**1. Introduction**

Fasciolosis is a major parasitic disease of livestock caused by the trematode *Fasciola* spp. [1]. Nowadays, the number of infected people around the world is increasing, which makes fasciolosis an emerging zoonosis [1]. The World Health Organization (WHO) estimates that approximately 180 million are at risk of infection and 17 million people are infected, with a high prevalence in humans in Africa and South America [2]. Moreover, the economic losses caused by fasciolosis are estimated at around 3 billion US dollars per year due to livestock infection [1].

During infection, *Fasciola hepatica* modulates the host immune response characterized by the presence of regulatory dendritic cells (DC) [2–5], alternative activated macrophages [6], and

**Citation:** Costa, M.; da Costa, V.; Frigerio, S.; Festari, M.F.; Landeira, M.; Rodríguez-Zraquia, S.A.; Lores, P.; Carasi, P.; Freire, T. Heme-Oxygenase-1 Attenuates Oxidative Functions of Antigen Presenting Cells and Promotes Regulatory T Cell Differentiation during *Fasciola hepatica* Infection. *Antioxidants* **2021**, *10*, 1938. https:// doi.org/10.3390/antiox10121938

Academic Editors: Elias Lianos and Maria G. Detsika

Received: 2 October 2021 Accepted: 10 November 2021 Published: 3 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 adaptive immune response characterized by Th2 and regulatory T cell (Treg)-associated cytokines [7–10]. Macrophages and DC comprise a highly heterogeneous myeloid cell population specialized in antigen presentation. DC play a crucial role in orchestrating the adaptive immune response by activating naïve T cells and inducing their differentiation into different effector T cells depending on the pathogen [11,12]. Furthermore, both macrophages and DC can promote inflammation by secreting pro-inflammatory cytokines and reactive oxygen or nitrogen species (ROS/RNS), although they can also inhibit inflammation through anti-inflammatory cytokine production [13–15]. Thus, they exhibit functional plasticity that enables them to adapt to various local conditions in order to restore homeostasis after inflammation [11,12,16].

Heme-oxygenase-1 (HO-1) is the inducible rate-limiting enzyme involved in the catabolism of free heme. It participates in several processes, by providing cytoprotection, protecting from heme-induced toxicity, and regulating the host inflammatory response [14,17]. In fact, HO-1 acts as a stress-responsive enzyme induced by the nuclear factor NF-E2-related factor 2 (NRF2), to provide defense against oxidative-induced injury during inflammatory processes [14]. HO-1 also limits the secretion of pro-inflammatory cytokines [18,19] and promotes anti-inflammatory cytokines [20,21], in a process triggered, at least in part, by ROS in macrophages, although its effects depend on the model of study [14]. In the same line, expression of HO-1 inhibits DC-maturation and the production of ROS [22]. In addition, HO-1 expression by DC induces the production of IL-10, an antiinflammatory cytokine, that inhibits T-cell proliferation [23]. Additionally, IL-10-producing anti-inflammatory M2c macrophages express HO-1 [24], especially on those that express CD163, a hemoglobin scavenger receptor, that in fact mediates IL-10 production and HO-1 synthesis [25]. Therefore, HO-1 acts as a key mediator of anti-inflammatory pathways in both macrophages and DC.

Several reports demonstrate that HO-1 can have both beneficial and detrimental effects for the host immunity against different pathogens [26]. Furthermore, the antioxidant role of HO-1 in infectious diseases is still unclear, especially in helminth parasites. Interestingly, bilirubin, one of the enzymatic byproducts of HO-1, suppresses the killing of bacteria by reducing the neutrophil burst via its antioxidant activity [27]. In addition, HO-1 promotes bacteria survival inside macrophages by decreasing ROS production, as demonstrated for S*almonella typhimurium* [28] and *Mycobacterium abscessus* [29] via its ROS-diminishing properties. Recently, we showed that the anti-inflammatory effects of HO-1 induction are detrimental in *F. hepatica* infection, which is dominated by a Th2/Treg differentiation profile [17]. However, the antioxidant mechanisms induced by HO-1 during *F. hepatica* infection have not been approached so far.

Thus, in this work, we investigated the role of ROS/RNS production by myeloid HO-1+ cells during *F. hepatica* experimental infection in mice, and characterized the adaptive cell immune response. Our results indicate that HO-1 expression by myeloid cells during *F. hepatica* infection negatively correlates with the production of ROS or RNS and the increase of antioxidant molecules. Furthermore, the pharmacological inhibition of HO-1 by a well-characterized inhibitor of HO-1 enzymatic activity, tin protoporphyrin IX (SnPP), in *F. hepatica*-infected mice was associated with lower levels of Tregs, in a process that was mediated by IL-10 biological activity. However, the gene expression of the NADPH oxidase subunit gp91phox was decreased when SnPP was administrated to infected mice. Moreover, parasite infection carried out in gp91phox knockout mice with inactive NADPH oxidase showed decreased levels of peritoneal HO-1<sup>+</sup> cells, splenic Tregs, and partially protected mice from the hepatic damage induced by the parasite, revealing that a more complex molecular mechanism involving ROS production participates in the intricate pathology induced by this helminth. Altogether, these results contribute to the elucidation of the immunoregulatory and antioxidant role of HO-1 during *F. hepatica* infection, providing interesting molecular checkpoints that might control fasciolosis.
