Recognize non-self pathogens or tissues

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The immune system is highly specialized to recognize non-self pathogens or tissues

And efficiently eliminate them, but tumors can often escape host immunity in various ways. Tumors generate an immunosuppressive microenvironment that protects the tumor from host immunity, enhance tumor growth, and attenuates immunothereaputic efficacy.(y)

Indoleamine 2,3-dioxygenase (IDO) is an interferon (IFN)-γ- inducible, extrahepatic, intracellular heme enzyme that catalyses initial rate limiting step in essential amino acid tryptophan breakdown along the kynurenine pathway.(1) It catalyses conversion of L-tryptophan to N-formulkunurenine, L-tryptophan is necessary for cell protein synthesis. It is an immunomodulatory enzyme produced by some alternatively activated macrophages and other immunoregulatory cells. IDO causes halted growth of microbes as well as T cells by causing depletion of tryptophan.(2) IDO is a heme-containing dioxygenase enzyme discovered by Osamu Hayaishis group. It exhibits substrate specificity for various indoleamines including L-Trp, and serotonin.(3)

Structure of Indoleamine 2,3-dioxygenase.

The structure of Indoleamine 2,3-dioxygenase is folded into small and large domain. The large domain comprised of 13α- helices and two 310 helices. The small domain is comprised of six-α-helices two short β- sheets and three 310 helices. Contact between these two domains is very extensive with a buried surface area of 3100Å.

Crystal structure of recombinant human Indoleamine 2,3-dioxygenase (45 kDa), complexes with the legend inhibitor 4-phenylimidazole (PI) and cyanide (CNˉ) at resolution of 2.3 and 3.4 Ǻ, respectively.

The catalytic reaction heme containing dioxygenase enzyme was showed by tertiary structure of Indoleamine 2,3-dioxygenase.

Discovery of Indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase was discovered in 1950s by Hayaishi and co-workers. They reported that Indoleamine 2,3-dioxygenase a heme containing dioxygenase, catalyses the first and rate limiting step of L-tryptophan catabolism in kynurenine pathway. This step results in production of L-formyl kynurenine by oxidative cleavage of 2,3 double bond in the indole moitiety of L-tryptophan. L-tryptophan is a precursor of a neurotransmitter serotonin and hormone melatonin.

Due to lack of structural information, physiological role and chemistry of IDO reaction is still poorly understood. But on the other hand, on the basis of the model system and crystal structures of other heme contaning monooxygenase like cytochrome P450 and peroxidase or nonheme type dioxygenase have been extensively studied.

Biology of Indoleamine 2,3-dioxygenase.

In humans over 90% of tryptophan was catabolised bu kynurenine pathway. Indoleamine 2,3-dioxygenase is a cytosolic monomeric hemoprotein of 403 aa long (MW 45, 324 kDa) that catalyses the first step in tryptophan catabolism by the kynurenine pathway. (4)

N-formyl kynurenine was produced during oxidative cleavage of the tryptophan 2-3 double bond.

Indoleamine 2,3-dioxygenase catalyses the conversion of tryptophan to N-formyal kynurenine. Initially Indoleamine 2,3-dioxygenase is bound by substrate and secondly IDO-tryptophan complex and finally bound to oxygen to form IDO-O2 complex which is then converted to N-formylkynurenine pathway.

Mis 346 and Asp 247 plays a crucial role in binding of heme and thus for enzymatic activity of IDO. (5)

The promoter region of human and murine IDO contains regulatory motifs conferring responsiveness to inflammatory eytokins. These motifs include ISRE a two interferon- stimulated response elements, GAS a single interferon active site and IFN-γ interferon gamma. From these studies it is suggested that Indoleamine 2,3-dioxygenase was primarily used as an inflammatory agent, limiting microbial infections and only later during evaluation it acquired an immunoregulatory role.

Reaction mechanism of Indoleamine 2,3-dioxygenase.

Number of substrates and inhibitors are known for IDO. L-tryptophan is an example os substrate that have electron donating group at C-5 or C-6 position.

Recently proposed mechanism involving proton abstraction by the iron bound O2, followed by electrophillic addition of the dioxygen-attacking double bound between C-2 and C-3 of the pyrrole ring.

Lack of H at first position of the indole derivatives, was caused by the inhibitory effect of either methylation (1-methyl D-Trp)or substitution of the indole N with O or S (furan or thiophene analogs).

Indoleamine 2,3-dioxygenase suppressive mechanism in immune system.

Indoleamine 2,3-dioxygenase is an evolutionary ancient enzyme and was originally postulated to serve as a host defense mechanism against certain microorganisms. However recently, IDO has also been shown to have potent immunosuppressive effects on T cell activation and inflammatory responses.

The immunologic role of IDO was initially recognized when it was shown that Indoleamine 2,3-dioxygenase expression in plecenta was necessary in order to maintain maternal tolerance towards allogenic fetus. From this observation two major implications come in light that were potentially of relevance for tumor immunology. First, the developing fetus appeared to have adopted a normal, preexisting suppressive mechanism in the immune system in order to create tolerance towards itself. Second blocking this mechanism by administrating the IDO inhibitor drug 1-methyl tryptophan (1MT) to mothers, allowed the fetus to be rejected by the maternal immune system.

