Propionibacterium acnes Induces
Autophagy in Keratinocytes: Involvement of Multiple Mechanisms
Q5 Kla´ra Megyeri1,La´szlo´Orosz1,7,SzilviaBolla2,Lilla Erdei2,ZsoltRa´zga3,Gyo¨rgySepre´nyi4,8, EditUrba´n5,Korne´liaSzabo´6and Lajos Keme´ny2,6
Propionibacterium acnesis a dominant member of the cutaneous microbiota. Herein, we evaluate the effects of differentP. acnesstrains and propionic acid on autophagy in keratinocytes. Our results showed thatP. acnes strain 889 altered the architecture of the mitochondrial network; elevated the levels of light chain 3B-II, Beclin-1, and phospho-50-adenosine-monophosphate-activated protein kinase
a
; stimulated autophagic flux; facilitated intracellular redistribution of light chain 3B; increased average number of autophagosomes per cell; and enhanced development of acidic vesicular organelles in the HPV-KER cell line. Propionic acid increased the level of phospho-50-adenosine-monophosphate-activated protein kinasea
, enhanced lipidation of light chain 3B, stimulated autophagic flux, and facilitated translocation of light chain 3B into autophagosomes in HPV-KER cells. P. acnes strains 889 and 6609 and heat-killed strain 889 also stimulated autophagosome formation in primary keratinocytes to varying degrees. These results indicate that cell wall components and secreted pro- pionic acid metabolite ofP. acnesevoke mitochondrial damage successively, thereby triggering 50-adenosine- monophosphate-activated protein kinase-associated activation of autophagy, which in turn facilitates the removal of dysfunctional mitochondria and promotes survival of keratinocytes. Thus, we suggest that low-level colonization of hair follicles with noninvasive P. acnes strains, by triggering a local increase in autophagic activity, might exert a profound effect on several physiological processes responsible for the maintenance of skin tissue homeostasis.Journal of Investigative Dermatology(2017)-,-e-;doi:10.1016/j.jid.2017.11.018
INTRODUCTION
Propionibacterium acnes is a dominant member of the cutaneous microbiota that is composed of highly variable and topographically diverse microbial communities. The skin microbiota provides colonization resistance, and thereby hampers invasion of virulent microbes. The structural com- ponents, secreted products, and metabolites of normal flora members have the potential to decrease pH; to modulate
inflammation, cell viability, and differentiation; and to manipulate the virulence of pathogenic microbes. In contrast, an altered cutaneous microbiota may contribute to diseases, including acne vulgaris (Belkaid and Hand, 2014;
Bouslimani et al., 2015; Christensen and Bru¨ggemann, 2014; Oh et al., 2014; Schommer and Gallo, 2013; Szabo´
et al., 2017; Weyrich et al., 2015).
The skin commensal P. acnesis a Gram-positive, anaer- obic rod that predominates in the anoxic, lipid-rich envi- ronment of sebaceous glands.P. acnesproduces propionic acid, which protects the skin from virulent microbes.
P. acnes carries several pathogen-associated molecular patterns, which bind to Toll-like receptor 2 (TLR2) and TLR4, leading to the production of cytokines and b-defen- sins (Drott et al., 2010; Kim et al., 2002; Nagy et al., 2005;
Thiboutot et al., 2014). Some invasive strains interact with intracellular pathogen-associated molecular pattern sensors and trigger inflammasome assembly, stimulate a proin- flammatory response, and facilitate the establishment of persistent infection (Qin et al., 2014; Tanabe et al., 2006).
