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. 2019 May 13;5(5):2631-2646.
doi: 10.1021/acsbiomaterials.9b00332. Epub 2019 Mar 26.

Dual-Sized Microparticle System for Generating Suppressive Dendritic Cells Prevents and Reverses Type 1 Diabetes in the Nonobese Diabetic Mouse Model

Jamal S Lewis  1   2   3 , Joshua M Stewart  1 , Gregory P Marshall  2 , Matthew R Carstens  1 , Ying Zhang  1 , Natalia V Dolgova  1 , Changqing Xia  4 , Todd M Brusko  4 , Clive H Wasserfall  4 , Michael J Clare-Salzler  4 , Mark A Atkinson  4   5 , Benjamin G Keselowsky  1   4
Affiliations

Dual-Sized Microparticle System for Generating Suppressive Dendritic Cells Prevents and Reverses Type 1 Diabetes in the Nonobese Diabetic Mouse Model

Jamal S Lewis et al. ACS Biomater Sci Eng. .

Abstract

Antigen specificity is a primary goal in developing curative therapies for autoimmune disease. Dendritic cells (DCs), as the most effective antigen presenting cells in the body, represent a key target to mediate restoration of antigen-specific immune regulation. Here, we describe an injectable, dual-sized microparticle (MP) approach that employs phagocytosable ∼1 μm and nonphagocytosable ∼30 μm MPs to deliver tolerance-promoting factors both intracellularly and extracellularly, as well as the type 1 diabetes autoantigen, insulin, to DCs for reprogramming of immune responses and remediation of autoimmunity. This poly(lactic-co-glycolic acid) (PLGA) MP system prevented diabetes onset in 60% of nonobese diabetic (NOD) mice when administered subcutaneously in 8 week old mice. Prevention of disease was dependent upon antigen inclusion and required encapsulation of factors in MPs. Moreover, administration of this "suppressive-vaccine" boosted pancreatic lymph node and splenic regulatory T cells (Tregs), upregulated PD-1 on CD4+ and CD8+ T cells, and reversed hyperglycemia for up to 100 days in recent-onset NOD mice. Our results demonstrate that a MP-based platform can reeducate the immune system in an antigen-specific manner, augment immunomodulation compared to soluble administration of drugs, and provide a promising alternative to systemic immunosuppression for autoimmunity.

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Conflict of interest statement

The authors declare the following competing financial interest(s): J.S.L. and G.P.M. conducted a portion of the reported experiments while employed by OneVax, LLC. C.H.W., T.M.B., M.A.A., and B.G.K. are cofounders of OneVax, LLC, a preclinical biotechnology company with interest in this technology. The authors declare that no other relevant conflicts of interest exist.

