Recent discoveries in dendritic cell tolerance-inducing pharmacological molecules

International Immunopharmacology 81 (2020) 106275

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Recent discoveries in dendritic cell tolerance-inducing pharmacological molecules

T

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Urban Švajger , Primož J. Rožman Blood Transfusion Center of Slovenia, Šlajmerjeva 6, 1000 Ljubljana, Slovenia

A R T I C LE I N FO

A B S T R A C T

Keywords: Dendritic cells Tolerance Tolerogenic Pharmacological Drugs Molecules

Dendritic cells (DCs) represent one of the most important biological tools for cellular immunotherapy purposes. There are an increasing number of phase I and II studies, where regulatory or tolerogenic DCs (TolDCs) are utilized as negative vaccines, with the aim of inducing tolerogenic outcomes in patients with various autoimmune or chronic-inflammatory diseases, as well as in transplant settings. The induction of tolerogenic properties in DCs can be achieved by altering their activation state toward expression of immunosuppressive elements and/or by achieving resistance to maturation, which leads to insufficient co-stimulatory signal delivery and inability to efficiently present antigens. In the past, one of the most efficient ways to induce DC tolerance has been the application of selected pharmacological agents which actively induce a tolerogenic transcription program or inhibit major pro-inflammatory transcription factors such as Nf-κB. Important examples include immunosuppressants such as different corticosteroids, vitamin D3, rapamycin and others. The quality of TolDCs induced by different approaches is becoming a vital issue and recent evidence suggests substantial heterogeneity between variously-generated TolDCs as evidenced by their transcriptomic profile and function. The possibility of various “flavors” of TolDCs encourages future research in discovery of Tol-DC inducing agents to enrich various ways of DC manipulation. This would enable a broader range of tools to manipulate DC toward specific characteristics desirable in different disease settings. In recent years, several novel small molecules have been identified with the capacity to promote DC tolerogenic characteristics. In this review, we will present and discuss these novel findings and also highlight novel understandings of tolerogenic mechanisms by which DC tolerogenicity is induced by already established agents.

1. Introduction Dendritic cells (DCs) are a diverse and unique leukocyte population, which possesses extensive functional plasticity. Their central position within the immunological network is granted by their evolutionary positioning between the innate and adaptive immune systems and the capacity to control and maintain both immunogenic and tolerogenic adaptive responses [1]. The extreme difference in ways in which a particular antigen (Ag) can be presented by DCs to responding T cells is in great part orchestrated by DCs' activation state. The two archetypal examples are either immature or mature DCs. In their immature state, the DCs typically reside near body surfaces and only slowly migrate to the draining lymph nodes [2]. In this way, Ag presentation is possible, although at a lower level due to reduced presence of MHC class II molecules on the DC surface. However, the absence of co-stimulatory molecules and lack of cytokine production by immature DCs result in induction of T cell anergy instead of activation. Contrary, in response to

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pathogen-associated molecular patterns (PAMPs), inflammatory cytokines or danger-associated molecular patterns (DAMPs), the DCs mature rapidly and can elicit strong effector responses. Dendritic cell tolerance can therefore be strongly associated with their immature state, however, as we well know today, immature DCs play only a certain part in peripheral tolerance induction. The tolerogenic function of DCs is not only conferred by the absence of co-stimulatory signals or absence of maturation, but can be greatly accelerated by more active elements such as extensive expression of various surface inhibitory molecules and production of anti-inflammatory cytokines, both being major denominators of tolerogenic DCs (TolDCs) [3]. Consequently, efficient induction of such elements of active tolerance represents the holy grail of ex vivo/in vitro TolDC generation for therapeutic purposes such as regulatory cellular immunotherapy [4]. In this manner, we will also need efficient strategies to set the benchmark for what represents a quality TolDC cellular therapy product. Recently, a European initiative has gone underway to describe a minimum information model for TolDCs,

Corresponding author at: Department of Therapeutic Services, Šlajmerjeva 6, 1000 Ljubljana, Slovenia. E-mail address: [email protected] (U. Švajger).

https://doi.org/10.1016/j.intimp.2020.106275 Received 15 November 2019; Received in revised form 31 January 2020; Accepted 31 January 2020 1567-5769/ © 2020 Elsevier B.V. All rights reserved.