P. acnes expansion in the pilosebaceous unit can trigger tissue damage during the course of acne vulgaris (Weyrich et al., 2015). P. acnes has elaborated a strain-specific variability manifesting in the production of virulence determinants, cellular effects, invasiveness, and pathogenic potential. It is now widely accepted that truly commensal and pathogenic lineages of P. acnes exist, which can be useful members of skin microbiota and causative agents
1Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged, Hungary;2Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary;3Department of Pathology, University of Szeged, Szeged, Hungary;4Department of Medical Biology, University of Szeged, Szeged, Hungary;5Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary; and6MTA-SZTE Dermatological Research Group, Szeged, Hungary
7Current address: Public Health and Food Chain Safety Service of Government Office for Csongra´d County, Laboratory Department, Derkovits fasor 7-11, Szeged, Hungary.
8Current address: Department of Anatomy, University of Szeged, Kossuth Lajos sgt. 40, Szeged, Hungary.
Correspondence: Lajos Keme´ny, Department of Dermatology and
Allergology, University of Szeged, Kora´nyi fasor 6, Szeged, Hungary. E-mail:
kemeny.lajos@med.u-szeged.hu
Abbreviations: AMPK, 50-adenosine-monophosphate-activated protein kinase; BFLA, bafilomycin A1; LC3B, light chain 3B; ROS, reactive oxygen species; TLR, Toll-like receptor
Received 13 August 2017; revised 4 November 2017; accepted 6 November 2017; accepted manuscript published online 27 November 2017; corrected proof published online XXX XXXX
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of acne vulgaris or systemic infections, respectively (Achermann et al., 2014; Beylot et al., 2014; McDowell et al., 2012).
Previous observations demonstrated that bacterial pathogen-associated molecular patterns, exotoxins, and some type 3 or type 4 secretion system effector proteins are powerful activators of autophagy. The autophagic process can function as an early antimicrobial defense pathway by targeting bacteria for autolysosomal destruction, a process known as xenophagy. Several intracellular bacteria have developed strategies with which to evade the degradative power of autophagy. These interesting studies have revealed the importance of autophagy in bacterial infections (Deretic et al., 2013; Deretic and Levine, 2009;
Mathieu, 2015).
A recent study has demonstrated that a cell-invasive P. acnes strain triggers the accumulation of autophago- somes in Raw 264.7 macrophages, mesenchymal cells, and the HeLa cell line (Nakamura et al., 2016). Further ob- servations have indicated that propionic acid is a powerful autophagy inducer in the HCT116 cell line. Autophagy, in response to propionic acid, was shown to develop by a succession of hierarchical steps involving mitochondrial dysfunction, reactive oxygen species (ROS) overproduction, and 50-adenosine-monophosphate-activated protein kinase (AMPK)-mediated inhibition of the mechanistic target of rapamycin. It has also been revealed that propionic-acid- associated autophagy helps to overcome energy crisis, and promotes cell survival by blocking apoptotic demise (Tang et al., 2011). However, investigations of the pro-autophagic effects of extracellular P. acnes strains have not yet been reported in keratinocytes.
In this study, therefore, we investigated the effects of differentP. acnesstrains on autophagy in keratinocytes, and, in parallel, measured the involvement of the AMPK- associated autophagic pathway.
RESULTS
P. acnesinduces autophagy in keratinocytes
To elucidate how live P. acnes strains 889 and 6609 and heat-killed strain 889 (HK-889) affect the cellular autophagic cascade, we incubated keratinocytes with bacteria in vitro at a multiplicity of infection of 100 and measured (i) the levels of microtubule-associated protein 1 light chain 3B-I (LC3B-I) and LC3B-II, (ii) autophagic flux, (iii) subcellular localization of LC3B and Beclin-1, (iv) the ultrastructural features of autophagic vacuoles, and (v) cytoplasmic acidification.
To study the effects of P. acnes strain 889 on basal autophagy, the levels of LC3B-I and LC3B-II were deter- mined by western blot analysis in the HPV-KER cell line.
The control cells displayed endogenous expression of both the lipidated and the nonlipidated forms of LC3B.P. acnes- treated cells displayed elevated LC3B-II and decreased LC3B-I levels compared with controls at each time point (Figure 1a). Furthermore, P. acnes strains also increased LC3B-II/LC3B-I ratios in normal human keratinocytes, live P. acnes strain 889 being the most powerful trigger (Supplementary Figure 1a online).
