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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Invest Dermatol. Author manuscript; available in PMC Nov 3, 2009.
Published in final edited form as:
PMCID: PMC2772209
NIHMSID: NIHMS152657
Antimicrobial Property of Lauric Acid Against Propionibacterium acnes: Its Therapeutic Potential for Inflammatory Acne Vulgaris
Teruaki Nakatsuji,1,2 Mandy C. Kao,1,2 Jia-You Fang,3 Christos C. Zouboulis,4 Liangfang Zhang,5,6 Richard L. Gallo,1,2 and Chun-Ming Huang1,2,6
1Division of Dermatology, Department of Medicine, University of California, San Diego, California, USA
2VA San Diego Healthcare Center, San Diego, California, USA
3Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan
4Department of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany
5Department of Nanoengineering, University of California, San Diego, California, USA
6Moores Cancer Center, University of California, San Diego, California, USA
Correspondence: Dr Chun-Ming Huang, Department of Medicine, University of California, Rm2317A, 3350 La Jolla Village, San Diego, California 92161, USA. chunming/at/ucsd.edu
The strong bactericidal properties of lauric acid (C12:0), a middle chain-free fatty acid commonly found in natural products, have been shown in a number of studies. However, it has not been demonstrated whether lauric acid can be used for acne treatment as a natural antibiotic against Propionibacterium acnes (P. acnes), which promotes follicular inflammation (inflammatory acne). This study evaluated the antimicrobial property of lauric acid against P. acnes both in vitro and in vivo. Incubation of the skin bacteria P. acnes, Staphylococcus aureus (S. aureus), and Staphylococcus epidermidis (S. epidermidis) with lauric acid yielded minimal inhibitory concentration (MIC) values against the bacterial growth over 15 times lower than those of benzoyl peroxide (BPO). The lower MIC values of lauric acid indicate stronger antimicrobial properties than that of BPO. The detected values of half maximal effective concentration (EC50) of lauric acid on P. acnes, S. aureus, and S. epidermidis growth indicate that P. acnes is the most sensitive to lauric acid among these bacteria. In addition, lauric acid did not induce cytotoxicity to human sebocytes. Notably, both intradermal injection and epicutaneous application of lauric acid effectively decreased the number of P. acnes colonized with mouse ears, thereby relieving P. acnes-induced ear swelling and granulomatous inflammation. The obtained data highlight the potential of using lauric acid as an alternative treatment for antibiotic therapy of acne vulgaris.
Acne vulgaris is the most common disorder of human skin that affects up to 80% of individuals in their lives (Dreno and Poli, 2003). Acne has many different symptoms including comedones, papules, pustules, nodules, cysts and pilosebaceous inflammation. Among these, inflammatory lesions of acne are of the greatest concern to patients because they may lead to acne scarring, thereby inducing adverse psychological effects. propionibacterium acnes (P. acnes) is a Gram-positive anaerobic bacterium that mostly resides in the pilosebaceous follicles of the skin. Although P. acnes is a member of the normal skin commensal bacterial flora, it plays a critical role in the development of inflammatory acne when it overgrows and colonizes the pilosebaceous unit (Leeming et al., 1988; Cunliffe and Gollnick, 2001; Leyden, 2001; Bojar and Holland, 2004). Genomic analysis of P. acnes has shown a variety of P. acnes pathogenicity (Bruggemann et al., 2004). Further studies on the entire genome of P. acnes has revealed numerous genes that regulate products involved in degrading host molecules and triggering inflammation (Bruggemann, 2005; Rosen, 2007). It has also been widely accepted that inflammatory acne is induced by host immune reactions to P. acnes. P. acnes releases chemoactive factors that attract the immune system cells such as neutrophils, monocytes, and lymphocytes (Webster and Leyden, 1980; Burkhart et al., 1999). Previous studies have found that P. acnes stimulates the production of proinflammatory cytokines such as interleukins−1β, −8, −12, and tumor necrosis factor-α. P. acnes-induced cytokines are mediated by toll-like receptor 2 (Kim et al., 2002; Kim, 2005; Nagy et al., 2006).
