CED

Experimental dermatology • Review article

Clinical and Experimental Dermatology

CPD

Acne, quorum sensing and danger S. M. Lwin,1 I. Kimber2 and J. P. McFadden1 1

St John’s Institute of Dermatology, St Thomas’ Hospital, London, UK; and 2Faculty of Life Sciences, University of Manchester, Manchester, UK

doi:10.1111/ced.12252

Summary

Propionibacterium acnes is a ubiquitous skin commensal bacterium, which is normally well tolerated by the immune system in healthy human skin. However, there is increasing evidence to suggest a pivotal role for P. acnes in the inflammatory process underlying the acne pathogenesis. With its features of inflammation and pustulation, acne vulgaris resembles the skin’s normal reaction to bacterial pathogens. P. acnes flourishes when sebum production increases in the follicles. Bacteria may undergo behavioural changes based on the surrounding bacterial population, a process called quorum sensing (QS). Evidence from in vitro studies suggests that QS enables P. acnes to upregulate its hydrolysis of sebum triglycerides by its bacterial lipases, secreting free fatty acids (FFAs) such as oleic, palmitic and lauric acids. These FFAs act as danger-associated molecular patterns (DAMPs), and activate Toll-like receptor (TLR) 2 and TLR4, leading to selective T-helper (Th)-driven immunity, with subsequent expression of Th1/Th17-associated inflammatory cytokines. To our knowledge, there is currently no explanation as to what determines the shift of recognition by the immune system of P. acnes from being symbiotic to pathogenic. We present a novel hypothesis based on the essence of QS and DAMPs. P. acnes sends no or only ‘safety’ signals when present in ‘controlled’ quantities under commensal conditions, but becomes pathogenic and sends ‘danger’ signals via QS in the form of excess FFA production, which stimulates TLR2 and TLR4 as the bacterial population flourishes.

Introduction Acne is a common chronic inflammatory disorder of the pilosebaceous follicle (PSF). The microbe that is usually isolated and implicated in acne is P. acnes, an anaerobic Gram-positive rod, which forms the predominant commensal in the PSF.1–4 This article explores and presents a novel hypothesis on how the commensal P. acnes can invoke danger-associated molecular pattern (DAMP) signalling, immune activation and subsequent inflammation in acne vulgaris.

Correspondence: Dr Su M. Lwin, St John’s Institute of Dermatology, St Thomas’ Hospital, Westminster Bridge Road, Lambeth, London SE1 7GN, UK E-mail: [email protected] Conflict of interest: none declared. Accepted for publication 10 September 2013

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Propionibacterium acnes, the commensal: when does P. acnes become ‘dangerous’? Increases in circulating androgens correlate with the presence of acne vulgaris, and androgens increase sebocyte proliferation and lipid production.5 The microcomedone theory suggests that the comedone enlarges with continual keratinocyte shedding and sebum accumulation within the PSF.6 The resulting anaerobic micro-environment is rich in various lipids, providing P. acnes with abundant nutrients, enabling its over-proliferation.5 This high correlation between sebum production and P. acnes levels suggests a symbiotic relationship.7,8 On reaching a certain density, P. acnes is capable of forming biofilms:9,10 macro-colonies of bacteria encased in an extracellular matrix of DNA, proteins and polysaccharides, adhering to a surface in order to optimize their survival11 through increased

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Acne, quorum sensing and danger  S. M. Lwin et al.

antimicrobial resistance.2,6,12 P. acnes biofilms are more prevalent in the PSF of acne samples compared with control samples,2 and this may explain the increased levels of P. acnes virulence factors, such as lipase activity, in human acne lesions. In vitro studies suggest that antibiotic resistance is more common in cultures of P. acnes biofilms compared with planktonic cells.11 Burkhart and Burkhart6 have proposed that the adhesive property of the P. acnes biofilm is responsible for the production of a biological ‘glue’ that holds corneocytes together in the infundibulum, forming the keratinaceous plug. The enlarging comedone beneath the strongly adhesive plug causes rupture of the sebaceous gland, allowing extrusion of immunogenic keratin, sebum and P. acnes into the surrounding tissues, with subsequent cytokine production.1,6,13 The immune system may be more sensitive to certain P. acnes strains; strains IA, IB and II have been detected in biofilms of human acne lesions, and induced expression of human b-defensin-2 and cytokines/chemokines.2,14,15 Evidence suggests a positive correlation between the number of P. acnes bacteria and the level of acne inflammation. Firstly, teenagers with acne can have numbers of P. acnes bacterial cells on their skin 100 times greater than in healthy, age-matched controls.16 Secondly, mild to moderate acne can be treated effectively with antibiotics, and is associated with a reduction in P. acnes numbers and inflammation, while the therapeutic effect of antibiotics fails in patients with erythromycin-resistant strains.17

Danger signals and immune responses The danger model of immunity proposes that responses to antigens are not dependent solely upon recognition of ‘non-self’ by the immune system. As first proposed by Matzinger,18 with subsequent compelling experimental support, initiation of the optimal immune response requires, in addition to an encounter with a foreign antigen, a sense of tissue damage or evidence of a pathogenic micro-organism. This prevents the full and unnecessary deployment of the immune system against ‘trivial’ antigens unlikely to be associated with any tangible threat to the host. Tissue damage or pathogenic microbials are sensed by so-called ‘danger signals’. One main group of danger signals is provided via ligation of the Toll-like receptors (TLRs), both by proteins associated with pathogenic bacteria (pathogen-associated molecular patterns; PAMPs) and by proteins associated with tis-

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Androgenic hormones

Increased sebum production

Blockage of pilosebaceous unit

Anaerobic environment within the pilosebaceous unit and grease excess

Increased population of P. acnes Quorum sensing via AI-2 Biofilm formation & increased production of virulence factors (increased lipases)

Increased FFAs

FFAs act as DAMPs & Danger signalling

TLR2 & TLR4

hBD-2, IL-8 IL-1β, TNF, IL-6, IL-12, IL-18, IL-23, α-IFN

Immune response to P. acnes

Figure 1 Androgen-induced increased sebum production causes

blockage of the pilosebaceous units, giving rise to a lipid-rich anaerobic environment where the population of Propionibacterium acnes flourishes. This leads to the formation of biofilms via the production of autoinducer (AI)-2 molecules and quorum sensing, with an increased release virulence factors such as free fatty acids (FFAs). FFAs in turn act as danger-associated molecular patterns (DAMPs), activating Toll-like receptor (TLR)2 and TLR4, with subsequent release of inflammatory cytokines and danger signalling, leading to immune cascades. hBD, human b-defensin; IFN, interferon; IL, interleukin; TNF, tumour necrosis factor.

sue trauma (DAMPs).19 Stimulation of such extracellular TLRs leads to expression of pro-inflammatory cytokines, and recruitment and activation of various immune cells, characteristically including T-helper (Th)1/Th17 cells.19

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Some evidence supports acne inflammation being mediated via TLR2 and TLR4.1,3,6,13 However, there is an apparent problem implicating danger theory in acne pathogenesis. TLRs recognize PAMPs expressed by pathogenic bacteria; how can P. acnes, a commensal bacterium, stimulate danger signalling? Here, we suggest that this comes about via a mechanism called quorum sensing (QS).

Quorum sensing: the en masse effect A specific bioluminescent bacterial strain, Vibrio fischeri, lives in symbiosis within the organs of the squid Euprymna scolopes;20 the luminescent property of V. fischeri protects the squid from its predators. V. fischeri bioluminescence is achieved only by colonies rather than by individual bacteria.20 This process of population-dependent changes in bacterial behaviour, including gene expression and processes optimal for survival in a given situation, is termed ‘quorum sensing’. Each bacterium detects the presence of any surrounding bacteria either of the same strain or different strains, via extracellular signalling of QS molecules called autoinducers (AIs), leading to synchronous control of gene expression of the bacterial group based on its population density.21 AIs are low-molecular-weight signalling molecules synthesized by the bacterial cells, and these molecules are then secreted into the extracellular matrix.20 The extracellular concentration of AI molecules increases as the bacterial cell population increases. Once the AI level rises above the minimal detectable threshold, the AIs bind to and activate the cognate cell receptors, leading to signal transduction cascades, with subsequent synchronous changes in gene expression and phenotypes required for optimal survival.21 Different bacteria utilize different AIs; for example, AI-2 is produced by the LuxS enzyme found in both Gram-positive and Gram-negative bacteria, including P. acnes.9,10 Although there is currently no in vivo evidence of QS by P. acnes, in vitro studies have shown increased production of AI-2 by P. acnes, and upregulation of its virulent activity in the presence of biofilms.9,12 Furthermore, P. acnes-induced TLR2 and TLR4 expression has been detected directly in human acne lesions in vivo and keratinocytes in vitro.3 P. acnes biofilms and virulence factors have also been detected more frequently in PSFs in biopsies taken from patients with acne compared with those of controls.2 AI-2 production has been identified in four different strains of P. acnes, two of which were derived from

