Rosacea is a chronic skin disorder characterized by painful episodes of facial inflammation provoked by a number of sensory stimuli (Two et al., 2015Two A.M. Wu W. Gallo R.L. Hata T.R. Rosacea.J Am Acad Dermatol. 2015; 72: 749-758Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). It is known that cathelicidin is highly expressed in rosacea skin and that in combination with increased serine protease activity, its cleavage product, LL37, can trigger a number of inflammatory processes, including mast cell (MC) activation (Yamasaki et al., 2007Yamasaki K. Di Nardo A. Bardan A. Murakami M. Ohtake T. Coda A. et al.Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.Nat Med. 2007; 13: 975-980Crossref PubMed Scopus (609) Google Scholar). Having previously shown that MCs are the key mediators of LL37-induced rosacea inflammation (Muto et al., 2014Muto Y. Wang Z. Vanderberghe M. Two A.M. Gallo R.L. Di Nardo A. Mast cells are key mediators of cathelicidin-initiated skin inflammation in rosacea.J Invest Dermatol. 2014; 134: 2728-2736Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), we sought to determine what factors lead to the increased activation of MCs and the increased release of their mediators in rosacea skin. Transient receptor potential vanilloid (TRPV) channels are known to play a role in a variety of sensory pathologies. From monitoring blood pressure (Sun et al., 2009Sun H. Li D.P. Chen S. Hittelman W.N. Pan H.L. Sensing of blood pressure increase by transient receptor potential vanilloid 1 receptors on baroreceptors.J Pharmacol Exp Ther. 2009; 331: 851-859Crossref PubMed Scopus (58) Google Scholar), to promoting both serotonergic and histaminergic itch responses (Akiyama et al., 2016Akiyama T. Ivanov M. Nagamine M. Davoodi A. Carstens M.I. Ikoma A. et al.Involvement of TRPV4 in serotonin-evoked scratching.J Invest Dermatol. 2016; 136: 154-160Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, Chen et al., 2016Chen Y. Fang Q. Wang Z. Zhang J.Y. MacLeod A.S. Hall R.P. et al.Transient receptor potential vanilloid 4 ion channel functions as a pruriceptor in epidermal keratinocytes to evoke histaminergic itch.J Biol Chem. 2016; 291: 10252-10262Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), these channels provide an influx of cations by which a variety of downstream cellular responses can be initiated. TRPV channels are also upregulated in rosacea, and more specifically, TRPV2 and TRPV4 were shown to colocalize with MCs in rosacea skin (Sulk et al., 2012Sulk M. Seeliger S. Aubert J. Schwab V.D. Cevikbas F. Rivier M. et al.Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.J Invest Dermatol. 2012; 132: 1253-1262Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Thus, we hypothesized that LL37 may also drive the expression of TRPV channels in the MCs of rosacea skin. To determine whether LL37 plays a role in TRPV expression, we used our previously described rosacea mouse model by intradermally injecting the backs of mice with 50 μl LL37 (320 μM) or phosphate buffered saline twice daily for 2 days. All animal experiments were approved by the UCSD Institutional Animal Care and Use Committee. Real-time quantitative polymerase chain reaction analysis showed that expression of both TRPV2 (Figure 1a) and TRPV4 (Figure 1d) increased dramatically in response to LL37 injection. Immunofluorescent staining of LL37-injected mouse skin also revealed a significant increase in colocalization between MC chymase (monoclonal antibody, Abcam, Cambridge, MA) and both TRPV2 (polyclonal antibody, Santa Cruz Biotechnology, Dallas, TX; Figure 1b and c) and TRPV4 (polyclonal antibody, Abcam; Figure 1e and f). To validate these findings in vitro, we cultured primary murine MCs with various concentrations of LL37 for either 6 or 24 hours. TRPV2 (Figure 1g) and TRPV4 (Figure 1h) mRNA expression increased significantly in response to LL37 after 24 hours; however, when these experiments were repeated in primary human MCs (hMCs), no expression change was noted for TRPV2, while a dose-dependent increase in TRPV4 expression was clearly observed (Figure 1i and j). These trends were confirmed at the protein level with cytospins of murine MCs stained for TRPV2 (Figure 1k and l) and hMCs stained for TRPV4 (Figure 1m and n). Having established a connection between LL37 and TRPV4 expression in hMCs, we sought to determine whether TRPV4 plays a role in regulating hMC activity. We first treated hMCs with 0.5 μM of a known TRPV4 inhibitor, HC 067047 (Tocris, Bristol, United Kingdom), for 24 hours and then measured hMC activity by inducing degranulation with compound 48/80, as previously described (Wang et al., 2012Wang Z. Lai Y. Bernard J.J. MacLeod D.T. Cogeh A.L. Moss B. et al.Skin mast cells protect mice against vaccinia virus by triggering mast cell receptor S1PR2 and releasing antimicrobial peptides.J Immunol. 