As reduction in P. acnes numbers in the hair follicle by antimicrobial agents correlates with clinical improvement of acne in patients, antibiotics have been used to treat acne for several decades and are still widely prescribed for acne patients. The oxidizing agent benzoyl peroxide (BPO) has been one of the most frequently used topical medications for acne treatment. It is often used as the first line treatment for patients suffering from mild to moderate acne. BPO is also used to treat severe acne in combination with a systemic medication such as an antibiotic agent and isotretinoin. The principal action mechanism of BPO is thought to be antibacterial activity, though slight anti-inflammatory effects and very mild anti-comedogenic effects may also exist (Gollnick and Schramm, 1998).
Self-disinfection of the human skin surface through various modes of action has long been suggested (Burtenshaw, 1945). Free fatty acids (FFAs) and antimicrobial peptides have been known to be responsible for at least part of the self-antimicrobial disinfecting activity of the skin surface against microbial colonization (Gallo and Huttner, 1998; Drake et al., 2008). FFAs are secreted from sebaceous glands as sebum triacylglycerides and subsequently released through hydrolyzation by lipases of the commensal bacterial flora, including P. acnes (Holland et al., 1981). Various FFAs have shown antibacterial activity against a range of Gram-positive bacteria, but not Gram-negative bacteria (Wille and Kydonieus, 2003; Georgel et al., 2005; Skrivanova et al., 2005; Drake et al., 2008). Lauric acid (C12: 0), a minor component in the sebum, is the most potent antimicrobial saturated fatty acid (Wille and Kydonieus, 2003). It is commonly found in natural products like coconut palm and milk. Although lauric acid exerts strong antimicrobial activity against many Grampositive bacteria (Kabara et al., 1972; Kitahara et al., 2004; Rouse et al., 2005; Skrivanova et al., 2005), it is unknown whether it has a similar effect on P. acnes in vivo; therefore, the potential of lauric acid to be used as a natural antibacterial agent in acne therapy is also unknown. In this study, we have demonstrated the potential of lauric acid as an alternative option for antibacterial therapy in acne treatment. Interestingly, lauric acid showed stronger antimicrobial activity as compared with BPO against skin bacteria, including P. acnes, in vitro. It also showed therapeutic potential against P. acnes-induced inflammation in vivo.
Antimicrobial effects of lauric acid and BPO against skin bacteria
To compare dose-response effects of lauric acid and BPO on the growth of bacteria that are present in the skin flora, P. acnes, Staphylococcus aureus (S. aureus), and Staphylococcus epidermidis (S. epidermidis) were co-cultured with either agent at various concentrations for 72, 24, and 48 hours, respectively. Bacterial growth was evaluated by measuring absorbance at 600 nm (Figure 1). The length of cultivation period was determined based on the growth speed of the bacteria. Both lauric acid and BPO were tested at concentrations of twofold serial dilutions from 0.24 to 500 μg ml−1. Higher concentrations of both components were not evaluated because they precipitated during incubation (Figure S1) and interfered with the measurement of optical density at 600 nm (OD600). The minimal inhibitory concentration (MIC), the lowest concentration to prevent bacterial growth of lauric acid on the bacterial growth, is over 15 times lower than that of BPO. This suggests that lauric acid has much stronger antimicrobial activity than BPO (Table 1). The half maximal effective concentration of lauric acid on P. acnes growth was the lowest among the bacteria tested, suggesting that P. acnes is more sensitive than S. aureus and S. epidermidis (Table 1). The values of MIC (1.95 μg ml−1) and the half maximal effective concentration (1.5 μg ml−1) of lauric acid were also determined using a different strain of P. acnes (ATCC 11827) (Figure S2).
Figure 1
Figure 1
Inhibitory effects of lauric acid on bacterial growth
Table 1
Table 1
MICs and EC50s of lauric acid and BPO on P. acnes, S. aureus, and S. epidermidis
To determine minimal bactericidal concentration (MBC) of lauric acid, P. acnes was anaerobically incubated in PBS with several concentrations of lauric acid for 5 hours at 37 °C. After incubation, the bacteria were diluted with PBS and spotted on an agar plate to count a colony-forming unit (CFU) (Figure 2). We found that when lauric acid concentration is higher than 60 μg ml−1, it started killing P. acnes. In contrast, no killing was observed when lauric acid is at 50 μg ml−1. Therefore, we estimated the MBC of lauric acid against P. acnes (ATCC 6919) was 60 μg ml−1. The same experiments were carried out to another strain of P. acnes (ATCC 11827) leading to an MBC of 80 μg ml−1 (Figure S3).
