Abstract
Enterotoxin-based proteins are powerful manipulators of mucosal immunity. The A1 domain of heat-labile enterotoxin from E. coli, or LTA1, is a newer adjuvant from this family under investigation for intranasal vaccines. Although LTA1 has been examined in mouse vaccination studies, its ability to directly stimulate immune cells compared to related adjuvant proteins has not been well explored. Here, we perform the first rigorous examination of LTA1’s effect on antigen presenting cells (APC) using a human monocyte cell line THP-1. To better understand LTA1’s stimulatory effects, we compared it to dmLT, or LT-R192G/L211A, a related AB5 adjuvant in clinical trials for oral or parenteral vaccines. LTA1 and dmLT both activated APCs to upregulate MHC-II (HLA-DR), CD86, cytokine secretion (e.g., IL-1β) and inflammasome activation. The effect of LTA1 on surface marker changes (e.g., MHC-II) was highly dose-dependent whereas dmLT exhibited high MHC-II expression regardless of dose. In contrast, cytokine secretion profiles were similar and dose-dependent after both LTA1 and dmLT treatment. Cellular activation by LTA1 was independent of ganglioside binding, as pre-treatment with purified GM1 blocked the effect of dmLT but not LTA1. Unexpectedly, while activation of the inflammasome and cytokine secretion by LTA1 or dmLT was blocked by the protein kinase A inhibitor H89 (similar to previous reports), these responses were not inhibited by a more specific PKA peptide inhibitor or antagonist; thus Indicating that a novel and unknown mechanism is responsible for inflammasome activation and cytokine secretion by LT proteins. Lastly, LTA1 stimulated a similar cytokine profile in primary human monocytes as it did in THP1 cells, including IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, and TNFα. Thus, we report that LTA1 protein programs a dendritic cell-like phenotype in APCs similar to dmLT in a mechanism that is independent of PKA activation and GM1 binding and entry.
Highlights
Adjuvants are useful additives that promote vaccine induced immunity, only a few adjuvant compounds can be used at mucosal surfaces or to induce mucosal immunity
To understand how observed changes compared to an labile toxin E. coli (LT) adjuvant containing an intact B subunit, we included a comparison to dmLT protein, which has never been evaluated in this model but has been reported to activate monocytes, bone marrow-derived DCs (BM-DCs), and in vivo DCs [11, 14, 15, 21]
To compare the effects of LTA1 and dmLT in THP-1 cells compared with dibutyryl cAMP (d-cAMP) and forskolin we evaluated cAMP levels and changes to surface activation markers and secreted cytokines (Fig 8). cAMP levels were significantly altered by dmLT, LTA1, d-cAMP or forskolin treatments compared to untreated cells; significant difference were observed between dmLT and LTA1 treatments and d-cAMP and forskolin (Fig 8A)
Summary
Adjuvants are useful additives that promote vaccine induced immunity, only a few adjuvant compounds can be used at mucosal surfaces or to induce mucosal immunity. Best known mucosal adjuvants are enterotoxin proteins, including heat-labile toxin E. coli (LT), cholera toxin (CT), and their detoxified derivatives like LT-R129G/L211A or dmLT [1,2,3,4] The latter is an advanced adjuvant candidate for both oral and parenteral vaccines [1]. CT, LT, dmLT and related mutant adjuvants activate APCs (e.g., monocytes, monocyte-derived dendritic cells [moDC], macrophages and DCs) in a process critical for the generation of post-vaccination responses, including upregulation of MHC-II, activation markers, and cytokine secretion [7,8,9,10,11,12]. PBMCs or human monocytes stimulated with dmLT exhibited similar responses, including inflammasome gene expression and IL-1β cytokine secretion [14, 15] The latter was required for antigen-specific IL-17A responses and was controlled by cAMP accumulation and PKA activation. We tested the hypothesis that LTA1 stimulates immunity through a mechanism that requires PKA activation but is GM1-independent, as expected
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