Aminated nanomicelles as a designer vaccine adjuvant to trigger inflammasomes and multiple arms of the innate immune response in lymph nodes

Figures & data

Figure 1 Lymph node targeting aPNMs.

Notes: Size and size distribution of (A) carboxyl-terminated and (B) amine-terminated γ-PGA nanomicelles measured by dynamic light scattering and transmission electron microscopy. In vivo trafficking of aPNMs to lymph nodes. (C) In vivo NIR fluorescence image of IRDye800-labeled aPNMs 20 minutes after injection into the footpad (triangle: footpad, circle: lymph node). (D) Immunohistofluorescence analysis of the dissected lymph nodes of a mouse injected with FITC-labeled aPNMs. The slides were stained with anti-CD169, anti-F4/80, or anti-CD205. Scale bar is 250 µm. Magnification ×4 (Olympus IX 71, Olympus, Tokyo, Japan).

Abbreviations: aPNMs, amine-terminated γ-PGA nanomicelles; FITC, fluorescein isothiocyanate; γ-PGA, poly-(γ-glutamic acid); NIR, near-infrared.

Figure 2 Induction of inflammasome response.

Notes: (A) Scheme of ex vivo experiment. (B, C) Secretion of IL-1β after incubation of BMDMs and BMDCs with various concentrations of aPNMs or carboxyl-terminated γ-PGA nanomicelles for 4 hours and after priming with LPS (400 ng mL⁻¹) for 3 hours. The concentration unit of the X-axis is µg mL⁻¹. ***p<0.001. Scale bar is 15 µm. (1: control, 2: 1 µg mL⁻¹, 3: 2 µg mL⁻¹, 4: 5 µg mL⁻¹, 5: 10 µg mL⁻¹). Immunofluorescent images (100×) of the inflammasome complex (NLRP3/ASC) of BMDM (D) and BMDC (E).

Abbreviations: APC, antigen-presenting cells; aPNMs, amine-terminated γ-PGA nanomicelles; BMDCs, bone marrow-derived dendritic cells; BMDMs, bone marrow-derived macrophages; γ-PGA, poly-(γ-glutamic acid); LPS, lipopolysaccharide.

Figure 3 Effect of inflammasome inhibitors.

Notes: Secretion of IL-1β after incubation of BMDMs (A) and BMDCs (B) with a caspase-1 inhibitor. Secretion of IL-1β after incubation of BMDMs (C) and BMDCs (D) with a cathepsin B inhibitor. All data were obtained in triplicate and are presented as the mean ± SD. ***p<0.001. The concentration unit of the X-axis is µg mL⁻¹, and poly-(dA:dT) is 2 µg mL⁻¹ (1: control, 2: 5 µg mL⁻¹, 3: 10 µg mL⁻¹, 4: poly-(dA:dT)).

Abbreviations: BMDCs, bone marrow-derived dendritic cells; BMDMs, bone marrow-derived macrophages; poly-(dA:dT), poly(deoxyadenylic–deoxythymidylic).

Figure 4 In vitro and in vivo induction of inflammasomes in lymph nodes.

Notes: Secretion of IL-1β after incubation of BMDMs (A) and BMDCs (B) isolated from wild-type or NLRP3-KO mice (NLRP3^−/−) with aPNMs. The concentration unit of the X-axis is µg mL⁻¹, and poly-(dA:dT) is 2 µg mL⁻¹. (1: control, 2: 5 µg mL⁻¹, 3: 10 µg mL⁻¹, 4: poly-(dA:dT)). (C) Scheme of in vivo and ex vivo experiment. (D) Secretion of IL-1β in lymph nodes at different time points after injection of aPNMs. (E) Different degrees of inflammasome induction between wild-type and NLRP3-KO mice. All data were obtained in triplicate and are presented as the mean ± SD. ***p<0.001. NS, not significant.

Abbreviations: aPNMs, amine-terminated γ-PGA nanomicelles; BMDCs, bone marrow-derived dendritic cells; BMDMs, bone marrow-derived macrophages; γ-PGA, poly-(γ-glutamic acid); KO, knock out; poly-(dA:dT), poly(deoxyadenylic–deoxythymidylic).

Figure 5 Triggering of multiple arms of the innate immune response in lymph nodes by aPNM-IC.

Notes: Secretion levels of (A) IL-1β, (B) TNF-α, (C) IL-6, (D) IFN-β. All data were obtained in triplicate and are presented as the mean ± SD. *p<0.05, ***p<0.001. NS, not significant.

