Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans

Linda M Sircy,James B Hayes,Lauren E Heusinkveld,Erin E Mcclelland,Wandi Ding,Rachel N Leander,David E Nelson

doi:10.1074/jbc.m116.738187

Linda M Sircy, James B Hayes + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m116.738187

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Abstract

Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection.

Highlights

LPS is detected by Toll-like receptor-4 (TLR4) at the macrophage cell surface, stimulating a cascade of intracellular signaling events that culminate in the phosphorylation of NF-␬B1⁄7I␬B complexes by I␬B kinase (IKK)
These p65-containing transcription factors regulate the expression of genes associated with the immune response, including pro-inflammatory cytokines like TNF-␣ [17,18,19] and inducible nitric oxide synthase [20, 21], the latter of which is associated with M1 or “classical” activation of macrophages
GXM Attenuates LPS-induced Nuclear Translocation of NF-␬B—The anti-inflammatory capacity of Cryptococcus neoformans (Cn) capsular polysaccharide was first illustrated by its ability to block LPS-induced TNF␣ production by monocyte-derived macrophages [10]

Summary

Introduction

We present detailed single-cell analysis of NF-␬B signaling dynamics in cultured macrophages and show that purified extracellular GXM and intracellular GXMpositive Cn have contrasting effects on the behavior of the pathway in the context of LPS-induced activation. The duration of LPS-induced p65 nuclear accumulation is significantly increased in macrophages containing relatively low numbers of ingested GXM-expressing Cn but not GXM-null Cn. A combination of computational modeling and experimental methods indicated that this extended response was caused by translational interference in cells infected with GXM-expressing Cn, which acts to decrease the strength of negative feedback.

Results

Conclusion