Hyaluronan regulates chemical allergen-induced IL-18 production in human keratinocytes

Highlights

• IL-18 has a key role in allergic contact dermatitis progression.

• Contact allergens enhance hyaluronan (HA) degradation in keratinocytes.

• Modulation of HA content reduces IL-18 production.

• Oxidative stress is involved in HA degradation and IL-18 production.

• HA fragments are mediators in contact sensitisation process.

Abstract

Interleukin-18 (IL-18) has been shown to play a key proximal role in the induction of allergic contact dermatitis. Low molecular weight hyaluronan (LMWHA), an endogenous molecule and a member of the so-called damage associated molecular patterns (DAMPs), has been suggested to elicit immune-stimulatory effects. The purpose of this study was to examine the role of hyaluronan (HA) degradation in IL-18 production in human keratinocytes following stimulation with the contact sensitizers 2,4-dinitrochlorobenzene (DNCB) and PPD. IL-18 production in the human keratinocyte cell line NCTC2544 was measured by ELISA, whereas changes in HA metabolism were determined by Real-time PCR and immunofluorescence. Both contact allergens were able to enhance hyaluronidase (HYAL) 1 and 2 expression inducing HA degradation. Modulation of HA production, by HYAL or aristolochic acid pre-treatment, resulted in a significant reduction of contact allergen-induced IL-18 production. Oxidative stress appears to be the initial step in KC activation, as all the sequels of events can be blocked using antioxidants. This is the first indication that LMWHA can act as a DAMP in keratinocytes. In conclusion LMWHA fragments are important mediators in the process of contact sensitisation leading to IL-18 dependent responses.

Introduction

Irritant and allergic contact dermatitis ¹(CD) are undesired side effects after contact with environmental or industrial chemicals as well as in the development of drugs and cosmetics. Due to a high prevalence of contact allergy in the general population, making it a common and important health hazard (Peiser et al., 2012), new approaches in mapping sensitisation pathways have been developed (Hartung and Corsini, 2013). Epidermal keratinocytes (KC) sense haptens, which in turn initiate a program of enhancement or de novo expression of inflammatory molecules, which ultimately represent the starting point of primary inflammation. IL-18, formerly known as IFN-γ-inducing factor (IGIF), which belongs to the IL-1 cytokine family, provides an early signal for development of Th1 lymphocyte responses (Dinarello et al., 1998). IL-18 has been shown to play a key proximal role in the induction of allergic contact dermatitis and to favor Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-α, IL-8, and IFN-γ (Okamura et al., 1995, Cumberbatch et al., 2001, Antonopoulos et al., 2008). IL-18 also enhances the production of granulocyte macrophage-colony stimulating factor and IL-12, potentiates anti-CD3-induced T cell proliferation, and increases Fas-mediated killing of NK cells (Micallef et al., 1996, Tsutsui et al., 1996). Haptens, acting as danger signals, stress the cells, stimulate the assembly of the inflammasome and NF-κB activation, resulting in IL-18 neosynthesis and release (Corsini et al., 2009). Therefore, Il-18 production is a representative biomarker of the initial phase “local trauma-proinflammatory cytokine production” necessary for maturation and migration of dendritic cells, and T cells activation (Corsini et al., 2009, Corsini et al., 2013a, Galbiati et al., 2011, Galbiati et al., 2014a).

Extracellular matrices (ECM) represent a complex network of macromolecules, including collagens, elastin, laminins, and glycosaminoglycans (GAGs), constituting the cell microenvironment. ECM provides the bulk, shape and strength of tissues in vivo reviewed by Nikitovic et al., (2013a). In addition to its structural role the ECM is critically important for cell growth, survival, differentiation and key to various disease processes including inflammation and cancer (Hynes, 2009, Sorokin, 2010, Afratis et al., 2013, Nikitovic et al., 2012). Importantly, it has been shown that the early/chronic inflammation stages of CD are associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components including HA (Esser et al., 2012) as well as fibronectin, tenascin-C, fibulin-1, and 2 and various matrix metalloproteinase (MMP) (Kusubata et al., 1999). Moreover, a participation of increased oxidative stress and generation of radical oxygen species (ROS) have also been correlated to CD-related alterations in ECM constitution (Esser et al., 2012, Corsini et al., 2013b). Indeed, ROS induced ECM¹ remodelling in general seems to perpetrate a crucial role during the progression of pathological conditions (Nikitovic et al., 2013a; Nikitovic et al., 2014).

HA is a free unbranched GAG composed of repeating disaccharides of N-acetyl-glucosamine and glucuronic acid units, synthesized in a unique manner by a family of hyaluronan synthases (HAS 1–3), degraded by hyaluronidases (HYAL 1 and 2) and exerts its biological effects by binding to families of cellular receptors, the hyaladherins (Antonio and Iozzo, 2001; Nikitovic et al., 2013b). HA is ubiquitously distributed in the ECM (Noble, 2002), and it is primarily produced by dermal fibroblasts and epidermal keratinocytes and to a smaller amount by other cell types including smooth muscle cells (Nusgens, 2010). A strict dependence on size is evident on HA biological roles as high molecular weight HA (HMWHA) (<1 × 106 kDa) is anti-angiogenic, anti-inflammatory and immunosuppressive (Stern et al., 2006, Kim et al., 2008). On the other hand low molecular weight HA (LMWHA) breakdown products, occurring in the range from 1.2 to 500 kDa, induce pro-inflammatory innate immune responses (Noble, 2002), presumably via TLR2 and/or TLR4 in immune cells like dendritic cells (DCs) or macrophages (Termeer et al., 2002a) or via CD44 in other cells (Bourguignon et al., 2011). LMWHA fragments are generated during inflammation or tissue damage (Jiang et al., 2005), either enzymatically by HYAL 1 and 2 or non-enzymatically by ROS (Agren et al., 1997, Galbiati et al., 2014b, Monzon et al., 2014). HA breakdown in CD seems to be initially directly mediated by ROS, given the rapid induction of ROS by contact sensitizers (Galbiati et al., 2014b). Early chemical fragmentation (Agren et al., 1997) is followed by enzymatic cleavage, since ROS also regulate p38 MAPK dependent up-regulation of hyaluronidase, as recently described for lung inflammation (Monzon et al., 2014). In continuation, LMWHA fragments can regulate the activation of crucial immunomodulatory pathways (Scheibner et al., 2006) expression of proinflammatory genes (Collins et al., 2011) as well as cytokines, such as IL-8, IL-10, and IL-12 (Boodoo et al., 2006, Hodge-Dufour et al., 1997, Hodge-Dufour et al., 1998). Importantly, these molecules, including TNF-α, IL-18, and IFN-γ (Okamura et al., 1995, Cumberbatch et al., 2001, Antonopoulos et al., 2008), have been demonstrated in vivo to be directly responsible for driving the effector phases of chemical skin sensitisation (Martin et al., 2011). The participation of HA in the induction of skin inflammation by contact sensitizers like 2,4,6-trinitrochlorobenzene (TNCB), was demonstrated by significantly reduced contact sensitisation in germ-free mice pre-treated with an inhibitor of HA function (Pep-1) (Martin et al., 2008). This process is crucial for CD since sensitisation was completely prevented by pre-treatment of the skin with antioxidants or the hyaluronidase inhibitor, aristolochic acid (Esser et al., 2012). The correlation of HA-metabolism with IL-18 production in keratinocytes stimulated by contact sensitizers has not previously been studied. Our results demonstrate that HA fragments are important mediators in the process of contact sensitisation leading to keratinocyte IL-18 dependent responses.