International Immunopharmacology 81 (2020) 106275

U. Švajger and P.J. Rožman

which are controlled by the PI3K/Akt/mTOR pathway, has been shown to have an important impact on DC tolerogenic phenotype and function [30]. In this manner, normal activity of the PI3K/Atk/mTOR pathway was imperative to maintenance of DC tolerogenic phenotype. The association between the role of metabolic processes and regulatory DC function has also been demonstrated in case of Dex [31]. Several genes associated with fatty acid oxidation, zinc homeostasis and reactive oxygen species were found to be strongly enriched in comparison to untreated cells, particularly in DCs treated with the combination of Dex and monophsophoryl lipid A (MPLA). The upregulation of genes involved in tolerance (such as il-10 and ido1) and down-regulation of genes involved in DC maturation (namely il-1b, mmp-2 and others) was more or less expected, when comparing untreated, immature DCs to TolDCs, in this case generated by Dex/MPLA treatment. Perhaps more informative is the fact that major differences in the transcriptional profile have also been observed when comparing variously generated TolDCs. In an exhaustive overview published by Navarro-Barriuso et al., comparison of transcriptomic and proteomic biomarkers identified for various TolDCs revealed much greater differences than similarities. For example, comparing TolDCs generated using vit D3, Dex or the combination of both, found only 1 commonly induced gene between all three groups amidst 29, 64 and 102 differentially induced genes for vit D3, Dex and vit D3/Dex treatment alone, respectively [17]. Similar extent of differences was found for other TolDC-inducing agents, namely Rapa, retinoic acid and others. The diversity of various biomarkers induced by different pharmacological molecules supports future efforts to investigate the potential synergy by using their combinations to achieve superior tolerogenic effects. This kind of approach has been already well executed in the quest for optimal DC activation in the opposite direction, toward type 1 DC maturation protocols for anti-cancer vaccine purposes. Indeed, new generation DC maturation protocols represent multi-component “cocktails” such as toll-like receptor (TLR) ligands in combination with type I and type II interferons, as well as additional pro-inflammatory cytokines [32]. In the tolerogenic manner, some combinations have been demonstrated to be successful, such as vit D3/Dex or vitD3/IL-10 [33], however, a great part of TolDC-inducing protocols still rely on the use of a single agent. Perhaps in greater extent than discovering potential synergy between various pharmacological immunosuppressants, several protocols have focused on so called “alternative activation” of DCs. This is achieved by additional activation of DCs with chosen maturation stimuli. For example Dex- or vit D3-treated DCs have been activated with TLR ligands [31,34]. Such activation can lead to additional important TolDC characteristics such as responsiveness to CCR7mediated migration. Other combinations are less well researched, though it is likely they could lead to important tolerogenic effects. For example, Leong et al. have shown the importance of crosstalk between glucocorticoids (GCs) and IL-4 for alternative activation of bone marrow-derived DCs and macrophages [35]. In another study, Rapa and aspirin, both of them being agents with previously confirmed TolDC-inducing characteristics [36 37], have been shown to act synergistically in reducing both Th1 and Th17 effector responses, while at the same time maintaining the CD4+FoxP3+ repertoire [38]. Very recently, we have shown that there is an important synergy in signaling between vit D3 and IFN-γ in inducing a TolDCs with unique tolerogenic characteristics. Termed γ/ D3DCs, they were characterized by extensive expression of ILT-3 and PDL-1 inhibitory molecules, which was significantly greater than that of TolDCs generated with vit D3 alone[39]. We have found that this synergy can be regulated via MEK/ERK and PI3K/Akt/mTOR pahthways and was reversed when either pathway was inhibited using specific inhibitors. Although γ/D3DCs increased the percentage of FoxP3+ during co-cultures with naive CD4+ T cells, the resuting “Tregs” were not functionally suppressive. Interestingly, in a DC-suppressive assay, where active suppression of TolDCs was evaluated by assessing their capacity to suppress T cell proliferation by mDCs, γ/D3DCs were not

which is a great start toward development of quality standards across different centers [5]. It is now more than 20 years since the first demonstration that when treated with interleukin (IL)-10, DCs obtain resistance to maturation and certain tolerogenic characteristics [6]. Shortly afterwards, other tolerance-inducing drugs or biomolecules have been designated with tolerance-inducing effects on DCs, namely dexamethasone (Dex), the active metabolite of vitamin D (vit D3), rapamycin (Rapa), transforming growth factor (TGF)-β and several others in the years to follow [7–11]. To this day, the immunosuppressive agents highlighted above still represent the golden standard in TolDC induction, either alone or in combination (e.g. vitD3/Dex or IL-10/vitD3). Although at a first glance, all of them induce generally similar characteristics regarding DC biology, namely resistance to strong immunogens such as LPS or CD40L, induction of tolerogenic elements (inhibitory molecules), as well as induction of regulatory T cells (Tregs), they achieve this by acting on biologically distant targets. In this manner, vitD3 binds its corresponding nuclear receptor VDR [12], Dex acts via glucocorticoid receptor (GR) [13], Rapa blocking the PI3K/Akt/mTOR pathway by directly inhibiting the mTOR [14] and both IL-10 and TGF-β activate signaling via their corresponding cytokine receptors [15,16]. A recent study has shown that just by looking at the transcriptomic profile of variously-generated TolDCs, we can easily observe more differences than similarities, leading to a strong speculation that such diversity could likely be noticed on the functional level as well [17]. There is still no clear consensus on what can yet be achieved in context of TolDC induction, the activation or blockade of which pathways (or combinations thereof) would lead to superior DC tolerogenicity or which exact approach should we use to generate TolDCs with specific characteristics that would best serve as TolDC-inducing protocols intended for specific pathological setting. The quest for discovery of new ways and pathways which allow for the induction of DC tolerogenic potential is therefore ongoing and necessary, wherein the use of pharmacological agents plays a major role. In this review, we will discuss most recent findings of newly discovered, TolDC-inducing small molecules or their combinations, that haven’t been discussed previously. Additionally, we will reflect on newly discovered mechanisms regarding classical ways of DC tolerance induction (see Fig. 1. and Table 1). 2. Old tricks, new perspectives In the last 20 years, a relatively large number of pharmacological agents have been shown to affect DC biology in various ways to achieve a reduced maturation potential with low stimulatory capacity and/or further induction of immunosuppressive elements such as production of anti-inflammatory cytokines and the capacity to induce Tregs. For previous discussions we refer the reader to several comprehensive reviews made on this subject [18–20]. In more recent years, several classical immunosuppressants have been revisited in terms of DC tolerogenicity and some of them have been implemented in combination protocols with other agents. Besides being a fundamental regulator of bone and mineral homeostasis, 1,25-dihydroxyvitamin D3 (vit D3) is widely recognized as an important regulator of both innate and adaptive immunity [21]. In DCs, it has been well demonstrated to induce a strong tolerogenic potential, affecting both differentiation and maturation [22–25]. Dendritic cells treated with vit D3 up-regulate tolerogenic elements, namely surface inhibitory molecules such as the immunoglobulin-like transcripts (ILT)-2 and ILT-3 [26], as well as programmed death ligand (PDL)-1 [27] and have the capacity to induce regulatory T cells [28]. The exact mechanisms of how TolDCs are induced by vit D3 treatment have not been fully addressed until recently, when Ferreira et al. demonstrated the correlation between metabolic regulation of DC function and vit D3 [29]. They have shown that vit D3 causes metabolic reprogramming with direct consequence to glucose metabolism. The availability of glucose as well as glucose metabolism, 2