To investigate autophagic flux in cells incubated with P. acnesstrains at 6 hpi, LC3B-II levels were measured under
conditions where autophagosome degradation was blocked by bafilomycin A1 (BFLA), a pharmacological inhibitor of autophagosome-lysosome fusion and lysosomal hydrolase activity. The cultures were treated with bacteria first, and incubated with BFLA for a 4-hour period just before the preparation of cell lysates. BFLA elevated the level of LC3B-II as compared with the untreated control cells, indicating that this drug efficiently blocked autophagic flux under the experimental conditions used. In the presence of BFLA, P. acnes triggered a higher increase in the LC3B-II/LC3B-I ratio than that observed in the corresponding drug control (Figure 1b andSupplementary Figure 1b).
Indirect immunofluorescence assay to determine the intracellular localization of LC3B revealed that the control cells displayed a faint cytoplasmic LC3B staining; the fluo- rescence intensity profiles consisted of a few peaks of low height. In contrast,P. acnes-treated cells exhibited very bright LC3B staining; the fluorescence intensity profiles consisted of numerous robust peaks (Figure 1c, d, f, g andSupplementary Figure 2aed online).
To investigate the effects ofP. acnesstrains on autophago- some formation, the abundances of LC3B-positive vesicles were determined. The average numbers of LC3B-positive vesicles per cell in P. acnes-treated cultures were significantly higher than that observed in the control cultures (Figure 1e, h andSupplementary Figure 2e, f). The liveP. acnes strain 889 was again more efficient than strain 6609 and HK-889 in promoting the autophagic process (Supplementary Figure 2e).
Indirect immunofluorescence assay to determine the intracellular localization of Beclin-1 revealed that the control HPV-KER cells displayed a faint cytoplasmic Beclin-1 stain- ing. In contrast, cells incubated with liveP. acnesstrain 889 exhibited very bright Beclin-1 staining; the fluorescence in- tensity profiles consisted of numerous robust peaks (Figure 2).
Transmission electron microscopy to investigate the ultra- structural features of autophagic compartments revealed that the control HPV-KER cells displayed a few autophagic vac- uoles. In contrast, cells incubated with live P. acnes strain 889 exhibited a significant rise in the number of autopha- gosomes as early as 3 hpi and an accumulation of autoly- sosome stage vacuoles at 24 hpi (Figure 3). Furthermore, this test also revealed the intracytoplasmic presence ofP. acnes partially surrounded by extensions bulging out of the endo- plasmic reticulum membrane (Figure 3a). However, bacterial invasion of HPV-KER cells seems to be a rare event occurring only in the late phase of incubation.
To determine the effects of liveP. acnesstrain 889 on the formation of acidic vesicular organelles, acridine orange staining was used. In the control HPV-KER cultures, the cytoplasm stained green. In P. acnes-treated cells, the cytoplasm exhibited bright-red staining with a marked punctate structure (Figure 4a and b). Analysis of the fluo- rescence intensities in green, red, and overlapping spectral regions revealed an enhancement in red and a reduction of green fluorescence in response to P. acnes treatment (Figure 4c). Moreover, the average numbers of acidic vesicular organelles per cell in P. acnes-treated cultures were significantly higher than that observed in control cultures (Figure 4d).
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P. acnestriggers abnormal mitochondrial dynamics, entailing AMPK activation and induction of autophagy To gain some insight into the mechanism ofP. acnes-medi- ated induction of autophagy, we incubated HPV-KER cells with the live strain 889 in vitro at a multiplicity of infection of 100, and measured (i) the levels of AMPKa and phospho- AMPKa (Thr172) and (ii) the ultrastructural features of mitochondria.
Western blot analysis revealed that the control cells dis- played endogenous expression of both AMPKaand phospho- AMPKa (Thr172). Phosphorylation of AMPKa at Thr172 is known to be essential for the activation of AMPK (Stein et al., 2000).P. acnestriggered a pronounced increase in the level of phospho-AMPKa; the phospho-AMPKa/AMPKa ratio in P. acnes-treated cultures was considerably higher than that observed in controls (Figure 5a).
P. acnes induced spherical and swollen mitochondria displaying destructive changes of their cristae (Figure 5b). The median aspect ratio and form factor values inP.acnes-treated
cultures were significantly lower than that observed in con- trols at the 3- and 6-hour time points (Figure 5c). To investi- gate further the effect ofP. acneson mitochondria, dot plots of aspect ratios versus form factors were generated, and divided into four quadrants defined by the 25th percentiles of the corresponding controls. The analyses ofP.acnes-treated cells indicated a dramatic increase in the mitochondrial compartment with <25th percentile values for both aspect ratios and form factors at 3 and 6 hpi (Figure 5d). There were no significant differences in the morphological features of mitochondria between the control cells andP. acnes-treated cultures at 24 hpi.
Propionic acid induces autophagy in the HPV-KER cell line To investigate the effect of propionic acid on autophagy, we treated HPV-KER cells with 10 mM propionic acid, and measured (i) the levels of LC3B-I, LC3B-II, AMPKa, and phospho-AMPKa; (ii) autophagic flux; and (iii) subcellular localization of LC3B.
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Figure 1. P. acnestreatment increases the LC3B-II/LC3B-I ratio, stimulates autophagic flux, and triggers autophagosome formation.(a) Western blot analysis showing the kinetics of LC3B-I and LC3B-II expression in control andP. acnes-treated cells. (b) Western blot analysis showing increased autophagic flux inP. acnes-treated cells. (c,d,f,g) Immunofluorescence assays showing the fluorescence intensities of LC3B-positive autophagic vacuoles. The line intensity scan graphs depict the intensity values along the arrows drawn across the images, whereas the 3D surface plots represent the intensity values of the whole image. (e, h) Immunofluorescence assays showing the average numbers of LC3B-positive autophagic vacuoles. Data are meansstandard error of the mean, n¼500. Scale bar, 10mm. ****P<0.0001. BFLA, bafilomycin A1; LC3B, light chain 3B.
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Western blot analysis revealed that propionic acid stimu- lated the lipidation of LC3B at the 6- and 24-hour time points, and increased autophagic flux (Figure 6a and b).
Indirect immunofluorescence assay to determine the intracellular localization of LC3B revealed that the control cells displayed a faint cytoplasmic LC3B staining at the 6-hour time point. In contrast, propionic-acid-treated cells exhibited very bright LC3B staining; the fluorescence in- tensity profile consisted of numerous robust peaks (Figure 6c). The average number of LC3B-positive vesicles per cell in propionic-acid-treated cultures at the 6-hour time point was significantly higher than that observed in the control cultures (Figure 6d).
To examine the involvement of AMPK in the propionic- acid-mediated induction of autophagy, the levels of AMPKa and phospho-AMPKa(Thr172) were determined by western blot analysis. Propionic acid induced moderate increases in AMPKalevels at the 3- and 6-hour time points, and triggered pronounced increases in the level of phospho-AMPKa at each time point. The phospho-AMPKa/AMPKa ratios in propionic-acid-treated cultures were considerably higher than that observed in the control cultures (Figure 6e and f).
DISCUSSION
Compelling evidence indicates that autophagy functions as an important cellular defense mechanism against the
invasion of pathogenic microorganisms (Benjamin et al., 2013). Commensal bacteria were shown to exert complex effects on the autophagic activities of tissues located at the entry sites of pathogens (Benjamin et al., 2013). However, we are just beginning to understand the protective role of the pro-autophagic effect exerted by the skin microbiota. Thus, in this study, we considered the question of whether different P. acnesstrains are able to stimulate the autophagic process in keratinocytes.
