DNA released from dying host cells mediates aluminum adjuvant activity

Journal name:
Nature Medicine
Volume:
17,
Pages:
996–1002
Year published:
DOI:
doi:10.1038/nm.2403
Received
Accepted
Published online

Abstract

Aluminum-based adjuvants (aluminum salts or alum) are widely used in human vaccination, although their mechanisms of action are poorly understood. Here we report that, in mice, alum causes cell death and the subsequent release of host cell DNA, which acts as a potent endogenous immunostimulatory signal mediating alum adjuvant activity. Furthermore, we propose that host DNA signaling differentially regulates IgE and IgG1 production after alum-adjuvanted immunization. We suggest that, on the one hand, host DNA induces primary B cell responses, including IgG1 production, through interferon response factor 3 (Irf3)-independent mechanisms. On the other hand, we suggest that host DNA also stimulates 'canonical' T helper type 2 (TH2) responses, associated with IgE isotype switching and peripheral effector responses, through Irf3-dependent mechanisms. The finding that host DNA released from dying cells acts as a damage-associated molecular pattern that mediates alum adjuvant activity may increase our understanding of the mechanisms of action of current vaccines and help in the design of new adjuvants.

At a glance

Figures

  1. Alum induces cell death and release of host DNA at sites of injection.
    Figure 1: Alum induces cell death and release of host DNA at sites of injection.

    (a) Quantity of free dsDNA in the acellular fraction of the peritoneal lavage fluid of mice treated i.p. with increasing doses of alum, measured over time using quantitative fluorescent dsDNA stain. (b) Confocal microscopic imaging of extracellular DNA deposition in alum macroscopic i.p. depots stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 25 μm. (c) Cell death rate in the peritoneal lavage fluid of mice treated i.p. with increasing doses of alum, assessed by staining with 7-aminoactinomycin D (7-AAD) and flow cytometry. n = 5 (a,c). Data are representative of one of three independent experiments. Error bars show means ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001.

  2. Host DNA released by alum cytotoxicity mediates alum adjuvant activity on humoral and TH2 cell responses.
    Figure 2: Host DNA released by alum cytotoxicity mediates alum adjuvant activity on humoral and TH2 cell responses.

    (af) Serum titers of OVA-specific IgM (OVA-IgM; a,d), IgG1 (OVA-IgG1; b,e) and IgE (OVA-IgE; c,f). Titers were measured on the indicated days in (ac) mice immunized i.p. with OVA alone, OVA and alum, or OVA and DNA on days 0 and 14, and boosted with OVA on d 21; or in (df) mice immunized i.p. with OVA or OVA and alum, treated i.p. with DNase I both 3 and 18 h later, and then boosted with OVA 10 d later. (g) Proliferation profile (top) and division index (bottom) of adoptively transferred CFSE-labeled OVA-specific CD4+ OT-II cells in the bronchial lymph nodes of mice treated i.p. with OVA, OVA and DNA, OVA and alum, or OVA and alum followed by DNase I treatment. Inserted numbers indicate division index values. n = 5; data are representative of one of two (ac) or three (dg) independent experiments. Error bars show means ± s.d. *, OVA versus OVA and adjuvant; °, OVA and alum versus OVA and alum followed by DNase I treatment; *P < 0.05, **,°°P < 0.01, ***,°°°P < 0.001. a.u., arbitrary unit. NS, not significant.

  3. Alum and host genomic DNA trigger type I IFN secretion and IgE responses through activation of the Tbk1-Irf3 axis.
    Figure 3: Alum and host genomic DNA trigger type I IFN secretion and IgE responses through activation of the Tbk1-Irf3 axis.

    (a,b) Quantities of (a) IFN-β1 and (b) IL-1β in the peritoneal lavage fluid over time by ELISA in WT mice treated i.p. with OVA, OVA and DNA, or OVA and alum. (cf) Serum titers of OVA-specific IgE (c,e) and IgG1 (d,f) measured on day 28 in WT and Irf3−/− mice immunized i.p. with OVA, OVA and alum (c,d), or OVA and DNA (e,f) on days 0 and 14, and then boosted with OVA on day 21. (g,h) Serum titers of OVA-specific IgE (g) and IgG1 (h) measured on day 28 in Tbk1+/−/Tnf−/− and Tbk1−/−/Tnf−/− mice immunized i.p. with OVA and alum on days 0 and 14, and then boosted with OVA on day 21. n = 5; data are representative of one of three independent experiments. Error bars show means ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. a.u., arbitrary unit.

  4. Irf3 is essential for the boosting of canonical TH2 cells by alum and genomic DNA.
    Figure 4: Irf3 is essential for the boosting of canonical TH2 cells by alum and genomic DNA.

    (a) Proliferation profile (left) and division index (right) of adoptively transferred OVA-specific CD4+ OT-II cells in the BLNs of WT and Irf3−/− mice treated i.p. with OVA, OVA and DNA, or OVA and alum. Inserted numbers indicate division index values. (b) Cytokine concentrations in the supernatant of OVA-stimulated BLN cells isolated from WT and Irf3−/− mice treated with OVA, OVA and DNA, or OVA and alum. (ce) Assessment of allergic airway inflammation in OVA- or OVA and alum–sensitized WT and Irf3−/− mice challenged with aerosolized OVA. (c) Total and differential immune cell counts in the bronchoalveolar lavage fluid (BALF). (d) Representative section and inflammatory scores of hematoxylin-eosin staining of lung sections. Scale bar, 50 μm. (e) Representative staining and percentage of periodic acid Schiff (PAS)-stained goblet cells per total epithelial cells in randomly selected bronchi. Scale bar, 50 μm. n = 5; data are representative of one of two (a) or three (be) independent experiments. Error bars show means ± s.d. *, OVA versus OVA and adjuvant; °, WT OVA and adjuvant versus Irf3−/− OVA and adjuvant; *P < 0.05, **,°°P < 0.01, ***P < 0.001.

