Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2-Biased Immunity

et al. (2009) Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2- Biased Immunity. PLoS ONE 4(2): e4519. doi:10.1371/journal.pone.0004519 Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2-Biased Immunity Luciana Pereira Ruas 0 Emerson Soares Bernardes 0 Marise Lopes Fermino 0 Leandro Licursi de Oliveira 0 Daniel K. Hsu 0 Fu-Tong Liu 0 Roger Chammas 0 Maria-Cristina Roque-Barreira 0 Marta Feldmesser, Albert Einstein College of Medicine, United States of America 0 1 Departamento de Biologia Celular e Molecular e Bioagentes Patogenicos, Faculdade de Medicina de Ribeira o Preto, Universidade de Sa o Paulo, Ribeira o Preto, Brasil, 2 Departamento de Biologia Geral, Universidade Federal de Vic osa, Vic osa, Brasil, 3 Department of Dermatology, School of Medicine, University of California Davis, Sacramento, California, United States of America, 4 Laborato rio de Oncologia Experimental, Faculdade de Medicina, Universidade de Sa o Paulo , Sa o Paulo , Brasil There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal32/2) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal32/2 macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2polarized type of response. - Funding: This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Pesquisa Cientfica e Tecnologica (CNPq). L.P.R. is a PhD student with a scholarship from FAPESP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Paracoccidioides brasiliensis, a thermally dimorphic fungus, is the etiological agent of human paracoccidioidomycosis, one of the most frequent systemic mycosis in Central and South America [1,2]. The main host defense against P. brasiliensis is the cellmediated immune response [35]. Macrophage activation and granuloma formation characterize the inflammatory response induced by the fungus, and protect the host against parasite dissemination [6]. Macrophages and lymphocytes are generally considered the major effector cells controlling the disease in vivo [7,8] through TNF-a and IFN-c production [9,10]. In the past few years, the immunity to several infections has been demonstrated to be influenced by galectin-3, the most studied member of the galectin family. These investigations were performed by comparing the course of experimental infections in mice that were genetically deficient, or not, in galectin-3 [11 13]. The studies of infection with the intracellular bacteria Rhodococcus equi have demonstrated that galectin-3 may regulate the innate immune response by diminishing IL-1b production by macrophages [13]. On the other hand, during Toxoplasma gondii and Schistosoma mansonii infection, the absence of galectin-3 drives the development of a heightened Th1-type immune response, suggesting that the lectin exerts a profound effect on the development of the adaptive immune response against pathogens [11,12]. Together with results obtained from a murine model of asthma [14], the studies provide consistent evidences that gal32/2 mice develop a lower Th2 response but a higher Th1 response compared with gal3+/+ mice. In consequence, galectin-3 emerges as a fine regulator of Th1/Th2 balance. Since protection against P. brasiliensis infection depends on adequate inflammation, cellular immune response and cytokine production, and because galectin-3 is important in the regulation of the Th1/Th2 balance, the aim of this study was to analyze the immunological aspects of P. brasiliensis infection in galectin-3deficient (gal32/2) mice. We demonstrate that gal32/2 mice present increased susceptibility to P. brasiliensis infection, associated with the inability to mount an adequate inflammatory response, the impairment of DTH response, high serum levels of specific antibodies, and the development of a Th2-polarized immune response. Such a picture is possibly due to the fact that, following contact with P. brasiliensis antigens, macrophages from gal32/2 mice have higher TLR2 transcript levels and produce higher levels of the deactivating cytokine IL-10. Our results indicate that galectin-3 exerts a protective and immunoregulatory role in the host response to P. brasiliensis infection. Materials and Methods Experimental Animals Galectin-3-deficient mice (gal32/2) were generated as previously described and backcrossed to C57BL/6 mice for nine generations [15]. Age-matched wild-type mice C57BL/6 (gal3+/+) were used as controls in all the experiments. Mice were housed under approved conditions in the Animal Research Facilities of Faculdade de Medicina de Ribeirao Preto - USP. All of the animals used in the experiments were male, at 6 to 8 week-old. The Ethics Committee on Animal Research of the University of Sao Paulo approved all the procedures performed in the studies described here. Parasite, Mice Infection, and Mortality The P. brasiliensis isolate 18 (Pb18), which is highly virulent, was used throughout this study. Yeasts cells were maintained by weekly subcultivation in a semisolid culture medium [16] at 36uC and were used on day 7 of culturing. They were harvested and washed three times with phosphate-buffered saline (PBS), pH 7.2. Cell viability was determined as previously described [17]. Mice were infected intraperitoneally (i.p.) with 56106 viable yeast cells in 500 mL PBS and control animals received PBS only. Animal death was registered daily until 120 days after infection. For the intratracheal (i.t.) infection assay, mice were anesthetized by intraperitoneal injection with 2.5% of tribromoethanol and infected with 56106 yeast cells suspended in PBS. Antigen Preparation P. brasiliensis-antigen (PbAg) was obtained as previously described [16]. Briefly, Pb18 yeast cells were harvested, washed with PBS, disrupted by ultrasonic treatment (5 cycles of 30 seconds), and centrifuged for 10 min at 20006g. The supernatants were collected and the protein conc (...truncated)