Finally, Cx43 is also expressed in ECs of the human SC. Taken together, our findings indicate that gap junction mediated ICWs play an important role in the maintenance of ependymal motile cilia, and suggest that the enhancement of functional gap junctions by pharmacological or genetic manipulations may be adopted to ameliorate motile ciliopathy.

Uncontrolled activation of transforming growth factor (TGF)-β results in a wide range of pathologic conditions. Therapeutic interventions to regulate TGF-β signaling during fibrosis have been developed but the effectiveness is still limited. Here, we show that developmental endothelial locus-1 (Del-1) ameliorates fibrosis in mice by inhibiting α_v integrin-mediated activation of TGF-β. Del-1 bound to α_vβ₆ integrin, an important activator of TGF-β, and inhibited the binding of α_vβ₆ integrin to the latency-associated peptide (LAP), thereby suppressing α_v integrin-mediated activation of TGF-β. Lack of Del-1 increased colocalization of α_v integrin and LAP in the lungs, which was reversed by Del-1 supplementation. The crucial role of Del-1 in regulating TGF-β activity was recapitulated in a mouse model of fibrosis using an adenovirus expressing inactive TGF-β1. Del-1 supplementation improved the pathological characteristics of the mice and reduced mortality. Thus, we propose that Del-1 is a negative regulator of TGF-β activation and a potential anti-fibrotic factor.

Disruption of the endothelial glycocalyx has a prominent role in the pathophysiology of sepsis. Developmental endothelial locus-1 (Del-1) is an endothelial-derived anti-inflammatory factor. We hypothesized that degradation of the endothelial glycocalyx during sepsis may increase serum Del-1. A mouse model of sepsis was created using cecal ligation and puncture. In septic mice, the endothelial glycocalyx was nearly completely degraded, with less formation of Del-1 in the endothelium and extracellular matrix than in control mice. Serum Del-1 levels were significantly increased in the septic mice with increasing severity of sepsis. Serum Del-1 levels were also measured in 84 patients with sepsis and septic shock and in 20 control subjects. The median serum Del-1 level in patients with sepsis was significantly higher than that in healthy controls. The high Del-1 group had higher illness severity scores and contained more patients with organ dysfunction than the low Del-1 group. The 90-day mortality rate was significantly higher in the high Del-1 group than in the low Del-1 group. Multivariate analysis indicated a tendency for a high serum Del-1 level to be associated with a higher mortality risk. Increased serum Del-1 may be a novel diagnostic biomarker of sepsis and an indicator of disease severity.

Connexin 43 (Cx43) deficiency was found to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is upregulated in macrophages by LPS treatment. In this study, we characterized a novel signaling pathway for LPS-induced Cx43 expression in RAW264.7 cells and thioglycolate-elicited peritoneal macrophages (TGEMs). LPS alone or LPS-containing conditioned medium (CM) upregulated Cx43. Overexpression or silencing of Cx43 led to the enhancement or inhibition, respectively, of CM-induced TGEM migration. This response involved the inducible NO synthase (iNOS)/focal adhesion kinase (FAK)/Src pathways. Moreover, CM-induced migration was compromised in TGEMs from Cx43^+/− mice compared with TGEMs from Cx43^+/+ littermates. Cx43 was upregulated by a serum/glucocorticoid-regulated kinase 1 (SGK) activator and downregulated, along with inhibition of CM-induced TGEM migration, by knockdown of the SGK gene or blockade of the SGK pathway. LPS-induced SGK activation was abrogated by Torin2, whereas LPS-induced Cx43 was downregulated by both Torin2 and rapamycin. Analysis of the effects of FK506 and methylprednisolone, common immunosuppressive agents following organ transplantation, suggested a link between these immunosuppressive drugs and impaired macrophage migration via the Cx43/iNOS/Src/FAK pathway. In a model of Escherichia coli infectious peritonitis, GSK650349-, an SGK inhibitor, or Torin2-treated mice showed less accumulation of F4/80⁺CD11b⁺ macrophages in the peritoneal cavity, with a delay in the elimination of bacteria. Furthermore, following pretreatment with Gap19, a selective Cx43 hemichannel blocker, the survival of model mice was significantly reduced. Taken together, our study suggested that Cx43 in macrophages was associated with macrophage migration, an important immune process in host defense to infection.