*Choera T, Zelante T, Romani L and Keller NP (2018) A Multifaceted Role of Tryptophan Metabolism and Indoleamine 2,3-Dioxygenase Activity in Aspergillus fumigatus–Host Interactions. Front. Immunol. 8:1996. doi: 10.3389/fimmu.2017.01996*

**295**

invasive pulmonary aspergillosis (IPA) in the hypo-immune, or in individuals with other susceptibilities such as patients unable to mount the necessary oxidative defenses such as in individuals with chronic granulomatous disease (CGD) (1).

The manifestation of disease is dependent not only on the host's immune status but also fungal factors including strain heterogeneity (2). *A. fumigatus* growth in the mammalian lung, following survival of resident pulmonary defenses, requires the fungus to adapt to a hypoxic and nutritionally scarce environment. *Aspergillus* mutants unable to synthesize primary metabolites necessary for growth are generally impaired in virulence. For example, deletion of *cpcA*, a transcription factor that globally modulates amino acid biosynthesis in the fungus led to a less virulent phenotype in a murine model of IPA (3). Additional studies have shown that mutants in sulfur utilization (4), uracil/ uridine synthesis (5), zinc uptake, iron acquisition, and many more (6, 7) are also decreased in virulence.

To complicate disease progression further, there is an alarming rise in antifungal resistance strains of *A. fumigatus* (8, 9). Therefore, an understanding of *A. fumigatus* and host metabolic pathways is important in identifying nutrient limitations. One critical metabolic pathway is the biosynthesis of aromatic amino acids [AAAs, tryptophan (Trp), phenylalanine, and tyrosine], which are required not only for growth of *A. fumigatus* but are also precursors for several toxins (**Table 1**). The host relies on dietary sources for all AAAs while *A. fumigatus* synthesizes all

Table 1 | *Aspergillus fumigatus* non-ribosomal peptides containing aromatic amino acids (AAAs) in their peptide structure.

*IPA, invasive pulmonary aspergillosis; HNEC, human nasal epithelial cells. a Only the AAA is designated. Other amino acids are also in the structure of these metabolites.*

*b–iBiosynthesis and host interactions based from the following sources: b(11, 12); c (13); d(14); e (15, 16); f (17); g (18, 19); h (7, 20); i (21, 22).*

three. However, the host and *A. fumigatus* both possess AAA catabolic enzymes. In particular, one key enzyme important in immune homeostasis is indoleamine 2,3-dioxygenase (IDO), which converts Trp to kynurenine and related metabolites in both organisms. Historically, host IDOs activity has been described as an effective antimicrobial control for pathogens that are natural Trp auxotrophs such as *Staphylococcus aureus*, *Chlamydia* spp., and *Toxoplasma gondii*, presumably by Trp starvation (10). However, *A. fumigatus* can synthesize its own Trp and thus the Trp starvation may not be an effective pathogen control for those microbes able to synthesize their own Trp pools*.* Although, IDOs also play additional roles in host defenses through modifying kynurenine levels and subsequent cytokine responses as described below. In this review, we will summarize the recent studies describing the anabolic and the catabolic pathways of Trp metabolism, the implications for therapeutics, and the host–pathogen interaction.

### Trp SYNTHESIS AND POTENTIAL THERAPEUTIC TARGETING

Chorismic acid derived from the shikimic acid pathway is a key intermediate in producing Trp, phenylalanine (Phe), and tyrosine (Tyr) in microorganisms including *A. fumigatus* (**Figure 1**). Trp and Phe are classified as essential amino acids, whereas mammals acquire them from diet, whereas Tyr is synthesized *via* the hydroxylation of Phe (23, 24). The absence of the AAA biosynthetic enzymes and the low bioavailability of Trp in humans makes the Trp biosynthetic enzymes attractive targets for antifungals (25).

### Fungal Trp Anabolic Pathway

Aromatic amino acid synthesis has been extensively studied in *Saccharomyces cerevisiae* and provides the basis for the functional characterization of orthologous enzymes in filamentous fungi (23, 24, 26–28). The shikimic acid pathway is a 7-enzymatic step reaction that initiates with two substrates, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), which are intermediates of glycolysis and pentose phosphate pathways, respectively (29) (**Figure 1**). The first step of the shikimic acid pathway is catalyzed by 3-deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthase to convert PEP and E4P to DAHP. In *S. cerevisiae* and *A. nidulans*, there are two DAHP synthases, Aro3 and Aro4, which are allosterically inhibited by phenylalanine and tyrosine, respectively (24). Steps 2–6 in filamentous fungi such as *A. nidulans* and *A. fumigatus* are completed by the pentafunctional enzyme AroM, or Aro1 in the model organism *S. cerevisiae* (30). The shikimate pathway culminates in the production of chorismic acid synthesized by the enzyme chorismate synthase (AroB) from 5-enolpyruvylshikimate-3-phosphate (EPSP) (31).

The synthesis of Trp from chorismate is initiated by an anthranilate synthase (AS), which converts chorismate to anthranilate, followed by three enzymatic steps as presented in **Figure 1** with the respective functions outlined in **Table 2**. AS(s) in *S. cerevisiae* have been characterized, and it consists of two subunits: anthranilate synthase subunit I (AAS-I), which binds chorismate and is subject to feedback inhibition by Trp and anthranilate synthase

subunit II (AAS-II) which is a glutamine amidotransferase (32). The *A. nidulans trpC*, an AAS-II encoding gene, was characterized in 1977 (33) and found exchangeable with an *A. fumigatus trpC* in 1994 (34). Wang et al. (28) recently characterized *trpE*, the AAS-I

uncharacterized reactions; however, putative orthologs are present in *A.*

encoding gene in *A. fumigatus*. Wang et al. (28) explored the functions of two putative AAS-Is termed *trpE* and *icsA* by creating null mutants*.* The deletion of *trpE* led to a Trp auxotrophic strain, whereas the deletion of *icsA* did not. To ensure that *icsA* did not serve a redundant role for Trp synthesis, the group overexpressed *icsA* in a *trpE* deletion and showed that the overexpression of *icsA* does not reverse the Trp auxotrophy concluding that TrpE is the only AS in *A. fumigatus*. Interestingly, the group showed that IcsA is an active enzyme in *A. fumigatus* as the precursor-chorismate pool is altered in the absence or overproduction of IcsA; however, the product is not known (28). Sasse et al. also confirmed that deletion of *trpE* (they termed *trpA*) results in an *A. fumigatus* Trp auxotrophy (31).

#### The Shikimate Pathway As Potential Antifungal Targets

Currently, there are four major classes of antifungals: azoles and amphotericin B targeting ergosterol, 5-fluorocytosine targeting DNA synthesis, and echinocandins targeting cell wall synthesis. These antifungals either exhibit high toxicity to the mammalian cell (particularly amphotericin B and 5-fluorocytosine) or lose efficacy due to the emergence of drug resistant strains (azoles and echinocandins) (9). With *A. fumigatus* being a eukaryotic pathogen and sharing many proteins with mammalian hosts, there are limitations to developing effective and safe antifungals and therefore a great need for treatments that are fungal specific. Since Trp is a human-essential amino acid and the enzymes in the biosynthesis are fungal specific, several studies have suggested utilizing and finding drugs to target the enzymes of this pathway (35–38).

Targeting essential amino acid pathways have already shown potential for new classes of antifungals. Several groups have explored inhibitors of genes or enzymes involved in methionine biosynthesis. Azoxybacillin, a compound isolated from *B. cereus* targets methionine biosynthesis by interfering with expression of homoserine transacetylase and sulfite reductase encoding genes (39–41). Whereas azoxybacillin displayed a broad spectrum antifungal activity *in vitro*, *in vivo* activity was low possibly due to bioavailability in the host (41). R1-331, a natural product from *Streptomyces akiyoshiensis*, is an effective inhibitor of homoserine dehydrogenase involved in both methionine and threonine biosynthesis (42, 43). Yamaguchi et al. show that R1-331 was active against medically important fungi such as *Candida albicans* and *Cryptoccocus neoformans* and proved to be effective in the treatment of systemic murine candidiasis (42, 44).

Compounds targeting AAA pathways are limited with the most famous being the herbicide Roundup, where the active ingredient glyphosate inhibits EPSP synthase, one of the first enzymes initiating the shikimate pathway (45) (**Figure 1**). Glyphosate has shown to inhibit growth of several fungi including *Candida maltose* (46), *Pneumocystis* (47), and *Cryptococcus neoformans* where glyphosate delayed fungal melanization *in vitro* and *in vivo* and prolonged mice survival during infection (48). Another inhibitor of AAA pathway is a fluorinated anthranilate moiety, 6-FABA, which targets the TrpE enzyme and showed bactericidal activity when used on *Mycobacterium tuberculosis* (49). The studies of

*fumigatus.*

Table 2 | *Aspergillus fumigatus* tryptophan (Trp) metabolism genes and putative protein function.

*a Prediction of protein function based on AspGD (http://www.aspgd.org/) and KEGG (http://www.genome.jp/kegg/kegg2.html).*

*DAHP synthase, 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase; EPSP synthase, enolpyruvylshikimate-3-phosphate synthase; AFMID, arylformamidase; KYNU, kynureninase; LAAO, l-amino-acid oxidase; AADC, aromatic-l-amino-acid decarboxylase, MAOA, monoamine oxidase; ALDH2, aldehyde dehydrogenase family.*

these inhibitors suggest that the Trp biosynthetic pathway could be fruitful in future antifungal drug design.

The value of AAA pathways as drug targets is supported by the findings that AAA auxotrophic mutants are less virulent in animal infection models. Sasse et al. explored the possibility of these pathways as potential drug target by testing the virulence of several AAA auxotrophic mutants in a murine IPA model (31). This study demonstrated that AroM (**Figure 1**) was required for *A. fumigatus* viability. The group also constructed a conditional AroB repression strain that was attenuated virulence. Both a Trp auxotroph (TrpE mutant) and Tyr/Phe auxotroph (AroC mutant) were severely attenuated in virulence for pulmonary infection. Interestingly, the group also unveiled a putative difference in AAA distribution within the host by conducting a systemic infection showing that in a bloodstream infection the TrpE and the AroC mutants although less virulent, can establish some infection (31). Taken together, these results suggest that inhibitors of AAA biosynthetic pathways can potentially be used against *A. fumigatus* as a standalone treatment in a localized pulmonary infection or as an additive treatment in a systemic infection. The result of the bloodstream infection observed by Sasse et al. also suggests that there are mechanisms for the fungus to sense Trp in its environment and utilize it. In *S. cerevisiae*, the Trp specific permease, Tat2, is required for Trp uptake in yeast (50) and its closest homolog in *A. fumigatus* (Afu7g04290) is upregulated during fungal encounters with neutrophils (51) and dendritic cells (52). In *A. nidulans*, the G-protein coupled receptor (Gpr) H may be responsible for sensing Trp and glucose and GprH is conserved in *A. fumigatus* (53, 54). Perhaps, for a systemic infection, inhibition of specific permeases or development of a GprH antagonist would be useful in reducing infections by *Aspergillus*.

#### CATABOLIC Trp METABOLITES IN FUNGI AND HOST

Although the anabolic Trp pathway is absent in mammals, the common catabolic pathways exist in the mammalian host with possession of the same enzymes as *A. fumigatus* (**Figure 2**). Through the expression of Trp degradation enzymes, immune cells are both controlling inflammation and combating microbial infection. In addition to Trp degradation pathways conserved with animals, *A. fumigatus* can also direct Trp pools to secondary metabolites that may impact host health and response (**Table 1**).

#### Fungal Trp Catabolism

There are three putative pathways (**Figure 2**; **Table 2**) for the degradation of Trp in *A. fumigatus*. The kynurenine branch is catalyzed by IDOs that convert Trp into formylkynurenine. In *A. fumigatus*, there are 3 putative *ido* genes: *idoA*, *idoB*, and *idoC*, the orthologs of *A. oryzae idoα*, *idoβ*, and *ido*γ, respectively (55). Enzymatic studies of *Aspergillus oryzae* IDO enzymes suggest two of the three enzymes, IDO*α*, and IDO*β*, may participate in Trp degradation as they have a higher affinity of its substrate. However the recent study by Wang et al., suggested IDOb might be the more dominant enzyme than IDOa as determined by gene expression of *A. fumigatus* grown on Trp amended media (28). Additionally, *idoC* gene expression was slightly induced by the addition of Trp, but the authors note that IDOc had a closer relationship to bacterial IDOs than to fungal IDOs (28, 55). In *S. cerevisiae*, formylkynurenine is further oxidized to the immunomodulatory product kynurenine by a kynurenine formamidase denoted as Bna7, which has been described in *A. nidulans* and is predicted to be involved in NAD (+), biosynthesis (56). The kynurenine branch in fungi is involved in the *de novo* biosynthesis of NAD (+), a coenzyme that is required for oxidation-reduction reactions (57).

Tryptophan can also be metabolized via the indole pyruvate pathway, initiated through the transamination of Trp by aromatic aminotransferases (termed Aro8 and Aro9 in *S. cerevisiae*). These aromatic aminotransferases are also involved in the synthesis of Phe and Tyr in *S. cerevisiae*, and their orthologs are present in *A. fumigatus* (28). In *S. cerevisiae*, the deletion of both Aro8 and Aro9 results in Phe and Tyr auxotrophies (24, 58, 59). In *Candida* spp.*,* where filamentation and pigment production play a role in virulence, the products of these enzymes have been described to influence both phenotypes. The deletion of *aro*8 in *C. glabrata* results in a reduced pigment production and leads to an increased sensitivity to hydrogen peroxide (27). The *aro*8 and *aro*9 mutants of *C. albicans* results in a decreased conversion of Trp to indole acetaldehyde, which is formed via decarboxylation of indole pyruvate. Filamentation of *C. albicans* increased with the exposure to indole acetaldehyde (60).

Indole acetaldehyde can also be produced via the third putative product of Trp degradation: tryptamine. Tryptamine is most famously known as the active compound in psilocybin and for its similarity to serotonins (61). Although the production of tryptamine has yet to be described in *A. fumigatus*, the downstream product of the tryptamine and indole pyruvate pathway, indole acetic acid has been described in several *Aspergillus* spp. including *A. fumigatus* (62, 63). Downstream metabolism of kynurenine, indole pyruvate, and tryptamine has not been explored further, but *A. fumigatus* does possess putative enzymes for the re-synthesis of anthranilate, the precursor to Trp (as denoted in **Figures 1** and **2**).

#### AAA Incorporation into *Aspergillus* Toxins

Many filamentous fungi, including *A. fumigatus*, produce bioactive small molecules that can have detrimental impacts on human health. Subsets of these toxins are small peptides synthesized by non-ribosomal peptide synthetases (NRPS). Several pathogenic *Aspergillus* species synthesize AAA derived peptides including gliotoxin (*Phe* and serine) (64), fumiquinazoline (*Trp*, *Anthranilate*, and Alanine) (13), fumigaclavine (*Trp*) (65), fumitremorgin (*Trp* and Proline) (66), hexadehydroastechrome (*Trp* and Alanine) (21), fumisoquin (*Tyr*, Serine, and Methionine) (14), DPP-IV inhibitor WYK-1 (*Trp*, *Tyr*, and Leucine) (67), cyclopiazonic acid (*Trp*) (68) and benzomalvin (*Phe* and *Anthranilate*) (69). **Table 1**

summarizes the known AAA derived secondary metabolites of *A. fumigatus* and their effect on the host.