International Immunopharmacology 81 (2020) 106275

U. Švajger and P.J. Rožman

Fig. 1. Tolerogenic DC-inducing molecules described in the last 5 years.

efficient at suppressing CD4+ T cell proliferation. However, their active suppression of CD8+ T cells was extensive and approximately 2-fold stronger compared to TolDCs obtained by vit D3 treatment, suggesting a selective tolerogenic activity directed toward CD8+ T cell compartment [39]. A partial explanation could be based on the tolerogenic contribution by IFN-γ signaling. It has been shown recently that when IFNγ is present in DC microenvironment, particularly at high doses, such DCs obtain regulatory properties and are very efficient at silencing cytotoxic CD8+ T cell immune responses [40].

cells, minocycline treatment resulted in enhancement of DCs’ regulatory properties. Such cells were resistant to maturation with LPS or pro-inflammatory cytokines, with significant down-regulation of CD54, CD80 and CD86 [44]. In this manner they also displayed a reduced production of pro-inflammatory cytokines with no changes in IL-10 production. The allostimulatory capacity of minocycline-treated DCs was low and in addition, they possessed a moderate capacity to induce FoxP3+ Tregs. Pre-treatment of animals in a murine experimental autoimmune encephalomyelitis (EAE) model with Ag-loaded minocyclinetreated DCs ameliorated disease severity [44]. The same group subsequently studied the potential tolerogenic synergy between minocycline and different immunosuppressants, namely vit D3, Dex, IL-10 and rapamycin [45]. They discovered that particularly the combination of minocycline with Dex generates the highest numbers of DCs with increased tolerogenic properties than single treatment alone. Dendritic cells treated with both agents had the capacity to induce significantly higher percentage of FoxP3+ T cells and also exerted the highest PDL1/CD86 ratio, which represents one of the hallmarks regarding TolDC potency [46]. However, since the authors singled out the combination of minocycline + Dex solely based on DC yield during differentiation, it would be interesting to also observe the tolerogenic effects exerted on DCs by minocycline combinations with vit D3, IL-10 or Rapa, which hasn’t been performed. Macrolides are another class of antibiotics that has been attributed with immunosuppressive properties. While active against Gram-positive bacteria and several others, they also exhibit numerous

3. Antibiotics as DC-targeting immunosuppressants Various classes of antibiotics have been demonstrated to possess anti-inflammatory properties in the past. The anti-inflammatory activity of tetracyclines was first observed in certain dermatological conditions, where improvements in inflammatory conditions such as rosacea were observed without associations with causative bacterial infections [41]. Today, tetracyclines are known to possess several antiinflammatory actions including inhibition of chemotaxis, granuloma formation, cytokine production by lymphocytes, as well as in vivo documented anti-inflammatory activity in pathological settings [42,43]. Their potential in manipulating DC biology has been recently appreciated. Minocycline is a second generation tetracycline, which has been shown to significantly augment the generation of bone marrow DCs in the presence of GM-CSF and IL-4. In comparison to non-treated 3

↓IL-12p70 ↑ IL-10 ↓IL-12p70, IL-23 ↑ IL-10, TGF-β ↓IL-12p70 ↑IL-10 N/A

↓IL-1β, IL-6, IL-12, TNF-α ↓IL-1β, IL-6, IL-12, TNF-α* ↑IL-10 ↓IL-6, IL-12, TNF-α ↓IL-10 ↓IL-6, IL-10, IL-12, TNF-α

↑ILT-2, ILT-3, PDL-1

↓CD80, CD83, CD86 ↑ CCR7 ↑ ILT-3, PDL-1

↓CD40, CD86

↓CD54, CD80, CD86

↓CD80, CD86*

↓CD80, CD83, HLA-DR

vit D3

vit D3 and(or) Dex/TLR ligands

Rapa + Aspirin

Minocycline

Minocycline + Dex *- synergistic effect compared to minocycline alone Azythromycin