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human acne isolates, and the levels of AI-2 productin were three times higher in mature P. acnes biofilms compared with planktonic cells.12 Interestingly, an increase in the lipase activity of P. acnes was also detected in biofilms.12 This suggests that AI-2 levels and lipase activity increase as the population of P. acnes increases. However, no signal-transduction system nor any receptor responding to AI-2 in P. acnes has been described to date, and so at this time, without confirmatory evidence, no direct causal relationship between AI-2 and lipase activity can be claimed. P. acnes lipases, via hydrolysis of sebum triglycerides, produce free fatty acids (FFAs) such as oleic, palmitic and lauric acids.22,23 These FFAs act as DAMPs on TLR2 and TLR4,13 with subsequent increase in expression of human β-defensin-2 by human sebocytes in vitro,13,15,22,24 promoting comedogenesis and inflammation.23 TLR2 activation induces innate immunity by activating downstream pathways, leading to upregulation of transcription factors such as nuclear factor jB, activator protein-1, interferon regulatory factor (IRF)3 and IRF7, and mitogen-activated protein kinases.25 This results in the production of Th1/Th17-associated inflammatory mediators such as interleukin (IL)-1b, IL-6, IL-12p70, and tumour necrosis factor (TNF)-a, as well as antimicrobial peptides such as b-defensins (TLR4 ligands),5,15,19,24,26 with subsequent danger signalling to the host defence system, resulting in tissue inflammation and destruction (Fig. 1), presenting as the inflammatory lesions and scarring seen in acne.

Conclusion There is mounting evidence for the apparently paradoxical roles of P. acnes both as a commensal bacterium, and in the pathogenesis of acne. To our knowledge, there has not yet been any explanation as to what determines the shift of recognition of P. acnes by the immune system from being symbiotic to pathogenic. We present a novel hypothesis based on the essence of QS and DAMPs. P. acnes may drive inflammation via differential activation of TLR2 and TLR4, defined danger signalling, and expression of proinflammatory cytokines. One plausible mechanism by which P. acnes might achieve the inflammatory response is by increased lipase production through QS, which increases FFAs, TLR2 and TLR4. Further research into this possible mechanism may provide novel therapeutic targets.

ª 2014 British Association of Dermatologists

Acne, quorum sensing and danger  S. M. Lwin et al.

Learning points  Acne vulgaris is a common chronic inflamma-

tory disorder of the pilosebaceous glands.  P. acnes is a ubiquitous human skin commen-

sal bacterium.  It is the predominant micro-organism residing

within the PSF, and is implicated in acne pathogenesis.  QS is a process by which bacterial cells communicate with each other to obtain information such as population density, through the production, detection and response to extracellular signalling molecules called AIs.  QS leads to a change in the bacterial group behaviour, including gene expression and processes optimal for survival in a given situation.  Biofilms are macro-colonies of bacteria encased in an extracellular matrix of carbohydrate adhering to a surface in order to optimize survival through increased antimicrobial resistance.  The danger model of immunity proposes that danger signals are provided via ligation of TLRs, which can recognize both proteins associated with pathogenic bacteria (PAMPs) and proteins associated with tissue trauma or damage (DAMPs).  Danger signals may involve innate immune stimulation.

References 1 Burkhart CG, Burkhart CN, Lehmann PF. Acne: a review of immunologic and microbiologic factors. Postgrad Med J 1999; 75: 328–31. 2 Jahns AC, Lundskog B, Gancevicience R et al. An increased incidence of Propionibacterium acnes biofilms in acne vulgaris: a case–control study. Clin Lab Invest 2012; 167: 50–8. 3 Jugeau S, Tenaud I, Knol AC et al. Induction of toll-like receptors by Propionibacterium acnes. Br J Dermatol 2005; 153: 1105–13. 4 Shaheen B, Gonzalez M. A microbial aetiology of acne: what is the evidence? Br J Dermatol 2011; 165: 474–85. 5 Thiboutot D. Regulation of human sebaceous glands. J Invest Dermatol 2004; 123: 1–12. 6 Burkhart CG, Burkhart CN. Expanding the microcomedone theory and acne therapeutics: Propionibacterium acnes biofilm produces biological glue that holds corneocytes together to form plug. J Am Acad Dermatol 2007; 57: 722–4. 7 Leyden JJ, McGinley KJ, Vowels B. Propionibacterium acnes colonization in acne and non-acne. Dermatology 1998; 196: 55–8.

