Xenotransplantation and immune response (173)Immunotherapy and Immune Responses (57)T-cell and B-cell Immunology (56)Glycosylation and Glycoproteins Research (36)Monoclonal and Polyclonal Antibodies Research (35)
An ancient evolutionary regenerative mechanism of injured myocardium in vertebrates has been conserved in zebrafish, urodeles (salamander, newt, and axolotl) and neonatal mice. This innate regenerative mechanism is characterized by extensive migration of pro-regenerative macrophages into the injured myocardium and non-immune activation of parts of the complement system. Loss of regenerative activity in neonatal mice within a few days after birth implies that it is suppressed and replaced by fibrotic repair and scar formation. Fibrosis prevents ventricular wall rupture following myocardial infarction (MI), but it compromises contractility and can lead to heart failure and premature death. Reactivation of the suppressed regenerative mechanism in post-MI adult mice may be feasible by localized immune activation of the complement system, resulting in extensive recruitment of pro-regenerative macrophages into the injured myocardium, recapitulating neonatal mechanisms. Localized complement activation can be achieved by a new method of harnessing the natural anti-Gal antibody, which constitutes ~1% of human immunoglobulins and binds the carbohydrate antigen "α-gal epitope". α-Gal nanoparticles (small liposomes presenting multiple α-gal epitopes) bind anti-Gal when administered into reperfused myocardium post-MI in anti-Gal-producing mice, thereby inducing localized complement activation. In this novel approach, macrophages recruited into the ischemic myocardium by complement cleavage chemotactic peptides, and binding anti-Gal-coated α-gal nanoparticles, polarize to become pro-regenerative macrophages that produce pro-regenerative cytokines and recruit stem cells. This process results in near-complete regeneration of the injured myocardium within 14 days. Future evaluation of this novel approach in larger animal models will help in determining whether trans-endocardial delivery by catheter of α-gal nanoparticles into ischemic myocardium warrants clinical application in acute MI.
The human natural anti-carbohydrate antibodies anti-Gal, anti-Neu5Gc, and anti-Forssman are "living-fossils" that appeared in ancestral apes, monkeys and hominins millions of years ago. These antibodies appeared at various evolutionary periods in few mutated-offspring that lost the ability to synthesize the corresponding dispensable (i.e., nonessential) carbohydrate-antigens, α-gal epitope, Neu5Gc (N-glycolyl neuraminic acid) and Forssman-antigen, respectively. Production of these antibodies is stimulated by environmental antigens such as those of the human microbiota. Elimination of carbohydrate-antigens in the few mutated-offspring was caused by accidental nonsense or missense mutations that inactivated genes encoding enzymes involved in their biosynthesis, while most individuals in parental-populations continued synthesizing these carbohydrate-antigens. It has been suggested that dispensable carbohydrate-antigens which are absent in some mammalian species were evolutionary eliminated due to selective pressure by lethal viruses using these carbohydrate-antigens as "docking" receptors. An alternative selective mechanism which is based on the distribution of anti-Gal, anti-Neu5Gc and anti-Forssman in mammals, is presented in this review and is associated with the protective effects of these natural antibodies. It is suggested that epidemics of lethal enveloped-viruses caused the extinction of parental-populations synthesizing the corresponding carbohydrate-antigens of these antibodies, independent of the cell adhesion mechanisms of such viruses. However, the few mutated offspring were protected by these natural antibodies which bound to carbohydrate-antigens synthesized on viruses as a result of their replication in individuals of the parental-populations. Recent studies suggest that these antibodies continue to contribute to the immune protection of humans against zoonotic infections by viruses presenting α-gal, Neu5Gc or Forssman antigens.
