Poster O4 - Engineered Polysaccharide A Nanoparticles for Inflammatory Bowel Disease Therapy

Introduction: Inflammatory bowel disease (IBD) is a series of chronic inflammation of the gastrointestinal tract including ulcerative colitis and Crohn’s disease, which is characterized by disrupted tract mucosa structure and altered gut microbial composition. It affects more than 3 million Americans and is estimated to cost more than $500M annually (Kahn-Boesel et al., 2022). Existing therapeutic interventions mainly focus on anti-inflammatory drugs like mesalamine and immunosuppressives such as methotrexate, but they are limited by short-term efficacy and unwanted adverse side effects such as increased risk of infection. Thus, there is an urgent need to develop an alternative IBD treatment that is effective and not immune suppressive. Recently, polysaccharide A (PSA), a molecule derived from the gut commensal Bacteroides fragilis (B.F.) has been reported to show great therapeutic potential in many mouse models of inflammation, relying on its tolerogenic properties by inducing regulatory T cells (Tregs) (Ramakrishna et al., 2019, Ochoa-Reparaz et al., 2010) The tolerogenic properties, as well as its polymeric structure, qualify PSA as a promising biomaterial for the formulation of tolerogenic nanoparticles. Here, we hypothesized that PSA nanoparticles (PSA NPs) encapsulating an IBD-relevant autoantigen - ovalbumin (OVA), will be a more efficacious and safer therapy due to the PSA NP's ability to induce OVA-specific Treg differentiation and to protect the OVA from mast cell activation mediated by IgE receptor. Overall, we hypothesized that this nanoplatform could promote antigen-specific tolerance by activating dendritic cells (DCs) to induce Treg differentiation, ultimately alleviating inflammation associated with IBD.

Materials and

Methods: Isolation and Preparation of PSA: PSA crude product was extracted from B.F. strain ΔmpiM44, an engineered strain of B.F. that was modified to only express PSA in its capsule, using water-phenol extraction (Alvarez et al., 2019). Next, the crude product was treated with RNase, DNase, and protease to remove impurities like nucleic acids. Finally, the size exclusion chromatography (SEC) column was used to separate PSA from LPS, and the collected fraction was visualized via gel electrophoresis, staining with ProQ Emerald 300 Lipopolysaccharide gel stain kit (P20495). Gel images were obtained using Biorad Gel Doc system.
Characterization of PSA: The NMR analysis was performed to determine the chemical composition structure of the PSA final product by comparing it with the previously published data (Baumann et al., 1992). As a complement, the microplate-based phenol sulfuric assay was performed to validate the lyophilized PSA weight by comparing the absorbance (λ=490nm) to the standard curve constructed using PSA of known amounts. mTLR2 assay was performed based on the protocol available from Invivogen using HEK-BlueTM mTLR2 cells. The specificity towards TLR2 was determined by calculating the absorbance (λ=623nm) difference of treatments with anti-mTLR2 antibody versus treatments without the antibody.
DC phenotype: BMDCs were harvested from C57LB/6 mouse bone marrow following a previously described DC culture protocol (Sauter et al., 2022). At day 10, DCs in 6-well plates were treated in triplicate with PBS, LPS and PSA respectively. All treatments were added to a final concentration of 50ug/mL and incubated for 2 days before flow cytometry.

Results, Conclusions, and Discussions: PSA is purified from LPS through the SEC Column: Analysis of the collected fractions from the SEC column via gel electrophoresis clearly showed that PSA was attained with high purity without LPS, and the fractions containing LPS-free PSA were combined and assessed for chemical structure. The 1H NMR spectrum was consistent with the previous literature data, confirming the chemical structure of extracted PSA.
PSA shows TLR2-specific activation in the mTLR2 assay: Different treatments of mTLR2 cells showed that extracted PSA could elicit a TLR2 response, proven by the significant color difference observed for extracted PSA in comparison with the untreated (DIW), while LPS could cause activation in the presence or absence of the mTLR2 antibody.
PSA promotes the expression of CD86 and MHCII on DC: Flow cytometry data revealed that the PSA-treated group had a higher expression of both CD86 and MHCII in CD11c+ cell populations compared to the untreated (PBS) group.
Current results collectively indicate that the PSA we extracted from B.F. is purified from LPS impurity and can specifically bind with TLR2. DCs treated with PSA promote the expression of CD86 and MHCII, which is necessary for the induction of antigen-specific Tregs. Overall, the completion of PSA extraction and characterization provides a material basis for the next step of constructing tolerogenic PSA NPs, and it is an important step for in vitro studies.
Although pure PSA was extracted from B.F., we will continue to optimize the yield and quality of PSA, e.g., to improve the TLR2 specificity of PSA as well as to examine the effect of PSA on other tolerogenic DC markers such as ICOSL and iNOS. In addition, our next step will be working on the fabrication of tolerogenic nanoparticles, PSA NPs, and the characterization of both their physiochemical properties and the TLR2-specific DC activation ability. Finally, we aim to understand the in vivo distribution of PSA NPs after oral gavage in mice model, and to explore the efficacy of PSA NPs loaded with the IBD-associated antigen OVA on IBD through DSS-colitis mice model studies.

Acknowledgements (Optional): Kahn-Boesel, O., Cautha, S., Ufere, N. N., Ananthakrishnan, A. N., & Kochar, B. (2022). A narrative review of financial burden, distress, and toxicity of inflammatory bowel diseases in the United States. Official journal of the American College of Gastroenterology| ACG, 10-14309.

Ramakrishna, C., Kujawski, M., Chu, H., Li, L., Mazmanian, S. K., & Cantin, E. M. (2019). Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis. Nature communications, 10(1), 2153.

Ochoa-Reparaz, J., Mielcarz, D. W., Wang, Y., Begum-Haque, S., Dasgupta, S., Kasper, D. L., & Kasper, L. H. (2010). A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease. Mucosal immunology, 3(5), 487-495.

Alvarez, C. A., & Cobb, B. A. (2019). Purification of capsular polysaccharide complex from gram-negative bacteria. Bacterial Polysaccharides: Methods and Protocols, 25-35.

Baumann, H., Tzianabos, A. O., Brisson, J. R., Kasper, D. L., & Jennings, H. J. (1992). Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy. Biochemistry, 31(16), 4081-4089.

Sauter, M., Sauter, R. J., Nording, H., Olbrich, M., Emschermann, F., & Langer, H. F. (2022). Protocol to isolate and analyze mouse bone marrow derived dendritic cells (BMDC). STAR protocols, 3(3), 101664.

Kakwere, H., Harriman, R., Pirir, M., Avila, C., Chan, K., & Lewis, J. S. (2022). Engineering immunomodulatory nanoplatforms from commensal bacteria-derived polysaccharide A. Journal of Materials Chemistry B, 10(8), 1210-1225.