Initially, we evaluated five distinct criteria for increased autophagic activity in keratinocytes incubated withP. acnes.
As LC3B is a well-characterized marker of autophagy (Klionsky et al., 2016), we first measured the levels of LC3B-I and LC3B-II.P. acneselevated LC3B-II and decreased LC3B-I levels, indicating that the lipidation of LC3B is stimulated by live P. acnes strains and heat-killed bacteria. We also assessed autophagic flux inP. acnes-treated cultures. In the presence of BFLA, the LC3B-II level of cells incubated with P. acnesstrains was higher than that seen in the drug control, demonstrating that autophagic flux is increased by this bac- terium. Next, we performed confocal imaging to investigate the intracellular distributions of LC3B and Beclin-1. These experiments revealed thatP. acnesraises the intensity levels of LC3B and Beclin-1 staining and stimulates translocation of these proteins into autophagosomes. Interestingly, the live P. acnesstrain 889 was more efficient than strain 6609 and
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Figure 2. P. acnestreatment alters the intracellular distribution of Beclin-1 protein.The samples were stained for the endogenous Beclin-1 protein, and images were obtained by confocal microscopy. The images were subjected to line scan fluorescence intensity analysis and 3D surface plotting using the Image J software (Schneider et al., 2012). The line intensity scan graphs depict the intensity values along the arrows drawn across the images, whereas the 3D surface plots represent the intensity values of the whole image. Scale bar, 10mm.
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HK-889 in promoting autophagosome formation. In addition, we determined the effects of P. acnes strain 889 on acidic vesicular organelle formation. The results showed that these bacteria increase cytoplasmic acidification and enhance the development of acidic vesicular organelles. Finally, we studied the ultrastructural features of autophagic compart- ments by transmission electron microscopy. The data demonstrated that, at 3 and 6 hpi,P. acnesstrain 889 triggers the accumulation of autophagosome-stage vacuoles, which subsequently evolve into degradative autolysosomes, sug- gesting that these bacteria trigger a transient increase in autophagic activity at the early phase of incubation, which declines thereafter. It was earlier reported that an invasive P. acnes strain induces autophagy in Raw 264.7
macrophages, mesenchymal cells, and HeLa cell line (Nakamura et al., 2016). Overall, our experiments demon- strate thatP. acnescan stimulate autophagy in keratinocytes when it is present extracellularly, as the level of bacterial invasion was negligible at the low multiplicity of infection used. These results together suggest that P. acnes-induced autophagy might exhibit significant cell-type specificity and bacterial-strain dependency.
The diversity of bacterial structural components, secreted virulence factors, and metabolic products involved suggests a highly intricate mechanism inP. acnes-mediated induction of autophagy. It has already been revealed that TLR4 ligands and complex TLR2 agonists, engaging additional receptors, are strong autophagy inducers, whereas individual TLR2
Figure 3.P. acnestriggers
enlargement of the early and the late autophagic compartments.(a) Representative transmission electron microscopy micrographs depicting the ultrastructural features of autophagic compartments in control andP. acnes-treated cells. The solid line boxes encompassing cytoplasmic portions of cells were further enlarged in the insets to show autophagosomes, autolysosomes, and internalized bacteria denoted by single, double, and triple arrows, respectively. Scale bar, 5mm.
(b) Quantification of autophagic vacuoles. The total cytoplasmic areas were determined using the point-counting method, and the autophagic vacuole profiles corresponding to autophagosomes or autolysosomes were scored and counted. Data are meansstandard error of the mean, n¼10. *P<0.05;
***P<0.001.
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ligands are unable to provoke autophagy (Delgado et al., 2008). Another interesting observation clearly demonstrated that the scavenger receptor CD36 is also implicated in autophagy induction (Sanjurjo et al., 2015). CD36 functions as a TLR coreceptor and participates in the formation of the CD36-CD14-TLR2/4-TLR6 signaling module, which is capable of evoking diverse biological responses, including the increased production of ROS (Di Gioia and Zanoni, 2015). CD36 stimulates ROS generation via the nicotin- amide adenine dinucleotide phosphate oxidase, whereas TLR2/4 trigger the TRAF6-ECSIT-NLRX1-dependent formation of mitochondrial ROS (Park et al., 2009; West et al., 2011).
