(W0930-02-12) Oral Tolerance Mediated by Phosphatidylserine Containing Lipid Nanoparticle

Purpose: Clinical conditions such as autoimmunity and allergies are caused by unwanted immune responses against self and non-self proteins. Further immunogenicity against therapeutic proteins in the form of anti-drug development ADA significantly impacts the clinical effectiveness of drug treatment. Therefore, a tolerance induction approach is a novel strategy to prevent and reverse this unwanted immune response. We have developed a Phosphatidylserine (PS) -based lipid nanoparticle, that emulates the surface properties of an apoptosis cell, to “teach” the immune system to tolerate a protein by converting an “Immunogen” into a “Tolerogen”. Previously, we have demonstrated PS liposome has regulated unwanted immune response across multiple antigens via subcutaneous, intravenous, or oral administration, even after repeated antigen rechallenge.(Figure 1). Interestingly, single-chain PS nanoparticles are effective in inducing oral tolerance induction but double-chain PS did not. However, the mechanistic basis of this observation is not clear. We hypothesize that M-cell mediated difference in uptake of double vs single chain PS contributes to the difference in oral tolerance. Therefore, this study aims to investigate the impact of double vs single-chain PS on the mechanism of uptake and oral tolerance.

Methods: Ovalbumina OVA is used as a model antigen to understand mechanism of oral tolerance mediated by PS. 18:1 PS nanoparticles using phosphatidylserine (either double chain or single chain) mixed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at 30%:70% mole ratio using thin film dehydration and rehydration methods, followed by high-pressure membrane extrusion, to generate uniformed and homogenous nanoparticle with 100nm diameter. To investigate the tolerogenic potential toward OVA antigen using PS nanoparticles, we immunized Swiss Webster mice continuously for 9 weeks. (Figure 2A) Starting on week 5, mice were rechallenged with 2ug of OVA protein subcutaneously for 4 weeks and then terminated on week 11. Blood samples were collected to measure anti-OVA antibodies. Spleen, mesenteric lymph nodes, and bone marrow were harvested at the terminal, and assessed frequency of Tregs and plasma cells after PS treatment. To investigate PS nanoparticle uptake into Microfold (M cells) for immune cell capture, we surgically ligated both ends of the intestine containing Peyer’s patches 1 cm apart, followed by the injection of 100ul of both fluorescently labeled PS nanoparticles and OVA protein for 1 hour. Peyer’s patches were harvested, digested, and analyzed using flow cytometry. Immune cell uptake including dendritic cells (CD11c), macrophages(F4/80), T cells (CD3) & B cells (B220) were measured and estimated using flow cytometry and confocal imaging. Statistical analysis was performed in GraphPad Prism using a non-parametric Mann-Whitney U test.

Results: PS-mediated oral tolerance was observed by elevated Tregs expression that negatively correlated with antibody responses. Our data showed that mice prophylactically treated with 18:1 LysoPS increased Foxp3 Tregs activation in the mesenteric lymph node (MLN) with a mean frequency of 70% of Foxp3 Treg activated compared to 58% of Foxp3 Treg in mice treated with BrainPS. In addition, mice that received 18:1 LysoPS also increased Lap expressing Treg in both MLN and spleen with a mean frequency of 5% in both MLN and Spleen compared to mice treated with BrainPS with a mean frequency of 1.8% in MLN and Spleen. (Figure 2) The increase in the Tregs population was also correlated with a reduced frequency of antibody-producing plasma cells. Mice treated with 18:1 LysoPS have a lower frequency of CD138-expressing plasma cells with 10% plasma cells in the bone marrow compared to BrainPS-treated mice with 23% of the plasma cells. Correspondingly, mice treated with LysoPS developed lower anti-ova IgG1 antibodies than the buffer, free ova, and BrainPS groups. Tregs can secret immunosuppressive cytokines il-10 and TGFb that suppress autoreactive T cell proliferation and engagement with B cells. Furthermore, we investigated the role of intestinal uptake on oral tolerance, especially M cells containing Peyer’s Patches (Figure 3A, B). Mice exposed to LysoPS nanoparticles increased immune cell uptake compared to mice that received BrainPS.

Conclusion: The M-cell mediated uptake and access to immune cells in Peyer’s patches contributes to the difference in oral tolerance between double and single-chain PS molecules.

References: 1. Dingman, R. and S.V. Balu-Iyer, Immunogenicity of Protein Pharmaceuticals. J Pharm Sci, 2019. 108(5): p. 1637-1654.
2. Nguyen, N.H., et al., Rational design of a nanoparticle platform for oral prophylactic immunotherapy to prevent immunogenicity of therapeutic proteins. Sci Rep, 2021. 11(1): p. 17853.
3. Glassman, F.Y., et al., Phosphatidylserine Is Not Just a Cleanup Crew but Also a Well-Meaning Teacher. J Pharm Sci, 2018. 107(8): p. 2048-2054.
4. Lim, H.W., et al., Cutting edge: direct suppression of B cells by CD4+ CD25+ regulatory T cells. J Immunol, 2005. 175(7): p. 4180-3.
5. Butera, A., et al., CD3+CD4+LAP+Foxp3-Regulatory Cells of the Colonic Lamina Propria Limit Disease Extension in Ulcerative Colitis. Front Immunol, 2018. 9: p. 2511.

Acknowledgements: Funding was provided by the National Institute of Allergy & Infectious Disease RO1 AI169296 to SVB. The authors would like to acknowledge the UB Optical Imaging and Analysis Facility for the use of the flow cytometer and confocal fluorescence imaging.


Figure 1: Following oral administration of PS nanoparticle with protein immunogen, PS nanoparticle will uptake into Peyer’s patch via M cells and expose to antigen-presenting cells, including dendritic cells (DC), B cells, and macrophages. DC will capture and process PS-containing antigens and then migrate to the MLN, present to T cells, and generate Tregs for tolerogenic responses. Tregs will induce tolerogenic effects by suppressing effector T cells and B cells and secreting immunosuppressive cytokine il-10 and Tgf-ᵝ.


Figure 2: PS nanoparticle-induced oral tolerance by generating Tregs and suppressed antibody-producing plasma cells. (A) Oral Prophalytic treatment strategy in Swiss Webster mice. (B) Frequency of CD4+Foxp3-LAP+ Tregs out of CD4+Foxp3- population in the spleen. Data are presented with 8 animals per group (mean ± SD). (C) Frequency of B220+CD138+plasma cells out of B220 population in Bone Marrow. Data are presented with 8 animals per group (mean ± SD). (D) Anti-OVA IgG1 antibody titer data were presented with 8 animals per group (mean ± SD). Statistical analysis was performed in GraphPad Prism using a non-parametric Mann-Whitney U test. * p<0.05, ** p<0.01, *** p<0.001


Figure 3: Structure difference in PS nanoparticle impacts M cell uptake to immune cells. (A) PS nanoparticle uptake into M cells containing Peyer’s patches for immune cell exposure was captured using intestinal gut loop assay using flow cytometry and with internal flow cytometry counting beads for internal control. We gated both fluorescently labeled PS nanoparticle and OVA protein as the target population and subgated in each immune cell population. Data presenting with 6 intestinal loops from 3 mice in each group. (mean ± SEM) (B) Intestinal gut loops assay to capture PS nanoparticle uptake into M cells containing Peyer’s patches using confocal fluorescence imaging techniques with z-stacking. Green represents GP-2 expressing FITC, a biomarker for M cells, Yellow represents PS nanoparticles, and red represents OVA protein.