Although, fumiquinazolines have yet to be assessed for virulence in an animal model, they are known to have cytotoxic properties (13). Fumigaclavines have been described to have immunosuppressive properties in several studies including the suppression of antifungal cytokines such as TNFα, IL-17, and IFN-γ (12). Fumitremorgin, verruculogen, and tryprostatin—all related products of the fumitremorgin pathway—induce tremorgenic activity in mice and act on the central nervous system (15, 66, 70, 71). Mutants in the hexadehydroastechrome pathway (21) and gliotoxin (20, 21) pathways have altered virulence in murine IPA models. Decreased virulence of the *gliP* mutant (GliP is the NRPS required for gliotoxin synthesis) is dependent on host immune status [reviewed in Ref. (7)]. Overexpression of *hasA* encoding the hexadehydroastechrome transcription factor and thus leading to increased hexadehydroastechrome production was more virulent than wild type *A. fumigatus* in a neutropenic model of IPA (21). Although the exact mechanism underlying the increased virulence of the OE::*hasA* strain is unknown, iron homeostasis and cross talk between metabolic pathways may contribute to the increased virulence of OE::*hasA* (22). These studies highlight the potential contribution of AAA derived toxins in virulence of *A. fumigatus*.

#### Host Trp Catabolism *via* IDO

The function of host IDO during mammalian infection was originally thought to center on the anti-proliferative effects of pathogenic microorganism via deprivation of Trp exerted by the host. IDO is up-regulated by interferon gamma (IFNγ) and depletes Trp (the least abundant essential amino acid) to inhibit pathogen expansion (72, 73), as demonstrated in the constraint of chlamydial growth (74). Numerous studies have since implicated IDO activity as important in fungal infections and have reported the relative outcomes of IDO expression on disease progression (**Table 3**). Accumulating data continues to support that IDO participates in the host–pathogen interaction in human epithelial cells; therefore, the co-evolution of host and microbe Trp metabolism has been investigated (75). The current consensus is that IDO activation is pivotal in regulating inflammatory processes directly

Table 3 | Summary of studies where IDO enzyme activity was found to be implicated in fungal infections.

via Trp depletion and indirectly via the IDO-mediated release of Trp catabolic secondary metabolites (namely, kynurenines).

Dietary Trp is catabolized by two different IDO protein isoforms, IDO1 and IDO2 that are expressed by immune cells, and TDO (Trp 2,3-dioxygenase) that is mainly expressed in the liver. Cells involved in the innate processes of the anti-microbial defense, such as dendritic cells (DCs), neutrophils, and macrophages express IDO1 upon microbial encounter mainly *via* toll-like receptor stimulation. How fungi specifically induce IDO expression is not known; however, induction by other pathogens is associated with pathogen associated molecular patterns, including lipopolysaccharides and CpG oligodeoxynucleotides (78, 90–92), underlining a role for kynurenine metabolism in microbial-induced inflammatory processes.

### IDO-Mediated Tolerance: Impacts on Antimicrobial Responses

Evolutionary studies have shown that the host immune defense against microbes is characterized by three different mechanisms: avoidance, resistance, and tolerance (93). Modules of immunity provide resistance to limit pathogen burden and tolerance and host damage caused by the immune reaction *per se*. However, the inflammatory reaction, although largely considered beneficial for its antimicrobial functions, may also contribute to pathogenicity. Thus, rescue from infection pathology may not only depend on microbial colonization (and inactivation of resistance mechanisms) but also on the resolution of tissue inflammatory pathology through tolerogenic responses to pathogens (94).

Studies using a mouse model of mucosal or invasive *C. albicans* infection found that systemic inhibition of IDO *in vivo* reduced gastrointestinal inflammation and unexpectedly, elevated the levels of fungal colonization compared to control mice (77). Notably, tolerogenic responses toward *C. albicans* were abrogated when IDO was antagonized *in vivo*, as shown in various models of inflammatory disorders (95, 96). As with *C. albicans*, IDO and kynurenine production during *A. fumigatus* infection contributes to fungal pathogen eradication and the regulation of an unacceptable level of tissue damage (97). Indeed, IDO can increase kynurenine host levels to induce adaptive Treg expansion while limiting Th17 polarization (83, 96). In this context, the Th17 pathway, which downregulates Trp catabolism, may instead favor pathology and better explain the paradoxical correlation between fungal infection and chronic inflammation (98).

Another example of this paradox was demonstrated in the context of CGD, in which an NADPH oxidase defect results in reduced host production of antimicrobial ROS and extreme susceptibility to *Aspergillus* infections (1, 83). Although human studies have excluded a role for IDO in CGD (99), further investigations into the IDO pathway are warranted as such studies have failed to demonstrate functional IDO activity at sites of chronic inflammation. Measures of IDO functional activity during IPA have, however, been made in mouse models, and implicate defective IDO activity as a key mediator of chronic inflammation in CGD (83). An exaggerated Th17 pulmonary response was associated with reduced fungal clearance in mouse models of CGD that develop IPA. Here, reduced IDO function was directly related to NADPH/ROS deficiency, as ROS is essential for IDO catalytic activity in mammals (100). ROS deficiency as a result of reduced NADPH function, significantly enhanced IL-17 inflammation and fungal germination in the lung, thus further reducing neutrophil-mediated antimicrobial activities (83).

Since regulation of homeostasis and peripheral tolerance are extremely important in prevention of invasive Aspergillosis or allergy to *Aspergillus* antigens (97, 101), the role of IDO has been extensively studied in this model of fungal infection (81, 82, 102). These studies highlight the induction of the IDO metabolic pathway at different site of fungal colonization as keratinocytes or lung as well as the important anti-inflammatory activity of IDO in the tissue microenvironment (80–82, 84).

#### Aryl Hydrocarbon Receptor (AhR) Activation by IDO Metabolites in Mammals: Biological Consequences

The AhR is a ligand-activated transcription factor first identified for its role during embryonic development and induction of xenobiotic metabolizing enzymes as a response to environmental toxins, such as dioxin (103). More recently, AhR has been shown to play a critical role in immunity by acting as an immune modulator during fungal infection (85). The connection between the AhR and the immune response lies in part in the endogenous Ahr ligands, which comprise many Trp metabolites, including kynurenine (104). Microbial Trp-derived metabolites can activate the AhR, leading to adjustments in the immune response that may hinder disease development (105). The AhR–IDO axis has been recently demonstrated in fungal infection, highlighting a role for IDO-derived metabolites to trigger AhR target genes (85, 106). For example, one AhR target gene, *Il22*, has been widely studied in the context of fungal/microbial infections (105, 107–109). AhR activation by IDO metabolites can also mediate the expansion of peripheral Treg with anti-inflammatory properties. Using IDO-deficient mice, increased pulmonary disease caused by *Paracoccidioides brasiliensis* was associated with decreased Treg expansion and reduced AhR protein expression (85). In murine models of IPA, distinct Treg populations capable of mediating anti-inflammatory effects expand following exposure to *Aspergillus* conidia (97). Late in infection, tolerogenic adaptive Treg (with shared phenotypic identity with the Treg controlling autoimmune diseases or diabetes) produce IL-10 and TGFβ, inhibit Th2 cells, and prevent an allergic reaction to *Aspergillus* (97).

## CONCLUSION

The interplay of Trp metabolic pathways and fungal/host interactions is intriguing with many unanswered questions of the exact nature of crosstalk of shared metabolites and consequences of activation of Trp degradative pathways. In *Aspergillus* infections in particular, not only does the pathogen synthesize and degrade Trp but it also can utilize this amino acid (and its precursor anthranilate or the other two AAAs Tyr and Phe) to yield several potentially damaging toxins (**Table 1**). Also, as both host and *Aspergillus* share catabolic IDO pathways, it is unclear which organism may generate immunomodulatory Trp degradation products and if they respond to each other's products (e.g., kynurenine). Development of *A. fumigatus* IDO mutants for investigation of disease development could yield valuable information on this front. The research on the expression host IDOs exhibit the importance of an extremely coordinated immune response to mount the right inflammatory response for clearance of spores. However, while an increased IDO expression in the host can control inflammation, the suppression of the IDOregulated antifungal Th17 responses can favor fungal growth. In this context, it will be critical to explore the entire IDO-mediated innate response, including the specific T cell regulatory subsets affected by IDO activity.

Although the Trp catabolic pathway is shared between host and pathogen, the anabolic pathway is unique to *A. fumigatus.* The antifungals currently used in treatment are becoming increasingly ineffective with emerging drug resistant strains; therefore, drugs targeting essential fungal specific pathways are needed. A proposal for fungal treatment has been highlighted through the

#### REFERENCES

studies of essential amino acids. As AAA mutants are auxotrophs and decreased in virulence (28, 31), investigations of drugs targeting these pathway enzymes could lead to novel antifungal compounds. Indeed, a few compounds have exhibited some efficacy in targeting Trp metabolic pathways in *M. tuberculosis* and several fungi and efforts to identify additional inhibitors are warranted*.*

#### AUTHOR CONTRIBUTIONS

TC, TZ, and NK have made a substantial, direct, and intellectual contribution to the work and LR provided intellectual insights to the revision. All authors have approved it for publication.

#### ACKNOWLEDGMENTS

The authors would like to thank support from Dalai Lama Trust MSN178745 to TC, The Italian Grant "Programma per Giovani Ricercatori - Rita Levi Montalcini 2013" to TZ, and NIH 5R01AI065728-10 to NK.

in *Klebsiella pneumoniae* pneumonia. *J Immunol* (2014) 192:1778–86. doi:10.4049/jimmunol.1300039

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Choera, Zelante, Romani and Keller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Disease Tolerance Mediated by Phosphorylated indoleamine-2,3 Dioxygenase confers resistance to a Primary Fungal Pathogen

*Eliseu Frank de Araújo1 , Flávio Vieira Loures1 , Cláudia Feriotti1 , Tania Costa1 , Carmine Vacca2 , Paolo Puccetti <sup>2</sup> , Luigina Romani <sup>2</sup> and Vera Lúcia Garcia Calich1 \**

*1Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil, 2Department of Experimental Medicine, University of Perugia, Perugia, Italy*

Resistance to primary fungal pathogens is usually attributed to the proinflammatory mechanisms of immunity conferred by interferon-γ activation of phagocytes that control microbial growth, whereas susceptibility is attributed to anti-inflammatory responses that deactivate immunity. This study challenges this paradigm by demonstrating that resistance to a primary fungal pathogen such as *Paracoccidiodes brasiliensis* can be mediated by disease tolerance, a mechanism that preserves host fitness instead of pathogen clearance. Among the mechanisms of disease tolerance described, a crucial role has been ascribed to the enzyme indoleamine-2,3 dioxygenase (IDO) that concomitantly controls pathogen growth by limiting tryptophan availability and reduces tissue damage by decreasing the inflammatory process. Here, we demonstrated in a pulmonary model of paracoccidioidomycosis that IDO exerts a dual function depending on the resistant pattern of hosts. IDO activity is predominantly enzymatic and induced by IFN-γ signaling in the pulmonary dendritic cells (DCs) from infected susceptible (B10.A) mice, whereas phosphorylated IDO (pIDO) triggered by TGF-β activation of DCs functions as a signaling molecule in resistant mice. IFN-γ signaling activates the canonical pathway of NF-κB that promotes a proinflammatory phenotype in B10.A DCs that control fungal growth but ultimately suppress T cell responses. In contrast, in A/J DCs IDO promotes a tolerogenic phenotype that conditions a sustained synthesis of TGF-β and expansion of regulatory T cells that avoid excessive inflammation and tissue damage contributing to host fitness. Therefore, susceptibility is unexpectedly mediated by mechanisms of proinflammatory immunity that are usually associated with resistance, whereas genetic resistance is based on mechanisms of disease tolerance mediated by pIDO, a phenomenon never described in the protective immunity against primary fungal pathogens.

Keywords: paracoccidioidomycosis, indoleamine-2,3 dioxygenase, pulmonary dendritic cells, disease tolerance, phosphorylated indoleamine-2,3 dioxygenase, signaling function

### INTRODUCTION

Indolamine-2,3 dioxygenase (IDO) is an intracellular enzyme of crucial importance in the tryptophan (trp) catabolism acting along the kynurenine (Kyn) pathway (1, 2). Although the cytokine IFN-γ is considered the major inducer of IDO expression, TGF-β, TNF-α, prostaglandins, and toll-like receptor ligands, among others, have also the ability of promoting the expression of the

#### *Edited by:*

*Steven Templeton, Indiana University School of Medicine – Terre Haute, United States*

#### *Reviewed by:*

*Babak Baban, Augusta University, United States Michael K. Mansour, Massachusetts General Hospital, United States*

> *\*Correspondence: Vera Lúcia Garcia Calich<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 12 August 2017 Accepted: 26 October 2017 Published: 13 November 2017*

#### *Citation:*

*de Araújo EF, Loures FV, Feriotti C, Costa T, Vacca C, Puccetti P, Romani L and Calich VLG (2017) Disease Tolerance Mediated by Phosphorylated Indoleamine-2,3 Dioxygenase Confers Resistance to a Primary Fungal Pathogen. Front. Immunol. 8:1522. doi: 10.3389/fimmu.2017.01522*

**306**

IDO gene (3–5). Among the cells of myeloid origin, dendritic cells (DCs) express the highest levels of IDO that is, however, also present in macrophages, neutrophils, endothelial cells, and fibroblasts (6–8).

Indolamine-2,3 dioxygenase was primarily associated with host defense against pathogens due to trp starvation caused by its enzymatic activity (9–11). However, the recent recognition of the immunoregulatory function of IDO and Kyn, put IDO to another level of knowledge and relevance (11, 12). Indeed, IDO has been recently described as an intracellular signaling molecule involved in the sustained tolerogenic activity of DCs. This signaling function is mediated by TGF-β signaling that promotes IDO phosphorylation that ultimately activates the non-canonical NF-κB pathway that triggers genes encoding IDO, TGF-β, IFNα, and IFN-β, that maintain the tolerogenic activity of murine plasmacytoid DCs (12). This likely accounts for the ability of IDO to favor microbial persistence and concomitant control of inflammation during chronic infections (13–15). Therefore, acting *via* trp depletion and Kyn production, IDO can inhibit the proliferation of T cells and induce their apoptosis. Furthermore, through the aryl hydrocarbon receptor (AhR), IDO directs the conversion of naive CD4<sup>+</sup> T cells into regulatory Foxp3<sup>+</sup> T cells (Tregs) (13, 16, 17).

Paracoccidioidomycosis (PCM), is a granulomatous disease caused by two species of the dimorphic fungus *Paracoccidioides*, *P. brasilienis*, and *P. lutzii* (18). Immunoprotection to PCM is mediated by prevalent Th1/Th17 immunity, whereas Th2 and Th9 responses are associated with severe forms of the disease (19–21). In a murine model of pulmonary infection, A/J and B10.A mice were described, respectively, as resistant and susceptible to PCM. The A/J mouse strain develops a chronic and regressive PCM restricted to the lungs. Its adaptive immunity is mainly mediated by Th1 and Th17 cells that are tightly controlled by elevated numbers and activity of Treg cells. In contrast, B10.A mice develop a progressive disseminated disease associated with large amounts of fungi in non-organized lesions that mimic the severe forms of PCM (22–27).

