Trivalent arsenic in mouse models of immune-mediate diseases

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PML and PML nuclear body regulation

It was initially found that the APL promotor promyelocytic leukemia-retinoic acid receptor-α (PML/RARα) was especially sensitive to As2O3-induced degradation, which was the critical step of disease eradication (Chen et al., 1996). The following comprehensive studies by de Thé et al reveal that As2O3 specifically targets the PML moiety (Lallemand-Breitenbach et al., 2018). PML nuclear bodies (NBs), nucleared by the PML protein, could recruit and sumoylate dozens of partner proteins, leading to a variety of biological processes (Lallemand-Breitenbach et al., 2001; Sahin et al., 2014). Notably, As2O3-induced oxidative stress could enhance formation of PML NBs, leading to p53 activation in vivo in normal mice (Niwa-Kawakita et al., 2017).
In APL, through both reactive oxygen species (ROS) production and direct binding, As2O3 exerts its dual-targeting effects (Jeanne et al., 2010). On one hand, As2O3 induces PML/RARα sumoylation, proteasomal degradation, and APL cell differentiation (Lallemand-Breitenbach et al., 2018; Fasci et al., 2015). On the other hand, As2O3 targets the wild-type PML proteins, leading to re-formation of PML NBs, subsequent p53 activation, and ultimately APL clearance (Niwa-Kawakita et al., 2017; Ablain et al., 2014). The mechanisms of action for As2O3-induced PML/RARα and PML NB regulation in the context of APL are shown in Figure 2.
AS, arsenic trioxide; RXR, retinoid X receptor; RARE, retinoic acid response elements; It is also found that in adult T-cell leukemia/lymphoma (ATL), As2O3, together with IFN-α, reaches disease complete remission both in mice and in human through degradation of the disease driver oncoprotein Tax (Kchour et al., 2009; El-Sabban et al., 2000; El Hajj et al., 2010). Interestingly, this process was also mediated through a As2O3 enforcement of PML NBs formation, Tax sumoylation and proteasomal degradation (Dassouki et al., 2015). In addition, it is also discovered that the As2O3/ATRA combination significantly decreases NPM1-mutant AML leukemia blast in patients though oxidative stress generation, p53 activation, and ultimately mutant NPM1 degradation (El Hajj et al., 2015; Martelli et al., 2015).
Apart from tumor suppression, PML and PML NBs are recently found to play key role in mediating innate immune responses (Lallemand-Breitenbach et al., 2001; Hsu et al., 2018; Scherer et al., 2016). Studies have identified PML as a direct, positive regulator of IFN-I signaling (Kim et al., 2015), and is implicated in the regulation of and extended spectrum of cytokines such as the pro-inflammatory IL-1 β and IL-6 (Scherer et al., 2016; Lo et al, 2013). It is thus with great interest to investigate if PML and PML NBs modulations explain As2O3’ efficacy in autoimmune/inflammatory diseases with IFN-I signature and abnormal cytokine profile.

The multifaceted effects of trivalent arsenic on immune cells

Chronic arsenic exposure leads to systemic immunosuppression (Dangleben et al., 2013). However, clinical use of As2O3 has shown good safety-profile, with limited side effects (Shen et al., 1997). No long-term treatment-related immune-mediated disease or tumorigenesis is reported. During the As2O3 single agent treatment for APL, there are inhibitory effects on hematopoietic progenitor cells, and it takes 145 and 265 days for circulating T and B cells, and 655 days for natural killer cells (NKs) to achieve the median normal levels (Alex et al., 2018). Comprehensive studies have revealed the multifaceted effects of As(III) on each immune subset, which will be introduced in the next section (summarized in Figure 3).

