Myeong Sup Lee

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway.

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.

OASL1 inhibits translation of the type I interferon-regulating transcription factor IRF7

The production of type I interferon is essential for viral clearance but is kept under tight control to avoid unnecessary tissue damage from hyperinflammatory responses. Here we found that OASL1 inhibited translation of IRF7, the master transcription factor for type I interferon, and thus negatively regulated the robust production of type I interferon during viral infection. OASL1 inhibited the translation of IRF7 mRNA by binding to the 5′ untranslated region (UTR) of IRF7 and possibly by inhibiting scanning of the 43S preinitiation complex along the message. Oasl1−/− mice were resistant to viral infection because of the greater abundance of type I interferon, which suggests that OASL1 could be a potential therapeutic target for boosting the production of type I interferon during viral infection.

Filamentous Artificial Virus from a Self‐Assembled Discrete Nanoribbon

The creation of virus-like nanomaterials (artificial viruses) has been the subject of intensive research in the field of gene/ drug delivery because of their huge therapeutic potential. Some progress has been made in the field; however, compared to the natural viruses, which are evolution-tailored experts in gene delivery, the synthetic system is still far behind the natural system. One of the critical reasons for this shortcoming is that the size and shape of the artificial viruses, among the most important determinants of their efficiency, are very difficult to control. As the polyanionic nature of nucleic acids (DNA and RNA) prevents them from crossing the same charged cytoplasmic membrane barrier, a vector system is necessary to neutralize their charge and to install other functions, such as cell binding, endosome escape, and nucleus localization. Although there are serious safety concerns, such as immunogenicity and carcinogenesis, in the use of viral vectors, they are still far more widely used for gene therapy than nonviral vectors (artificial viruses) because of their higher efficiency. The basic principle of artificial virus formation is a condensation reaction which is induced by the attraction between oppositely charged molecules (for example, between cationic polymer and DNA). However, this polyion coupling generally leads to the formation of huge nanoaggregates that are highly heterogeneous in size and shape, and is uncontrollable in most cases. In regard to the size issue, a certain optimal size exists for the nanoparticulate delivery systems to work best. The shape of the nanoparticle can also be a crucial factor. For example, sometimes the cylindrical (that is, filamentous) nanostructure has certain advantages over the spherical one in that it persists longer in vivo, which might explain why many filamentous viruses exist in nature. Therefore, it is imperative to find a general strategy to control the size and shape of artificial viruses. Recent advances in supramolecular chemistry have made it possible, by rational design of building blocks, to control supramolecular architectures from spherical micelles, cylindrical micelles, vesicles, and toroids to nanotubes. Inspired by this elaboration, we envisioned that the control of the size and shape of artificial viruses would be possible if the preorganized supramolecular architectures used were robust polycationic scaffolds that remain unchanged after the formation of an interpolyelectrolyte complex (IPC) with negatively charged nucleic acids. Herein, we report a potentially generalizable strategy to create an artificial virus that memorizes the size and shape of its precursor. By using a preorganized supramolecular nanostructure as a template, we show that well-defined, discrete artificial viruses can be elaborated after IPC formation between the nanostructure and nucleic acids. This controlled feature and appropriate surface functionalization with multivalent carbohydrate ligands make the artificial virus highly efficient in the intracellular delivery of genes and drugs. With the aim of constructing filament-shaped, discrete artificial viruses, a b-sheet peptide-based supramolecular building block (Glu-KW) was designed (Figure 1a). To our knowledge the filament-shaped artificial virus is unprecedented. It has been shown that the combination of hydrophobic and electrostatic interactions produced by the alternating placement of hydrophobic and charged amino acids in b-sheet peptides promotes b-sheet interaction and subsequent self-assembly into bilayered filamentous nanostructures (b ribbon). Coupling of hydrophilic segments, such as polyethylene glycol, hydrophilic peptides, or carbohydrates, to b-sheet peptides has been reported to stabilize b-ribbon nanostructures by suppressing lateral aggregate formation. The Glu-KW structure is characterized by a b-sheetforming self-assembly segment, two linker segments, a nucleic acid-binding cationic segment, and a carbohydrate ligand segment. The b-sheet peptide segment consists of tryptophan–lysine–tryptophan–aspartic acid repeats, the amino acid configuration of which promotes b-sheet formation. The linker segments are designed to be flexible and nonionic by using glycine and serine residues. The eight lysine residues are placed between two linker segments to shield the cationic segment, upon self-assembly, from the b-ribbon surface. dGlucose is positioned at the outermost part of Glu-KW to render the b-ribbon surface charge-neutral and to increase the chances of b-ribbon binding to the cell surface through multivalent interactions with cell-surface glucose transporters (GLUTs). GLUTs are present in nearly all mammalian cells and overexpressed in most cancer cells. To address the question of whether Glu-KW forms bsheet-mediated nanostructures, the self-assembly of Glu-KW was investigated by circular dichroism (CD) and transmission [*] Dr. Y.-b. Lim, E. Lee, Y.-R. Yoon, Prof. M. Lee Center for Supramolecular Nano-Assembly and Department of Chemistry Yonsei University, Seoul 120-749 (Korea) Fax: (+82)2-393-6096 E-mail: mslee@yonsei.ac.kr Homepage: http://csna.yonsei.ac.kr

