Human Monocyte-Derived Dendritic Cells Are the Pharmacological Target of the Immunosuppressant Flavonoid Silibinin

Silibinin, a natural polyphenolic flavonoid, is known to possess anti-inflammatory, anticancer, antioxidant, and immunomodulatory properties. However, the effects of Silibinin on the maturation and immunostimulatory functions of human dendritic cells (DC) remain to be elucidated. In this study, we have attempted to ascertain whether Silibinin influences the maturation, cytokine production, and antigen-presenting capacity of human monocyte-derived DC. We show that Silibinin significantly suppresses the upregulation of costimulatory and MHC molecules in LPS-stimulated mature DC and inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-12, IL-23, and TNF-α production. Furthermore, Silibinin impairs the proliferation response of the allogenic memory CD4 T lymphocytes elicited by LPS-matured DC and their Th1/Th17 profile. These findings demonstrate that Silibinin displays immunosuppressive activity by inhibiting the maturation and activation of human DC and support its potential application of adjuvant therapy in the treatment of autoimmune diseases.     

The Phagocytosis of Lacticaseibacillus casei and Its Immunomodulatory Properties on Human Monocyte-Derived Dendritic Cells Depend on the Expression of Lc-p75, a Bacterial Peptidoglycan Hydrolase

The human gut symbiont Lacticaseibacillus (L.) casei (previously Lactobacillus casei) is under intense research due to its wide range of immunomodulatory effects on the human host. Dendritic cells (DCs) are crucial players in the direct and indirect communication with lactobacilli in the gastrointestinal tract. Here, we demonstrate that human monocyte-derived DCs (moDCs) are able to engulf L. casei BL23, in which the intact bacterial cell wall and morphology have a key role. The absence of the bacterial cell-wall-degrading enzyme, Lc-p75, in L. casei cells causes remarkable morphological changes, which have important consequences in the phagocytosis of L. casei by moDCs. Our results showed that the Lc-p75 mutation induced defective internalization and impaired proinflammatory and T-cell-polarizing cytokine secretion by bacteria-exposed moDCs. The T helper (Th) 1 and Th17 cell activating capacity of moDCs induced by the mutant L. casei was consequently reduced. Moreover, inhibition of the phagocytosis of wild-type bacteria showed similar results. Taken together, these data suggested that formation of short bacterial chains helps to exert the potent immunomodulatory properties of L. casei BL23.     

Hypoxia Enhances the Expression of RNASET2 in Human Monocyte-Derived Dendritic Cells: Role of PI3K/AKT Pathway

Hypoxia is a key component of the tumor microenvironment (TME) and promotes not only tumor growth and metastasis, but also negatively affects infiltrating immune cells by impairing host immunity. Dendritic cells (DCs) are the most potent antigen-presenting cells and their biology is weakened in the TME in many ways, including the modulation of their viability. RNASET2 belongs to the T2 family of extracellular ribonucleases and, besides its nuclease activity, it exerts many additional functions. Indeed, RNASET2 is involved in several human pathologies, including cancer, and it is functionally relevant in the TME. RNASET2 functions are not restricted to cancer cells and its expression could be relevant also in other cell types which are important players in the TME, including DCs. Therefore, this study aimed to unravel the effect of hypoxia (2% O₂) on the expression of RNASET2 in DCs. Here, we showed that hypoxia enhanced the expression and secretion of RNASET2 in human monocyte-derived DCs. This paralleled the HIF-1α accumulation and HIF-dependent and -independent signaling, which are associated with DCs’ survival/autophagy/apoptosis. RNASET2 expression, under hypoxia, was regulated by the PI3K/AKT pathway and was almost completely abolished by TLR4 ligand, LPS. Taken together, these results highlight how hypoxia- dependent and -independent pathways shape RNASET2 expression in DCs, with new perspectives on its implication for TME and, therefore, in anti-tumor immunity.     

