**1. Introduction**

Infection of the mammalian vasculature and central nervous system (CNS) with the extracellular protozoan parasite *Trypanosoma brucei* (*T. brucei*) can lead to fatal human sleeping sickness, also known as African trypanosomiasis. Like most parasites, trypanosomes are continuously challenged by the host-immune system, however, they have evolved very effective evasion strategies in order to maintain infection and prolong the host's survival [1].

**Citation:** Fitzgerald, H.K.; O'Rourke, S.A.; Desmond, E.; Neto, N.G.B.; Monaghan, M.G.; Tosetto, M.; Doherty, J.; Ryan, E.J.; Doherty, G.A.; Nolan, D.P.; et al. The *Trypanosoma brucei*-Derived Ketoacids, Indole Pyruvate and Hydroxyphenylpyruvate, Induce HO-1 Expression and Suppress Inflammatory Responses in Human Dendritic Cells. *Antioxidants* **2022**, *11*, 164. https://doi.org/ 10.3390/antiox11010164

Academic Editors: Elias Lianos and Maria G. Detsika

Received: 20 December 2021 Accepted: 12 January 2022 Published: 15 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

An infection with *T. brucei* is accompanied by the excretion of high levels of ketoacids into the host's bloodstream [2,3], a phenomenon that was, until recently, largely unexplained. The ketoacids are derived from the conversion of aromatic amino acids (tryptophan, tyrosine, and phenylalanine) to indole pyruvate (IP), hydroxyphenylpyruvate (HPP), and phenyl pyruvate (PP), through the action of a cytoplasmic aspartate aminotransferase (TbcASAT), which is expressed by the parasite.

In recent years, we, and others, have demonstrated that these molecules have potent immunomodulatory properties, which likely contribute to the suppression of host immune responses, but that may also be exploited for potential therapeutic benefit [4–8]. For example, IP and HPP are potent inducers of the immunosuppressive enzyme heme oxygenase-1 (HO-1) in murine glia and macrophages [5]. This occurs in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2) dependent manner and leads to a reduction in LPSinduced pro-inflammatory cytokines and innate immune cell maturation. Indeed, HO-1 has well established anti-inflammatory properties and is induced by a vast number of stimuli during oxidative stress and inflammation. It catalyses the conversion of heme to biliverdin with the liberation of free iron and carbon monoxide (CO). Biliverdin is then converted to bilirubin by biliverdin reductase [9,10] and both biliverdin and bilirubin are considered powerful antioxidants, while many of the anti-inflammatory effects of HO-1 are attributed to CO. The potent anti-inflammatory properties of HO-1 are underlined by rare HO-deficiencies in humans and HO-1 knockout mice, which, along with the expected sensitivity to oxidative stress, show high levels of chronic inflammation [11–13]. Unsurprisingly, induction of HO-1 with known and novel HO-1 inducers is now being explored as a therapy for a number of autoimmune and inflammatory diseases and we have recently reviewed this in detail elsewhere [14].

In addition to HO-1 induction, *T. brucei*-derived ketoacids are also capable of inhibiting HIF-1α–induced pro-IL-1β expression, as well as prostaglandin production, an effect which was shown to be dependent on the activation of the aryl hydrocarbon receptor [4,5,8]. The immunomodulatory effects of IP and HPP have also been confirmed in animal models of disease. In a murine model of skin damage caused by exposure to ultraviolet B radiation, administration of IP resulted in a reduction in damage lesions and expression of the pro-inflammatory cytokines, IL-1β and IL-6 [6]. Furthermore, IP administration reduced disease severity in the DSS colitis model, and this was accompanied by a decrease in the expression of pro-inflammatory cytokines IL-12, TNF, IFNγ, and IL-1β, and an increase in the expression of the anti-inflammatory cytokine IL-10 [7]. A reduction in Th1 cells, as well as a reduced capacity for dendritic cells (DC) to activate T cells, was also observed [7].

Despite the recent interest in these ketoacids as immunomodulators, to date, they have been primarily studied in murine immune cells with very little evidence to support their mechanism of action in human immune cells. In this study, we investigate the immunomodulatory properties of the *T. brucei*-derived ketoacids, IP and HPP, in primary human immune cells and further examine their potential as a therapy for inflammatory diseases.

#### **2. Materials and Methods**

#### *2.1. Reagents and Chemicals*

4-hydroxyphenylpyruvic acid (HPP) and indole-3-pyruvic acid (IP) were purchased from Merck (Darmstadt, Germany) and dissolved in RPMI to a final concentration of 2 mM before use. Ultrapure lipopolysaccharide (LPS) from *E. Coli* O111:B4 was purchased from Enzo Life Sciences (Bruxelles, Belgium). Complete RPMI or complete IMDM were prepared by supplementing with 10% foetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, which were all purchased from Merck (Darmstadt, Germany). Lymphoprep is manufactured by Axis-Shield poC (Dundee, Scotland). GM-CSF and IL-4 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The protease inhibitor cocktail and High-Pure RNA Isolation Kit were purchased from Roche (Basel, Switzerland). The western blot antibodies for Nrf2, HK2, p-AMPK, t-AMPK, p62 and LC3 were all purchased from Cell Signalling Technology (Danvers, MA, USA), while

the antibody for HO-1 was purchased from Enzo Life Sciences (Bruxelles, Belgium), and the β-actin antibody and secondary anti-rabbit were purchased from Merck (Darmstadt, Germany). The Fixable Viability Dye, antibodies for CD40, CD80, CD83, and CD86, and Annexin V & PI staining kit were all purchased from eBioscience (San Diego, CA, USA). The DQ-Ovalbumin, CD3 and IL-17 flow antibodies, FIX & PERM™ Cell Permeabilization Kit, anti-CD3, and all ELISA kits used were purchased from Invitrogen (Waltham, MA, USA). The High-Capacity cDNA reverse transcription kit was purchased from Applied Biosystems (Beverly, MA, USA) and the iTaq Universal SYBR Green mastermix from Bio-Rad (Hercules, CA, USA). The MagniSort Human CD14 Positive Selection kit and MagniSort Human CD4 T cell Positive Selection kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Zombie NIRTM Fixable Viability kit was purchased from BioLegend (San Diego, CA, USA). The antibodies for CD8, ki67 and IFNγ were purchased from BD biosciences (East Rutherford, NJ, USA). The Complete XF assay medium was purchased from Agilent (Santa Clara, CA, USA). The Corning™ Cell-Tak Cell and Tissue Adhesive was purchased from Fisher Scientific (Waltham, MA, USA). Oligomycin was purchased from Cayman Chemicals (Ann Arbor, MI, USA) and carbonyl cyanide-p trifluoromethoxyphenylhydrazone (FCCP) from Santa Cruz biotechnology (Dallas, TX, USA). The HEK-BlueTM hTLR4 assay system was purchased from InvivoGen (San Diego, CA, USA). Dulbecco's phosphate buffered saline (PBS), enhanced chemiluminescent substrate, phosphatase inhibitor cocktail, Antioxidant assay kit, Phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, rotenone, antimycin A, and 2-deoxy-D-glucose (2-DG) were all purchased from Merck (Darmstadt, Germany).
