Innate Immune Training Initiates Efferocytosis to Protect against Lung Injury.

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 ß-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. ß-Glucan pretreatment induces neutrophil accumulation in the lungs and enhances efferocytosis. Training of mice with ß-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 ß-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.

Formulation of Glycyrrhizic Acid-based Nanocomplexes for Enhanced Anti-cancer and Anti-inflammatory Effects of Curcumin.

In this study, nanocomplexes composed of glycyrrhizic acid (GA) derived from the root of the licorice plant (Glycyrrhiza glabra) were formulated for the delivery of curcumin (CUR). Sonication of amphiphilic GA solution with hydrophobic CUR resulted in the production of nanosized complexes with a size of 164.8 ± 51.7 nm, which greatly enhanced the solubility of CUR in aqueous solution. A majority of the CURs were released from these GA/ CUR nanocomplexes within 12 h. GA/CUR nanocomplexes exhibited excellent intracellular uptake in human breast cancer cells (Michigan cancer foundation-7/multi-drug resistant cells), indicating enhanced anti-cancer effects compared to that of free CUR. In addition, GA/CUR nanocomplexes demonstrated high intracellular uptake into macrophages (RAW264.7 cells), consequently reducing the release of the pro-inflammatory cytokine tumor necrosis factor-α. Furthermore, GA/CUR nanocomplexes successfully reduced the levels of serum pro-inflammatory cytokines and splenomegaly in a rheumatoid arthritis model.

Enhanced intracellular uptake and stability of umbelliferone in compound mixtures from Angelica gigas in vitro.

Understanding how natural compounds work together for disease treatments can improve their clinical efficacy and therapeutic effects. To elucidate the mechanisms of synergistic biological effects in natural compound mixtures, umbelliferone (UMB, 7-hydroxycoumarin), derived from Angelica (A.) gigas, was selected as active compound with fluorescent characteristic to examine bioactivities in vitro in the presence of other compounds from Angelica gigas. Antioxidant effects of UMB in biochemical assays and cellular reactive oxygen species (ROS) levels in RAW264.7 cells were not significantly improved by addition of other compounds. However, intracellular uptake, inhibition of the efflux pump P-glycoprotein (P-gp), and physiological stability of UMB were greatly enhanced by the addition of other compounds, specifically Angelicin (ANG) and Byakangelicin (BYN). Taken together, enhanced intracellular localization and enzymatic stability in compound mixtures might lead to superior synergistic bioactivity of UMBs in compound mixtures.

Mannose-Modified Serum Exosomes for the Elevated Uptake to Murine Dendritic Cells and Lymphatic Accumulation.

The surface of bovine serum-derived exosomes (EXOs) are modified with α-d-mannose for facile interaction with mannose receptors on dendritic cells (DCs) and for efficient delivery of immune stimulators to the DCs. The surface of the EXOs is modified with polyethylene glycol (PEG) without particle aggregation (≈50 nm) via the incorporation of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) into the lipid layer of the EXO, compared to chemical conjugation by N-hydroxysuccinimide activated PEG (NHS-PEG). PEG modification onto the exosomal surface significantly decreases the non-specific cellular uptake of the EXOs into the DCs. However, the EXOs with mannose-conjugated PEG-DSPE (EXO-PEG-man) exhibit excellent intracellular uptake into the DCs and boost the immune response by the incorporation of adjuvant, monophosphoryl lipid A (MPLA) within the EXO. After an intradermal injection, a higher retention of EXO-PEG-man is observed in the lymph nodes, which could be used for the efficient delivery of immune stimulators and antigens to the lymph nodes in vivo.

Byakangelicin as a modulator for improved distribution and bioactivity of natural compounds and synthetic drugs in the brain.

BACKGROUND: The elucidation of the biological roles of individual active compounds in terms of their in vivo bio-distribution and bioactivity could provide crucial information to understand how natural compounds work together as treatments for diseases. PURPOSE: We examined the functional roles of Byakangelicin (Byn) to improve the brain accumulation of active compounds, e.g., umbelliferone (Umb), curcumin (Cur), and doxorubicin (Dox), and consequently to enhance their biological activities. METHODS: Active compounds were administered intravenously to mice, with or without Byn, after which organs were isolated and visualized for their ex vivo fluorescence imaging to determine the bio-distribution of each active compound in vivo. For the in vivo bioactivity, Cur, either with or without Byn, was administered to a lipopolysaccharide (LPS)-induced neuro-inflammation model for 5 days, and its anti-inflammatory effects were examined by ELISA using a brain homogenate and serum. RESULTS: We successfully demonstrated that the levels of active compounds (Umb, Cur, and Dox) in the brain, lung, and pancreas were greatly elevated by the addition of Byn via direct ex vivo fluorescence monitoring. In addition, sufficient accumulation of the active compound, Cur, greatly reduced LPS-induced neuro-inflammation in vivo. CONCLUSION: Byn could serve as a modulator to allow improved brain accumulation of diverse active compounds (Umb, Cur, and Dox) and enhanced therapeutic effects.

