Caspase-11–Mediated Cell Death Contributes to the Pathogenesis of Imiquimod-Induced Psoriasis

Inflammasomes are multiprotein complexes that respond to infection or injury to activate inflammation. Inflammatory caspases, caspase-1, -4, and -5 in humans, and their murine orthologues caspase-1 and -11, are crucial components of inflammasomes, responsible for the maturation and secretion of IL-1β and IL-18 and for pyroptosis (inflammatory cell death) (Creagh, 2014Creagh E.M. Caspase crosstalk: integration of apoptotic and innate immune signalling pathways.Trends Immunol. 2014; 35: 631-640Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Psoriasis is a chronic inflammatory skin condition with a range of clinical manifestations. The most common manifestation is chronic plaque psoriasis, where the adaptive immune response predominates. However, innate and autoinflammatory events, governed by IL-1β (Martinez-Quiles and Goldbach-Mansky, 2018Martinez-Quiles N. Goldbach-Mansky R. Updates on autoinflammatory diseases.Curr Opin Immunol. 2018; 55: 97-105Crossref PubMed Scopus (22) Google Scholar), prevail in pustular forms of psoriasis (Liang et al., 2017Liang Y. Sarkar M.K. Tsoi L.C. Gudjonsson J.E. Psoriasis: a mixed autoimmune and autoinflammatory disease.Curr Opin Immunol. 2017; 49: 1-8Crossref PubMed Scopus (134) Google Scholar). The IL-23–IL-17 axis is the main driver of pathogenesis in psoriasis, as evidenced by successful therapies that target these cytokines (Lowes et al., 2014Lowes M.A. Suárez-Fariñas M. Krueger J.G. Immunology of psoriasis.Annu Rev Immunol. 2014; 32: 227-255Crossref PubMed Scopus (973) Google Scholar). In murine skin, IL-1β stimulates keratinocytes to produce chemoattractants for immune cells, proliferation of γδ T cells, and production of IL-17 (Cai et al., 2019Cai Y. Xue F. Quan C. Qu M. Liu N. Zhang Y. et al.A critical role of the IL-1beta-IL-1R signaling pathway in skin inflammation and psoriasis pathogenesis.J Invest Dermatol. 2019; 139: 146-156Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Ghoreschi et al., 2010Ghoreschi K. Laurence A. Yang X.P. Tato C.M. McGeachy M.J. Konkel J.E. et al.Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling.Nature. 2010; 467: 967-971Crossref PubMed Scopus (1073) Google Scholar). IL-1β mRNA and protein levels in patient psoriatic skin correlate with disease progression and treatment response (Cai et al., 2019Cai Y. Xue F. Quan C. Qu M. Liu N. Zhang Y. et al.A critical role of the IL-1beta-IL-1R signaling pathway in skin inflammation and psoriasis pathogenesis.J Invest Dermatol. 2019; 139: 146-156Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and blocking IL-1β is effective in patients with pustular psoriasis and psoriatic arthritis (Tsai and Tsai, 2017Tsai Y.C. Tsai T.F. Anti-interleukin and interleukin therapies for psoriasis: current evidence and clinical usefulness.Ther Adv Musculoskelet Dis. 2017; 9: 277-294Crossref PubMed Scopus (78) Google Scholar). The involvement of IL-1β in cutaneous inflammation implies a central role for inflammatory caspases in the pathogenesis of psoriasis. Expression and activation of inflammatory caspases is upregulated in psoriatic lesions (Johansen et al., 2007Johansen C. Moeller K. Kragballe K. Iversen L. The activity of caspase-1 is increased in lesional psoriatic epidermis.J Invest Dermatol. 2007; 127: 2857-2864Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, Salskov-Iversen et al., 2011Salskov-Iversen M.L. Johansen C. Kragballe K. Iversen L. Caspase-5 expression is upregulated in lesional psoriatic skin.J Invest Dermatol. 2011; 131: 670-676Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, Zwicker et al., 2017Zwicker S. Hattinger E. Bureik D. Batycka-Baran A. Schmidt A. Gerber P.A. et al.Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation.PLOS ONE. 2017; 12: e0175153Crossref PubMed Scopus (21) Google Scholar). Imiquimod (IMQ), the active component of Aldara cream (5% IMQ), is a TLR7/8 agonist that induces the development of an inflammatory skin disease remarkably similar to psoriasis (van der Fits et al., 2009van der Fits L. Mourits S. Voerman J.S. Kant M. Boon L. Laman J.D. et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.J Immunol. 2009; 182: 5836-5845Crossref PubMed Scopus (1348) Google Scholar). Significant amelioration of IMQ-induced murine skin inflammation was recently demonstrated by the genetic deficiency or pharmacological inhibition of both caspase-1 and caspase-11 (Aira et al., 2019Aira L.E. Gonçalves D. Bossowski J.P. Rubio-Patiño C. Chiche J. Paul-Bellon R. et al.Caspase 1/11 deficiency or pharmacological inhibition mitigates psoriasis-like phenotype in mice.J Invest Dermatol. 2019; 139: 1306-1317Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). However, caspase-1 and caspase-11 may have nonredundant roles during psoriasis, and their individual contribution to its pathogenesis remains to be addressed. There are conflicting reports regarding the function of caspase-1 during psoriasis (Cho et al., 2012Cho K.A. Suh J.W. Lee K.H. Kang J.L. Woo S.Y. IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1beta by keratinocytes via the ROS-NLRP3-caspase-1 pathway.Int Immunol. 2012; 24: 147-158Crossref PubMed Scopus (108) Google Scholar, Rabeony et al., 2015Rabeony H. Pohin M. Vasseur P. Petit-Paris I. Jégou J.F. Favot L. et al.IMQ-induced skin inflammation in mice is dependent on IL-1R1 and MyD88 signaling but independent of the NLRP3 inflammasome.Eur J Immunol. 2015; 45: 2847-2857Crossref PubMed Scopus (47) Google Scholar); thus, we aimed to determine the specific role of caspase-11 during IMQ-induced skin inflammation. All experiments were performed under license and approval of the Trinity College Dublin animal research ethics committee and the Irish Health Protection Regulatory Agency. Following application of Aldara to the back skin of Casp-11+/+ and Casp-11-/- mice for four consecutive days, there was significantly less scaling and erythema in Casp-11-/- skin than Casp-11+/+ (Figure 1a–c). Less epidermal thickening in Casp-11-/- skin was also observed at early stages of disease (Supplementary Figure S1 online). Psoriatic lesions typically display increased acanthosis, immune cell infiltration, and angiogenesis. Histologic examination of IMQ-treated skin demonstrates that Casp-11-/- skin displays significantly less epidermal proliferation (Figure 1d and e) and leukocyte infiltration into the dermis (Figure 1f and g). The reduction in erythema seen in Casp-11-/- skin correlates with significantly fewer endothelial cells in the dermis (Figure 1h and i), decreased mRNA expression of the proangiogenic marker Ang-2 (Figure 1j), and increased expression of the antiangiogenic marker TSP-1 (Figure 1k). These results reveal that caspase-11 contributes to IMQ-induced skin pathology by promoting keratinocyte proliferation, immune cell infiltration, and neoangiogenesis to support the metabolic demands of the uncontrolled keratinocytes. Analysis of the epidermal layer revealed that IMQ-treated Casp-11-/- skin had significantly less TUNEL positivity than Casp-11+/+ skin (Figure 2a and b). TUNEL staining identifies both apoptotic and pyroptotic cells; thus, both pathways were further examined. Although caspase-11 has been reported to promote caspase-3 processing after lipopolysaccharide challenge (Kang et al., 2002Kang S.J. Wang S. Kuida K. Yuan J. Distinct downstream pathways of caspase-11 in regulating apoptosis and cytokine maturation during septic shock response.Cell Death Differ. 2002; 9: 1115-1125Crossref PubMed Scopus (91) Google Scholar), no differences in caspase-3 processing were observed (Figure 2c). Caspase-11 mediates pyroptosis via Gasdermin-D cleavage, resulting in pore formation and release of inflammatory mediators such as IL-1β and lactate dehydrogenase. We show that Gasdermin-D cleavage, and consequent lactate dehydrogenase release, are significantly lower in Casp-11-/- skin than Casp-11+/+ (Figure 2c and d), confirming a caspase-11 requirement for pyroptotic cell death during this disease. Decreased cell death in both the dermis and epidermis of Casp-11-/- skin was observed as early as 24 hours following Aldara treatment (Supplementary Figure S2a and b online). The absence of caspase-11 did not alter the secretion of inflammasome-dependent cytokines from IMQ-treated skin (Figure 2e and f). Aira et al., 2019Aira L.E. Gonçalves D. Bossowski J.P. Rubio-Patiño C. Chiche J. Paul-Bellon R. et al.Caspase 1/11 deficiency or pharmacological inhibition mitigates psoriasis-like phenotype in mice.J Invest Dermatol. 2019; 139: 1306-1317Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar recently reported decreased IL-1β and IL-18 secretion from caspase-1/caspase-11 double knockouts, suggesting that caspase-1, rather than caspase-11, is required for the secretion of inflammasome-mediated cytokines from skin. However, when healthy Casp-11+/+ and Casp-11-/- skin were stimulated with Aldara ex vivo, and conditioned media from Aldara-treated skin was subsequently applied to bone marrow–derived macrophages, less inflammasome-mediated cytokine secretion occurred in Casp-11+/+ bone marrow–derived macrophages (Figure 2g and h). Findings suggest that caspase-11–mediated pyroptosis in the skin induces the secretion of alarmins that serve to drive inflammasome activation in immune cells. Caspase-11 also contributes to IL-1β secretion