Gonçalves Milene, Furgiuele Alessia, Rasini Emanuela, Legnaro Massimiliano, Ferrari Marco, Luini Alessandra, Rodrigues-Santos Paulo, Caramelo Francisco, Marino Franca, Pereira Frederico C, Cosentino Marco
Univ Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, Coimbra, Portugal; Univ Coimbra, Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, Coimbra, Portugal; Univ Coimbra, CIBB - Centre for Innovative Biomedicine and Biotechnology, Coimbra, Portugal; Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal; Univ Coimbra, Institute for Interdisciplinary Research, Doctoral Programme in Experimental Biology and Biomedicine (PDBEB), Coimbra, Portugal.
Center for Research in Medical Pharmacology, Univ Insubria, Varese, Italy.
Eur J Pharmacol. 2024 Apr 5;968:176420. doi: 10.1016/j.ejphar.2024.176420. Epub 2024 Feb 16.
Proinflammatory cytokines powerfully induce the rate-limiting enzyme indoleamine 2, 3-dioxygenase-1 (IDO-1) in dendritic cells (DCs) and monocytes, it converts tryptophan (Trp) into L-kynurenine (KYN), along the kynurenine pathway (KP). This mechanism represents a crucial innate immunity regulator that can modulate T cells. This work explores the role of IDO1 in lymphocyte proliferation within a specific pro-inflammatory milieu.
Peripheral blood mononuclera cells (PBMCs) were isolated from buffy coats taken from healthy blood donors and exposed to a pro-inflammatory milieu triggered by a double-hit stimulus: lipopolysaccharide (LPS) plus anti-CD3/CD28. The IDO1 mRNA levels in the PBMCs were measured by RT-PCR; the IDO1 activity was analyzed using the KYN/Trp ratio, measured by HPLC-EC; and lymphocyte proliferation was measured by flow cytometry. Trp and epacadostat (EP) were used as an IDO1 substrate and inhibitor, respectively. KYN, which is known to modulate Teffs, was tested as a positive control in lymphocyte proliferation.
IDO1 expression and activity in PBMCs increased in an in vitro pro-inflammatory milieu. The lymphoid stimulus increased IDO1 expression and activity, which supports the interaction between the activated lymphocytes and the circulating myeloid IDO1-expressing cells. The addition of Trp decreased lymphocyte proliferation but EP, which abrogated the IDO1 function, had no impact on proliferation. Additionally, incubation with KYN seemed to decrease the lymphocyte proliferation.
IDO1 inhibition did not change T lymphocyte proliferation. We present herein an in vitro experimental model suitable to measure IDO1 expression and activity in circulating myeloid cells.
促炎细胞因子可有力地诱导树突状细胞（DCs）和单核细胞中的限速酶吲哚胺2,3-双加氧酶-1（IDO-1），其沿着犬尿氨酸途径（KP）将色氨酸（Trp）转化为L-犬尿氨酸（KYN）。该机制是一种关键的先天免疫调节因子，可调节T细胞。本研究探讨IDO1在特定促炎环境中淋巴细胞增殖中的作用。
从健康献血者的血沉棕黄层中分离外周血单个核细胞（PBMCs），并使其暴露于由双重刺激引发的促炎环境中：脂多糖（LPS）加抗CD3/CD28。通过RT-PCR测量PBMCs中IDO1 mRNA水平；使用通过高效液相色谱-电化学检测法（HPLC-EC）测量的KYN/Trp比值分析IDO1活性；并通过流式细胞术测量淋巴细胞增殖。分别使用Trp和依帕司他（EP）作为IDO1底物和抑制剂。已知可调节效应性T细胞（Teffs）的KYN作为淋巴细胞增殖的阳性对照进行测试。
在体外促炎环境中，PBMCs中IDO1的表达和活性增加。淋巴细胞刺激增加了IDO1的表达和活性，这支持了活化淋巴细胞与循环中表达IDO1的髓样细胞之间的相互作用。添加Trp可降低淋巴细胞增殖，但消除IDO1功能的EP对增殖没有影响。此外，与KYN孵育似乎可降低淋巴细胞增殖。
抑制IDO1不会改变T淋巴细胞增殖。我们在此提出一种适合测量循环髓样细胞中IDO1表达和活性的体外实验模型。
