Vaccine approaches to infectious diseases are widely applied and besides the number of diseases that can be prevented by vaccines is growing, little is known about vaccination strategies against fungal infections. In the last years, several studies have been focusing in the P. brasiliensis
experimental infection after immunization protocols. The vaccination with the recombinant protein PbHSP60 conferred partial protection in mice against a pulmonary PCM and allowed the identification of the cytokines and T CD4+
cells responsible for HSP60 protective properties 
. In addition, low virulent fungal cells (via extensive DNA fragmentation) have been used to induce immunoprotection during experimental PCM. The prior contact of BALB/c mice with radioattenuated yeast cells of P. brasiliensis
, which lost their virulence, promoted a long lasting protection associated with Th1 responses against highly infective yeast forms of P. brasiliensis 
On the contrary, studies have demonstrated that soluble antigens from Pb lead to immunossupression during experimental PCM. Mice inoculation with the antigens released from the surface of P. brasiliensis
(CFA), which can be recognized by receptors on the membrane of macrophages 
, repressed the cell-mediated immune responses against Pb cells 
. Considering that the immunosuppressive mechanisms and the identification of the responsible soluble immune factors remained unknown, in the present study we focused in the events involved in the immunoregulation triggered after a prior contact with CFA during a murine model of PCM.
We first evaluated the involvement of CFA in the modulation of granuloma formation, cytokine production, and control of fungal growth in Pb-infected mice. We showed that inoculation of CFA prior to infection resulted in severe lung pathology. The increased pulmonary CFU recovered from CFA-treated mice was accompanied by intense inflammatory infiltrate and severe granulomatous lesions. Moreover, CFA-treated mice showed granulomas containing central necrotic areas with diffuse cell distribution and a massive number of yeasts, which could be contributing to the intensity and the persistence of the inflammatory response in these mice.
The Th1/Th2 paradigm provides a basis for understanding T cell responses and is applicable to explain the resistant/susceptible pattern observed during experimental paracoccidioidomycosis. Cytokine studies, mainly in a pulmonary model of infection, have confirmed that Th1-biased immune response characterized by IFN-γ, TNF-α and IL-12 production, are linked with asymptomatic and mild forms of PCM 
. In contrast, a Th2 pattern has been associated with severe disease. Patients presenting the disseminated infection produce higher levels of type 2 cytokines, like IL-4, IL-5 and IL-10 
In the present study, we described that the injection of CFA prior to infection induced a Th2 profile, increasing IL-4 levels in the lung, liver and spleen homogenates, and decreased the IFN-γ production when compared with only Pb-infected mice. Using IL-4−/−
mice and their IL-4-sufficient counterparts, we observed that the absence of IL-4 resulted in increased inflammatory reaction in the lungs as showed before 
. These data corroborates with elevated numbers of PMN cells in the early phase of infection (data not shown) and lower CFU counts in the pulmonary tissue. In agreement, previous studies demonstrated that the severity of PCM is mild in IL-4−/−
mice compared with WT group, with increased IFN-γ release in lung homogenates and enhanced fungicidal activity of alveolar phagocytes 
When we pre-treated mice with anti-IL-4 monoclonal antibody one week after the last CFA-inoculation, we observed that the in vivo IL-4-depletion impaired the suppressive effects of CFA. It implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Moreover, IL-4 favors the development of a Th1 rather than Th2 immune response. Because M2-macrophage activation is mediated by IL-4 and/or IL-13 (Th2-type cytokines), these macrophages are normally associated with immune responses that possess a Th2-skewed cytokine environment, as observed in parasite infections and allergic inflammation 
. Future studies should explore the role of these cells during P. brasiliensis
Although IL-10 have an inhibitory effect during PCM by preventing fungal killing by IFN-γ or TNF-α-activated macrophages via a reduction in NO and H2
, in the present study we did not find a crucial role for this cytokine during the immunosuppression mediated by CFA during the late phase of the infection. Using IL-10 deficient mice, we observed the absence of this cytokine resulted in a similar or increased lung and liver fungal growth at day 30 post-infection when compared with WT controls also exposed to CFA. At day 15, the absence of IL-10 resulted in decreased fungal growth in all tissues analyzed. No differences were found in only infected WT and IL-10−/−
mice. Since suppressive effects of IL-10 during paracoccidioidomycosis is well established 
, we propose that the presence of IL-10 during the first 15 days is essential to establish a initial negative regulation of immune cells via down-regulation of APC function or via Treg induction. However, the fungal persistence and excessive pathology induced by CFA at day 30 seems to be independent of IL-10, and it is IL-4 that is the pivotal cytokine driving the long-term immunossupression in CFA-treated mice. Also, the absence of IL-4 regulated the production of IL-10 during Pb infection 
. Nevertheless, we cannot exclude the possibility that IL-10-related cytokines such as IL-22, IL-26, among others are compensating for the lack of IL-10 in IL-10-deficient mice 
The gp43, the major 43-kDa antigenic glycoprotein of P. brasiliensis
, and the one single short peptide P10 from gp43 are able to promote increased protective immunity in mice when inoculated in the presence of adjuvant 
. When we fractionated our CFA preparation by SDS-PAGE, gp43 was not observed. However, it is possible that it could be present in low concentrations and, in this case, was not able to mediate a protective immunity. In addition, our CFA inoculation protocol was performed in the absence of adjuvant.
In order to determine which antigenic fraction of CFA, proteins, glycoproteins, or glycosphingolipids was responsible for the immunossupression, mice were inoculated with intact, denaturated, deglycosylated or delipidated CFA. Only the denaturated CFA treatment resulted in a significant decrease of viable yeast cells recovered from infected mice, suggesting that the immunossupression is associated with to a conformational protein presented in CFA, and not require glycosylation of the presence of glycosphingolipids and glycoproteins, like gp43 
or paracoccin 
In summary, CFA amplify IL-4 production, which consequently impairs the Th1 response. Diminished control of fungal growth after CFA inoculation was dependent on IL-4 production. Moreover, the injection of CFA did not affect the course of infection in IL-4-deficient mice, suggesting the exacerbation of PCM after inoculation of CFA was mediated by IL-4 and not IL-10. In conclusion, our results suggest that CFA has an important role in the modulation of Th2-cytokines for the installation of primary infection in the hosts. Therefore, these data open new perspectives regarding anti-Th2 strategies, which might be considered in the treatment of PCM or other fungal infections.