The immunoregulatory mechanisms that control resistance to PCM are complex and not yet completely solved. In this aspect, our previous studies have shown that in pulmonary PCM, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity but only in susceptible mice IDO inhibition by 1-methyl-dl-tryptophan (1MT) caused progressive tissue pathology and increased mortality rates (28). This difference appeared to be mediated by the opposite innate immunity of resistant and susceptible mice. Indeed, in B10.A mice the innate immunity is preferentially proinflammatory with elevated production of IL-12 and IFN-γ, whereas in A/J mice the predominant TGF-β secretion provides an anti-inflammatory innate response (26–30). These findings led us to hypothesize that IDO has distinct immunoregulatory roles in pulmonary PCM. Here, we could demonstrate that in susceptible mice IDO activity is IFN-γ-dependent and mediated by its catalytic activity, whereas in resistant mice a prevalent TGF-β signaling triggered IDO phosphorylation imparting a signaling and tolerogenic function. The TGF-β-IDO-Treg interplay generates an early pathogen tolerance that allows A/J mice to interact with a primary fungal pathogen as a commensal microbe. Thus, the signaling function of pIDO may lead to an unusual fungus–host interaction that efficiently balances tolerance and resistance mechanisms to the benefit both the pathogen and the host.

#### MATERIALS AND METHODS

#### Mice

Susceptible (B10.A) and resistant (A/J) mouse strains to *Paracoccidiodes brasiliensis* infection were obtained from our Isogenic Unit (Immunology Department of Institute of Biomedical Sciences of University of São Paulo, Brazil) and used at 8–11 weeks of age. Specific pathogen free mice were fed with sterilized laboratory chow and water *ad libitum*.

#### Fungus

*Paracoccidiodes brasiliensis* 18 (Pb 18) was used throughout this investigation. To ensure the maintenance of its virulence, the isolate was used after three serial animal passages. Pb18 yeast cells were maintained by weekly sub cultivation in semisolid Fava Netto culture medium at 37°C and used on the seventh day of culture. Phosphate-buffered saline (PBS)-washed yeast cells were adjusted to 20 × 106 cells/mL based on hemocytometer counts. Viability was determined with Janus Green B vital dye (Merck) and was always higher than 85%.

#### 1MT Treatment and *In Vivo* Fungal Infection

Mice were anesthetized and submitted to intratracheal (i.t.) *P. brasiliensis* infection as previously described. Briefly, after intraperitoneal (i.p.) anesthesia the animals were infected with 1 × 106 Pb18 yeast cells, contained in 50 µL of PBS, by surgical i.t. inoculation, which allowed dispensing of the fungal cells directly into the lungs. The skins of the animals were then sutured, and the mice were allowed to recover under a heat lamp. Groups of infected B10.A and A/J mice were treated with daily i.p. injections of 5 mg/mL of 1MT or 1 mg/mL of rice starch (Sigma-Aldrich) as control. Mice were sacrificed after 96 h or 2 weeks of infection.

### Isolation of Pulmonary CD11c**<sup>+</sup>** Cells

Cell suspensions of the lungs were prepared as previously described (31). The lungs were removed and digested for 60 min in RPMI-1640 medium (Sigma) containing collagenase (2 mg/mL) and DNAse (30 mg/mL). The erythrocytes were lysed with lysis buffer (TRIS + ammonium chloride) and viability determined by the trypan blue exclusion test with viabilities at least to 95%. CD11c+ cells were isolated from total pulmonary cells by magnetic microbeads (Miltenyi Biotec, Cologne, Germany). Pellets of pulmonary cells were counted, washed and the pellet resuspended in 400 µL of buffer (PBS + 0.5% BSA + 2 mM EDTA). The Trypan Blue dye exclusion test was used to determine the number of viable cells present in the DCs cell suspension with viabilities at least to 95%. Then, 100 µL of anti-CD11c coated microspheres were added for each 108 cells and incubated for 15 min at 2–8°C. The cells were washed, the supernatant removed and the pellet resuspended in 500 µL of buffer. This suspension was fractionated using a magnetic separation column, and purified CD11c<sup>+</sup> DCs obtained. This fractionation methods resulted in 95% CD11c<sup>+</sup> DCs.

#### Culture of DCs

The viability of the CD11c<sup>+</sup> cells was evaluated by the trypan blue exclusion test, and was always higher than 85%. The cell suspensions were then centrifuged at 1,200 rpm at 4°C for 10 min and resuspended in 1.0 mL of culture medium (RPMI-1640 medium) supplemented with 10% fetal bovine serum. The cells were adjusted to 1 × 106 /mL and 500 µL were dispensed into each well of 24-well culture plates. The cultures were incubated at 37°C in an incubator containing 5% CO2–95% air for 24 h. After this period, the cells were removed and used in different assays.

### CFU Assay

After 24 h of culture, DCs suspensions were lysed and 100 µL of pellets plated onto BHI-agar medium containing 5% "fungus growth factor" and 4% horse serum (32). The plates were incubated at 35°C and colonies counted daily until no increase in CFU was observed. The numbers (log10) of viable *P. brasiliensis* colonies are expressed as the mean ± SE. Two to three experiments were performed separately.

#### NO and Cytokines Measurement

Supernatants from DC cultures and lymphoproliferation assays were separated and stored at −70°C. The levels of IL-12, IL-1β, TGF-β, IL-6, IFN-γ, and IL-12 were measured by a capture enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from eBioscience. The ELISA procedure was performed according to the manufacturer's protocol, and absorbance was measured with a Versa Max Microplate Reader (Molecular Devices). The concentrations of cytokines were determined based on a standard curve of serial twofold dilutions of murine recombinant cytokines. Nitric oxide production was quantified by the accumulation of nitrite in the supernatants from *in vitro* protocols by a standard Griess reaction (33). All determinations were performed in duplicate, and results were expressed as micromolar concentration of NO.

### Determination of IDO Enzymatic Activity

To monitor IDO enzymatic activity, Kyn were measured using a modified spectrophotometric assay (5). The amount of 50 µL of 30% trichloroacetic acid was added to 100 µL of DCs supernatants, vortexed, and centrifuged at 800 *g* for 5 min. A volume of 75 µL of the supernatant was then added to an equal volume of Ehrlich reagent (100 mg P-dimethylbenzaldehyde, 5 mL glacial acetic acid) in a 96-well microtiter plate. Optical density was measured at 492 nm, using a Multiskan MS (Labsystems) microplate reader. A standard curve of defined l-Kyn concentrations (0–100 mM) was used to determine unknown Kyn concentrations.

### Flow Cytometry

Pulmonary CD11c<sup>+</sup> cells were obtained from 1MT treated and untreated B10.A and A/J mice 96 h and 2 weeks after *P. brasiliensis* infection. For cell surface staining, pulmonary CD11c<sup>+</sup> cells were washed and resuspended at a concentration of 1 × 106 cells/mL in staining buffer (PBS 1×, 2% FBS, 0.5% NaN3). Fc receptors were blocked by the addition of unlabeled anti-CD16/32 (Fc block; BD Biosciences). The DCs cells were then stained for 30 min at 4°C with the optimal dilution of each labeled antibody [phycoerythrin tandem Cy7 (PE-Cy7)-labeled anti-CD11c, phycoerythrin tandem Cy5 (PE-Cy5)-labeled anti-B220, pacific blue (PB)-labeled anti-CD8, phycoerythrin (PE)-labeled anti-CD86, allophycocyanin (APC)-labeled anti-CD40, fluorescein isothiocyanate (FITC)-labeled MHCII (IAK), and allophycocyanin tandem Cy7 (APC-Cy7)-labeled anti-CD11b] monoclonal antibodies (mAbs) from BD Biociences. Cells were washed twice with staining buffer, fixed with 2% paraformaldehyde (Sigma), and acquired using a FACSCanto II equipment and FACSDiva® software (BD Biosciences) and analyzed by the FlowJo software (Tree-Star, Ashland, OR, USA).

#### Lymphocyte Proliferation Assay and Lymphocyte Phenotyping

Spleen lymphocytes from normal B10.A and A/J mice were resuspended (1 × 107 ) in 1 mL PBS-BSA (0.1%) and 1 µL of carboxyfluorescein diacetate, succinimidly ester (CFSE) at a concentration of 5 mM (Molecular Probes, USA). The cells were incubated at room temperature in the dark for 15 min (shaking the tube constantly). The cells were washed two times with 10 mL of supplemented RPMI, counted and resuspended at a concentration of 1 × 106 /mL. In parallel, purified DCs from A/J and B10.A of uninfected and infected mice, treated and untreated with 1MT were centrifuged and adjusted to 1 × 106 /mL in supplemented medium. Aliquots of 100 µL of DCs were placed in a 96-well plate (U-bottom) in the presence of lymphocytes previously stained with CFSE at a ratio 1:10 (100 µL of DCs + 100 µL of lymphocytes). CFSE stained lymphocytes (treated or not with 1MT), purified DCs and Concanavalin A (Sigma) stimulated lymphocytes were used as controls. The cells were incubated at 37°C in 5% CO2 for 3 days. After the incubation period the cells were washed and resuspended at a concentration of 1 × 106 cells/mL in staining buffer (PBS 1×, 2% FBS, 0.5% NaN3). Fc receptors were blocked by the addition of unlabeled anti-CD16/32 (Fc block; BD Biosciences). The leukocytes were then stained for 30 min at 4°C with the optimal dilution of each antibody labeled with the adequate fluorochrome (BD Biosciences). PE-labeled anti-CD44, PE-Cy7-labeled anti-CD8, and PerCP Cy5.5-labeled anti-CD4, and APC-Cy7-labeled anti-CD62L mAbs from BD Biosciences were used. Cells were washed twice with staining buffer, resuspended in 100 µL, and an equal volume of 2% paraformaldehyde was added to fix the cells. A minimum of 50,000 events was acquired on a FACSCanto II flow cytometer using FACSDiva® and FlowJo softwares. Lymphocytes were identified on forward-scatter (FSC) and side-scatter (SSC) analysis. Gated cells were measured for CD4<sup>+</sup> and CD8<sup>+</sup> expression followed by CD44 expression, and cells expressing high and low levels of this molecule were gated. Gated CD44high cells were then measured for expression of low levels of CD62L identifying the effector/ memory CD4<sup>+</sup>CD44highCD62Llow and CD8+CD44highCD62Llow subpopulations. Gated CD44low cells were then measured for the expression of high levels of CD62L identifying the naive CD4+CD44lowCD62Lhigh and CD8+CD44lowCD62Lhigh subpopulations (Figure S2A,B in Supplementary Material). Fifty thousand cells were acquired and the data expressed as the frequency of positive cells. The cell division index was calculated and was based on the number of CFSE<sup>+</sup>CD4<sup>+</sup> or CFSE<sup>+</sup>CD8<sup>+</sup> T cells found in the stimulated culture/number of CFSE<sup>+</sup>CD4<sup>+</sup> or CFSE<sup>+</sup>CD8<sup>+</sup> T cells in the unstimulated culture (34).

## Assay to Determine Regulatory CD4**+**CD25**+**FoxP3**+** T Cells

Culture supernatants from lymphoproliferation assays were removed and stored at 70°C for the determination of cytokines according to the manufacturer's protocol. The pellet cells were stained with PE-Cy7-labeled anti-CD4 and PerCP Cy5.5-labeled anti-CD25 for 30 min 4°C. Next, the cells were washed with staining buffer containing 2% fetal bovine serum, 0.1% azide, 100 µL of PBS. The permeabilization of the plasma membrane was made with the addition of 200 µL/well of Cytofix/Cytoperm (Fixation/ permeabilization kit BD Biosciences®) at room temperature for 30 min, protected from light. After washing the cells, the nuclear membrane permeabilization was performed using 130 µL/well of solution containing 750 µL of 1× PBS, 250 µL of 4% paraformaldehyde, 5 µL Tween 20 Sigma® and the cells then were incubated for 30 min at room temperature in the dark.

After washing the cells with cold PBS (1×), the nuclear transcription factor FoxP3 was stained with an anti-FoxP3 labeled specific antibody (APC-labeled anti-FoxP3) diluted 1/100 in staining buffer. The cells were incubated for 30 min at 4°C, then washed with ice-cold PBS, fixed with 2% paraformaldehyde and stored at 4°C in the dark until analyzed by flow cytometry. Gates for total lymphocytes, CD4<sup>+</sup>, CD4<sup>+</sup>CD25<sup>+</sup>, and finally for TCD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> T lymphocytes were made (Figure S2C in Supplementary Material). Cells were analyzed as above described.

#### Intracellular Cytokines Measurement

Dendritic cells obtained from the lungs were adjusted to 1 × 106 cells and stimulated for 6 h in complete medium in the presence of 100 ng/mL phorbol 12-myristate 13-acetate, 500 ng/mL ionomycin (both from Sigma-Aldrich; Germany), and monensin (3 mM, eBioscience). Cells were subsequently labeled for surface molecules (PerCP-labeled anti-CD11c) and then treated according to the manufacturer's protocol for intracellular staining using the Cytofix/Cytoperm kit (BD Biosciences). Cells were then stained with Alexa Fluor 488-labeled anti-IDO, PB-labeled anti-IL-10, PE-Cy7-labeled anti-TNF-α, APC-CY7-labeled anti-IL-12, and PE-labeled anti-TGF-β. Cells were washed twice with staining buffer, resuspended in 100 µL, and fixed with an equal volume of 2% paraformaldehyde. The flow cytometry data were acquired and analyzed as above described.

### Quantitative Analysis of mRNA Expression

RNA was extracted from DC cultures and the RNA concentrations were determined by spectrophotometer readings at an absorbance of 260 nm. First-strand cDNAs were synthesized from 2 µg RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer's instructions. Gene expression for the genes of IDO, IFN-γ, janus kinase 1 (JAK1), signal transducer and activator of transcription (STAT1), IL-6, suppressor of cytokine signaling 3 (SOCS3), nitric oxide synthase (NOS2), TGF-β, small mothers against decapentaplegic 2 and 3 (SMAD2 and SMAD3), inositol polyphosphate phosphatase (SHIP), Shp1, Sh22 (tyrosine phosphatases 1 and 2), IFN-α, IFN-β, and the endogenous gene glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was characterized by the real time polymerase chain reaction technique (qPCR) (Applied Biosystems, Foster City, CA, USA). The samples were placed in microcentrifuge tubes for specific qPCR testing of samples containing 2 µL cDNA + 10 µL TaqMan PCR Master Mix (Applied Biosystems, Foster City, CA, USA) according to the following thermal profile: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s. The sequences of the primers for Indo (Mm00492506\_m1), IFN-γ (Mm01168134\_m1), Jak1 (Mm00600614\_m1), Stat1 (Mm00439531\_m1), IL-6 (Mm00446190\_m1), SOCS3 (Mm00545913\_s1), NOS2 (Mm00440502\_m1), TGF-β1 (Mm01178820\_m1), Smad2 (Mm00487530\_m1), Smad3 (Mm01170760\_m1), Inpp5d (Ship—Mm00494987\_m1), Nr0b2 (Shp1—Mm00442278\_m1), PTPN11 (Shp2—Mm00448434\_m1), Ifna1 (Mm03030145\_gH), Ifnb1 (Mm00439552\_s1), and the gene endogenous transcripts in all cell types, GAPDH (Mm99999915\_g1) were obtained from Applied Biosystems ready for testing. Probes were labeled with 6-carboxyfluorescein (FAM) at their 5′-terminal end. Data were normalized to GAPDH gene expression. *Taq*Man PCR assays were performed on a Stratagene MxP3005P QPCR System and data were developed using the MxPro QPCR software (Agilent Technologies, USA).

