Toll-Like Receptor 4 Mediates Intracellular Signaling Without TNF-α Release in Response to Cryptococcus neoformans Polysaccharide Capsule

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Abstract
Toll-like receptors (TLR) 2 and 4 are cell surface receptors that in association with CD14 enable phagocytic inflammatory responses to a variety of microbial products. Activation via these receptors triggers signaling cascades, resulting in nuclear translocation of NF-κB and a proinflammatory response including TNF-α production. We investigated whether TLRs participate in the host response to Cryptococcus neoformans glucuronoxylomannan (GXM), the major capsular polysaccharide of this fungus. Chinese hamster ovary fibroblasts transfected with human TLR2, TLR4, and/or CD14 bound fluorescently labeled GXM. The transfected Chinese hamster ovary cells were challenged with GXM, and activation of an NF-κB-dependent reporter construct was evaluated. Activation was observed in cells transfected with both CD14 and TLR4. GXM also stimulated nuclear NF-κB translocation in PBMC and RAW 264.7 cells. However, stimulation of these cells with GXM resulted in neither TNF-α secretion nor activation of the extracellular signal-regulated kinase 1/2, p38, and stress-activated protein kinase/c-Jun N-terminal kinase mitogen-activated protein kinase pathways. These findings suggest that TLRs, in conjunction with CD14, function as pattern recognition receptors for GXM. Furthermore, whereas GXM stimulates cells to translocate NF-κB to the nucleus, it does not induce activation of mitogen-activated protein kinase pathways or release of TNF-α. Taken together, these observations suggest a novel scenario whereby GXM stimulates cells via CD14 and TLR4, resulting in an incomplete activation of pathways necessary for TNF-α production.