Heme-Oxygenase-1 Attenuates Oxidative Functions of Antigen Presenting Cells and Promotes Regulatory T Cell Differentiation during Fasciola hepatica Infection

Fasciola hepatica is a fluke that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. The parasite regulates the host immune system by inducing a strong Th2 and regulatory T (Treg) cell immune response through mechanisms that might involve the expression or activity of heme-oxygenase-1 (HO-1), the rate-limiting enzyme in the catabolism of free heme that also has immunoregulatory and antioxidant properties. In this paper, we show that F. hepatica-infected mice upregulate HO-1 on peritoneal antigen-presenting cells (APC), which produce decreased levels of both reactive oxygen and nitrogen species (ROS/RNS). The presence of these cells was associated with increased levels of regulatory T cells (Tregs). Blocking the IL-10 receptor (IL-10R) during parasite infection demonstrated that the presence of splenic Tregs and peritoneal APC expressing HO-1 were both dependent on IL-10 activity. Furthermore, IL-10R neutralization as well as pharmacological treatment with the HO-1 inhibitor SnPP protected mice from parasite infection and allowed peritoneal APC to produce significantly higher ROS/RNS levels than those detected in cells from infected control mice. Finally, parasite infection carried out in gp91phox knockout mice with inactive NADPH oxidase was associated with decreased levels of peritoneal HO-1+ cells and splenic Tregs, and partially protected mice from the hepatic damage induced by the parasite, revealing the complexity of the molecular mechanisms involving ROS production that participate in the complex pathology induced by this helminth. Altogether, these results contribute to the elucidation of the immunoregulatory and antioxidant role of HO-1 induced by F. hepatica in the host, providing alternative checkpoints that might control fasciolosis.


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].

Parasite Infections, Animal Treatments, and Sample Obtention
The infection was achieved by orally administrating 10 F. hepatica metacercariae (Montevideo, Uruguay) per mouse. Mice were bled and peritoneal exudate cells (PECs), spleens, and livers were removed after 1,8,15, and 21 days post-infection (dpi), while non-infected animals were used as controls. Each experimental group contained at least four mice. PECs and hepatic leukocytes were obtained as already described [31]. Red cells were lysed with ammonium chloride potassium buffer.
HO-1 activity was inhibited using SnPP (40 mg/kg), and vehicle (PBS, 200 µL) was used as a control. The SnPP dose was within a range of doses used in previous works [32,33]. Mice received intraperitoneal injections of SnPP 1 day before infection, 1 day after infection, and every 4 days until the end of the experimental protocol (between 19 and 21 dpi). When gp91 phoxand non-infected littermates were used (n = 6-8/group), infections were performed in the same conditions as previously described. In order to neutralize IL-10 receptor (IL-10R), BALB/c mice (n = 6-8/group) were intraperitoneally injected with 15 µg of monoclonal rat IgG2a anti-IL-10R (clone 1B1.3A from BioXcell, Lebanon, NH, USA) or an isotype-matched control antibody (clone HRPN from BioXcell, Lebanon, NH, USA), the day before and after infection with F. hepatica and every 3 days until animal sacrifice at 20 dpi. Blood samples were obtained, and PECs, spleens, and livers were removed. The infection severity was assessed with a defined clinical score according to the following parameters: presence or absence of peritoneal hemorrhage, presence of macroscopic liver damage and splenomegaly, and the amount of cell content in the peritoneal cavity [17], where the minimum score was 0 and the maximum was 10. The alanine aminotransferase (ALT) activity in sera was used to quantify liver damage, determined with a commercial kit (Spinreact, Girona, Spain) according to the manufacturer's instructions.

Statistical Analysis
Results of the experiments were expressed as mean ± SEM. GraphPad Prism version 6.04 for Windows (GraphPad Software, San Diego, CA, USA) was used to perform statistical analyses. Results were analyzed using one-way ANOVA followed by Tukey's test, or the two-tailed Student's t-test, depending on the experiment. Significant differences shown by an asterisk were considered when p < 0.05.