We and others have previously shown thatP. acnesactivates the TLR2/4 signal transduction mechanisms in keratinocytes (Drott et al., 2010; Kim et al., 2002; Nagy et al., 2005).
Moreover, the cell wall lipoproteins of this bacterium were shown to trigger the production of ROS through the CD36 pathway (Grange et al., 2009). Excessive ROS levels might lead to mitochondrial dysfunction, which in turn evokes ATP depletion, activation of AMPK, and induction of the auto- phagic cascade (Inoki et al., 2003; Wang and Klionsky, 2011;
Wu et al., 2014; Zhang et al., 2016; Zhao and Klionsky, 2011). It is widely accepted that AMPK-dependent auto- phagy functions as an important adaptive mechanism during oxidative stress by facilitating the removal of damaged mitochondria (He and Klionsky, 2009; Kroemer et al., 2010).
In light of these interesting observations, we investigated how P. acnesaffects the levels of phospho-AMPKa (Thr172) and the architecture of the mitochondrial network. Our studies have shown that the level of phospho-AMPKa(Thr172) was strongly increased in HPV-KER cells incubated with live P. acnesstrain 889, indicating that extracellular bacteria are powerful activators of AMPK. The ultrastructural features of mitochondria were significantly altered at the early phase of P. acnes treatment, whereas at the late stage, the mitochon- drial configurations and shape heterogeneities were largely
restored. The time course of the increased autophagy level correlated well with the changes in mitochondrial morphology inP. acnes-treated cells. These results indicate thatP. acnestriggers mitochondrial dysfunction and, in par- allel, activates AMPK-dependent autophagy that can function as an antioxidant defense mechanism promoting the removal of damaged mitochondria.
SCFAs produced by bacterial fermentation act as signalQ2 molecules between microbiota and host cells and regulate several specialized functions of various tissues (Ganapathy et al., 2013). The importance of commensal-derived metab- olites in the regulation of autophagy is highlighted by the greatly increased autophagic activity in SCFA-treated cells (Adom and Nie, 2013; Jan et al., 2002; Tang et al., 2011).
Although the level of propionic acid in the skin has not yet been determined, the propionic acid quantity in the large intestine varies between 1.5 and 26.7 mmol/kg contents (Cummings et al., 1987). Propionic acid levels can reach high concentrations at sites of bacterial colonization and infec- tion, the subgingival concentration of this SCFA was 9.5 mM in patients with periodontal disease (Al-Lahham et al., 2010).
P. acneshas been reported to produce 13.85 mM propionic acid during in vitro culture (Douglas and Gunter, 1946).
Thus, we additionally considered the question of whether propionic acid at 10 mM concentration affects autophagy in HPV-KER cells. We found that propionic acid activated AMPK via phosphorylating AMPKaat Thr172, and stimulated the lipidation of LC3B, increased autophagic flux, as well as enhanced translocation of LC3B into autophagosomes. These data demonstrate that propionic acid enhances the auto- phagic activity of keratinocytes via AMPK activation. Strik- ingly, the time course of autophagic response was different in propionic-acid- andP. acnes-treated keratinocytes. Thus, we suggest that this SCFA metabolite might also be implicated in the autophagic response of keratinocytes, but only after a short delay followingP. acnesencounter.
