Sequestering of damage-associated molecular patterns (DAMPs): a possible mechanism affecting the immune-stimulating properties of aluminium adjuvants

Immunol Res (2017) 65:1164–1175 https://doi.org/10.1007/s12026-017-8972-5 ORIGINAL ARTICLE Sequestering of damage-associated molecular patterns (DAMPs): a possible mechanism affecting the immune-stimulating properties of aluminium adjuvants Andreas Svensson 1 & Tove Sandberg 2 & Peter Siesjö 3 & Håkan Eriksson 2 Published online: 27 November 2017 # The Author(s) 2017. This article is an open access publication Abstract Aluminium-based adjuvants (ABAs) have been used in human and veterinary vaccines for decades, and for a long time, the adjuvant properties were believed to be mediated by an antigen depot at the injection site, prolonging antigen exposure to the immune system. The depot hypothesis is today more or less abandoned, and instead replaced by the assumption that ABAs induce an inflammation at the injection site. Induction of an inflammatory response is consistent with immune activation initiated by recognition of molecular patterns associated with danger or damage (DAMPs), and the latter are derived from endogenous molecules that normally reside intracellularly. When extracellularly expressed, because of damage, stress or cell death, a sterile inflammation is induced. In this paper, we report the induction of DAMP release by viable cells after phagocytosis of aluminium-based adjuvants. Two of the most commonly used ABAs in pharmaceutical vaccine formulations, aluminium oxyhydroxide and aluminium hydroxyphosphate, induced a vigorous extracellular expression of the two DAMP molecules calreticulin and HMGB1. Concomitantly, extracellular adjuvant particles adsorbed the DAMP molecules released by the cells whereas IL-1β, a previously reported inflammatory mediator induced by ABAs, was not absorbed by the adjuvants. Induction of extracellular expression of the two DAMP molecules was * Håkan Eriksson 1 Lund Stem Cell Center, BMC B10, Lund University, Lund, Sweden 2 Department of Biomedical Science, Faculty of Health and Society, Malmö University, SE-205 06 Malmö, Sweden 3 Glioma Immunotherapy Group, Neurosurgery, Department of Clinical Sciences, BMC D14, Lund University, SE-221 84 Lund, Sweden more prominent using aluminium hydroxyphosphate compared to aluminium oxyhydroxide, whereas the extracellular adsorption of the DAMP molecules was more pronounced with the latter. Furthermore, it is hypothesised how induction of DAMP expression by ABAs and their concomitant adsorption by extracellular adjuvants may affect the inflammatory properties of ABAs. Keywords Alarmins . Aluminium-based adjuvant . Damp . Lumogallion . Sterile inflammation Introduction Aluminium-based adjuvants, ABAs, have been used in pharmaceutical vaccine formulations for decades, and for many years, the prolonged release of antigen at the inoculation site was regarded as the mechanism of the immune-stimulating properties of ABAs [1]. However, it has also been proposed that ABAs induce inflammation, activating the innate immune system and thereby an adaptive response [2–4]. Several reports have verified that ABAs trigger an inflammatory response, and infiltration of immune cells at the inoculation site initiates activation and maturation of innate and adaptive immune cells [5–7], with a direct effect on antigen-presenting cells [8]. The consensus has been that the immune-stimulating effects of ABAs are pleotropic and involve multiple signalling pathways. The NLRP3-inflammasome has been reported to play an important role in the inflammatory response induced by ABAs. Activation of NLRP3-inflammasomes by ABAs initiates the cleavage and release of the pro-inflammatory cytokines IL-1β and IL-18 [2, 9, 10]. However, the NLRP3inflammasome can also be activated by endogenous dangerassociated molecular patterns, DAMPs [11]. ABA-induced Immunol Res (2017) 65:1164–1175 release of pro-inflammatory cytokines has so far only been reported for cytokines of the IL-1 family, and in the context of ABAs, release of DAMP molecules has been claimed to emanate from injured or necrotic cells, due to endocytosis of the adjuvant [3, 4, 12]. The most common types of aluminium salts used as adjuvants are aluminium oxyhydroxide, AlO(OH), in this paper referred to as Alhydrogel, or aluminium hydroxyphosphate, Al(OH)x (PO4)y, here referred to as Adju-Phos. Considering the extensive use of these two ABAs, knowledge and understanding regarding the mechanisms underlying the immune response induced are surprisingly limited. In this paper, we show that viable cells release DAMP molecules upon in vitro exposure to both Alhydrogel and Adju-Phos. Release of DAMP molecules is exemplified by calreticulin and the high mobility group box 1 protein (HMGB1). Calreticulin is an intracellular homeostasis regulator of Ca2+ as well as chaperone controlling the quality of newly synthesised proteins in the endoplasmic reticulum [13, 14], and cells undergoing immunogenic cell death have been shown to secrete calreticulin [13]. HMGB1, on the other hand, is a nonhistone chromosomal binding protein normally located in the nucleus, regulating chromosome stability [15]. Though calreticulin and HMGB1 have completely different intracellular locations, both have been shown to be secreted upon immunogenic cell death and studies have implicated a diagnostic value of both molecules in cancer therapy [13, 16, 17]. 1165 Co-culture with aluminium adjuvants and dealuminated zeolite Y The aluminium adjuvant preparations used in this study were Alhydrogel; AlO(OH) and Adju-Phos, Al(OH)x(PO4)y, purchased from Brenntag Biosector (Frederikssund, Denmark). Dealuminated zeolite Y (USY) was purchased from Tosoh Corporation, Japan. Lumogallion (CAS 4386–25-8) was purchased from TCI Europe N.V., Antwerp, Belgium, and lipopolysaccharide (LPS, from Escherichia coli O111:B4) was purchased from Sigma-Aldrich, St. Louis, MO, USA. Triplicates of THP-1 cells, 0.5 × 106 cells per ml, were cocultured in 96-well plates with Alhydrogel or Adju-Phos corresponding to final aluminium concentrations ranging from 25 to 100 μg/ml in a total volume of 200 μl R10 during 1 to 16 h (over night) at 37 °C. Cells cultured in R10 in the absence of aluminium adjuvant were used as control. Specified concentrations of aluminium and incubation periods of each experiment are described in the figure legends. Cells from three to five wells of each incubation were pooled and centrifuged for 5 min at 1000×g. The supernatants were collected, re-centrifuged for 10 min at 13,000×g and then divided into aliquots and stored at − 80 °C until DAMP or cytokine content were assayed. Collected cells were re-suspended in PBS containing 0.1% (w/v) BSA and 0.1% (w/v) human IgG at 1 × 106 cells per ml. The cells were sub-divided into aliquots and stained with APC-labelled anti-human calreticulin, anti-human HMGB1, anti-human IL-1β, or an APC labelled isotype control (all antibodies from R&D Systems, Minneapolis, MN, USA) by incubation for 30 min on ice. Finally, the cells were washed with PBS containing (...truncated)