Trivalent arsenic and monocytes/macrophages

Recent studies have shown that monocytes are not simply precursors of macrophages as previously thought, but in fact give rise to functionally distinct monocyte-derived cells during inflammation (Guilliams et al., 2018). As2O3 induces human monocyte apoptosis during macrophage differentiation through down-regulation of the Nf- κ B related pathway (Lemarie et al., 2006b). Moreover, As2O3 increases lipopolysaccharide (LPS) dependent expression of the inflammatory IL-8 gene, by stimulating a redox-sensitive pathway that strengthens p38-kinase activation (Bourdonnay et al., 2011).
For macrophages, high concentration of As2O3 induces cell apoptosis through a mitochondrial-dependent pathway (Sengupta et al., 2002; Srivastava et al., 2016). It changes human monocyte-derived macrophages’ morphology, reduces their adhesive capacity, decreases macrophagic surface marker expressions, and impairs phagocytosis of E.coli in vitro (Lemarie et al., 2006a). Interestingly, As2O3 potentiates macrophages’ ability to secrete inflammatory TNF-α, IL-1α, CCL18, and to induce allogeneic or autologous T cell responses (Lemarie et al., 2008; Sakurai et al., 2005). These in vitro observations are confirmed by ex vivo data (Banerjee et al., 2009; Bishayi et al., 2003). The functional changes are probably due to superoxide generation, activation of the Rho-kinase/p38-kinase pathway (Lemarie et al., 2006a), and modulation of the UPR signaling (Srivastava et al., 2013). Especially, activating transcription factor 4 (TCF4, or E2-2) protein, a UPR transcription factor, plays a key role in As2O3-mediated regulation of macrophage functions (Srivastava et al., 2016). In addition, As2O3 also globally regulates redox-sensitive gene expression in human macrophages (Bourdonnay et al., 2009a, b). Therefore, As(III) exerts double-sided effects on macropahges by imparing their clearance capacity while enhancing the pro-inflammatory functions.

Trivalent arsenic and dendritic cells

In vivo we distinguished two types of dendritic cells (DCs), namely conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). MDDCs are broadly used for research in vitro for availability reason. NaAsO2 inhibits dendritic differentiation of monocytes in vitro (Bahari et al., 2017). It also decreases MDDCs viability, maturation, phagocytic capacity, as well as their ability to secrete the pro-inflammatory cytokines IL-12 and IL-23, and to stimulate T helper cell (Th) to secrete IFN- γ (Macoch et al., 2013). The inhibition of IL-12 is mediated by the induced expression of Nrf2 (Macoch et al, 2015).

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Trivalent arsenic and T cells

T cells play a central role in cell-mediated immunity. High concentrations of As(III) induce apoptosis of T helper cells (CD4+ T cell), cytotoxic T cells (CD8+ T cell), and regulatory T cells (Treg) (Gupta et al., 2003; Thomas-Schoemann et al., 2012; Tenorio et al., 2005). Both mitochondrial-mediated and the receptor-mediated pathways are involved in T cell apoptosis. Gupta et al. revealed that As2O3 induces apoptosis of CD4+ and CD8+ T cells via the mitochondrial pathway by enhancing the generation of oxidative stress and by regulating the expression of Bcl-2 family proteins (Gupta et al., 2003). When CD4+ T cells were investigated within human peripheral blood mononuclear cells (PBMC), tumor necrosis factor α released from other mononuclear cells after NaAsO2 exposure induced CD4+ T cell apoptosis through TNF-R1 apoptotic signaling (Yu et al., 2002). There is different sensitivity to As(III) toxicity among the T cell subsets. CD4+ are more sensitive than CD8+ T cells to the pro-apoptotic toxicity of NaAsO2, as discovered both in vitro and in vivo in human and mouse model (Yu et al., 2002; Duan et al., 2017; Vega et al., 2004). This difference leads to a reduced CD4+/CD8+ ratio, which is associated with immunodysfunction (Lu et al., 2015). Tregs are important CD4+ T cells mediating immune tolerance. In vitro tests show that As2O3, at the same concentration, preferentially induces apoptosis of human purified CD4+CD25+ Tregs than CD4+CD25- effector T cells, and decreases the Treg frequency in APL patients’ peripheral blood (Xu et al., 2018). As2O3-mediated selective Treg depletion is also discovered in vivo in mouse model (Thomas-Schoemann et al., 2012). As2O3 induces Treg apoptosis through ROS and reactive nitrogen species (RNS) generation, and the differential effect of As2O3 on Treg versus other CD4+ cells may be related to differences in the cells’ redox status (Thomas-Schoemann et al., 2012). However, some other studies report opposite results (Zhao et al., 2018a; Tohyama et al., 2013). After exposure to NaAsO2, the mRNA level of the Treg specific transcription factor forkhead box P3 (Foxp3) is upregulated in the spleen and thymus of the experimental mice (Duan et al., 2017). Interestingly, Tohyama et al report that in mitogen activated human PBMC, 5μM As2O3 decreases the Treg frequency after treatment for 48h and, conversely, increases its frequency after 96h of culture (Tohyama et al., 2013). These results indicate that short-term exposure to As(III) may deplete Treg, in contrast to long-term exposure leading to Treg percentage increase.