Thapsigargin-Stimulated LAD2 Human Mast Cell Line Is a Potent Cellular Adjuvant for the Maturation of Monocyte-Derived Dendritic Cells for Adoptive Cellular Immunotherapy

The preparation of dendritic cells (DCs) for adoptive cellular immunotherapy (ACI) requires the maturation of ex vivo-produced immature(i) DCs. This maturation ensures that the antigen presentation triggers an immune response towards the antigen-expressing cells. Although there is a large number of maturation agents capable of inducing strong DC maturation, there is still only a very limited number of these agents approved for use in the production of DCs for ACI. In seeking novel DC maturation agents, we used differentially activated human mast cell (MC) line LAD2 as a cellular adjuvant to elicit or modulate the maturation of ex vivo-produced monocyte-derived iDCs. We found that co-culture of iDCs with differentially activated LAD2 MCs in serum-containing media significantly modulated polyinosinic:polycytidylic acid (poly I:C)-elicited DC maturation as determined through the surface expression of the maturation markers CD80, CD83, CD86, and human leukocyte antigen(HLA)-DR. Once iDCs were generated in serum-free conditions, they became refractory to the maturation with poly I:C, and the LAD2 MC modulatory potential was minimized. However, the maturation-refractory phenotype of the serum-free generated iDCs was largely overcome by co-culture with thapsigargin-stimulated LAD2 MCs. Our data suggest that differentially stimulated mast cells could be novel and highly potent cellular adjuvants for the maturation of DCs for ACI.     

Different Roles of Dendritic Cells for Chronic Rhinosinusitis Treatment According to Phenotype

Dendritic cells (DCs) are antigen-presenting cells derived from the bone marrow that play an important role in the association between the innate and adaptive immune responses. The onset and development of chronic rhinosinusitis (CRS) involve a serious imbalance in immune regulation and mechanical dysfunction caused by an abnormal remodeling process. Recent studies have shown that an increase in DCs in CRS and their function of shaping the nasal mucosal immune response may play an important role in the pathogenesis of CRS. In this review, we discuss DC subsets in mice and humans, as well as the function of DCs in the nasal sinus mucosa. In addition, the mechanism by which DCs can be used as targets for therapeutic intervention for CRS and potential future research directions are also discussed.     

Parathyroid Hormone Induces Human Valvular Endothelial Cells Dysfunction That Impacts the Osteogenic Phenotype of Valvular Interstitial Cells

Parathyroid hormone (PTH) is a key regulator of calcium, phosphate and vitamin D metabolism. Although it has been reported that aortic valve calcification was positively associated with PTH, the pathophysiological mechanisms and the direct effects of PTH on human valvular cells remain unclear. Here we investigated if PTH induces human valvular endothelial cells (VEC) dysfunction that in turn could impact the switch of valvular interstitial cells (VIC) to an osteoblastic phenotype. Human VEC exposed to PTH were analyzed by qPCR, western blot, Seahorse, ELISA and immunofluorescence. Our results showed that exposure of VEC to PTH affects VEC metabolism and functions, modifications that were accompanied by the activation of p38MAPK and ERK1/2 signaling pathways and by an increased expression of osteogenic molecules (BMP-2, BSP, osteocalcin and Runx2). The impact of dysfunctional VEC on VIC was investigated by exposure of VIC to VEC secretome, and the results showed that VIC upregulate molecules associated with osteogenesis (BMP-2/4, osteocalcin and TGF-β1) and downregulate collagen I and III. In summary, our data show that PTH induces VEC dysfunction, which further stimulates VIC to differentiate into a pro-osteogenic pathological phenotype related to the calcification process. These findings shed light on the mechanisms by which PTH participates in valve calcification pathology and suggests that PTH and the treatment of hyperparathyroidism represent a therapeutic strategy to reduce valvular calcification.     

Regulatory Macrophages and Tolerogenic Dendritic Cells in Myeloid Regulatory Cell-Based Therapies

Myeloid regulatory cell-based therapy has been shown to be a promising cell-based medicinal approach in organ transplantation and for the treatment of autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, Crohn’s disease and multiple sclerosis. Dendritic cells (DCs) are the most efficient antigen-presenting cells and can naturally acquire tolerogenic properties through a variety of differentiation signals and stimuli. Several subtypes of DCs have been generated using additional agents, including vitamin D3, rapamycin and dexamethasone, or immunosuppressive cytokines, such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β). These cells have been extensively studied in animals and humans to develop clinical-grade tolerogenic (tol)DCs. Regulatory macrophages (Mregs) are another type of protective myeloid cell that provide a tolerogenic environment, and have mainly been studied within the context of research on organ transplantation. This review aims to thoroughly describe the ex vivo generation of tolDCs and Mregs, their mechanism of action, as well as their therapeutic application and assessment in human clinical trials.     

