Poster K18 - Brefeldin A causes cell death and loss of function in islet cells while preventing host immune response and overgrowth of capsules in vivo

Thursday, October 24, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Alexandra J. Gorham (she/her/hers)

Undergraduate Researcher
Johns Hopkins University
Plymouth, Massachusetts, United States

Co-Author(s)

Victor M. Quiroz, Jr. (he/him/his)

PhD Candidate
Johns Hopkins University, United States
NK

Neta Kutner

MSC Student
Technion Israel Institute of Technology, United States
MS

Merna Shaheen-Mualim

Senior Researcher and Lab Manager
Technion Israel Institute of Technology, United States
EO

Edwar Odeh

MSC Student
Technion - Israel Institute of Technology, United States
Shady Farah, PhD

Neubauer Asst. Professor
Technion - Israel Institute of Technology, United States

Primary Investigator(s)

Joshua C. Doloff, MA, PhD (he/him/his)

Assistant Professor
Johns Hopkins University, Depts. of Biomedical Engineering and Materials Science & Engineering, Translational Tissue Engineering Center (TTEC), and the Institute for NanoBioTechnology (INBT)
Baltimore, Maryland, United States

Introduction: For patients with type 1 diabetes, allogeneic islet cell transplants offer a promising alternative treatment to exogenous insulin and even a potential cure for their disease. However, current islet transplants require lifelong immunosuppression, which can lead to severe complications. Thus, there is a need to eliminate the rejection of transplanted islets without the use of systemic immunosuppressants. One method being explored by researchers is islet cell encapsulation in alginate microspheres that protect them from direct immune killing. While alginate alone induces a foreign body response (FBR), we previously reported on the ability of crystallized, slow-release GW2580, a CSFR1 inhibitor, in preventing the FBR to empty alginate capsules for over 1.3 years. However, encapsulation does not protect against indirect immune killing, as rejection eventually occurs when islet cells are added to the capsules. We believe that this rejection is due to an indirect immune response that is graft-dependent, and we aim to identify a slow-release drug that can eliminate this second response. Brefeldin A (BFA), an inhibitor of Golgi apparatus transport, was identified as a candidate drug, as it is known to prevent antigen presentation by dendritic cells (DCs) and has previously been found to eliminate CD8+ T cell activation when alginate-encapsulated OVA cells are cultured with CD8+ T cells and CD8+ DCs in vitro. Thus, the presented work investigates the effects of the encapsulation of slow-release BFA crystals on islet cells in vitro and in vivo.

Materials and

Methods: Primary islets were isolated from Sprague Dawley rats (Charles River) and incubated overnight at 5% CO2 and 37ºC before resuspension in SLG20 alginate (sterile low viscosity high guluronic acid content, PRONOVA) at a concentration of 1000 islet equivalents (IEQ, 150 µm diameter) per 1mL. For non-control conditions, crystallized drug was added to the suspension to a final concentration of 10 mg/mL. The suspension was loaded into a flat-tip syringe for electrospray into a BaCl2 crosslinking solution bath, with voltage adjusted to produce capsules around 1.5 mm in diameter (12 capsules per 10 seconds). For in vitro assays, islets were maintained in RPMI media (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep, with media changes every other day. Viability was measured with either PrestoBlue Cell Viability Reagent (Thermo Fisher) or CellTiter-Glo 3D Cell Viability Assay (Promega) according to the manufacturer instructions. Glucose-Stimulated Insulin Secretion (GSIS) in vitro assays were performed by collecting supernatant from encapsulated islet cells after 1 hour in sequential low (2 mM), high (20 mM), and low glucose solution incubations. Supernatants were stored at -80ºC until their insulin content was quantified using a High Range Rat Insulin ELISA (Mercodia). For in vivo study, capsules were transplanted into the intraperitoneal space of C57BL/6 male mice by laparotomy. After 2 weeks, the capsules were retrieved and stored in paraformaldehyde at 4ºC until imaged via brightfield or immunofluorescence. Viability stains Ethidium homodimer (dead) and Calcein AM (live) were used at 4μM and 2μM respectively.

Results, Conclusions, and Discussions: The effects of BFA on islet cell functionality and viability were evaluated in vitro. Encapsulated islets with either no drug, GW2580 crystals, or BFA crystals were assessed for viability on days 1, 3, and 7 by the PrestoBlue assay and day 10 by the terminal CellTiter-Glo assay. While all three groups demonstrated a decline in viability, the islet alone control and the GW2580 groups sustained viable cells over the 10-day course, whereas the BFA group showed no significant difference in viability with plain media at 3 days and onwards (Fig. 1A-B). GSIS was also performed on days 1 and 3 for the islet alone control and BFA crystal groups since GW2580 has previously been shown to not interfere with GSIS. Control islets demonstrated glucose responsiveness, though producing less insulin overall at day 3 likely due to the drop in viability (Fig. 1C). In contrast, the BFA group was unable to respond to glucose levels at either day 1 or day 3, indicating an acute decline in islet cell functionality with the crystallized BFA (Fig. 1C). However, in vivo studies differ from in vitro by having increased diffusion and extracellular fluid turnover. Thus, encapsulated islets with BFA or GW2580 crystals were transplanted in vivo, and islet viability as well as immune response to the graft was evaluated. Capsules were imaged before transplant into C57BL/6 male mice and then retrieved and imaged again after 2 weeks. Those with GW2580 sustained some immune cell overgrowth while those with BFA largely did not, but BFA continued to have cytotoxic effects on islet cells (Fig. 2). While encapsulated BFA prevented capsule overgrowth, its efficacy for use with islet cell transplants is minimal given its negative effects on islet viability and function. Experiments are now underway to evaluate other compounds that can more specifically prevent the indirect killing of islet cells while also preserving necessary islet cell processes and functionality.

Acknowledgements (Optional):