**4. Discussion**

In this work, we have examined the cellular and molecular mechanisms that govern the expansion or differentiation of Tregs induced by HO-1<sup>+</sup> cells in *F. hepatica* infection. We presented evidence showing that HO-1 activity results in decreased ROS/RNS production by F4/80+ antigen-presenting cells, thereby enhancing the pathological effects caused by *F. hepatica* and promoting parasite infection. Furthermore, apart from its antioxidant capacity, HO-1 has other functions, such as its immunoregulatory properties and controlling gene expression as a transcription factor [14,21,26,32,38]. Indeed, HO-1 inhibition promotes IFNγ- and NOS2-mediated control of *M. tuberculosis* infection in mice [39]. Furthermore, it has been previously reported that HO-1 has a role in suppressing pro-inflammatory Th1 immune responses in experimental colitis, and sickle cell alloimmunization has been reported, and it protects from atherosclerosis [40,41]. Finally, HO-1 can impair the immunity against other pathogens, such as *Plasmodium yoelii* [42].

Indeed, we demonstrated that during *F. hepatica* experimental infection in mice, there is an increase in the expression of HO-1 in F4/80<sup>+</sup> cells in the peritoneal cavity and it inversely correlates with ROS/RNS production. Furthermore, we demonstrated an association between the expression of HO-1 and the presence of putative Tregs in the spleens of infected animals (Figure 9A). These results were also confirmed when using the HO-1 inhibitor SnPP, which inhibits its enzymatic activity. At first sight, the inhibition of HO-1 activity by SnPP would suggest that its effects are caused by the heme-catabolizing activity rather than by its expression and function as a transcription factor. Indeed, F4/80<sup>+</sup> peritoneal cells from SnPP-treated mice did not show a decrease in HO-1 expression, although a significant increase in ROS/RNS production was detected. SnPP is a metalloporphyrin that acts as a competitive inhibitor of HO-1 both in vitro and in vivo. Its efficiency can be explained by its higher binding affinity to HO-1/2 than to heme [43,44]. However, enzymatically inactive HO-1 can still mediate protection against hydrogen peroxide-induced toxicity, probably by promoting the gene expression of antioxidant proteins [14,45], although the mechanisms underlying these effects are still unclear. Thus, the possibility that HO-1 would act as transcription factor cannot be discarded, since the nuclear localization of HO-1 in F4/80<sup>+</sup> cells derived from *F. hepatica*-infected mice with or without SnPP treatment was not investigated. Furthermore, it is unlikely that the protective outcome of SnPP treatment represents a direct effect on *F. hepatica*, since the degree of infection and pathological effects induced by the parasite were also related to an increase in Tregs, evidencing that HO-1 activity influences the host adaptive immunity in vivo. Indeed, our results indicate that the increase of the mRNA levels of *nrf2*, a transcription factor responsible for the regulation of cellular redox balance and protecting antioxidant responses [46,47], is accompanied by an increase in some antioxidant enzyme genes, demonstrating that the infection, HO-1, Tregs, and the Nrf2 master regulator comprise a complex axis of antioxidant and immunoregulatory properties in *F. hepatica* infection. However, the function of these enzymes should be determined in order to confirm their antioxidant role during *F. hepatica* infection. On the other hand, heme-activated murine macrophages have functional anti-inflammatory features that are dependent on the enzymatic activity of HO-1 [38]. Thus, the immunoregulatory and immunosuppressive properties of HO-1 together with its antioxidant properties demonstrate that its function during *F. hepatica* infection goes far beyond heme degradation itself.

The role of ROS/RNS in helminth parasite killing is still controversial. Some reports showed that the infection by *Strongyloides papillosus* induced an oxidative/nitrosative stress in sheep [48], although its effect on the parasite itself has not been demonstrated. On the other hand, *Schistosome* infection relates to an immense oxidative stress by the host that is not sufficient to control infection [49]. Further data demonstrated that excretory/secretory factors from *Mesocestoides corti* inhibit ROS-induced neutrophil extracellular traps, showing that the parasite could use this mechanism to attenuate the effects induced by ROS [50]. It should be highlighted, however, that although oxidative mechanisms are induced by helminth parasite infections, their detrimental role in the parasite itself as well as in the host surroundings is not well-established yet [51–53]. A recent report has demonstrated

a high oxidative status in serum and liver in rabbits infected with *F. gigantica*, together with a decline in the SOD and catalase gene expression and enzyme activity in sera from infected animals [54], which is not in agreement with data from our work in *F. hepatica* experimentally infected mice. However, the authors came to the conclusion that the disruption of antioxidant and detoxification cascades by *F. gigantica* likely leads to the pathogenic response from the host [54].

**Figure 9.** General hypothesis. (**A**) ICOSL<sup>+</sup> F4/80<sup>+</sup> APC express HO-1 induced by parasite infection that promotes IL-10 production and differentiation or expansion of CTLA4+ Tregs. (**B**) SnPP treatment inhibiting HO-1 activity in IL-33R<sup>+</sup> F4/80+ APC allows ROS/RNS production, that induce parasite damage in early stages of the infection.