HO-1 Expression in F4/80 + Peritoneal Cells Inversely Correlate with ROS/RNS Production
In order to confirm the recruitment of PECs expressing HO-1 to the peritoneum of F. hepatica-infected mice, we identified HO-1 + cells by flow cytometry at different time points of the infection. As seen in Figure 1A, the clinical score increased upon infection, although ALT in serum significantly increased only after 21 dpi, demonstrating liver dysfunction. In addition, HO-1 + cells significantly increased in the peritoneal cavity during the infection ( Figure 1B,C and Supplementary Figure S1). These cells were mainly composed by F4/80 + cells ( Figure 1D,E and Supplementary Figure S1), and their increase also correlated with the advanced stages of the infection (after 15 dpi). The expression of HO-1 in F4/80 + cells slightly increased after 1 dpi, while it considerably increased during the infection ( Figure 1F). On the other hand, the production of ROS/RNS was significantly increased only at 1 dpi, and decreased during infection ( Figure 1G), suggesting that the expression of HO-1 in peritoneal F4/80 + cells inversely correlated with the production of ROS/RNS. In order to provide more evidence in this regard, we incubated RAW 264.7 macrophages with parasite components (FhTE) in the presence of CoPP or SnPP, and analyzed the production of ROS/RNS by these cells. FhTE slightly increased the production of ROS/RNS, while CoPP and SnPP significantly decreased and increased the production of ROS/RNS by FhTEtreated macrophages, respectively ( Figure 1H). Of note, FhTE per se induced ROS/RNS expression, which could be the result of an active respiratory burst, such as that seen in F4/80 + cells from PECs of infected mice at 1 dpi ( Figure 1G). Altogether, these results might indicate that F. hepatica induces the expression of HO-1 in F4/80 + cells recruited to the peritoneum, inhibiting ROS/RNS production during the course of the infection.

The
Presence of Peritoneal HO-1 + Cells Associates with Increased Splenic CD4 + CD25 + and CD8 + CD25 + Cells during Infection Considering that HO-1 can induce regulatory T cells [34][35][36][37], we analyzed the presence of both CD4 + and CD8 + cells in spleens of infected mice. Although we could not find any significant differences between the percentage of CD4 + and CD8 + cells during the infection, we did observe that they significantly increased in number after 15 dpi (Figure 2A and Supplementary Figure S2). We also analyzed the presence of splenic CD25 + CD4 + ( Figure 2B and Supplementary Figure S2) or CD8 + ( Figure 2C and Supplementary Figure S2) T cells. Again, no significant differences were found in the percentage of these cells during the infection, while their number significantly increased after 15 dpi. Finally, we analyzed the presence of CD4 + T cells in livers from infected animals and did not find any difference in their percentage nor their number ( Figure 2D and Supplementary Figure S3). However, the number, but not the frequency, of hepatic CD25 + CD4 + T cells was increased in advanced infected mice ( Figure 2E and Supplementary Figure S3). Further analyses demonstrated that the number of splenic CD25 + CD4 + and CD25 + CD8 + cells positively correlated with the number of peritoneal HO1 + cells ( Figure 2F).

HO-1 Activity Decreases the Production of ROS/RNS by F4/80 + Cells and Correlates with an Increase of Splenic Regulatory CD4 + T Cells Induced by F. hepatica Infection
In order to evaluate whether HO-1 interferes with the production of ROS/RNS, we treated F. hepatica-infected mice with the HO-1 inhibitor SnPP. SnPP treatment was associated with a decrease in the clinical signs of infected mice ( Figure 3A). In addition, SnPP treatment of infected mice abrogated the increase of HO-1 + cells, both in frequency and number, induced by the infection, since no significant difference was found in infected mice with respect to the control group with SnPP treatment ( Figure 3B and Supplementary Figure S4). Surprisingly, a significant increase in F4/80 + cell number, but not frequency, was found in both SnPP-treated and non-treated infected mice ( Figure 3C and Supplementary Figure S4). Indeed, the F4/80 + cell number was higher in SnPP-treated infected mice. Nevertheless, F4/80 + cells of SnPP-treated infected mice produced higher levels of ROS/RNS than control infected mice ( Figure 3D), although they expressed similar levels of HO-1 ( Figure 3E). Of note, the expression of ICOSL in peritoneal F4/80 + cells of infected mice was significantly reduced with SnPP treatment ( Figure 3F). Lastly, SnPP treatment during F. hepatica infection did not induce an increase in the number of splenic CD4 + T cells ( Figure 3G) or CD4 + /CD25 + T cells ( Figure 3H), although these cells expressed higher levels of CTLA4 in the absence of SnPP treatment ( Figure 3H). Altogether, these results suggest that HO-1 activity inhibited by SnPP decreases the production of ROS/RNS during fasciolosis and correlates with an increase of splenic regulatory CD4 + T cells in a process that might involve ICOSL in antigen-presenting cells or CTLA4 expression in Tregs.
To complement these results, we evaluated whether the inhibition of HO-1 by SnPP treatment affected the recruitment or the phenotypical characteristics of peritoneal F4/80 + cells at the early stages of F. hepatica infection. To this end, we analyzed F4/80 + cells in the peritoneal cavity of SnPP-treated mice at 1 dpi and compared them with both non-treated infected and control mice. We observed the presence of two different cell populations according to F4/80 expression ( Figure 4A and Supplementary Figure S1). SnPP treatment increased both the frequency and number of F4/80 int cells, while it reduced F4/80 hi cells in the peritoneal cavity of infected mice at 1 dpi ( Figure 4B). Nevertheless, F4/80 int cells expressed similar levels of HO-1 ( Figure 4C) and ROS/RNS ( Figure 4D) in F4/80 int cells regardless of SnPP treatment. Interestingly, peritoneal F4/80 int cells expressed higher levels of CCR2 ( Figure 4E), while only those from SnPP-treated infected mice expressed significantly increased levels of IL-33R ( Figure 4F), which could be related to the initiation of an early immune response against the parasite. Thus, these results suggest that the presence of F4/80 int IL-33R + cells in the peritoneum is induced by SnPP treatment, which in turn protects mice from infection.  Figure 2B), CD8 CD25 + (in Figure 2C), and HO-1 + cells (in Figure 1C) was plotted. Gate analyses by flow cytometry are shown in Supplementary Figures S2 and S3. The results shown represent one experiment. Asterisks indicate significant differences with p < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.