Figures

Figure 1
Figure 1
Schematic of the dual-sized microparticle (dMP) system. The dMP formulation is an injectable platform that provides sustained extracellular release of a DC chemokine, GM-CSF, and a protolerogenic factor, TGF-β1, via ∼30 μm nonphagocytosable MPs to recruit and condition DCs at a subcutaneous injection site. Concurrently, ∼1 μm phagocytosable MPs encapsulating antigen, denatured insulin, and a tolerizing agent, vitamin D3, provide targeted intracellular delivery to the locally recruited DCs in order to promote presentation of the T1D autoantigen in a tolerogenic context.
Figure 2
Figure 2
Characterization of fabricated microparticles. Representative SEM images of (A) phagocytsoable MPs and (B) nonphagocytosable MPs show size and surface morphology. (C) Size distributions of phagocytosable MPs (vitamin D3 or insulin) and nonphagocytosable MPs (TGF-β1 or GM-CSF) were confirmed by dynamic light scattering, reporting mean and standard deviation in the legend (n = 5). (D) Release kinetics of encapsulated factors from biodegradable PLGA MPs over 28 days, as determined by ELISA or spectrophotometry (n = 3–5). (E) The loading efficiencies of encapsulated agents and mass delivered per 2.5 mg of PLGA injection is calculated. Data are represented by the mean ± SEM.
Figure 3
Figure 3
Co-incubation of vitamin D3(VD3) MPs and TGF-β1 MPs induce DCs with suppressive phenotypes in vitro. Dendritic cells were incubated with 10 mg of nonphagocytosable TGF-β1 MPs, and phagocytosable VD3 MPs were added at a 10:1 MP to DC ratio. Microparticles were incubated with bone marrow-derived DCs for 48 h and subsequently washed with PBS to remove MPs. Untreated, immature DCs (iDC), DCs stimulated with LPS (1 μg/mL), and DCs incubated with unloaded MPs were included as controls. (A) Maturation markers CD80, CD86, and MHC-II were characterized by flow cytometry on MP-treated DCs and controls (n = 3). Surface expression is normalized to iDCs. (B) Maturation resistance in response to LPS was quantified (n = 3). Dendritic cells were stimulated with LPS (1 μg/mL) for 24 h following MP treatment. Flow cytometric analysis quantified expression of CD80, CD86, and MHC-II. Surface expression is normalized to LPS stimulated DCs. (C) Dendritic cell expression of the immunosuppressive enzyme IDO was quantified in response to MP treatment (n = 3). P-values (∗ = ≤0.05, ∗∗ = ≤0.01, ∗∗∗ = ≤0.001) were obtained by one-way ANOVA with Dunnett’s multiple comparisons test against the iDC control (A, C) or the LPS-stimulated control (B). Data are represented by the mean ± SEM.
Figure 4
Figure 4
Co-incubation of vitamin D3 (VD3) MPs and TGF-β1 MPs generates suppressive DCs that inhibit proliferation of allogeneic T cells and induces a modestly higher Treg frequency in vitro. Dendritic cells were incubated with 10 mg of nonphagocytosable TGF-β1 MPs, and phagocytosable VD3 MPs were added at a 10:1 MP to DC ratio. Microparticles were incubated with DCs for 48 h and subsequently washed with PBS to remove MPs. Balb/c splenic CD4+ T cells were then added to the MP-treated C57Bl/6 bone marrow-derived DCs at a 150 000:25 000 ratio. Untreated, immature DCs (iDC) and T cells only were included as controls. After 72 h, flow cytometry assessed T cell proliferation via BrdU incorporation (A) and CD25+FoxP3+ Treg frequency (B) (n = 3). P-values (∗ = ≤0.05, ∗∗ = ≤0.01, ∗∗∗ = ≤0.001) were obtained by one-way ANOVA with Tukey’s significance test. Data are represented by the mean ± SEM.
Figure 5
Figure 5
Subcutaneously injected MPs traffic to lymph nodes primarily by DCs with a suppressive phenotype in vivo. NOD mice were subcutaneously injected in the abdominal region with either the dMP or unloaded MPs. Encapsulated agents (VD3, insulin, or unloaded) in phagocytosable MPs were concomitantly loaded with fluorescent dye (DiI). (A) Axillary lymph nodes (ALN) were excised 48 h after dMP administration, and IHC was performed to identify MP localization. (B) Lymphoid organs (ALN, inguinal lymph nodes (ILN), and spleen) were excised from NOD mice 48 h after subcutaneous MP injection and cells characterized by flow cytometry for MP presence. The ratio of MP+ frequency in CD11b+CD11c+ DCs relative to CD11b+CD11c– MΦs was assessed and compared to unloaded MP-treated mice (n = 3–4). (C, D) PD-L1 and BTLA mean fluorescent intensity (MFI) was evaluated in ALNs on dMP+ DCs, dMP– DCs, unloaded MP+ DCs, unloaded MP– DCs, and DCs from untreated mice (n = 3–4). P-values (∗ = ≤0.05, ∗∗ = ≤0.01, ∗∗∗ = ≤0.001) were obtained by two-tailed unpaired Student’s t tests (B) and one-way ANOVA (C, D) with Tukey’s significance test. Data are represented by the mean ± SEM.