4 ↓IL-6, TNF-α, IL-12 ↑IL-10, TGF-β ↑IL-10 ↓IL-12p40 ↓IL-6, IL-12, IL-23, TNF-α ↑IL-10 no negative effect on pro-inflammatory cytokine production ↓IL-6, IL-12, TNF-α

↓CD40, CD86

↓CD80, CD86, MHC class II

↓CD11b, CD11c

↓CD80, CD83, CD86, MHC class II ↓CD40, CD80, CD86

no negative effect on co-stimualtory molecule expression ↓CD86, MHC class II

semi-maturation, moderate increase in CD40, CD80, CD86

ethyl pyruvate

captopril

atorvastatin

haloperidol cobalt (III) protoporphyrin-IX-chloride

carbon monoxide

pegylated TLR7 ligand 1Z1

thimerosal

↓IL-1β, IL-6, IL-12, TNF-α

↓IL-10, IL-12p70 ↓TNF-α, IL-12p70

butyrate γ-Res

↓CD40, CD80, CD86, PDL-1, HLA class II ↓CD40, CD80, CD83 ↓CD80, CD86, MHC class I/II

I-BET151

vit D3 + IFN-γ

Cytokine production

Surface molecules

Agent

Tregs

Tregs loss of dendrite formation ↑Treg induction ↓ allo-stimulatory potential ↑FoxP3+ Treg induction ↓allo-stimulatory capacity ↓ Ag processing ↓ naive CD4+ T cell priming inhibiton of Th1 polarization from naive CD4+ T cells inhibition of T cell proliferation

↑induction of FoxP3

+

↑induction of Tr1 cells ↑ induction of FoxP3+ T cells ↓Th1 and Th17 responses in vivo ↓allo-stimulatory capacity

↑induction of FoxP3

+

↓allo-stimulatory capacity

↓Ag-specific T cell proliferation ↑ CCR7-mediated migratory capacity Active suppression of CD8+ cytotoxic T cells ↓allo-stimulatory capacity (Foxp3+ T cell induction lower than with using Rapa alone) ↓allo-stimulatory capacity - increased FoxP3+ T cell induction ↓Ag-specific T cell proliferation* ↑induction of FoxP3+ Tregs*

Treg induction

Functional characteristics

[116]

N/A

[120]

[114,115]

type 1 diabetes model disease inhibition

induction of PDL-1+ DCs in vivo, delayed onset of diabetes

[109] [112]

[104]

[100]

[89]

[71] [86]

[58,59]

[51]

[45]

[44]

[38]

[39]

[25,32,33,34]

[22–24]

Refs.

↓immune response in hypersensitivity model ↑Treg induction after adoptive transfer

attenuation of atherosclerosis in rats correlated with TolDC induction reduced lymph-node homing

N/A reduced disease progression in DSS colitis model ↑ induction of IL-10+ T cells

moderate effects in mouse colitis model

N/A

amelioration of disease severity in EAE

amelioration of disease severity in EAE

N/A

N/A

suppression of autoreactive T cells

In vivo evidence

Table 1 Basic phenotypic and functional characteristics of TolDCs generated by various agents. Where applicable, the in vivo evidence of TolDCs after their ex vivo generation and subsequent adoptive transfer has been described.

U. Švajger and P.J. Rožman

International Immunopharmacology 81 (2020) 106275

International Immunopharmacology 81 (2020) 106275

U. Švajger and P.J. Rožman

molecules was seen with administration of 1 µM of I-BET151, which corresponds to previously calculated IC50 of 0.79 µM determined by ligand displacement assay against Brd4 [54]. The tolerogenic properties of I-BET151-treated DC were determined in co-culture experiments with naive T cells. In this manner, treated DCs significantly increased the numbers of FoxP3-expressing T cells after a 6 day co-culture. Such FoxP3+ T cells were functionally suppressive and inhibited T cell proliferation in vitro. However, when I-BET151 was administered in vivo to mice using a colitis model, only moderate effects on disease amelioration was seen, with no particular enrichment of FoxP3+ populations in vivo.

pharmacological effects including anti-inflammatory activity [47]. They have been shown to modulate lymphocyte activation as well as cytokine production [48,49]. Perhaps the most well-known macrolide affecting DC function is Rapa, which has been extensively reviewed elsewhere [18]. In contrast to vit D3 and Dex, Rapa does not induce increased IL-10 production, nor has it been documented to induce inhibitory molecule expression. It does however significantly affect Ag up-take, presentation and co-stimulatory molecule expression. Other macrolides, such as clarithromycin and roxithromycin have also been shown to importantly affect DC stimulatory capacity and cytokine production in a negative manner [50]. More recently, azithromycin is the last demonstrated member to show important effects to augment DCs tolerogenic characteristics. In a study by Lin et al., monocyte-derived DCs were treated with azithromycin during differentiation [51]. This led to a down-regulation of maturation markers and inhibition of cytokine production upon LPS-induced maturation, particularly the production of IL-12. The endocytosis of azithromycin-treated cells was up-regulated upon maturation, suggesting an inhibitory effect on the maturation process.