PLGA Microspheres Coated with Cancer Cell-Derived Vesicles for Improved Internalization into Antigen-Presenting Cells and Immune Stimulation.

Microspheres (MS; 1-3 µm) with different degrees of surface roughness were prepared to assess the effects of surface topology on internalization into antigen-presenting cells (APCs; macrophages and dendritic cells). In this study, we demonstrated that the intracellular uptake of MS is readily enhanced by surface modification with nanoparticles or cancer cell-derived vesicles (VE) to modulate their surface topology. MS coated with nanovesicles (MS-VE) with high surface roughness was more successfully and efficiently engulfed by APCs, compared with bare MS and those with low surface roughness. Incorporated MPLA within MS-VEs (M/MS-VE) triggered greatly elevated release of immune stimulating cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), from macrophages and dendritic cells, compared to free MPLA. Taken together, this MS-VE could serve as a platform system for the delivery of immune stimulators and antigens to APCs with negligible toxicity.

Platelet-derived nanovesicles for hemostasis without release of pro-inflammatory cytokines.

Natural platelet-derived nanovesicles with a vacant core were prepared by hypotonic sonication. The nanovesicles efficiently formed platelet-like aggregates using membrane protein in the presence of thrombin and calcium chloride without a notable release of pro-inflammatory cytokines. These natural and biocompatible platelet-derived nanovesicles have great potential as biomaterials for inflammation-free injectable hemostasis.

Correction: CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells.

[This corrects the article DOI: 10.1039/C7RA13293J.].

Efficient Delivery of Tyrosinase Related Protein-2 (TRP2) Peptides to Lymph Nodes using Serum-Derived Exosomes.

Exosomes (EXO) are considered to be versatile carriers for biomolecules; however, the delivery of therapeutic peptides using EXOs poses several challenges. In this study, the efficiency of serum-derived EXOs in delivering tyrosinase-related protein-2 (TRP2) peptides to lymph nodes is determined. TRP2 peptides are successfully incorporated into EXOs, which show a uniform and narrow size distribution of around 45 nm. The TRP2-incorporated exosomes (EXO-TRP2) are efficiently internalized into macrophages and dendritic cells, and are seen to display a punctate distribution. EXOs loaded with TRP2 together with MPLA, (EXO-MPLA-TRP2) result in a strong release of proinflammatory cytokines (TNF-α and IL-6) from both RAW264.7 and DC2.4 cells. Finally, subcutaneous injection of fluorescently labeled EXO-TRP2 followed by ex vivo imaging using in vivo imaging system (IVIS) show a strong fluorescent signal in the lymph nodes after only 1 h, which is maintained until at least 4 h after injection. Taken together, the findings suggest that serum-derived EXOs can serve as promising carriers to deliver therapeutic peptides to lymph nodes for immunotherapy.

Comparative evaluation of cell- and serum-derived exosomes to deliver immune stimulators to lymph nodes.

To determine whether exosomes are efficient carriers for immune stimulating molecules into lymph nodes, comparative studies of exosomes (EXOs) derived from different origins (cells and serums) in terms of physicochemical properties and delivery efficiency were performed. Serum-derived EXOs were of a preferable size and generated higher yields than RAW264.7 cell-derived exosomes (RAW-EXO). In particular, fetal bovine serum-derived exosomes (bo-EXO), with a size below 50 nm, were delivered not only to surface zones (subcapsular sinus (SCS) macrophage zone) but also to inner paracortex zones (T cell zone) of lymph nodes, which allowed an efficient delivery of immune stimulating molecules to antigen presenting cells and T cells. The encapsulation of immune stimulating biomolecules (monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN)) within EXOs greatly increased intracellular delivery to macrophages via phagocytic pathways, which induced higher TNF-α and IL-6 secretion than free MPLA and free CpG ODN. MPLA-incorporated exosomes activated and differentiated T cells after subcutaneous injection, which elevated cytokine IFN-γ and TNF-α induction for CD3+ T cells. Taken together, bo-EXOs might serve as efficient carrier systems of immune stimulators to lymph nodes for desired immune responses.

CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells.

As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used. For efficient induction of immune responses, current issues affecting the use of nucleic acid-based adjuvants, e.g. stability in physiological conditions, delivery to immune cells, and successful release within the phagolysosome, should be addressed. Here, we present CpG-based DNA microparticles (DNA-MPs) fabricated by complementary rolling circle amplification (cRCA) as adjuvants for enhancing immune response and production of selective antibody production. Using cRCA method, the sizes of CpG-based DNA-MPs were finely controlled (0.5 and 1 µm) with superior and provided mismatched single stranded form of CpG ODN region for specific cleavage site by DNase II within the phagolysosome. Fabricated CpG-based 1 µm DNA-MPs (DNA-MP-1.0) were successfully internalized into primary macrophages and macrophage cell line (RAW264.7 cells), and elicited superior cytokine production e.g. TNF-α and IL-6, compared to conventional CpG ODNs. After in vivo administration of DNA-MP-1.0 with model antigen ovalbumin (OVA), significantly elevated OVA-specific antibody production was observed. With this in mind, DNA-MP-1.0 could serve as a novel type of adjuvant for the activation of macrophages and the following production of selective antibodies without any noticeable toxicity in vitro and in vivo.

Complexation of curcumin with 2-aminoethyl diphenyl borate and implications for spatiotemporal fluorescence monitoring.

In this study, we successfully determined spatiotemporal distribution of curcumin in mice via simple and fast fluorescence detection of native curcumin and stabilized curcumin. We used 2-aminoethyl diphenyl borate (DPBA) as a stabilizer of curcumin, which binds to curcumin and enhances its aqueous stability. After intravenous injection, curcumin and DPBA-curcumin complexes showed similar fluorescence intensities in the brain, pancreas, lungs, and kidneys at 15min. However, stabilized DPBA-curcumin complexes exhibited much stronger fluorescent signals at metabolically active sites such as liver tissues than native curcumin. After incubation for 1-3h, native curcumin showed significantly rapid reduction of fluorescent signals, compared to DPBA-curcumin complexes, probably due to degradation and reduction. In addition, complicate extraction procedures inhibited precise fluorescent monitoring of unstable curcumin, which result in different biodistribution of curcumin before and after extraction. Direct fluorescent monitoring could allow evaluation of in vivo distribution and fate of curcumin, which could be also applied to diverse natural polyphenols with fluorescent signals.

Submicron-sized hydrogels incorporating cyclic dinucleotides for selective delivery and elevated cytokine release in macrophages.

Despite the emerging evidences supporting the potential of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) as a vaccine adjuvant, few properly designed micro-/nanocarriers for the delivery of cyclic dinucleotides have been developed. In this study, we formulated cGAMP within linear polyethyleneimine (LPEI)/hyaluronic acid (HA) hydrogels via inverse water-in-oil (W/O) emulsion/crosslinking. Spherical and cationic LPEI/HA hydrogels (LH gels) with a size of 455.3±3.1nm and a surface charge of 48.7±3.7mV were selectively and efficiently delivered into phagocytic macrophage cells, which are one type of antigen-presenting cells (APCs), but not into non-phagocytic fibroblast cells. LH gels incorporating cGAMP (LH/cGAMP gels) elicited excellent induction of the cytokines interferon-ß (IFN-ß) and interleukin-6 (IL-6). In particular, the amount of IFN-ß released by LH hydrogels was significantly increased by 2.5-fold compared to that released by conventional cationic liposomes, such as Lipofectamine. In addition, fabricated LH gels showed superior biocompatibility in phagocytic cell lines and primary bone marrow-derived macrophages (BMDMs). After intramuscular injection with ovalbumin into C57BL/6 mice, LH/cGAMP gels exhibited significantly elevated levels of anti-ovalbumin total IgG in serum and IFN-ß mRNA in spleens. Thus, the newly designed cGAMP-incorporating hydrogels can serve as safe and potent adjuvants for vaccination and immunotherapy. STATEMENT OF SIGNIFICANCE: Since cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) was first found as a second messenger of immune signaling in human systems in February 2013 (Science, 15, 826), several scientific studies have been reported related to the potential of cGAMP as a vaccine adjuvant or additive for immunotherapy. However, only naked cGAMP without carriers were studied via intramuscular or intranasal administration so far. In our study, we first investigated the feasibility of polymeric hydrogels incorporating cGAMP in terms of selective uptake into phagocytic antigen presenting cells (APCs), induction of cytokines, production of target antibodies, and biocompatibility for vaccination and immunotherapy in vitro and in vivo. Therefore, we believe this manuscript would be of great interest to the biomaterial communities especially who are studying immunotherapy.

