Abstract
Aluminum-based adjuvants are the most widely used adjuvants in human vaccines. A comprehensive understanding of the mechanism of action of aluminum adjuvants at the molecular level, however, is still elusive. Here, we unravel the effects of aluminum hydroxide Al(OH)3 by a systems-wide analysis of the Al(OH)3-induced monocyte response. Cell response analysis by cytokine release was combined with (targeted) transcriptome and full proteome analysis. Results from this comprehensive study revealed two novel pathways to become activated upon monocyte stimulation with Al(OH)3: the first pathway was IFNβ signaling possibly induced by DAMP sensing pathways like TLR or NOD1 activation, and second the HLA class I antigen processing and presentation pathway. Furthermore, known mechanisms of the adjuvant activity of Al(OH)3 were elucidated in more detail such as inflammasome and complement activation, homeostasis and HLA-class II upregulation, possibly related to increased IFNγ gene expression. Altogether, our study revealed which immunological pathways are activated upon stimulation of monocytes with Al(OH)3, refining our knowledge on the adjuvant effect of Al(OH)3 in primary monocytes. SignificanceAluminum salts are the most used adjuvants in human vaccines but a comprehensive understanding of the working mechanism of alum adjuvants at the molecular level is still elusive. Our Systems Vaccinology approach, combining complementary molecular biological, immunological and mass spectrometry-based techniques gave a detailed insight in the molecular mechanisms and pathways induced by Al(OH)3 in primary monocytes. Several novel immunological relevant cellular pathways were identified: type I interferon secretion potentially induced by TLR and/or NOD like signaling, the activation of the inflammasome and the HLA Class-I and Class-II antigen presenting pathways induced by IFNγ. This study highlights the mechanisms of the most commonly used adjuvant in human vaccines by combing proteomics, transcriptomics and cytokine analysis revealing new potential mechanisms of action for Al(OH)3.
Highlights
Since 1926, colloidal aluminum salts are known for their adjuvant features, when Glenny et al discovered that diphtheria toxoid adsorbed to aluminum salts showed a significantly higher antibody titer against the toxoid compared to antigen alone [1]
When immature monocyte-derived dendritic cells were stimulated with Al(OH)3, no significant changes in cell surface marker expression were observed, as determined by flow cytometry, whereas LPS stimulation resulted in the upregulation of CD40, CD80 and CD83 (Supplementary S4)
Gene expression of CD14, a typical monocyte marker that is lost upon differentiation into immature MDDCs (iMDDCs) and linked to LPS stimulations [31], was downregulated in Al(OH)3-stimulated monocytes after 24 h (Fig. 1), while gene expression was upregulated in LPS-stimulated monocytes (Supplementary S3, S5)
Summary
This study was conducted using blood donations, provided by the National Institute for Public Health and the Environment (Bilthoven, The Netherlands), for primary cell isolation. The blood donations for this research goal were approved by the accredited Medical Research Ethics Committee (MREC), METC, Noord-Holland in The Netherlands. The study was conducted according to the principles expressed in the Declaration of Helsinki and written informed consent was obtained from all blood donors before collection and use of their samples. All human primary cells described in this study were obtained by the rules of this ethical statement
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