Kihyuk Shin

Designed Nanocage Displaying Ligand-Specific Peptide Bunches for High Affinity and Biological Activity

Protein-cage nanoparticles are promising multifunctional platforms for targeted delivery of imaging and therapeutic agents owing to their biocompatibility, biodegradability, and low toxicity. The major advantage of protein-cage nanoparticles is the ability to decorate their surfaces with multiple functionalities through genetic and chemical modification to achieve desired properties for therapeutic and/or diagnostic purposes. Specific peptides identified by phage display can be genetically fused onto the surface of cage proteins to promote the association of nanoparticles with a particular cell type or tissue. Upon symmetrical assembly of the cage, peptides are clustered on the surface of the cage protein in bunches. The resulting PBNC (peptide bunches on nanocage) offers the potential of synergistically increasing the avidity of the peptide ligands, thereby enhancing their blocking ability for therapeutic purposes. Here, we demonstrated a proof-of-principle of PBNCs, fusing the interleukin-4 receptor (IL-4R)-targeting peptide, AP-1, identified previously by phage display, with ferritin-L-chain (FTL), which undergoes 24-subunit assembly to form highly stable AP-1-containing nanocage proteins (AP1-PBNCs). AP1-PBNCs bound specifically to the IL-4R-expressing cell line, A549, and their binding and internalization were specifically blocked by anti-IL-4R antibody. AP1-PBNCs exhibited dramatically enhanced binding avidity to IL-4R compared with AP-1 peptide, measured by surface plasmon resonance spectroscopy. Furthermore, treatment with AP1-PBNCs in a murine model of experimental asthma diminished airway hyper-responsiveness and eosinophilic airway inflammation along with decreased mucus hyperproduction. These findings hold great promise for the application of various PBNCs with ligand-specific peptides in therapeutics for different diseases, such as cancer.

TH2 cells and their cytokines regulate formation and function of lymphatic vessels.

Lymphatic vessels (LVs) are critical for immune surveillance and involved in the pathogenesis of diverse diseases. LV density is increased during inflammation; however, little is known about how the resolution of LVs is controlled in different inflammatory conditions. Here we show the negative effects of T helper type 2 (TH2) cells and their cytokines on LV formation. IL-4 and IL-13 downregulate essential transcription factors of lymphatic endothelial cells (LECs) and inhibit tube formation. Co-culture of LECs with TH2 cells also inhibits tube formation, but this effect is fully reversed by interleukin (IL)-4 and/or IL-13 neutralization. Furthermore, the in vivo blockade of IL-4 and/or IL-13 in an asthma model not only increases the density but also enhances the function of lung LVs. These results demonstrate an anti-lymphangiogenic function of TH2 cells and their cytokines, suggesting a potential usefulness of IL-4 and/or IL-13 antagonist as therapeutic agents for allergic asthma through expanding LV mediated-enhanced antigen clearance from the inflammatory sites.

Innate type 2 immunity is associated with eosinophilic pleural effusion in primary spontaneous pneumothorax.

RATIONALE Eosinophilic pleural effusion (EPE) is characterized by greater than 10% eosinophilia and is frequently associated with air and/or blood in the pleural cavity. Primary spontaneous pneumothorax (PSP), defined as the spontaneous presence of air in the pleural space, is one of the most common causes of EPE. Recent studies have shown that type 2 immune responses play important roles in eosinophilic airway inflammation resulting in pleural pathology. OBJECTIVES To determine the predominant immune responses associated with PSP in humans, and to examine whether IL-33, thymic stromal lymphopoietin (TSLP), or type 2 innate lymphoid cell (ILC2)-mediated immune responses are associated factors. METHODS Eosinophil-associated cytokines were measured in the pleural fluid of patients with PSP and control subjects. Th2 cell and ILC2 responses in the pleural cavity and peripheral blood were also evaluated by in vitro restimulation and intracellular cytokine staining of T cells and ILC2s in patients with PSP (n = 62) and control subjects (n = 33). IL-33-mediated IL-5 production by ILC2s was also evaluated. MEASUREMENTS AND MAIN RESULTS Significantly higher concentrations of IL-5 and eotaxin-3 were detected in the pleural fluid of patients with PSP, in addition to significantly higher concentrations of IL-33 and TSLP. Although IL-5 production was induced by IL-33 treatment of ILC2s, other Th2 cell-mediated immune responses were not detected. CONCLUSIONS Our results indicate that innate immune responses characterized by the production of IL-33, TSLP, and IL-5 are associated with the development of EPE in PSP by an ILC2-dependent and Th2-independent mechanism.

