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SPADE Analysis Reveals The Recruitment Of Rare Immune Cell Subtypes To Site Of Injury Following Treatment With Immunomodulatory Hydrogels
Nusaiba Baker*, MS1,2, Mary Caitlin P. Sok*, PhD1,2, Claire McClain2, Hong Seo Lim2, Thomas Turner2, Lauren Hymel2, Molly Ogle2, Claire Segar, PhD1, Andrés Garcia, PhD2, Peng Qiu, PhD2, Edward Botchwey, PhD2.
1Emory University, Atlanta, GA, USA, 2Georgia Institute of Technology, Atlanta, GA, USA.

PURPOSE - Wound care following surgical procedures involves careful management, reducing risk of infection, and maximizing tissue oxygenation. Even with new strides in biological and synthetic products, achieving adequate wound care remains an important topic of interest. In this study, we develop an injectable hydrogel to promote wound resolution while increasing polarization of immune subsets towards healing phenotypes. These poly(ethylene glycol)-maleimide (PEG-MAL) hydrogels are capable of locally delivering therapeutic doses of the specialized pro-resolving mediator aspirin-triggered resolvin D1 (AT-RvD1) and the immunomodulatory cytokine interleukin 10 (IL-10) to enrich the injured tissue niche. To objectively uncover cellular heterogeneity from flow cytometry data, we utilized Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a computational dimensionality reduction technique. This method highlighted established responses to wound healing, and, more notably, revealed novel aspects of the wound healing cascade in response to immunomodulatory treatment.
METHODS - The murine dorsal skinfold window chamber model was used to monitor vascularization and wound healing in response to hydrogel treatment. Each animal received an unloaded control hydrogel on the caudal side of the window chamber, and a loaded hydrogel rostrally. Loaded gels contained either IL-10, AT-RvD1, or both. At days 1, 3, and 7, dorsal tissue was excised and digested for flow cytometry. SPADE analysis was performed on manually gated single cell events to identify proportions of cells from the innate and adaptive immune system.
RESULTS - We show that AT-RvD1 and IL-10 alone are able to modulate the recruitment of various pro-inflammatory and pro-regenerative immune cells, but dual delivery of these factors enhances the recruitment of pro-regenerative immune cells, including M2 macrophages and tolerogenic dendritic cells, suggesting a synergistic interplay. Moreover, novel computational methods revealed the recruitment of rare immune cell subtypes, particularly from the adaptive immune system, to the site of injury following immunomodulatory hydrogel treatment. These findings suggest a promising method to target pro-regenerative cells from different branches of the immune system, and this treatment has the potential to enhance tissue regeneration and prevent wound healing complications after skin tissue injury.
CONCLUSIONS - Biomaterial implants to deliver cells or molecules capable of recruiting and promoting the host immune response after injury can be instrumental to the restoration of tissue homeostasis and the promotion of wound healing. This dual-delivery system has the potential to improve therapeutic healing outcomes via synergy of cellular recruitment and polarization processes. Moreover, dimensionality reduction techniques, such as SPADE, provide novel and objective analytical approaches for analyzing high-dimensional data. We applied SPADE to mouse flow cytometry data and demonstrated that SPADE can be used to identify functional changes in response to treatment. Interestingly, SPADE enabled the identification of unexpected immune cell populations. This allows for the future development of immunomodulatory treatments that tailor the immune response and enhance the process of healing.


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