Figure 2
Figure 2
Bactericidal effects of lauric acid on P. acnes
Cytotoxicity of lauric acid
Human sebocyte is one of the major target cells of P. acnes in acne patients. So cytotoxicity of lauric acid was examined using the human immortalized sebaceous gland cell line, SZ95 (Zouboulis et al., 1999) (Figure 3). In the study, SZ95 sebocytes were incubated with lauric acid at various concentrations from 0.24 to 500 μg ml−1 for 18 hours at 37 °C and cell viability was determined subsequently. It was found that lauric acid did not affect sebocyte viability at the concentrations tested. Notably, lauric acid did not influence the viability of SZ95 sebocytes even at the high concentration of 125 μg ml−1, at which P. acnes was completely killed as shown in Figure 2.
Figure 3
Figure 3
Cytotoxicity of lauric acid on human sebocytes
Inflammatory acne model using mouse ear
To induce inflammation in vivo using P. acnes, 1 × 107 CFU of living P. acnes were intradermally injected into the ear of Institute of Cancer Research (ICR) mice. Significant ear swelling (Figure 4b) was observed in P. acnes-injected ear 24 hours after the injection. In contrast, no swelling was observed in the PBS-injected ear (Figures 4a). Histological observation revealed that injection with P. acnes induced a considerable increase in the number of infiltrated inflammatory cells (Figure 4b). Transmission electron microscopy showed colonized and/or phagocytized P. acnes in the macrophage-like cells 24 hours after bacterial injection (Figure 4c and d), but no bacteria were observed in the PBS-injected ear (Figure 4e). In addition, fluorescent immunohistochemistry was employed to visualize P. acnes injected ear and PBS-injected ear, respectively, using antimouse CD11b IgG, a conventional macrophage marker. The infiltration of CD11b+ macrophages was observed at the site where P. acnes was administrated (Figure 4f), whereas no CD11b+ macrophages were observed in PBS-injected controls (Figure 4g).
Figure 4
Figure 4
Inflammatory acne model using mouse ears
Effects of lauric acid on P. acnes in vivo and P. acnes-induced inflammation
To examine the antimicrobial effect of lauric acid in vivo, mouse ears were intradermally injected with P. acnes (1 × 107 CFU). The P. acnes-injected site was then injected with lauric acid (2 μg) (Figure 5) for 1 day. Injection of lauric acid significantly reduced P. acnes-induced ear swelling (Figure 5a) and the number of P. acnes colonized within the ear (Figure 5b). Histological images showed that injection of lauric acid reduced granulomatous response to P. acnes (Figure 5d) compared with injection of an equal amount of vehicle (Figure 5e), whereas sole injection of lauric acid (without pre-injection of P. acnes) did not induce inflammatory reaction (Figure 5c). Furthermore, a TUNEL assay illustrated that the injection of lauric acid did not trigger skin cells to undergo apoptosis (Figure S4). To examine a therapeutic potential of lauric acid by epicutaneous application for acne therapy, lauric acid (150 μg) in Vaseline was applied epicutaneously on the ear injected with P. acnes (1 ± 107 CFU). Epicutaneous application of lauric acid for 1 day noticeably reduced P. acnes-induced ear swelling (Figure 6a) and the number of P. acnes colonized within the ear (Figure 6b). Furthermore, data from TUNEL assays indicated that epicutaneous application of lauric acid did not result in the apoptosis of differentiated keratinocytes (Figure 6c). These data suggest that epicutaneous application of lauric acid can effectively relieve P. acnes-induced inflammation without detrimental effects on skin cells.