Lactate Rewrites the Metabolic Reprogramming of Uveal Melanoma Cells and Induces Quiescence Phenotype

Uveal melanoma (UM), the most common primary intraocular cancer in adults, is among the tumors with poorer prognosis. Recently, the role of the oncometabolite lactate has become attractive due to its role as hydroxycarboxylic acid receptor 1 (HCAR1) activator, as an epigenetic modulator inducing lysine residues lactylation and, of course, as a glycolysis end-product, bridging the gap between glycolysis and oxidative phosphorylation. The aim of the present study was to dissect in UM cell line (92.1) the role of lactate as either a metabolite or a signaling molecule, using the known modulators of HCAR1 and of lactate transporters. Our results show that lactate (20 mM) resulted in a significant decrease in cell proliferation and migration, acting and switching cell metabolism toward oxidative phosphorylation. These results were coupled with increased euchromatin content and quiescence in UM cells. We further showed, in a clinical setting, that an increase in lactate transporters MCT4 and HCAR1 is associated with a spindle-shape histological type in UM. In conclusion, our results suggest that lactate metabolism may serve as a prognostic marker of UM progression and may be exploited as a potential therapeutic target.     

Argon Plasma Exposure Augments Costimulatory Ligands and Cytokine Release in Human Monocyte-Derived Dendritic Cells

Cold physical plasma is a partially ionized gas expelling many reactive oxygen and nitrogen species (ROS/RNS). Several plasma devices have been licensed for medical use in dermatology, and recent experimental studies suggest their putative role in cancer treatment. In cancer therapies with an immunological dimension, successful antigen presentation and inflammation modulation is a key hallmark to elicit antitumor immunity. Dendritic cells (DCs) are critical for this task. However, the inflammatory consequences of DCs following plasma exposure are unknown. To this end, human monocyte-derived DCs (moDCs) were expanded from isolated human primary monocytes; exposed to plasma; and their metabolic activity, surface marker expression, and cytokine profiles were analyzed. As controls, hydrogen peroxide, hypochlorous acid, and peroxynitrite were used. Among all types of ROS/RNS-mediated treatments, plasma exposure exerted the most notable increase of activation markers at 24 h such as CD25, CD40, and CD83 known to be crucial for T cell costimulation. Moreover, the treatments increased interleukin (IL)-1α, IL-6, and IL-23. Altogether, this study suggests plasma treatment augmenting costimulatory ligand and cytokine expression in human moDCs, which might exert beneficial effects in the tumor microenvironment.     

Dendritic Cells and Cancer Immunotherapy: The Adjuvant Effect

Dendritic cells (DCs) are immune specialized cells playing a critical role in promoting immune response against antigens, and may represent important targets for therapeutic interventions in cancer. DCs can be stimulated ex vivo with pro-inflammatory molecules and loaded with tumor-specific antigen(s). Protocols describing the specific details of DCs vaccination manufacturing vary widely, but regardless of the employed protocol, the DCs vaccination safety and its ability to induce antitumor responses is clearly established. Many years of studies have focused on the ability of DCs to provide overall survival benefits at least for a selection of cancer patients. Lessons learned from early trials lead to the hypothesis that, to improve the efficacy of DCs-based immunotherapy, this should be combined with other treatments. Thus, the vaccine’s ultimate role may lie in the combinatorial approaches of DCs-based immunotherapy with chemotherapy and radiotherapy, more than in monotherapy. In this review, we address some key questions regarding the integration of DCs vaccination with multimodality therapy approaches for cancer treatment paradigms.     

