Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America (1). The pattern recognition receptors (PRRs) expressed on phagocytic cells recognize pathogen-associated molecular patterns (PAMPs) on P. brasiliensis surface and regulate the innate and adaptive phases of immunity. The different signaling pathways used by these PRRs to activate immune cells result in different patterns of cell activation, production of diverse pools of cytokines and chemokines triggering diverse immune mechanisms. In human PCM and experimental models, the prevalent expansion of Th1/Th17 cells with reduced expansion of Treg cells characterize the benign disease, whereas elevated proliferation of Th2/Th9 and Treg cells is associated with severe PCM (2–11).

The toll-like receptors (TLRs) play a crucial role in the recognition of fungal pathogens such as P. brasiliensis, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and others (8, 12–16). TLR2 and TLR4 sense P. brasiliensis mediated by MyD88-dependent signaling that leads to cytokines production by alveolar and peritoneal macrophages (6, 7, 17, 18). These components were also seen to control the adaptive immunity and severity of disease developed by P. brasiliensis infected mice (6, 7, 18). In addition, C-type lectin receptors (CLRs) like mannose receptor (MR) and dectin-1 as well as the complement receptors 3 play an important role in P. brasiliensis recognition and activation of immune cells (8, 19–22). Altogether, these studies have demonstrated that in pulmonary PCM, as in other infectious pathologies, PRRs are key elements that govern protective immunity, which must be tightly controlled by anti-inflammatory mechanisms as those mediated by Treg cells and their products (5–7).

The NOD-like receptor P3 (NLRP3) belongs to the NOD family of cytosolic PRRs that detect intracellular PAMPs well as danger signals, named danger associated molecular patterns (DAMPs). Under cell activation, components of NLR family can aggregate into large cytoplasmic complexes called inflammasomes (23). The members of this family have a LRR (leucine-rich repeat) domain in the C-terminal structure, a nucleotide-binding and oligomerization domain containing a neuronal apoptosis inhibitory protein, and a N-terminal caspase recruitment domain (CARD) or pyrin (PYD) domain. In the canonical pathway of NLRP3 inflammasome activation, NLRP3 oligomerization is initiated through the apoptosis-associated speck-like protein (ASC) containing C-terminal CARD that is recruited to the complex via NLRP3-PYD-ASC-PYD interaction. ASC associates with procaspase-1 via CARD interaction, leading to caspase-1 activation that promotes the processing of pro-IL-1β and pro-IL-18 into their mature forms (24–26). The NLRP3 inflammasome is activated by a large repertoire of PAMPs and DAMPs, including ATP, uric acid crystals, silica, aluminum hydroxide, asbestos, reactive oxygen species (ROS), and bacterial or viral RNA (27, 28). Besides the canonical pathway, a non-canonical pathway that utilizes caspase-11 has been shown to activate NLRP3 inflammasome (29, 30). The non-canonical NLRP3 inflammasome can be directly activated by LPS derived from Gram-negative bacteria and by some fungi, which are delivered into the cytosol and activate caspase-11. This, in turn, triggers the opening of the pannexin-1 channel that induces the K + efflux required for NLRP3 inflammasome activation and release of mature IL-1β (30, 31). The first signal for pro-IL-1β processing is through pro-IL-1β and NLRP3 expression mediated by NF-kB transcription that is potentially activated by TLRs and CLRs, and the second signal is the proteolytic processing of pro-caspase-1 by activated NLRP3 (25). NLRP3 is the NLR mostly involved in the immunity against fungal infections (32–34). In C. albicans infection, the canonical and non-canonical pathways of NLRP3 inflammasome activation were shown to be essential for mediating IL-1β secretion (29, 35). In vivo studies of disseminated C. albicans infection showed that NLRP3, Syk, ASC, and caspase-1 are fundamental to control disease severity and regulate the adaptive antifungal immune response through the induction of Th1 and Th17 development (36, 37). In an invasive pulmonary model of aspergillosis, NLRP3 and AIM2 were required to engage the inflammasome to trigger innate immune responses against A. fumigatus; mice lacking both AIM2 and NLRP3, but not mice lacking a single inflammasome receptor, were hyper susceptible to invasive aspergillosis (38). The pioneer study of Tavares et al. demonstrated that following P. brasiliensis infection, the production of mature IL-1β by bone marrow-derived dendritic cells depends on NLRP3 and caspase-1 activation (39), although the fungal molecules associated with this process are still unknown. However, K + efflux, ROS generation, lysosomal acidification, and cathepsin B release to the cytosol were seen to be required to the NLRP3 inflammasome activation by P. brasiliensis (39). Furthermore, a recent work of our lab showed that a dectin-1-Syk mediated mechanism controls NLRP3 inflammasome activation by P. brasiliensis infected macrophages of resistant A/J mice (21). In another study using a murine model of systemic PCM induced by intravenous infection, the susceptibility of NLRP3 and caspase-1 knockout mice to P. brasiliensis infection was evaluated and their increased susceptibility was associated with reduced IL-18 production and Th1 immunity (40).