## NF-**κ**B DNA-Binding ELISA-Based Assay

Nuclear extracts from DCs were obtained using the Nuclear Extract Kit (Active Motif, USA). DNA binding activity of NF-κB was measured by an ELISA-based assay using a TransAM NF-κB kit (Active Motif, USA) following manufacturer's instructions. This assay detects binding of p65, p50, p52, and Rel-B proteins to oligonucleotides containing an NF-κB consensus-binding site immobilized onto 96-well plates by specific primary antibodies that recognize an epitope that is accessible only when NF-κB is activated and bound to its target DNA. The binding activity was quantified by spectrophotometry, the OD at 450 nm.

### Western Blot Analysis

Dendritic cells were lysed in ice-cold NP40 buffer [1% NP40, 50 mmol/L Tris–HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA containing 5 mmol/L NaF, 2 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL leupeptine, and 5 µg/mL aprotinine], supplemented with a protease inhibitor (Sigma Aldrich). After incubating for 30 min at 4°C, the samples were centrifuged, and the supernatants were kept as the NP40-soluble fraction. The pellets were resuspended in SDS buffer [2% SDS, 80 mmol/L Tris (pH 6.8), 100 mmol/L DTT, and 10% glycerol]. Protein concentration was determined using a BCA assay (Pierce), and 40–60 µg of protein was run on a polyacrylamide gel and transferred to a 0.45 µm nitrocellulose membrane (Millipore, Bedford, MA, USA). Western blotting was carried out according to a standard procedure using horseradish peroxidase–conjugated secondary antibodies (Thermo Scientific and Sigma Aldrich). mAbs used were as follows: Jak1 (1:500; Sigma-Aldrich), Stat1 (1:500; Sigma-Aldrich), IKK-β (1:1,500; Sigma-Aldrich), IDO1 (1:200; Santa Cruz Biotechnology), Smad2 (1:2,500; Sigma-Aldrich), Ship (1:2,000; Sigma-Aldrich), Shp-2 (1:1,500; Sigma-Aldrich), and IKK-α (1:500; Sigma-Aldrich). Mouse monoclonal antimouse β-tubulin antibody (1:2,000; Sigma-Aldrich) was used as an internal control. The immunoreactive proteins were visualized with ECL plus reagents (ECL Western blotting Detection Reagents; Amersham) and enhanced chemiluminescence apparatus (ImageQuant LAS 4000). Densitometry was performed using ImageQuant TL 8.1 software.

### Detection of Phosphorylated IDO1 (pIDO1) by Western Blotting

Lung extracts from susceptible and resistant mice infected with *P. brasiliensis*, treated or not with 1MT were obtained after 96 h and 2 weeks of infection and analyzed by Western Blot for detection of pIDO1. Protein concentration was determined using a BCA assay (Pierce), and 20 µg of protein in Ripa Buffer and Sample buffer 4× were heated at 100°C for 4 min, then on ice for 2 min and loaded 60 μL per well in 10% SDS PAGE, 1.5 mm. Electrophoresis was then performed by transferring the gel to the nitrocellulose membrane with the membranes incubated in blocking solution [5% non-fat dry milk, 0.1% Tween 20 in Tris buffer saline (TBS) for 1 h at room temperature on an orbital shaker]. After two washings steps with TBS-Tween 20 0.1% buffer, the membrane was incubated overnight with polyclonal rabbit antimouse pIDO (CV223 AP8), 2 μg/mL in 5% non-fat dry milk, TBS at 0.1%. The membrane was washed three times for 5 min and incubated with HRP-labeled secondary antibody (anti-rabbit HRP–Pierce) for 1 h at room temperature. The revelation was performed using the ECL chemiluminescent substrate for enzymatic activity detection of peroxidase (HRP) on photographic film. The same method was used with a monoclonal rabbit anti-mouse IDO1 (CV152) and monoclonal antimouse β-tubulin antibody (1:1,000).

#### Statistical Analysis

Data are expressed as the mean ± SEM. Differences between groups were analyzed by analysis of variance followed by the Bonferroni test. Differences between survival times were determined with the LogRank test. Data were analyzed using GraphPad Prism 6.2 software for Windows (GraphPad). A *p* value of ≤0.05 was considered statistically significant.

### RESULTS

#### IDO Inhibition Increases the Fungal Loads of DCs from Susceptible and Resistant Mice and Down Regulates NO, Kyn, and IDO mRNA Production

Initially, we sought to investigate the effects of IDO inhibition on the interaction of DCs, from susceptible and resistant mice, with *P. brasiliensis*. To explore the effect of IDO inhibition *in vivo*, A/J and B10.A mice were treated or not with 1MT and subsequently subjected to i.t. infection with *P. brasiliensis* yeasts cells. Pulmonary DCs from both mouse strains were isolated 96 h and 2 weeks after infection and *in vitro* cultivated for 24 h. Using a CFU assay it was verified that in both postinfection periods 1MT-treated DCs presented increased fungal loads than untreated cells (**Figures 1A,B**). The levels of NO and Kyn in the supernatants of 1MT-treated DCs from both mouse strains was significantly lower than those detected in untreated groups (**Figures 1A,B**). In agreement with the reduced levels of Kyn observed, a reduced expression of IDO mRNA was found in 1MT-treated groups of both mouse strains (**Figures 1A,B**). The reduced IDO mRNA expression was further

Figure 1 | 1-Methyl-dl-tryptophan (1MT) treatment reduces fungicidal activity, NO, kynurenine (Kyn), and indoleamine-2,3 dioxygenase (IDO) production by pulmonary dendritic cells (DCs) of *Paracoccidiodes Brasiliensis*-infected mice. Pulmonary DCs were obtained from 1MT treated or untreated A/J and B10.A mice at 96 h (A,C,E) and 2 weeks (B,D,F) after infection with 1 × 106 yeasts and cultivated for 24 h at 37°C in 5% CO2. The cells were centrifuged, resuspended in 100 µL of culture medium and assayed for the presence of viable yeasts by a CFU assay. Supernatants from DCs cultures were used to determine the levels of nitrite and Kyn. In the same experimental conditions, DCs of A/J and B10.A mice were mixed with TRizol reagent for RNA extraction. IDO mRNA was measured using TaqMan real-time PCR assay. IDO was also characterized by immunoblotting. DCs were lysed, supernatants supplemented with a protease inhibitor and protein concentration determined by a BCA assay. After electrophoresis in polyacrylamide gel, proteins were transferred to nitrocellulose membranes and stained with anti-IDO antisera. Densitometry of bands was performed using ImageQuant TL 8.1 software. NO production was measured by Griess reagent, and Kyn were evaluated using Ehrlich's reagent. The data represent the mean ± SEM of three independent experiments where the asterisk (\*) represents a statistically significant difference between treatments (\**p* < 0.05, \*\**p* < 0.01, and \*\*\**p* < 0.001) and the hashtag (#) represents the difference between the strains (# *p* < 0.05, ##*p* < 0.01, and ###*p* < 0.001).

confirmed by the decreased protein production as detected by Western Blotting (**Figures 1C–F**).

### IDO Inhibition Promotes Increased Influx and Activation of Pulmonary DCs

We have then asked whether the *in vivo* treatment with 1MT could interfere with the number and activation of pulmonary DCs. We verified that 96 h and 2 weeks after infection, 1MT-treated A/J and B10.A mice presented a higher number of myeloid, lymphoid and plasmacytoid DCs (CD11chighCD11b+, CD11chighCD8<sup>+</sup>, and CD11clowB220<sup>+</sup>, respectively) in their lungs than control mice (**Figures 2A,B**; Figure S1 in Supplementary Material). In addition, 1MT treatment increased the expression of MHC class II (IAK) and costimulatory molecules (CD86, CD40, CD11b) in pulmonary DCs obtained at both periods and mouse strains studied (**Figures 2C,D**). Thus, the increased fungal loads induced by 1MT treatment were associated with increased migration and activation of pulmonary DCs.

### IDO Inhibition Reduces the Secretion of DCs Cytokines

To better evaluate the function of pulmonary DCs from 1MT-treated and untreated A/J and B10.A mice, the levels of cytokines present in the supernatants of cultivated cells were measured by ELISA. First, we could confirm our previous findings (28, 30) demonstrating that DCs from A/J mice produce higher levels of TGF-β and IL-1β than DCs from B10.A mice that, in contrast, are better producer of IL-12 and IL-6. We have found that 1MT treatment reduced the levels of TGF-β and IL-1β produced by A/J DCs at both postinfection periods. In contrast, reduced levels of IL-12, IL-6, and IL-1β were observed in 1MT-treated B10.A mice at both time points (**Figures 3A,B**).

### IDO Inhibition Reduces the Levels of Intracellular IDO and TGF-**β** in A/J DCs, whereas in B10.A DCs Diminishes the Levels of IDO, IL-12, and IL-6

We have also characterized the effect of IDO inhibition on intracellular cytokine (TGF-β, IL-12, and IL-6) content and IDO. Purified DCs from 1MT-treated and -untreated A/J and B10.A mice obtained at two postinfection periods were cultivated and intracellular proteins analyzed by flow cytometry. Compared with untreated controls, decreased numbers of TGF-β+ DCs were observed in the lungs of 1MT treated A/J mice. In contrast, reduced numbers of IL-12<sup>+</sup> and IL-6<sup>+</sup> DCs were detected in 1MT treated B10A mice (**Figures 4A,B**). In addition, reduced numbers of IDO positive DCs were observed in 1MT-treated DCs from both mouse strains and postinfection periods analyzed (**Figures 4A,B**).

### IDO Inhibition Enhances the Immunogenic Activity of DCs

Dendritic cells were isolated from 1MT-treated and untreated A/J and B10.A mice 96 h and 2 weeks after *P. brasiliensis* infection.

Figure 2 | 1-Methyl-dl-tryptophan (1MT) treatment increases the influx of activated dendritic cells (DCs) to the lungs. Number of myeloid (CD11chigh11b+), plasmacytoid (CD11clowB220+), and lymphoid (CD11chighCD8+) DCs obtained 96 h (A) and 2 weeks (B) after infection of 1MT-treated and -untreated A/J and B10.A mice with 1 × 106 viable *Paracoccidiodes brasiliensis* yeasts. Median fluorescence intensity (MFI) of IAK, CD86, CD11b, and CD40 expressed by DCs obtained from lungs of A/J and B10.A mice 96 h (C) and 2 weeks (D) after infection. DCs were obtained by fractionation with anti-CD11c magnetic beads and DCs phenotyping determined by flow cytometry. The data represent the mean ± SEM of three independent experiments where the asterisk (\*) represents a statistically significant difference between treatments (\**p* < 0.05, \*\**p* < 0.01, and \*\*\**p* < 0.001) and the hashtag (#) represents the difference between the strains (# *p* < 0.05, ##*p* < 0.01, and ###*p* < 0.001).

In parallel, splenic lymphocytes from uninfected A/J and B10. A mice were obtained, labeled with CFSE, and cocultured with purified DCs. After 3 days, the proliferation of lymphocytes was analyzed by flow cytometry and the levels of IFN-γ and IL-2 were measured in culture supernatants.

As previously described with untreated pulmonary DCs (30), DCs from 1MT-treated and untreated A/J mice obtained at both postinfection periods induced higher lymphoproliferation than those of B10.A mice (**Figures 5A,B**). However, for both mouse strains at both periods assayed, the inhibition of IDO activity resulted in increased proliferation of lymphocytes. In cultures of 1MT treated or untreated naive lymphocytes no cell proliferation was observed. In the same way, no observe lymphoproliferation were detected when 1MT treated or untreated DCs from uninfected mice were used as APC. The levels of IFN-γ and IL-2 were measured in the supernatants of lymphoproliferation assays. Compared with untreated controls, decreased levels of IFN-γ were observed in 1MT-treated DCs from B10.A mice, whereas increased levels of IL-2 were detected in 1MT-treated DCs of both mouse strains and postinfection periods (**Figures 5A,B**).

#### IDO Inhibition Expands CD4**+** and CD8**<sup>+</sup>** T Cells and Reduces Treg Cells

Treatment of B10.A and A/J mice with 1MT increased the differentiation of T cells induced by isolated DCs. Indeed, increased frequency of naive CD4<sup>+</sup> and CD8<sup>+</sup> (CD4<sup>+</sup>CD44lowCD62Lhigh and CD8<sup>+</sup>CD44lowCD62Lhigh, respectively) as well as memory/ effector CD4<sup>+</sup> and CD8<sup>+</sup> (CD4<sup>+</sup>CD44highCD62Llow and CD8<sup>+</sup>CD44highCD62Llow) T cells were detected when 1MT-treated DCs were cocultivated with naive lymphocytes (**Figures 6A,B**).

Accordingly, reduced frequencies of Treg (CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup>) cells were observed when DCs were obtained from 1MT-treated resistant and susceptible mice (**Figures 6C,D**).

### IDO Is Regulated by IFN-**γ** in B10.A Mice and TGF-**β** in A/J Mice

Two opposite pathways were shown to regulate the production and activity of IDO. The IDO-IFN-γ axis is associated with the catalyst function of this enzyme, leads to trp starvation and Kyn production that in turn control pathogen growth and the inflammatory response. In contrast, the TGF-β-IDO axis has been linked with a signaling function of pIDO that promotes the differentiation of tolerogenic DCs and increases the expansion of Treg cells (12, 35, 36).

We speculated that in B10.A mice IDO is mainly catalyst and IFN-γ-regulated, whereas in A/J mice IDO has a predominant signaling function induced by TGF-β. To explore this hypothesis, the expression of genes and proteins related to IFN-γ and TGF-β signaling pathways was characterized in DCs of 1MT-treated and untreated B10.A and A/J mice obtained at 96 h (Figures S3 and S4 in Supplementary Material) and 2 weeks of infection. As shown in **Figure 7A**, DCs from B10.A mice expressed higher levels of mRNA from genes associated with IFN-γ signaling. Therefore, 1MT treatment led to a marked reduction in the expression of IFN-γ, Jak1, Stat1, IL-6, SOCS3, and NOS2 mRNA by DCs from B10.A mice but had a minor effect on DCs of A/J mice (**Figure 7A**).