Figure 6
Figure 6
dMP administration prevents diabetes onset in NOD mice. A cohort of 8-week-old NOD mice (n = 10/group) were injected at a subcutaneous site anatomically proximal to the pancreas with the described MP formulations over 16 weeks. Animals received MP injections (arrows) once a week for the first 3 weeks (8, 9, and 10 weeks of age) and a booster injection once monthly thereafter for 4 months (12, 16, 20, and 24 weeks of age). Unloaded MPs, a soluble bolus of factors without MPs, and omission of factors were investigated. When a factor-loaded MP was omitted, unloaded MPs were delivered to deliver an equivalent PLGA mass. Animals were monitored weekly until week 28, and mice were considered diabetic when blood glucose levels were ≥240 mg/dL on 2 consecutive days. The full dMP (solid line with solid tilted square, VD3/TGF-β1/GM-CSF/insulin MPs) and unloaded MPs groups were replotted alongside different experimental groups to highlight the requirement of MP encapsulation (A), antigen (B), and the full dMP formulation (C) in order to see maximum therapeutic effect. Survival data are fit using the Kaplan–Meier nonparametric survival analysis model, and statistical analysis was performed via log-rank test (Mantel–Cox method). Statistical significance was not realized when accounting for multiple comparisons via Bonferroni correction, as the study was not powered to resolve this large number of groups. However, pairwise comparison between survival curves of mice that received the dMP and mice that received unloaded MPs resulted in a P-value of <0.05, suggesting a difference between treatments.
Figure 7
Figure 7
Diabetes prevention in dMP-treated mice is associated with an increase in Tregs, upregulation of PD-1 on CD4+ and CD8+ T cells, and an increase in DCs. Eight-week-old prediabetic NOD mice received MP injections at identical time points as in the prevention study and were euthanized at 10, 12, or 14 weeks of age. Mice analyzed at 12 weeks of age were sacrificed before receiving the week 12 monthly booster injection. As before, MP injections were administered subcutaneously on the right side of the abdomen, proximal to the pancreas. Ipsilateral inguinal and axillary LNs from dMP-treated mice and unloaded MP-treated mice were excised and stained for flow cytometry. Contralateral inguinal and axillary LNs from unloaded MP-treated mice were excised as a control. (A) Frequency of FoxP3+CD4+ T cells isolated from spleen and pancreatic LNs (pLNs) of 10-, 12-, and 14-week-old dMP-treated, unloaded MP-treated, and untreated naïve mice of total CD4+ T cells was quantified (n = 5–6). (B) Lymphoid organs (draining lymph nodes (dLNs; combined axillary and inguinal LNs), pLNs, and spleen) from animals euthanized at 12 weeks of age were analyzed for PD-1 expression on both CD4+ and CD8+ T cells (n = 5). (C) Frequency of DCs in dLNs as a percent of total cells (n = 5). P-values (∗ = ≤0.05, ∗∗ = ≤0.01, ∗∗∗ = ≤0.001) were obtained by one-way ANOVA with Tukey’s significance test. Data are represented by the mean ± SEM.
Figure 8
Figure 8
dMP administration in recent-onset NOD mice reverses T1D for a limited time period. Newly diabetic NOD mice (≥240 mg/dL on consecutive days) were serially enrolled in a recent-onset diabetes reversal study. Upon enrollment, animals received a sustained release insulin pellet to temporarily (∼2 weeks) control glycemia and immediately began MP treatment (left: study timeline). Mice received the dMP (n = 11), a soluble bolus of the four factors plus unloaded MPs (n = 8), or no treatment (n = 10) (right). Animals received MP injections (arrows) three times in the first week and three weekly booster injections. Blood glucose levels were monitored twice weekly, and mice were removed from the study upon diabetes recurrence. Survival data are fit using the Kaplan–Meier nonparametric survival analysis model, and statistical analysis was performed via log-rank test (Mantel–Cox method).
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