Because the human disease is thought to be acquired by the pulmonary route, and diverse routes of infection induce different patterns of immunity and disease severity (41), in the present study, we sought to investigate the role of NLRP3 inflammasome in the pulmonary infection caused by P. brasiliensis. Therefore, several C57BL/6 mouse strains deficient for NLRP3 inflammasome components (Nlrp3^−/−, Casp1/11^−/−, Asc^−/− mice) as well as deficient for the ATP receptor (P2x7r^−/− mice) were intratracheally infected with 1 × 10⁶ P. brasiliensis yeasts and compared with their wild type (WT) controls. Several parameters of pulmonary infection and immune response were evaluated at the acute and chronic phases of the disease. Our results indicated that NLRP3 inflammasome plays a crucial role in the control of the innate and adaptive immunity against P. brasiliensis infection. This control was associated with IL-1β and IL-18 secretion induced by caspase-1 activity starting at an early phase of infection. The activation of NLRP3 inflammasome was seen to be essential to control fungal growth and activate protective T cell immunity. This protective inflammatory response was composed of increased numbers of PMN leukocytes and activated CD4⁺ and CD8⁺ T lymphocytes that migrate to the site of infection. Furthermore, NLRP3 inhibited the expansion and migration of regulatory T (Treg) cells resulting in a well-balanced and protective Th1/Th17 immunity.

