This study showed, for the first time, that experimental pulmonary mucormycosis in three widely used mouse strains resulted in fungal dissemination in the absence of immunosuppression. We also identified the partial role of IFN-γ in the efficient elimination of R. oryzae during pulmonary infection.
In mucormycosis, experimental models are widely used for the evaluation of antifungal therapy and, in these studies, immunosuppression was used to induce dissemination in murine models [27–30]. Nevertheless, the effect of immunosuppressive drugs restricts the evaluation of the immunological mechanisms involved in fungal resistance. Recent studies exploring host-parasite relationships in mucormycosis have used immunocompetent models, such as disseminated mucormycosis models, using BALB/c, C57BL/6, and Swiss mice [23]; mucormycosis in a skin model using BALB/c mice [31]; and a pulmonary mucormycosis model using C57BL/6 mice [12]. Although pulmonary mucormycosis in immunocompetent C57BL/6 mice has been reported once [12], dissemination was not investigated, and the study focused on the innate immune response against R. oryzae, while the adaptive immune response in pulmonary infection is still unknown. Considering this, our study was the first to characterize a model of a pulmonary infection of R. oryzae that induces angioinvasion and dissemination in immunocompetent mice. Angioinvasion has been associated with the ability of the pathogen to disseminate hematogenously from the primary site of infection to other organs [32]. In humans, pulmonary mucormycosis occurs due to the inhalation of fungal spores into the bronchioles and alveoli, which typically results in the rapid progression of pneumonia or endobronchial disease, hemoptysis with vascular invasion causing necrosis, and invading tissue to spread locally or disseminate systemically [4]. Our R. oryzae-infected mice were characterized via the recovery of viable fungal growth, hemorrhage, vessel alteration, and inflammatory cell infiltration in the lungs as well as in the brain, kidney, liver, and spleen after seven days of infection, which showed an angioinvasive infection, indicating that our models are similar to the human pattern of infection [33].
In a murine model of mucormycosis, angioinvasion was frequently observed following the establishment of pulmonary mucormycosis after intranasal challenge with Mucorales of corticosteroid-treated mice [34]. In the study above, it was observed that mice infected with up to 106 spores of Lichtheimia corymbifera were susceptible to infection until 10 days p.i. [34]. In addition to the angioinvasion observed in our immunocompetent models of pulmonary mucormycosis, no fatality was observed in the mice after administration with an inoculum of 2 × 106 spores of R. oryzae. The lack of immunosuppression may explain the absence of susceptibility in our model; in addition, an intranasal inoculum of 2 ×106 spores of R. oryzae in BALB/c, C57BL/6, and Swiss immunocompetent mice did not lead to the recovery of viable fungal or histological tissue alterations (data not shown). These data suggest that for the establishment of R. oryzae pulmonary infection in immunocompetent mice, intratracheal inoculation is more efficient than intranasal inoculation. Similar to our study, in C57BL/6 mice intratracheally infected with R. oryzae established as an immunocompetent model of pulmonary mucormycosis, considerable tissue edema, neutrophil infiltration, and viable R. oryzae in the lungs were observed five days p.i. [12]. In addition to the study above, we compared three different strains of mice and observed that differences in the capacity for fungal clearance was related to the genetic background.
It is well-established that inbred mouse strains have varying dynamics of infection. The differences in the type of immune response developed and the resulting severity of disease allows the applicability of mice with different genetic bases as effective tools for studying the dynamics of infections [35–38]. In this study, we observed that under pulmonary R. oryzae infection, C57BL/6 and BALB/c mice were more resistant, while Swiss mice were less resistant . Inbred lines of highly susceptible (A/J, A/Sn, DBA / 2) and highly resistant (BALB/c, C57BL/6J) mice have been described to be infected with Aspergillus fumigatus [35], Candida albicans [36], Cryptococcus neoformans [37], Paracoccidoides brasiliensis [38], and Rhizopus oryzae [23]. Some genetic targets such as C5-deficiency (Hc0 allele, hemolytic complement) and C5-independent pathways (such as the loci Carg3 and Carg4) have been identified to modulate the host’s initial response and cause an ineffective inflammatory response against Candida albicans in some inbred mouse strains [36]. In Swiss mice, C5 deficiency is the cause of the high susceptibility to fungal infections [39–41]. In addition, in experimental models of disseminated mucormycosis, Swiss mice were the less resistant strain, compared with the other two inbred strains, showing an anti-inflammatory response during intravenous R. oryzae infection.
