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Cathelicidin LL-37 Ignites Primed NLRP3 Inflammasomes in Rosacea

  • David O. Croitoru
    Affiliations
    Division of Dermatology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada
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  • Vincent Piguet
    Correspondence
    Correspondence: Vincent Piguet, Division of Dermatology, Department of Medicine, Women’s College Hospital, Toronto, Ontario M5S 1B2, Canada.
    Affiliations
    Division of Dermatology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada

    Division of Dermatology, Department of Medicine, Women’s College Hospital, Toronto, Ontario, Canada
    Search for articles by this author
Published:September 22, 2021DOI:https://doi.org/10.1016/j.jid.2021.04.024
      Microbes and commensal mites contribute to the development of inflammation and neurovascular dysregulation in rosacea. Cathelicidin family proteins are epithelial antimicrobial peptides expressed in higher-order mammals. In humans, mature LL-37 is cleaved from its precursor in response to microbial infection, UV light, and injury. In their new article in the Journal of Investigative Dermatology, Yoon et al. expand on existing evidence supporting LL-37 proinflammatory activity in lipopolysaccharide (LPS)- and UV-primed models of rosacea. They show in vitro that LL-37 promotes NLRP3-mediated inflammasome activation through lysosomal destabilization in the presence of LPS and that the injection of LL-37 in vivo leads to skin inflammation that is abrogated by direct NLRP3 inhibition and homozygous knockout in a murine model.

       The role of LL-37 in cutaneous immune regulation

      The pleiotropic role of LL-37 in the cutaneous response to pathogen-associated molecular patterns (PAMPs) is rapidly emerging. LL-37 is the C-terminal domain of the sole human cathelicidin precursor, hCAP18, which is cleaved by kallikrein 5 in response to UV, tissue injury, and microbial infection (
      • Moreno-Angarita A.
      • Aragón C.C.
      • Tobón G.J.
      Cathelicidin LL-37: a new important molecule in the pathophysiology of systemic lupus erythematosus.
      ). The effector cathelicidin, LL-37, has been shown to directly impair microbial function as a pore-forming toxin (
      • Xhindoli D.
      • Pacor S.
      • Benincasa M.
      • Scocchi M.
      • Gennaro R.
      • Tossi A.
      The human cathelicidin LL-37 — a pore-forming antibacterial peptide and host-cell modulator.
      ). In addition to its bactericidal activity, LL-37 both inhibits IL-1B and amplifies proinflammatory signaling through lipopolysaccharide (LPS) binding in vitro and in vivo (
      • Dombrowski Y.
      • Peric M.
      • Koglin S.
      • Kammerbauer C.
      • Göss C.
      • Anz D.
      • et al.
      Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions.
      ;
      • Hu Z.
      • Murakami T.
      • Suzuki K.
      • Tamura H.
      • Kuwahara-Arai K.
      • Iba T.
      • et al.
      Antimicrobial cathelicidin peptide LL-37 inhibits the LPS/ATP-induced pyroptosis of macrophages by dual mechanism.
      ). LL-37 has also been shown to be upregulated in the skin of patients with psoriasis, suggesting an overarching relevant role in the mediation of cutaneous inflammatory disease (
      • Lande R.
      • Gregorio J.
      • Facchinetti V.
      • Chatterjee B.
      • Wang Y.H.
      • Homey B.
      • et al.
      Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.
      ).
      “Repeated exposure to LL-37 can activate the NLRP3-mediated inflammasome pathway, which in turn promotes the recruitment of inflammatory cells and skin inflammation, triggering rosacea-like phenotypes.”
      Rosacea is a chronic, inflammatory disorder typically involving centrofacial skin, which may have a profound effect on QOL through pruritus, pain, cosmesis, and potentially, permanent disfigurement in certain clinical variants (
      • Elssner A.
      • Duncan M.
      • Gavrilin M.
      • Wewers M.D.
      A novel P2X7 receptor activator, the human cathelicidin-derived peptide LL37, induces IL-1 beta processing and release.
      ). It is a common skin disease, with an estimated prevalence of 5.5% among the global adult population. There are mixed reports of female preponderance, with typical onset after the age of 30 years (
      • Gether L.
      • Overgaard L.K.
      • Egeberg A.
      • Thyssen J.P.
      Incidence and prevalence of rosacea: a systematic review and meta-analysis.
      ). Commensal microbes (Staphylococcus epidermidis) and mites (Demodex follicularum) have been implicated in the pathogenesis from both translational and clinical standpoints, supported by the efficacy of antimicrobials (tetracycline, nitroimidazole) and antimite therapies (ivermectin) (
      • Lazaridou E.
      • Giannopoulou C.
      • Fotiadou C.
      • Vakirlis E.
      • Trigoni A.
      • Ioannides D.
      The potential role of microorganisms in the development of rosacea.
      ). Despite this, the mechanisms of action of exogenous triggers and the associated host inflammatory mediators remain unknown, and no targeted host-directed therapies yet exist.

       LL-37 promotes inflammasome activation with LPS costimulation

      On the basis of the previous conflicting reports of LL-37 action on IL-1 signaling and inhibition as well as the synergistic amplification of LPS and other PAMPs,
      • Yoon S.-H.
      • Hwang I.
      • Lee E.
      • Cho H.-J.
      • Ryu J.H.
      • Kim T.-G.
      • et al.
      Antimicrobial peptide LL-37 drives rosacea-like skin inflammation in an NLRP3-dependent manner.
      treated bone marrow‒derived macrophages with LL-37 in the presence and absence of LPS. They found that LPS priming followed by treatment of LL-37 induced caspase-1 cleavage and IL-1B production, whereas treatment with LL-37 and LPS alone as well as with LL-37 primed by TNF-α and IFN-γ was insufficient in activating inflammasomes. This was also seen in dendritic cells (DCs) and bone marrow‒derived neutrophils, suggesting a bona fide role for LL-37 in immune cell inflammasome activation through toll-like receptor (TLR) 3 and/or TLR4 priming. Furthermore, they show that cytosolic entry of LL-37 may be mediated by P2X7 receptor‒mediated endocytosis, which is disrupted by oxidized adenosine triphosphate.