in response to conditioned media from Aldara-treated skin (Figure 2g and h). The role of pyroptosis has not been studied in detail in psoriasis pathology; however, our results suggest that inhibiting inflammasome activation could be beneficial for patients with psoriasis. Dataset analysis reveals that inflammatory caspase-4 and -5, pro–IL-1β, and the pyroptotic mediator Gasdermin-D are all significantly elevated in human psoriatic lesions (Supplementary Figure S3 online). Alarmins (including heat shock proteins, mitochondrial components, and S100 proteins) are enriched in secretomes from human psoriatic lesions (Williamson et al., 2013Williamson J.C. Scheipers P. Schwämmle V. Zibert J.R. Beck H.C. Jensen O.N. A proteomics approach to the identification of biomarkers for psoriasis utilising keratome biopsy.J Proteomics. 2013; 94: 176-185Crossref PubMed Scopus (30) Google Scholar), suggesting that proinflammatory cell death is also occurring in human lesions. Treatment with NLRP3 (Irrera et al., 2017Irrera N. Vaccaro M. Bitto A. Pallio G. Pizzino G. Lentini M. et al.BAY 11–7082 inhibits the NF-kappaB and NLRP3 inflammasome pathways and protects against IMQ-induced psoriasis.Clin Sci (Lond). 2017; 131: 487-498Crossref PubMed Scopus (70) Google Scholar) or inflammatory caspase (Aira et al., 2019Aira L.E. Gonçalves D. Bossowski J.P. Rubio-Patiño C. Chiche J. Paul-Bellon R. et al.Caspase 1/11 deficiency or pharmacological inhibition mitigates psoriasis-like phenotype in mice.J Invest Dermatol. 2019; 139: 1306-1317Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) inhibitors has already been shown to ameliorate IMQ-induced murine psoriasis. This study identifies a clear and singular role for caspase-11 in mediating IMQ-induced skin inflammation and proposes further investigation of caspase-4/5 inhibition for the treatment of inflammatory skin conditions. All data generated or analyzed during this study are included in this published article (and its supplementary information files). Sinéad Kenealy: https://orcid.org/0000-0003-4666-346X Joan Manils: https://orcid.org/0000-0001-8429-1295 Mathilde Raverdeau: https://orcid.org/0000-0002-6088-6902 Natalia Munoz-Wolf: https://orcid.org/0000-0002-5173-4026 Gillian Barber: https://orcid.org/0000-0002-6909-6042 Alex Liddicoat: https://orcid.org/0000-0002-1841-2033 Ed C. Lavelle: https://orcid.org/0000-0002-3167-1080 Emma M. Creagh: https://orcid.org/0000-0001-7631-4370 The authors state no conflict of interest. We thank the J. Yuan laboratory (Harvard Medical School) for gifting Casp-11-/- mice. This study was supported by an SFI Investigator program grant (12/IP/1400). Conceptualization: SK, JM, EMC; Data Curation: SK, JM, MR, NMW, GB, AL; Funding Acquisition: EMC; Resources: ECL; Supervision: EMC; Writing - Original Draft Preparation: SK, JM, EMC; Writing - Review and Editing: SK, JM, EMC, ECL. Download .xml (.0 MB) Help with xml files Data ProfileSupplementary Figure S2Less cell death is observed in FFPE skin of Casp-11-/- mice at early stages of experimental psoriasis. Casp-11+/+ and Casp-11-/- mice were treated with Aldara (50 mg) for 24 hours. Back whole skin tissue was fixed in formalin and paraffin embedded. (a) Representative images of TUNEL-stained control and Aldara-treated back skin taken at 24 hours. (b) TUNEL positive cells were counted/field in the epidermis in an average of three images per piece of tissue, and three pieces of tissue per mouse. Control (n = 2) and Aldara (n = 5), error bars represent mean ± SEM. Two-way ANOVA followed by Bonferroni post-test found *P < 0.05. ANOVA, analysis of variance; SEM, standard error of the mean; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. 10× magnification, Bar = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3Enhanced expression of pyroptosis-related genes in biopsy tissue from human psoriatic lesions. Relative mRNA expression levels of (a) caspase-4, (b) caspase-5, (c) IL-1β, and (d) Gasdermin-D in normal, uninvolved, and lesioned human psoriatic skin were analyzed using the published dataset, GDS4602 (Nair et al., 2009). For each indicated gene, graphs indicate the gene expression values in individual samples. One-way ANOVA test was used to evaluate the significant differences among the three groups. *P < 0.05, ****P < 0.0001. ANOVA, analysis of variance.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Caspase 1/11 Deficiency or Pharmacological Inhibition Mitigates Psoriasis-Like Phenotype in MiceJournal of Investigative DermatologyVol. 139Issue 6PreviewInflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1β/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. 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