Allergy-Inducing Chromium Compounds Trigger Potent Innate Immune Stimulation Via ROS-Dependent Inflammasome Activation  Christian Adam, Jonas Wohlfarth, Maike Haußmann, Helga Sennefelder, Annette Rodin, Mareike Maler, Stefan F. Martin, Matthias Goebeler, Marc Schmidt  Journal of Investigative Dermatology  Volume 137, Issue 2, Pages 367-376 (February 2017) DOI: 10.1016/j.jid.2016.10.003 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Chromium (VI) compounds fail to trigger direct proinflammatory activation but potently activate the NLRP3 inflammasome. (a) IL-8 ELISA of human THP1 monocytes stimulated for 16 hours with the indicated chromium (VI) compounds, Ni2+, or medium (ctrl). (b) IL-1β ELISA and IL-1β immunoblot of culture supernatants (SNs) from unprimed or TPA-primed THP1 stimulated as indicated. (c) IL-1β release from TPA-primed THP1 stimulated ± the caspase-1 inhibitor Z-YVAD-fmk. (d) IL-1β ELISA and immunoblots of culture supernatants (SN, left) or lysates (right) from TPA-primed parental THP1 (THP1-WT) and functionally NLRP3-deficent THP1 cells (THP1-defNLRP3) stimulated as indicated. Bar diagrams show means ± standard deviations of n ≥ 3 experiments. ∗∗P < 0.01, ∗∗∗P < 0.001, nonsignificant P > 0.05. One-way analysis of variance (in a) or two-way analysis of variance (in b–d). Immunoblots are representative of n = 3–4 experiments. ATP, adenosine triphosphate; ctrl, control; CrO42–, potassium chromate; Cr2O72–, potassium dichromate; n.s., not significant; SN, supernatant; TPA, 12-O-tetradecanoylphorbol-13-acetate; WT, wild type; YVAD, Z-YVAD-fmk. Journal of Investigative Dermatology 2017 137, 367-376DOI: (10.1016/j.jid.2016.10.003) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Chromium (VI)-induced IL-1β release is licensed by TLR stimulation and occurs independently of P2X7. (a) IL-1β ELISA of THP1 culture supernatants, showing CrO42– or Cr2O72–-induced IL-1β upon priming with medium or the TLR4 agonist LPS. (b) IL-8 ELISA and IL-1β immunoblots of lysates from NHEKs stimulated with tumor necrosis factor or the indicated TLR ligands for 6 hours. (c) IL-1β ELISA of supernatants from medium-, tumor necrosis factor-, or MALP-2–primed NHEKs exposed to the indicated stimuli. (d) Cr2O72–-induced IL-1β secretion from murine wild-type BMDCs (P2x7+/+) and P2x7-deficient BMDCs (P2x7–/–) with and without LPS priming. Upper panel shows IL-1β ELISA, and lower panel shows IL-1β immunoblot of culture supernatants (SN). Diagram data show mean cytokine release ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, nonsignificant (n.s.) P > 0.05; two-way analysis of variance; n = 3. ATP, adenosine triphosphate; ctrl, control; BMDC, bone marrow-derived dendritic cell; DNCB, 2,4-dinitrochlorobenzene; LPS, lipopolysaccharide; NHEK, normal human epidermal keratinocytes; n.s., not significant; SN, supernatant; TLR, toll-like receptor; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2017 137, 367-376DOI: (10.1016/j.jid.2016.10.003) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Chromium (VI)-induced inflammasome activation is associated with dose-dependent cytotoxicity. THP1 were primed with TPA and stimulated with the indicated doses of CrO42– or Cr2O72–, ATP, or medium (ctrl), respectively. (a) LDH release assay, showing mean percentile cytotoxicity ± standard deviation in relation to a lysed control set to 100. ∗∗P < 0.01, ∗∗∗P < 0.001; n = 3–4, two-way analysis of variance. (b) Dose correlation between chromium (VI)-induced IL-1β p17 release, caspase-1 cleavage, and tubulin secretion. Upper panel shows IL-1β ELISA, showing mean IL-1β secretion ± standard deviation of n = 3–5 independent experiments. ∗∗∗P < 0.001, one-way analysis of variance. Lower panel shows representative (n = 3) immunoblots of culture supernatants (SN) stained for the indicated proteins. ATP, adenosine triphosphate; Cr, chromium; ctrl, control; LDH, lactate dehydrogenase; M, mol/L; SN, supernatant; TPA, 12-O-tetradecanoylphorbol-13-acetate. Journal of Investigative Dermatology 2017 137, 367-376DOI: (10.1016/j.jid.2016.10.003) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Chromium (VI)-induced cytotoxicity is inhibited by high extracellular K+ and NLRP3 depletion. (a, b) TPA-primed THP1 (THP1-WT) were stimulated with Cr2O72– or medium (ctrl) in the absence or presence of the indicated KCl concentrations or exposed to the indicated stimuli in parallel to TPA-primed THP1 lacking functional NLRP3 expression (THP1-defNLRP3). (a, d) LDH release assays, (b, top) IL-1β ELISA, or (b, bottom, c) representative immunoblots of culture supernatants (SNs) show the impact of the individual treatments on cytotoxicity or release of IL-1β, caspase-1 p10, and tubulin, respectively. Bar diagrams represent (a, d) mean percentile cytotoxicity ± standard deviation related to a lysed control set to 100% (n = 3) (b) or mean IL-1β supernatant concentration ± standard deviation (n = 4). ∗∗P < 0.01, ∗∗∗P < 0.001; two-way analysis of variance. ATP, adenosine triphosphate; ctrl, control; LDH, lactate dehydrogenase; M, mol/L; SN, supernatant; TPA, 12-O-tetradecanoylphorbol-13-acetate; WT, wild type. Journal of Investigative Dermatology 2017 137, 367-376DOI: (10.1016/j.jid.2016.10.003) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Differential outcomes of chromium (VI) and chromium (III) stimulation and roles of oxidation and phagocytosis in Cr2O72–-induced inflammasome activation. (a–e) TPA-primed THP1 were incubated with medium (ctrl), ATP, alum, 100 μmol/L of the specified chromium (VI) compounds, or (a) Cr3+ at the indicated concentration, and IL-1β processing ± 2 μmol/L cytochalasin D, caspase-1 cleavage, cytotoxicity, chromium (VI) supernatant concentration, or mROS production were determined by (a, d) IL-1β ELISA and immunoblot of supernatants (SN), (b) LDH release assay, (c) quantitative substrate oxidation by absorption measurement, or (e) flow cytometric quantification of percentile MitoSox fluorescence, respectively. Immunoblots and histogram overlays are representative of n = 3 experiments. Bar diagrams show averages ± standard deviations from n ≥ 3 experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, nonsignificant (n.s.) P > 0.05; one-way analysis of variance. ATP, adenosine triphosphate; ctrl, control; h, hours; LDH, lactate dehydrogenase; M, mol/L; n.s., not significant; SN, supernatant; TPA, 12-O-tetradecanoylphorbol-13-acetate. Journal of Investigative Dermatology 2017 137, 367-376DOI: (10.1016/j.jid.2016.10.003) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Chromium (VI)-induced inflammasome activation and cytotoxicity is mediated by mROS. TPA-primed THP1 (THP1-WT) were treated with (b, c) medium (ctrl), 100 μmol/L Cr2O72–, or (a) the indicated Cr2O72– concentrations ± (a) 10 mmol/L of the ROS inhibitor NAC, (b) 50 mmol/L KCl, or (c) 10 μmol/L of the caspase-1 inhibitor Z-YVAD-fmk, respectively. In c, additionally NLRP-depleted THP1-defNLRP3 were stimulated with Cr2O72– or medium. (a) Top shows IL-1β ELISA, showing mean IL-1β secretion ± standard deviation. ∗∗∗P < 0.001, nonsignificant (n.s) P > 0.05, two-way analysis of variance, n = 3. Bottom shows representative immunoblots of supernatants (SN, n = 3), showing release of IL-1β, caspase 1 p10, and tubulin protein ± NAC. (b, c) Representative quantification of n = 3 FACS experiments, analyzing percentile MitoSox positivity of the indicated cells upon Cr2O72– (red) or medium stimulation (black) ± YVAD. ctrl, control; M, mol/L; NAC, N-acetyl cysteine; n.s., not significant; SN, supernatant; TPA, 12-O-tetradecanoylphorbol-13-acetate; WT, wild type; YVAD, Z-YVAD-fmk. Journal of Investigative Dermatology 2017 137, 367-376DOI: (10.1016/j.jid.2016.10.003) Copyright © 2016 The Authors Terms and Conditions