title: Increased expression of TLR9 associated with pro-inflammatory S100A8 and IL-8 in diabetic wounds could lead to unresolved inflammation in type 2 diabetes mellitus (T2DM) cases with impaired wound healing.
abstract: BACKGROUND
  Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia which causes a chain of abrupt biochemical and physiological changes. Immune dys-regulation is the hallmark of T2DM that could contribute to prolonged inflammation causing transformation of wounds into non-healing chronic ulcers. Toll like receptor -9 (TLR9) is a major receptor involved in innate immune regulation. TLR9 activation induces release of pro-inflammatory molecules like S100A8 and interleukin-8 (IL-8) by myeloid cells causing migration of myeloid cells to the site of inflammation. We hypothesized that pro-inflammatory S100A8 and IL-8 proteins could cause persistent inflammation in chronic wounds like diabetic foot ulcer (DFU) and may contribute to impaired wound healing in T2DM patients.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Molecular & Human Genetics, Banaras Hindu University, Varanasi, 221005, India.
authors.forename: Kanhaiya
authors.initials: K
authors.lastname: Singh
authors.affiliation: Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India.
authors.forename: Neeraj K
authors.initials: NK
authors.lastname: Agrawal
authors.affiliation: Department of Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Gupta
authors.affiliation: Department of Biological Sciences, University of Maryland Baltimore County Baltimore, MD, USA. Electronic address: sinhapratima2008@gmail.com.
authors.forename: Pratima
authors.initials: P
authors.lastname: Sinha
authors.affiliation: Department of Molecular & Human Genetics, Banaras Hindu University, Varanasi, 221005, India. Electronic address: singhk4@rediffmail.com.
authors.forename: Kiran
authors.initials: K
authors.lastname: Singh
mesh_terms: D000328:Adult; D000368:Aged; D001706:Biopsy; D039481:CD11b Antigen / Q000378:metabolism; D040501:Calgranulin A / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000150:complications / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D017719:Diabetic Foot / Q000276:immunology / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007113:Immunity, Innate; D016209:Interleukin-8 / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D022423:Myeloid Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D063268:Sialic Acid Binding Ig-like Lectin 3 / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D014945:Wound Healing; D014946:Wound Infection / Q000276:immunology / Q000378:metabolism / Q000382:microbiology / Q000473:pathology
chemical_list: D039481:CD11b Antigen; C571163:CD33 protein, human; C508606:CXCL8 protein, human; D040501:Calgranulin A; C509295:ITGAM protein, human; D016209:Interleukin-8; D063268:Sialic Acid Binding Ig-like Lectin 3; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9
keywords: CD11b(+) CD33(+) myeloid cells; IL-8; S100A8; TLR9; Type 2 diabetes mellitus; Wound healing impairment