5. Modulators of epigenetic modification mechanisms Both innate and adaptive immune pathways can be regulated by epigenetic mechanisms. The machinery of epigenetic modifications, particularly DNA methylation and various histone modifications, such as acetylation, methylation, phosphorylation and ubiquitylation have important consequences on gene expression and function of other signaling proteins [60]. Histone acetylation and methylation, as well as DNA methylation have been previously associated with development of immune-mediated diseases such as rheumatoid arthritis or multiple sclerosis [61–63]. Additionally, firm evidence exists that underlying mechanisms of epigenetic changes can also modulate cytokine production and inflammatory pathways, such as Nf-κB and p38MAPK pathway in immune cells [64,65]. Histone deacetylase (HDAC) enzymes remove the acetyl group from lysine residues. While generally associated with histone modification, their functional role is now considered much wider and can affect other proteins as well [66]. Innate, as well as adaptive immune pathways have been shown to be importantly regulated by HDAC enzymes and can influence immune cells in various stages of their life cycle. For example, differentiation of macrophages from monocytes is accompanied by up-regulation of HDAC5 [67] and can on the other hand, be negatively regulated by interaction of HDAC3 with the transcription factor PU.1 [68]. In terms of DC function, HDACs can regulate their inflammatory state by interfering with TLR and IFN signaling [65]. Due to their anti-inflammatory activity, the effects of HDAC inhibitors on DC maturation and function have been well studied in the past. It has been demonstrated that HDAC inhibition importantly reduces the expression of co-stimulatory molecules, particularly CD40 and CD80, while CD86 expression remains less affected [69]. Nencioni and colleagues observed skewed DC differentiation from monocytes in the presence of HDAC inhibitor, with down-regulation of CD1a marker [70]. After maturation, they observed reduced CCL19-guided migration along with attenuated immunostimulatory capacity and cytokine secretion. More recently, additional discoveries have been presented in context of HDAC inhibition on DC function. Kaisar et al. have performed an in depth study of the effects of butyrate on human DCs [71]. Butyrate is a short-chain fatty acid previously recognized with the capacity to condition DCs toward a tolerogenic profile and the ability to induce Tregs [72]. In their study, Kaisar and colleagues have shown that butyrate antagonizes the LPS-induced maturation of DCs by simultaneous inhibition of HDACs and G protein-coupled receptor 109A signaling. Similarly to above mentioned studies, the down-regulation of co-stimulatory molecules was mostly evident for CD40, CD83 and CD80, with CD86 being much less affected. Butyrate-treated DCs displayed low production of both IL-12p70 and IL-10. However, despite low IL-10 production, they readily induced IL-10-secreting type 1 Tregs (Tr1) from naive CD4+ T cells [71]. Their capacity to induce Tr1 cells was shown to be dependent on increased activity of RALDH1 in DCs, an enzyme that converts vitamin A into retinoic acid. In this manner, the competence of DCs to produce retinoic acid has been previously associated with the capacity to induce IL-10-secreting T cells [73].

4. Inhibitors of BET bromodomain proteins In the last 10 years, proteins that contain bromodomains have gained a significant biological interest alongside the development of specific inhibitors, which target the acetyl-binding pockets of bromoand extraterminal-domain (BET) proteins [52–54]. In humans, 41 different proteins contain 57 bromodomains altogether and they represent different components of transcription factor complexes with additional involvement in epigenetic memory [55]. The inhibitors of BET family of proteins, which regulates transcription by interactions with lysine residues of histones, have been recently recognized as important antitumor drugs and regulators of inflammation [52]. The expression of three members of BET family, namely Brd2, Brd3 and Brd4 is ubiquitously expressed, while BrdT is primarily found in testis and ovary [56]. These have also been recognized as the major members of the BET family. From these, Brd4 seems to be particularly important in regulating gene transcription via interaction with histones 3, 4 and can increase inflammatory gene expression by binding to acetylated RelA protein, thereby serving as a Nf-κB co-activator [57]. First evidence that inhibition of Brd proteins significantly affects DC function was demonstrated in a pre-clinical allogeneic bone marrow transplantation (BMT) model. Administration of Brd inhibitor I-BET151 at the beginning of BMT resulted in an ameliorated GvHD severity and lower mortality, with no inhibition of graft-versus-leukemia (GvL) effect [58]. After examining the characteristics of DCs cultured in the presence of various Brd inhibitors, treatment significantly lowered the production of IL-6, TNF-α and IL-12. Upon stimulation with LPS, treated DCs displayed reduced expression of CD40, CD80, CD86, PDL-1 and MHC class II molecules in a dose-dependent manner, peaking at 1 µM of inhibitor as the highest concentration used in the study. In a mixed lymphocyte reaction, treatment with I-BET151 significantly reduced the percentage of proliferating cells. Similarly, when T cells were treated with I-BET151 for 24 h and activated via T-cell receptor (TCR), there was a marked down-regulation of IFN-γ and IL-17 production. Mechanistically, Brd inhibition did not change either NF-κB expression or acetylation of RelA. However, they have shown that acetylated RelA associated with Brd4 and treatment with Brd inhibitors disrupted this association. In a later study by Schilderink and colleagues, the effect of Brd inhibition on maturation and function of both bone marrow and monocyte-derived DCs was further evaluated [59]. The addition of Brd inhibitors to already differentiated DCs prior to maturation (I-BET151 at 1 µM) resulted in a significant reduction of IL-12p70, IL-6, as well as IL10. In a similar manner, treatment with I-BET151 negatively affected the surface expression of CD80, CD83, CD86 and MHC class II molecules. Extensive down-regulation of both cytokines and co-stimulatory 5

