Gabriela B Ferreira

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

Surface‐exposed calreticulin (ecto‐CRT) and secreted ATP are crucial damage‐associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)‐based (reactive oxygen species (ROS)‐regulated) pathway for ecto‐CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS‐mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC‐IIhigh) and functional stimulation (NOhigh, IL‐10absent, IL‐1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto‐CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK‐orchestrated pathways that require a functional secretory pathway and phosphoinositide 3‐kinase (PI3K)‐mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase‐8 signalling are dispensable for this ecto‐CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto‐CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase‐8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK‐dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS‐mediated ER stress.

The vitamin D receptor gene FokI polymorphism: Functional impact on the immune system

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) has important effects on the growth and function of multiple cell types. These pleiotropic effects of 1,25(OH)2D3 are mediated through binding to the vitamin D receptor (VDR). Several polymorphisms of the human VDR gene have been identified, with the FokI polymorphism resulting in VDR proteins with different structures, a long f‐VDR or a shorter F‐VDR. The aim of this study was to investigate the functional consequences of the FokI polymorphism in immune cells. In transfection experiments, the presence of the shorter F‐VDR resulted in higher NF‐κB‐ and NFAT‐driven transcription as well as higher IL‐12p40 promoter‐driven transcription. Marginal differences were observed for AP‐1‐driven transcription, and no differential effects were observed for transactivation of a classical vitamin D‐responsive element. Concordantly, in human monocytes and dendritic cells with a homozygous short FF VDR genotype, expression of IL‐12 (mRNA and protein) was higher than in cells with a long ff VDR genotype. Additionally, lymphocytes with a short FF VDR genotype proliferated more strongly in response to phytohemagglutinin. Together, these data provide the first evidence that the VDR FokI polymorphism affects immune cell behavior, with a more active immune system for the short F‐VDR, thus possibly playing a role in immune‐mediated diseases.

ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death

Calreticulin surface exposure (ecto-CALR), ATP secretion, maturation of dendritic cells (DCs) and stimulation of T cells are prerequisites for anticancer therapy-induced immunogenic cell death (ICD). Recent evidence suggests that chemotherapy-induced autophagy may positively regulate ICD by favoring ATP secretion. We have recently shown that reactive oxygen species (ROS)-based endoplasmic reticulum (ER) stress triggered by hypericin-mediated photodynamic therapy (Hyp-PDT) induces bona fide ICD. However, whether Hyp-PDT-induced autophagy regulates ICD was not explored. Here we showed that, in contrast to expectations, reducing autophagy (by ATG5 knockdown) in cancer cells did not alter ATP secretion after Hyp-PDT. Autophagy-attenuated cancer cells displayed enhanced ecto-CALR induction following Hyp-PDT, which strongly correlated with their inability to clear oxidatively damaged proteins. Furthermore, autophagy-attenuation in Hyp-PDT-treated cancer cells increased their ability to induce DC maturation, IL6 production and proliferation of CD4+ or CD8+ T cells, which was accompanied by IFNG production. Thus, our study unravels a role for ROS-induced autophagy in weakening functional interaction between dying cancer cells and the immune system thereby helping in evasion from ICD prerequisites or determinants.

1,25-Dihydroxyvitamin D3 Promotes Tolerogenic Dendritic Cells with Functional Migratory Properties in NOD Mice

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is able to promote the generation of tolerogenic mature dendritic cells (mDCs) with an impaired ability to activate autoreactive T cells. These cells could represent a reliable tool for the promotion or restoration of Ag-specific tolerance through vaccination strategies, for example in type 1 diabetes patients. However, successful transfer of 1,25(OH)2D3-treated mDCs (1,25D3-mDCs) depends on the capacity of 1,25(OH)2D3 to imprint a similar tolerogenic profile in cells derived from diabetes-prone donors as from diabetes-resistant donors. In this study, we examined the impact of 1,25(OH)2D3 on the function and phenotype of mDCs originating from healthy (C57BL/6) and diabetes-prone (NOD) mice. We show that 1,25(OH)2D3 is able to imprint a phenotypic tolerogenic profile on DCs derived from both mouse strains. Both NOD- and C57BL/6-derived 1,25D3-mDCs decreased the proliferation and activation of autoreactive T cells in vitro, despite strain differences in the regulation of cytokine/chemokine expression. In addition, 1,25D3-mDCs from diabetes-prone mice expanded CD25+Foxp3+ regulatory T cells and induced intracellular IL-10 production by T cells in vitro. Furthermore, 1,25D3-mDCs exhibited an intact functional migratory capacity in vivo that favors homing to the liver and pancreas of adult NOD mice. More importantly, when cotransferred with activated CD4+ T cells into NOD.SCID recipients, 1,25D3-mDCs potently dampened the proliferation of autoreactive donor T cells in the pancreatic draining lymph nodes. Altogether, these results argue for the potential of 1,25D3-mDCs to restore Ag-specific immune tolerance and arrest autoimmune disease progression in vivo.

