Matrix Metalloproteinase 9 Plays a Crucial Role in Inflammation and Itch in Allergic Contact Dermatitis by Regulating Toll-Like Receptor 2/1 Signaling

Atopic dermatitis (AD) and allergic contact dermatitis (ACD) are two common but clinically distinct itchy conditions with significant impacts on skin health and overall well-being (Owen et al., 2018Owen J.L. Vakharia P.P. Silverberg J.I. The Role and Diagnosis of Allergic Contact Dermatitis in Patients with Atopic Dermatitis.Am J Clin Dermatol. 2018; 19: 293-302Crossref PubMed Scopus (95) Google Scholar). ACD is triggered by direct contact with allergens, causing T-cell mediated hypersensitivity and skin inflammation (Johansen et al., 2022Johansen J.D. Bonefeld C.M. Schwensen J.F.B. Thyssen J.P. Uter W. Novel insights into contact dermatitis.J Allergy Clin Immunol. 2022; 149: 1162-1171Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). AD, a chronic inflammatory skin condition, results from genetic, environmental factors, and immune dysregulation. Triggers include allergens, irritants, infections, stress, and hormonal changes, involving both allergic and non-allergic pathways (Steinhoff et al., 2022Steinhoff M. Ahmad F. Pandey A. Datsi A. AlHammadi A. Al-Khawaga S. et al.Neuroimmune communication regulating pruritus in atopic dermatitis.J Allergy Clin Immunol. 2022; 149: 1875-1898Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Toll-like receptor 2 (TLR2) plays a crucial role in the immune response for AD and ACD (Jin et al., 2009Jin H. Kumar L. Mathias C. Zurakowski D. Oettgen H. Gorelik L. et al.Toll-like receptor 2 is important for the T(H)1 response to cutaneous sensitization.J Allergy Clin Immunol. 2009; 123: 875-882 e1Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), yet specific pathways by which TLR2 influences the development and progression of itch remains largely undefined. TLR2 expression is elevated in AD, contributing to the progression to chronic AD through IL-4 mediation in IL-10 suppression (Kaesler et al., 2014Kaesler S. Volz T. Skabytska Y. Koberle M. Hein U. Chen K.M. et al.Toll-like receptor 2 ligands promote chronic atopic dermatitis through IL-4-mediated suppression of IL-10.J Allergy Clin Immunol. 2014; 134: 92-99Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In mice, TLR2 ablation influences ACD via an IL-12 dependent mechanism (Kaplan et al., 2012Kaplan D.H. Igyarto B.Z. Gaspari A.A. Early immune events in the induction of allergic contact dermatitis.Nat Rev Immunol. 2012; 12: 114-124Crossref PubMed Scopus (409) Google Scholar), yet its role in itch induction remains unclear. Our previous study demonstrated that inhibiting the neuronal TLR2-IL-13Rα2 pathway prevents AD itch by alleviating IL-13-induced scratching behavior (Xiao et al., 2021Xiao S. Lu Z. Steinhoff M. Li Y. Buhl T. Fischer M. et al.Innate immune regulates cutaneous sensory IL-13 receptor alpha 2 to promote atopic dermatitis.Brain Behav Immun. 2021; 98: 28-39Crossref PubMed Scopus (13) Google Scholar). However, the role of epidermal TLR2 activation in itch regulation in ACD remains poorly understood. Matrix metalloproteinase 9 (MMP9), also known as gelatinase B, is a complex enzyme in the MMP family. Under the influence of IL-13 Keratinocytes can secrete MMP-9, which cleaves gelatins, type IV collagen, fibronectin, laminin, and elastin (Birkedal-Hansen, 1995Birkedal-Hansen H. Proteolytic remodeling of extracellular matrix.Curr Opin Cell Biol. 1995; 7: 728-735Crossref PubMed Scopus (0) Google Scholar), therefore facilitating leukocyte migration into the epidermis (Purwar et al., 2008Purwar R. Kraus M. Werfel T. Wittmann M. Modulation of keratinocyte-derived MMP-9 by IL-13: a possible role for the pathogenesis of epidermal inflammation.J Invest Dermatol. 2008; 128: 59-66Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In AD, elevated plasma MMP9 levels are reported (Devillers et al., 2007Devillers A.C. van Toorenenbergen A.W. Klein Heerenbrink G.J. Muldert P.G. Oranje A.P. Elevated levels of plasma matrix metalloproteinase-9 in patients with atopic dermatitis: a pilot study.Clin Exp Dermatol. 2007; 32: 311-313Crossref Scopus (29) Google Scholar), while reduced levels are observed in the chronic phase of allergic eczemas. Despite its potential role in AD, the involvement of MMP9 in itch propagation remains unknown. This study aims to elucidate the role of MMP9 in itch, spanning AD and ACD. We have identified the TLR2/TLR1 signaling pathway as downstream component of MMP9 in itch propagation, highlighting MMP9 as a potential therapeutic target for itch-related conditions. At the outset of this study, our primary focus was to investigate the correlation between MMP9 transcription and skin conditions associated with AD, ACD, and dry skin itch. Utilizing skin samples from three distinct mouse models: the MC903-induced AD-like model, the DNFB-induced ACD model, and the AEW-induced dry skin model, we found significantly elevated MMP9 transcription levels in both DNFB and MC903 models compared to their respective controls (Figure 1a). Conversely, no significant change in MMP9 transcription levels was observed in the AEW model (Figure 1a). Notably, the DNFB-induced ACD model exhibited a more pronounced upregulation of MMP9 compared to the MC903-induced AD-like model (Figure 1a). This finding suggests a potential association between MMP9 and AD and ACD. Further research is needed to