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6. Non-steroidal anti-inflammatory molecules and TolDCs

potency of EP-induced TolDCs was also tested in an in vivo setting, where mice were administered variously treated DCs one day prior to administration of complete Freund's adjuvant. After 4 days, various immunological parameters were measured, the major difference showing that mice that received EP-induced TolDCs had an increased percentage of IL-10-secreting CD4+ T cells and less IFN-γ-producing T cells in comparison to administration of mature DCs [89]. Although not sufficient, such evidence highlights the potential use of EP as a candidate for development of future TolDC-inducing protocols.

Aspirin (acetylsalicylic acid) was one of the first non-steroidal antiinflammatory drugs shown to potently inhibit in vitro maturation of myeloid DCs, as well as their in vivo stimulatory function [37]. To this day several compounds with anti-inflammatory properties have been shown to exert their effects by modulating DC function including ibuprofen [74], niflumic acid [75] and resveratrol (Res) [76]. Although inhibition of prostaglandin synthesis via cyclooxygenase-2 inhibition is one of main mechanisms underlying anti-inflammatory effects of such drugs, the effects exerted on DCs were mostly associated with direct inhibition of the Nf-κB and mitogen-activated protein kinase (MAPK) pathway. Resveratrol is a polyphenol with low molecular weight that has been attributed with multiple therapeutic effects such as protection against cardiovascular disease, inflammatory bovel disease and cancer [77,78]. Its popularity greatly increased with evidence that it can increase health and lifespan of mice by activating sirtuins, a class of proteins involved in aging [79]. In terms of its effects on immune function, Res was mostly described to possess anti-inflammatory and immunosuppressive activity, with some evidence that it can work in an immunostimulatory fashion at low concentrations [80,81]. We have demonstrated that DCs treated with Res display down-regulation of maturation-associated markers with increased production of IL-10 and surface inhibitory molecules ILT-3 and ILT-4 [76]. The beneficial effects of Res are intensively studied in various clinical trials, however its poor bioavailability represents a major obstacle [82]. More recently, formulation of Res incorporated into lipid nanostructures was shown to be efficiently internalized by both immature and mature monocyte-derived DCs. In such manner, Res successfully inhibited DC maturation in terms of surface markers and IL-12 production at concentrations 2-fold lower than free Res [83], making lipid nanocarriers a viable option to target Res to native DCs thereby increasing its bioavailability. Previously, it has been shown that function of certain polyphenols can be improved by structural modifications induced by γ-irradiation [84,85]. In a recent study, Kim et al. induced a structural modification of Res to investigate the tolerogenic potential of its new radiolysis product. Irradiation of Res with a dose of 50 kGy resulted in isolation of a product with an approximate 2-fold lower molecular weight identified as γ-Res [86]. Treatment of bone marrow-derived DCs with γ-Res significantly down-regulated TNF-α and IL-12p70 production by LPS-stimulated DCs while simultaneously inhibiting CD80 and CD86 co-stimulatory molecule, as well as MHC class I and II expression. In terms of DC tolerogenicity, γ-Res-treated DCs had an induced capacity to produce IL-10 and were able to suppress T cell activation. In short-term in vitro cocultures, γ-Res-treated DCs significantly promoted the induction of FoxP3+ Tregs, although without evidence of their direct immunosuppressive capacity. However, administration of γ-Res to mice with DSS-induced colitis resulted in reduced disease progression and protective immunity reflected in decreased percentage of Th1 and Th17 effector cells [86]. Importantly, compared to natural Res, γ-Res was not cytotoxic at higher concentrations and therefore presents a more suitable option for TolDC induction. Another pharmacological agent with strong anti-inflammatory potential [87], namely ethyl pyruvate (EP), has been recently investigated for its effects on DC biology. Previously, EP has already been shown to have a beneficial effect on disease progression in EAE model, where its therapeutic effects were correlated with attenuation of Th1/Th17 responses [88]. Within the same study, the inhibitory effect of EP on costimulatory molecule expression on macrophages was confirmed, prompting further studies in context of DCs. When added to cultures of bone marrow-derived DCs, EP inhibited the expression of CD40 and CD86 and the production of IL-6, TNF-α and IL-12 [89]. In in vitro MLR cultures, EP-treated DCs displayed poor allogeneic stimulatory capacity. These basic inhibitory effects on DC biology were demonstrated to similar extent also for human monocyte-derived DCs. The tolerogenic