Heat Shock Alters the Proteomic Profile of Equine Mesenchymal Stem Cells

The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was observed, and many of the specific differentially expressed proteins were involved in cell-signaling pathways such as Notch and Wnt signaling, which are known to regulate cellular development. Furthermore, cytoskeletal proteins profilin, DSTN, SPECC1, and DAAM2 showed increased protein levels in osteoblasts differentiated at 42 °C as compared with 37 °C, and these cells, while they appeared to accumulate calcium, did not organize into a whorl agglomerate as is typically seen at physiological temperatures. This altered proteome composition observed suggests that heat stress could have long-term impacts on cellular development. We propose that this in vitro stem cell culture model of cell differentiation is useful for investigating molecular mechanisms that impact cell development in response to stressors.     

Central Role of Dendritic Cells in Pulmonary Arterial Hypertension in Human and Mice

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old Tnfaip3^DNGR1-KO mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 (Tnfaip3) gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of Tnfaip3^DNGR1-KO mice, particularly in the right ventricle (RV). Secondly, in young Tnfaip3^DNGR1-KO mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in Tnfaip3^DNGR1-KO mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (Bmpr2), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the Bmpr2 gene.     

Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen

Macrophages (MΦ) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and MΦs in immunity against Mycobacterium bovis Bacillus Calmette-Guérin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic MΦs compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in MΦs were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic MΦs were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic MΦs participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation.     

Shaping of Monocyte-Derived Dendritic Cell Development and Function by Environmental Factors in Rheumatoid Arthritis

Dendritic cells (DC) are heterogeneous cell populations essential for both inducing immunity and maintaining immune tolerance. Chronic inflammatory contexts, such as found in rheumatoid arthritis (RA), severely affect the distribution and the function of DC, contributing to defective tolerance and fueling inflammation. In RA, the synovial fluid of patients is enriched by a subset of DC that derive from monocytes (Mo-DC), which promote deleterious Th17 responses. The characterization of environmental factors in the joint that impact on the development and the fate of human Mo-DC is therefore of great importance in RA. When monocytes leave the blood and infiltrate inflamed synovial tissues, the process of differentiation into Mo-DC can be influenced by interactions with soluble factors such as cytokines, local acidosis and dysregulated synoviocytes. Other molecular factors, such as the citrullination process, can also enhance osteoclast differentiation from Mo-DC, favoring bone damages in RA. Conversely, biotherapies used to control inflammation in RA, modulate also the process of monocyte differentiation into DC. The identification of the environmental mediators that control the differentiation of Mo-DC, as well as the underlying molecular signaling pathways, could constitute a major breakthrough for the development of new therapies in RA.     

Functional Ambivalence of Dendritic Cells: Tolerogenicity and Immunogenicity

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) and inducers of T cell-mediated immunity. Although DCs play a central role in promoting adaptive immune responses against growing tumors, they also establish and maintain peripheral tolerance. DC activity depends on the method of induction and/or the presence of immunosuppressive agents. Tolerogenic dendritic cells (tDCs) induce immune tolerance by activating CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and/or by producing cytokines that inhibit T cell activation. These findings suggest that tDCs may be an effective treatment for autoimmune diseases, inflammatory diseases, and infertility.     

In vivo Targeting of DNA Vaccines to Dendritic Cells via the Mannose Receptor Induces Long-Lasting Immunity against Melanoma

Herein, we report effective, C-type lectin mannose receptor (MR)-selective, in vivo dendritic cell (DC)-targeting lipid nanoparticles (LNPs) of a novel lipid-containing mannose-mimicking di-shikimoyl- and guanidine head group and two n-hexadecyl hydrophobic tails (DSG). Subcutaneous administration of LNPs of the DSG/p-CMV-GFP complex showed a significant expression of green fluorescence protein in the CD11c⁺ DCs of the neighboring lymph nodes compared to the control LNPs of the BBG/p-CMV-GFP complex. Mannose receptor-facilitated in vivo DC-targeted vaccination (s.c.) with the electrostatic complex of LNPs of DSG/pCMV-MART1 stimulated long-lasting (270 days post B16F10 tumor challenge) antimelanoma immunity under prophylactic conditions. Remarkably, under therapeutic settings, vaccination (s.c.) with LNPs of the DSG/pCMV-MART1 complex significantly delayed melanoma growth and improved the survival of mice with melanoma. These findings demonstrate that this nonviral delivery system offers a resilient and potential approach to deliver DNA vaccines encoding tumor antigens to DCs in vivo with high efficacy.     

