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.2008 Apr 14;205(4):869-82.
doi: 10.1084/jem.20071087. Epub 2008 Mar 24.

Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells

Affiliations

Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells

Mirjam Kool et al. J Exp Med..

Abstract

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)-resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c(+) monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid.

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Figures

Figure 1.
Figure 1.
The mediastinal and ipsilateral ILN drain the peritoneal cavity after i.p. injection. Mice were injected with CFSE-labeled DO11.10 OVA-TCR Tg cells 1 d before the i.p. injection of OVA in the right lower quadrant. (A) 2 d after OVA injection, different LNs and spleen were taken and T cell proliferation was assessed with flow cytometry after CFSE dilution. Only KJ1-26 Ag-reactive T cells divide. (B) Immediately after the administration of OVA in the right lower quadrant, a sterile puncture was made at the left lower quadrant. 4 d later, proliferation was measured in the left and right ILN. An example is shown of four mice (representative of at least two independent experiments).
Figure 2.
Figure 2.
Addition of alum adjuvant to OVA leads to a stronger, more persistent and recirculating Th2 immune response. Mice were injected with CFSE-labeled DO11.10 OVA-TCR Tg cells 1 d before the i.p. injection of OVA or OVA-alum. (A) 4 and 7 d after the injection the DLN (MLN), nondraining LN (CLN), and the spleen were analyzed for T cell proliferation with flow cytometry (n = 4 mice; experiment performed three times). (B) CFSE content was calculated as described in Materials and methods, and is shown for DLN (MLN) and nondraining LN (ALN). Open symbols represent the OVA-injected mice, and the filled symbols the OVA-alum injected mice. (C) 7 d after the i.p. injection, LN cells (DLN: MLN, nondraining LN: ALN) were taken and restimulated in vitro for 4 d with OVA. Cytokines were measured in the supernatants by ELISA. Open bars represent OVA-injected mice, and closed bars represent the OVA-alum–injected mice. Data are shown as the mean ± the SEM, *, P < 0.05.n = 4–6 mice per group.
Figure 3.
Figure 3.
Alum recruits innate immune cells to the peritoneal cavity. Mice were injected i.p. with OVA or OVA alum. (A) 6, 12, and 24 h after injection, the peritoneal lavage was taken and the number of macrophages (F4/80highCD11b+SSChigh), monocytes (CD11b+Ly6ChighLy6G−F4/80int), myeloid DCs (MHCIIhighCD11c+F4/80low), plasmacytoid DCs (120G8+CD11bdimCD11cint), neutrophils (CD11b+Ly6C+Ly6GhighF4/80−), and eosinophils (CD11b+Ly6CintLy6GintF4/80int) was determined. Open symbols represent the OVA-injected mice, and filled symbols the OVA-alum injected mice. (B) 2 h after injection, the peritoneal lavage was taken and chemokine levels were determined in the supernatant by ELISA. Data shown are the mean ± the SEM. *, P < 0.05.n = 4–6 mice per group.
Figure 4.
Figure 4.
Alum adjuvant stimulates DC function in vivo. (A) Mice were injected i.p. with OVA-Alexa Fluor 647 (OVA-AF647) or OVA-AF647-alum. 24 h after injection, the peritoneal lavage was taken and the uptake of OVA-AF647 was assessed in F4/80−MHCII+CD11c+ DCs. (B) Mice were injected with OVA-DQ or OVA-DQ-alum i.p., and 24 h later, the mDCs (F4/80−MHCII+CD11c+) in the peritoneal lavage were analyzed for the uptake and processing of DQ by flow cytometry. OVA-DQ fluoresces green when processed in acidified lysosomes. Red fluorescence is caused by accumulation of OVA-DQ in endosomal processing compartments in the cell. (C) The CD11c+ cells were also analyzed for the expression of MHCII in the DQ-negative or –double-positive gate. Gray-filled histograms represent OVA-DQ–negative, and black line histograms represent OVA-DQ–double-positive CD11c+ cells. (D) Maturation of mDCs in the peritoneal lavage was assayed 6, 12, and 24 h after injection of OVA or OVA-alum by flow cytometry. BM-derived DCs (BM-DCs) were pulsed for 16 h with OVA or OVA-alum. Gray filled histograms represent naive mice or unpulsed BM-DCs, black dotted line histograms represent OVA-injected mice or OVA-pulsed BM-DCs, and black solid line histograms represent OVA-alum–injected mice or OVA-alum–pulsed BM-DCs. (E) Mice were injected with OVA or OVA-alum, and 6 h later, the F4/80−MHCII+CD11c+ DCs were sorted from the peritoneal lavage and placed in co-culture with CFSE-labeled DO11.10 Tg CD4+ T cells. After 4 d, cells were analyzed for proliferation and gated for CD4+, KJ1-26+, and CD25+.