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8 McGinley KJ, Webster GF, Ruggieri MR et al. Regional variations in density of cutaneous propionibacteria: correlation of Propionibacterium acnes populations with sebaceous secretion. J Clin Microbiol 1980; 12: 672–5. 9 Bruggeman H, Henne A, Hoster F. The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Science 2004; 305: 671–3. 10 Burkhart CN, Burkhart CG. Genome sequence of Propionibacterium acnes reveals immunogenic and surface-associated genes confirming existence of the acne biofilm. Int J Dermatol 2006; 45: 872. 11 Olson ME, Ceri H, Morck DW et al. Biofilm bacteria: formation and comparative susceptibility to antibiotics. Can J Vet Res 2002; 66: 86–92. 12 Coenye T, Peeters E, Nelis HJ. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res Microbiol 2007; 158: 386–92. 13 Nagy I, Pivarcsi A, Koreck A et al. Distinct strains of Propionibacterium acnes induce selective human beta-defensin-2 and interleukin-8 expression in human keratinocytes through toll-like receptors. J Invest Dermatol 2005; 124: 931–8. 14 Bruggemann H, Lomholt HB, Kiian M. The flexible gene pool of Propionibacterium acnes. Mob Genet Elements 2012; 2: 145–8. 15 Nagy I, Pivarcsi A, Kis K et al. Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes. Microbes Infect 2006; 8: 2195–205. 16 Leyden JJ, McGinley KJ, Mills OH et al. Propionibacterium levels in patients with and without acne vulgaris. J Invest Dermatol 1975; 65: 382–4. 17 Eady EA, Cove JH, Holland KT et al. Erythromycin resistant propionibacteria in antibiotic treated acne patients: association with therapeutic failure. Br J Dermatol 1989; 146: 840–8. 18 Matzinger P. Tolerance, danger and the extended family. Annu Rev Immunol 1994; 12: 991–1045. 19 McFadden JP, Dearman RJ, White JML et al. The hapten-atopy hypothesis II: the ‘cutaneous hapten paradox’. Clin Exp Allergy 2011; 41: 327–37. 20 Lazdunski AM, Ventre I, Sturgis JN. Regulatory circuits and communication in Gram-negative bacteria. Nat Rev Microbiol 2004; 2: 581–92. 21 Ng WL, Bassler BL. Bacterial quorum-sensing network architectures. Annu Rev Genet 2009; 43: 197–222. 22 Nakatsuji T, Kao MC, Zhang L et al. Sebum free fatty acids enchance the innate immune defense of human sebocytes by upregulating beta-defensin-2 expression. J Invest Dermatol 2010; 130: 985–94. 23 Perisho K, Wertz PW, Madison KC et al. Fatty acids of acylceramides from comedones and from the skin surface of acne patients and control subjects. J Invest Dermatol 1988; 90: 350–3.

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24 Biragyn A, Ruffini PA, Leifer CA et al. Toll-like receptor 4-dependent activation of dendritic cells by beta-1. defensin 2. Science 2002; 298: 1025–9. 25 Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 2010; 11: 373–84.

26 Pivarcsi A, Bodai L, Rethi B et al. Expression and function of toll-like receptors 2 and 4 in human keratinocytes. Int Immunol 2003; 15: 721–30.

CPD questions

Question 4

Learning objectives To show an understanding of basic concepts involved in the pathogenesis of acne.

Question 1 Which of the following are characteristic of Toll-like receptors (TLRs)? a) They recognize PAMPs from tissue damage and DAMPs from pathogenic bacteria. b) They are endocrine receptors. c) They recognize PAMPs from pathogenic bacteria and DAMPs from tissue damage. d) Once activated, they elicit an inflammatory response through production of PAMPs and DAMPs. e) They recognize insulin and glucagon.

Which of the following statements best describes a biofilm? a) An aggregate of free-swimming planktonic bacteria with increased anti microbial resistance. b) An aggregate of static planktonic bacteria with increased antimicrobial susceptibility. c) An aggregate of bacteria encased in an extracellular matrix of polysaccharides with increased antimicrobial susceptibility. d) An aggregate of a variety of micro-organisms adhered to a surface matrix of proteins and DNA with increased antimicrobial susceptibility. e) An aggregate of bacteria encased in an extracellular matrix of polysaccharides, DNA and proteins adhering to a surface with resultant increase in antimicrobial resistance.

Question 5 Question 2 Which of the following would commonly initiate danger signalling? a) Insulin. b) Free fatty acids. c) Thyroxine. d) Testosterone. e) Glucagon.

Which of the following statements about quorum sensing (QS) is correct? a) QS is a process of stimulus and response correlated to bacterial morphology. b) QS was first identified in Vibrio cholerae. c) AI-2 is the only QS molecule discovered to date. d) AI-2 is produced by LuxS, an enzyme found in Gram-negative bacteria only. e) QS is a process of stimulus and response, which correlates with bacteria population density.

Question 3 Which of the following is the predominant commensal bacterium found in the pilosebaceous unit? a) Staphylococcus epidermidis. b) Staphylococcus aureus. c) Propionibacterium freudenreichii. d) Propionibacterium acnes. e) Streptococcus pyogenes.

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Acne, quorum sensing and danger.

Propionibacterium acnes is a ubiquitous skin commensal bacterium, which is normally well tolerated by the immune system in healthy human skin. However...
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