Particles from the Echinococcus granulosus Laminated Layer Inhibit CD40 Upregulation in Dendritic Cells by Interfering with Akt Activation

The larval stage of the cestode Echinococcus granulosus causes cystic echinococcosis in humans and livestock. This larva is protected by the millimeter-thick, mucin-based laminated layer (LL), from which materials have to be shed to allow parasite growth. We previously reported that dendritic cells (DCs) respond to microscopic pieces of the mucin gel of the LL (pLL) with unconventional maturation phenotypes, in the absence or presence of Toll-like receptor (TLR) agonists, including lipopolysaccharide (LPS).

FIG S1. Summary of the known participation of the NF-κB, MAP kinases, Ca 2+calcineurin-NFAT and PI3K-Akt-GSK3 pathwasy in the activation of GMCSF-BMDCs and other myeloid cell models in response to LPS.
For clarity, a general view of the pathways is presented in (a) and the PI3K-Akt-GSK3 pathway is detailed in (b). Only major points relevant to the present paper are shown. Red and blue lines represent activating and inhibitory effects respectively. Similarly, red and blue circles represent activating and inhibitory phosphorylation events respectively, and only phosphorylation events analyzed in the paper are represented.
LPS is mainly sensed by TLR4, in cooperation with CD14. TLR4 activation leads, through a complex series of steps, to the largely parallel activation (with certain early steps in common) of each of the MAP kinases (p38, JNK and ERK) and of the canonical NF-κB pathway. The central step in canonical NF-κB activation is the proteolytic degradation of IκB-α, which releases active NF-κB subunits for their nuclear translocation. Canonical NF-κB activation is required for up-regulation of the co-stimulatory molecules CD86 and CD40 and for expression of the cytokines IL-10 and IL-12. The p38 MAP kinase is needed for full IL-12 expression; contradictory data are available about its effects on CD86, CD40 and IL-10. JNK downregulates IL-12 expression whereas its effects on CD86, CD40 and IL-10 are not well established. ERK is dispensable for CD86 expression, whereas its effects on CD40 expression are not established; ERK is needed for IL-10 expression and dispensable for IL-12 expression.
Also in response to LPS, CD14 initiates, independently of TLR4 and dependent on Syk and phospholipase Cγ2 (PLCγ2), Ca 2+ influx leading to calcineurin activation and therefore the activating dephosphorylation of NFAT. Information on the role of the NFAT pathway on expression of CD86, CD40, IL-10 and IL-12 in LPS-stimulated DCs is lacking or contradictory.
TLR4 activation additionally leads to activation of PI3K, through complex steps that are omitted for simplicity. PI3K catalyzes the conversion of phosphatidylinositol(4,5) bisphosphate (PIP2) into phosphatidylinositol (3,4,5)trisphosphate (PIP3). PIP3 promotes the recruitment of PDK1 and Akt to the plasma membrane, facilitating the activation by phosphorylation of both these kinases. Active PDK1 and the mTORC2 complex doubly phosphorylate Akt, causing its full activation. The kinases TBK-1 and IKK-ε are also required for LPS-driven Akt phosphorylation in BMDCs, but the exact mechanism by which they act is unclear. Akt directly phosphorylates GSK3, inactivating this kinase. Akt additionally, and indirectly, activates the mTORC1 complex, which also leads to GSK3 phosphorylation through the p70S6K kinase. Active GSK3 has negative effects on CD86 and CD40 and IL-10 expression, and a positive effect on IL-12 expression. Thus, activation of the PI3K pathway enhances the expression of CD40, CD86 and IL-10, through a "double-negative" (inhibition-inhibition) mechanism; in contrast, activation of the pathway inhibits expression of IL-12.
The diagram is based on references (1-22); see "References in Supplementary Materials", at the end of this document.
FIG S3. The effects of pLL on GMCSF-BMDCs are not associated with gross alteration in the activation of the canonical NF-kB pathway. GMCSF-BMDCs were exposed to pLL, LPS, or both stimuli together, for the indicated times. Cell lysates were analyzed for total IκB-α. The Western blot shown is representative of 2 independent experiments. The graph shows the quotients of the IkB-a amounts (normalized over loading controls) for cells treated with pLL plus LPS over cells exposed to LPS only, for the 60-min timepoint; values are the mean and range of the 2 independent experiments. The cell-surface interaction and signaling envisaged is similar to membrane affinity-triggered signaling (MATS). MATS requires actin remodeling, PI3K and Syk, although the precise mechanistic relationships between these players have not been elucidated (23)(24)(25); the role of Syk in MATS would be independent from its role in LPS-stimulated, CD14-dependent signaling shown in Fig. S1a. We propose that in the context of a MATS-like interaction with pLL particles, receptor-activated PI3K class I activity is diminished, possibly as a consequence of decreased availability of its PIP2 substrate (24,26).
Diminished PI3K activity in the presence of pLL blunts Akt activation, and hence enhances GSK3 activity, which in turn blunts CD40 up-regulation. Parallel, unidentified signaling changes downstream of the MATS-like interaction, independent of the NF-κB, MAP kinases and Ca 2+calcineurin-NFAT pathways, are responsible for the effects of pLL on CD86 and IL-10; these unidentified changes would overwhelm the opposite effects on CD86 and IL-10 brought about by the enhanced GSK3 activity. The effects of pLL on IL-12 expression would be brought about by Akt-dependent and Akt-independent signaling changes interacting in complex ways that we do not have a model for, as indicated by the question mark in the diagram.
Although not represented in the diagram, the MATS-like interaction with pLL and the blunting of receptor-activated PI3K activity would also arise in the absence of LPS stimulation, explaining that pLL also inhibits Akt activation in response to IL-4 or GM-CSF (27). FIG S7 (related to Figs. 2 and 3). Effects of mTORC1 inhibitors on CD86, IL-10 and IL-12 expression in GMCSF-BMDCs, in the absence and presence of pLL. GMCSF-BMDCs were exposed to pLL, LPS, or both stimuli together, for 18 h, in the absence or presence of the mTORC1 inhibitors rapamycin or torin1. Cell surface expression of CD86, and IL-10 and IL-12p70 levels in supernatants were measured. All data shown correspond to means +/-SD of triplicate wells. Results shown are representative of 3 independent experiments.