Differential Protein Pathways in 1,25-Dihydroxyvitamin D-3 and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

Tolerogenic dendritic cells (DC) that are maturation-resistant and locked in a semimature state are promising tools in clinical applications for tolerance induction. Different immunomodulatory agents have been shown to induce a tolerogenic DC phenotype, such as the biologically active form of vitamin D (1,25(OH)(2)D(3)), glucocorticoids, and a synergistic combination of both. In this study, we aimed to characterize the protein profile, function and phenotype of DCs obtained in vitro in the presence of 1,25(OH)(2)D(3), dexamethasone (DEX), and a combination of both compounds (combi). Human CD14(+) monocytes were differentiated toward mature DCs, in the presence or absence of 1,25(OH)(2)D(3) and/or DEX. Cells were prefractionated into cytoplasmic and microsomal fractions and protein samples were separated in two different pH ranges (pH 3-7NL and 6-9), analyzed by 2D-DIGE and differentially expressed spots (p < 0.05) were identified after MALDI-TOF/TOF analysis. In parallel, morphological and phenotypical analyses were performed, revealing that 1,25(OH)(2)D(3)- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)(2)D(3) is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)(2)D(3) with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)(2)D(3) effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)(2)D(3), rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, which may affect the production of or the response to ROS generation. These findings provide new insights on the molecular basis of DC tolerogenicity induced by 1,25(OH)(2)D(3) and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation.

1,25‐Dihydroxyvitamin D3 alters murine dendritic cell behaviour in vitro and in vivo

Differentiation and maturation of dendritic cells yield a cell type with the ability to prime immune responses towards defence and destruction. 1,25(OH)2D3, the active form of vitamin D3, fosters the development of tolerogenic dendritic cells. This study aimed to evaluate the effects of 1,25(OH)2D3 on murine dendritic cell behaviour in vitro and in vivo.

Proteome analysis demonstrates profound alterations in human dendritic cell nature by TX527, an analogue of vitamin D.

Structural analogues of vitamin D have been put forward as therapeutic agents able to exploit the immunomodulatory effects of vitamin D, without its undesired calcemic side effects. We have demonstrated that TX527 affects dendritic cell (DC) maturation in vitro, resulting in the generation of a tolerogenic cell. In the present study, we aimed to explore the global protein changes induced by the analogue in immature DC (iDC) and mature human DC and to correlate them with alterations in DC morphology and function. Human CD14+ monocytes were differentiated toward iDC or mature DCs, in the presence or absence of TX527 (10−8 M) (n=4). Protein samples were separated into two different pH ranges (pH4–7 and 6–9), analyzed by 2‐D DIGE and differentially expressed spots (p<0.01) were identified by MALDI‐TOF/TOF (76.3 and 70.7% in iDC and mature DCs, respectively). Differential protein expression revealed three protein groups predominantly affected by TX527 treatment, namely proteins involved in cytoskeleton structure, in protein biosynthesis/proteolysis and in metabolism. Moreover, protein interactome‐network analysis demonstrated close interaction between these different groups (p<0.001) and morphological and functional analyses confirmed the integrated effect of TX527 on human DCs, resulting in a cell with altered morphology, cell surface marker expression, endocytic and migratory capacity.

Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen‐capturing cell towards a professional antigen presenting cells. In this study, a 2‐D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14+ peripheral blood monocytes was investigated using two pH ranges (pH 4–7 and 6–9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein‐interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune‐associated disorders.

Understanding dendritic cell biology and its role in immunological disorders through proteomic profiling.

Dendritic cells (DC) have always been present on the bright spot of immune research. They have been extensively studied for the last 35 years, and much is known about their different phenotypes, stimulatory capacity, and role in the immune system. During the last 15 years, great attention has been given to studies on global gene and protein expression profiles during the differentiation and maturation processes of these cells. It is well understood that studying the proteome, together with information on the role of protein post‐translational modifications (PTM), will reveal the real dynamics of a living cell. The rapid increase of proteomic studies during the last decade describing the differentiation and maturation process in DCs, as well as modifications brought by the use of different compounds that either increase or decrease their immunogenicity, reflects the importance of understanding the molecular processes behind the functional properties of these cells. In the present review, we will give an overview of proteomic studies focusing on DCs. Thereby we will concentrate on the importance of these studies in understanding DC behavior from a molecular point of view and how these findings have aided in understanding the differences in functional properties of these cells.

The phenotype and function of murine bone marrow-derived dendritic cells is not affected by the absence of VDR or its ability to bind 1α,25-dihydroxyvitamin D3

The nuclear vitamin D receptor (VDR) is generally recognized as a ligand-dependent transcription factor that mediates the actions of its natural ligand, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on multiple target genes involved in mineral homeostasis, bone development, as well as immune reactivity. As the VDR is widely distributed in nearly all cells of the body, it implies that the vitamin D endocrine system may regulate many cell types and functions. Experiments in VDR null mice established that the VDR has intrinsically critical roles in skin and keratinocyte biology but not in immune responses. Oppositely, absence of the VDR ligand is linked to susceptibility to autoimmunity, illustrating a potential role for the unliganded VDR in the immune system. This discrepancy stimulated us to further investigate the impact of the VDR on the phenotype and function of myeloid dendritic cells (DCs) generated ex vivo from bone marrow precursors of VDR null (with a truncated VDR) and VDR ΔAF2 mice (with a mutated C-terminal activation factor 2 domain thus rendering ligand-induced gene transcription impossible). Absent or unliganded VDR did not affect bone marrow-derived myeloid DC generation. DCs obtained from VDR null and VDR ΔAF2 bone marrow cells had comparable MHC-II, and costimulatory molecule CD86, CD80 and CD40 expression than DCs from wild-type bone marrow cells. Additionally, an unliganded VDR did not affect the cytokine production nor the antigen-specific T cell stimulatory capacity of bone marrow-derived DCs. In conclusion, we showed that although clear effects of 1α,25-dihydroxyvitamin D3 are described on DC generation, absence of VDR or presence of an unliganded VDR does not affect the profile and function of ex vivo generated bone marrow-derived DCs.