Tumor-Localized Administration of α-GalCer to Recruit Invariant Natural Killer T Cells and Enhance Their Antitumor Activity against Solid Tumors

Invariant natural killer T (iNKT) cells have the capacity to mount potent anti-tumor reactivity and have therefore become a focus in the development of cell-based immunotherapy. iNKT cells attack tumor cells using multiple mechanisms with a high efficacy; however, their clinical application has been limited because of their low numbers in cancer patients and difficulties in infiltrating solid tumors. In this study, we aimed to overcome these critical limitations by using α-GalCer, a synthetic glycolipid ligand specifically activating iNKT cells, to recruit iNKT to solid tumors. By adoptively transferring human iNKT cells into tumor-bearing humanized NSG mice and administering a single dose of tumor-localized α-GalCer, we demonstrated the rapid recruitment of human iNKT cells into solid tumors in as little as one day and a significantly enhanced tumor killing ability. Using firefly luciferase-labeled iNKT cells, we monitored the tissue biodistribution and pharmacokinetics/pharmacodynamics (PK/PD) of human iNKT cells in tumor-bearing NSG mice. Collectively, these preclinical studies demonstrate the promise of an αGC-driven iNKT cell-based immunotherapy to target solid tumors with higher efficacy and precision.     

Benzyl Isothiocyanate Attenuates Inflammasome Activation in Pseudomonas aeruginosa LPS-Stimulated THP-1 Cells and Exerts Regulation through the MAPKs/NF-κB Pathway

Inflammasomes are a group of intracellular multiprotein platforms that play important roles in immune systems. Benzyl isothiocyanate (BITC) is a constituent of cruciferous plants and has been confirmed to exhibit various biological activities. The modulatory effects of BITC on inflammasome-mediated interleukin (IL)-1β expression and its regulatory mechanisms in Pseudomonas aeruginosa (P. aeruginosa) LPS/ATP-stimulated THP-1 cells was investigated. Monocytic THP-1 cells were treated with phorbol myristate acetate (PMA) to induce differentiation into macrophages. Enzyme-linked immunosorbent assays (ELISA) were performed to measure the levels of IL-1β produced in P. aeruginosa LPS/ATP-exposed THP-1 cells. Western blotting was performed to examine the BITC modulatory mechanisms in inflammasome-mediated signaling pathways. BITC inhibited IL-1β production in P. aeruginosa LPS/ATP-induced THP-1 cells. BITC also inhibited activation of leucine-rich repeat protein-3 (NLRP3) and caspase-1 in P. aeruginosa LPS/ATP-induced THP-1 cells. Furthermore, we show that mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation in P. aeruginosa LPS was attenuated by BITC. These BITC-mediated modulatory effects on IL-1β production may have therapeutic potential for inflammasome-mediated disorders such as a nasal polyp.     

Pseudomonas aeruginosa DnaK Stimulates the Production of Pentraxin 3 via TLR4-Dependent NF-κB and ERK Signaling Pathways

Microbe-derived factors trigger innate immune responses through the production of inflammatory mediators, including pentraxin 3 (PTX3). PTX3 is a soluble pattern recognition molecule that stimulates the clearance of clinically important bacterial pathogens such as Pseudomonas aeruginosa. However, the P. aeruginosa factors responsible for the production of PTX3 have not been elucidated. In this study, we found that P. aeruginosa DnaK, a homolog of heat shock protein 70, induced PTX3 production. Induction was mediated by intracellular signals transmitted through the Toll-like receptor 4 (TLR4) signaling pathway. Following receptor engagement, the stimulatory signals were relayed initially through the nuclear factor kappa B (NF-κB) signaling pathway and subsequently by extracellular signal-regulated kinases (ERK), which are mitogen-activated protein kinases. However, ERK activation was negatively controlled by NF-κB, implying the existence of negative crosstalk between the NF-κB and the ERK pathways. These data suggest that P. aeruginosa DnaK acts as a pathogen-associated molecular pattern to trigger modulation of host defense responses via production of PTX3.     

