Kanhaiya, Agrawal NK, Gupta SK and Kiran Singh
Toll like Receptor 4 (TLR4), is known for its key role in initiation of innate immunity and regulation of adaptive immune responses. TLR4 is also an important regulator of wound inflammation, stimulator of growth factors like vascular endothelial growth factor (VEGF) and plays an important role in restoring damaged tissue integrity during normal wound healing. In the present study we have investigated 78 Diabetic Foot ulcer (DFU) patients with Type 2 diabetes (T2DM) and 8 foot ulcers patients without T2DM as controls for the differential expression of TLR4 both at mRNA level and protein level. Expression analysis was done using semi-quantitative RT-PCR, quantitative Real-time PCR and western blot. TLR4 message and protein were significantly down-regulated in DFU patients as compared to controls. Similarly down regulation of VEGF was also observed in DFU patients as compared to controls. DNA Methylation is an important regulator of gene expression therefore Methylation status of promoter of TLR4 gene was analyzed in DFU and control wounds using specific methylation-sensitive restriction enzyme which suggested higher methylation of TLR4 promoter in DFU with respect to controls. TLR4 expression was not influenced by the infection status and wound grade of the subjects however TLR4 was significantly affected by the gender of the subjects. These results clearly suggests that TLR4 down regulation in the wounds of T2DM subjects could be one of the causes contributing to impairment in healing of diabetic wounds and eventually into the development of chronic, non healing ulcers in T2DM subjects.