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Figure 4. Propionibacterium acnesstimulates AVO formation.(a,b) Representative fluorescence micrographs and correlation plots showing the fluorescence intensity and intracellular localization of AVOs. Fluorescence intensities were determined and analyzed using an “apoptosis correlator”
plugin (Mironova et al., 2007) operated in Image J. Thresholds indicated by dashed lines were chosen empirically so as to separate visible fluorescence from the dark pixels. (c) Distribution of fluorescence measured in green, red, and overlapping spectral regions. Fluorescence intensities were quantified, and the average distribution of fluorescence within the green, red, and overlapping regions was calculated. Data are meansstandard error of the mean, n¼50. (d) The average numbers of AVOs. Data are meansstandard error of the mean, n¼500. ****P<0.0001. AVO, acidic vesicular organelle.
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Figure 5.Propionibacterium acnes treatment increases the level of AMPKaphosphorylated at Thr172and alters mitochondrial morphology.
(a) Western blot analysis showing increased levels of phospho-AMPKa inP. acnes-treated cells. Data are meansstandard error of the mean, n¼3. (b) Representative transmission electron microscopy micrographs depicting the ultrastructural features of mitochondria corresponding to the numbered data points in (d).
(c) Graphical representation of mitochondrial form factor and aspect ratio values. The dot plots depict the form factor and the aspect ratio values of each individual mitochondrion. The median values with interquartile ranges are shown within the graphs. (d) Graphical representation of mitochondrial form factor versus aspect ratio values. **P<0.01, ***P<0.001.
AMPK, 50-adenosine-monophosphate- activated protein kinase; AU, arbitrary unit.
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On the basis of the present results, we propose that P. acnesinduces autophagy via its complex interactions with keratinocytes. We hypothesize that P. acnes stimulates the CD36-CD14-TLR2/4-TLR6 signaling module, triggers ROS generation through nicotinamide adenine dinucleotide phosphate oxidase and TRAF6-ECSIT-NLRX1 pathway, and evokes mitochondrial dysfunction. The P. acnes-derived propionic acid causes mitochondrial damage and aggravates oxidative stress. ROS, generated via multiple mechanisms, trigger AMPK-dependent activation of autophagy, which in
turn facilitates the removal of damaged mitochondria and promotes the survival of keratinocytes (see Supplementary Figure S3 online). Thus, P. acnes-induced autophagy may increase the adaptive potential of keratinocytes to cope with oxidative damage.
The human skin provides an extremely potent barrier against microbial invasion because its outermost layer is composed of dead cells formed as a result of the epidermal cornification process, whereas the hair follicles can function as convenient entry sites for pathogenic bacteria (Galluzzi
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Figure 6. Propionic acid increases the LC3B-II/LC3B-I ratio, stimulates autophagic flux, and induces autophagosome formation.(a) Western blot analysis showing the kinetics of endogenous LC3B-I and LC3B-II expression in control and propionic-acid-treated cells. (b) Western blot analysis showing increased autophagic flux in propionic-acid-treated cells. (c) Immunofluorescence assays showing the fluorescence intensities of LC3B-positive autophagic vacuoles. (d) Immunofluorescence assays showing the average numbers of LC3B-positive autophagic vacuoles. Data are meansstandard error of the mean, n¼500. (e,f) Western blot analysis showing increased levels of phospho-AMPKain propionic-acid-treated cells. Data are meansstandard error of the mean, n¼3. Scale bar, 10mm. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. AMPK, 50-adenosine-monophosphate-activated protein kinase;
BFLA, bafilomycin A1; LC3B, light chain 3B.
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et al., 2015; Schommer and Gallo, 2013; Szabo´ et al., 2017).
Thus, low-level colonization of hair follicles with noninvasive P. acnes strains might confer remarkable antimicrobial pro- tection by triggering a local increase in autophagic activity of keratinocytes. In addition to infectious agents, keratinocytes are also exposed to other harmful environmental stimuli, such as the UVR, chemicals, and temperature variations that lead to various pathological conditions via triggering exten- sive oxidative damage. Interestingly, P. acnes is endowed with the ability to decrease oxidative damage of bacteria and keratinocytes via the secretion of the RoxP (radical oxygenase of P. acnes) antioxidant enzyme (Allhorn et al., 2016). In view of the importance of autophagy in keratinocyte physi- ology (Li et al., 2016), the pro-autophagic effect of P. acnes might represent another indirect mechanism for how this commensal bacterium exerts a beneficial role in cutaneous homeostasis.
