Elucidating the spatial and temporal immune response to immunomodulatory materials

Abstract

Immune modulation therapy has come to the forefront of basic science and clinical research to either interrupt immune dysregulation or induce a specific immune response. This dissertation presents the results of using hydrogel-based biomaterial delivery of therapeutic factors to modulate endogenous mechanisms of innate and adaptive immunity. Our studies integrate pre-clinical mouse models of chronic inflammation and inflammation driven disease states with multi-dimensional flow cytometry analysis to characterize how the local immune cell responses can be manipulated by altering the spatial and temporal presentation of immune modulatory factors. In Aim 1, we designed a synthetic poly(ethylene) glycol (PEG)-based hydrogel to release specialized pro-resolving lipid mediator aspirin triggered resolvin D1 (AT-RvD1) and recombinant human interleukin 10 (IL-10). We hypothesized that an initial quick release of AT-RvD1 after in situ hydrogel gelation would target the myeloid first responders followed by a more gradual release of thiolated IL-10 to influence the activity of cells involved in the later stages of the inflammatory response, mimicking the natural progression of the immune cascade. Here, we utilized a side-by-side internally controlled design wherein bioactive hydrogels were injected adjacent to control hydrogels in the dorsal skinfold window chamber model. We profiled the recruitment of circulating immune cell subsets using flow cytometry, followed by analysis using Spanning-tree Progression Analysis of Density-normalized Events (SPADE), an unbiased clustering algorithm used for dimensionality reduction of high-throughput data. SPADE creates a 2D visualization of complex datasets in a branched “tree” without much needed oversight from the user, which allows for the elucidation of cellular heterogeneity of the immune response, as well as for the identification of rare cell subsets. Aim 1 studies show that the recruitment and re-education of mononuclear phagocytes by dual hydrogel therapy localizes pro-regenerative immune subsets to the hydrogel. The manipulation of innate and adaptive immune phenotypes by IL-10 and AT-RvD1 in the local microenvironment is a promising strategy for enhanced healing in the post-surgical wound. In Aim 2, we utilized alginate and chitosan microparticles to deliver Salmonella AvrA to the site of intestinal inflammation. AvrA is an effector molecule that inhibits activation of pro-inflammatory immune factors. These anti-inflammatory properties make AvrA a promising therapeutic for the amelioration of inflammation. We hypothesized that oral delivery of AvrA microparticles would inhibit the immune response in the gut-associated lymphoid tissue (GALT) but would not lead to a systemic immune suppression. Here, we observed reduced neutrophil infiltration and intracellular TNF expression in the gastrointestinal epithelium. Moreover, SPADE revealed decreased pro-inflammatory T-cell effector markers following AvrA treatment. These data demonstrate the use of biomaterial-based methods to deliver bioactive molecules capable of directing the host immune response after injury, thus modulating processes critical to the restoration of tissue homeostasis. Furthermore, we demonstrate the importance of analytical techniques like SPADE for elucidating cellular heterogeneity. In summary, we show how the purposeful manipulation of immune cell fates could therefore be useful for promoting an immune response or inhibiting inflammation and promoting healing.