       Intradermal LL-37 induces NLRP3-dependent inflammasome activation and skin inflammation

      Cleavage of caspase 1 and IL-1β was detected in lesional skin of wild-type (WT) mice (C57BL/6) but not in that of NLRP3–/– mice after intradermal injection of LL-37, and it was absent in nonlesional control skin in both strains. Phenotypically, this correlated with rosacea-like inflammation (modified skin thickness and erythema) and lesional accumulation of Gr1+ macrophages and DCs in WT mice compared with that in NLRP3–/– mice. There was limited inflammation in both NLRP3–/– and WT mice with phosphate-buffered intradermal controls. Further supporting the necessary role of NLRP3 in vivo, ASC protein oligomerization was detected in the same pattern after injection, and this was also replicated by a caspase-1 cleavage‒activated fluorophore.

       Lysosomal rupture and potassium leakage may underly LL-37 NLRP3 activation

      Potassium efflux is a unique feature of NLRP3 inflammasome activation, and transmembrane concentration gradients were found to be significantly associated with inflammasome activation, attenuated by glibenclamide and high extracellular potassium hydrochloride. Lysosomal rupture was also detected through lysosomal protease cathepsin B, which was additionally associated with inflammasome activation.

       Inhibition and knockout of NLRP3 attenuates the murine LL-37 rosacea phenotype

      Selective inhibition of NLRP3 with MCC950, a small molecule inhibitor, showed a pronounced decrease in the LL-37 murine rosacea-like phenotype and marked decrease in IL-1B, with normal endocytosis of LL-37 in vitro. Taken together, the inflammasome-mediated effects of LL-37 appear to be initiated by TLR3 and TLR4 priming and downstream of NLRP3 inflammasome activation, which may be mediated by impaired lysosomal trafficking and cytosolic rupture.

       LL-37 as an inflammatory network regulator with multiple effectors

      The detection of LL-37 in high concentrations in the skin of patients with rosacea compared with that of healthy controls (
      • Yamasaki 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.
      ), its implication in other inflammatory diseases of the skin, and the preserved immune function of homologous cathelicidin family proteins across species support LL-37 as a potential link between exogenous PAMPs and host autoinflammation. This work by
      • Yoon S.-H.
      • Hwang I.
      • Lee E.
      • Cho H.-J.
      • Ryu J.H.
      • Kim T.-G.
      • et al.
      Antimicrobial peptide LL-37 drives rosacea-like skin inflammation in an NLRP3-dependent manner.
      aims to reconcile the previously reported inhibitory and synergistic roles of LL-37 in LPS-induced IL-1B and caspase cleavage. They show that only in the setting of LPS-primed TLRs does LL-37 achieve significant cytosolic access through P2X7 receptors and activate the inflammasome through NLRP3 in a mechanism that implicates lysosomal rupture and potassium efflux. Further validation of this work using gram-positive and gram-negative microbes will provide additional insight into the physiologic relevance of LPS in the context of LL-37 and, subsequently, into the taxa of bacteria most responsible for LL-37‒driven rosacea inflammation. The observation that LL-37 alone was sufficient to induce inflammasome activation and a rosacea-like phenotype in vivo but not in vitro suggests that other signals occurring in the murine cutaneous microenvironment may fulfill the role of LPS TLR priming. This is another attractive area for future investigation. The limitations of this model must also be recognized, and ultimately, the physiologic role of LL-37 in rosacea, while potentially still mediated by NLRP3, may be shifted toward different driving factors and mediators in humans. The identification of NLRP3 and the inflammasome as therapeutic host targets may have immediately translatable effects in the treatment of rosacea, generally, or may be of particular relevance to certain clinical variants (i.e., granulomatous). Future animal and human models will be critical to exploring the relevance of this interaction and the utility of the pathway as a clinical target.

      ORCIDs

      Conflict of Interest

      VP has no personal financial ties with any pharmaceutical company. He undertakes advisory work for Pfizer, AbbVie, Janssen, UCB, Novartis, Almirall, and Celgene. He has received honoraria from Kyowa Kirin. In his role as Department Division Director of Dermatology at the University of Toronto (Ontario, Canada), VP has received departmental support from AbbVie, Bausch Health, Celgene, Janssen, LEO Pharma, Lilly, NAOS, Novartis, Pfizer, Pierre-Fabre, and Sanofi in the past 36 months. DOC states no conflict of interest.

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      Linked Article

      • Antimicrobial Peptide LL-37 Drives Rosacea-Like Skin Inflammation in an NLRP3-Dependent Manner
        Journal of Investigative DermatologyVol. 141Issue 12
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          Rosacea is a chronic inflammatory skin disease characterized by immune response–dependent erythema and pustules. Although the precise etiology of rosacea remains elusive, its pathogenesis is reportedly associated with an increased level of antimicrobial peptide LL-37. However, molecular mechanisms underlying the progression of rosacea via LL-37 remain poorly understood. Here, we examined the potential role of LL-37 in rosacea-like skin inflammatory phenotypes at a molecular level. Our in vitro data demonstrated that LL-37 promotes NLRP3-mediated inflammasome activation in lipopolysaccharide-primed macrophages, indicated by the processing of caspase-1 and IL-1β.
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