#### Figure 5 | Continued

1-Methyl-dl-tryptophan (1MT) treatment increases the immunogenicity of dendritic cells (DCs). Pulmonary DCs were obtained from lungs of 1MT-treated and -untreated A/J and B10.A mice 96 h (A) and 2 weeks (B) after infection with 1 × 106 *Paracoccidiodes brasiliensis* yeasts using anti-DC11c magnetic beads. Spleen lymphocytes from non-infected A/J and B10.A mice were obtained, labeled with CFSE (5 mM) and cocultivated with DCs, in a ratio of DC:lymphocyte of 1:10. In parallel, pulmonary DCs from uninfected A/J and B10.A mice were obtained using anti-DC11c magnetic beads and cultured with 1MT treated or untreated naive lymphocytes from non-infected A/J and B10.A mice. After 3 days of cocultivation, the cells were adjusted to 1 × 106 , labeled with specific anti-CD4 (A) and anti-CD8 (B) antibodies and analyzed by flow cytometry. The cell division index (CDI) was calculated as previously described by Mannering et al. (34) and was based on the number of CFSE+CD4+ or CFSE+CD8+ T cells found in the stimulated culture/number of CFSE+CD4+ or CFSE+CD8+ T cells in the unstimulated culture. The lymphocyte population was gated by FSC/SSC analysis. Fifty thousand cells were counted, and the data expressed as frequency of positive cells. Results are representative of three independent experiments. Cytokines were measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA). The data represent the mean ± SEM of three independent determinations where the asterisk (\*) represents a statistically significant difference between treatments (\**p* < 0.05 and \*\**p* < 0.01) and the hashtag (#) represents the difference between the strains (##*p* < 0.01 and ###*p* < 0.001).

Figure 6 | 1-Methyl-dl-tryptophan (1MT) increases proliferation of CD4<sup>+</sup> and CD8+ T cells and reduces regulatory T cells (Tregs). Spleen lymphocytes from uninfected A/J and B10.A mice were labeled with CFSE (5 mM) and cocultivated with purified dendritic cells (DCs) obtained from 1MT treated and untreated A/J and B10.A mice infected i.t. with 1 × 106 of *Paracoccidiodes brasiliensis* yeasts. DCs were obtained at 96 h and 2 weeks postinfection, and cocultivated with CFSE labeled lymphocytes at a ratio of DC:lymphocyte of 1:10. After 3 days, the cells were adjusted to 1 × 106 , labeled with specific anti-CD4, -CD8, -CD44, -CD62L, -CD25, and -FoxP3 antibodies. The frequency of naive (CD62LhighCD44low) and effector/memory (CD62LlowCD44high) CD4+ and CD8+ T cells as well as CD4+CD25+FoxP3 regulatory T cells (Tregs) was measured by flow cytometry. Values are the mean of three independent experiments. The asterisks represent statistically significant differences between treatments (\**p* < 0.05, \*\**p* < 0.01, and \*\*\**p* < 0.05). The hashtag marks represent statistically significant differences between strains (# *p* < 0.05, ##*p* < 0.01, ###*p* < 0.001).

Figure 7 | Indoleamine-2,3 dioxygenase (IDO) is regulated by IFN-γ signaling in B10.A dendritic cells (DCs). 1-Methyl-dl-tryptophan (1MT) treated or untreated B10.A and A/J mice were infected i.t. with 1 × 106 yeast cells of *P. brasiliensis*, and 2 weeks after infection total lung inflammatory cells were obtained and DCs purified with anti-CD11c magnetic beads. (A) The relative expression of mRNA of IFN-γ, Janus kinase 1 (Jak1), signal transducer and activator of transcription (STAT1), IL-6, suppressor of cytokine signaling 3 (SOCS3), and nitric oxide synthase (NOS2) was measured by real-time PCR. (B) Presence of Jak1, Stat1, IKK-β, and IDO1 proteins was assessed by western blot in supernatants of lysed DCs. Proteins were estimated by analyzing the intensity of each band normalized by β-tubulin, used as control. (C) Densitometry of bands was performed using ImageQuant TL 8.1 software. The asterisks represent statistically significant differences between treatments (\**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001). The hash marks represent statistically significant differences between strains (# *p* < 0.05, ##*p* < 0.01, ###*p* < 0.001).

These findings indicate that in B10.A mice the IDO function is mainly catalyst and regulated by IFN-γ. Immunoblotting assays confirmed these results, and increased amounts of JAK1, STAT-1, and IKK-β proteins were found in DCs extracts of B10.A DCs (**Figures 7B,C**). In contrast to B10.A mice, DCs from A/J mice express higher levels of mRNA from genes associated with TGFβ signaling (**Figure 8**). Compared with B10.A DCs, cells from A/J mice expressed increased levels of TGF-β, SMAD2, SMAD3, SHIP1, SHP1, SHP2, IFN-α, and IFN-β mRNA. 1MT treatment significantly reduced these levels in A/J DCs but had no effect on B10.A cells (**Figure 8A**). Assessing protein production, increased

Figure 8 | Indoleamine-2,3 dioxygenase (IDO) is regulated by TGF-β signaling in A/J dendritic cells (DCs). 1-Methyl-dl-tryptophan (1MT)-treated or -untreated B10.A and A/J mice were infected i.t. with 1 × 106 *Paracoccidiodes brasiliensis* yeasts and 2 weeks after infection total lung inflammatory cells were obtained and DCs purified by anti-CD11c magnetic beads. (A) The relative expression of mRNA of TGF-β, Smad2, Smad 3, SHIP, SHP-1, SHP-2, IFN-α, and IFN-β was measured by real-time PCR. (B) Smad2, Ship, Shp-2, IKK-α and IDO1, and pIDO protein expression was assessed by western blot in supernatants of lysed DCs. (C) Proteins were estimated by analyzing the intensity of each band normalized by β-tubulin, used as control. Densitometry of bands was performed using ImageQuant TL 8.1 software. Values are the mean ± SEM of three independent experiments; the asterisks represent statistically significant differences between treatments (\**p* < 0.001, \*\**p* < 0.01, \*\*\**p* < 0.05). The hash marks represent statistically significant differences between strains (# *p* < 0.05, ##*p* < 0.01, ###*p* < 0.001).

amounts of SMAD2, SHIP1, SHP2, and IKK-α were observed in extracts of A/J DCs (**Figures 8B,C**).

### A/J Mice pIDO and Use the Non-Canonical Pathway of NF-**κ**B Activation, whereas B10.A Mice Express Non-pIDO and Use the Canonical Pathway of NF-**κ**B Activation

Because IFN-γ signaling is mainly associated with the canonical pathway of NF-κB activation, the transcriptional activity of p65 and p50 were measured by ELISA in the nuclear extracts of 1MT-treated and -untreated DCs. As depicted in **Figure 9A**, DCs from B10.A mice showed increased expression of nuclear

Figure 9 | Phosphorylated indoleamine-2,3 dioxygenase (pIDO) of A/J mice activate the non-canonical pathway of NF-κB, whereas in B10.A dendritic cells (DCs) non-phosphorylated IDO use the canonical pathway of NF-κB activation. (A,B) Quantitation of p65, p50, p52, and RelB by enzyme-linked immunosorbent assay (ELISA) in nuclear extracts of DCs. Results are presented as absorbance at 450 nm (A450). Values are the mean ± SE of three independent experiments. The asterisks represent statistically significant differences between treatments (\**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001). The hash marks represent statistically significant differences between strains (# *p* < 0.05, ##*p* < 0.01, ###*p* < 0.001). (C) Expression of IDO and phosphorylated IDO was assessed by Western blot in lung homogenates of 1MT treated and untreated B10.A and A/J mice i.t. infected with 1 × 106 *Paracoccidiodes brasiliensis* yeasts. pIDO was revealed by a specific antibody that recognizes the phosphorylated ITIM moiety of IDO (CV223 AP8) and the enzyme IDO by a specific monoclonal antibody (CV152). (D) In susceptible B10.A mice, IDO is induced by IFN-γ that activates the canonical pathway of NF-κB and enhanced iNOS and IDO expression resulting in anergy of T cell immunity. In resistant A/J mice, TGF-β signaling induces the non-canonical pathway of NF-κB activation and continuous synthesis of TGF-β that confers an stable tolerogenic behavior to DCs that control immunity and tissue pathology.

p65 and p50 than those of A/J mice. Furthermore, 1MT treatment reduced this production by B10.A DCs (**Figure 9A**). An equivalent result was observed with DCs obtained at 96 h of infection (Figure S1 in Supplementary Material). The prevalent non-canonical activation of NF-κB by A/J DCs was confirmed by the increased levels of nuclear p52 and RelB produced in comparison with B10.A DCs (**Figure 9A**). Treatment with 1MT caused a significant reduction of p52 and RelB only in A/J DCs. Again, an equivalent result was observed in pulmonary DCs obtained at 96 h of infection (Figure S2 in Supplementary Material). To further confirm the signaling function of IDO, lung extracts from 1MT treated and untreated B10.A and A/J mice were obtained 2 weeks after infection and the expression of pIDO was assessed by immunoblotting using a specific mAb (CV223 AP8 Ab) that recognizes the phosphorylated moiety of ITIM. As depicted in **Figure 9B**, only A/J mice express pIDO and 1MT treatment did not affect the production of this molecule. Using a polyclonal antibody to IDO (CV152 Ab) two isoforms of IDO, and the elevated expression of the isoform 1 by B10.A DCs were seen. The **Figure 9C** summarizes the main findings obtained in this study. In susceptible B10.A mice IDO is induced by IFN-γ that activates the canonical pathway of NF-κB and enhanced iNOS and IDO expression resulting in anergy of T cell immunity. In resistant A/J mice, TGF-β signaling induces the non-canonical pathway of NF-κB activation and continuous synthesis of TGF-β that confers an stable tolerogenic behavior to DCs that control immunity and tissue pathology.

### DISCUSSION

The antimicrobial and immunoregulatory functions of IDO have been recognized as central mechanisms used by infected hosts to control pathogen growth and excessive inflammatory responses (37, 38). Both IDO activities were used by resistant and susceptible mice to control pulmonary PCM. However, only in susceptible mice IDO inhibition caused a sustained fungal growth, exacerbated tissue pathology and increased mortality rates. This is the worst scenario for host fitness because it allies uncontrolled pathogen growth with exacerbated, but inefficient, inflammation resulting in warful tissue destruction. These findings led us to further investigate the role of IDO in the resistance mechanisms to pulmonary PCM. We found the IDO inhibitor 1-MT as two stereoisomers, 1-D-MT and 1-L-MT. Most of the studies using the IDO enzyme model employed the racemic mixture 1-DL-MT to inhibit the enzime, independent of the known IDO isoforms, IDO1 and IDO2, as we did in this work. Indeed, recent studies have shown that IDO1 is the preferentially inhibited by1-L-MT, while 1-D-MT inhibits IDO2 (39, 40). These findings directed our subsequent studies on the inhibition of IDO using deficient mice, as we have shown in recent work with IDO1-deficient (IDO1<sup>−</sup>/<sup>−</sup>) C57BL/6 mice (41). Using isolated pulmonary DCs, we verified that IDO inhibition led to increased recovery of yeasts from both A/J and B10.A cells. This finding could be attributed to the increased trp availability (42–44), but also to the reduced NO production, an important fungicidal mechanism of macrophages and DCs (26, 27, 30).

Interestingly, IDO inhibition by 1MT reduced the typical cytokines that characterize the innate response of B10.A and A/J mice (26, 27, 30). Furthermore, the assessment of intracellular cytokines has further emphasized the higher expression of IDO and TGF-β by A/J DCs, whereas the proinflammatory cytokines IL-12 and IL-6 were more expressed by B10.A DCs. This led us to suppose that IDO activity in B10.A mice was mainly catalytic, whereas in A/J mice was predominantly signaling and involved in signal transduction (12). These investigators clearly demonstrated that TGF-β signaling activates the tyrosine kinase Fyn that phosphorylates ITIM domains of IDO. This promotes the association of IDO with tyrosine phosphatases (SHP1 and SHP2) that results in the activation of the non-canonical NF-κB pathway (p52/RelB), which translocates to the nucleus and induces the production of type I IFNs (IFNα/β), TGF-β, and IDO (12).

An increased number of activated DCs with enhanced immunogenic activity was detected in the lungs of 1MT treated mice. The increased proliferation of naive lymphocytes was concomitant with increased levels of IL-2 in A/J cocultures and decreased IFN-γ production by B10.A cells mirroring the main suppressive mechanisms developed by susceptible and resistant mice (25, 26, 30, 45). This enhanced immunogenic activity was further confirmed by the increased proliferation of naive and effector/ memory CD8<sup>+</sup> and CD4<sup>+</sup> T cells concomitant with a reduced expansion of CD4<sup>+</sup>CD25<sup>+</sup>Foxp3<sup>+</sup> Treg cells, a finding that may be also associated with the reduced levels of suppressive Kyn produced (46, 47). This tolerogenic function of Kyn on T cells has been linked to the activation of the AhR, a ligand activated transcription factor (46, 48) that participates in the pIDO-tolerogenic DCs-Treg loop of immunoregulation.

Further assessing the mechanisms involved in the divergent activities of IDO, a preferential IFN-γ signaling pathway was observed in B10.A DCs which expressed increased levels of IFN-γ and Jak1-Stat1 mRNA. In addition, elevated expression of IL-6, SOCS3, and iNOS2 were also observed indicating a prevalent IFN-γ-mediated catalytic activity. These data were further confirmed by the increased synthesis of Stat1 and IKKβ proteins involved in IFN-γ signaling. The striking expression of IL-6 and SOCS3 suggests that in susceptible mice IDO degradation may occur *via* proteasomal activity (49). Our data have also confirmed the prevalent TGF-β-mediated activation of A/J DCs which express high levels of TGF-β, Smad2, Smad3, Ship1, Shp1, Shp2, IFN-α, and IFN-β mRNA. The high levels of IKK-α, Smad2, Ship1, and Sph-2 proteins expressed by these cells have further confirmed the use of the TGF-β signaling by DCs from resistant mice. These data agree with the signaling cascade that promotes a tolerogenic activity in plasmacytoid DCs previously described (12) and explain the continuous production of TGF-β by A/J mice that favors the conversion of naive CD4<sup>+</sup> T cells into Treg cells (25). Interestingly, the inhibition of IDO by 1MT showed little interference with the TGF-β-mediated pathway used by A/J DCs, demonstrating that the signaling function of the IDO has predominance over the catalytic activity.

The predominant expression of p65 and p50 by B10.A DCs and the higher levels of p52 and RelB by A/J DCs showed that B10.A and A/J mice predominantly use, respectively, the canonical and non-canonical pathways of NF-κB activation by their pulmonary DCs. More importantly, we could demonstrate the presence of pIDO in the lungs of A/J mice, whereas the non-phosphorylated forms of IDO were observed in B10.A mice. Therefore, we could clearly demonstrate that in B10.A mice IDO has a catalytic function, whereas in A/J mice, besides its enzymatic activity, IDO has a signaling function.

Similarly with other infection models, our studies in experimental PCM showed that the balanced activation of the immune system is fundamental for the protection of hosts, and the excessive activation of pro- or anti-inflammatory mechanisms leads to severe disease (23, 29, 50, 51). In susceptible mice, the suppression of adaptive immunity is a consequence of excessive proinflammatory activity of innate immunity, which, however, is able to control the initial fungal burden. In resistant mice, in contrast, an anti-inflammatory, TGF-β dominated activity is observed impairing the initial control of fungal growth. This activity, however, is balanced by the marked dectin-1 and NLRP3 activation and synthesis of TNF-α and IL-1β by A/J macrophages. This pattern of response leads to the subsequent differentiation of Th1/Th17 mixed responses, always strongly controlled by elevated numbers and activity of Treg cells that control tissue pathology (24–27, 29). This is an unusual but interesting model of resistance and susceptibility to a primary fungal pathogen not previously described.

Host defenses are governed by resistance and tolerance mechanisms, the first involved in reducing pathogen burden during infection and the latter associated with mechanisms that protected the hosts from pathogen- or immune-induced damage. These mechanisms are not mutually exclusive and both can contribute to host fitness (52, 53). Our studies clearly demonstrate that the resistance mechanisms used by A/J mice are governed by resistance to disease and not resistance to pathogen growth. Although not intensely explored in infectious diseases, tolerance to disease is well known and described in plant pathology where host protection is not achieved by the elimination of the pathogen, but it is based on the control of host fitness. This response allows pathogen persistence, but its excessive growth is partially controlled by immune mechanisms that are tightly regulated to preserve homeostasis (52). This is also observed in the host responses to commensal fungi such as *Aspergillus fumigatus* and *Candida albicans* that concomitantly control fungal burden and tissue inflammation (50). The important influence of the IDO-AhR-Treg-Th17 axis on the control of this well-balanced response to commensal microorganisms has been well described in the literature (44, 54, 55). The IFN-γ-mediated activation of IDO and Kyn synthesis by tissue DCs confers a tolerogenic profile to these cells that promote the differentiation of naive T cells into Foxp3<sup>+</sup> Treg cells *via* activation of AhR that prevents the excessive expansion of Th17 lymphocytes (44, 56). This tightly regulated immunity confers immune homeostasis that results in microbial resistance but avoids tissue pathology. The signaling property of pIDO induced by TGF-β activation of DCs was more recently described and has been viewed as potent immunoregulatory mechanism that confer stable tolerogenic activity to plasmacytoid DCs (12, 36). This tolerogenic mechanism was validated in an *in vivo* model of delayed-type hypersensitivity response and in the protective activity of endotoxin-tolerant state of mice against Gram-positive and Gram-negative infections (12, 54). However, to our knowledge, this mechanism of disease tolerance was never described in the host responses to primary fungal pathogens. Indeed, pIDO-mediated tolerance was poorly explored in infectious pathologies. Therefore, we believe that the findings here described open new perspectives to understand resistance against PCM and other primary fungal pathogens where excessive inflammation has been associated with severe disease (57, 58). Previous results obtained in our experimental model, not well understood when originally obtained, can now be re-evaluated in face of the IDO mediated mechanism here described. The subcutaneous immunization with viable yeasts confers sterile immunity to i.p. challenged B10.A mice but partial immunoprotection in A/J mice that remain with persistent fungal loads (59, 60). We have also previously described that *P. brasiliensis* infection induces an elevated production of TGF-β and M2 differentiation of A/J macrophages which express high levels of arginase (27), another finding which is not in line with the mechanisms of pathogen resistance usually described. However, this finding may now be better appreciated and understood because arginase expression by innate immune cells was reported to be essential for the differentiation of tolerogenic DCs, whose signaling activity is mediated by pIDO (61).

In conclusion, the studies here reported clearly demonstrate that susceptibility to PCM is mainly mediated by the proinflammatory IFN-γ-IDO axis of innate responses, whereas resistance relies on the initial anti-inflammatory TGF-β-pIDO-Treg axis that confers a sustained tolerogenic phenotype in pulmonary DCs allowing A/J mice to interact with a primary fungal pathogen as a commensal microbe. Therefore, the signaling function of pIDO leads to an efficient fungus–-host interaction that conditions advantages to both partners, and may be explored in further innovative anti-fungal therapy.

#### ETHICS STATEMENT

The experiments were performed in strict accordance with the Brazilian Federal Law 11,794 establishing procedures for the scientific use of animals, and the State Law establishing the Animal Protection Code of the State of São Paulo. All efforts were made to minimize animal suffering. The procedures were approved by the Ethics Committee on Animal Experiments of the Institute of Biomedical Sciences of University of São Paulo (Proc.180/11/ CEEA).

#### AUTHOR CONTRIBUTIONS

EA designed, conducted, and analyzed all experiments. FL, CF, and TC performed and analyzed experiments. CV characterized IDO and IDO phosphorylation. LR provided conceptual help and wrote the article. PP provided conceptual help and precious reagents for IDO analysis. VC planned experiments, supervised the overall study, and wrote the manuscript.

#### FUNDING

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) research grants no. 2011/51258-2 (VLGC) and 2016/04783-2 (FVL); FAPESP postdoctoral felowships no. 2013/02396-9 (CF) and 2014/18668-0 (EFA); and Conselho Nacional de Pesquisas (CNPq) research grant no.<PHONE_NUMBER> (VLGC).

#### REFERENCES

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01522/ full#supplementary-material.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 de Araújo, Loures, Feriotti, Costa, Vacca, Puccetti, Romani and Calich. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The iDO–ahr axis controls Th17/ Treg immunity in a Pulmonary Model of Fungal infection

*Eliseu Frank de Araújo, Claudia Feriotti, Nayane Alves de Lima Galdino, Nycolas Willian Preite, Vera Lúcia Garcia Calich† and Flávio Vieira Loures\*†*

*Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil*

In infectious diseases, the enzyme indoleamine 2,3 dioxygenase-1 (IDO1) that catalyzes the tryptophan (Trp) degradation along the kynurenines (Kyn) pathway has two main functions, the control of pathogen growth by reducing available Trp and immune regulation mediated by the Kyn-mediated expansion of regulatory T (Treg) cells *via* aryl hydrocarbon receptor (AhR). In pulmonary paracoccidioidomycosis (PCM) caused by the dimorphic fungus *Paracoccidioides brasiliensis*, IDO1 was shown to control the disease severity of both resistant and susceptible mice to the infection; however, only in resistant mice, IDO1 is induced by TGF-β signaling that confers a stable tolerogenic phenotype to dendritic cells (DCs). In addition, in pulmonary PCM, the tolerogenic function of plasmacytoid dendritic cells was linked to the IDO1 activity. To further evaluate the function of IDO1 in pulmonary PCM, IDO1-deficient (IDO1−/−) C57BL/6 mice were intratracheally infected with *P. brasiliensis* yeasts and the infection analyzed at three postinfection periods regarding several parameters of disease severity and immune response. The fungal loads and tissue pathology of IDO1−/− mice were higher than their wild-type controls resulting in increased mortality rates. The evaluation of innate lymphoid cells showed an upregulated differentiation of the innate lymphoid cell 3 phenotype accompanied by a decreased expansion of ILC1 and NK cells in the lungs of infected IDO1−/− mice. DCs from these mice expressed elevated levels of costimulatory molecules and cytokine IL-6 associated with reduced production of IL-12, TNF-α, IL-1β, TGF-β, and IL-10. This response was concomitant with a marked reduction in AhR production. The absence of IDO1 expression caused an increased influx of activated Th17 cells to the lungs with a simultaneous reduction in Th1 and Treg cells. Accordingly, the suppressive cytokines IL-10, TGF-β, IL-27, and IL-35 appeared in reduced levels in the lungs of IDO1−/− mice. In conclusion, the immunological balance mediated by the axis IDO/AhR is fundamental to determine the balance between Th17/Treg cells and control the severity of pulmonary PCM.

Keywords: paracoccidioidomycosis, indolemine 2,3 dioxygenase, aryl hydrocarbon receptor, Th17 cells, regulatory T cells, innate lymphoid cells

## INTRODUCTION

Indoleamine 2,3 dioxygenase-1 (IDO1) is an enzyme that catalyzes the degradation of tryptophan (Trp) along the kynurenines (Kyn) pathway. This enzyme plays a critical role to host defenses against a wide range of pathogens by inducing Trp starvation and controlling inflammatory

#### *Edited by:*

*Steven Templeton, Indiana University School of Medicine, United States*

#### *Reviewed by:*

*Anamelia Lorenzetti Bocca, University of Brasília, Brazil Agostinho Carvalho, University of Minho, Portugal*

> *\*Correspondence: Flávio Vieira Loures<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 19 May 2017 Accepted: 10 July 2017 Published: 24 July 2017*

#### *Citation:*

*de Araújo EF, Feriotti C, Galdino NAdL, Preite NW, Calich VLG and Loures FV (2017) The IDO–AhR Axis Controls Th17/ Treg Immunity in a Pulmonary Model of Fungal Infection. Front. Immunol. 8:880. doi: 10.3389/fimmu.2017.00880*

**320**

and immunological reactions. Several types of cells, such as macrophages, epithelial, and dendritic cells (DCs), express IDO1 that can be induced by proinflammatory cytokines (e.g., IFN-γ), toll-like receptor ligands (e.g., lipopolysaccharide), and interactions between immune cells through the engagement of costimulatory molecules (CD80, CD86) on antigen-presenting cells by cytotoxic T-lymphocyte antigen-4 on T cells (1–3). It is known that IDO1 can affect immunity through two nonexclusive mechanisms: (a) establishment of a local response with "amino acid deprivation" that inhibits cell and pathogen proliferation (2); (b) cascade generation of Trp metabolites with specific immunomodulatory or cytotoxic functions that inhibit T cell activation and modulate the differentiation of naïve T cells into regulatory T (Treg) cells (1, 4, 5). Furthermore, Kyn has immunomodulatory effects in the absence of Trp starvation, *via* activation of the transcription factor aryl hydrocarbon receptor (AhR) (6, 7).

Indeed, in fungal infections, IDO1 modulates the mechanisms of host resistance and disease tolerance by inducing Treg cells and inhibiting Th17 differentiation (1, 8–10). Importantly, Treg and Th17 cells share similar developmental pathways (11) and may arise from a common progenitor depending on the balance of inflammatory and anti-inflammatory cytokines (12), presence of retinoic acid (13), and the activation of the AhR, a ligandactivated transcription factor (14, 15).

Paracoccidioidomycosis (PCM), a systemic granulomatous disease caused either by the dimorphic fungi *Paracoccidioides brasiliensis* or *P. lutzii*, is considered the most prevalent deep mycosis in Latin America (16). The inhalation of conidia usually leads to an asymptomatic infection but a few infected individuals evolve to overt disease. The diverse patterns of T cell responses of *P. brasiliensis*-infected individuals are associated with different clinical manifestations. The resistance to infection observed in asymptomatic individuals is mediated by a predominant Th1 response, which is responsible for macrophage activation and fungal killing. The most severe form of the disease, the juvenile form, presents a prevalent Th2/Th9 response and enhanced antibody production. In the chronic inflammatory response characteristic of the adult form of the disease, a prominent Th17 immunity with important participation of Th1 cells was described (17, 18). The polar patterns of the disease could be reproduced in a murine model of pulmonary PCM (19). Resistant mice (A/Sn) develop a chronic regressive disease allied with prevalent Th1/Th17 immunity concomitant with protective CD8<sup>+</sup> T cells that synthesize large amounts of IFN-γ. The relative protection of susceptible mice (B10.A) is mediated by CD8<sup>+</sup> T cells that, however, are not able to compensate the CD4<sup>+</sup> T cell anergy induced by excessive proinflammatory innate response (20–23).

Recent studies of our group have demonstrated that in pulmonary PCM, IDO1 is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. Actually, only in the susceptible mouse strain IDO1 inhibition by 1-methyl-dl-tryptophan (1MT) caused progressive tissue pathology and increased mortality rates. In addition, as previously reported in candidiasis and aspergillosis (24, 25), an IDO1-mediated immunomodulatory function of plasmacytoid dendritic cells (pDCs) has been described in pulmonary PCM. The *in vivo* depletion of IDO1 expressing pDCs resulted in less severe disease and increased T cell immunity (26), demonstrating that in pulmonary PCM pDCs have a tolerogenic function as previously described by Pallotta et al. (27). These findings led us to further elucidate the role of IDO1 in pulmonary PCM by comparing the disease developed by IDO1-deficient (IDO1<sup>−</sup>/<sup>−</sup>) C57BL/6 mice with their normal wild-type (WT) controls. IDO1<sup>−</sup>/<sup>−</sup> mice developed a more severe infection with elevated fungal burdens, accompanied by increased inflammatory reactions mediated by prevalent Th17 responses insufficiently controlled by Treg cells. The absence of IDO1 expression has also influenced the expression of AhR and the differentiation of pulmonary innate lymphoid cells (ILCs) by increasing the presence of innate lymphoid cell 3 (ILC3) and reducing NK cells. Both the uncontrolled fungal growth and the exuberant inflammatory reactions appear to have contributed to the exacerbated tissue pathology that resulted in increased mortality rates of IDO1<sup>−</sup>/<sup>−</sup> mice.

### MATERIALS AND METHODS

#### Ethics Statement

The experiments were performed in strict accordance with the Brazilian Federal Law 11,794 establishing procedures for the scientific use of animals, and the State Law establishing the Animal Protection Code of the State of São Paulo. All efforts were made to minimize animal suffering. The procedures were approved by the Ethics Committee on Animal Experiments of the Institute of Biomedical Sciences of University of São Paulo (Proc.180/11/CEEA).

#### Mice

Eight- to twelve-week-old male C57Bl/6 WT and C57Bl/6 IDO1<sup>−</sup>/<sup>−</sup> mice originally obtained from Jackson Laboratories and bred as the specific pathogen-free mice at the Isogenic Breeding Unit of the Department of Immunology, Institute of Biomedical Sciences, University of São Paulo were used throughout this study.

#### Fungus and Infection

The highly virulent *P. brasiliensis* 18 isolate (Pb18) was used throughout this study. Yeast cells were maintained by weekly cultivation in Fava Netto culture medium at 36°C and used on days 6–8 of culture. The viability of fungal suspensions, determined by Janus Green B vital dye (Merck), was always higher than 95%. Mice were anesthetized and submitted to intra-tracheal (i.t.) infection as previously described (19). Briefly, after intraperitoneal (i.p.) injection of ketamine and xylazine, animals were infected with 1 × 106 yeast cells, contained in 50 µL of PBS, by surgical i.t. inoculation, which allowed dispensing of the fungal cells directly into the lungs.

### Colony-Forming Unit (CFU) Assays, Mortality Rates, and Histological Analysis

The numbers of viable yeasts in infected organs (lung and liver) were determined by counting the number of CFUs as previously described (28). Mortality studies were done with groups of 10–12 mice. Deaths were registered daily. For histological examinations, the left lung of infected mice was removed and fixed in 10% formalin. Five-micrometer sections were stained by hematoxylin-eosin for an analysis of the lesions and were silver stained (Grocott stain) for fungal evaluation. Morphometrical analysis was performed using a Nikon DXM 1200c camera and Nikon NIS AR 2.30 software. The areas of lesions were measured (in square micrometers) in 10 microscopic fields per slide in 5 mice per group as previously described (29). Results are expressed as the mean ± SEM of total area of lesions for each mouse.

#### Assessment of Leukocyte Subpopulations and Flow Cytometric Analysis

The lungs from *P. brasiliensis*-infected WT and IDO1<sup>−</sup>/<sup>−</sup> mice were collected after 96 h, 2, and 10 weeks of infection and digested enzymatically for 45 min with collagenase (1 mg/mL; Sigma) in RPMI culture medium (Sigma). Total lung leukocyte numbers were assessed with trypan blue, and viability was always >95%. For cell-surface staining, lung cells were washed and suspended at 1 × 106 cells/mL in staining buffer (PBS, 2% fetal calf serum and 0.1% NaN3). Fc receptors were blocked by the addition of unlabeled anti-CD16/32 (eBioscience). The cells were then stained in the dark for 20 min at 4°C with the optimal dilution of each monoclonal antibody. To myeloid cells: anti-CD11b, CD11c, CD40, CD80, CD86, and MHC-II; lymphocytes: CD4, CD25, CD8, CD44, and CD62L; NK: NK1.1, CD49b, NPK46, and Eomes; ILC1: CD127, CD49a, IL-12Rβ1, and Tbet; ILC2: CD127, ICOS, IL-17RB, IL-33, and GATA3; ILC3: CD127, IL-23R, IL-22, and RORC (eBiosciences or BioLegend). Cells were washed twice with staining buffer, fixed with 2% paraformaldehyde (PFA; Sigma). For intracellular detection of cytokines, leukocytes obtained from lungs were stimulated for 6 h in complete RPMI medium containing 50 ng/mL phorbol 12-myristate 13-acetate, 500 ng/mL ionomycin (Sigma), and 3 mM monensin (eBioscience). Next, cells were labeled for surface molecules and then treated according to the manufacturer's protocol for intracellular staining using the Cytofix/Cytoperm kit (BD Biosciences) and specifics antibodies anti-17, IL-4, IFNγ, IL, 22, IL-1β, IL-12, TNF-α, IL-6, TGF-β, FoxP3, IDO1, and AhR. Cells were washed twice with staining buffer, suspended in 100 µL, and an equal volume of PFA was added to fix the cells. A minimum of 50,000 events was acquired on FACScanto II flow cytometer (BD Biosciences) using the FACSDiva software (BD Biosciences). Lymphocytes, myeloid cells, NK cells, and ILCs were gated as judged from forward and side light scatter. For Treg cell characterization, FACS plots or histograms were gated on live CD4<sup>+</sup> CD25<sup>+</sup> cells and the expression of FoxP3<sup>+</sup> were determined. The cell-surface expression of leukocyte markers as well as intracellular cytokine expression was analyzed using the FlowJo software (Tree Star).

#### RNA Isolation and cDNA Synthesis

Lungs were homogenized in TRIzol reagent using tissue grinders. Phase separation was realized following addition of 0.2 mL chloroform per mL of TRIzol and centrifugation at 12,000 × *g* for 15 min at 4°C. The upper aqueous RNA phase was removed to a new tube and further purified using Ultraclean Tissue and Cells RNA Isolation Kit (MO BIO Laboratories) according to the manufacturer's protocol. RNA purity and concentration were assessed on a NanoDrop ND-1000 spectrophotometer. An amount of 1 µg total RNA was reverse transcribed in a 20 µL reaction mixture using the High Capacity RNA-tocDNA kit (Applied Biosystems) following the manufacturer's directions.

#### Real-time Quantitative Polymerase Chain Reaction (RT-PCR)

The cDNA was amplified using TaqMan Universal PCR Master Mix (Applied Biosystems) and pre-developed TaqMan assay primers and probes (*Ahr*, Mm00478932\_m1, *Ifng*, Mm001168134\_m1, *Tnf*, Mm99999068\_m1, *Il-6*, Mm00446190\_m1, *Il-10*, Mm00439614\_ m1, *Tgfb1*, Mm00117882\_m1, *Il-17*, Mm00439618\_m1, *Il-22*, Mm01226722\_m1, *Tbx21*, Mm00450960\_m1; *GATA3*, Mm00484683\_m1; *Rorc*, Mm01261022\_m1; *Foxp3*, Mm 00475162\_m1; all from Applied Biosystems). PCR assays were performed on an MxP3000P qPCR System and data were developed using the MxPro qPCR software (Stratagene). The average threshold cycle (*C*T) values of samples were normalized to *C*<sup>T</sup> value of *Gapdh* gene. The relative expression was determined by the 2−ΔΔ*C*T method.

### Cytokines and Nitric Oxide (NO) Detection

The lungs from *P. brasiliensis*-infected WT and IDO1<sup>−</sup>/<sup>−</sup> mice were collected after 96 h, 2, and 10 weeks of infection with one million *P. brasiliensis* yeasts. The organs were aseptically removed and individually disrupted in 7 mL of PBS. Supernatants were separated from cell debris by centrifugation at 3,000 × *g* for 10 min and stored at −80°C. The levels of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-22, IL-27, IL-35, IL-23, TNF-α, IFN-γ, and TGF-β were measured by capture enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from eBioscience or PBL. NO production was quantified by the accumulation of nitrite in the supernatants from *in vitro* protocols by a standard Griess reaction. All determinations were performed in duplicate, and results were expressed as micro molar concentration of NO. Plates were read using a spectrophotometric plate reader (VersaMax, Molecular Devices).

#### Statistical Analysis

Differences between groups were analyzed by the Student's *t*-test or analysis of variance (ANOVA) followed by the Tukey test. For comparisons of greater than two groups, significance was determined using the one- or two-way ANOVA with Tukey's multiple correction. Differences between survival times were determined with the log-rank test. Calculations were performed using statistical software (GraphPad Prism 7.03). Data are expressed as the mean ± SEM. *p*-Values of ≤0.05 were considered statistically significant.

## RESULTS

### Absence of IDO1 Increases Mortality Rates Associated With Increased Fungal Loads and Tissue Damage

C57Bl/6 WT and C57Bl/6 IDO1<sup>−</sup>/<sup>−</sup> mice were infected with one million yeasts of *P. brasiliensis* by the i.t. route. After 96 h, 2, and 10 weeks, the severity of fungal infection was assessed by a CFU assay. As shown in the **Figure 1**, pulmonary (**Figure 1A**) fungal burdens were increased in IDO1<sup>−</sup>/<sup>−</sup> mice at 96 h, 2, and 10 weeks after infection, whereas significant increases in the liver (**Figure 1B**) after 10 weeks of infection showed that the lung of both IDO1<sup>−</sup>/<sup>−</sup> mice were concomitant with increased levels of pulmonary NO (**Figure 1C**), considered a potent fungicidal mediator in pulmonary PCM. The histopathology analysis (**Figures 1D–G**) after 10 weeks of infection showed that the lung of both IDO1<sup>−</sup>/<sup>−</sup> and WT control mice exhibited large granulomas containing many yeasts and large damaged areas. Comparatively, lung lesions from IDO1<sup>−</sup>/<sup>−</sup> were more severe than those of WT mice, involved extensive areas of the lung parenchyma and were composed of isolated or confluent granulomas containing a huge number of fungi (**Figures 1F,G**). To assess the influence of IDO1 deficiency in the disease outcome, the area of lung lesions and mortality of infected mice were evaluated. The absence of IDO1 expression led to increased lung pathology (**Figure 1H**) and mortality rates (**Figure 1I**). At day 127 of infection, all IDO1<sup>−</sup>/<sup>−</sup> mice were dead; whereas in the same period, 5 of 10 WT mice were still alive and apparently healthy.

### IDO-1 Expression Increases the Expansion of ILC3 but Reduces ILC1 and NK Cells in the Lungs of *P. brasiliensis*-Infected Mice

Innate lymphoid cells are a new family of lymphocytes that lack specific antigen receptors, produce significant amounts of cytokines, and may be cytotoxic upon activation. The distinct ILC subsets exhibit transcription factors and cytokine signatures found in the CD4<sup>+</sup> T helper (Th) subpopulations in their responses to specific antigens (30, 31). These features include the shared expression of Tbet and IFN-γ by ILC1 and Th1 cells; GATA-3, IL-5, and IL-13 by Th2 and ILC2 cells; RORC, IL-17, and IL-22 by ILC3 and Th17/Th22 cells, as well as Eomes, IFN-γ, and cytolytic molecules by CD8<sup>+</sup> T cells and conventional NK cells (30). The previously described influence of IDO/AhR in the differentiation of T and ILC cells (30–33) led us to investigate the influence of IDO1 in the differentiation of pulmonary ILC subsets after *P. brasiliensis* infection of WT and IDO1<sup>−</sup>/<sup>−</sup> mice. Therefore, ILC subpopulations were characterized by their expression of specific surface molecules and transcription factors. The NK cells are classified as NK11<sup>+</sup> CD49b<sup>+</sup> NKp46<sup>+</sup> Eomes<sup>+</sup>, the ILC1 are CD127<sup>+</sup> CD49a<sup>+</sup> IL-12Rβ1<sup>+</sup> Tbet<sup>+</sup>, the ILC2 are CD127<sup>+</sup> ICOS<sup>+</sup> IL-17RB<sup>+</sup> IL-33<sup>+</sup> GATA3<sup>+</sup>, and the ILC3 are CD127<sup>+</sup> IL-23R<sup>+</sup> RORC<sup>+</sup>, or the so-called NCR<sup>+</sup> ILC3<sup>+</sup>, producing IL-22 that are CD127<sup>+</sup> IL-23R<sup>+</sup> RORC<sup>+</sup> IL-22<sup>+</sup>. After 96 h and 2 weeks of infection, a significant decrease in NK cells was detected in the lung infiltrating lymphocytes of IDO1<sup>−</sup>/<sup>−</sup> mice. However, at all periods of infection studied, a significant increase in the number of ILC3 and NRC IL-22 was observed in the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice. Differences in the influx of ILC1 and ILC2 to the lungs were only observed at week 10 postinfection, when ILC1 appeared in reduced numbers whereas ILC2 appeared in increased numbers in the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice (**Figure 2**).

#### IDO1 Controls the Expression of Activation Markers and Intracellular Cytokines of Pulmonary CD11b**+** and CD11c**<sup>+</sup>** Cells

IDO1<sup>−</sup>/<sup>−</sup> and WT C57BL/6 mice were infected with one million yeasts of *P. brasiliensis* by the i.t. route. After 96 h, 2, and 10 weeks of infection, the lung infiltrating leukocytes were obtained and analyzed for the expression of surface molecules by flow cytometry. We analyzed the expression of some activation molecules (IAb , CD40, CD80, and CD86) expressed by CD11b+ and CD11c+ (**Figure 3**). When compared with the WT control group, CD11b<sup>+</sup> (**Figure 3A**) and CD11c<sup>+</sup> (**Figure 3B**) cells from IDO1<sup>−</sup>/<sup>−</sup> mice expressed increased levels of IAb , CD80, and CD86 at all postinfection periods studied. CD40 was also upregulated in CD11b<sup>+</sup> cells of IDO1<sup>−</sup>/<sup>−</sup> mice.

To better define the activation profile of the lung infiltrating leukocytes of both infected mouse strains, the expression of intracellular cytokines (IL-12, TNF-α, IL-1β, IL-6, TGF-β, and IL-10) was characterized by flow cytometry. At all periods after infection analyzed, a decreased number of CD11b<sup>+</sup> cells expressing intracellular IL-12, TNF-α, IL-1β, TGF-β, and IL-10 was detected in the lungs of IDO1−/− mice when compared to the WT counterparts. By contrast, increased numbers of CD11b<sup>+</sup> IL-6<sup>+</sup> cells were found at all postinfection periods in the lungs of IDO<sup>−</sup>/<sup>−</sup> mice (**Figure 4A**). When CD11c<sup>+</sup> cells were studied, similar results were obtained (**Figure 4B**), but the differences between IDO1 sufficient and deficient mice was much more evident.

### IDO1 Controls the Expression of AhR in CD11b**+** and CD11c**<sup>+</sup>** Cells

The influence of IDO1 in the expression of the transcription factor AhR was also investigated in our experimental model. Thus, after 96 h, 2, and 10 weeks of infection with yeasts, the lung infiltrating leukocytes from the IDO1<sup>−</sup>/<sup>−</sup> and WT mice were obtained, and the presence of intracellular IDO1 and AhR in CD11b<sup>+</sup> and CD11c<sup>+</sup> cells detected by flow cytometry. As depicted in **Figure 5**, only WT mice expressed intracellular IDO1. The AhR was detected in both mouse strains, but this transcription factor was markedly downregulated in IDO1<sup>−</sup>/<sup>−</sup> mice. These results clearly showed that the expression of AhR is dependent on the enzyme IDO1 (**Figure 5**).

#### The Absence of IDO1 Increases the Influx of Naïve and Activated TCD4**+** and TCD8**<sup>+</sup>** Lymphocytes to the Lungs

We have also assessed whether the absence IDO1 modulates the influx of T cell subpopulations to the lung of *P. brasiliensis*infected mice. As showed in the **Figure 6**, after 2 and 10 weeks of infection, a higher number of both naive (CD4<sup>+</sup> CD44low CD62Lhigh) and effector/activated (CD4+ CD44high CD62Llow) CD4<sup>+</sup> T cells were found in the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice when compared with WT counterparts. Similarly, a larger number of naïve and activated CD8<sup>+</sup> T cells (CD8<sup>+</sup> CD44low CD62Lhigh and

infected i.t. with 1 × 106 of *Paracoccidioides brasiliensis* yeasts. The colony-forming units (CFUs) from lungs (A) and liver (B) were determined 96 h, 2, and 8 weeks after infection. The bars represent means ± SEs of the mean (SEM) of log10 CFU counts obtained from groups of 4 to 5 mice. (C) Supernatants of lung homogenates were obtained from infected mice (*n* = 4–5/time point) and used to determine the levels of nitrite by the Griess reagent. (D–G) Photomicrographs of lung lesions of WT control mice (D,E) and IDO1−/− mice (F,G) at week 10 of infection. Lesions were stained with hematoxylin-eosin (left panels) and Grocott (right panels). (H) Morphometrical analysis of lung lesions. The areas of lesions were measured (in square micrometers) in 10 microscopic fields per slide in five mice per group and results expressed as the mean ± SEM of total area of lesions for each mouse. (I) Survival curves of WT and IDO1−/− infected mice (*n* = 10) were determined in a period of 175 days. Data represent the means ± SEM and are representative of two independent experiments with equivalent results (\**p* < 0.05; \*\**p* < 0.01, and \*\*\**p* < 0.001).

Figure 2 | Influence of indoleamine 2,3 dioxygenase-1 (IDO1) expression on the presence of innate lymphoid cells (ILCs) in the lungs of *Paracoccidioides brasiliensis*-infected mice. The phenotypic analysis of ILCs in the lungs of C57Bl/6 wild-type and IDO1−/− mice was performed after 96 h, 2, and 10 weeks after *P. brasiliensis* infection. The lung infiltrating leukocytes were labeled with specific antibodies and analyzed for the phenotypes of NK, ILC1, ILC2, innate lymphoid cell 3 (ILC3), and ILC3 (NCR IL-22) subsets according to the respective markers: for NK: NK1.1, CD49b, NKp46, and Eomes; ILC1: CD127, CD49a, IL-12Rβ1, and Tbet; ILC2: CD127, ICOS, IL-17RB, IL-33, and GATA3; ILC3: CD127, IL-23R, IL-22, and RORC (A). The cell surface and intracellular markers were measured by flow cytometry. One hundred thousand cells were counted and the data expressed by number of positive cells (B). Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (\**p* < 0.05; \*\**p* < 0.01, and \*\*\**p* < 0.001).