explore the precise mechanisms by which MMP9 contributes preferentially to AD and ACD skin conditions particularly in their related itch. We then investigated the potential involvement of MMP9 in itch pathogenesis in the MC903 model using MMP9-IN-1, a selective inhibitor (Supplementary Figure S1a). Compared to vehicle, intraperitoneal (i.p.) administration of MMP9-IN-1 did not significantly reduce spontaneous scratching behavior in the MC903-induced ear model (Figure 1b). In contrast, the identical dose of MMP9-IN-1 provided relief from itch during the elicitation phase in the DNFB-induced ear model (Supplementary Figure S1b), specifically from day 6 to 8 (Figure 1d), indicating a crucial role for MMP9 in ACD- rather than AD-related itch in mice. In the DNFB model, MMP9-IN-1 treatment improved skin redness (Figure 1e), decreased epidermal thickness (Figure 1f), and reduced skin inflammation (Figure 1h), as observed in H&E staining (Supplementary Figure S1c). MMP9-IN-1 also led to a significant decrease in skin MMP9+ cells (Figure 1g and Supplementary Figure S1d). Moreover, MMP9-IN-1-treated mice exhibited reduced inflammatory cell infiltration, as evidenced by labeling with antibodies targeting mMCP8, CD68, and CD11b (Figure 1h and Supplementary Figure S1e). Immunofluorescence dual staining revealed that MMP9+ cells are predominantly mMCP8+, with some also being CD68+ (Supplementary Figure S1f). These finding confirm the effective reduction by MMP9-IN-1 treatment in inflammation of the whole skin. To investigate the role of MMP9 in gene transcription regulation, RNA-seq analysis was performed during the elicitation phase of DNFB-induced models with or without MMP9-IN-1 treatment. The DNFB-elicited skin exhibited an upregulation of various itch-related genes (Supplementary Figure S2a), which were significantly reduced following MMP9-IN-1 treatment (Supplementary Figure S2b). The downregulated transcripts included itch-related receptors (Plaur, Ccr1, Ccr3), pruritogenic genes (Osm, Tslp, Nppb, Tac1), and inflammation-related genes (Cxcl3, Cxcl1, Cxcl2, Mmp3, Mmp9, Mmp10, Mmp12, Mmp13, Ccl3, Ccl4, Il1b, Il6, Il10, Tnf, Mcpt2, and Mcpt8). These findings suggest that MMP9 activation contributes to the upregulation of itch receptors and mediators in the DNFB-induced ACD model. Importantly, in the DNFB model a significant upregulation of Tlr2 hub genes , including Tlr1, Tlr2, Tlr4, Tlr6, Dusp6, Junb, Fos, and Nifkb2 and Nifkb1, was observed (Figure 2a). Treatment with MMP9-IN-1 resulted in the downregulation of Tlr2, Tlr6, Nifkbia, Junb, and Fos (Figure 2b), indicating potential involvement of the TLR2 pathway in MMP9-regulated downstream signaling during the elicitation phase of DNFB-induced ACD. This effect seems specific to the elicitation phase, as TLR2 remained unaffected during the sensitization phase of DNFB-model following MMP9-IN-1 treatment (Supplementary Figure S3a). In the MC903 model, despite upregulation of Tlr1, Tlr2, Tlr4, Tlr6, Dusp4, Dusp6, Junb, and Fos (Supplementary Figure S3b), MMP9-IN-1 did not impact the transcription levels of Tlr1, Tlr2, Tlr4, and Tlr6 (Supplementary Figure S3c). These findings suggest potential involvement of the TLR2-associated pathway downstream of MMP9-regulated gene transcription in DNFB-elicited ACD, not in MC903-induced AD-like model. To further explore the impact of interfering with the MMP9 pathway on itch triggered by TLR2 activation, we established a cheek model by intradermally administering a TLR2/TLR1 agonist, Pam3CSK4, following intraperitoneal administration of either MMP9-IN-1 or its vehicle control (Supplementary Figure S3d). The MMP9-IN-1 group exhibited significantly reduced scratching bouts compared to the vehicle control group (Figure 2c), with the peak effect observed at 30 minutes (Figure 2d). Thus, these results confirm that MMP9 signaling contributes to the TLR2/TLR1-promoted itch in the elicitation phase in DNFB-induced model. This mechanism is not an innate immune response to DNFB as it occurs only in previously sensitized mice. We also examined whether MMP9-IN-1 alleviates itch in another ACD model induced by methylisothiazolinone (MI). Treatment by MMP9-IN-1 during the elicitation phase of MI significantly reduced itch-like behaviors (Figure 2e). RT-q-PCR analysis of the MMP9-IN-1 treated MI-challenged skin indicated a trend of downregulation of TLR2 and MMP9 transcription levels, although the reduction in MMP9 did not reach statistical significance (Figure 2f). Taken together, these findings emphasize potential role of MMP9 in itch regulation in ACD and its involvement in itch promotion via the TLR2/TLR1 pathway, highlighting MMP9 as a promising and innovative therapeutic target for addressing ACD-associated itch. Housing, handling and experimental procedures involving mice were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Henan University and approved by the Animal Ethics Committee of Henan University. This study did not involve human research participants. All data generated or analysed during this study are included in this manuscript. RNA-seq datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/gds/, hosted by GEO database (accession number: GSE232349). The authors declare that there are no conflicts of interest. This worked is supported by a fund from National Natural Science of Foundation of China (NSFC 82273509), and a fund from LEO Foundation to JM.