Allogeneic organ/stem cell transplantation

During organ or allogeneic stem cell transplantation, human leukocyte antigen (HLA) mismatches lead to severe T- and B-cell-mediated alloreactivity, which may eventually lead to severe inflammatory transplantation complications of allograft rejection or GVHD, respectively. As2O3 is shown to prolong the allograft survival in immunocompetent mouse heart transplant models, with reduction of the proportions of CD4+ and CD8+ memory T cells, and increase of Tregs in recipient spleen and lymph nodes (Yan et al., 2009; Xu et al., 2010; Lin et al., 2011). Meanwhile, the IFN-γ expression is reduced and TGF-β expression is increased in both the recipient serum and the graft (Xu et al., 2010; Lin et al., 2011). Further studies show that As2O3 inhibits accelerated allograft rejection mediated by alloreactive CD8+ and/or CD4+ memory T cells, and prolongs allograft survival (Yan et al., 2013; Li et al., 2015a). Moreover, in two models (one allo- and one xeno-) of islet transplantation, As2O3 is shown to prolong the graft survival by inhibiting inflammatory reactions and T cell responses (Gao et al., 2015; Zhao et al., 2018b). GVHD occurs when immunocompetent T cells in the graft recognize the recipient as foreign, and attacks the target organs. A study reveals that As2O3 prevents disease occurrence in a murine sclerodermatous GVHD model mediated by overproduction of H2O2 which kills activated CD4+ T cells and pDCs (Kavian et al., 2012a). To summarize, As(III) could be a promising drug for allo-reactivity through effector-cell modulations.

Clinical trials of trivalent arsenic on immune-mediated diseases

Given the discoveries in basic research and pre-clinical models, interests grow on As(III) as a clinical therapeutic agent in immune-mediated diseases. An on-going randomized clinical trial (NCT02966301) is currently testing As2O3 as first-line treatment of chronic GVHD. Results of a phase 2a randomized clinical trial (NCT01738360) aiming to evaluate the therapeutic efficacy of As2O3 in SLE are expected soon.

Table of contents :

1. Introduction
1.1. Immunomodulatory properties of trivalent arsenic
1.1.1. Pharmaceutical mechanisms of action
1.1.2. The multifaceted effects of trivalent arsenic on immune cells
1.1.3. Trivalent arsenic in mouse models of immune-mediate diseases
1.1.4. Trivalent arsenic and tumor immunotherapy
1.2. Plasmacytoid dendritic cells in autoimmunity/alloreactivity
1.2.1. Definition of pDCs
1.2.2. Development of pDCs
1.2.3. Functions of pDCs
1.2.4. pDCs in autoimmunity
1.2.5. pDCs in alloreactivity
1.3. Mouse models of acute GVHD
1.3.1. Acute GVHD (aGVHD)
1.3.2. Challenges in aGVHD prophylaxis/treatment
1.3.3. Translational value of existing aGVHD mouse models
2. Results
2.1. Part 1. Immunomodulatory effects of arsenic trioxide on plasmacytoid dendritic cells and study of mechanism
2.1.1. Article 1: Arsenic trioxide induces regulatory functions of plasmacytoid dendritic cells through interferon-alpha inhibition
2.2. Part 2. Effects of arsenic trioxide and other immunomodulatory drugs in a novel mouse models of acute graft-versus-host disease
2.2.1. Results
2.2.2. Materials and Methods
3. Discussions
3.1. As2O3, a promising drug for diseases with IFN-I signature
3.2. A novel aGVHD model with chemotherapy-based conditioning and G-CSF mobilized graft: advances and limitations
4. Conclusion
5. References
6. Annex

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