Figure 5
Figure 5
Effects of intradermal injection of lauric acid on P. acnes growth in vivo and P. acnes-induced inflammation
Figure 6
Figure 6
Effects of epicutaneous application of lauric acid on P. acnes growth in vivo and P. acnes-induced inflammation
P. acnes produces a number of extracellular enzymes and metabolites that can directly damage host tissues (Hoeffler, 1977; Holland et al., 1981; Cove et al., 1983; Hoffler et al., 1985). One of the well-known enzymes is extracellular triacylglycerol lipase that produces FFAs by hydrolyzing triglycerides in sebum (Miskin et al., 1997). Sebum FFAs, if overproduced, induce very mild inflammation and assist bacterial adherence and colonization in sebaceous follicles (Puhvel and Sakamoto, 1977; Gribbon et al., 1993). On the other hand, FFAs of various chain lengths (C8- C18) have antibacterial activity against a range of Gram-positive bacteria, but not against a number of Gram-negative bacteria (Wille and Kydonieus, 2003; Georgel et al., 2005; Skrivanova et al., 2005; Drake et al., 2008). Lauric acid is a minor sebum component (1-2% of total sebum FFA) (Bach and Babayan, 1982), but it is the most active antimicrobial FFA (Kabara et al., 1972; Kitahara et al., 2004; Rouse et al., 2005; Skrivanova et al., 2005). Palmitoleic acid isoforms (C16:1Δ6 or C16:1Δ9), a major FFA in the sebum, causes a reduction in the growth of the Gram-positive bacteria Staphylococcus, Streptococcus, and Fusobacterium, but is ineffective against P. acnes (Wille and Kydonieus, 2003). Previous studies have demonstrated antimicrobial activities of middle chain saturated FFAs, including lauric acid, against P. acnes in vitro (Puhvel and Reisner, 1970; Higaki, 2003). Despite a number of studies on antimicrobial activity of FFAs in vitro have been made, little effort has been conducted to demonstrate the antimicrobial activity of FFAs against P. acnes in vivo to evaluate its potential for acne therapy.
Our results indicate that lauric acid exerts the inhibitory effect on the growth of skin bacteria such as P. acnes, S. aureus, and S. epidermidis at a concentration 15 times lower than that of BPO, a frequently used oxidizing agent in acne treatment (Figure 1, Table 1). In addition, ED50 values from dose-response curves showed that P. acnes is the most sensitive bacteria to lauric acid among the skin bacteria tested (Figure 1 and Figure S2, Table 1). Lauric acid exerts antimicrobial activity on two P. acnes strains (ATCC 6919 and ATCC 11827) (Figure 1 and Figure S2) and other strains isolated from human sebaceous material (Puhvel and Reisner, 1970). Because acne patients may harbor different skin microbes and P. acnes strains, it is worthwhile to test the potency of lauric acid on a variety of clinical bacteria isolates in the future. In addition, although it has been known that FFAs have antibacterial activities against many Gram-positive bacteria (Drake et al., 2008), it is worthwhile to investigate if lauric acid exhibits antibacterial activity against Gramnegative bacteria in skins. It has been demonstrated that FFAs can be integrated into liposomes (Araseki et al., 2002). Engineering a lauric acid-integrated liposome may circumvent the precipitation problem of water-insoluble lauric acid at high concentrations. The MBC assay indicated that 5 hours incubation of 100 μg ml−1 of lauric acid completely killed two P. acnes strains at (1 × 107 CFU per ml) (Figures 2 and Figure S3). Determination of the bactericidal effects of lauric acid as a function of bacterial numbers may provide valuable information for treatments of various stages of acne vulgaris. On the other hand, when lauric acid was incubated with human sebocytes for 18 hours, whose activity is highly involved in the development of acne, lauric acid did not show cytotoxicity at the concentrations exerting antimicrobial activity (Figure 3). In incubations of the Gram-positive bacteria Clostridium perfringens or S. aureus with lauric acid or capric acid (C10:0), respectively, separation in inner and outer membranes and complete cytoplasmic disorganization were observed in the treated cells despite the lack of alteration in cell wall structure and cell size (Bergsson et al., 2001; Skrivanova et al., 2005). Taken together, lauric acid may specifically disrupt bacterial membranes but not mammalian cell membranes.
Significant antibiotic resistance and multiple drug resistance have been identified for P. acnes strains from acne patients with long-term antibiotic treatments (Eady et al., 2003; Nord and Oprica, 2006). Moreover, biofilm formation by P. acnes increases its resistance against antimicrobial agents (Coenye et al., 2007). These problems of resistance may cause the failure of antibiotic treatment against acne. Lauric acid, however, has been shown to exhibit antimicrobial activity against methicillin-resistant S. aureus (Kitahara et al., 2004). A previous study showed that antimicrobial FFA treatment led to less frequent development of spontaneous resistant bacteria strains compared with treatments with the frequently used antibiotics, tetracycline and metronidazole (Petschow et al., 1996). In addition, bactericidal effects of lauric acid was equally observed on both planktonic and biofilm-formed bacteria (Chavant et al., 2004). Thus, lauric acid may have the potential to be used as an effective antibacterial treatment for antibiotic-refractory acne.
The study of immune responses to P. acnes required an acne animal model, which would be an important tool in the evaluation of the antimicrobial activity of lauric acid against P. acnes in vivo. Proliferation of P. acnes starts in the microcomedone, which is the precursor acne lesion characterized by hyperkeratinization, formation of a keratin plug, and increase in sebum secretion by the sebaceous gland (Leeming et al., 1988; Leyden et al., 1998; Cunliffe and Gollnick, 2001; Leyden, 2001). Overgrowth of P. acnes in the microcomedones results in the rupture of the epithelium of the sebaceous follicle, allowing bacteria to enter the dermis. Consequently, bacteria contact with the host immune system, causing granulomatous inflammation (typical inflammatory acne) (Kligman, 1974; Toyoda and Morohashi, 2001; Degitz et al., 2007). To create an animal model for P. acnes-induced inflammation, the ear of ICR mouse was intradermally injected with P. acnes. Ear swelling was then measured according to a rat ear model as described earlier (De Young et al., 1984, 1985). Our data show that intradermal challenge of mouse ear with P. acnes attracts numerous macrophages to the site of P. acnes (Figure 4). The profile of the granulomatous inflammation in the mouse ear is similar to that of the inflammatory acne in the human sebaceous follicle because numerous P. acnes have been observed inside phagosomes of an infiltrating macrophage in an inflammatory acne lesion (Toyoda and Morohashi, 2001). P. acnes is resistant to phagocytes and is able to survive in macrophages (Webster et al., 1985). In our previous data obtained using a tissue chamber model integrated with a dermis-based cell-trapped system to mimic the in vivo microenvironment of acne lesions, injection of living P. acnes into the intradermally implanted tissue chamber attracted Gr-1+ neutrophils and CD11b+ macrophages into the chamber (Nakatsuji et al., 2008d) and increased the level of proinflammatory cytokine and macrophage inflammatory protein-2 in the chamber fluid (Nakatsuji et al., 2008a, b). We have recently developed effective vaccines for P. acnes associated inflammation as an alternative treatment for acne. The vaccines consist of killed-whole organism of P. acnes and a cell wall-anchored P. acnes sialidase (Nakatsuji et al., 2008a, b, c). The vaccine may be effective against P. acnes outside of a cell but not against phagocytized and/ or invading bacteria. However, P. acnes inside cells may be susceptible to the antimicrobial activities of lauric acid, which is cell membrane permeable. Our data demonstrate that either intradermal or epicutaneous administration of lauric acid with P. acnes decreased the number of detected bacteria in the ear, thereby relieving P. acnes-induced ear swelling and granulomatous inflammation (Figures (Figures55 and and6).6). In addition, application of lauric acid did not trigger the skin cells to undergo apoptosis (Figures 6 and Figure S4). These data suggest that lauric acid can suppress pathogenicity of P. acnes in vivo, but is harmless to host cells. As human skin is in general thicker than mouse ear skin, a higher dosage with repeated applications of lauric acid may be required for acne treatments in clinics.
Epicutaneous application of lauric acid enhances the activity of antibiotic agents, skin permeability, and transdermal drug delivery of antibiotic chemicals (Lee et al., 1994; Kravchenko et al., 2003). Conjugation of lauric acid with synthetic antimicrobial peptide results in an increased ability of the compound to permeabilize bacteria membranes (Chu-Kung et al., 2004). Thus, a combination of topical antimicrobial therapies for acne with lauric acid may induce synergistically enhanced effectiveness. In conclusion, we demonstrated the antimicrobial property of lauric acid against P. acnes in vitro and its therapeutic effects on P. acnes-induced inflammation in vivo using the ICR mouse ear model. The obtained data highlight the potential of using lauric acid as an alternative treatment option to the antibiotic therapy of acne vulgaris.