Generation of knockout mice by Cpf1-mediated gene targeting

VOLUME 34 NUMBER 8 AUGUST 2016 NATURE BIOTECHNOLOGY target sites by four mismatches. We also electroporated an AsCpf1 RNP targeting Tyr, the gene encoding tyrosinase, into mouse embryos and observed mutations in 4 out of 12 blastocysts (33%) (Supplementary Fig. 6). Taken together, these results show that Cas9 and Cpf1 RNPs can be delivered efficiently by electroporation into animal embryos, resulting in high mutation frequencies. We next transplanted mouse embryos after Cpf1 microinjection or electroporation into surrogate mothers and obtained mice with targeted mutations in Foxn1 (Fig. 1d and Supplementary Fig. 7a) or Tyr (Supplementary Table 2 and Supplementary Fig. 8a). Three out of seven mice carried mutations at the Cpf1 cleavage site in the Foxn1 gene. One Tyr mutant mouse showed a partial coat color change, consistent with its mosaic genotype (Supplementary Fig. 8b). To investigate whether Cpf1 had offtarget effects, we performed whole genome sequencing using genomic DNA isolated from one Foxn1 mutant mouse and its wildtype sibling (Supplementary Note). The sequence analysis showed that no off-target mutations were introduced at homologous sites with up to 7-nucleotide mismatches. Notably, the mutant allele in a female Foxn1 mutant mouse was transmitted to embryos (Supplementary Fig. 7b,c). In summary, our results show that electroporation of AsCpf1 RNPs resulted in efficient and specific genome editing in mouse embryos. RNPs7 are as effective as mRNA8 or plasmids9, but are degraded rapidly by endogenous proteases and RNases in cells, and have been previously shown to reduce off-target effects9 and mosaicism10. Unlike microinjection, electroporation is easy to carry out, fast, and scalable. Up to 50 embryos can be electroporated simultaneously. We propose that electroporation of Cpf1 RNPs is a potentially useful new method for genome editing in animals.

Early-stage formation of an epigenetic field defect in a mouse colitis model, and non-essential roles of T- and B-cells in DNA methylation induction.

Epigenetic fields for cancerization are involved in development of human cancers, especially those associated with inflammation and multiple occurrences. However, it is still unclear when such field defects are formed and what component of inflammation is involved in induction of aberrant DNA methylation. Here, in a mouse colitis model induced by dextran sulfate sodium (DSS), we identified three CpG islands specifically methylated in colonic epithelial cells exposed to colitis. Their methylation levels started to increase as early as 8 weeks after DSS treatment and continued to increase until colon cancers developed at 15 weeks. In contrast to the temporal profile of DNA methylation levels, infiltration of inflammatory cells spiked immediately after the DSS treatment and then gradually decreased. Exposure of cultured colonic epithelial cells to DSS did not induce DNA methylation and it was indicated that inflammation triggered by the DSS treatment was responsible for methylation induction. To clarify components of inflammation involved, severe combined immunodeficiency (SCID) mice that lack functional T- and B-cells were similarly treated. Even in SCID mice, DNA methylation, along with colon tumors, were induced at the same levels as in their background strain of mice (C.B17). Comparative analysis of inflammation-related genes showed that Ifng, Il1b and Nos2 had expression concordant with methylation induction whereas Il2, Il6, Il10, Tnf did not. These results showed that an epigenetic field defect is formed at early stages of colitis-associated carcinogenesis and that functional T and B cells are non-essential for the formation.

