Mechanisms of immunosuppression induced by cryptococcal capsular polysaccharide

CHIAPELLO L S

Foz do Iguazú

Congreso; 10th International Conference on Cryptococcus and Cryptococcosis.; 2017

ISHAM

The prominent polysaccharide capsule from Cryptococcus spp is an essential virulence factor that has multiple effects on host immunity. This acidic and viscous polysaccharide, which is comprised mainly of glucuronoxylomannan (GXM), is continuously released by encapsulated yeasts during their replication. High levels of GXM in the body fluids of patients have a direct relationship on the severity of cryptococcosis. During different infectious diseases, as a crucial event of innate immunity, microbial molecules interactions with host phagocytic cells trigger inducible nitric oxide synthase (iNOS or NOS2) expression and nitric oxide (NO) release. This NO may have a dual role during infections, with both anti-microbial effector functions and immunosuppressive properties mediated by the apoptosis of inflammatory cells. Apoptosis is the most common and well-studied type of programmed cell death that helps to reduce the inflammatory stress, but it can also be manipulated by pathogens as a survival strategy. Two pro-apoptotic signal transduction pathways have been described: an extrinsic/receptor-linked apoptotic pathway and an intrinsic/mitochondria-mediated pathway, both activate downstream effector caspase-3. However, mitochondrial damage can also lead to a caspase-independent death through activation of death effectors, such as the apoptosis-inducing factor (AIF) and endonuclease G.In our laboratory, we have developed experimental models of cryptococcosis in rats, which have similarities with human infections showing tisular granulomas associated with inducible iNOS expression and NO production by macrophages. In this presentation, I will discuss experimental data describing mechanisms involved in macrophage apoptosis promoted by cryptococcal capsular polysaccharide (GXM) through NO generation. In vitro experiments using peritoneal macrophage cultured with purified GXM from C. neoformans demonstrated that GXM promotes iNOS expression with NO production in a dose-dependent manner. Likewise, capsular polysaccharide from C. gattii also elicited macrophage NO production, but to a lesser extent than GXM from C. neoformans. In vivo, intraperitoneal GXM administration in Wistar rats showed that most peritoneal cells were able to internalize polysaccharide and these cells produced elevated NO release. Furthermore, immunohistochemistry of lung tissue from GXM-treated rats also demonstrated that GXM induces iNOS expression in vivo.Experiments using blocking antibodies and flow cytometric or immunofluorescence analysis further revealed that NO production involves GXM binding by CD18 on macrophages. In contrast, GXM did not stimulate NO production via mannose or β-glucan receptors. Capsular polysaccharide also binds macrophages through Fcγ receptor II (FcRII), but this interaction provided inhibitory signaling for NO production. Engagement of CD18 on cellular surface induces activation of serine/threonine kinases, protein kinases C (PKC). According to this, our experiments show that PKC activation, but not tyrosine kinases (TK) or mitogen-activated protein kinases (MAPK), are involved in GXM-induced iNOS and NO production by rat macrophages.On the other hand, our data depicted that GXM-loaded macrophages undergo apoptosis after culture, and this phenomenon was fully prevented by the iNOS inhibitor, aminoguanidine (AG). Furthermore, the pathways involved in GXM-mediated NO production: CD18, FCγRII and PKC, also modulated in the same way the macrophage apoptosis. In agreement to the in vitro observations, polysaccharide-loaded tisular macrophages also presented apoptotic nuclei during disseminated cryptococcosis in rats. Strinkly, GXM-induced macrophage apoptosis was not prevented by the presence of a pan-caspase inhibitor (Z-VAD-fmk) in cultures, suggesting that apoptosis was independent of caspases activation. In contrast, GXM increased the mitochondrial membrane permeability and cytosolic AIF expression, showing that GXM triggers a caspase-independent cell death by promoting depolarization of mitochondria membrane potential.Taken together, these findings describe novel immunomodulatory mechanisms by the main cryptococcal capsular polysaccharide, which could contribute to limit inflammation during the infection