Abbreviations: aPNM-IC, amine-terminated γ-PGA nanomicelles + poly-(I:C); poly-(I:C), polyinosinic–polycytidylic acid; γ-PGA, poly-(γ-glutamic acid); IFN-β, interferon-β; TNF-α, tumor necrosis factor-alpha.

Scheme 1 Schematic illustrations of γ-PGA nanomicelles and inflammasome pathway.

Notes: Aminated γ-PGA nanomicelles facilitate efficient migration through lymphatic vessels, targeting of APCs in the lymph nodes, and induction of inflammasomes after lysosomal destabilization. When aPNMs combine with anionic poly-(I:C), a TLR3 agonist, they trigger multiple arms of the innate immune response (IL-1β secretion by inflammasomes, inflammasome-independent secretion of proinflammatory [TNF-α and IL-6], and type I interferon [IFN-β]).

Abbreviations: APCs, antigen-presenting cells; aPNM, amine-terminated γ-PGA nanomicelles; γ-PGA, poly-(γ-glutamic acid); IFN-β, interferon-β; LPS, lipopolysaccharide; poly-(I:C), polyinosinic–polycytidylic acid; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-alpha.

Figure S1 The stability of aPNM in PBS and serum at body temperature.

Note: The stability test results conducted at body temperature showed that the size of aminated micelles was kept during a time span of 6 days.

Abbreviations: aPNM, amine-terminated γ-PGA nanomicelles; γ-PGA, poly-(γ-glutamic acid); PBS, phosphate-buffered saline.

Figure S2 Intracellular location of aPNM-FITC measured by fluorescence microscopy.

Notes: BMDMs and BMDCs were stained with 50 nM LysoTracker^® red (Thermo Fisher Scientific, Waltham, MA, USA) and treated with aPNM-FITC overnight. Some of aPNM-FITC was colocalized with LysoTracker, while others were in the cytosol. Scale bar is 5 µm. Magnification ×60 (Delta vision PD instrument; GE Healthcare, Little Chalfont, UK).

Abbreviations: aPNM, amine-terminated γ-PGA nanomicelles; BMDCs, bone marrow-derived dendritic cells; BMDMs, bone marrow-derived macrophages; DIC, differential interference contrast; FITC, fluorescein isothiocyanate; γ-PGA, poly-(γ-glutamic acid).

Figure S3 Effect of inflammasome inhibitors.

Notes: Secretion of IL-1β after incubation of BMDMs (A) and BMDCs (B) with caspase-1 inhibitor. Secretion of IL-1β after incubation of BMDMs (C) and BMDCs (D) with cathepsin B inhibitor. All data were obtained in triplicate and are presented as a mean value ± SD. ***p<0.001. The concentration unit of X-axis is µg mL⁻¹ (1: control, 2: 1 µg mL⁻¹, 3: 2 µg mL⁻¹, 4: 5 µg mL⁻¹, 5: 10 µg mL⁻¹).

Abbreviations: BMDCs, bone marrow-derived dendritic cells; BMDMs, bone marrow-derived macrophages.

Figure S4 In vivo induction of inflammasomes in lymph nodes.

Notes: Secretion of IL-1β in lymph nodes at different time points after injection of carboxyl-terminated γ-PGA nanomicelles. All data were obtained in triplicate and are presented as the mean ± SD. NS, not significant.

Abbreviation: γ-PGA, poly-(γ-glutamic acid).

Scheme S1 Mechanism study of inflammasome activation by inhibitors of inflammasome signaling pathway.

Notes: Cathepsin B was inhibited by CA-074, and caspase-1 was inhibited by Ac-YVAD-cmk. NLRP3 was depleted in NLRP3-KO mice.

Abbreviations: aPNM, amine-terminated γ-PGA nanomicelles; γ-PGA, poly-(γ-glutamic acid); IFN-β, interferon-β; KO, knock out; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-alpha.

Scheme S2 Signaling pathway of different types of inflammasomes.

Notes: Poly-(dA:dT) stimulates the AIM2 inflammasome. The final pathway for the activation of caspase-1 to produce IL-1β is the same.

Abbreviations: aPNM, amine-terminated γ-PGA nanomicelles; γ-PGA, poly-(γ-glutamic acid); poly-(dA:dT), poly(deoxyadenylic–deoxythymidylic); TLR4, toll-like receptor 4.