MATERIALS AND METHODS
An extended description of materials and methods is given in Supplemental Materials and Methodsonline.
Cell culture
The HPV-KER cell line was established and grown as previously described (Tax et al., 2016). Primary keratinocytes were obtained from healthy individuals who underwent plastic surgery after written informed consent according to the institutional review board pro- tocol. The Medical Research Council Ethics Committee of Hungary approved the use of skin samples (ETT-TUKEB 39361). Human epidermal keratinocytes were isolated and cultured as described previously (Nagy et al., 2005). For experimental purposes, kerati- nocytes were cultured in antibiotic-free medium for a 24-hour period beforeP. acnestreatment.
P. acnesstrain and growth conditions
P. acnes strain 889 was isolated and cultured as previously described, whereas the strain 6609 was obtained from ATCC (Tax et al., 2016). For experiments, keratinocytes were incubated with P. acnes strains at a multiplicity of infection of 100 CFU/cell. To prepare heat-killed suspensions of bacteria,P. acnesstrain 889 was killed by incubation at 60C for 30 minutes.
Indirect immunofluorescence assay
Cytospin cell preparations were fixed in methanol-acetone (1:1). The slides were incubated with rabbit polyclonal antibodies to LC3B or Beclin-1 (Sigma-Aldrich, St. Louis, MO). After washing, the samples were reacted with CF640R- or CF488A-conjugated anti-rabbit antibodies (Sigma-Aldrich). The cells were visualized by confocal microscopy using an Olympus FV1000 confocal laser scanning microscope. LC3B-positive vacuoles were quantified as previously described (Orosz et al., 2016; Pa´sztor et al., 2014). The fluorescence intensities were determined using the line scan analysis and surface plot functions of Image J
Q3 (Schneider et al., 2012).
Transmission electron microscopy
The samples were fixed, dehydrated, and embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a JEOL JEM-1400Plus transmission electron microscope (JEOL USA, Peabody, MA). Autophagosomes and autolysosomes were scored according to their morphology. The results are presented as number
of organelles/cytoplasmic areastandard error of the mean. Mito- chondrial shape descriptors were determined using Image J.
Western blot assays
Protein samples were prepared for SDS-PAGE and western blot assay as previously described (Orosz et al., 2016; Pa´sztor et al., 2014). The membranes were developed using a chemiluminescence detection system, the autoradiographs were scanned with a GS-800 densi- tometer (Bio-Rad), and band intensities were quantified using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Acridine orange staining
Cytoplasmic acidification was assessed by the acridine orange staining procedure as previously described (Pa´sztor et al., 2014). The fluorescence intensities were analyzed by using an “apoptosis correlator” plugin operated in the Image J software.
Statistical analysis
Differences in autophagic vacuole numbers and protein levels between control and P. acnes-treated cells were evaluated with Student’s unpaired t-test, and the values are expressed as means standard error of the mean. Mitochondrial shape de- scriptors followed nonnormal distributions as determined by the Shapiro-Wilk normality test. Differences in aspect ratios and form factors therefore were evaluated by the Kolmogorov-Smirnov test, and the values are expressed as medians with interquartile ranges.
P-values of less than 0.05 were considered statistically significant.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
This work was supported by research grants OTKA 105369, GINOP-2.3.2-15- 2016-00015, GINOP-2.2.1-15-2016-00007, GINOP-2.3.3-15-2016-00007, GINOP-2.3.3-15-2016-00012, and EFOP-3.6.1-16-2016-00008.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper atwww.
jidonline.org, and athttps://doi.org/10.1016/j.jid.2017.11.018.
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