1. IL-1β Drives Inflammatory Responses to Propionibacterium acnes In Vitro and In Vivo 
Magdalena Kistowska, Samuel Gehrke, Dragana Jankovic, Katrin Kerl, Antonia Fettelschoss, Laurence Feldmeyer, Gabriele Fenini, Antonios Kolios, Alexander Navarini, Ruta Ganceviciene, Jürgen Schauber, Emmanuel Contassot, Lars E. French 
Journal of Investigative Dermatology 
Volume 134, Issue 3, Pages (March 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
P. acnes induces cytokine secretion from human myeloid cells. (a) Mature-IL-1β expression in human sections of papulo-pustular acne or normal skin. Bottom left panel, high magnification. Bar=100 μM. (b) Quantitative PCR of IL-1β in acne (AV, n=12) and normal skin (NS, n=6). Relative IL-1β abundance against RPL27 analyzed and normalized with average of normal skin. (c) Monocytes exposed to live or heat-inactivated (HI) P. acnes (multiplicity of infection (MOI)=100). Western blotting of pro- and mature-IL-1β-p17 in lysates and supernatants (SN), respectively. (d–g) Monocytes stimulated with live or HI P. acnes for 24 hours. IL-1β, IL-6, tumor necrosis factor-α (TNFα), and IL-8 release was determined by ELISA. (h) ELISA of IL1-β secreted from ultra-pure lipopolysaccharide (upLPS)-primed monocytes exposed to P. acnes. (i) IL-1β ELISA of P. acnes-exposed or UVB-irradiated keratinocytes supernatant. Results are mean±SD, representative of three experiments. **P≤0.001; ***P≤0.0001; ****P≤ NS, not significant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
P. acnes activates caspase-1 and NLRP3 inflammasome. (a) Ultra-pure lipopolysaccharide (upLPS)-primed human monocytes, exposed to medium, live P. acnes, or monosodiumurate crystals (MSU) in the presence of DMSO or Z-VAD-fmk for 24 hours. IL-1β and mature-IL-1β levels determined in supernatants by ELISA and western blotting, respectively. (b–d) ELISA of secreted IL-1β from PMA (phorbol-12-myristate-13-acetate)-differentiated THP-1 cells transfected with caspase-1-shRNA (shCASP1), shASC, shNLRP1, or shNLRP3 and exposed to live P. acnes. shLamin A/C served as control. (e–g) ELISA of IL-1β from wild-type (WT), Casp1−/−, Asc−/−, Nlrp3−/−, upLPS-pulsed murine bone marrow–derived dendritic cells (BMDC) exposed to medium, P. acnes (multiplicity of infection=300), or MSU in the presence of DMSO or Z-VAD-fmk. (h) Quantitative PCR of NLRP1 and NLRP3 in human acne (AV, n=12) and normal skin (NS, n=6). (i) Correlation between NLRP3 and IL-1β expression in human acne. (a–g) Mean±SD are presented, n=3. *P≤0.05; **P≤0.001; ***P≤ NS, not significant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Inflammasome activation by P. acnes requires ROS, K+ efflux, phagocytosis, and lysosomal destabilization. (a) THP1 cells DMSO- or cytochalasin D (Cyt. D)-treated were exposed to CFSE (carboxyfluorescein succinimidyl ester)-labeled P. acnes and analyzed for bacteria uptake. (b) IL-1β secretion from ultra-pure lipopolysaccharide (upLPS)-primed human monocytes pre-incubated with DMSO or Cyt. D and exposed to live P. acnes. (c) Non-upLPS-primed human monocytes were exposed P. acnes in the presence of DMSO, Cyt. D, or Z-VAD-fmk. ELISA of IL-1β (supernatant) and western blotting of pro- and mature-IL-1β (lysates) are presented. (d–g) IL-1β or tumor necrosis factor-α (TNFα) release from upLPS-primed human monocytes pre-incubated with the indicated inhibitors and exposed to live P. acnes or control stimuli. (h) IL-1β from wild-type (WT) or P2X7−/− upLPS-primed bone marrow–derived dendritic cells (BMDC) and exposed to medium or P. acnes was measured by ELISA. Means±SD, n=3. **P≤0.001; ***P≤ NS, not significant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
P. acnes induces NLRP3-inflammasome-dependent inflammation in vivo. (a) Wild-type (WT; n=13), Asc−/− (n=13), and Nlrp3−/− (n=7) mice were injected intradermally with live P. acnes or phosphate-buffered saline (PBS). Ear swelling after 24 hours is presented. (b) Pro- and mature-IL-1β western blotting of ear extracts from P. acnes−challenged WT, Asc−/−, and Nlrp3−/− mice. (c) Histology of WT or Asc−/− mice injected with P. acnes, PBS, or P. acnes with anti-IL-1β. Bar=500 μM or 50 μm (magnification panel). (d) Ear swelling in WT mice treated with vehicle (n=11), intraperitoneal IL-1Ra (n=13), anti-IL-1β antibody (n=12), or etanercept (n=12) 1 hour before P. acnes or PBS challenge. (e) Ear swelling in WT (n=7) and Il-1rI −/− mice (n=7) injected with P. acnes or PBS. Means±SEM are presented. P≤ NS, not significant.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Cells of myeloid origin mediate immune responses to P. acnes in vivo. (a, b) Wild-type (WT) mice reconstituted with WT (n=12), Asc−/− (n=11), or Nlrp3−/− (n=8) bone marrow were challenged with P. acnes or phosphate-buffered saline (PBS) as described above. The resulting swelling (a) and histology of the injected ear (b) are presented. Bar=500 μm. (c) Ear swelling in WT (n=6), Asc−/− (n=6), or Nlrp3−/− (n=6) reconstituted with WT bone marrow and challenged P. acnes or PBS as above. Means±SEM are presented. P≤ NS, not significant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions