Immuno-modulatory Role of Indoleamine 2, 3-Dioxygenase in Allogeneic Islet and Skin Transplantation

Elevated levels of IDO and downstream metabolites such as N-formylkynurenine are usually what occur when the tryptophan metabolism pathway is deregulated. N-formylkynurenine is readily hydrolyzed to kynurenine and enzymatically converted to many different metabolites. One of these metabolites includes quinolinic acid, an excitotoxin whose production has been linked to the pathogenesis of neuroinflammatory, neurodegenerative disorders such as Alzheimer’s and depression, age-related cataract, and HIV encephalitis (Heyes et al 1992, Guillemin et al 2005, Wichers and Maes 2004, Vazquez et al 2004, and Sardar and Reynolds 1995).

Expression and Regulation

IDO is primarily an inducible intracellular protein and is expressed in different tissues. IDO is most prominently expressed by some subsets of antigen presenting cells (APCs) of lymphoid organs and the placenta (Jalili et al 2007). However, it is also expressed in eosinophils (Odemuyiwa et al 2004), endothelial cells (Varga et al 1996 and Beutelspacher et al 2006), and lung epithelial cells (Hayashi et al 2004) suggesting that it may play an important role in allergic inflammation. IDO is only constitutively expressed in the male epididymis and lower gastrointestinal tract (Yoshida et al 1981). The actual presence or absence of functional IDO enzymatic activity is tightly regulated by specific maturation and activation signals. Conceptually this ability to up regulate or down regulate IDO in response to external stimuli seems logical, given the need for APCs to sometimes present antigens in an activating fashion and sometimes in a tolerating fashion, depending on the context (Munn and Mellor 2007).

A single gene located on the short arm of chromosome 8 (8p12-8p11) encodes the IDO protein (Burkin et al 1993). 10 exons are spread over 1.5 kbp of DNA (Burkin et al 1993). In cell types such as dendritic cells (DCs), macrophages, eosinophils, epithelial and endothelial cells, the IDO gene promoter is driven by a combination of locally produced potent pro-inflammatory stimuli. It is most strongly activated by the T helper 1 (Th-1) type cytokine interferon gamma (IFN-γ) (Jalili et al 2007). In addition to IFNs, CD40 ligand (CD40) up regulation on the T lymphocytes is also important for IDO expression (Munn et al 1999 and Hwu et al 2000).

Cells usually do not express IDO unless they are induced to do so by treating the cells with IFN- γ, which is produced in response to inflammation. All mammalian IDO genes studied so far have IFN-γ response elements including one interferon-gamma activated sequence (IGAS) and two interferon stimulated response elements (ISREs) (Jalili et al 2007 and Johnson et al 2009). IGAS is specific for IFN-γ, whereas the ISREs are nonspecific and can respond to IFN-α, IFN-β, and IFN-γ. IFN-γ is usually up to 100 times more potent at inducing IDO expression than either IFN-α or IFN-β but this depends on the cell type being cultured (Taylor and Feng 1991). Following ligation of IDO inducers, intracellular signaling occurs along the JAK-STAT pathway and nuclear factor kB (NF-kB) (Mellor and Munn 2004 and Thomas et al 1999) to result in the expression of the IDO protein (Tone et al 1990). After the IDO protein has been encoded, further signals are needed for the IDO protein to be activated. 

IDO gene transcription can also occur in response to cytokines such as toll-like receptor (TLR) ligation (e.g. through lipopolysaccharide) (Mahanonda et al 2007 and Furset et al 2008), tumour necrosis factor α (TNF-α) (Werner-Felmayer et al 1989), glucocorticoid-induced TNF receptor (GItR) ligand (Grohmann et al 2007), and histone deacetylase inhibitors (HDACS) (Reddy et al 2008). T helper 2 (Th-2) type cytokines such as interleukin 4 (IL-4) and interleukin 13 (IL-13) inhibit the expression of IDO (Chaves et al 2001 and Musso et al 1994).

