Paracoccin Induces M1 Polarization of Macrophages via Interaction with TLR4

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.

Paracoccin overexpression in Paracoccidioides brasiliensis reveals the influence of chitin hydrolysis on fungal virulence and host immune response

“…* p < 0.1. (B) Western blot analysis of a recombinant PCN protein, expressed in Pichia pastoris [48] and the PCN content in the supernatant of yeast cells that were co-cultured or not with THP-1 macrophages. Reactions were developed by IgY anti-PCN conjugated to HRPO.…”

“…The response is triggered by the PCN lectin domain interaction with N-glycans of TLR2 and TLR4 [45, 47]. Both receptors are expressed on the surface of macrophages, which undergo M1 polarization followed by high production of pro-inflammatory mediators, such as the Th1 polarizing cytokine IL-12 [48]. We demonstrated that the developed Th1 immune response accounts for the host resistance to the P. brasiliensis infection conferred by the PCN administration [45].…”

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection

“…Thus, the original use of lectins in vaccination protocols has been expanded to other applications. Hence, besides its use as therapeutic agents in infectious and parasitic diseases, lectins have been applied as new potential antineoplastic agents targeting apoptosis, autophagy, and anti-angiogenesis in preclinical or clinical trials for cancer therapeutics, and in neurodegenerative disorders, as well (Alegre-Maller et al, 2014; Bogoeva et al, 2014; Aminou et al, 2016; Barbosa-Lorenzi et al, 2016; Dar et al, 2016; Freitas et al, 2016; Ricci-Azevedo et al, 2016). …”

Paracoccina: uma quitinase importante para a patobiologia e virulência de Paracoccidioides brasiliensis

“…Esses resultados contribuíram importantemente para elucidar os possíveis mecanismos responsáveis pelo efeito imunomodulador da PCN. A alta produção de IL-12 pelos macrófagos M1 e a decorrente diferenciação de células Th1, produtoras de IFNɣ, são responsáveis pela geração de resposta protetora contra P. brasiliensis, quando PCN é administrada na forma profilática ou terapêutica (Freitas, Oliveira et al 2016).…”

“…Estudos subsequentes utilizaram uma forma recombinante, denominada p-rPCN, que induziu macrófagos peritoneais murinos a produzirem mediadores pró-inflamatórios (IL-12p40, TNF-α, IL-6 e NO) e assumirem um perfil M1 de polarização. Tais efeitos de p-rPCN foram mediados por ligação da lectina a N-glicanos ligados ao exodomínio do receptor TLR-4, mas não de TLR2 (publicação inserida no capítulo I) (Freitas, Oliveira et al 2016). Além disso, p-rPCN induziu neutrófilos humanos a formarem NETs e liberarem IL-8 (publicação inserida no capítulo II) (Ricci-Azevedo, Goncales et al 2017).…”