The healing of skin wounds, myocardial, and spinal cord injuries in salamander, newt, and axolotl amphibians, and in mouse neonates, results in scar-free regeneration, whereas injuries in adult mice heal by fibrosis and scar formation. Although both types of healing are mediated by macrophages, regeneration in these amphibians and in mouse neonates also involves innate activation of the complement system. These differences suggest that localized complement activation in adult mouse injuries might induce regeneration instead of the default fibrosis and scar formation. Localized complement activation is feasible by antigen/antibody interaction between biodegradable nanoparticles presenting α-gal epitopes (α-gal nanoparticles) and the natural anti-Gal antibody which is abundant in humans. Administration of α-gal nanoparticles into injuries of anti-Gal-producing adult mice results in localized complement activation which induces rapid and extensive macrophage recruitment. These macrophages bind anti-Gal-coated α-gal nanoparticles and polarize into M2 pro-regenerative macrophages that orchestrate accelerated scar-free regeneration of skin wounds and regeneration of myocardium injured by myocardial infarction (MI). Furthermore, injection of α-gal nanoparticles into spinal cord injuries of anti-Gal-producing adult mice induces recruitment of M2 macrophages, that mediate extensive angiogenesis and axonal sprouting, which reconnects between proximal and distal severed axons. Thus, α-gal nanoparticle treatment in adult mice mimics physiologic regeneration in amphibians. These studies further suggest that α-gal nanoparticles may be of significance in the treatment of human injuries.
Gopalakrishnan B, Galili U, Saenger M, Burket NJ, Koss W, et al.
Tissue engineering and regenerative medicine •
Previous investigations have shown that local application of nanoparticles presenting the carbohydrate moiety galactose-α-1,3-galactose (α-gal epitopes) enhance wound healing by activating the complement system and recruiting pro-healing macrophages to the injury site. Our companion in vitro paper suggest α-gal epitopes can similarly recruit and polarize human microglia toward a pro-healing phenotype. In this continuation study, we investigate the in vivo implications of α-gal nanoparticle administration directly to the injured spinal cord. α-Gal knock-out (KO) mice subjected to spinal cord crush were injected either with saline (control) or with α-gal nanoparticles immediately following injury. Animals were assessed longitudinally with neurobehavioral and histological endpoints. Mice injected with α-gal nanoparticles showed increased recruitment of anti-inflammatory macrophages to the injection site in conjunction with increased production of anti-inflammatory markers and a reduction in apoptosis. Further, the treated group showed increased axonal infiltration into the lesion, a reduction in reactive astrocyte populations and increased angiogenesis. These results translated into improved sensorimotor metrics versus the control group. Application of α-gal nanoparticles after spinal cord injury (SCI) induces a pro-healing inflammatory response resulting in neuroprotection, improved axonal ingrowth into the lesion and enhanced sensorimotor recovery. The data shows α-gal nanoparticles may be a promising avenue for further study in CNS trauma.
Gopalakrishnan B, Galili U, Dunbar A, Solorio L, Shi R, et al.
Tissue engineering and regenerative medicine •
Macrophages and microglia play critical roles after spinal cord injury (SCI), with the pro-healing, anti-inflammatory (M2) subtype being implicated in tissue repair. We hypothesize that promoting this phenotype within the post-injured cord microenvironment may provide beneficial effects for mitigating tissue damage. As a proof of concept, we propose the use of nanoparticles incorporating the carbohydrate antigen, galactose-α-1,3-galactose (α-gal epitope) as an immunomodulator to transition human microglia (HMC3) cells toward a pro-healing state. Quiescent HMC3 cells were acutely exposed to α-gal nanoparticles in the presence of human serum and subsequently characterized for changes in cell shape, expression of anti or pro-inflammatory markers, and secretion of phenotype-specific cytokines. HMC3 cells treated with serum activated α-gal nanoparticles exhibited rapid enlargement and shape change in addition to expressing CD68. Moreover, these activated cells showed increased expression of anti-inflammatory markers like Arginase-1 and CD206 without increasing production of pro-inflammatory cytokines TNF-α or IL-6. This study is the first to show that resting human microglia exposed to a complex of α-gal nanoparticles and anti-Gal (from human serum) can be activated and polarized toward a putative M2 state. The data suggests that α-gal nanoparticles may have therapeutic relevance to the CNS microenvironment, in both recruiting and polarizing macrophages/microglia at the application site. The immunomodulatory activity of these α-gal nanoparticles post-SCI is further described in the companion work (Part II).
Macrophages play a pivotal role in the process of healing burns. One of the major risks in the course of burn healing, in the absence of regenerating epidermis, is infections, which greatly contribute to morbidity and mortality in such patients. Therefore, it is widely agreed that accelerating the recruitment of macrophages into burns may contribute to faster regeneration of the epidermis, thus decreasing the risk of infections. This review describes a unique method for the rapid recruitment of macrophages into burns and the activation of these macrophages to mediate accelerated regrowth of the epidermis and healing of burns. The method is based on the application of bio-degradable "α-gal" nanoparticles to burns. These nanoparticles present multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R), which bind the abundant natural anti-Gal antibody that constitutes ~1% of immunoglobulins in humans. Anti-Gal/α-gal nanoparticle interaction activates the complement system, resulting in localized production of the complement cleavage peptides C5a and C3a, which are highly effective chemotactic factors for monocyte-derived macrophages. The macrophages recruited into the α-gal nanoparticle-treated burns are activated following interaction between the Fc portion of anti-Gal coating the nanoparticles and the multiple Fc receptors on macrophage cell membranes. The activated macrophages secrete a variety of cytokines/growth factors that accelerate the regrowth of the epidermis and regeneration of the injured skin, thereby cutting the healing time by half. Studies on the healing of thermal injuries in the skin of anti-Gal-producing mice demonstrated a much faster recruitment of macrophages into burns treated with α-gal nanoparticles than in control burns treated with saline and healing of the burns within 6 days, whereas healing of control burns took ~12 days. α-Gal nanoparticles are non-toxic and do not cause chronic granulomas. These findings suggest that α-gal nanoparticles treatment may harness anti-Gal for inducing similar accelerated burn healing effects also in humans.
Two human natural anti-carbohydrate antibodies appeared in critical evolutionary events that brought primates and hominins to brink of extinction. The first is the anti-Gal antibody, produced in Old-World monkeys (OWM), apes and humans. It binds the carbohydrate-antigen 'α-gal epitope' (Galα1-3Galβ1-4GlcNAc-R) on carbohydrate-chains (glycans) synthesized by non-primate mammals, lemurs and New-World monkeys (NWM). The second is anti-N-glycolylneuraminic-acid (anti-Neu5Gc) antibody binding Neu5Gc on glycans synthesized by OWM, apes and most non-primate mammals. Ancestral OWM and apes synthesized α-gal epitopes and were eliminated ~20-30 million-years-ago (mya). Only few accidentally mutated offspring lacking α-gal epitopes, produced anti-Gal and survived. Hominin-populations living ~3 mya synthesized Neu5Gc and were eliminated, but few mutated offspring that accidently lost their ability to synthesize Neu5Gc, produced natural anti-Neu5Gc antibody. These hominins survived and ultimately evolved into present-day humans. It is argued that these two near-extinction events were likely to be the result of epidemics caused by highly virulent and lethal enveloped viruses that killed parental-populations. These viruses presented α-gal epitopes or Neu5Gc synthesized in host-cells of the parental-populations. Mutated offspring survived the epidemics because they were protected from the lethal virus by the natural anti-Gal or anti-Neu5Gc antibodies they produced due to loss of immune-tolerance to α-gal epitopes or to Neu5Gc, respectively.
This review discusses a novel experimental approach for the regeneration of original tissue structure by recruitment of endogenous stem-cells to injured sites following administration of α-gal nanoparticles, which harness the natural anti-Gal antibody. Anti-Gal is produced in large amounts in all humans, and it binds the multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) presented on α-gal nanoparticles. In situ binding of anti-Gal to α-gal nanoparticles activates the complement system and generates complement cleavage chemotactic-peptides that rapidly recruit macrophages. Macrophages reaching anti-Gal coated α-gal nanoparticles bind them via Fc/Fc receptor interaction and polarize into M2 pro-reparative macrophages. These macrophages secrete various cytokines that orchestrate regeneration of the injured tissue, including VEGF inducing neo-vascularization and cytokines directing homing of stem-cells to injury sites. Homing of stem-cells is also directed by interaction of complement cleavage peptides with their corresponding receptors on the stem-cells. Application of α-gal nanoparticles to skin wounds of anti-Gal producing mice results in decrease in healing time by half. Furthermore, α-gal nanoparticles treated wounds restore the normal structure of the injured skin without fibrosis or scar formation. Similarly, in a mouse model of occlusion/reperfusion myocardial-infarction, near complete regeneration after intramyocardial injection of α-gal nanoparticles was demonstrated, whereas hearts injected with saline display ~20% fibrosis and scar formation of the left ventricular wall. It is suggested that recruitment of stem-cells following anti-Gal/α-gal nanoparticles interaction in injured tissues may result in induction of localized regeneration facilitated by conducive microenvironments generated by pro-reparative macrophage secretions and "cues" provided by the extracellular matrix in the injury site.