Topical Modulation of Immune-Mediated Wound Healing Using the Naturally Occurring Antigen Alpha-Gal
Omer Kaymakcalan, MD1, Andrew Abadeer, MEng1, Alice Harper, BA1, Sarah Karinja, BA1, Julia Jin, BS1, Jaime Bernstein, BS1, Yoshiko Toyoda, BA1, Alexandra Lin, BA1, John Morgan, PhD1, Uri Galili, PhD2, Jason Spector, MD1.
1Weill Cornell Medical College, New York, NY, USA, 2University of Massachusetts Medical School, Worcester, MA, USA.
BACKGROUND: Optimizing the rate and quality of wound healing remains a top clinical priority. The most crucial phase of wound healing is the inflammatory stage, with the microscopic debridement and control of foreign microbes and directing the transition to the proliferative phase. An accumulating body of evidence indicates that this process is guided by macrophages, which are in turn attracted to and activated by antibodies and the products of compliment cleaved by antibody-antigen complexes. Furthermore it has been shown that humans are continually and safely exposed to the natural antigen α-gal (Galalpha1-3Galbeta1-(3)4GlcNAc-R), which is ubiquitous in our animal food sources, and approximately 1% of IgG and IgM in human serum are directed against it. We therefore theorized that topical application of α-gal nanoparticles on wound surfaces would stimulate an early, enhanced, and beneficial local immune response and therein accelerate wound
METHODS: Since mice normally produce the antigen α-gal, α-1,3galactosyltrasferase knockout mice (which do not produce the antigen and therefore can be stimulated to produce antibodies against it) were used in all experiments. Mice were immunized to produce anti α-gal antibodies at titers comparable to those in humans. Two 6-mm bilateral dorsal splinted wounds were genreated, which were then treated with α-gal nanoparticles or PBS in a 2% carboxymethyl cellulose carrier, immediately after wounding and again on postoperative day 1. Wounds were harvested on days 1, 2, 3, 6, and 9, sectioned, studied with immunohistochemical (IHC) staining with CD11b and CD31 for macrophage invasion and neovascularization respectively, and studied with hematoxylin and eosin (H&E) staining to determine the extent of keratinocyte migration and granulation tissue deposition. Flow cytometry was performed to evaluate for macrophage phenotypes on day 3, as M2/M1 phenotype have been demonstrated to favorably aid wound healing.
RESULTS: IHC staining demonstrated 2.6, 5.2, and 2.4 fold greater rates of macrophage invasion into α-gal nanoparticle treated wound beds compared to PBS treated controls for postoperative days 1, 2, and 3 respectively, p<0.05. Flow cytometry revealed stable levels of 90% of M2 macrophages in experimental and control arms demonstrating, however when taken into account the two-fold increase in overall macrophages, this indicates twice the ‘healing' M2 macrophages at this time. Furthermore the α-gal nanoparticle treated wound normalized to control levels, demonstrating that the elevation was transient and non-pathologic. Increased vascularization was noted in the treated wounds by post-op day 9. H&E staining demonstrated an accelerated rate of healing, with 4.9% and 100% wound coverage by keratinocytes for α-gal nanoparticle treated wounds on days 3 and 6 respectively compared to 0.4% and 61% coverage for saline treated wounds, p<0.05. An increased rate of deposition of granulation tissue was demonstrated as well in the α-gal treated wounds.
CONCLUSIONS: Topical treatment of wound surfaces with of α-gal containing nanoparticles yielded an early and enhanced macrophage invasion into wound beds which resulted in increased vascularization and accelerated wound closure. This safe and naturally occurring agent shows great promise to enhance both the rate and the quality of wound healing in both normal and pathological states.
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