The role of NLRP3 inflammasome and the cooperation of TLRs and CLRs in the modulation of immune response against P. brasiliensis infection in macrophages and dendritic cells have been studied in our lab (8, 15, 18, 22). Using our murine model of resistance and susceptibility to PCM, it was verified that the activation of NLRP3 inflammasome by P. brasiliensis infection of macrophages from resistant A/J mice is dependent on dectin-1-Syk signaling (21). Other studies showed the role of TLR2, TLR4, dectin-1, and MyD88 signaling in the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) and control of protective Th1/Th17 immune responses (5, 18, 20). Mice and patients infected with P. brasiliensis secrete IL-1β and IL-18, suggesting that NLRP3 is an important player in the recognition of this fungal pathogen (55, 56). The role of NLRP3 using in vivo models of PCM were less explored; however, a recent study using mice systemically infected with P. brasiliensis demonstrated that NLRP3 controls host resistance by inducing a prevalent Th1 immunity associated with IL-18 secretion (40). Because human PCM is acquired by the pulmonary route of infection, in the present study, we sought to characterize the role of NLRP3 inflammasome in i.t. infected mice whose response involves pulmonary-mediated immunological mechanisms. Several parameters of disease severity and host response were evaluated at several postinfection periods. Increased numbers of viable yeasts were recovered from lungs and livers of Nlrp3^−/−, Casp1/11^−/−, P2x7r ^−/−, and Asc^−/− mice. Our CFU data corroborate with those of Ketelut-Carneiro et al. that found increased fungal loads in the lungs and livers of Asc^−/− and Casp1/11^−/− mice infected by the i.v. route (40). Our study has also revealed for the first time the importance of the purinergic P2X7 receptor (P2X7R) in NLRP3 activation by P. brasiliensis infection, in agreement with recent studies that demonstrated the participation of this receptor in the activation of the NLRP3 inflammasome and immunity against intracellular infections caused by Mycobacterium, Chlamydia, Leishmania, and Toxoplasma (57–60). In our pulmonary model of PCM, a marked influence of P2X7R in the immune response and disease severity of mice was clearly shown by the deficient fungicidal activity, decreased cytokines production, high tissue damage and decreased survival rates developed by P2X7R deficient mice. As expected, NLRP3 activation exerted a profound influence in the levels of IL-1β and IL-18 present in the lung tissue and subsequent polarization of immune response. This finding indicates that the new equilibrium in the pool of pulmonary cytokines were responsible by the diverse immunity developed by NLRP3 deficient mice, once the levels of other cytokines such as TNF-α and IL-6 remained in the control levels. It is well known that IL-1β and IL-18 contribute to the induction of the adaptive immune response by stimulation of T cell proliferation, differentiation of Th1/Th17 immunity and increased activity of NKT cytotoxic cells (61). NLRP3 and pro-IL-1β mRNAs are highly expressed in the first signal of NLRP3 activation, as a result of the engagement of PRRs and PAMPs on phagocytes (25, 62). Our findings demonstrated that the genetic deficiency of NLRP3, Casp1/11, and Asc totally impaired the expression of IL-1β, IL-18, NLRP3, Casp1, and ASC mRNA in the lungs of infected mice at week 4 of infection. Besides the control of NLRP3 inflammasome components, P. brasiliensis infection induced an increased expression of Syk, confirming our previous studies demonstrating the crucial role of dectin-1-Syk signaling in the activation of this receptor (21). It is well known that the definition of the inflammatory infiltrates that accompanies an infectious process helps to define the type and intensity of the established immune response. Here, the expression of NLRP3 inflammasome-associated components was concomitant with increased infiltration of PMNs but not macrophages into the lungs of mice, indicating the selective influence of NLRP3 activation on this important effector cell in fungal infections. PMN-rich inflammatory reactions characterize the function of Th17 cells that, besides the typical production of IL-17 and IL-22, are committed with the secretion of chemokines involved in the migration and activation of PMN leukocytes (8, 63–66). In agreement, an enhanced differentiation of Th17 and Tc17 cells were seen during the infection of NLRP3 sufficient mice. The NLRP3 inflammasome activation has also induced an increased influx and activation of CD4⁺ and CD8⁺ T cells to the lungs of mice that were probably involved in the control of fungal loads, and subsequent control of lung pathology. Indeed, decreased numbers of CD4⁺- and CD8⁺-activated T cells were consistently seen in the lungs of Nlrp3^−/−, Casp1/11^−/−, and Asc^−/− mice that developed increased fungal burdens associated with increased lung pathology and mortality rates. In pulmonary PCM, both T cell subsets participate in the protective mechanisms, with an intense and prominent contribution of CD8⁺ T cells (67, 68). Although in less number, CD8⁺ T cells were here shown to participate in the immunity associated with NLRP3 activation, a relevant observation added to the role of this receptor in the host defense mechanisms against P. brasiliensis infection.

The caspase-1-dependent cytokines exert important effects in the initiation of the adaptive Th1 and Th17 cellular responses to fungal infections (37). IL-1β has been suggested to promote inflammatory diseases by inducing the expansion of differentiated T cells (69) and has been implicated, alone or in combination with IL-6 and TGF-β, in driving murine and human Th17 priming and phenotype stabilization (66, 69). IL-18 was initially discovered as an IFN-γ-inducing factor, which together with IL-12 promotes the development of Th1 cells (70). Here, we assessed the intracellular cytokines production to define the effect of NLRP3 activation in the Th1, Th2, and Th17 defense mechanisms of P. brasileinsis infected mice. A new balance of Th cells subsets was seen in the absence of NLRP3 inflammasome components: an increased expression of IL-4 and TGF-β that accompanied a reduced number of IFN-γ⁺ and IL-17⁺ CD4⁺ and CD8⁺ T cells. In murine PCM, we previously demonstrated that IL-4 can exert protective or deleterious roles, depending on the mouse strain studied (71). It can dampen excessive inflammatory reactions of B10.A mice but suppresses the protective Th1 immunity of C57BL/6 mice. A recent report showed that IL-4 suppresses NLRP3-dependent caspase-1activation and subsequent IL-1β secretion (72). These data show a mutual control between NLRP3 inflammasome and IL-4, as here observed. Our study also showed a clear association between NLRP3 activation and decreased synthesis of TGF-β and Treg cells development suggesting that the predominant pro-inflammatory milieu produced by this receptor significantly reduced the regulatory anti-inflammatory mechanisms. Interestingly, in a model of chronic kidney disease, the expression of NLRP3 was linked to the control of TGF-β-dependent signaling (73) although in our model enhanced TGF-β expressing cells were allied with absence of NLRP3 activation.

