Abstract
The fungal human pathogen Paracoccidioides brasiliensis contains paracoccin (PCN), a multi-domain protein that has lectin and N-acetyl-glucosaminidase activities, which account for its effects on the growth and morphogenesis of the fungus and on the activation of host macrophages through its interaction with TLR N-glycans. With the purpose of detailing the knowledge on the effects of PCN on macrophages, we used recombinant PCN expressed in Pichia pastoris (p-rPCN) to stimulate isolated murine peritoneal macrophages. The activation of these cells manifested through the release of high levels of inflammatory mediators, such as nitric oxide, TNF-α, IL-12p40, and IL-6. Furthermore, peritoneal macrophages stimulated with p-rPCN increased the relative expression of STAT1, SOCS3, and iNOS2 mRNA (M1 polarization markers). However, the expression of Arginase-1, Ym-1, and FIZZ1 (M2 polarization markers) remained at basal levels. Interestingly, the observed M1 macrophages’ polarization triggered by p-rPCN was abolished in cells obtained from knockout Toll-like receptor-4 mice. In this case, the p-rPCN-induced production of pro-inflammatory mediators was blocked too. These results demonstrate that the classical activation of macrophages induced by paracoccin depends on TLR4. Taken together, the results of our study indicate that paracoccin acts as a TLR agonist able to modulate immunity and exerts biological activities that favor its applicability as an immunotherapeutic agent to combat systemic fungal infections.
Highlights
Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermally dimorphic fungi and the causal agents of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America
Paracoccin purified from Paracoccidioides brasiliensis has both lectin and enzymatic properties, which are mimicked by a recombinant form of the protein, expressed in Escherichia coli (b-rPCN) (Alegre et al, 2014)
Given that recombinant paracoccin expressed in E. coli interacts with the N-glycans of TLR2 and TLR4 on antigen-presenting cells and promotes IL-12 production (Alegre-Maller et al, 2014), we examined the relevance of TLR2 and TLR4 in the production of inflammatory mediators induced by p-rPCN
Summary
Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermally dimorphic fungi and the causal agents of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. The PCM course depends on factors inherent to the fungus, such as its virulence and antigenic composition, as well as on environmental conditions and the host’s immune state (Kurokawa et al, 2005). In this scenario, macrophages are essential in establishing the first barrier to the invading pathogens and in guiding the ensuing development of adaptive immunity (Hussell and Bell, 2014). The M1 and M2 subsets are discriminated by the production of nitric oxide (NO) and arginase activity, respectively, as well as by the expression of particular genes, such as iNOS2, STAT1, and SOCS3 for M1, and Arginase, FIZZ1, YM1, STAT3, and SOCS1 for M2 (Lawrence and Natoli, 2011)
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