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Healing ahead with Hsp60?
BETHESDA, Md.—A newly elucidated function of a well-known gene, heat shock protein 60 (Hsp60), might offer a new approach to wound healing. Researchers from the National Institutes of Health—specifically the National Human Genome Research Institute (NHGRI), the National Eye Institute (NEI) and their colleagues—found that Hsp60 plays a key role in the body’s inflammatory response in the face of injury. The results of their work appeared in the paper “Extracellular Hsp60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation,” which was published in Regenerative Medicine.
The proteins produced by the Hsp60 gene are generally known for ensuring that other proteins fold properly, and the Hsp60 protein is known to function as a signaling molecule to induce an inflammatory response to bacterial infection in wounds.
Dr. Shawn Burgess, head of NHGRI’s Developmental Genomics Section and senior author on the study, tells DDNews this was the first time his lab had worked with Hsp60, and that this work has its roots in a study published four years ago. His lab focuses primarily on studying zebrafish (known for their ability to regenerate a variety of tissues) and hearing regeneration. In the aforementioned study, the team used transcriptional profiling to find all the genes that turn on or off in zebrafish during hearing regeneration, he explains, and identified 2,000 genes. They then started using CRISPR/Cas9 to systematically knock out all of those 2,000 genes. Burgess reports that they have gone through 250 genes so far, and found eight genes that affect regeneration, one of which was Hsp60.
“Because HSP60 has a well-known role as a protein chaperonin, we were a little surprised how strong the block in regeneration was when we KO’ed Hsp60,” Burgess admits. “There was no regeneration at all, and that extended to other tissues such as the tail fins, which also did not regenerate in the mutants. We found an old paper from Mark Keating’s lab that showed a similar effect in zebrafish tail regeneration in an Hsp60 mutant they identified in a screen for regeneration mutants … We did some research in the literature and found out that extracellular Hsp60 was shown to cause inflammation. We checked the inflammation response in our Hsp60 mutant fish and saw that it was lower than normal. Inflammation has been shown to be essential for wound healing, so we wondered if Hsp60 was sending a specific signal to the innate immune system to stimulate wound healing.”
The team knocked out Hsp60 in zebrafish and found that while the mutant fish developed normally, when they faced a wound—either an injury to the cells involved in hearing or the amputation of a caudal fin—they could not regenerate the damage. Burgess and his team then used fluorescently tagged leukocytes to show that when Hsp60 was knocked out, there were noticeably fewer of these immune cells at the injury site.
However, when Hsp60 was reintroduced to the area, either injected directly to the injury site or as a topical treatment, regeneration started up again. When the topical treatment was applied to a puncture wound in animal models, the diabetic mice saw complete healing after just 21 days.
Wound healing is a chronic problem for diabetic individuals, as roughly 15 percent of diabetics develop a foot ulcer at some point. These ulcers result from a variety of factors, including reduced sensation, poor circulation and skin irritation. High blood glucose levels can result in diabetic neuropathy, in which damaged nerves lead to a loss of sensation, and atherosclerosis, which can hamper circulation to extremities. Foot ulcers can become serious and in some cases lead to amputation; in fact, diabetes is the top cause of non-traumatic lower limb amputation in the country.
As for why diabetics suffer from impaired wound healing, Burgess explains that “There is some recent evidence that the diabetic wound response is a hyperactive inflammation response. In particular, there seems to be a near-catastrophic overstimulation of neutrophils at the wound site, but macrophages are also involved. A key concept in inflammation is ‘inflammatory resolution,’ i.e., the immune response needs to know when to stop attacking the wound site (primarily an anti-infection response) and let it heal.”
“It does seem counterintuitive that a protein that increases inflammation could help injuries that are a result of overactive inflammation but again, resolution of the inflammatory response is the important idea,” he adds. “Generally speaking, there are two ‘phases’ for macrophages: M1 macrophages, which are an attack mode for injury sterilization, and M2 macrophages, which stimulate wound healing and tissue regeneration. It turns out that Hsp60 appears to bring in M2 macrophages (or convert M1 to M2). So we think in the case of diabetes, the Hsp60 is allowing the inflammation response, which has been locked into M1 phase, to resolve into M2 and continue normally along the wound-healing process.”
Burgess says the team plans to explore multiple avenues as they move forward, including if the results seen with topical Hsp60 treatment will translate to humans and help heal any wound. They hope to identify “the exact receptor (or receptors) that HSP60 is being bound by that triggers this wound healing response,” and hope to find researchers interested in moving this work into the clinic to see if it could help individuals with diabetic ulcers. They also aim to identify the downstream responses Hsp60 triggers in macrophages, he notes, and intend to continue their work in knocking out the rest of the 2,000 genes in hopes of identifying others that play a role in triggering regeneration.