Recently another protein involved in tryptophan metabolism called IDO2 was also discovered (Ball et al 2007). The IDO2 gene is also located on chromosome 8 and its amino acid sequence is 43% similar to that of IDO (which is now also known as IDO1) (Ball et al 2007). However, it only shows 3-5% of the enzymatic activity of IDO when expressed as a transgene in bacteria or eukaryotic cells (Ball et al 2007).

IDO and Immunosuppression

The immune system continuously modulates the balance between responsiveness to pathogens and tolerance to non-harmful antigens, although the exact mechanisms are not understood.  IDO has been suggested in taking part in the counter-regulatory mechanisms that prevent the immune system from causing inappropriate or excessive responses, which could potentially lead to damaging effects (Kyewski and Klein 2006). Munn et al (1998) were the first to report the potential role of IDO in immunosuppression. It was seen that in mammals during pregnancy, the expression of IDO at the interface between the fetus and the mother prevents abortion of the allogeneic fetus. IDO prevents maternal T cells from causing potentially harmful responses to paternally inherited fetal alloantigens by inhibiting the proliferation of these T cells in the placenta (Munn et al 1998). Munn et al (1998) also showed that the administration of 1-methyl tryptophan (1-MT), a commonly used competitive inhibitor of IDO, led to fetal rejection. 1-MT causes a reversal of the IDO induced suppression of T cells. IDO has also been shown to play an important role in setting up immunologically privileged parts of the body, such as the anterior chamber of the eye (Malina and Martin 1993) and the brain (Sardar and Reynolds 1995 and Hansenet al 2000).

Previous studies showed that IDO plays an important role in the induction of T cell unresponsiveness, thereby establishing immune tolerance and controlling autoreactive immune responses. The regulatory effect of IDO on T cells is due to the expression of this enzyme by DCs, monocytes, and macrophages. An inverse correlation has been found between IDO expression and the number of CD8 + (Brandacher et al 2006 and Inaba et al 2009) and CD3+ (Ino et al 2008) T lymphocytes.  In particular, DCs, which are specialized to acquire, process, and present antigens to stimulate naïve T cells, can cause potent and dominant T cell and natural killer (NK) cell suppression (Johnson et al 2009). Exactly how IDO causes immunosuppression is unclear, but it has been proposed that IDO causes toxic tryptophan metabolites to accumulate and a tryptophan deficient microenvironment to form (Jalili et al 2007). The tryptophan metabolites, in particular kynurenine, quinolinic acid, and picolinic acid as shown in Figure 1, are directly toxic to T cells and NK cells (Soliman et al 2010). Depletion of the essential amino acid tryptophan causes immunosuppression by making the T lymphocytes more prone to cell cycle arrest at the G1 phase (Munn et al 1999, Frumento et al 2009 and Terness et al2002), anergy (Munn et al 2004), and apoptosis (Fallarino et al 2002). Tryptophan starvation also causes naïve CD4+ T lymphocyte conversion to immunosuppressive regulatory T cells (Tregs) (Fallarino et al 2006) and activation of mature CD4+ CD25+ Foxp3+ Tregs (Sharma et al 2007). An increase in the number of Tregs causes an antigen-specific impairment of T-cell priming (Jalili et al 2010). The IDO overexpression also induces a Th2 immune response shift and generates an anti-inflammatory cytokine profile (Jalili et al 2010). IDO has also been known to be an innate defense mechanism. By depleting the local microenvironment of tryptophan, it has been able to help limit the growth of viruses, bacteria, and intracellular pathogens (de Jong et al 2012).
 
Our research group has been interested in the immune-protective role of IDO for allogeneic engraftment of a skin substitute. Li et al also showed that immune rejection of an allogeneic skin substitute could be prevented by using IDO expressing fibroblasts used as the cellular component of this skin substitute (Li et al 2004). It was then shown that the expression of this enzyme suppresses the major histocompatibility (MHC) class I antigens due to depletion of tryptophan (Li et al 2004). When tryptophan or an IDO inhibitor was added, MHC class I levels were restored (Li et al 2004). MHC class I antigens serve as targets for allogeneic immune rejection and as such, down regulation of these antigens seems to be a part of the mechanism underlying IDO mediated local immunosuppressive effects (Li et al 2004).

