(M1230-07-37) Local Controlled Delivery of an A3 Receptor Agonist Reduces Inflammation Associated with Contact Hypersensitivity

Purpose: Allergic contact dermatitis (ACD), a T cell-mediated inflammatory skin disease, impacts almost 20% of the adult population (1, 2). In ACD, repetitive exposure to chemical allergens (e.g., nickel) causes inflammation, resulting in cutaneous pruritis and edema (3). To prevent and/or treat ACD, patients are first encouraged to remove and/or avoid environmental allergens (1, 2). Otherwise, ACD can be treated through repetitive application of topical corticosteroids and/or calcineurin inhibitors (1, 2). However, these approaches suppress immune responses non-specifically and can result in a plethora of side effects (e.g., striae, atrophy). Thus, there is a clinical need for novel approaches that suppress immune responses to chemical allergens while limiting side effects. Our bodies, in contrast, employ extremely sophisticated strategies to regulate immune responses. For instance, a native cell population known as regulatory T cells (Tregs) promotes immunosuppression through the conversion of extracellular ATP (eATP), an inflammatory cue released by dying or active cells, to adenosine (Ado), an anti-inflammatory signal. Ado signaling can reduce secretion of pro-inflammatory cytokines (IL-12, TNFa, IL-6, IL-8) from monocytes and monocyte-derived dendritic cells (DCs), potentially blocking naïve T cell differentiation toward effector T cell subtypes (Th1, Th17, Tc1) (4, 5). In this study, we first sought to study the immunosuppressive effects of soluble IB-MECA, an adenosine receptor agonist, on immune cells implicated in the pathogenesis of ACD (e.g., dendritic cells, T cells, etc.) in vitro. Following, we sought to assess the effects of enhancing adenosine signaling in contact hypersensitivity (CHS), an in vivo model of ACD, through local, controlled delivery of IB-MECA.

Methods: Microparticle fabrication and characterization – Microparticles were fabricated using a well-documented single emulsion solvent evaporation method. Microparticle size, surface morphology, and release kinetics were characterized through volume impedance measurements (Beckman Coulter Counter), scanning electron microscopy (SEM), and absorbance assays, respectively. In vivo studies – For all in vivo studies, a murine model of CHS was used. Briefly, microparticles were injected -1 days prior to sensitization. On day 0, mice were sensitized with 0.5% dinitrofluorobenzene (DNFB) painted topically on the ears. Ear thickness measurements were taken with calipers during the sensitization period (days 0 to 5). On day 10, mice were challenged with 0.2% DNFB painted topically on the ears. Ear thickness measurements were taken after challenge to assess swelling (days 10 to 12). For histology, cryosectioned ear sections from challenge (D12) were stained with hematoxylin and eosin and imaged. For phenotypic analysis of T cells in draining lymph nodes (DLNs), ear-draining cervical lymph nodes were harvested and processed to create a single cell suspension, stained for T-cell markers (CD4, CD8, Tbet, Foxp3, etc.) and analyzed with a flow cytometer (BD LSR II).

Results: IB-MECA was encapsulated in polymeric microparticles for controlled release (Figure 1). IBMECA-MPs demonstrate spherical morphology and an average size distribution of ~10 µm (Figure 1A, 1B). Release assays demonstrate controllable release of IBMECA for a period of at least two weeks (Figure 1C). Local administration of IBMECA-MPs in an in vivo model of CHS was found to reduce ear swelling during sensitization when compared to Blank-MPs, as evidenced by reduce ear thickness measurements (Figure 2B). IBMECA-MPs also increased the ratio of CD4+ Foxp3+ Tregs to effector T cells (Th1 and/or Tc1) in cervical draining lymph nodes after sensitization when compared to Blank-MPs (Figure 2C). Comparably, local administration of IBMECA-MPs also reduced ear swelling after challenge when compared to Blank-MPs (Figure 3B). Histology images demonstrate reduced epidermal thickening for IBMECA-MPs when compared to Blank-MPs (Figure 3C).

Conclusion: Altogether, these results demonstrate the efficacy of IBMECA-MPs in a model of allergic contact dermatitis. It can be concluded that IB-MECA can be encapsulated within polymeric microparticles for controlled release. Furthermore, IBMECA-MPs were shown to reduce inflammation associated with allergic contact dermatitis when compared to Blank-MPs, as evidenced by reduced ear thickness measurements during allergen sensitization and after allergen challenge. Results from this study also suggest possible mechanisms for the reduction in inflammation, as IBMECA-MPs increased the ratio of Tregs to effector T cells when compared to Blank-MPs.

References: . S. F. Martin, T. Rustemeyer, J. P. Thyssen, Recent advances in understanding and managing contact dermatitis. F1000Res 7, (2018).
2. S. C. Balmert et al., In vivo induction of regulatory T cells promotes allergen tolerance and suppresses allergic contact dermatitis. J Control Release 261, 223-233 (2017).
3. D. H. Kaplan, B. Z. Igyarto, A. A. Gaspari, Early immune events in the induction of allergic contact dermatitis. Nat Rev Immunol 12, 114-124 (2012).
4. W. I. Effendi, T. Nagano, K. Kobayashi, Y. Nishimura, Focusing on Adenosine Receptors as a Potential Targeted Therapy in Human Diseases. Cells 9, (2020).
5. C. Silva-Vilches, S. Ring, K. Mahnke, ATP and Its Metabolite Adenosine as Regulators of Dendritic Cell Activity. Front Immunol 9, 2581 (2018).

Acknowledgements: The first and corresponding authors would like to thank the Nanofabrication and Characterization Facility at the University of Pittsburgh for training on and access to scanning electron microscopy machines used in this study.

Figure 1. IB-MECA-MPs characterization. (A) Scanning electron microscopy image of IBMECA-MPs (scale bar = 10 um, 901x magnification). (B) Size distribution of IBMECA-MPs. (C) Cumulative release profile of IBMECA-MPs demonstrating sustained release of IB-MECA over 14 days.

Figure 2. Prophylactic treatment with IBMECA-MPs reduces inflammation during sensitization. (A) Experimental timeline. (B) Ear thickness measurements for Blank-MPs (n = 5) and IBMECA-MPs (n = 5) taken during sensitization. (C) Ratio of Foxp3+ Treg to Tbet+ Tc1 and/or Th1 effector T cells in DLNs 5 days post-sensitization.

Figure 3. Prophylactic treatment with IBMECA-MPs reduces inflammation after challenge. (A) Experimental timeline. (B) Change in ear thickness measurements for Blank-MPs (n = 5) and IBMECA-MPs (n = 5) taken after challenge. (C) Representative ear skin histology (H&E) from 2 days post-challenge for Blank-MPs and IBMECA-MPs.