Introduction
For individuals with end-stage organ disease, solid organ transplantation is the gold standard therapy. The field of transplantation still faces several unique challenges, including a shortage of transplantable organs and an increasing pool of extended criteria donor (ECD) organs, which are extremely susceptible to ischemia-reperfusion injury (IRI), the risk of graft rejection, and immune regulation challenges.
Furthermore, there are no precise and specific biomarkers that can predict allograft malfunction and/or rejection in a timely manner. The important amino acid tryptophan and, in particular, its metabolites produced via the kynurenine pathway, have been extensively investigated as a contributor and therapeutic target in a variety of illnesses, including neuropsychiatric, autoimmune, allergies, infections, and cancer.
The tryptophan-kynurenine pathway has also piqued attention in solid organ transplantation, with a number of experimental investigations looking into its involvement in IRI and immune modulation following allograft implantation. The latest information on the function of tryptophan and its metabolites in solid organ transplantation is provided in this review, which provides insight into molecular processes as well as therapeutic and diagnostic/prognostic potential.
The Tryptophan-Kynurenine Pathway, Its Enzymes and Metabolites
Firstly, tryptophan is converted to N-formyl-L-kynurenine. The reaction is catalyzed by one of three rate-limiting enzymes: tryptophan-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase 1 or 2 (IDO1 or IDO2).
All the three enzymes are hemoproteins and use molecular O2 as a co-substrate, which also allows them to utilize reactive oxygen species (ROS) and regulate the redox balance in the cell. Further N-formyl-kynurenine form amidase hydrolyzes N-formyl-L-kynurenine to L-kynurenine into three alternative metabolites with different properties regarding oxidative stress and organ toxicity:
- Kynurenic acid (KYNA) by kynurenine aminotransferase (KAT)
- Anthranilic acid (AA) by kynureninase (KYNU)
- 3-hydroxykynurenine (3-HK) by kynurenine-3- monooxygenase (KMO).
The extrahepatic way is responsible for only 5–10% of the overall tryptophan degradation in physiologic conditions. The extrahepatic tryptophan kynurenine pathway does not provide all the necessary enzymes; therefore, its intermediate metabolites and their properties become critical in the pathogenesis and modulation of these conditions (Fig 1).
Role of Tryptophan and Its Metabolites in IRI
IRI is a substantial and unavoidable threat in Tx. Adenosine triphosphate (ATP) depletion, impaired ATPases activity, cellular calcium overload, deterioration of the mitochondrial membrane potential via opened mitochondrial permeability transition pores, the promotion of proapoptotic mechanisms, endothelial dysfunction, increased thrombogenicity and the induction of inflammatory responses are examples for the consequences of IRI. The histidine-tryptophan-ketoglutarate (HTK) organ preservation solution contains 2-mmol/L tryptophan due to its antioxidant capacity and membrane stabilizing potential.
It has been previously demonstrated that short-term dietary restrictions, i.e., reductions of specific food intakes without calorie depletion, increases the resistance to acute stress, including IRI. Besides the impact of tryptophan and its metabolites on IRI of solid organ grafts, the role of tryptophan derivatives has recently been under investigation.
Role of the Tryptophan-Kynurenine Pathway in Immune Regulation after Tx
The immune system plays a key role in Tx. Immunological mechanisms, which normally act as a protective organism response against foreign pathogens, pose a significant challenge to successful Tx. Several types or rejections exist, ranging from hyperacute and acute to chronic, all representing not only different rates of response but, also, different pathophysiological mechanisms in which both cellular and humoral immunity are involved. Despite constantly increasing the knowledge and broadened opportunities in immunosuppressive regimens, the full view of the immune response against allografts is still not fully understood, and the survival of transplanted organs remains time-limited.
Indoleamine-2,3-dioxygenase (IDO) is probably the most thoroughly investigated tryptophan-kynurenine pathway enzyme, which acts as a natural tolerogenic factor regulating immune responses. IDO is expressed in various cells, such as APCs, fibroblasts, endothelial, epithelial, smooth muscle cells, etc., in response to proinflammatory mediators.
Due to IDO activity, decreased levels of tryptophan activate the GCN2 kinase, which leads to T-cell anergy, T-cell cycle arrest and apoptosis. The deactivated mTOR pathway results in suppressed T-cell proliferation and differentiation. Increased levels of toxic tryptophan-kynurenine pathway metabolites induce effector T-cell apoptosis and promote Treg formation and activation (Fig 2).
Heart
Reported that the intracoronary administration, as well as intramyocardial injection, of an adenoviral vector encoding for IDO cDNA (Ad-IDO) resulted in significantly prolonged cardiac graft survival in rats. Among tryptophan metabolites, contrary to AA and QUIN, 3-HAA, 3-HK and L-kynurenine showed the best ability to inhibit the proliferation of unstimulated spleen-derived T cells by the induction of apoptosis in vitro.