7. Other molecules that induce DC-related immunological tolerance A number of structurally and pharmacologically unrelated molecules have been studied in recent years for their ability to affect DC biology, many of them regularly used for treatment of various conditions such as antihypertensives, cholesterol-lowering drugs, antipsychotics, as well as others. In atherosclerosis, the main pathological mechanisms consist of chronic inflammatory processes and accumulation of lipids. One of key cellular events is the accumulation of macrophages which scavenge oxidized lipids and contribute to plaque formation as foam cells [90]. However, a number of other cells including DCs can be found within atherosclerotic lesions and they can exert both pathogenic and protective roles [91]. Interestingly, mature DCs have been associated with advanced lesions where they readily interact with T cells [92,93]. On the contrary, reduction of atherosclerotic plaques has been associated with increased presence of immature DCs [94]. Furthermore, the importance of active DC tolerogenic function for protection against atherosclerosis has been demonstrated previously [95]. Captopril, an angiotensin-converting enzyme (ACE) inhibitor, is used as a regular medication for the treatment of congestive heart failure and hypertension. It has also been shown to possess anti-atherosclerotic effects [96], particularly in combination with anti-lipemic therapy using statins [97]. Demonstrations regarding additional involvement of captopril in down-regulation of inflammatory responses [98,99] prompted further research to reveal cell-specific activity. Recently, in vivo administration of captopril to atherosclerotic rats has shown that disease attenuation is significantly correlated with inhibiton of DC maturation, down-regulating expression of co-stimulatory molecules CD80 and CD86, as well as MHC class II expression [100]. The tolerogenic properties of splenic DCs were induced as shown by upregulation of IL-10 and TGF-β expression and increased induction of Tregs, registered as an increase of FoxP3 expression within aortic tissue. The synergy in anti-atherosclerotic effects observed between ACE inhibitors and statins could be in part due to their concomitant inhibition of inflammatory processes. Indeed, anti-inflammatory activity has been shown as an important feature of various statins [101]. The effects of statins on DC function has been demonstrated in the past, showing predominantly an immunosuppressive effect, although treatment prior to maturation can cause increase in inflammatory cytokine production [102,103]. Most recently, atorvastatin was studied in-depth for its effects on the tolerogenic function of human and murine DCs [104]. Using monocyte-derived and bone marrow-derived DCs, the authors demonstrated that treatment with atorvastatin causes a dosedependent alterations in DCs' morphological structure resulting in loss of dendrite formation. This was reflected in reduced in vivo lymph nodehoming ability and could be reversed by mevalonate, the end product of statin's target enzyme. Atorvastatin treatment caused a general decrease in co-stimulatory molecule expression. Although differentiation markers CD11b and CD11c were down-regulated, the expression of CD14 remained low after differentiation. Dendritic cells differentiated in the presence of atorvastatin produced significantly more IL-10 upon activation and had the capacity to polarize naive CD4+ T cells into regulatory T cells that were functionally suppressive in vitro. In contrast to previous studies, a characteristic anti-inflammatory cytokine profile 6

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the tolerogenic effects of a pegylated TLR7 ligand 1Z1. Treatment of DCs with 1Z1 resulted in semi-maturation with minimal increase in CD40, CD80 and CD86 co-stimulatory molecules. Such DCs displayed functional T cell suppressive capacity in vitro, as well as after adoptive transfer into diabetic NOD mice [120]. In case of daily administrations of 1Z1 in span of one week there was a delay in disease onset and after examination of pancreatic lymph nodes, the percentage of PDL-1 expressing DCs increased significantly.

was observed for atorvastatin-treated DCs. Interestingly, psychiatric drugs such as antipsychotics or sedatives have been frequently associated with various immunomodulatory effects. For example, haloperidol, a classical antipsychotic drug used to treat symptoms of bipolar disorders, schizophrenia and others, has been associated with effects on immune cell proliferation and cytokine production [105,106]. Furthermore, changes in immunological parameters have been observed for patients suffering from schizophrenia in comparison to healthy controls, with an increase in Th1- vs Th2-type cytokine concentrations in plasma [107]. In addition, patients treated with antipsychotic drugs displayed attenuated signs of Th1 responses, such as lower IL-12 plasma levels [108]. It is therefore safe to speculate the importance of antipsychotic therapy on the adaptive immune response. In this manner, Matsumoto and colleagues studied the effect of haloperidol on murine bone marrow-derived DCs [109]. Haloperidol inhibited DC maturation in terms of CD80, CD83 and CD86 molecule expression. Treated DCs also displayed lower expression of MHC class II molecules and possessed lower allo-stimulatory potential compared to control DCs. In correlation with clinical effects of haloperidol on Th1type immunity, adoptive transfer of haloperidol-treated DCs in a contact hypersensitivity model failed to induce a normal immune response, compared to adoptively transfered control DCs. The immunosuppressive effect of haloperidol was associated with direct inhibition of D2 dopamine receptors, since blocking the D2 receptor using a synthetic antagonist also caused suppression of DC maturation. Similar findings were reported for berberine, a natural D1 and D2 receptor antagonist. Berberine ameliorated disease symptoms in a murine colitis model with simultaneous reduction in both IFN-γ and IL-17 expression [110]. Drawing to conclusion, in the last 5 years, induction of DC tolerance and/or inhibition of their activation has been demonstrated or revisited for a few additional molecular entities with pharmacological acitivity. The expression of heme oxygenase 1 (HO-1) has been confirmed as of importance for DC tolerogenic function, stemming from previous reports on association of HO-1 competence with inhibition of DC maturation and development of IL-10-producing capacity [111]. Wong and colleagues have shown that induction of HO-1 by cobalt (III) protoporphyrin-IX-chloride was sufficient for increased DC capacity to induce FoxP3+ Tregs in vitro and in vivo after adoptive transfer of treated DCs in an airway inflammation model [112]. In a similar manner, transfered DCs were capable of reducing DC-mediated airway inflammation. Degradation of heme, a natural substrate for HO-1, leads to the release of biliverdin, free iron and carbon monoxide (CO) [113]. Carbon monoxide in particular has been established as one of major factors driving the immunosuppressive effects exerted on DCs [114]. In a recent study, the tolerogenic impact of CO on DCs was shown to be mediated by effects of CO on mitochondrial destabilization, thereby blocking Ag processing and T cell-mediated immunity [115]. The importance of mitochondrial stability for efficient Ag presentation, demolished by CO treatment, was evident in vivo in a DC-induced type 1 diabetes model where adoptive transfer of DCs treated by CO-releasing reagents inhibited disease onset. Among atypical molecules affecting DC function in an immunosuppressive manner is thimerosal, a mercury-based perservative used in multidose vials vaccine formulations. At nanomolar concentrations, thimerosal was shown to inhibit LPS-induced DC maturation and suppressed the production of IL-12, TNF-α nad IL-6 in vitro [116]. Another atypical inducer of DC tolerogenicity in terms of mechanisms, was recently discovered to work by TLR7 activation. While activation of various TLRs is generally associated with immunogenicity, it can lead to induction of tolerogenic responses in certain circumstances, e.g. in type 1 diabetes or by Treg activation [117]. This is most likely associated with partial- or semi-maturation of DCs that has been previously shown to result in tolerance induction and is also known to be induced by other factors such as TNF-α or short-term exposure to LPS [118,119]. More recently, Hayashi and colleagues demonstrated