Considering the lack of knowledge in the adaptive immune response in the context of pulmonary mucormycosis, we used the differences in response of three strains of mice against R. oryzae to identify relevant resistance mechanisms, and we observed that greater resistance against pulmonary infection was associated with the Th-1 response mediated by higher IFN-γ and IL-2 levels, as observed in the more resistant BALB/c and C57BL/6 strains, while less resistance was associated with an absolute adaptive immune response observed in the Swiss strain. It is known that IFN-γ-producing Th-1 cells confer protective immunity against fungi, whereas Th-2 responses increase susceptibility to fungal infections [42]. In addition, Th-17 cells have also been implicated in mucosal immunity against fungi [42]. IL-2 induces T cell clonal expansion and is a marker of adaptive immune response development [43].
In patients with invasive mucormycosis, Mucorales-specific T-cells showed high levels of IL-4, IFN-γ, IL-10, and IL-17 [17]. In the same study, it was observed that all patients showed a high expression of IL-10 at the time of diagnosis and close to the time of their death. Interestingly, the absence of Mucorales-specific T cells producing IL-10 was found in one patient at the time of complete resolution [17]. In contrast to our results, in an immunocompetent BALB/c and C57BL/6 model of disseminated mucormycosis, resistance against R. oryzae was associated with higher levels of IL-17.
It has been noticed that different routes of inoculation may, at times, give rise to different host responses, which could be another explanation for the observed differences [44]. In this context, during intravenous inoculation of C. albicans, it was observed that Th-1 and Th-17 responses mediated protective adaptive immunity [45], while in experimental C. neoformans intratracheal infection, a type of Th-1 response mediated by IFN-γ was mainly developed [46]. In aspergillosis, major T-helper cell lineages, Th-1, Th-2, and Th-17, have been demonstrated to play important roles in patients and animal models depending on the type of infection [47–51]. For invasive aspergillosis, the Th-17 type of response was essential for fungal control [52,53], but in pulmonary infections caused by A. fumigatus, the Th-1 response profile is more effective [54]. In pulmonary infections, the Th-17 type of response seems to be pathogenic to the lung due to an uncontrolled or prolonged inflammatory response that caused persistent inflammation (50–52). For mucormycosis, the pattern of adaptive immune response seems to be similar to that observed in aspergillosis. In disseminated mucormycosis, IL-17 signaling is crucial to control the infection and, as observed here in R. oryzae pulmonary infection, the Th-1 response was developed as evidenced by higher levels of IFN-γ (Fig. 4).
It has been shown that the Th-1 response is crucial for fungal clearance since it potentiaties the fungicidal activity of innate immune cells through IFN-γ release [54–57]. To elucidate the possible involvement of IFN-γ signaling in the protection against R. oryzae pulmonary infection, we induced pulmonary mucormycosis in IFN- γ-/- mice and found that they were incapable of eliminating R. oryzae in the lungs until 60 days of infection. These results indicate that, as observed in other fungal infections [45,46,54], the Th-1 response through IFN-γ signaling is partially important for fungal clearance in mucormycosis [17,27,58,59].
In contrast to our study, in an experimental model of invasive aspergillosis, the absence of IFN-γ resulted in an increase in susceptibility to infection [60], while our IFN-γ-/- mice did not succumb to R. oryzae infection. Two hypotheses can explain these results. First, there was an absence of an immunosuppressive drug in our model to induce mucormycosis, while in the study above, a neutropenic model of mice induced to have invasive aspergillosis was used. Second, other immune signaling may be involved in the prevention of death in IFN-γ-/- mice infected with R. oryzae [23]. For exemplo, IL-2 has already been demonstrated to be capable of activating human NK cells to damage R. oryzae hyphae, but it did not affect resting conidia in a previous in vitro study (58).
Although responses other than Th-1 are capable of preventing death in infected IFN-γ-/- mice, they were not as efficient as the response mediated by IFN-γ, since the IFN-γ-/- mice were unable to eliminate live R. oryzae conidia in the lungs. The absence of additional experiments to explore the mechanisms of survival of infected IFN-γ-/- mice was the main limitation of the present study, and we suggest that more studies should be done to clarify the survival mechanisms observed here.
In summary, we introduced three immunocompetent models of pulmonary R. oryzae infection resulting in angioinvasive mucormycosis, which permits new studies to explore infection dynamics in this context. Using the differences in capacity of murine models in eliminationg R. oryzae, we gathered new insights into the immune response during pulmonary mucormycosis, and, in addition, we recognized that IFN-γ contributes to a more effiective mechanism of eliminating R. oryzae.