Genetic and epigenetic alterations in Toll like receptor 2 and wound healing impairment in type 2 diabetes patients

Abstract

Aim

Persistent hyperglycemic microenvironment in type 2 diabetes mellitus (T2DM) leads to the development of secondary complications like wound healing impairment. Proper co-ordination of innate immune system plays an integral role in wound healing. Toll like receptors (TLRs) are prominent contributors for the induction of the innate immune and inflammation response. TLR2 is an important extracellular member in mammalian TLR family and has been shown to be a potent player in the wound healing mechanism.

Methods

Expressional status of TLR2 was seen in wounds of T2DM cases with respect to the severity of wounds in 110 human lower extremity wounds. The methylation status of TLR2 promoter was also examined.

Results

Although TLR2 transcripts were downregulated in T2DM wounds compared to control, their levels tend to increase with the severity of T2DM wounds. The methylation status of TLR2 gene promoter was not significantly different among different grades of wounds in T2DM subjects. The CpG sites investigated were totally or partially methylated in majority of DFU cases.

Conclusion

TLR2 down regulation in wounds of T2DM patients compared to non diabetic patients may lead to development of non healing chronic ulcers in them.

Introduction

The cascade of wound healing in higher organisms generally exhibits an integration of several mutually coherent steps ranging from (i) wound homeostasis through (ii) acute inflammation, (iii) proliferation and finally leading to (iv) remodeling of the affected part (Singh, Agrawal, Gupta, & Singh, 2013a). These steps should be tightly regulated spatially and temporally as any type of imbalance may lead to non healing chronic ulcers (Whitney, 2005). The immune system, both innate and adaptive, plays an integral function in the process of wound healing, as evident by the secretion of signaling molecules like cytokines, lymphokines and growth factors (Singer and Clark, 1999, Werner and Grose, 2003). Innate immune system provides the first line of defense against foreign invaders during the process of wound healing (Park & Barbul, 2004). Signaling receptors like Toll like receptors (TLRs) are one of the most prominent contributors for the induction of the innate immune and inflammation response (Takeda, Kaisho, & Akira, 2003). TLRs are the family of transmembrane proteins, expressed on almost every immune cell like macrophages, neutrophils and dendritic cells, where their main function is to serve as a pathogen recognizing receptor (PRR) and to sense the pathogen associated molecular patterns (PAMPs) over a plethora of microbes invading during open wounds (Akira, Uematsu, & Takeuchi, 2006). After binding with these PAMPs, TLRs initiate signaling pathways that ultimately lead to activation of two main transcription factors: Nuclear factor kB (NF-kB) or type I Interferon (IFN) (Dasu & Isseroff, 2012). The TLR induced inflammation may be either agonist or antagonist of wound healing, depending upon the timing and the extent of these transcription factors which critically determine the fate of the healing wound (Dasu & Isseroff, 2012).

TLR2 is an important extracellular member in mammalian Toll family of leucin rich receptors. TLR2 is known to be a signaling receptor for many microbial products including whole gram positive bacteria, microplasma, peptidoglycan and lipoteichoic acid derived from Gram-positive bacteria (Flo, Halaas, & Torp, 2001). Anti infectious property of TLR2 is evident from the fact that the TLR2-deficient mouse strain is more prone to infection with Gram-positive bacteria S. aureus and shows defective clearance of spirochetes after infection by Borrelia burgdorferi as compared to their wild type counterparts (Kuo et al., 2013). TLR2 has an ability to form heterophilic dimers with other structurally related TLRs like TLR1 and TLR6, due to which it can recognize a wide spectrum of microbial components. Alteration in the methylation pattern of TLR2 has been also described in certain epithelial diseases including carcinoma and cyst formation (Furuta et al., 2008). TLR2 has been shown to be a potent player in the wound healing mechanism. TLR2 activation after acute ischemic injury promotes the process of angiogenesis by inducing endothelial cell migration and adhesion to the wound site (Xu et al., 2013). TLR2 has been also shown to modulate the synthesis of Connexin-43 (Cx43), a gap junction protein, another potent wound healing agent (Ey, Eyking, & Gerken, 2009).

Persistent hyperglycemic microenvironment in type 2 diabetes mellitus (T2DM) leads to the development of secondary complications like cardiovascular disease, neuropathy, nephropathy, retinopathy and impairment of wound healing in patients. Wound healing impairment is a serious secondary complication of T2DM which contributes to a huge percentage of total amputations performed worldwide. As per recent data, around 25% of T2DM patients develop non healing wounds once in their life time (Singh, Agrawal, Gupta, & Singh, 2013b). The reason for this observation is that the hyperglycemic conditions in T2DM cases lead to decrease in cytokines and growth factors essential for healing of wounds (Werner & Grose, 2003). The immune response is also compromised in T2DM cases which generally leads to prolonged inflammation and unresolved infection in the wound microenvironment, thereby resulting in chronic wound which either takes a long time to heal or does not heal at all. TLRs are one of important members of immune system which is affected significantly in the T2DM individuals (Kanhaiya, Agrawal, Gupta, & Singh, 2013). Recently our group has shown that genetic and epigenetic alterations in TLRs, especially, TLR4 lead to impairment of wound healing in T2DM cases (Kanhaiya et al., 2013, Singh, Singh, Agrawal, Gupta and Singh, 2013, Singh, Singh, Agrawal, Gupta and Singh, 2014). In the present study we tried to look upon the expressional status of TLR2 in wounds of T2DM cases with respect to the severity of wounds. The methylation status of many CpG dinucleotides situated near the regulatory sequence in the promoter region of TLR2 gene was also examined to see the relationship of epigenetic regulation of TLR2 in the patho-physiology of diabetic wound healing impairment.