(M1230-10-54) 3D-Printed Microneedles Delivering Regulatory Immune Cues to Antigen-Presenting Cells in the Skin Promote Immune Tolerance

Purpose: Autoimmune diseases, including multiple sclerosis (MS), type 1 diabetes, and lupus, impact at least 50M people in the US.¹ In healthy individuals, immune tolerance prevents attacks against host tissues. However, during autoimmune diseases such as MS, dysfunctional immune cells attack host self-molecules termed self-antigens; this process leads to inflammation and tissue destruction. There is no cure for these diseases, and the current therapies are non-specific, leaving patients susceptible to immunosuppressive side effects. An experimental idea in treating autoimmune diseases is to drive tolerance specific to self-antigen (e.g., myelin in MS) without hindering normal immunity.^2,3 One strategy to achieve this goal is to co-deliver self-antigen with immune cues that regulate cellular pathways in specialized immune cells called antigen-presenting cells (APCs). Specialized APC subsets, dendritic cells (DCs), are highly concentrated in the skin, and these DCs are powerful mediators of immune response. DCs can migrate to secondary lymphoid organs such as lymph nodes (LNs) to bias self-reactive T cells away from inflammatory phenotypes.⁴ These characteristics make the skin a rich immunological niche for driving tolerance. Microneedles (MNs) are micron-sized needles that deliver cargo to the skin and are an efficient, pain-free way to target the DCs in the skin. Here, we show 3D-printed MNs delivering self-antigen and specific immune cues to DCs in the skin bias these populations towards tolerogenic phenotypes to promote selective immune tolerance.

Methods: We started by loading self-antigen (myelin peptide: MOG) along with two distinct regulatory immune cues (structures omitted due to University IP processes) known to promote tolerogenic phenotypes in DCs into MNs. Subsequently, to check the effect of the immune cues on the phenotypes of DCs, we cultured monocyte-derived DCs (moDCs) with these immune cues. We characterized their inflammatory and tolerogenic profiles using flow cytometry and ELISAs. In order to demonstrate the role of these DCs in transporting the cargo to the draining lymph nodes and thus modulating the immune response, we employed a combination of in vivo imaging system (IVIS) analysis and clodronate depletions. Clodronate depletion depletes APC populations at the injection site, enabling us to analyze the necessity of DCs in trafficking cargo. In brief, clodronate liposomes were injected intradermally on both sides of the flank exactly in the area where the MNs will be applied. 1hr after clodronate liposomes were injected, the mice were treated with the cargo-loaded MNs. 3 days later, 1hr after clodronate injection, mice skin and inguinal LNs were harvested and imaged using IVIS to measure cargo accumulation and trafficking.  Next, to test the therapeutic efficacy of these MNs, we used a pre-clinical model of MS (experimental autoimmune encephalomyelitis (EAE)). In this model, we treated mice on days 8,11, and 14 and compared efficacy to untreated mice.

Results: We show that MNs can be loaded with distinct immune cues and self-antigen (Figure 1 A, B). Subsequently, using ELISAs we illustrate that treatment with these immune cues leads to an increase in regulatory cytokines such as IL-10 by moDCs that were first activated by LPS (an inflammatory signal), in contrast to moDCs treated only with LPS (Figure 1C). Additionally, treated DCs exhibit lower expression of CD80, a marker of DC activation, indicating a shift towards more tolerogenic phenotypes. Furthermore, clodronate depletion and IVIS imaging revealed that these DCs are, to some extent, responsible for trafficking the cargo to the LNs. Specifically, mice treated with clodronate did not exhibit any cargo signal, while those without the clodronate treatment showed cargo signals, as measured by IVIS imaging (Figure 1D). Finally, we observe that treatment with these cargo-loaded MNs leads to increased efficacy compared to untreated controls in a pre-clinical model of MS. This is shown by reduced clinical scores in the treated mice, where higher clinical scores indicate a higher level of paralysis (Figure 1E, F).

Conclusion: This data indicates that delivering specific regulatory immune cues and self-antigen to the skin resident DCs via MNs promotes immune tolerance in a pre-clinical model of MS. The findings also suggest that these immune cues bias the DCs towards a tolerogenic phenotype. Our data suggests that these tolerogenic DCs then migrate to the draining LNs to present self-antigen and potentially biased T cells towards a more regulatory phenotype.  Overall, we show that targeting the skin using MNs to deliver self-antigens and immune cues that activate regulatory pathways in DCs in the skin may offer a potential approach to combat autoimmune diseases.

References: [1] The American Autoimmune Related Diseases Association (AARDA), https://autoimmune.org/resource-center/about-autoimmunity/, accessed 7/9/2024
[2] Filippi, M. et al. Multiple sclerosis. Nat. Rev. Dis. Prim. 4, 1–27 (2018)
[3] Brandstadter, R. & Sand, I. K. Neuropsychiatr. Dis. Treat. 13, 1691 (2017)
[4] Wang, X. N. et al. J. Invest. Dermatol. 134, 965–974 (2014)

Acknowledgements: This work was supported by the National Institutes of Health (NIH) # R01AI144667 (C.M.J.), the United States Department of Veterans Affairs (VA) #IK2BX005061(R.S.O.), and the PhRMA Foundation Award #PDDL-1155845 (S.A.S.).
C.M.J. and R.S.O. are employees of the VA Maryland Health Care System. The views reported here do not reflect the views of the VA or the United States Government. C.M.J. has an equity position with Cartesian Therapeutics, Barinthus Biotherapeutics, and Nodal Therapeutics. Beyond these declarations, there is no other Conflicts of Interest to report.
All animal care and experiments were carried out using protocols approved and overseen by the University of Maryland IACUC committee in compliance with local, state, and federal guidelines.

Figure 1. MNs delivering specific regulatory cues along with self-antigen lead to improved efficacy in pre-clinical models of MS. MNs offer an ability to be loaded with different immune cues, A) MOG + Immune cue 1, and B) MOG + Immune cue 2. C) Il-10 production (pg/ml) by moDCs after treatment with these immune cues, measured using ELISA. * represents p<0.05. Statistics were carried out using one-way ANOVA with Tukey post-correction for multiple comparisons. D) IVIS images of draining LNs 3 days after treatment with MNs loaded with fluorescently labeled antigen and immune cues, with or without clodronate (clod) depletion (N=5 per group). Mean clinical scores of mice after treatment with MNs loaded with E) MOG + Immune cue 1 and F) MOG + Immune cue 2. Increasing scores indicate an increased level of paralysis. Arrows indicate treatment timepoints. Scores were assigned using a standard scale: 0-asymptomatic, 1-flaccid tail, 2-hind limp paresis/partial paralysis, 3-total hind limb paralysis, and 4-hind and front limb paralysis. * represents p<0.05. Statistics were carried out using multiple t-tests at each time point for the clinical scores.