THERAPEUTIC IMMUNOMODULATION OF MICROGLIA WITH α-GAL NANOPARTICLES: IMPLICATIONS FOR SPINAL CORD INJURIES

<p><a>The pathophysiology of spinal cord injury (SCI) is complex, involving immune cells like macrophages. In their classical activated M1 phenotype, macrophages help in phagocytosis of cell debris, secretion of pro-inflammatory cytokines (IL-6, TNF-α) and generation of reactive oxygen/nitrogen species. </a>On the other hand, anti-inflammatory M2 macrophages aid in tissue repair and remodeling. It has been suggested that the improper transition from the M1 to M2 phenotype post-SCI may lead to an inhibitory microenvironment that prevents proper wound resolution and axonal regeneration. Previous attempts to increase the anti-inflammatory macrophages using external addition of cytokines post-injury, produced limited results as it involved cross talk between cell signaling pathways. Alternatively, we suggest to use an antigen based approach with galactose-alpha-1,3-galactose (α-gal) nanoparticles (liposomes containing α-gal epitopes) to mediate the SCI immune response. α-Gal epitope is a carbohydrate antigen, synthesized in non-primate mammals (mice, rats, pigs, rabbits) and New World monkeys via the enzyme α1,3galactosyltransferase (α1,3GT). On the other hand, humans, apes and Old-World monkeys do not synthesize α-gal antigen but instead produce anti-Gal antibody. Previous wound healing dermal studies have shown that the application of α-gal nanoparticles in anti-Gal producing mice (α1,3GT knock out) improved healing without scarring. The α-gal nanoparticles bind to anti-Gal antibody and activate complement cascade to release chemotactic peptides such as C3a, C5a. These chemotactic peptides increased macrophage recruitment and the bound immune complex further polarized the macrophages via Fcγ receptors to an anti-inflammatory state. As SCI pathophysiology parallels some aspects of wound healing, we hypothesize that α-gal nanoparticles may enhance the recruitment of anti-inflammatory macrophages to the injury site post SCI and facilitate tissue repair. But, the SCI lesion has both macrophages and resident microglia. As a first step, we assessed if the resident microglia can be activated by α-gal nanoparticles in the presence of serum containing anti-Gal antibody. Using an <i>in-vitro</i> model with human microglia cells (HMC3), we found that α-gal nanoparticles with serum activated HMC3 cells resulting in an increase in expression of CD68. Additionally, the activated cells exhibited a morphology change from circular in resting HMC3 to an amoeboid form in activated cells. Moreover, these cells exhibited increased anti-inflammatory markers like Arginase-1 and CD206 and VEGF production while reducing IL-6 levels. Collectively, these results suggest that the HMC3 cells were polarized towards the M2 phenotype. Subsequently, intracord injection of α-gal nanoparticles into a crush injured 1,3GT knock out mice demonstrated positive outcomes. Initial experiment showed a significant increase in macrophage recruitment in the treated cohort compared to saline controls. The α-gal nanoparticles injected mice showed an increase in anti-apoptotic Bcl-2 expression, decreased iNOS production and decreased pro-apoptotic Bax expression at 24h and 72h post-injury, suggesting neuroprotection. At 7, 14 and 21 days post-injury, the α-gal treated mice expressed higher levels of M2 markers (Arginase-1, CD206) while the pro-inflammatory M1 marker, CD16/CD32 was reduced. We also observed an increase in neurofilament staining and decreased astrocyte (GFAP) expression and increased angiogenesis in α-gal injected mice at 45 days post-injury. Behavioral experiments showed improved fine-skill locomotor recovery, increased open-field activity and faster paw withdrawal response in the α-gal treated group against saline controls. Collectively, these results indicate that α-gal nanoparticles induce a pro-healing inflammatory response and has therapeutic potential in SCI tissue repair.</p>