| Abstract |
Allergic contact dermatitis (ACD) is the most common dermatopathy and is caused by topical exposure to chemical allergens. ACD is a type IV (delayed) hypersensitivity response resulting in sterile inflammation (Tan et al., 2014). The term “sterile” characterizes inflammatory responses not caused by pathogens/ microbes (Chen and Nunez, 2010; Kurbet et al., 2016). Importantly, almost 20% of the general population presents ACD to at least one or more contact allergens (Peiser et al., 2012). The symptoms include erythema, vesiculation, scaling and itchiness. Contact allergens are haptens, low molecular weight chemicals (<1000Da) (Martin, 2012) normally unable to trigger immune responses (Corsini and Kimber, 2007). Hence, binding of haptens to self- macromolecules is crucial for the initiation of inflammation (Murphy et al., 2019). The complexity of the immune system may sometimes result in a “mistaken” response to haptens (McFadden et al., 2012). P-phenylenediamine (PPD) and 2,4 dinitrochlorobenxene (DNCB) are common contact sensitizers, responsible for many cases of ACD (Milam et al., 2019; Saarnilehto et al., 2014). They were previously shown to trigger the production of IL-18, a reliable marker of keratinocyte sensitization (Galbiati et al., 2014; Nikitovic et al., 2015). Epidermal keratinocytes play a key role in innate immunity, affect the skin inflammatory responses and are crucial for ACD progression (Corsini et al., 2013). The extracellular matrix (ECM) that provides tissue structure, specificity and organization, regulates as well cellular functions and responses, such as inflammation by triggering specific signaling pathways (Nikitovic et al., 2013; Kavasi et al., 2017). Hyaluronic acid or Hyaluronan (HA), an important ECM component, is a linear non-sulfated glycosaminoglycan, composed of repeating units of N-acetylglucosamine GlcNAc) and glucuronic acid. HA is well established to participate in a wide variety of biological functions. The metabolism of HA has been demonstrated to play an important role in sterile inflammation (Kavasi et al., 2017). Indeed, HA deposition has been demonstrated to be altered, upon keratinocyte treatment with PPD and DNCB contact sensitizers. Notably, Low molecular weight Hyaluronan (LWMHA) is able to trigger IL-18 production (Nikitovic et al., 2015). HA is synthesized by specific enzymes called Hyaluronan Synthases (HAS1, HAS2 and HA3). The process takes place on the plasma membrane where these enzymes are located, with simultaneous secretion of the HA chain to the ECM. HA degradation is performed by enzymes denominated, Hyaluronidases (HYAL1 and HYAL2). In the skin HA is synthesized mainly by dermal fibroblasts, but also by epidermal keratinocytes (Kavasi et al., 2017). Toll- like receptors (TLRs) are a family of transmembrane receptors, widely known for their roles in innate immunity. Toll-like receptor 4 (TLR4), is strongly implicated in skin inflammation with the ability to bind Damage Associated Molecular Patterns (DAMPs), including LMWHA (Termeer et al., 2002; Kuzmich et al., 2017). DAMPs are endogenous molecules, with the ability to act as “danger signals,” that trigger cellular responses and they have strongly been correlated to the process of sterile inflammation (Chen and Nunez, 2010; Hernandez et al., 2016). A category of DAMPs are the Reactive Oxygen Species (ROS), chemically active small molecules, resulting from incomplete reduction of oxygen (Kubert et al., 2016; Nastase et al., 2017). Indeed, ROS have been characterized as mediators of inflammation (Mittal et al., 2014) and noteworthy they can degrade HA chemically. TLR4 activation results in the downstream triggering of nuclear factor kappa-beta (NF-κΒ), the transcription factor, established to regulate the transcription of pro- inflammatory cytokines (Kaplan et al., 2012). In its inactive form this transcription factor is located to the cytoplasm, while in its activated form, translocates to the nucleus engaged in the transcription of several genes (Akira and Takeda, 2004). NF-κΒ has been strongly correlated to innate immunity and inflammation (Kaplan et al., 2012). In fact, several therapeutic approaches for inflammation target the inhibition of TLR4/ NF-κΒ signaling (Huang et al., 2014; Gomes et al., 2015). In the present study, the putative co- operation of HA and TLR4 in the process of contact allergen- induced keratinocyte activation was investigated. Specifically, contact sensitizers PPD and DNCB were shown, in NCTC2544 human keratinocytes, to significantly increase the expression of the TLR4 receptor in a concentration dependant manner, as demonstrated by western blot and Real-time PCR. The contact allergens were also demonstrated to induce HAS1, HAS2 and HAS3 overexpression at both the protein and mRNA level. These data suggest that the contact sensitizers enhance HMWHA production by the upregulation of HAS isoforms. Thus, the previously shown contact sensitizer mediated HYALs upregulation (Nikitovic et al., 2015) in combination to ROS action, will increase endogenous LMWHA release. Furthermore, LMWHA treatment of keratinocytes induced increased expression of not only the TLR4 receptor, but also of the HAS1 and HAS3 enzymes. This observation implies that the increased release of LMWHA by the activated keratinocytes will result in enhanced HMWHA deposition, available for degradation to LMWHA and the subsequent TLR4 signaling, forming a loop for the sustaining of inflammation. Moreover, downregulation of TLR4, upon RNA interference utilization, resulted in an attenuation of HAS1 and HAS3 basal levels, suggesting a direct correlation between HA synthesis and TLR4 signaling. PPD and DNCB effects on HA metabolism, were shown to be partly executed through the TLR4 downstream signaling. Indeed, blocking of the TLR4 receptor with a neutralizing antibody, resulted in attenuated contact allergen mediated HYAL and HAS upregulation. Furthermore, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB as well as its translocation to the nuclei of keratinocytes. Blocking of TLR4 activities reduced NF-κΒ activation. Additionally, LMWHA treatment enhanced NF-κB activation, whereas blocking HYALs’ action with an inhibitor (aristolochic acid) attenuated the contact allergen mediated NF-κΒ activation. In conclusion, keratinocyte sensitization by the PPD and DNCB contact allergens is partly mediated via a LMWHA/ TLR4/ NF-κB signaling axis.
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The role of Hyaluronic acid in keratinocyte activation by contact allergens