Interplay between Inflammatory Responses and Lymphatic Vessels

Lymphatic vessels are routes for leukocyte migration and fluid drainage. In addition to their passive roles in migration of leukocytes, increasing evidence indicates their active roles in immune regulation. Tissue inflammation rapidly induces lymphatic endothelial cell proliferation and chemokine production, thereby resulting in lymphangiogenesis. Furthermore, lymphatic endothelial cells induce T cell tolerance through various mechanisms. In this review, we focus on the current knowledge on how inflammatory cytokines affect lymphangiogenesis and the roles of lymphatic vessels in modulating immune responses.

Macelignan attenuated allergic lung inflammation and airway hyper-responsiveness in murine experimental asthma

AIMS Macelignan isolated from Myristica fragrans Houtt. is widely used for spice and flavoring for foods, and has been reported to have anti-inflammatory activity. The aim of this study was to investigate the effects of macelignan on allergic lung inflammation with a murine model of experimental asthma. MAIN METHODS Fungal protease mixed with chicken egg ovalbumin allergen was used as a challenge to induce murine experimental asthma. To determine its effects on allergy and inflammation, macelignan was administered orally during allergen challenge, and the symptoms of allergic asthma and its underlined mechanisms were examined. KEY FINDINGS Treatment with macelignan attenuated eosinophilic airway inflammation and airway hyper-responsiveness. With the administration of macelignan, interleukin-4 (IL-4) producing cells, but not interferon-γ (IFN-γ) or IL-17 producing cells, were diminished in the lungs. Additionally, activation of the T helper type 2 (Th2) cell-specific master transcription factor, GATA3 was decreased with macelignan treatment. Finally, production of IL-4 but not IFN-γ or IL-17, by CD4(+) T cells was reduced with stimulation when combined with the administration of macelignan. SIGNIFICANCE Our data show that macelignan has anti-inflammatory effects on Th2 cell-mediated allergic lung inflammation and could potentially provide a novel preventative and/or therapy for the treatment of allergic diseases.

Leukotriene enhanced allergic lung inflammation through induction of chemokine production

The leukotrienes (LTs) enhance allergen- and interleukin (IL)-13-dependent allergic lung inflammatory disease. However, the precise requirement of LTs and the mechanism by which they elicit allergic lung responses remain uncertain. To clarify the involvement of LTs in respiratory allergen- and IL-13-induced experimental asthma and elucidate the underlying mechanisms of LTs-mediated enhanced allergic asthma, we investigated the role of LTs in two models of allergic inflammation: intranasal Aspergillus protease allergen and recombinant IL-13-induced T helper type 2 (Th2) cell-mediated inflammation, and also examined Th2-related chemokines downstream of LTs signaling. 5-Lipoxygenase (5-LO)-deficient mice exposed to short-term intranasal Aspergillus protease allergen showed attenuated airway inflammation, decreased airway hyper-responsiveness and reduced bronchoalveolar eosinophilia when compared to wild-type mice. However, this phenotype was less apparent using long exposure to the same allergen. 5-LO-deficient mice exposed to intranasal rIL-13 also showed attenuated phenotypes of allergic asthma via significant reduction in Th2-specific chemokines, CCL7 and CCL17 production and decreased Th2 cells recruitment to the lungs. Addition of leukotriene B4 (LTB4) and LTC4 to the airways of 5-LO-deficient mice resulted in the rescue of rIL-13-induced experimental asthma. Furthermore, LTs addition to rIL-13 synergistically enhanced the production of Th2-specific chemokines in the lung and inflammatory responses. Therefore, our findings suggest that LTs complement allergens and their downstream cytokine (e.g., IL-13) induced Th2 inflammation by enhancing the induction of Th2 chemokines.