HSP70-Homolog DnaK of Pseudomonas aeruginosa Increases the Production of IL-27 through Expression of EBI3 via TLR4-Dependent NF-κB and TLR4-Independent Akt Signaling

IL-27, a heterodimeric cytokine composed of the p28 subunit and Epstein–Barr virus-induced gene 3 (EBI3), acts as a potent immunosuppressant and thus limits pathogenic inflammatory responses. IL-27 is upregulated upon Pseudomonas aeruginosa infection in septic mice, increasing susceptibility to the infection and decreasing clearance of the pathogen. However, it remains unclear which P. aeruginosa-derived molecules promote production of IL-27. In this study, we explored the mechanism by which P. aeruginosa DnaK, a heat shock protein 70-like protein, induces EBI3 expression, thereby promoting production of IL-27. Upregulation of EBI3 expression did not lead to an increase in IL-35, which consists of the p35 subunit and EBI3. The IL-27 production in response to DnaK was biologically active, as reflected by stimulation of IL-10 production. DnaK-mediated expression of EBI3 was driven by two distinct signaling pathways, NF-κB and Akt. However, NF-κB is linked to TLR4-associated signaling pathways, whereas Akt is not. Taken together, our results reveal that P. aeruginosa DnaK potently upregulates EBI3 expression, which in turn drives production of the prominent anti-inflammatory cytokine IL-27, as a consequence of TLR4-dependent activation of NF-κB and TLR4-independent activation of the Akt signaling pathway.     

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1β, IL-4 and IL-17 are associated with different phenotypes of T helper (T_H) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to T_H cells and macrophages. We studied the effects of IFNγ, IL-1β, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1β [in the C(IL-1β) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1β) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1β) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFβ signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to T_H cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.     

Skin Resident γδ T Cell Function and Regulation in Wound Repair

The skin is a critical barrier that protects against damage and infection. Within the epidermis and dermis reside γδ T cells that play a variety of key roles in wound healing and tissue homeostasis. Skin-resident γδ T cells require T cell receptor (TCR) ligation, costimulation, and cytokine reception to mediate keratinocyte activity and inflammatory responses at the wound site for proper wound repair. While both epidermal and dermal γδ T cells regulate inflammatory responses in wound healing, the timing and factors produced are distinct. In the absence of growth factors, cytokines, and chemokines produced by γδ T cells, wound repair is negatively impacted. This disruption in γδ T cell function is apparent in metabolic diseases such as obesity and type 2 diabetes. This review provides the current state of knowledge on skin γδ T cell activation, regulation, and function in skin homeostasis and repair in mice and humans. As we uncover more about the complex roles played by γδ T cells in wound healing, novel targets can be discovered for future clinical therapies.     

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.     

Evaluation of the Anti-Atopic Dermatitis Effects of α-Boswellic Acid on Tnf-α/Ifn-γ-Stimulated HaCat Cells and DNCB-Induced BALB/c Mice

Boswellic acids, triterpenoids derived from the genus Boswellia (Burseraceae), are known for their anti-inflammatory and anti-tumor efficacy. Atopic dermatitis is a chronic, non-infectious inflammatory skin disease. However, the effects of α-boswellic acid on atopic dermatitis have not been studied. Therefore, in this study we examined the expression level of pro-inflammatory cytokines, histopathological analysis, and physiological data from BALB/c mice with atopic-like dermatitis induced by 2,4-dinitrochlorobenzene and TNF-α/IFN-γ-stimulated HaCaT cells to better understand the agent’s anti-atopic dermatitis efficacy. First, we found that α-boswellic reduced the epidermal thickening, mast cell numbers, and dermal infiltration of 2,4-dinitrochlorobenzene-induced atopic-like dermatitis in BALB/c mice. Furthermore, we also found that α-boswellic acid can restore transepidermal water loss and skin reddening in mice. In human keratinocytes inflamed by TNF-α/IFN-γ, α-boswellic acid inhibited MAP kinase activation and showed a reduction in NF-κB nuclear translocation. Finally, α-boswellic acid can reduce the expression level of cytokines (IL-1β, IL-6, and IL-8) following the stimulation of TNF-α/IFN-γ in HaCaT cells. Taken together, our study suggests that α-boswellic acids are a potential component for the development of anti-atopic dermatitis drugs.     

Elucidating the Ability of CGRP to Modulate Microvascular Events in Mouse Skin

Oedema formation and polymorphonuclear leukocyte (neutrophil) accumulation are involved in both acute and chronic inflammation. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that is released from stimulated sensory nerves. CGRP is a potent vasodilator neuropeptide, especially when administered to the cutaneous microvasculature, with a long duration of action. Here, we have investigated the ability of vasodilator amounts of CGRP to modulate oedema formation and neutrophil accumulation induced in the cutaneous microvasculature of the mouse. To learn more about the mechanism of action of endogenous CGRP, we have investigated the response to the inflammatory stimulants tumour necrosis factor alpha (TNFα) and carrageenan in three different murine models: a model where sensory nerves were depleted by resiniferatoxin (RTX); a pharmacological method to investigate the effect of a selective CGRP receptor antagonist; and a genetic approach using wildtype (WT) and αCGRP knockout (KO) mice. Our results show that exogenous CGRP potentiates oedema formation induced by substance P (SP) and TNFα. This is further supported by our findings from sensory nerve-depleted mice (in the absence of all neuropeptides), which indicated that sensory nerves are involved in mediating the oedema formation and neutrophil accumulation induced by TNFα, and also carrageenan in cutaneous microvasculature. Furthermore, endogenous CGRP was shown to contribute to this inflammatory response as carrageenan-induced oedema formation is attenuated in WT mice treated with the CGRP receptor antagonist, and in αCGRPKO mice. It is therefore concluded that CGRP can contribute to inflammation by promoting oedema formation in skin, but this response is dependent on the pro-inflammatory stimulus and circumstance.     

