Phosphatidylserine-mediated oral tolerance

Abstract

Phosphatidylserine (PS) is an anionic phospholipid exposed on the surface of apoptotic cells. The exposure of PS typically recruits and signals phagocytes to engulf and silently clear these dying cells to maintain tolerance via immunological ignorance. However, recent and emerging evidence has demonstrated that PS converts an "immunogen" into a "tolerogen", and PS exposure on the surface of cells or vesicles actively promotes a tolerogenic environment. This tolerogenic property depends on the biophysical characteristics of PS-containing vesicles, including PS density on the particle surface to effectively engage tolerogenic receptors, such as TIM-4, which is exclusively expressed on the surface of antigen-presenting cells. We harnessed the cellular and molecular mechanistic insight of PS-mediated immune regulation to design an effective oral tolerance approach. This immunotherapy has been shown to prevent/reduce immune response against life-saving protein-based therapies, food allergens, autoantigens, and the antigenic viral capsid peptide commonly used in gene therapy, suggesting a broad spectrum of potential clinical applications. Given the good safety profile of PS together with the ease of administration, oral tolerance achieved with PS-based nanoparticles has a very promising therapeutic impact.

Figure 1:. Uptake of PS and Lyso-PS nanoparticles by different immune cell subsets in the Peyer’s patches.

Twenty μg of OVA-AF488 was loaded into rhodamine-labeled PS and Lyso-PS nanoparticles. One-cm section of the mouse’s intestinal loop containing the Peyer’s patches was surgically ligated and formulations were injected into this loop. The Peyer’s patches were harvested 1 h post-injection, digested, and stained with viability dye, B220, CD3, F4/80, CD11c, and CD103 antibodies prior to flow cytometry analysis. Data were presented as the frequency of cells double-positive for OVA and nanoparticle signal at respective cell populations (mean ± SD). Statistical analysis was performed using a one-way ANOVA test.

Figure 2:. Lyso-PS nanoparticles reduce immunogenicity of multiple antigens.

Swiss Webster mice were continuously pre-exposed to either 1 μg antigens alone or antigens associated with Lyso-PS nanoparticles via oral gavage for 9 weeks. Starting at week 6, mice also received four weekly subcutaneous rechallenge injections with 1 μg free antigens 24 h post oral treatment. After a 2-week wash-out period, blood was collected for titer analysis. Titer levels against (A) type II collagen and (B) AAV8 immunogenic peptide were presented as mean ± SEM. Statistical significance was denoted by P < 0.05 (*) or P < 0.005 (**) by an unpaired t-test.

Figure 3:. Proposed mechanism of Lyso-PS-mediated oral tolerance.

Lyso-PS nanoparticles gain entry into the laminar propria (1) through intact enterocytes and (2) by M cells. In the laminar propria, antigens associated with Lyso-PS nanoparticles can be processed directly by TIM4⁺CD103⁺ DCs or long-lived TIM-4⁺CX3CR1^hi macrophages, which are then transferred to the neighbor migratory CD103⁺ DCs in a connexin 43-dependent manner. In the Peyer’s patches, Lyso-PS nanoparticles exiting M cells are processed by CD103⁺ DCs, CX3CR1^hi macrophages, and TIM-4⁺ CX3CR1^hi macrophages. The third route of antigen presentation likely occurs is via the extended dendritic projections of CX3CR1^hi macrophages across the mucosal epithelium. Following antigen presentation, tolerogenic CD103⁺ DCs migrate to the mesenteric lymph nodes and promote the expansion of antigen-specific T_regs. These T_regs then either migrate back to the laminar propria for local tolerance induction or enter the periphery to induce systemic tolerance. T_regs reduce anti-drug antibody formation by suppressing the effector T cells, B cells, and antibody-producing plasma cells via the production of anti-inflammatory cytokines.