HO-1 Activity Decreases the Production of ROS/RNS by F4/80 + Cells and Correlates with an Increase of Splenic Regulatory CD4 + T Cells Induced by F. hepatica Infection
In order to evaluate whether HO-1 interferes with the production of ROS/RNS, we treated F. hepatica-infected mice with the HO-1 inhibitor SnPP. SnPP treatment was associated with a decrease in the clinical signs of infected mice ( Figure 3A). In addition, SnPP treatment of infected mice abrogated the increase of HO-1 + cells, both in frequency and  Figure 2B), CD8 CD25 + (in Figure 2C), and HO-1 + cells (in Figure 1C) was plotted. Gate analyses by flow cytometry are shown in Supplementary Figures S2 and S3. The results shown represent one experiment. Asterisks indicate significant differences with p < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.     Figure S1. Representative experiments are shown. Asterisks indicate significant differences with p < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.

The Inhibition of HO-1 Activity by SnPP Controls the Gene Expression of Antioxidant Molecules
To deeply analyze the relationship between HO-1 expression in peritoneal cells induced by F. hepatica infection with the production of ROS/RNS, the gene expression of different molecules involved in the oxidative and antioxidative responses was evaluated. PECs of SnPP-treated infected mice were characterized by a significant decrease in the mRNA levels of the transcription factor nrf2 ( Figure 5A). Moreover, the SnPP-induced decrease in nrf2 gene expression levels was associated with decreased mRNA levels in the antioxidant enzymes catalase, glutathione peroxidase 2 (gpx2), and superoxide dismutase 2 (sod2) ( Figure 5B). However, no differences were found in the gene expression of gpx1, while an increase in sod1 expression was observed ( Figure 5B). Finally, an unexpected decrease in the mRNA levels of the NADPH oxidase subunits gp91 phox and p47 phox was observed with SnPP treatment of infected mice ( Figure 5C).

Deficiency of Functional NADPH Oxidase Partially Protects Mice from Liver Damage Induced by F. hepatica and Limits the Production of IL-10
Considering the fact that SnPP treatment protected mice from parasite infection and that reduced levels of gp91 phox mRNA were found in PECs from infected mice, we analyzed the infection in gp91 phox knockout mice. gp91 phox deficiency reduced the clinical signs and liver damage induced by F. hepatica infection ( Figure 6A and Supplementary Figure S5). This partial protection was associated with a lower increase of HO-1 + peritoneal cells, both in frequency and number ( Figure 6B). Moreover, HO-1 + cells from gp91 phox knockout infected mice expressed lower levels of MHCII ( Figure 6C) and CD40 ( Figure 6D), but not CD80 ( Figure 6E), than those from wildtype mice, indicating that NADPH oxidase may play a role both in the immune response and the pathogenesis induced by F. hepatica infection. Further characterization of the peritoneal cells from these mice indicated that the increase of F4/80 + cells was abrogated in the absence of gp91 phox ( Figure 7A), and as expected, very low levels of ROS/RNS produced by these cells ( Figure 7B). Additionally, these cells expressed lower levels of Sirpα ( Figure 7C), ICOSL ( Figure 7D), and IL-10 ( Figure 7E). Finally, lower numbers of CD4 + ( Figure 7F and Supplementary Figure S6) and CD4 + /CD25 + FoxP3 + Tregs ( Figure 7G and Supplementary Figure S6) were found in gp91 phox knockout infected mice with respect to wildtype mice. However, no significant differences in the production of IFNγ by splenocytes stimulated with parasite components (FhTE) between gp91 phox knockout and wildtype infected mice were detected ( Figure 7H). antioxidant enzymes catalase, glutathione peroxidase 2 (gpx2), and superoxide dism 2 (sod2) ( Figure 5B). However, no differences were found in the gene expression of while an increase in sod1 expression was observed ( Figure 5B). Finally, an unexp decrease in the mRNA levels of the NADPH oxidase subunits gp91 phox and p47 phox wa served with SnPP treatment of infected mice ( Figure 5C).

