Expression of TLR9 and its downstream effector molecules S100A8, and IL-8 were analyzed in chronic diabetic wound and non-diabetic control wound tissue samples by semiquantitative reverse transcriptase - polymerase chain reaction (RT-PCR), quantitative RT-PCR, western blot and immunofluorescence. CD11b+CD33+ myeloid cells were analyzed by flow cytometry.
TLR9 message and protein were higher in diabetic wounds compared to control wounds (p = 0.03, t = 2.21 for TLR9 mRNA; p = < 0.001, t = 4.21 for TLR9 protein). TLR9 down-stream effector molecules S100A8 and IL-8 were also increased in diabetic wounds (p = 0.003, t = 3.1 for S100A8 mRNA; p = 0.04, t = 2.04 for IL-8). CD11b+ CD33+ myeloid cells were decreased in T2DM as compared to non-diabetic controls (p = 0.001, t = 3.6). DFU subjects had higher levels of CD11b+ CD33+ myeloid cells as compared to non-DFU T2DM control (p = 0.003, t = 2.8). Infection in the wound microenvironment could be the cause of increase in CD11b+CD33+ myeloid cells in DFU (p = 0.03, t = 2.5).
The up-regulation of myeloid cell-derived pro-inflammatory molecules S100A8 and IL-8 in combination with lower levels of CD11b+ CD33+ myeloid cells may cause the impairment of wound healing in T2DM subjects leading to chronic ulcers.