Date Published: March 12, 2008
Publisher: Public Library of Science
Author(s): Leandro Licursi de Oliveira, Kely Cristine Coltri, Cristina Ribeiro Barros Cardoso, Maria-Cristina Roque-Barreira, Ademilson Panunto-Castelo, Edgar Carvalho
Abstract: Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1–stimulating adjuvant in P. brasiliensis–infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund’s adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund’s adjuvant (IFA – control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.
Partial Text: Paracoccidioides brasiliensis is a thermally dimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), the most prevalent human systemic mycosis in Latin America, being endemic in Brazil, Argentina, Venezuela and Colombia. This infection is acquired by inhalation of airborne propagules found in nature, which reach the lungs and are converted to the yeast form ,. The yeasts can either be eliminated by immune-competent cells or disseminated into tissues through lymphatic or hematogenous routes. PCM is characterized by granulomatous inflammation, intense immunological involvement with suppression of cellular immunity and high levels of non-protective antibodies in serum . The disease may present a broad spectrum of clinical and pathological manifestations ranging from asymptomatic pulmonary infection to severe and disseminated forms ,. The chronic progressive form of the disease (CF) is the most common clinical presentation and predominantly affects adult males, with frequent pulmonary, mucosal, cutaneous and adrenal involvement. Although the outcome of the infection can be due to several factors, it is especially dependent on the protective capacity of the host immune system. The cell-mediated immune response represents the main mechanism of defense in PCM . Conversely, it has been reported that a high level of humoral immune response is associated with increased disease dissemination .
The depression of cell-mediated immune responses has been associated with severe PCM in humans and in the experimental host ,,,. However, the propensity for persistence of the fungus in infected tissues appears to be consequence of cell-mediated immune dysregulation with suppression of Th1 and overexpression of Th2 responses –.To evaluate whether therapeutic immunostimulation is able to interfere in experimental murine PCM and restore the host immune response, we selected immunomodulators for therapy strategy based on the induction of Th1 or Th2 immune response. Since CFA supports a Th1 status, while incomplete Freund’s adjuvant (IFA) promotes a Th2 status , BALB/c mice were divided into two groups and treated with CFA or IFA on day 20 after infection with P. brasiliensis. The progression of P. brasiliensis infection was determined by lung histopathology and analysis of colony-forming unity (CFU), parameters that are considered trustworthy to discriminate susceptible and resistant mice to systemic fungal infection ,,,. At 20 days of infection the mice presented 5.8×104 CFU/g of lung tissue (Figure 1A, dashed line) and compact granulomas (data not shown), for this reason, this time was chosen for the treatment regimens. On day 30 after treatment (50 days postinfection), the lungs from IFA-treated mice presented multiple sites of focal and confluent epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells (Figure 2A, C and E). Morphometric analysis of the lungs from IFA-treated mice revealed a number of granulomas of 41±5.2, with a relative area of 40.7±6.2%. These granulomas presented 12.2±1.8% of yeast cells and 6±0.6% collagen (data not shown). In contrast, in the P. brasiliensis-infected mice treated with CFA, no granulomas or yeast cells were seen in the pulmonary sections examined and a well-preserved alveolar architecture was observed on day 30 after treatment (Figure 2B, D and F). Most importantly, the treatment with CFA induced a 2.4 log reduction in the fungal burden when compared to the IFA-treated mice, corresponding to 99% less CFU (Figure 1A). The CFU data are in agreement with the histopathology analyses, pointing out that therapeutic immunostimulation led to an increased clearance of fungal burden from lungs.