The analysis of intracellular cytokines revealed that NLRP3 played an important role in the induction of Th1 and Th17 responses with concomitant reduction of Th2/Treg expansion, a process of immune regulation associated with regressive disease in both humans and experimental models of PCM (2, 3, 5–8, 10). In addition, an important contribution of CD8⁺ T cells to IL-17 and IFN-γ production was also seen. Although no studies have directly addressed the role of IL-17 in PCM, several indirect findings clearly demonstrated that a well-balanced production of pro- and anti-inflammatory cytokines is crucial to determine a protective immunity. The evaluation of cytokines production by patients with polar forms of PCM has shown that the prevalent synthesis of Th1/Th17 cytokines characterized the mild forms of chronic PCM, whereas an elevated production of IL-4 and IL-9, associated with high numbers of Treg cells, were linked with the severe forms of the disease (10). Our previous studies have also demonstrated that a balanced differentiation of Th1/Th17/Treg cells is fundamental to achieve immunoprotection in pulmonary PCM. Indeed, the absence of TLR2 signaling induces excessive pathology associated with increased Th17 differentiation an impaired Treg development (6). In contrast, in TLR4 deficiency the excessive proliferation of Treg cells and reduced Th17 differentiation led to more severe disease (7). Importantly, the absence of dectin-1 signaling induced a critical reduction in all IL-17 secreting cells, particularly IL-17⁺CD8⁺ T cells and a severe PCM (20). These findings, and those here reported clearly indicate that the equilibrium in the expansion of T cell subpopulations is fundamental to immunoprotection against PCM.

The findings here reported are in contrast with those of Ketelut-Carneiro et al. showing that an enhanced Th1 immunity controlled by increase production of IL-18 was responsible by the protective role of NLRP3 inflammasome in a murine model of PCM (40). These discrepancies, however, could be attributed to the route of infection used. Indeed, the study of Ketelut-Carneiro et al. used i.v. infected mice (40), whereas our studies were done with i.t. infected animals. In this aspect, our previous studies have clearly shown the influence of the route of infection in the severity of the disease and immune response of P. brasiliensis infected mice (41). We verified that the s.c. route induces a self-healing infection in several mouse strains allied with elevated delayed hypersensitivity reactions. Interestingly, the previous s.c. infection of B10.A mice led to immunoprotection or disease exacerbation depending on the route of fungal challenge. Immunoprotection was achieved after intraperitoneal challenge and was associated with persistent cell-mediated immunity and a mixed type-1/type-2 immunity. Exacerbated disease was found after intravenous challenge and was linked with anergy of cellular immunity and prevalent type-2 immune response (41). In contrast to the prevalent Th1/Th17 immunity induced by NLRP3 inflammasome activation in WT mice, Nlrp3^−/−, Casp1/11^−/−, and Asc^−/− mice showed increase expansion and migration of Treg cells to the site of infection that possibly have contributed to the deficient control of fungal loads and non-protective inflammatory reactions. In agreement, the survival studies showed increased mortality rates among Nlrp3^−/−, Casp1/11^−/−, Asc^−/−, and P2x7r^−/− mice accompanying increased fungal burdens and areas of lung lesions. In conclusion, the more efficient immunity mediated by Th1 and Th17 cells associated with NLRP3 inflammasome activation is highly protective to pulmonary PCM and this intracellular receptor appears to be significantly involved in the control of signaling pathways that led to the integration of innate and adaptive host immune responses to P. brasiliensis infection.

Animal 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 suffering, and all animal procedures were approved by the Ethics Committee on Animal Experiments of the Institute of Biomedical Sciences of University of São Paulo (Proc.76/04/CEUA).

Conceived and designed experiments: VC and CF. Contributed with reagent: DZ. Performed the experiments: CF, TC, EA, FL, and NG. Analyzed the data: CF, VC, FL, EA, and DZ. Wrote the paper: CF and VC.

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.