The Immunomodulatory Effects Of Indoleamine2,3-dioxygenase In Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation

[Objective] To evaluate the expression levels of IDO and interferon (IFN)y in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify the correlation between IDO activity, IFNy and acute graft-versus-host disease (aGVHD),[Methods] We measured IDO mRNA expression in peripheral blood mononuclear cells in89allo-HSCT patients by reverse transcriptionpolymerase chain reaction. The IDO activity in plasma was also performed by reverse-phase high-performance liquid chromatography (HPLC); plasma IFNy was detected by a standard enzyme-linked immunosorbent assay (Elisa)[Result] IDO mRNA was detected in55of74patients (74.32%) with aGVHD. Of patients without aGVHD, only2of26expressed IDO mRNA (7.69%); none of8healthy volunteers was positive for IDO expression. Plasma IDO activity was much higher in aGVHD patients than in those without aGVHD (4.74±3.35vs1.79±1.02, respectively; P <0.0001) or in healthy control subjects (4.74±3.35vs1.77±.22; P<0.0001). Patients with severe (grade Ⅲ/Ⅳ) aGVHD had much higher IDO activity than those with mild (grade Ⅰ/Ⅱ) aGVHD (6.57±3.34vs2.46±1.41; P<0.0001). Meanwhile, there was a significant increase in plasma IFNy level in aGVHD patients (P=0.0043). IDO activity decreased after alleviation of aGVHD, whereas fluctuation of plasma IDO was also observed upon the recurrence of aGVHD. Plasma IDO activity was correlated with the level of plasma IFNy(r=0.8288; P<0.0001). Using receiver-operating characteristic curves analysis, the sensitivity and specificity for evaluation of aGVHD were determined. The area under the curve of IDO activity was higher than that of IFNy(0.852vs0.694) with a sensitivity and specificity for IDO of81%and78%, respectively, whereas the sensitivity and specificity for IFNy were41%and93%, respectively.[Conclusion] IDO mRNA was expressed in blood mononuclear cells of patients with aGVHD. Plasma IDO activity was elevated in aGVHD patients and was correlated with the severity of aGVHD. In combination with plasma IFN-g, IDO activity may represent a potential biomarker for the diagnosis and evaluation of aGVHD after allo-HSCT. Intervention of the IDO pathway may also represent an alternative way to overcome steroid-resistant aGVHD. [Objective] To explore the relationship between IDO and the damage of aGVHD target organs,we detected the expression level of indoleamine2,3dioxygenase (IDO) in various organs of mice aGVHD models, Provide preliminary exploration for regulating IDO pathway may beneficial to the prevention of aGVHD and induction of tolerance.[Method] Lethally (8.0Gy) irradiated female BALB/c (H-2b) recipients were transplanted with bone marrow cells and splenic cells from male C57BL/6(H-2d) donors. The recipients were observed for the features of GVHD, established syngeneic transplant control group, observed the signs and the survival time of the mice, aGVHD clinical score, aGVHD score(?)7, the mice were sacrificed, reversed-phase high performance liquid chromatography (HPLC) measured the concentration of plasma tryptophan (Trp) and kynurenine (KYN);detected the IDOmRNA protein expression levels of the organ,by RT-PCR and immunohistochemical, flow cytometric was used to detect CD4+/CD25+T cells ratio. In peripheral blood[Results] Plasma Trp concentrationin in allogeneic transplant mouse was significantly lower than syngeneic transplant mice (P<0.05), the KYN concentration higher than that of control mice (P<0.05), IDO activity was significantly higher than control group (P<0.01). IDO mRNA expression levels of colon and lung higher than the control mice, small intestine IDO expression level in two groups mice is very low, the difference is no significant, in skin and liver, IDO mRNA expression was not obvious. Immunohistochemical results showed that:in aGVHD mice, lung and colon epithelial cells and endothelial cells highly expressed IDO; vascular endothelial cells of skin express IDO,meanwhile scattered IDO-positive mononuclear cells in skin; the scattered distribution of IDO-positive mononuclear cells in liver around blood vessels; in syngeneic transplant mice,IDO expression was very low or no expression. [Conclusion] IDO expression play an important role in the immune regulation of tissue level in aGVHD target organs, vascular endothelial cells as a first recipient cells that contact to the allo-reactive T cell were involved in this role; regulate IDO pathway maybe a effective way for treating aGVHD [Background] Local catabolism of tryptophan (Trp) by indoleamine2,3-dioxygenase (IDO) is considered an important mechanism of regulating T cell immunity.N-(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) is an active synthetic anthranilic acid derivative which was proved to be effective to treat type I helper T lymphocytes (Thl) mediated autoimmune diseases such as multiple sclerosis. In this report, we investigated the effects of3,4-DAA on the acute graft versus host disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) and its potential mechanism of action.[Methods] Using a murine aGVHD model,3,4-DAA was injected intraperitoneally at200mg/kg/day per mouse immediately after allo-BMT or at the onset of aGVHD for14consecutive days; the signs of aGVHD and the survival were recorded periodically; the histological changes of target organs were evaluated with hematoxylin-eosin staining; the IDO activity and cytokine levels in plasma were measured by reverse-phase high-performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay(ELISA), respectively.[Results] Administration of3,4-DAA after allo-BMT significantly reduced the severity and the histological score of aGVHD; The survival for mice receiving3,4-DAA prophylaxis and treatment were prolonged in comparison to the vehicle control mice. The plasma levels of IFN-y and IL-2in3,4-DAA treatment group were found to be decreased, while the IDO activity and the IL-10level elevated in these mice. In consistent with the in vivo results,3,4-DAA also inhibited IFN-y and IL-2production of spleen T lymphocytes in vitro.[Conclusions]3,4-DAA diminishes the murine experimental aGVHD through inhibiting Th1responses, this property makes it a potential alternative agent for prevention and treatment of GVHD in the clinic. [Objective] To investigate the indoleamine2,3-dioxygenase expression in pulmonary microvascular endothelial cells (PMVEC)induced by IFNy and its role in T cell proliferation[Method] Mouse PMVEC were cultivated by Tissue block method and observed of their morphological features. After series of identification and Passage purification, they were dealed with different concentrations of IFNy. Primary EC or the EC treated with IFNy or VP16was detected the IDO expression by RT-PCR and Western blot, reversed phase high-performance liquid chromatography (HPLC) was used to evaluate the the levels of tryptophan and kynurenine and the activity of IDO. after co-cultivating PMVEC and T lymphocyte derived from mice spleen, FMC was applied to detect the proliferation of T cell by labeled with carboxy fluorescein succinimidyl ester(CFSE).[Results] The EC acquired exposed the regular features of Cobblestone in morphology was positive in CD31expression with indirect immunofluorescence staining, and can be observed of the Weilel—palade globule by Transmission electron microscopy. There was higher level expression of IDOmRNA and protein in IFNy-treated PMVEC, and with the raising of the concentration of IFNy, the activity of IDO increased.After dealed with VP-16, with the stimulation of IFNy, a reduced IDO expression in PMVEC was evaluated, while no IDO expressed in without IFNy stimulation group, the PMVEC treated with IFNy inhibited the T cell proliferation, while the untreated showed no significant inhibition.[Conclusion] PMVEC could suppress T-lymphocyte responses via IDO enzyme activity in vitro.