Current preclinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics.

Recent promising clinical results of RNA therapeutics have drawn big attention of academia and industries to RNA therapeutics and their carrier systems. To improve their feasibility in clinics, systemic evaluations of currently available carrier systems under clinical trials and preclinical studies are needed. In this review, we focus on recent noticeable preclinical studies and clinical results regarding siRNA-based conjugates for clinical translations. Advantages and drawbacks of siRNA-based conjugates are discussed, compared to particle-based delivery systems. Then, representative siRNA-based conjugates with aptamers, peptides, carbohydrates, lipids, polymers, and nanostructured materials are introduced. To improve feasibility of siRNA conjugates in preclinical studies, several considerations for the rational design of siRNA conjugates in terms of cleavability, immune responses, multivalent conjugations, and mechanism of action are also presented. Lastly, we discuss lessons from previous preclinical and clinical studies related to siRNA conjugates and perspectives of their clinical applications.

Multivalent aptamer-RNA based fluorescent probes for carrier-free detection of cellular microRNA-34a in mucin1-expressing cancer cells.

In this study, multivalent carrier-free aptamer-RNA based fluorescent probes (CF-probes) were designed as a simpler, more reliable, timesaving strategy for cellular miRNA detection. CF-probes spontaneously delivered into cells without the need for additional carriers and visualized target microRNA-34a specifically.

Deficiency of developmental endothelial locus-1 (Del-1) aggravates bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is a lung disease wherein lung parenchyma is gradually and irreversibly replaced with collagen. The molecular pathogenesis of pulmonary fibrosis is not fully understood and the only effective treatment available is lung transplantation. To test if Del-1, an endogenous anti-inflammatory molecule, may be implicated in the development of pulmonary fibrosis, we induced pulmonary fibrosis in wild type (WT) and Del-1(-/-) mice by intratracheal administration of bleomycin. Del-1 expression in the lung was decreased in the WT mice treated with bleomycin compared to control mice. In addition, bleomycin-induced pulmonary fibrosis increased collagen deposition and TGF-ß production in the lung of Del-1(-/-) mice. Finally, Del-1(-/-) mice treated with bleomycin displayed higher weight loss and greater mortality than did WT mice identically treated. These findings suggest that Del-1 may negatively regulate development of pulmonary fibrosis. Further delineation of a role for Del-1 in the development of pulmonary fibrosis will broaden our understanding of the molecular pathogenesis of this disease and hopefully help develop potential therapeutics.

Developmental endothelial locus-1 inhibits MIF production through suppression of NF-κB in macrophages.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that regulates leukocyte recruitment, thereby playing a pivotal role in the regulation of innate and adaptive immunity and tumor progression. Elevated levels of MIF are associated with numerous inflammatory disorders and cancers. To determine whether developmental endothelial locus-1 (Del-1) regulated MIF, RAW264.7 macrophages were treated with Del-1 and assessed using ELISA. The results showed that MIF was downregulated in macrophages by Del-1, an endogenous anti-inflammatory protein that was previously shown to limit leukocyte adhesion and migration. Treatment of RAW264.7 macrophages with Del-1 inhibited constitutive and lipopolysaccharide (LPS)-induced MIF secretion. Recombinant Del-1 protein attenuated the phosphorylation of IκBα induced by a relatively low concentration of LPS in THP-1 monocytes, but did not inhibit IκBα phosphorylation in response to a relatively high concentration of LPS. Concomitantly, translocation of NF-κB to the nucleus was inhibited by Del-1 in LPS-activated macrophages. In addition, conditioned medium harvested from cells transfected with a Del-1 expression plasmid suppressed NF-κB activation in response to relatively low concentrations of TNF-α, albeit not the activation that was induced by a relatively high concentration of TNF-α. On the other hand, although Del-1 enhanced the macrophage expression of p53, a known negative regulator of MIF production, MIF production was not significantly affected by the level of p53 in mouse bone marrow-derived macrophages. These findings suggested that Del-1 controls NF-κB-activated MIF production in macrophages, and the potential application of Del-1 to therapeutic modalities for chronic inflammation-associated cancers.