It is worth noting that in our work, we used a DCFDA fluorescent probe that does not distinguish between ROS and RNS. Therefore, these studies should be complemented with others using ROS-specific probes such as DHE or specific inhibitors of nitric oxide production (such as L-Name). In order to analyze the ROS produced by NADPH-oxidase, we used, instead, gp91phox knockout mice. Interestingly, the fact that mice that are deficient in NADPH oxidase function, with a considerable decrease in ROS production, were partially protected against *F. hepatica* infection, suggests that the moment when ROS is produced by NADPH oxidase might be crucial to limit *F. hepatica*-induced damage (Figure 9B). Indeed, an exacerbated ROS production induced by a pro-inflammatory immune response can be detrimental to leukocyte cell function or viability and induced damage to the immune system [54]. Thus, a prolonged and not regulated production of ROS by F4/80<sup>+</sup> cells could benefit the parasite, and not the host. Of note, these cells expressed higher levels of ICOSL and IL-10 than those from gp91phox knockout mice, which could be associated with the differentiation or expansion of a higher number of splenic Tregs, which in turn express higher levels of CTLA4. Indeed, both ICOSL [55,56] and CTLA4 [57] are key mediators of Treg differentiation. In the same line, macrophages can suppress T cell responses and favor the expansion of Tregs [58]. Furthermore, ROS levels on T cell activation seem to be important, since small quantities of ROS result in antigen hyporesponsiveness, while high doses lead to oxidative stress-induced apoptosis [59]. Further analysis of the role of IL-10 produced by antigen-presenting cells in the differentiation or expansion of Tregs showed that IL-10 signaling is essential to increase HO-1 expression in peritoneal F4/80<sup>+</sup> cells and likely the production of Tregs. Interestingly, it would seem that the parasite exploits the host defense mechanisms, on the one hand by recruiting HO-1+ cells with less antioxidative functions that produce IL-10, and on the other hand by in

turn inducing the differentiation to Tregs. Nevertheless, the production of IL-10 by the host would also protect host cells in the acute pro-inflammatory immune response, caused either by damage induced by the parasite in the early state of the infection or by liver damage, at least in this experimental model. However, more experiments are needed in order to confirm these results, and to determine the role of ROS in the induction of Tregs and its relationship with IL-10.

One hypothesis that can explain these results might be the fact that ROS/RNS production is (partially) effective only during early stages of *F. hepatica* infection (Figure 9B). After ingestion of metacercariae by the mammalian host, juvenile flukes penetrate the host intestine wall and reach the liver through the peritoneal cavity between 4 and 6 days in livestock, although it is thought that it takes around 24 h in mice [3]. To further understand the early events that take place during *F. hepatica* infection in mice, we analyzed HO-1 expression and F4/80+ cell recruitment at 1 dpi, finding that two different populations expressing different levels of F4/80 are present in the peritoneum, and those elicited in SnPP-treated mice expressed higher levels of IL33R (Figure 9B). IL33 is an alarmin that participates in the type 2 innate immune response, promoting innate lymphoid cells type 2. However, during *Schisotosoma* infection, IL-33 seems to contribute to the development of pathology via the induction of type 2 innate lymphoid cells and alternative activation of macrophages, thus favoring the infection [60–62]. Therefore, the functions of IL-33 during *F. hepatica* infection in mice, and in particular the overexpression of its receptor in antigen-presenting cells at the early events of the infection, remain to be elucidated.

In conclusion, our work showed that HO-1 is a key molecule that favors *F. hepatica* infection, by which HO-1 could control ROS/RNS production and Treg differentiation and how the parasite elicits/triggers these mechanisms. Altogether, these results contribute to the elucidation of the immunoregulatory and antioxidant roles of HO-1 induced by *F. hepatica* in the host, providing interesting checkpoints that might control fasciolosis.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/antiox10121938/s1, Figure S1: Gates used for flow cytometry analyses of PECs from infected and control mice corresponding to Figures 1B,D and 4A; Figure S2: Gates used for flow cytometry analyses of splenocytes from infected and control mice corresponding to Figures 2A–C and 3G,H; Figure S3: Gates used for flow cytometry analyses of hepatic leukocytes from infected and control mice corresponding to Figure 2D,E; Figure S4: Gates used for flow cytometry analyses of splenocytes from infected and control mice corresponding to Figure 3B,C; Figure S5: Representative images of livers from gp91phox KO and C57BL/6 liitermates infected and control mice; Figure S6: Gates used for flow cytometry analyses of splenocytes from infected and control mice corresponding to Figure 7F,G; Figure S7: Gates used for flow cytometry analyses of splenocytes from IL 10R or isotype treated infected and control mice corresponding to Figure 8D,E.

**Author Contributions:** M.C. performed all the experiments, with the exception of the anti-IL-10R experiment, and wrote the original draft of the manuscript; V.d.C. and S.F. performed biological sample collection and flow cytometry analyses, and reviewing the manuscript; M.F.F., M.L., S.A.R.-Z. and P.L. helped with cell cultures, and performed flow cytometry analyses and interpretation; M.F.F. critically reviewed the manuscript; P.C. performed the IL-10R experiment, conceptualization, and reviewed the manuscript; T.F. performed conceptualization, experiment design and supervision, analyses of data, and writing, review, and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Programa de Desarrollo de Ciencias Básicas (PEDECIBA), Agencia Nacional de Investigación e Innovación (SNI-ANII, FCE\_1\_2017\_1\_136094 and FCE\_ 1\_2019\_1\_156295) to Teresa Freire.

**Institutional Review Board Statement:** Animal experimentation was carried out according to the International Guiding Principles for Biomedical Research Involving Animals, as issued by the Council for the International Organizations of Medical Sciences. Procedures involving animals were approved by the Universidad de la República's Committee on Animal Research (Comisión Honoraria de Experimentación Animal, CHEA Protocol Number 07153-000817-18).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary materials.

**Acknowledgments:** We are grateful to Unidad de Reactivos Biológicos de experimentación at Facultad de Medicina, UdelaR, for animal housing and care and to Ignacio Anegón for his help.

**Conflicts of Interest:** The authors declare no conflict of interest.