Preparation of bacteria
P. acnes (ATCC 6919 and ATCC 11827) (American Type Culture Collection, Manassas, VA) was cultured on Brucella broth agar (BD, Sparks, MD), supplemented with 5% (v v−1) defibrinated sheep blood (LAMPIRE Biological Laboratories, Pipersville, PA), vitamin K (5 μg ml−1, Remel, Lenexa, KS), and hemin (50 μg ml−1, Remel), under anaerobic conditions using Gas-Pak (BD) at 37 °C. Single colonies were inoculated in Reinforced Clostridium Medium (Oxford, Hampshire, England) and cultured at 37 °C until reaching around OD600=1.0 (logarithmic growth phase) under anaerobic conditions. S. aureus (ATCC 35556) or S. epidermidis (ATCC 12228) was cultured on tryptic soy broth (Sigma, St. Louis, MO) agar overnight at 37 °C. The bacteria from single colonies were cultured in tryptic soy broth overnight at 37 °C. The overnight culture was diluted 1:20 and cultured until reaching around OD600=1.0. These bacteria were harvested by centrifugation at 5,000 g for 10 minutes, washed with PBS, and suspended to an appropriate amount of PBS for the experiments.
In vitro antimicrobial assays
MIC of lauric acid was compared with that of BPO, which has long been used clinically in acne treatment. Lauric acid (Sigma) or BPO (Fisher scientific, Pittsburgh, PA) were dissolved in DMSO and then added to bacteria suspension to obtain the final concentration of 5% (v v−1) DMSO. P. acnes (1 × 106 CFU per ml) was incubated with lauric acid or BPO at the concentrations of twofold serial dilution (0.24-500 μg ml−1) in Reinforced Clostridium Medium on a 96-well microplate (100 μl per well) under anaerobic conditions for 72 hours. S. aureus or S. epidermidis (1 × 106 CFU per ml) were incubated with the same concentrations of lauric acid and BPO in tryptic soy broth under aerobic conditions for 24 or 48 hours, respectively. The control received only 5% (v v−1) DMSO. After incubation, the plates were mixed well and then absorbance at 600 nm was measured by a microplate reader to estimate bacterial growth.
To determine MBC of lauric acid against P. acnes, P. acnes (1 × 107 CFU per ml) was incubated with lauric acid at various concentrations (12.5-100 μg ml−1) in PBS on a 96-well microplate (100 μl per well) under anaerobic conditions. The control received only 5% (v v−1) of DMSO. The growth of P. acnes was decreased 1 hour after incubation and completely eliminated 3 and 5 hours after incubation with lauric acid (Figure S5). The reaction mixture was diluted 1:10-1:106 with PBS. MBC was determined by spotting the dilution (5 μl) on a Brucella broth agar plate for the counting of CFUs.
Cytotoxicity of lauric acid on human sebocytes
The immortalized human sebaceous gland cell line SZ95 (Zouboulis et al., 1999), was cultured on 96-well plates in Sebomed basal medium (Biochrom, Berlin, Germany) supplemented with 5 ng ml−1 human recombinant epidermal growth factor (Sigma), 10% (v v−1) heat-inactivated fetal bovine serum (Mediatech Inc., Herndon, VA), at 37 °C under an atmosphere of 5% (v v−1) CO2 in air. Lauric acid was prepared in DMSO and added to the culture medium at various concentrations for the final concentration of 5% DMSO. SZ95 sebocytes (1 × 105 cells per well) were incubated with lauric acid for 18 hours at 37 °C. As a negative control, an equal amount of DMSO was added to the culture medium. Triton X-100 (0.01%) was used to achieve 100% of cell cytotoxicity. After incubation, cell viability of sebocytes was determined with acid phosphatase assays (Martin and Clynes, 1991). Cells were washed with PBS three times and incubated with 100 μl of 10mM p-nitrophenyl phosphate in acid phosphatase assay buffer [1 M sodium acetate buffer, pH 5.5, containing 0.1% (w v−1) Triton X-100] for 1 hour at 37 °C. After that, 10 μl of 1N NaOH was added to stop the reaction and absorbance at 405 nm was measured. Cytotoxicity of lauric acid was calculated as the percentage of cytotoxicity of Triton X-100 ((the OD405 difference without and with lauric acid treatment) ÷ (the OD405 difference without lauric acid and with Triton X-100 treatment) × 100 (%)).
