1. Propionibacterium acnes Activates the NLRP3 Inflammasome in Human Sebocytes 
Zheng Jun Li, Dae Kyoung Choi, Kyung Cheol Sohn, Min Seok Seo, Hae Eul Lee, Young Lee, Young Joon Seo, Young Ho Lee, Ge Shi, Christos C. Zouboulis, Chang Deok Kim, Jeung Hoon Lee, Myung Im 
Journal of Investigative Dermatology 
Volume 134, Issue 11, Pages (November 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression of IL-1β and caspase-1 in sebaceous glands of acne lesions. Paraffin-embedded skin specimens of normal human facial skin (n=4) and facial inflammatory acne lesions (n=6) were immunohistochemically stained with (a) anti-IL-1β and (b) caspase-1 (Casp1). The percentage of sebocytes with cytoplasmic immunostaining was evaluated in several sebaceous glands. Bars=100μm. Data represent mean±SEM. Data were analyzed by the Student’s t-test (**P<0.01, ***P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
P. acnes-induced IL-1β processing and secretion in SZ95 sebocytes. (a) Immunofluorescence labeling of IL-1β (green) in human SZ95 sebocytes after exposure to Propionibacterium acnes at a multiplicity of infection (MOI) of 10 for 24hours. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (blue). Bars=100μm. (b) Quantitative reverse-transcriptase–PCR analysis of IL-1β messenger RNA expression after P. acnes stimulation (MOI of 1 and 10). (c) Mature IL-1β secretion in supernatants at a MOI after P. acnes stimulation was assessed using ELISA. (d) Pro-IL-1β protein in the cell extracts (CE) and mature IL-1β protein (mIL-1β) in supernatants (SN) were determined by western blot. Data represent mean±SEM. Data were analyzed by the Student’s t-test (n=3; *P<0.05, **P<0.01, ***P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
P. acnes-induced IL-1β secretion requires caspase-1 activation in SZ95 sebocytes. (a) The expression of the active caspase-1 p20 subunit (p20) was determined in SZ95 sebocytes by western blot after exposure to Propionibacterium acnes. The p20 protein levels were expressed relative to the actin control (lower panel). (b) SZ95 sebocytes were transduced with an adenovirus expressing caspase-1 specific microRNA (miR-Casp1) and scrambled microRNA (miR-Scr) as the control. (c) Supernatants (SN) and cell extracts (CE) from miR-Scr- and miR-Casp1-transduced sebocytes after P. acnes (multiplicity of infection (MOI) of 10) stimulation were analyzed for expression of IL-1β by western blot. (d) Supernatants were also analyzed for IL-1β by ELISA. NS, no significant difference. Data represent mean±SEM. Data were analyzed by the Student’s t-test (n=3; *P<0.05, **P<0.01, ***P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
P. acnes induction of IL-1β is NLRP3-dependent in SZ95 sebocytes. (a) SZ95 sebocytes were transduced with an adenovirus expressing microRNA specific for NLRP3 (miR-NLRP3) and absent in melanoma 2 (AIM2) (miR-AIM2). (b) MicroRNA-transduced SZ95 sebocytes were stimulated with Propionibacterium acnes (multiplicity of infection (MOI) of 10) for 24hours, and then supernatants (SN) and cell extracts (CE) were analyzed by western blot. (c) Supernatants were also analyzed for IL-1β by ELISA. NS, no significant difference. Data represent mean±SEM. Data were analyzed by the Student’s t-test (n=3; **P<0.01).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Metalloprotease activity and reactive oxygen species generation are required for P. acnes-induced inflammasome activation. Propionibacterium acnes (multiplicity of infection (MOI) of 10) was incubated at 65°C for 30minutes and then applied to SZ95 sebocytes. (a) Supernatants (SN) and cell extracts (CE) were analyzed by western blotting. (b) Supernatants were also analyzed for IL-1β by ELISA. (c, d) SZ95 sebocytes were treated with different concentrations of EDTA and were then stimulated with P. acnes. After SZ95 sebocytes were exposed to P. acnes in the presence of (e) glibenclamide and (f) N-acetyl-L-cysteine (NAC), IL-1β release in supernatants was measured by ELISA. Data represent mean±SEM. Data were analyzed by the Student’s t-test (n=3; **P<0.01 vs P. acnes-unstimulated cells; †P<0.05, ††P<0.01 vs only P. acnes-treated cells).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
P. acnes-induced inflammation is reduced in NALP3-deficient mice. (a) Wild-type (WT) (n=10) and NLRP3-deficient mice (n=10) were injected intradermally with 107 colony-forming units of P. acnes (right ear) or an equal volume of phosphate-buffered saline (PBS) (left ear). Inflammation-induced redness of the ear was visualized 24hours after injection. (b) Hematoxylin and eosin (HE)-stained frozen tissue sections of the ear injected with PBS alone or P. acnes in wild-type (WT) mice or with P. acnes in NLRP3-deficient mice. Bars=200μm. (c) Each specimen was immunohistochemically stained with anti-caspase-1 (Casp1) and IL-1β monoclonal antibodies. Bars=50μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions