Background: Diabetic foot ulcers (DFU) are a leading cause of lower limb amputations. Current DFU treatments rely on pharmacological drugs and standard of care which often results in poor prognosis. The role of bacterial biofilm and its interaction with the host immune system in the context of wound healing is poorly understood.
Purpose: This study delves into the connection between DFU and bacterial biofilms, exploring the role of biofilm-derived oxylipins, particularly 10-hydroxy-8-octadecenoic acid (10S-HOME) and 9- hydroxy-octadecadienoic acid (9S-HODE) in immune cell impairment. Oxylipins have been reported to be immunomodulatory and capable of polarizing CD4+T cells.
Methods: To test our hypothesis, we collected wound samples (N=27) from consenting subjects under a protocol approved by IRB. The samples underwent biofilm analysis using scanning electron microscopy (SEM) and 16S rRNA next-generation sequencing (NGS). Metabolite profiling of 10(S)-HOME and 9(S)-HODE was conducted via liquid chromatography-mass spectrometry (LC-MS/MS) using deuterated analogs of 10(S)-HOME and 9(S)-HODE as internal standard. T-cells from a healthy donor were studied for their immunological role and correlated with human keratinocytes-derived cell line mimicking wound under oxylipin-challenged conditions. Quantitative gene expression and protein abundance were performed using RT-qPCR, ELISA, and dot-blot techniques.
Results: Our data from a cohort of 27 patient specimens (wound 17 and control 10) showed a discernible augmentation in bacterial biofilm abundance in the wound samples relative to the control sample. NGS unveiled the presence of microorganisms, specifically Finegoldia magna, Pseudomonas aeruginosa, Staphylococcus aureus, and Anaerococcus vaginalis within the wound tissue. Moreover, oxylipin profiling revealed the upregulation of 10(S)-HOME and 9(S)-HODE in the wound tissue compared to normal unwounded skin. Treatment with 10(S)-HOME induced a discernible retardation in human keratinocyte proliferation compared to sham control (n=10, p<0.0003). Additionally, dysregulation in the expression of the immune checkpoint gene CTLA4 was observed after treatment with the oxylipins 10(S)-HOME (n=7) and 9(S)-HODE (n=7) in CD4+T-cells derived from healthy donors.
Conclusion: These findings suggest that host-pathogen interaction mediated via biofilm-derived oxylipins may interfere with the healing outcomes of DFU. This alteration seems to be mediated through the regulation of intracellular immune checkpoint genes. Therefore, CTLA4 and other intracellular checkpoints may hold promising candidates in controlling DFUs by targeting specific immune cell subsets. In addition, other checkpoint genes like PD-1 and CISH are under investigation. Further research and verification are essential to establish the potential of this approach in DFU management.