Generally, Indoleamine 2,3-dioxygenase functions as a normal regulatory mechanism in immune system. Endogenous IDO expression has been suggested to contribute to self tolerance in NOD mice, as well as to control autonomic encephalitis experimental asthma, and inflammatory bowel disease.

Indoleamine 2,3-dioxygenase may serve as a down stream suppressive mechanism used by Tregs and by certain tolerance inducing agents such as CTLA-4-Ig and activating Anti-4-BB antibody. In most of the preceding studies, the mechanistic role of IDO was demonstrated in vivo by treating a mice with the IDO inhibitor drug 1MT and showing a consequent loss of immune regulation or tolerance controversially, viral mediated gene transfer of recombinant IDO into grafted tissues delays the destruction of pancreatic islet cells and protects heptomismatched lung transplants from rejection. Thus mediator of certain forms of self tolerance towards new antigens.

Number of cell types, including epithelial cells in the gut and lung, as well as professional antigen cells (APCs) such as microphages and dendrite cells express IDO in response to proinflamatory stumli.

IDO mediated inhibition of T cell prolification has now been demonstrated in atopic human monocytes. (6), human monocyte derived macrophages, monocyte derived DCs (7) when studied in vitro.

During in vitro studies, it was found that during adoptive T cell responses (DTH) to antigen and this tolerogenic activity was blocked if DCs were treated with IOD inhibitor during IFN exposure.(8)

Other potential ways to target the Indoleamine 2,3-dioxygenase pathway: GCN2 kinase.

Past studies behind use of IDO inhibitors in cancer therapy rests on the hypothesis that IDO (whether expressed by APCs or tumor cells) finally acts to inhibit T cell responses. Therefore, it might be reasonable to target downstream pathway in T cells, in addition to targeting IDO itself.

Therefore, for inhibiting the immunosuppressive effect of IDO, this may provide an alternative target for drug discovery or perhaps an additional strategy. Studies showed that T cells deficient in the stress response kinase GCN2 become refectory to IDO mediated suppression and anergy induction.(9)

Down stream responses such as protein synthesis and cell division in yeast are regulated by GCN2 senses. Recently the regulatory domain of GCN2 binds to tRNA (uncharged form). During amino acid insufficiency leads to activation of GCN2 kinase domain and initiates downstream singling.(10)

Recently, the mammalian homolog of GCN2 has been identified and shown to have similar singling properties. (11)

In the case of IDO, it is hypothesize that, the GCN2 kinase pathway in responding to T cells was activated by expression of IDO by APCs. This leads to generation of an intracellular signal in the t cells that is responsible for certain of the key biologic effects of IDO. (12)

To elucidate the biology of the GCN2 pathway in T cells, much additional work will be required. Till now no pharmacologic inhibitors of this pathway are known.

Role ofIndoleamine 2,3-dioxygenase in ovarian cancer

Over 190,000 women worldwide each year affected by ovarian cancer (International agency of research on cancer). Front line treatment (cytoreductive surgery followed by taxaen and platinum based chemotherapy) showed highly response in over 80% patients. However, 20-30% of ovarian cancer patients survive 5 years or more after diagnosis.

From the past few year studies it has been shown that host immune system too has a strong influence on survival from ovarian cancer. In numerous studies, it has been found that, presence of CD8+T cells in tumor epithelium has been associated with with prolonged disease free and overall survival (13).Intratumoral levels of interferon-γ and its receptor, TNF-α, IL-18, MHC class I molecules, IRF-1 and antigen processing machinery, are features of CD8+t cell responses are also associated with favourable prognosis in ovarian cancer. Inaba and colleagues (14), assessed expression of enzyme IDO in ovarian tumors, by using a cohort of 60 ovarian cancer cases representing a range of histological subtypes. According to their report, IDO expression is associated with high grade, significantly fewer intraepithelial CD8+T cells infiltrates, and decreased overall and progression free survival.

The association between IDO expression and reduced T cell infiltrates fits with work by this group and others showing that IDO can have immunosuppressive effects (15). IDO expression can be induced in a variety of tissues by activated lymphocytes (by releasing IFN-γ). Several mechanisms come in light by which IDO can inhibit T cell prolification and function. As mentioned above, tryptophan was degraded by IDO yielding breakdown products called kynurenines. Therefore, IDO expression can deplete tryptophan locally, leaving T cell starve for this amino acid. Additionally, T cell apoptosis can directly caused by knurenines. Finally, inhibition of activation of naïve T cells in tumor draining lymph nodes, by induced expression of IDO on dendraitic cells (15).

Inaba and colleagues transfected IDO into the ovarian cancer line SKOV3 and assessed the in vitro and in vivo consequences on cell behavior. IDO expressing cells showed normal morphology, prolification, mitigation, inverse activity and sensitivity to the chemotherapeutic agent paclitexal during in vitro evaluation. However, a marked increase in the volume and extent of dissemination of IDO expressing tumors compared to the parentral tumor line was resulted when an IDO-expressing clone was injected intraperitoneally into nude mice.

