Perforin-2: a New Molecule on the Block That Kills Bacteria Inside Human Cells, But Not In Diabetic Foot Ulcers

Bacterial infection contributes to impediment of wound healing process, however the mechanisms by which this occurs are not fully understood. Bacteria can reside inside of human cells hiding away from immune cell attack, also evading effects of antibiotics. Our collaborative team examined the role of recently-discovered anti-microbial protein, Perforin-2 (P-2), which is responsible for killing intracellular bacteria(1). We analyzed P-2 expression in diabetic foot ulcer (DFU; n=12) and human ex vivo wounds (n=6) by using PrimeFlow, a new technique capable of simultaneous detection of P-2 RNA and cell-specific proteins at a single cell resolution(2). This approach identified P-2 expression in both immune and skin cells, including gamma-delta T cells, keratinocytes, and fibroblasts. Furthermore, keratinocytes acted as potent killers of intracellular bacteria, while P-2 siRNA knock-down resulted in a loss of keratinocyte microbicidal ability allowing intracellular bacterial replication. To evaluate P-2 expression in acute wounds, cells and mRNA were isolated from ex vivo human wounds daily during a course of seven days. P-2 levels were increased in immune (CD45+) cells post-wounding, with peak at 48 hours post-wounding. The wound response to infection was measured by experimental infection of ex vivo human wounds with Staphylococcus aureus isolate from DFU patients. The presence of common pathogen, S. aureus, in acute wound resulted in striking suppression of P-2 expression and delayed healing. Interestingly, further analyses of tissue from non-healing DFUs revealed striking suppression of P-2 in a specific subset of immune cells and keratinocytes, suggesting their inability to eliminate intracellular pathogens. Indeed, we confirmed the presence of S. aureus inside the skin cells obtained from the ulcer tissue. Our data suggest that P-2 has dual role in wound healing: as anti-microbial against intracellular pathogens and as wound healing simulator. Thus, P2 maybe new target for prevention of chronic wound infections and associated complications.