8. Conclusion In the last few years we have witnessed evidence for a fair amount of pharmacological entities that have been attributed with TolDC-inducing properties. It is becoming increasingly clear that TolDC induction, although with the main purpose of inducing an immunosuppressive DC type, is not a one way street and can lead to important biological differences in final cell characteristics. Such different »flavors« of TolDCs could be seen in differential expression of surface and soluble inhibitory molecules, production of various inflammatory cytokines, as well as specific migratory abilities. Precise konwledge of how to manipulate DCs toward a particular tolerogenic state has important implications for their future use as a cellular therapy product in context of different immune-mediated pathologies, where certain TolDC characteristics would be favoured in treatment of a specific disease. This has been clearly shown by revisiting the mechanisms and biological effects of TolDC induction by »gold standard« pharmacological drugs such as vit D3, dexamethasone and rapamycin. In the future, more emphasis should be given to specific combinations of various pharmacologicals and also their combinations with tolerance-inducing biomolecules. Furthermore, since many of the described agents are already used for the treatment of various pathological conditions, their technological formulation could be designed in a way to directly tolerize DCs in vivo, e.g. by using DC-targeting nanoparticles. This would allow for discovery of potential tolerogenic synergies and/ or the exploitment of full therapeutic potential of TolDCs. Funding This work was supported by the Slovenian national research agency, grant no.: P3-0371. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] C. Audiger, M.J. Rahman, T.J. Yun, K.V. Tarbell, S. Lesage, The importance of dendritic cells in maintaining immune tolerance, J. Immunol. 198 (6) (2017) 2223–2231. [2] R.M. Steinman, S. Turley, I. Mellman, K. Inaba, The induction of tolerance by dendritic cells that have captured apoptotic cells, J. Exp. Med. 191 (3) (2000) 411–416. [3] M.C. Takenaka, F.J. Quintana, Tolerogenic dendritic cells, Semin Immunopathol 39 (2) (2017) 113–120. [4] A. Ten Brinke, M. Martinez-Llordella, N. Cools, C.M.U. Hilkens, S.M. van Ham, B. Sawitzki, E.K. Geissler, G. Lombardi, P. Trzonkowski, E. Martinez-Caceres, Ways forward for tolerance-inducing cellular therapies- an AFACTT perspective, Front. Immunol. 10 (2019) 181. [5] P. Lord, R. Spiering, J.C. Aguillon, A.E. Anderson, S. Appel, D. Benitez-Ribas, A. Ten Brinke, F. Broere, N. Cools, M.C. Cuturi, J. Diboll, E.K. Geissler, N. Giannoukakis, S. Gregori, S.M. van Ham, S. Lattimer, L. Marshall, R.A. Harry, J.A. Hutchinson, J.D. Isaacs, I. Joosten, C. van Kooten, A. Lopez Diaz de Cerio, T. Nikolic, H.B. Oral, L. Sofronic-Milosavljevic, T. Ritter, P. Riquelme, A.W. Thomson, M. Trucco, M. Vives-Pi, E.M. Martinez-Caceres, C.M.U. Hilkens, Minimum information about tolerogenic antigen-presenting cells (MITAP): a first step towards reproducibility and standardisation of cellular therapies, PeerJ 4 (2016). [6] K. Steinbrink, H. Jonuleit, G. Muller, G. Schuler, J. Knop, A.H. Enk, Interleukin-10-

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