2012; 188: 345-357Crossref PubMed Scopus (78) Google Scholar). The hMCs pretreated with the TRPV4 antagonist showed a reduced capacity for degranulation when compared with control cells (Figure 2a). Hoping to confirm this effect with a more sustained TRPV4 inhibition, we transfected CD34+ cord blood cells with a TRPV4 CRISPR/Cas9 knockout plasmid in combination with the TRPV4 HDR plasmid (Santa Cruz Biotechnology). The transfection was performed 5 days after plating the CD34+ cord blood cells (Astarte Biologics, Bothell, WA), while the cells were highly proliferative. These cells were then differentiated into hMCs for 10 weeks through a previously established protocol (Kirshenbaum and Metcalfe, 2006Kirshenbaum A.S. Metcalfe D.D. Growth of human mast cells from bone marrow and peripheral blood-derived CD34+ pluripotent progenitor cells.Methods Mol Biol. 2006; 315: 105-112PubMed Google Scholar). Real-time quantitative polymerase chain reaction analysis of these hMCs revealed a significant drop in TRPV4 expression, whereas expression of other genes involved in rosacea inflammation (cathelicidin antimicrobial peptide, chymase1, Mas-related gene X2 [MRGX2]) remained unchanged (Figure 2b). A decrease in TRPV4 protein expression was also observed in the knockdown cells by western blot (data not shown). To validate the effect of TRPV4 on hMC activation, we measured degranulation after stimulating hMCs with either LL37 (5 μM) or compound 48/80 for 45 minutes. We observed that degranulation in the TRPV4 knockdown population was significantly reduced after both LL37 and 48/80 stimulation, which is consistent with the results of the previous TRPV4 antagonist experiment (Figure 2c). Given the dramatic effect that TRPV4 expression appears to play in MC activation, we investigated a potential mechanism by which LL37 can promote TRPV4 upregulation. Many publications have shown various Mas-related G-protein coupled receptors to be upstream in the expression of other TRP channels (Solinski et al., 2014Solinski H.J. Gudermann T. Breit A. Pharmacology and signaling of MAS-related G protein-coupled receptors.Pharmacol Rev. 2014; 66: 570-597Crossref PubMed Scopus (95) Google Scholar), and thus, we hypothesized that Mas-related G-protein coupled receptors may play a role in LL37’s induction of TRPV4 expression. To determine whether uncoupling Gi/o from the G-protein coupled receptor with pertussis toxin can attenuate TRPV4 upregulation, we treated hMCs with 1 ng/ml pertussis toxin for 16 hours followed by a 24-hour exposure to 2.5 μM LL37. hMCs that were pretreated with pertussis toxin showed no increase in TRPV4 mRNA expression after LL37 stimulation when compared with controls (Figure 2d). Subramanian et al., 2011Subramanian H. Gupta K. Guo Q. Price R. Ali H. Mas-related gene X2 (MrgX2) is a novel G protein coupled receptor for the antimicrobial peptide LL-37 in human mast cells: resistance to receptor phosphorylation, desensitization and internalization.J Biol Chem. 2011; 286: 44739-44749Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar previously showed that MRGX2 is a pertussis toxin-sensitive G-protein coupled receptor that mediates LL37-initiated degranulation in MCs. Therefore, to more specifically determine whether MRGX2 is involved in this pathway, we transfected CD34+ cord blood cells with MRGX2 CRISPR/Cas9 knockout and HDR plasmids (Santa Cruz Biotechnology) as described above. Real-time quantitative polymerase chain reaction analysis revealed a significantly reduced mRNA expression of MRGX2 in the knockdown hMCs (Figure 2e), and this was confirmed at the protein level by western blotting (data not shown). Of note, there is also a reduction of cathelicidin antimicrobial peptide mRNA observed with MRGX2 knockdown (Figure 2e). When these MRGX2-deficient hMCs were stimulated with LL37 for 24 hours, no accompanying increase in TRPV4 expression was observed (Figure 2f), suggesting that MRGX2 is involved in the pathway of TRPV4 upregulation. Taken together, our work suggests that TRPV4 plays a key role in LL37-driven MC activation in rosacea. We demonstrate that LL37 can directly increase TRPV expression in hMCs and that TRPV4 expression is needed for full degranulation of the MC in the setting of rosacea. Furthermore, LL37-induced upregulation of TRPV4 appears to be dependent on MRGX2 activity. We propose that LL37 activates MRGX2, which signals to increase expression of TRPV4 in the MC. Considering that higher intracellular Ca2+ levels have been shown to promote MC activity (Neher, 1988Neher E. The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum.J Physiol. 1988; 395: 193-214Crossref PubMed Scopus (193) Google Scholar), TRPV4 expression likely allows for a greater influx of cations, which can prime the MC for continuous degranulation. Furthermore, our work provides a basis for TRPV4-targeted therapies, which have already been shown to reduce pain-associated behavior in mouse models (Kanju et al., 2016Kanju P. Chen Y. Lee W. Yeo M. Lee S.H. Romac J. et al.Small molecule dual-inhibitors of TRPV4 and TRPA1 for attenuation of inflammation and pain.Sci Rep. 2016; 6: 26894Crossref PubMed Scopus (48) Google Scholar). Similarly, TRPV4 antagonism may allow for MC inhibition with the potential to reduce cutaneous inflammation in patients with rosacea. The authors state no conflict of interest. This work was supported by a grant from the National Rosacea Society and National Institutes of Health 5R01AI093957 to ADN. TRPV4 Moves toward Center-Fold in Rosacea PathogenesisJournal of Investigative DermatologyVol. 137Issue 4PreviewMascarenhas et al. report that TRPV4 expression is upregulated in mast cells in response to the proteolytic cathelicidin fragment LL37 in a murine rosacea model and that TRPV4 loss of function attenuates mast cell degranulation. These findings render TRPV4 a translational-medical target in rosacea. However, signaling mechanisms causing increased expression of TRPV4 await elucidation. Moreover, we ask whether TRPV4-mediated Ca++-influx evokes mast cell degranulation. Full-Text PDF Open Archive