Effect of Vitamin C/Hydrocortisone Immobilization within Curdlan-Based Wound Dressings on In Vitro Cellular Response in Context of the Management of Chronic and Burn Wounds

Bioactive dressings are usually produced using natural or synthetic polymers. Recently, special attention has been paid to β-glucans that act as immunomodulators and have pro-healing properties. The aim of this research was to use β-1,3-glucan (curdlan) as a base for the production of bioactive dressing materials (curdlan/agarose and curdlan/chitosan) that were additionally enriched with vitamin C and/or hydrocortisone to improve healing of chronic and burn wounds. The secondary goal of the study was to compressively evaluate biological properties of the biomaterials. In this work, it was shown that vitamin C/hydrocortisone-enriched biomaterials exhibited faster vitamin C release profile than hydrocortisone. Consecutive release of the drugs is a desired phenomenon since it protects wounds against accumulation of high and toxic concentrations of the bioactive molecules. Moreover, biomaterials showed gradual release of low doses of the hydrocortisone, which is beneficial during management of burn wounds with hypergranulation tissue. Among all tested variants of biomaterials, dressing materials enriched with hydrocortisone and a mixture of vitamin C/hydrocortisone showed the best therapeutic potential since they had the ability to significantly reduce MMP-2 synthesis by macrophages and increase TGF-β1 release by skin cells. Moreover, materials containing hydrocortisone and its blend with vitamin C stimulated type I collagen deposition by fibroblasts and positively affected their migration and proliferation. Results of the experiments clearly showed that the developed biomaterials enriched with bioactive agents may be promising dressings for the management of non-healing chronic and burn wounds.     

Lactosamine‐Based Derivatives as Tools to Delineate the Biological Functions of Galectins: Application to Skin Tissue Repair

Galectins have been recognized as potential novel therapeutic targets for the numerous fundamental biological processes in which they are involved. Galectins are key players in homeostasis, and as such their expression and function are finely tuned in vivo. Thus, their modes of action are complex and remain largely unexplored, partly because of the lack of dedicated tools. We thus designed galectin inhibitors from a lactosamine core, functionalized at key C2 and C3′ positions by aromatic substituents to ensure both high affinity and selectivity, and equipped with a spacer that can be modified on demand to further modulate their physico‐chemical properties. As a proof‐of‐concept, galectin‐3 was selectively targeted. The efficacy of the synthesized di‐aromatic lactosamine tools was shown in cellular assays to modulate collective epithelial cell migration and to interfere with actin/cortactin localization.     

Immunogenicity and Immunoprotection of PCV2 Virus-like Particles Incorporating Dominant T and B Cell Antigenic Epitopes Paired with CD154 Molecules in Piglets and Mice

Porcine circovirus type 2 (PCV2) is capable of causing porcine circovirus-associated disease (PCVAD) and is one of the major threats to the global pig industry. The nucleocapsid protein Cap encoded by the PCV2 ORF2 gene is an ideal antigen for the development of PCV2 subunit vaccines, and its N-terminal nuclear localization sequence (NLS) structural domain is essential for the formation of self-assembling VLPs. In the present study, we systematically expressed and characterized full-length PCV2 Cap proteins fused to dominant T and B cell antigenic epitopes and porcine-derived CD154 molecules using baculovirus and found that the Cap proteins fusing epitopes were still capable of forming virus-like particles (VLPs). Both piglet and mice experiments showed that the Cap proteins fusing epitopes or paired with the molecular adjuvant CD154 were able to induce higher levels of humoral and cellular responses, particularly the secretion of PCV2-specific IFN-γ and IL-4. In addition, vaccination significantly reduced clinical signs and the viral load of PCV2 in the blood and tissues of challenged piglets. The results of the study provide new ideas for the development of a more efficient, safe and broad-spectrum next-generation PCV2 subunit vaccine.