CD11b<sup>+</sup> (A) and CD11c<sup>+</sup> (B) leukocytes were measured in lung infiltrating leukocytes of C57Bl/6 wild-type and IDO1−/− after 96 h, 2, and 10 weeks of infection with 1 × 106 *Paracoccidioides brasiliensis* yeasts. The lung cells were obtained as described in Section "Materials and Methods" and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. The cells were gated for CD11b+ or CD11c+ expression and then for the presence of IAb, CD40, CD80, and CD86. One hundred thousand cells were acquired on FACS CANTO II and, subsequently, analyzed by FlowJo software. Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (\**p* < 0.05; \*\**p* < 0.01, and \*\*\**p* < 0.001).

CD8<sup>+</sup> CD44high CD62Llow, respectively) were detected in the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice when compared with WT controls (**Figure 6**).

#### IDO1 Controls the Secretion of Pulmonary Cytokines

Lung homogenates were obtained from infected IDO1−/− and control WT mice at 96 h, 2, and 10 weeks of infection. Cytokines were measured by ELISA. Similar patterns of cytokines production were seen at all postinfection periods studied. Most cytokines (TNF-α, IL-1β, IFN-γ, TGF-β, IL-27, IL-10, and IL-35) appeared in decreased levels, whereas IL-2, IL-6, IL-17, and IL-22 were detected in higher concentrations than those detected in WT mice (**Figure 7**).

### IDO1 Influences the Expression of Cytokines and Transcription Factors Genes

We have also assayed the gene expression of transcription factors and cytokines involved in Th1, Th2, Th17, Th22, and Treg differentiation as well as the transcription of AhR gene

Figure 4 | Indoleamine 2,3 dioxygenase-1 (IDO1) expression controls the presence of intracellular cytokines in pulmonary CD11b+ and CD11c+ cells. The intracellular cytokines were determined in CD11b<sup>+</sup> (A) and CD11c<sup>+</sup> (B) lung infiltrating leukocytes of wild-type and IDO1−/− C57BL/6 mice after 96 h, 2, and 10 weeks of infection with 1 × 106 *Paracoccidioides brasiliensis* yeasts. The lung cells were obtained as described in Section "Materials and Methods" and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. The cells were gated for CD11b+ or CD11c<sup>+</sup> expression and then for the presence of cytokines IL-12, TNF-α, IL-1β, IL-10, IL-6, and TGF-β. One hundred thousand cells were acquired on FACS CANTO II and subsequently analyzed by FlowJo software. Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (\**p* < 0.05; \*\**p* < 0.01, and \*\*\**p* < 0.001).

Figure 5 | Indoleamine 2,3 dioxygenase-1 (IDO1) expression controls the levels of aryl hydrocarbon receptor (AhR) present in CD11b+ e CD11c+ cells. The number of IDO1 and AhR expressing cells was determined in lung infiltrating leukocytes of wild-type and IDO1−/− mice 96 h, 2, and 10 weeks after infection with 1 × 106 viable *Paracoccidioides brasiliensis* yeasts. The lung cells were obtained as described in Section "Materials and Methods" and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. The cells were gated for CD11b+ or CD11c+ and then for IDO1 and AhR expression. One hundred thousand cells were acquired on FACS CANTO II and subsequently analyzed by FlowJo software. Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (\*\**p* < 0.01, and \*\*\**p* < 0.001).

associated with IDO1 activity. Thus, the mRNA expression in whole lung cells of WT and IDO1<sup>−</sup>/<sup>−</sup> mice was determined at weeks 2 and 10 after infection by RT-PCR using appropriate primers. As shown in **Figure 8**, the absence of the IDO1 led to a marked decrease in the relative expression of IFN-γ mRNA, a cytokine associated with the induction and catalytic function of the enzyme. The mRNA expression for IFN-γ, TNF-α, TGFβ, and IL-10 also appeared in decreased levels, whereas IL-6, IL-17 and IL-22 mRNAs were upregulated in IDO1<sup>−</sup>/<sup>−</sup> mice. In both postinfection periods, a concomitant decrease in mRNA for AhR and Foxp3 transcription factors were observed in IDO1 deficient mice. By contrast, RORC and GATA3 mRNAs were significantly upregulated IDO1−/− mice, but minor differences were seen in Tbet expression.

Figure 6 | The absence of indoleamine 2,3 dioxygenase-1 (IDO1) induces an increased influx of naïve and activated TCD4+ and TCD8+ lymphocytes to the lungs. The phenotypic analysis of lung infiltrating lymphocytes from wild-type and IDO1−/− mice was performed at 2 and 10 weeks of *Paracoccidioides brasiliensis* infection. The lung cells were obtained as described in Section "Materials and Methods" and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. The cells were then gated for CD4+ or CD8<sup>+</sup> expression and for membrane markers that characterize naïve and effector/ activated cells (CD44low CD62Lhigh and CD44high CD62Llow, respectively). For gate strategy, see Figures S1 and S2 in Supplementary Material. One hundred thousand cells were acquired on FACS CANTO II and subsequently analyzed by FlowJo software. Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (\**p* < 0.05; \*\**p* < 0.01, and \*\*\**p* < 0.001).

#### The Absence of IDO1 Increases the Number of Th17 but Reduces the Presence of Th1 and Treg Cells

It has been demonstrated in fungal infections the fundamental role of IDO1 in promoting the balance between T cell subpopulations (8, 9). IDO1 and Kyn, have the ability of inducing Treg and inhibit Th17 cells, contributing to the regulatory mechanisms that govern immunity or tolerance to pathogens (1, 2, 10). Therefore, we have also characterized the Th subpopulations in the course of

*P. brasiliensis* infection of IDO1-deficient and -sufficient mice. As depicted in **Figure 9A**, the IDO1 enzyme consistently regulates the differentiation of Th lymphocytes. In the absence of IDO1, decreased numbers of CD4<sup>+</sup> IFN-γ+ T cells, which define the Th1 subset, were found. Regarding the Th2 subpopulation, only at the 10th week postinfection, a significant increase in the number of CD4<sup>+</sup> IL-4<sup>+</sup> cells were detected in the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice. After 2 and 10 weeks of infection, CD4<sup>+</sup> IL-17<sup>+</sup> cells appeared in higher numbers in the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice when compared with their WT controls. Furthermore, a marked reduction in pulmonary Treg (CD4<sup>+</sup> CD25<sup>+</sup> Foxp3<sup>+</sup>) cells was observed in IDO1<sup>−</sup>/<sup>−</sup> mice at weeks 2 and 10 of infection (**Figure 9B**).

#### DISCUSSION

The characterization of the infectious environment is particularly important to define how the innate immunity influences the subsequent development of adaptive immunity. The regulatory mechanisms that operate at both phases of immunity are also fundamental to determine how the immune response will dictate the severity of an infectious process. In this context, we have explored the regulatory mechanism exerted by IDO1 in both phases of immunity against *P. brasiliensis* infection. It was quite interesting to verify the close association between T cell phenotypes and those of ILCs, a class of innate cells present at the site of infection at the early phase of infection that plays an important role in the further definition of adaptive immunity (34). We could demonstrate for the first time that ILC participate in the immune response against pulmonary PCM. The expansion of these ILCs was influenced by the IDO1 activity that controls the initial cytokines microenvironment and the induction of transcription factors that determine ILC and T cell phenotypes. Reduced Eomes and Tbet led, respectively, to diminished NKp46 and ILC1, whereas increased RORC resulted in increased ILC3, including those associated with increased production of IL-22, despite the reduced AhR mRNA and protein, which were previously shown to be involved in the synthesis of IL-22 (15).

Previous studies demonstrated that IDO1 plays an important role in the immunity against commensal fungi such as *Aspergillus fumigatus* and *Candida albicans* by regulating fungal tolerance mediated by prevalent activity of IDO and Treg cells and fungal immunity mediated by Th1/Th17 cells properly controlled by Treg cells (24, 35, 36). Here, we demonstrated that in pulmonary PCM, IDO1 controls both, fungal burdens and inflammatory reactions, functions that were concomitant with the expression of AhR. The absence of IDO1 expression increases fungal burdens potentiating the expression of activation markers in macrophages and DCs resulting in exacerbated Th17 immunity poorly controlled by Treg cells. This is the worst scenario for a host response where the uncontrolled fungal growth is accompanied by intense but inefficient inflammatory reactions causing exaggerated tissue pathology that ultimately leads to the precocious death of infected mice.

In vaginal candidiasis, the selective deficiency of IDO1 significantly increased fungal loads and tissue damage mediated by inflammatory cells (37) and here a similar result was observed. Our finding of elevated fungal loads may be attributed to the inability of IDO1<sup>−</sup>/<sup>−</sup> mice to deplete Trp, which possibly contributed to the enhanced *P. brasiliensis* growth. However, the more severe infection of IDO1<sup>−</sup>/<sup>−</sup> mice was accompanied by increased levels of NO, a well-recognized fungicidal mediator in *P. brasiliensis* infection (38–44). Actually, a dual role regarding NO activity has been observed in PCM. It can enhance the killing ability of IFN-γ activated macrophages exerting a protective effect (40–42) but is also suppressive as previously demonstrated (39, 43, 44). Interestingly, Bernardino et al. using iNOS<sup>−</sup>/<sup>−</sup> mice clearly showed that absence of NO production is accompanied by an early reduction in fungal loads and increased T cell responses. At the chronic phase of the disease, iNOS<sup>−</sup>/<sup>−</sup> mice showed increased numbers of viable yeasts circumscribed, however, by elevated presence of activated

\*\**p* < 0.01, and \*\*\**p* < 0.001).

Figure 8 | Indoleamine 2,3 dioxygenase-1 (IDO1) influences the gene expression of cytokines and transcription factors. Relative expression of mRNA for aryl hydrocarbon receptor (AhR), IFN-γ, TNF-α, IL-6, RORC, Tbet, GATA3, FoxP3, IL-10, TGF-β, IL-17, and IL-22 in whole lung cells of wild-type and IDO1−/− mice after 10 weeks of *Paracoccidioides brasiliensis* infection. The level of gene transcription was determined by real-time PCR. Bars show mean ± SEM from at least four mice per group and are representative of three independent experiments (\**p* < 0.05; \*\**p* < 0.01, and \*\*\**p* < 0.001).

Figure 9 | The absence of indoleamine 2,3 dioxygenase-1 (IDO1) increases the number of pulmonary Th17 but reduces the presence of Th1 and regulatory T (Treg) cells. The phenotypic analysis of lung infiltrating lymphocytes from wild-type and IDO1−/− mice was performed at 2 and 10 weeks of *Paracoccidioides brasiliensis* infection. The lung cells were obtained as described in Section "Materials and Methods" and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. (A) The cells were gated for CD4+ and then for intracellular expression of IL-4, IL-17, IL-22, or IFN-γ. (B) For Treg cells characterization, the FoxP3+ cells were counted on CD4+ CD25+ doublepositive cells. For gate strategy, see Figure S3 in Supplementary Material. One hundred thousand cells were acquired on FACS CANTO II and subsequently analyzed by FlowJo software. Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (\**p* < 0.05 and \*\*\**p* < 0.001).

macrophages and T cells (39). Importantly, the inducible isoform of nitric oxide synthase (iNOS) responsible for NO production and the enzyme IDO1 are induced by IFN-γ (45, 46), but NO and IDO1 are mutually inhibitory (47–49). This reciprocal regulation was here detected, since the absence of IDO1 expression enhanced NO production that, however, was insufficient to control the fungal growth. These results were consistent with our previous studies in resistant and susceptible mice where increased NO synthesis paralleled the IDO1 inhibition by 1MT (50). More than that, these results demonstrate that, contrary to the well accepted paradigm, in murine PCM the control of fungal growth by IDO1 activity is more relevant than that mediated by NO production.

At all postinfection periods analyzed, cells from IDO1<sup>−</sup>/<sup>−</sup> mice showed increased levels of activation molecules that can explain the increased expansion of T cell that migrate to the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice. Despite its statistical significance, it is difficult to point out the biological relevance of the increased expression of costimulatory molecules by DC11b+ cells. However, our previous report (26) showed that CD11c<sup>+</sup> cells from IDO<sup>−</sup>/<sup>−</sup> mice have an enhanced ability of inducing T cell proliferation. DCs from IDO<sup>−</sup>/<sup>−</sup> mice have an increased ability of expanding CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes in parallel with decreased proliferation of Treg cells (CD4<sup>+</sup> CD25<sup>+</sup> Foxp3<sup>+</sup> T cells). This study has also shown that the CD4<sup>+</sup> and CD8<sup>+</sup> T cells exhibited an activated phenotype as revealed by the expression of CD25 and DC69. Interestingly, both CD11b<sup>+</sup> and CD11c<sup>+</sup> cells showed significantly reduced levels of pro- and anti-inflammatory cytokines, in parallel with increased presence of IL-6<sup>+</sup> cells. These effects were more prominent in CD11c<sup>+</sup> than in CD11b<sup>+</sup> cells as expected by the major expression and activity of IDO1 by the former leukocyte subpopulation (2, 51). Furthermore, the new cytokines balance in CD11b<sup>+</sup> and CD11c<sup>+</sup> cells of IDO1<sup>−</sup>/<sup>−</sup> mice led to increased proliferation of ILC3 and Th17 lymphocytes that are known to be expanded by IL-6 and TGF-β signaling, STAT3 phosphorylation, and expression of RORγτ, the molecular signature of IL-17 secreting cells.

It has been reported that IDO1 and AhR expression are mutually regulated (1, 52), and here we could demonstrate the profound impairment of AhR production caused by IDO1 deficiency. These findings can explain the increased inflammatory reactions developed by IDO1<sup>−</sup>/<sup>−</sup> mice and the phenotype of T cells and ILC developed by *P. brasiliensis*-infected IDO1<sup>−</sup>/<sup>−</sup> mice: increased presence of ILC3 and Th17 cells associated with reduced numbers of Treg cells.

The elevated numbers of naïve and activated lymphocytes found in the lung of IDO1<sup>−</sup>/<sup>−</sup> mice were consistent with the higher levels of IL-2 produced by IDO1<sup>−</sup>/<sup>−</sup> mice, a fact also observed in our studies with IDO1 inhibition by 1MT (50). In addition, the decreased levels of IL-12, TNF-α, and IFN-γ reflect the reduced expansion of Th1 cells as shown by the low numbers of CD4<sup>+</sup> IFN-γ+ lymphocytes present in the lungs of IDO<sup>−</sup>/<sup>−</sup> mice. The reduced levels of IL-10, TGF-β, and IL-35 are also concordant with the drastic reduction in the expansion and migration of Treg cells to the lungs of IDO1<sup>−</sup>/<sup>−</sup> mice. Furthermore, the blockade of the IDO/AhR axis increased the levels of IL-17 and IL-22 secreted into the lungs parenchyma and reflected the major development of Th17 cells that have the ability of secreting both cytokines. The same shift for the IL-17/IL-22 production was reported by De Luca et al. (37) studying the effect of genetic IDO1 deficiency in vaginal candidiasis.