A single 3-HAA and allogeneic bone marrow-derived DC injection of recipient rats resulted in significantly prolonged cardiac graft survival and lowered the pathological grade of rejection, while a 3-HAA injection alone had only minimal protective effects. Importantly, the tolerogenic state, achieved by direct IDO gene transfer to cardiac cells or by IDO-overexpressing DC infusion, is allograft-specific and not only limited to the local milieu but is rather systemic, as shown by the increased survival of secondary implanted skin allografts that were genetically identical to implanted hearts.
Lungs
Importantly, it has been found that IDO is induced and the tryptophan-kynurenine pathway is promoted not only in antigen-presenting but, also, in nonimmune pulmonary cells, such as epithelial cells, which is important in the local protection of lung tissue from collateral damage.
Therefore, among a bunch of therapeutic strategies, targeting the tryptophan-kynurenine pathway has also been investigated in several in vivo animal models of lung Tx. T cells from treated allografts produced significantly less IL-2 and TNF-α, while the INF-γ production remained obvious. In recent experiments, the IDO gene was transferred into tissue-engineered lung allografts, which were constructed from decellularized rat lungs, differentiating medium and rat bone marrow mesenchymal stem cells.
IDO gene transfer into such composites allowed to achieve tolerogenic status by the reduction of inflammatory cytokines levels and upregulation of regulatory T cells. Thus, IDO overexpression may be considered as one of the possible methods allowing to go one step further in setting up a nonimmunogenic lung tissue construct and to overcome the problem of graft shortage.
Liver
First investigators demonstrated that, unless IDO mRNA is not expressed in the mouse liver in physiological conditions, it is induced after allogeneic liver Tx, and the inhibition of IDO activity and tryptophan metabolism leads to the rejection of otherwise spontaneously accepted liver grafts.
The IDO gene was found to be significantly upregulated in allogeneic spontaneously accepted liver tissue, while naïve and syngeneic livers express IDO mRNA. Several studies suggested that TDO, which is a liver-specific tryptophan-kynurenine pathway enzyme, may also play a role in immune response regulation after allogeneic liver Tx.
Inhibiting IDO activity by means of the IDO inhibitor 1-metyltryptophan (1-MT), in allogeneic liver-transplanted rats for the first week after Tx, did not cause acute rejection.
Kidneys
The group of Vavrincova-Yaghi investigated the role of IDO gene therapy in kidney allograft preconditioning after organ retrieval. IDO transgene delivery directly into the kidney graft resulted in a significantly attenuated increase in plasma creatinine and improved allograft function, as well as reduced levels of kidney injury markers KIM-1 and alpha smooth muscle actin (α-SMA).
Significantly reduced inflammation and upregulated Treg cell markers were found in IDO-preconditioned kidneys, which was accompanied by preserved tubular morphology and reduced interstitial pre-fibrosis. Reduced angiotensin-converting enzyme mRNA expression in IDO-preconditioned renal grafts can be another mechanism through which IDO may reduce arterial blood pressure.
Moreover, kidney graft preconditioning with IDO in a long time period protected against transplant vasculopathy, which is one of the most important pathophysiologic elements in chronic transplant rejection, as proliferated neointima and narrowed vascular lumen lead to hypoperfusion, subsequent graft fibrosis and chronic transplant dysfunction.
Small Bowel
A murine small bowel Tx model revealed that a recipient treatment with IDO-transfected DCs, intravenous 3-HAA or a combination of these approaches results in a significantly prolonged graft survival, reduced inflammation and diminished morphological graft distortion.
Despite that in vitro 3-HAA enhanced the immunosuppressive effect of IDO-DCs when used in combination, in vivo, no significant improvement in allograft survival was observed compared to the IDO-DC treatment alone.
Intensifying the investigation on novel organ preservation techniques, such as normothermic or sub-normothermic machine perfusion, will potentially provide an attractive platform for gene therapy by direct IDO gene transfer to graft cells or IDO-transfected APC delivery directly to the organ.
Diagnostic and Prognostic Role of Tryptophan and Its Metabolites in Solid Organ Tx
The increased levels of kynurenine in the context of acute graft rejection was dependent on the severity of rejection. In another targeted metabolomics study that investigated urinary metabolites, kynurenine was found as one of the top 10 metabolites able to
identify acute T-cell-mediated rejection in pediatric kidney Tx recipients. However, they found that the increased serum kynurenine/tryptophan ratio, but not tryptophan or kynurenine alone, reflects episodes of acute rejection in children after renal Tx.
Serum tryptophan was decreased and kynurenine increased in the group of patients experiencing no acute graft rejection in comparison to recipients who did. The authors explained this finding as a reflection of the increased IDO activity and its probable tolerogenic role in relieving acute rejection.
Conclusions
It seems that the tryptophan metabolism via the kynurenine pathway may have multiple clinical effects in solid organ Tx, depending on the timepoint and the dominant pathophysiological mechanism.
However, this evidence grants some interesting insights into therapeutic possibilities by offering several attractive pharmacological and/or genetic modification targets, probably paving the way for protection from deleterious pathological effects in solid organ Tx and the improvement of short- and long-term outcomes.
Metabolites of the tryptophan-kynurenine pathway may potentially serve as diagnostic and prognostic tools allowing improved care in transplanted patients.
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