Mycobacterium tuberculosis RpfE-Induced Prostaglandin E2 in Dendritic Cells Induces Th1/Th17 Cell Differentiation

Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Currently, there are no reports on the mycobacterial components that regulate PGE2 production. Previously, we have reported that RpfE-treated dendritic cells (DCs) effectively expanded the Th1 and Th17 cell responses simultaneously; however, the mechanism underlying Th1 and Th17 cell differentiation is unclear. Here, we show that PGE2 produced by RpfE-activated DCs via the MAPK and cyclooxygenase 2 signaling pathways induces Th1 and Th17 cell responses mainly via the EP4 receptor. Furthermore, mice administered intranasally with PGE2 displayed RpfE-induced antigen-specific Th1 and Th17 responses with a significant reduction in bacterial load in the lungs. Furthermore, the addition of optimal PGE2 amount to IL-2-IL-6-IL-23p19-IL-1β was essential for promoting differentiation into Th1/Th17 cells with strong bactericidal activity. These results suggest that RpfE-matured DCs produce PGE2 that induces Th1 and Th17 cell differentiation with potent anti-mycobacterial activity.     

New Therapeutic Approaches for Conjunctival Melanoma—What We Know So Far and Where Therapy Is Potentially Heading: Focus on Lymphatic Vessels and Dendritic Cells

Conjunctival melanoma (CM) accounts for 5% of all ocular melanomas and arises from malignantly transformed melanocytes in the conjunctival epithelium. Current therapies using surgical excision in combination with chemo- or cryotherapy still have high rates for recurrences and metastatic disease. Lately, novel signal transduction-targeted and immune checkpoint inhibitors like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein-1 (PD-1) receptor inhibitors, BRAF- or MEK-inhibitors for systemic treatment of melanoma have improved the outcome even for unresectable cutaneous melanoma, improving patient survival dramatically. The use of these therapies is now also recommended for CM; however, the immunological background of CM is barely known, underlining the need for research to better understand the immunological basics when treating CM patients with immunomodulatory therapies. Immune checkpoint inhibitors activate tumor defense by interrupting inhibitory interactions between tumor cells and T lymphocytes at the so-called checkpoints. The tumor cells exploit these inhibitory targets on T-cells that are usually used by dendritic cells (DCs). DCs are antigen-presenting cells at the forefront of immune response induction. They contribute to immune tolerance and immune defense but in the case of tumor development, immune tolerance is often prevalent. Enhancing the immune response via DCs, interfering with the lymphatic pathways during immune cell migration and tumor development and specifically targeting tumor cells is a major therapeutic opportunity for many tumor entities including CM. This review summarizes the current knowledge on the function of lymphatic vessels in tumor growth and immune cell transport and continues to compare DC subsets in CM with related melanomas, such as cutaneous melanoma and mucosal melanoma.     

Ficus carica Polysaccharides Promote the Maturation and Function of Dendritic Cells

Various polysaccharides purified from plants are considered to be biological response modifiers and have been shown to enhance immune responses. Ficus carica L. is a Chinese traditional plant and has been widely used in Asian countries for its anti-tumor properties. Ficus carica polysaccharides (FCPS), one of the most essential and effective components in Ficus carica L., have been considered to be a beneficial immunomodulator and may be used in immunotherapy. However, the immunologic mechanism of FCPS is still unclear. Dectin-1 is a non-toll-like pattern recognition receptor, predominately expressed on dendritic cells (DCs). Activation of DCs through dectin-1 signaling can lead to the maturation of DC, thus inducing both innate and adaptive immune responses against tumor development and microbial infection. In our study, we found that FCPS could effectively stimulate DCs, partially through the dectin-1/Syk pathway, and promote their maturation, as shown by the up-regulation of CD40, CD80, CD86, and major histocompatibility complex II (MHCII). FCPS also enhanced the production of cytokines by DCs, including IL-12, IFN-γ, IL-6, and IL-23. Moreover, FCPS-treated DCs showed an enhanced capability to stimulate T cells and promote T cell proliferation. Altogether, these results demonstrate that FCPS are able to activate and maturate DCs, thereby up-regulating the immunostimulatory capacity of DCs, which leads to enhanced T cell responses.