  5. Deficient migration of inflammatory monocytes impairs alum-induced TH2 and IgE responses in Irf3-/- mice.
    Figure 5: Deficient migration of inflammatory monocytes impairs alum-induced TH2 and IgE responses in Irf3−/− mice.

    (a) Flow cytometric assessment of the numbers of iDCs, cDCs and pDCs in the BLNs of WT and Irf3−/− mice 24 h after i.p. injection of OVA or OVA and alum. (b) Recruitment of iDCs to the BLNs of WT and Irf3−/− mice treated i.p. with OVA, OVA and DNA, or OVA and alum. (c) Correlation between the percentage of cell death (assessed as in Fig. 1c), DNA release (assessed as in Fig. 1a), and the recruitment of iDCs to the BLN (assessed as in b, at 24 h) of WT mice treated with alum for 24 h. (e) Proliferation profile (left) and division index (right) of adoptively transferred OVA-specific CD4+ OT-II cells in the BLNs of Irf3−/− mice that received OVA and alum and an adoptive transfer of WT iMonos obtained from the peritoneal cavity of OVA and alum–treated WT mice. Inserted numbers indicate division index values. (f) Cytokine concentrations in the supernatant of OVA-stimulated BLN cells isolated from Irf3−/− mice that received OVA and alum and an adoptive transfer of WT iMonos obtained as in e. (g,h) Serum titers of OVA-specific IgE (g) and IgG1 (h) in Irf3−/− mice treated with OVA and alum, transferred with WT iMonos as in e on days 0 and 14, and then boosted with OVA on day 21. As controls, we used WT and Irf3−/− mice that received PBS with OVA alone or OVA and alum. (i) Numbers of CFSE+ iDCs in the BLNs of OVA and alum–treated WT mice that received CFSE-labeled WT or Irf3−/− iMonos 18 h earlier. Control mice received PBS alone. n = 5; data are representative of one of four (a,c), one of three (fi) or one of two (b,d,e) independent experiments. Error bars show means ± s.d. #, WT OVA and adjuvant versus Irf3−/− OVA and adjuvant (a,b); *P < 0.05, **,°°P < 0.01, ***,°°°P < 0.001. a.u., arbitrary unit.

  6. Alum-induced iMono migration depends on IL-12p40 homodimer signaling.
    Figure 6: Alum-induced iMono migration depends on IL-12p40 homodimer signaling.

    (a) Gating strategy for iMonos and flow cytometric analysis of their surface expression of CCR7 and co-stimulatory molecules in OVA and alum–treated WT and Irf3−/− mice. Bottom right, percentage of CCR7hi iMonos in OVA and alum–treated WT and Irf3−/− mice is indicated. (b) ELISA measurement of IL-12p70, IL-23 and IL-12p80 in the acellular phase of the peritoneal lavage fluid of WT and Irf3−/− mice treated overnight with OVA or with OVA and alum. #, WT OVA and alum versus Irf3−/− OVA and alum. (c,d) Recruitment of iDCs to the BLNs of WT and Irf3−/− mice treated with OVA and alum and recombinant IL-12p80 (rIL12p80) (c) or p40-specific neutralizing antibody (d). Control OVA- and OVA and alum–treated mice received PBS only. (e,f) Serum titers of OVA-specific IgE (e) and IgG1 (f) measured on day 17 in WT mice treated i.p. with OVA and alum and p40-specific neutralizing antibody, and then boosted with OVA i.p. 10 d later. n = 5; data are representative of one of three (a) or two (bf) independent experiments. Error bars show means ± s.d. *P < 0.05, **°P < 0.01, ***P < 0.001. a.u., arbitrary unit.

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Author information

  1. These authors contributed equally to this work.

    • Ken J Ishii,
    • Fabrice Bureau &
    • Christophe J Desmet

Affiliations

  1. Laboratory of Cellular and Molecular Physiology, Groupe Interdisciplinaire de Génoprotéomique Appliquée, University of Liège, Liège, Belgium.

    • Thomas Marichal,
    • Denis Bedoret,
    • Claire Mesnil,
    • Catherine Sabatel,
    • Pierre Lekeux,
    • Fabrice Bureau &
    • Christophe J Desmet
  2. Laboratory of Biochemistry, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.

    • Thomas Marichal,
    • Denis Bedoret,
    • Claire Mesnil,
    • Catherine Sabatel,
    • Pierre Lekeux,
    • Fabrice Bureau &
    • Christophe J Desmet
  3. World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan.

    • Keiichi Ohata,
    • Kouji Kobiyama,
    • Cevayir Coban,
    • Shizuo Akira &
    • Ken J Ishii
  4. Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Osaka, Japan.

    • Kouji Kobiyama &
    • Ken J Ishii

Contributions

T.M., K.J.I., F.B. and C.J.D. designed the experiments; C.C., K.J.I., F.B. and C.J.D. supervised the project; T.M. and D.B. made the initial observation; T.M. did most of the experiments and compiled the data; T.M., K.O. and K.K. did the experiments involving Tbk1/Tnf double-knockout mice, Zbp1−/−, Ifnar2−/− and Mavs−/− mice; C.M. and C.S. did the FACS analyses; S.A. provided the Tbk1/Tnf double-knockout mice and Zbp1−/− mice; P.L., S.A., K.J.I. and F.B. secured funding; K.J.I. and F.B. provided feedback on the manuscript; and C.J.D. wrote the manuscript.

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The authors declare no competing financial interests.

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