However, by administrating the IDO-inhibitor 1-methyl tryptophan (1-MT), this effect could be suppressed. While increased survival of mice dosent lead by alone administration of 1-MT. The role of 1-MT is to potentate the therapeutic effect of pacllitaxel (16).

However, the therapeutic effects of 1-MT and paclitexal speak for themselves; the immunological basis of these observations is unclear. It has been showed that 1-MT potentates the effect of paclitexal by a T-cell dependent mechanism, in a spontaneous mammary tumor model (17). But in the study by Inaba and colleagues, the nude mice lack T cell. Nonetheless, the nude mice contain NK cells, which could be the target of immune suppression by IDO and rescue by 1-MT.

Role ofIndoleamine 2,3-dioxygenase in Prostate cancer

Around 31.5% of cancer related death in man is due to prostate cancer. It is the second leading cause of cancer (18). To understand prostate cancer induction and progression, a contemporary model should include a detailed analysis of mechanisms possibly inhibiting immune responsiveness to cancer. Factors such as arginase 2, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and TGF-β5, potentially imparing immune responses in PCA patients. Furthermore anti CD3 or PHA included T cell prolification can be inhibited by some of these immunosuppressive factors.

Production of immunosuppressive catabolites from indoleamine 2,3-dioxygenase known to inhibit T cell stimulation in vitro and to cause T cell apoptosis. IDO expression has thus been proposed as a possible mechanism facilating induction of immune tolerance towards cancer (19).

Indoleamine 2,3-dioxygenase has been proposed to induced tumoral immune resistance. Expression of IDO gene has been detected in PCA; its cancer specificity has not been investigated so far. Studies showed that IDO gene expression is also detectable in BPH (Benign prostatic hyperplasia) specimens, it strongly enhanced in a size able percentage of PCA specimens.

AMACR A, is a candidate biomarker for PCA, and overall IDO gene expression is significantly correlated with the expression of AMACR A (20). A significant increase of the kynurenine / tryptophan ratio is detectable in sera in the subgroup of PCA patients whose tumors express high levels of IDO gene expression could extend beyond tumor microenvironment and result in systemic effects (21).

In the study by Chantal Feder Mengus et al, endothelial cell express IDO protein more frequently, than by tumor cells in BPH and PCA. BPH and PCA tissue expressing low level of IDO gene. Furthermore, IDO inhibition activity is known to improve the ability of HUVEC cells to stimulate allogenic T cell responses, and HUVEC cells transfect with IDO gene induce anergy in allospecific T cells (22).

However, high level of IDO gene expression was observed in PCA specimen, IDO protein was also focally detectable in tumor cells. However, in the areas of inflammation and atrophic glands and ducts, prostate cancer cells expressing IDO protein were often located and were found to be positive to for IDO specific staining in PCA. Correlation between TGF-β1 gene expression with IDO gene expression in PCA expressing high levels of IDO gene contributes to identification of particular subset of tumors.

Role ofIndoleamine 2,3-dioxygenase in Lung cancer

Yuzo Suzuki and coworkers determine the IDO activity in patients with lung cancer by measuring the serum concentrations of Trp and Kyn using LC-ESI/MS/MS and calculate the Kyn/Trp ratio. From their studies they found, lower concentration of Trp and higher concentration of Kyn in patient with lung cancer as compared with healthy controls. This results in significantly IDO activity. Furthermore, increased level of IDO activity and degradation of Trp correlated with disease progression. The data suggest that, progression of lung cancer is associated with Trp catabolism by the IDO activity (23).

Uythenhove et al with immunochemistry found IDO expression in 9 of 11 non small cell lung cancer and 2 of 10 small cell lung cancer (24). Karanikas et al found indicated increased level of IDO mRNA expression, relative to the reference level in normal lung tissues, in lung cancer tissues as well as in adjacent non malignant tissues (25). By these studies it is concluded that, IDO is expressed constitutively by lung cancer cells, however higher level of IDO expression observed in lung cancer patients and can also be contribute to other cells, such as macrophages and DC expressing enzyme that recruited in the peri-tumoral lung tissues. An increased expression of IDO in lung cancer is associated with tumor induced immunosuppression or tolerance (24,25).

However, still a little is known about a correlation of IDO activity and clinicopathological parameters in lung cancer. Studies showed that a significantly higher IDO activity was found inpatients with advanced lung cancer. This indicates a correlation between this enzyme activity and disease progression.

Studies also suggested that higher IDO activity is associated with extent of lymph node metastasis. However IDO activity is not related with T factors, including tumor size.

Brandacher et al demonstrated that higher IDO protein expression in cancer tissues significantly correlated a worse prognosis, indicating that IDO protein expression in tumors was a significantly prognostic variable (26).

In contrast Karanikas et al found no significant correlation between IDO mRNA expressions by lung cancer tissue tissues and disease staging in 28 patients with lung cancer (25).

By these studies, it is suggested that increased IDO activity is involved in disease progression of lung cancer, possibly through its immunosuppressive effect. But more studies are required to use IDO as a target for lung cancer.