Gene Expression Profiling of Human Monocyte-derived Dendritic Cells – Searching for Molecular Regulators of Tolerogenicity

The ability of dendritic cells (DCs) to initiate and modulate antigen-specific immune responses has made them attractive targets for immunotherapy. Since DC research in humans is limited by the scarcity of DC populations in the blood circulation, most of our knowledge about DC biology and function has been obtained in vitro from monocyte-derived DCs (moDCs), which can be readily generated in sufficient numbers and are able to differentiate into distinct functional subsets depending on the nature of stimulus. In particular, moDCs with tolerogenic properties (tolDCs) possess great therapeutic potential for the treatment of autoimmune diseases. Several protocols have been developed to generate tolDCs in vitro, able to reinstruct auto-reactive T cells and to promote regulatory cells. While ligands and soluble mediators, by which DCs shape immune responses, have been vastly studied, the intracellular pathways and transcriptional regulators that govern tolDC differentiation and function are poorly understood. Whole-genome microarrays and proteomics provide useful strategies to dissect the complex molecular processes that promote tolerogenicity. Only few attempts have been made to understand tolDC biology through a global view on “omics” profiles. So far, the identification of a common regulator of tolerogenicity has been hampered by the fact that each protocol, used for tolDC generation, targets distinct signaling pathways. Here, we review the progress in understanding the transcriptional regulation of moDC differentiation, with a special focus on tolDCs, and highlight candidate molecules that might be associated with DC tolerogenicity.

Immature DCs continuously sample antigen, but represent poor inducers of immune responses (2). Upon recognition of pathogen-or danger-associated patterns and integration of pro-inflammatory signals from the environment, DCs undergo a maturation process, which enables them to migrate toward lymphoid tissues and initiate antigen-specific T cell responses (5,6). DCs instruct T cells through the presentation of antigen peptides on major histocompatibility complexes (MHC), co-stimulation, and the secretion of T cell-attracting chemokines and cytokines promoting T cell expansion and differentiation into effector cells with a particular cytokine profile (7). DCs are also able to induce and maintain tolerance to harmless and self-antigens, through deletion of auto-reactive T cells, induction of anergy, and/or generation of regulatory T cells (Tregs) (8)(9)(10)(11).
In vitro, DCs can be differentiated from human peripheral blood monocytes in the presence of granulocyte-macrophage colonystimulating factor (GM-CSF) and interleukin (IL)-4 (12). Human myeloid and plasmacytoid DC subsets can be obtained from CD34+ cord blood progenitors in stromal cell-containing cultures, supplemented with Flt3L, stem cell factor, and GM-CSF (13). Here, we focus on monocyte-derived DCs (moDCs), which are closely related to inflammatory DCs (4), and represent the model of choice for studies on human DC biology and function, and the design of cell-based immunotherapies targeting antigen-specific immune responses (14)(15)(16)(17). Autologous moDCs can be easily obtained in sufficient numbers from peripheral blood of patients, and either matured/activated to induce immunogenic properties (15), or modulated to promote immunoregulatory functions (18,19).
Several protocols have been developed for the in vitro generation of human moDCs with tolerogenic properties (tolDCs), able to silence or reprogram auto-reactive T cells and induce regulatory lymphocytes (18,20,21). Their immunoregulatory function has been corroborated in vivo in rodent models of autoimmune diseases (22)(23)(24)(25)(26), and first clinical trials using autoantigen-pulsed tolDCs in patients with autoimmune disorders confirmed their safety and efficacy (27,28). Studies on the mechanisms, by which tolDCs modulate T cell responses, indicate that cell-contact via co-stimulatory/ co-inhibitory signals (29), and anti-inflammatory cytokines, such as IL-10 and TGF-β (30) are required for tolerance induction. However, the intracellular molecular processes that govern DC differentiation toward a tolerogenic profile are scarcely understood (31).
Here, we review recent findings in gene expression analysis of moDCs, with special focus on approaches to unveil the molecular basis of DC tolerogenicity.