Figure 5.
Figure 5.
Inflammatory monocytes recruited by alum take up Ag, migrate to DLN and acquire a DC phenotype. Mice were injected with OVA-Alexa Fluor 647 (OVA-AF647) or OVA-AF647-alum, and 24 h later, the peritoneal lavage and DLN (MLN) were taken. (A) Presence of OVA-AF647 in inflammatory monocytes (defined as CD11b+Ly6ChighLy6G−F4/80int) is shown in the peritoneal lavage and MLN. (B) Inflammatory monocytes and mDCs (CD11b+MHCIIhighLy6C−) were sorted and placed in co-culture with CFSE-labeled DO11.10 Tg CD4+ T cells. T cell proliferation was assayed at day 4 and plots depict PI-negative CD4+ cells. (C) Expression of CD11c, MHC II, and CD86 on inflammatory monocytes determined by nine-color flow cytometry. Gray filled histograms represent the OVA-AF647–negative monocytes, whereas the black line histogram represents the OVA-AF647-positive ones. An example representative of four mice is shown. (D) CD45.1 mice were injected with OVA or OVA-alum. 2 h later, they received CD45.2+ monocytes sorted from bone marrow (purity >95%). 36 h later, the number of CD45.2+ cells in the MLN were determined by flowcytometry. Data shown are the mean ± the SEM. **, P < 0.01.n = 4–5 mice per group. (E) The CD11c and MHC II expression was assessed on the CD45.2+ cells before injection, and 36 h later, in the MLN.
Figure 6.
Figure 6.
Contribution of resident versus recruited CD11c+ DCs on Ag presentation and immunopotentiating effect of alum adjuvant. (A) CD11c-DTR Tg mice were depleted of resident MLN DCs by an i.t. injection of 100 ng DT or PBS as a control. 1 d before DT, they received a cohort of CFSE-labeled CD4+ DO11.10 T cells i.v. 1 d after DT, OVA or OVA-alum was given i.p. 3 d after the last injection, proliferation of Tg T cells were determined in the draining MLN and draining right ILN. Percentages in the plots are the percentage of Tg cells from total CD4+ T cells. (B) To deplete all CD11c+ cells (resident and recruited) CD11c-DTR Tg mice were injected i.p. with 100 ng DT or PBS as a control. 1 d before DT, they received a cohort of CFSE-labeled CD4+ DO11.10 T cells i.v. OVA or OVA-alum was given i.p. 4 d after the last injection, and proliferation of Tg T cells was determined in the DLN (MLN) and nondraining LN (CLN). Percentages in the plots are the percentage Tg cells from total CD4+ T cells. (C) CD11c-DTR Tg mice were depleted of DCs by an i.p. injection of 100 ng DT or PBS as a control. 1 d before DT, they received a cohort of CFSE-labeled CD4+ DO11.10 T cells i.v. 1 d after DT, OVA, or OVA-alum was given i.p. with or without sorted monocytes from BALB/c mice. 4 d after the last injection, proliferation of Tg CD4+ T cells was determined in the draining MLN. An example is shown in 4 mice; the experiment was repeated at least two times. (D) CD11c-DTR Tg mice and non-Tg mice were injected with PBS or DT and received an i.p. injection of OVA-alum, and 10 d later, serum samples were taken and OVA-specific IgG1 levels were determined by ELISA. Data are shown as the mean ± the SEM, * P < 0.05.n = 4–5 mice per group.
Figure 7.
Figure 7.
The alum response in mice depends on uric acid and MyD88 signaling. (A) Mice were injected with saline, OVA, or OVA-alum, and after 2 h, uric acid levels were determined in the peritoneal lavage. Data are shown as the mean ± the SEM. **, P < 0.01.n = 5–6 mice per group. (B) Mice were injected with uricase 1 d and 5 min before OVA-AF647 or OVA-AF647-alum, and 24 h later, the DLNs (MLN) were taken. The number of OVA-AF647+ inflammatory monocytes (defined as CD11b+Ly6ChighLy6G−F4/80int) are shown. Data are shown as the mean ± the SEM. *, P < 0.05.n = 4–5 mice per group. (C) At day 0, mice received a cohort of CFSE-labeled DO11.10 T cells i.v. and uricase i.p. At day 1, mice received another injection with uricase, and 5 min thereafter OVA or OVA-alum i.p. 4 d after the last injection, proliferation of Tg T cells were determined in the draining MLN. Percentages in the plots are the percentage of Tg cells from total CD4+ T cells. An example is shown from four mice, and the experiment was repeated twice. (D) Quantification of the number of Tg cells from 4 and 7 d after OVA or OVA-alum plotted in C. Data are shown as mean ± SEM. *, P < 0.05; **, P < 0.01.n = 4 mice per group. (E) MyD88−/− and WT mice were injected with OVA-AF647 or OVA-AF647-alum, and 24 h later, the DLN (MLN) were examined. The number of OVA-AF647+ inflammatory monocytes (defined as CD11b+Ly6ChighLy6G−F4/80int) are shown. Data are shown as mean ± SEM. *, P < 0.05.n = 4 mice per group.
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