Role of Citrullinated Fibrinogen Peptides in the Activation of CD4 T Cells from Patients with Rheumatoid Arthritis

This study was conducted to determine whether CD4 T cell responses to citrullinated fibrinogen occur in patients with rheumatoid arthritis (RA), especially in HLA-DR4-positive subjects. Whole peripheral blood mononuclear cells (PBMCs) of RA patients and control subjects were stimulated with citrullinated fibrinogen peptides, and T-cell production of proliferation and proinflammatory cytokines, such as interferon-γ(IFN-γ) and interleukin-17A (IL-17A), were measured. In addition, CD4 T cells from RA patients were stimulated with the citrullinated fibrinogen peptide, Fib-α R84Cit, identified as a DRB1*0401-restricted T cell epitope in HLA-DR4 transgenic mice, and the degree of T cell activation was examined similarly. No proliferative responses to the citrullinated fibrinogen peptides were observed in whole PBMCs or CD4 T cells from RA patients. Furthermore, no increased production of IFN-γ or IL-17A was found in whole PBMCs or CD4 T cells stimulated with the citrullinated fibrinogen peptides, although these cells responded to recall antigen, a mixture of tetanus toxoid, purified protein derivative (PPD) from Mycobacterium tuberculosis, and Candida albicans. The results of this study indicate that anti-citrulline immunity in RA patients may be mediated by fibrinogen because there is no evidence of CD4 T cell-mediated immune responses to citrullinated fibrinogen peptides.

Profiling of protein–protein interactions via single-molecule techniques predicts the dependence of cancers on growth-factor receptors

The accumulation of genetic and epigenetic alterations in cancer cells rewires cellular signalling pathways through changes in the patterns of protein–protein interactions (PPIs). Understanding these patterns may facilitate the design of tailored cancer therapies. Here, we show that single-molecule pull-down and co-immunoprecipitation techniques can be used to characterize signalling complexes of the human epidermal growth-factor receptor (HER) family in specific cancers. By analysing cancer-specific signalling phenotypes, including post-translational modifications and PPIs with downstream interactions, we found that activating mutations of the epidermal growth-factor receptor (EGFR) gene led to the formation of large protein complexes surrounding mutant EGFR proteins and to a reduction in the dependency of mutant EGFR signalling on phosphotyrosine residues, and that the strength of HER-family PPIs is correlated with the strength of the dependence of breast and lung adenocarcinoma cells on HER-family signalling pathways. Furthermore, using co-immunoprecipitation profiling to screen for EGFR-dependent cancers, we identified non-small-cell lung cancers that respond to an EGFR-targeted inhibitor. Our approach might help predict responses to targeted cancer therapies, particularly for cancers that lack actionable genomic mutations.The characterization of protein–protein interactions by combined single-molecule pull-down and single-molecule co-immunoprecipitation assays uncovers, for specific cancers, their dependence on signalling complexes of the human epidermal growth-factor receptor.

Circulating Anti-Elastin Antibody Levels and Arterial Disease Characteristics: Associations with Arterial Stiffness and Atherosclerosis

Purpose Elastin is a major arterial structural protein, and elastin-derived peptides are related to arterial change. We previously reported on a novel assay developed using aortic elastin peptides; however, its clinical implications remain unclear. In this study, we assessed whether anti-elastin antibody titers reflect the risk of coronary artery disease (CAD) or its characteristics. Materials and Methods We included 174 CAD patients and 171 age- and sex-matched controls. Anti-elastin antibody titers were quantified by enzyme-linked immunosorbent assay. Parameters of arterial stiffness, including the augmentation index (AI) and heart-to-femoral pulse wave velocity (hfPWV), were measured non-invasively. The clinical and angiographic characteristics of CAD patients were also evaluated. Associations between anti-elastin levels and vascular characteristics were examined by linear regression analysis. Results The median blood level of anti-elastin was significantly lower in the CAD group than in the controls [197 arbitrary unit (a.u.) vs. 63 a.u., p<0.001]. Levels of anti-elastin were significantly lower in men and in subjects with hypertension, diabetes mellitus, hyperlipidemia, or high hfPWV. Nevertheless, anti-elastin levels were not dependent on atherothrombotic events or the angiographic severity of CAD. In a multivariate analysis, male sex (β=-0.38, p<0.001), diabetes mellitus (β=-0.62, p<0.001), hyperlipidemia (β=-0.29, p<0.001), and AI (β=-0.006, p=0.02) were ultimately identified as determinants of anti-elastin levels. Conclusion Lower levels of anti-elastin are related to CAD. The association between antibody titers and CAD is linked to arterial stiffness rather than the advancement of atherosclerosis.