CHAnGeS in Gene eXPReSSiOn PROFiLeS UPOn DenDRiTiC CeLL MATURATiOn
Several stimuli are used to induce maturation of DCs in vitro, including pro-inflammatory cytokines and microbial products, leading to morphological changes, upregulation of MHC, and co-stimulatory molecules, as well as characteristic chemokine and cytokine profiles (38)(39)(40)(41)(42).
Alternative or partial activation of DCs has been considered as essential for the efficacy of tolDC-based immunotherapy and can be achieved by adding LPS (10,64,65), its non-toxic analog monophosphoryl lipid A (66), CD40L (66), or the standard cytokine cocktail for DC maturation (67). This endows tolDCs with enhanced IL-10 production, antigen presentation, and lymph node homing capacity, while preserving a stable tolerogenic profile upon exposure to activating stimuli (68).
Despite the diversity of stimuli used to obtain tolDCs, and although some properties vary amongst protocols, there is a consensus about fundamental features that tolDCs must possess, including low expression of co-stimulatory molecules, high production of anti-inflammatory cytokines, mainly IL-10, and low levels of pro-inflammatory cytokines, as well as the ability to induce T cell hyporesponsiveness or Tregs (67,69,70).

Global Gene expression Profiling of Tolerogenic Dendritic Cells
To date, few studies have attempted to unravel the molecular basis of DC tolerogenicity through transcriptome and proteome profiling ( Table 1).
Ferreira and colleagues explored global molecular changes induced in human moDCs by vitD3 and its analog TX527 through transcriptomic and proteomic approaches, and assigned differentially regulated genes to three functional categories: cytoskeleton structure, protein biosynthesis/proteolysis, and metabolism (73,75). VitD3 and TX527 reduced the expression of most cytoskeleton proteins, such as fascin, while enhancing the expression of metabolic proteins, e.g., CA2 and FBP1 (73,75). Protein proteolysis/biosynthesis comprised the main group of proteins that were upregulated in response to TX527, involving translation (eEF1G, eEF2, EIF3S3, EIF4H) and the MHCI/II pathway, particularly CTSD and CTSS, which mediate the degradation of MHCII invariant chain/CD74 (73). Additionally, TX527 treatment increased the expression of stress response proteins, including SOD2, ORP150, HSPD1, and TXNDC4, and proteins of the cellular defense response, such as LTB4 and NCF2 (73).
The comparison of protein profiles of tolDCs, modulated with vitD3, dexamethasone, or both, and subsequently activated by CD40L, revealed common functional groups that were regulated in all three tolDC types, but not in CD40L-matured DCs (75). These comprised lipid metabolism, i.e., fatty acid oxidation and elongation in mitochondria, glycerophospholipid metabolism and phospholipid degradation, as well as NRF2-mediated oxidative stress response (75). Protein interaction networks and pathway analysis indicated that vitD3, rather than dexamethasone, has a strong impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, as well as on mitochondrial processes, including alterations of the mitochondrial transmembrane potential (75). By contrast, dexamethasone was shown to affect predominantly proteins of the stress response, e.g., HSP71, and induced proteins of glutathione metabolism, acute phase response signaling, and MHCII antigen presentation pathways, including multiple isoforms of CTSB, CTSD, and CTSZ (75). Combined treatment with vitD3 and dexamethasone, which promotes a strong tolerogenic profile regarding the modulation of T cell responses (75), induced a unique protein signature, dominated by the metabolic effect of vitD3 (75). When compared to treatment with each stimulus alone, combination of vitD3 and dexamethasone upregulated proteins involved in the antiapoptotic response (HSPA9, PYCARD, and ANXA1), protein biosynthesis/proteolysis, protein binding/folding, and immune response (IL1RN, ANXA11, SAMHD1) (75).
Microarray analysis of intracellular processes, induced early during differentiation of monocytes to vitD3-tolDC, revealed an upregulation of genes related to glucose metabolism, tricarboxylic acid cycle, and oxidative phosphorylation, including GLU3, HK3, PFKFB4, PDHA1, LDHA, ATP5A1, and the transcription factor C-MYC (34). Glucose availability and glycolysis, controlled by the PI3K/Akt/mTOR pathway, were shown to dictate the induction and maintenance of the tolerogenic phenotype and function in vitD3-modulated tolDCs (34).