GalaxyPepDock: a protein–peptide docking tool based on interaction similarity and energy optimization

Protein–peptide interactions are involved in a wide range of biological processes and are attractive targets for therapeutic purposes because of their small interfaces. Therefore, effective protein–peptide docking techniques can provide the basis for potential therapeutic applications by enabling an atomic-level understanding of protein interactions. With the increasing number of protein–peptide structures deposited in the protein data bank, the prediction accuracy of protein-peptide docking can be enhanced by utilizing the information provided by the database. The GalaxyPepDock web server, which is freely accessible at http://galaxy.seoklab.org/pepdock, performs similarity-based docking by finding templates from the database of experimentally determined structures and building models using energy-based optimization that allows for structural flexibility. The server can therefore effectively model the structural differences between the template and target protein–peptide complexes. The performance of GalaxyPepDock is superior to those of the other currently available web servers when tested on the PeptiDB set and on recently released complex structures. When tested on the CAPRI target 67, GalaxyPepDock generates models that are more accurate than the best server models submitted during the CAPRI blind prediction experiment.

Rab35 Mediates Transport of Cdc42 and Rac1 to the Plasma Membrane during Phagocytosis

ABSTRACT Phagocytosis of invading microbes requires dynamic rearrangement of the plasma membrane and its associated cytoskeletal actin network. The polarization of Cdc42 and Rac1 Rho GTPases to the site of plasma membrane protrusion is responsible for the remodeling of actin structures. However, the mechanism of Rho GTPase recruitment to these sites and the identities of accessory molecules involved in this process are not well understood. In this study, we uncovered several new components involved in innate immunity in Drosophila melanogaster. Our data demonstrate that Rab35 is a regulator of vesicle transport required specifically for phagocytosis. Moreover, recruitment of Cdc42 and Rac1 to the sites of filopodium and lamellipodium formation is Rab35 dependent and occurs by way of microtubule tracks. These results implicate Rab35 as the immune cell-specific regulator of vesicle transport within the actin-remodeling complex.

IL-7 production in murine lymphatic endothelial cells and induction in the setting of peripheral lymphopenia

IL-7 is a required factor for T-cell homeostasis. Because of low expression levels and poor reagent availability, the cellular sources of IL-7 have proven challenging to characterize. In this study, we describe a reporter mouse in which enhanced GFP is expressed from the endogenous Il7 locus. We show that IL-7 is produced by lymphatic endothelial cells (LECs) distributed throughout the systemic lymphatic vasculature as well as by fibroblastic reticular cells, and that phosphorylation of STAT5 in lymphocytes is higher in lymphatics than in blood. Furthermore, in nodes depleted of lymphocytes, Il7 transcription is increased in stromal but not in myeloid subsets. These data support recent findings that lymphocyte homeostasis is influenced by access to secondary lymphoid organs and point to LECs as an important in vivo source of IL-7, bathing trafficking immune cells under both resting and lymphopenic conditions.

GalaxySite: ligand-binding-site prediction by using molecular docking

Knowledge of ligand-binding sites of proteins provides invaluable information for functional studies, drug design and protein design. Recent progress in ligand-binding-site prediction methods has demonstrated that using information from similar proteins of known structures can improve predictions. The GalaxySite web server, freely accessible at http://galaxy.seoklab.org/site, combines such information with molecular docking for more precise binding-site prediction for non-metal ligands. According to the recent critical assessments of structure prediction methods held in 2010 and 2012, this server was found to be superior or comparable to other state-of-the-art programs in the category of ligand-binding-site prediction. A strong merit of the GalaxySite program is that it provides additional predictions on binding ligands and their binding poses in terms of the optimized 3D coordinates of the protein–ligand complexes, whereas other methods predict only identities of binding-site residues or copy binding geometry from similar proteins. The additional information on the specific binding geometry would be very useful for applications in functional studies and computer-aided drug discovery.

Negative Regulation of Type I IFN Expression by OASL1 Permits Chronic Viral Infection and CD8+ T-Cell Exhaustion

The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2′–5′ oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected Oasl1 knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected Oasl1 KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2–3 days post-infection). Oasl1 deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.