Deficiency of Functional NADPH Oxidase Partially Protects Mice from Liver Damage Induced by F. hepatica and Limits the Production of IL-10
Considering the fact that SnPP treatment protected mice from parasite infectio that reduced levels of gp91 phox mRNA were found in PECs from infected mice, we ana the infection in gp91 phox knockout mice. gp91 phox deficiency reduced the clinical sign liver damage induced by F. hepatica infection ( Figure 6A and Supplementary Figur This partial protection was associated with a lower increase of HO-1 + peritoneal cells in frequency and number ( Figure 6B). Moreover, HO-1 + cells from gp91 phox knocko fected mice expressed lower levels of MHCII ( Figure 6C) and CD40 ( Figure 6D), bu CD80 ( Figure 6E), than those from wildtype mice, indicating that NADPH oxidase play a role both in the immune response and the pathogenesis induced by F. hepati fection. Further characterization of the peritoneal cells from these mice indicated th increase of F4/80 + cells was abrogated in the absence of gp91 phox (Figure 7A), and a pected, very low levels of ROS/RNS produced by these cells (Figure 7B). Additio these cells expressed lower levels of Sirpα ( Figure 7C

IL-10 Signaling Is Crucial for HO-1 Expression in F. hepatica-Infected Mice
Considering the fact that IL-10 induces HO-1 expression that can favor the production of IL-10, and that IL-10 is crucial for Treg differentiation [36], we analyzed whether there was a relationship between IL-10 signaling and HO-1 expression during F. hepatica infection. To this end, we treated infected mice with a neutralizing antibody of IL-10 receptor (IL-10R). The results demonstrate that IL-10R blocking reduced the clinical signs associated with parasite infection ( Figure 8A). Although the recruitment of F4/80 + cells in the peritoneum of infected mice was not affected by IL-10R neutralization ( Figure 8B), it abrogated the elevated expression of HO-1 induced by F. hepatica infection ( Figure 8C). Interestingly, IL-10R neutralization reduced the frequency, but not the number, of CD4 + ( Figure 8D and Supplementary Figure S7) and CD4 + CD25 + ( Figure 8E and Supplementary Figure S7) T cells in the spleens of infected animals. Altogether, these results indicate that IL-10 signaling is essential for HO-1 expression of F4/80 + cells during F. hepatica infection, likely affecting the differentiation of regulatory T cells.
Antioxidants 2021, 10, x FOR PEER REVIEW 14 of 22 gp91 phox knockout infected mice with respect to wildtype mice. However, no significant differences in the production of IFNγ by splenocytes stimulated with parasite components (FhTE) between gp91 phox knockout and wildtype infected mice were detected ( Figure 7H).  (G) Frequency and cell number of splenic CD4 +/ CD25 + Foxp3 + cells from infected and non-infected mice. Gate analyses by flow cytometry are shown in (A) and Supplementary Figure S1. (H) IFNγ levels in culture supernatants of splenocyte proliferation assay cultured with FhTE for 5 days at 37 °C. The results shown represent one experiment. Asterisks indicate significant differences with p < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.

IL-10 Signaling Is Crucial for HO-1 Expression in F. hepatica-Infected Mice
Considering the fact that IL-10 induces HO-1 expression that can favor the production of IL-10, and that IL-10 is crucial for Treg differentiation [36], we analyzed whether there was a relationship between IL-10 signaling and HO-1 expression during F. hepatica infection. To this end, we treated infected mice with a neutralizing antibody of IL-10 receptor (IL-10R). The results demonstrate that IL-10R blocking reduced the clinical signs associated with parasite infection ( Figure 8A). Although the recruitment of F4/80 + cells in the peritoneum of infected mice was not affected by IL-10R neutralization ( Figure 8B), it abrogated the elevated expression of HO-1 induced by F. hepatica infection ( Figure 8C). Interestingly, IL-10R neutralization reduced the frequency, but not the number, of CD4 + ( Figure 8D and Supplementary Figure S7) and CD4 + CD25 + ( Figure 8E and Supplementary Figure S7) T cells in the spleens of infected animals. Altogether, these results indicate that IL-10 signaling is essential for HO-1 expression of F4/80 + cells during F. hepatica infection, likely affecting the differentiation of regulatory T cells.

Discussion
In this work, we have examined the cellular and molecular mechanisms that govern the expansion or differentiation of Tregs induced by HO-1 + 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 + 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 + 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 + 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][52][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].
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][52][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]. 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 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 + cells could benefit the parasite, and not the host. Of note, these cells expressed higher levels of ICOSL and IL-10 than those from gp91 phox 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 + 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][61][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.