Similar results were reported by Malinarich and colleagues, who compared transcriptomes of tolDCs modulated by dexamethasone and vitD3, with or without activation by LPS, to those of immature and LPS-matured DCs (74). This study confirmed the upregulation of catabolic pathways, including oxidative phosphorylation, fatty acid metabolism, and glycolysis, in vitD3modulated tolDCs compared to immature DCs (74). However, LPS-induced activation was shown to decrease the metabolic plasticity in tolDCs and DCs, mainly by negatively regulating oxidative phosphorylation, without affecting mitochondrial function (74).
Using a different approach, Zimmer and colleagues analyzed proteomes of human moDCs, either modulated with dexamethasone or activated with LPS or peptidoglycan, using DIGE and label-free mass spectrometry to identify putative biomarkers of tolDCs (72). Proteomic analysis uncovered 14 potential marker candidates that were significantly upregulated in tolDCs compared to immature DCs and LPS-or peptidoglycan-matured DCs, including FKBP5, GPX1, C1QA, and STAB1 (72). Evaluation of candidate expression in other tolDC types, modulated by IL-10, rapamycin, vitD3, TGF-β, or Aspergillus oryzae protease, through qPCR and Western blot analysis, revealed substantial heterogenicity. Only ANXA1, CATC, and GILZ were upregulated in all tolDCs subtypes and therefore suggested as tolDC markers (72).
Different tolDC types share main phenotypic and functional characteristics, however, transcriptome and proteome studies demonstrated that each modulatory agent, used to promote tolDCs, induces a distinct transcriptional program in DCs ( Table 1). While IL-10 mainly affects immunological processes (71), vitD3 has a major impact on metabolic pathways, involving oxidative phosphorylation, fatty acid, and glucose metabolism (34,74). By contrast, dexamethasone exerts an influence on glutathione metabolism and upregulates genes related to stress response and redox homeostasis (72,74,75). There are only few common molecules found to be upregulated in different tolDC types, including the cytokine IL-1Ra (IL1RN), complement component 1q (C1Q), coagulation factor XIIIa (F13A), thrombospondin-1 (THBS1/TSP1), and superoxide dismutase (SOD2). IL-1Ra competes with IL-1 for binding to the IL-1 receptor, without inducing any intracellular response, and has been shown to inhibit DC maturation as well as T cell activation and polarization (76,77). C1q was proposed to render DCs tolerogenic, by reducing the expression of co-stimulatory molecules and promoting high levels of immunosuppressive IL-10 and TGF-β (78,79). F13A+ DCs were shown to produce retinoic acid and induce Foxp3+ Tregs (80). THBS1 is directly associated with tolerance induction, by impairing T effector cell proliferation while promoting Treg generation through ligation with its receptor CD47/ IAP (69,81,82). Only SOD2 was found to be upregulated in all tolDC types described herein, irrespective of subsequent activation via TLR or CD40 (34,69,73,75). This antioxidative enzyme is also expressed by immature and mature DCs (33,36,69), and is crucial for oxidative stress resistance and the regulation of inflammatory processes by attenuating the activity of NF-κB (83,84). Accordingly, in mice with heterozygous SOD2 deficiency, DCs accumulate reactive oxygen species under stress conditions, secrete higher amounts of IL-6, CXCL1, and CXCL2/MIP-2α, and show an impaired antigen-presenting and co-stimulatory capacity, and decreased TNF-α secretion upon activation (85).
It is to be noted that the expression profiles of tolDCs show some overlap with those of immature DCs, e.g., upregulation of C/EBP, c-myc, p53, and SOD2 transcripts, which might be due to the inhibition of maturation/activation (34,36). However, the transcriptome and proteome studies described herein unraveled distinctive molecular signatures of tolDCs, indicating that tolerogenic features emerge from a specific transcriptional program, rather than resulting from retention at an immature state.

COnCLUDinG ReMARKS
Knowledge about molecular mechanisms that govern DC differentiation and function has increased due to technological advances. However, molecular switches that "turn on" tolerogenic functions in DCs remain largely unknown. Comparative transcriptome studies confirmed that tolDCs possess a characteristic molecular signature rather than being retained at a phenotypic and functional immature/semi-mature state. Since diverse modulating agents used for the generation of human tolDCs target distinct signaling pathways, the identification of master regulators of DC tolerogenicity has been challenging. Further comparative "omics" studies are required to define which molecules induce an immunoregulatory profile and thus might be used as targets to render DCs tolerogenic and to enhance their stability, longevity, and resistance to stress or pro-inflammatory stimuli for immunotherapeutic application.

ACKnOwLeDGMenTS
The authors thank CONICYT and Millennium Institute on Immunology and Immunotherapy (IMII) for financial support.