The immune privilege created by locally expressed IDO may not always be beneficial to the host and can lead to pathological effects, similar to those resulting from the use of other long-term immunosuppressant agents. Suppression of T cells can cause chronic infections to escape attack even if T cell antigens are present (Johnson et al 2009). At the sites of chronic inflammation, a potent and dominant T cell suppression has been studied (Johnson et al 2009). The same problem can result with developing malignancies. Cancer cells expressing IDO can deplete their local microenvironment of tryptophan, preventing T cell proliferation (Mellor and Munn 1999), and cause toxic metabolites to accumulate, further inhibiting T cell activation (Mellor et al 2002). As in DCs, monocytes, and macrophages, an inverse correlation has been found in many tumour types between IDO expression and the number of tumour infiltrating CD8+ T cells (Brandacher et al2006, Inaba et al 2009, Ino et al 2008, and Uyttenhove et al 2003). Overexpression of IDO by malignant cells is an important cancer induced immune escape mechanism and has been associated with poor prognoses for survival (Brandacher et al 2006, Inaba et al 2009, Ino et al 2008, Risenberg et al 2007, Inaba et al 2010, and Pan et al 2008). This finding, however, has increased the potential of using IDO inhibitors, such as 1-MT, in cancer treatments. Studies in mice have shown that the combination of 1-MT with chemotherapeutic agents is more effective against tumours than the use of both therapies on their own (Hou et al 2007 and Qian et al 2009). It should be noted though that is has been shown in certain cancers, such as ovarian cancer, only the levo-1-MT (L-1MT) isomer is effective in blocking IDO expression (Qianet al 2009). The dextro-1-MT (D-1MT) isomer is inefficient (Qian et al 2009). IDO Increased expression of IDO has also been associated with inflammatory bowel disease (IBD) (Ciorba 2013).

In general, depending on the application of IDO, the expression and inhibition of IDO might be helpful in generating local immune privilege for allogeneic engraftment or disabling the cancer cells to protect themselves against the host immune system.  Our research group has used the expression of IDO in skin cells to immuno protect either allogeneic skin cells or insulin producing islets.  Using this approach, a tryptophan deficient environment has been generated within which the activated immune cells, mainly CD8+ T cells, cannot survive and attack the allogeneic cells.  

Role of IDO Expression in Allogeneic Islet Transplantation

IDO expression has great potential in helping patients suffering from autoimmune disorders such as type I diabetes.  Since the discovery of insulin, daily insulin injections have been the only way of treating patients with type I diabetes. Islet transplantation has been thought to be a feasible and attractive alternative therapeutic approach (Ricordi et al 2008 and Fiorina and Secchi 2007). However, patients need to receive a systemic immunosuppressive drug for their entire lifetime, which is problematic due to its side effects, and some immunosuppressive drugs are also prodiabetic (Froud et al 2006, Zahr et al 2007, Drachenberg et al 1999, and Vantyghem et al 2007). Our research group conducted a series of novel studies showing the potential use of three-dimensional islet grafts equipped with IDO-expressing dermal fibroblasts to prevent islet allograft rejection without systemic immunosuppressive agents. We showed that IDO genetically modified islets transplanted into mouse kidney capsules survive longer than those islets transplanted without IDO expressing fibroblasts (Jalili et al 2010). This prolongation was due to the expression of IDO preventing T cell proliferation and infiltration into transplanted allogeneic islets (Jalili et al 2010).

Later studies by our research group (Jalili et al 2010) investigated the potential use of composite 3-dimensional islet grafts. These grafts were engineered by embedding allogeneic mouse islets and adenoviral transduced IDO-expressing syngeneic bystander fibroblasts within collagen gel matrices (Jalili et al 2010). As shown in Figure 2 (Jalili et al 2010), T cells accumulate at the margins of IDO-expressing grafts but are not able to actually invade the islets.