Transmission electron microscopy
Ears of ICR mice were intradermally injected with 1 × 107 CFU per 20 μl of live P. acnes (ATCC 6919) suspension in PBS or PBS alone. The ear was excised 24 hours after bacterial challenge, fixed in Karnovsky’s fixative (4% paraformaldehyde, 2.5% glutaraldehyde, 5mM CaCl2 in 0.1 M Na Cacodylate buffer, pH 7.4) overnight at 4 °C followed by 1% OsO4 in 0.1 M Na Cacodylate buffer, pH 7.4, en bloc staining with 4% uranyl acetate in 50% ethanol, and subsequently dehydrated using a graded series of ethanol solutions followed by propylene oxide and infiltration with epoxy resin (Scipoxy 812, Energy Beam Sciences, Agawam, MA). After polymerization at 65 °C overnight, thin sections were cut and stained with uranyl acetate (4% uranyl acetate in 50% ethanol) followed by bismuth subnitrate. Sections were examined at an accelerating voltage of 60 kV using a Zeiss EM10C electron microscope (Carl Zeiss, Thornwood, NY).
Fluorescence immunohistochemistry
To induce inflammation, mouse ear was intradermally injected with live P. acnes (ATCC 6919) as described above. The ear was excised 24 hours after bacterial injection, fixed in Optimal Cutting Temperature (OCT) compound (Sakura Finetek, Torrance, CA), and frozen at −80 °C. The tissue block was cross-sectioned with Cryostat and fixed in 10% formamide in PBS. The sections were blocked with PBS containing 5% bovine serum albumin (BSA) and anti-mouse CD16/CD32 IgG (5 μg ml−1) (BD Biosciences Pharmingen, Sparks, MD) for 30 minutes, and incubated with biotinylated anti-mouse CD11b IgG (5 μg ml−1) (BD Biosciences), a conventional macrophage marker. Tetramethylrhodamine isothiocyanate (TRITC)-streptavidin conjugate (5 μg ml−1) (ZYMED, Carlsbad, CA) was applied to the section, which was incubated for 20 minutes at room temperature, followed by 4′-6-Diamidino-2-phenylindole (Sigma). Images were obtained using an Olympus BX41 fluorescent microscope (Olympus, Center Valley, PA).
To examine the toxicity of lauric acid in vivo, the ear was excised for cross-sections 24 hours after intradermal injection or epicutaneous application of lauric acid. To detect the apoptotic cells, the tissue sections were stained with a DeadEnd Fluorometric Terminal Deoxynucleotidyl Transferase TUNEL System (Promega, Madison, WI) followed by immuno-reactions to rabbit anti-mouse keratin 10 (K10) IgG (1:1,000 dilution) (Covance, Berkeley, CA) and goat anti-rabbit IgG-TRITC conjugate (5 μg ml−1).
P. acnes-induced inflammation
P. acnes (ATCC 6919) (1 × 107 CFU per 20 μl in PBS) was intradermally injected into left ears of ICR mice. Right ears received with an equal amount (20 μl) of PBS. Lauric acid (2 μg20μl−1 in 5% DMSO in PBS) was injected into the same location of both ears right after P. acnes or PBS injection. As a control, an equal volume (20 μl) of 5% DMSO in PBS was intradermally injected into both the ears. For epicutaneous application, lauric acid (150 μg in 5% acetone mixed with 15 mg of Vaseline (Sigma)) was applied on the surface of the ear skin right after intradermal injection with P. acnes (1 × 107 CFU per 20 μl) or PBS (20 μl). Epicutaneous application of 5% acetone mixed with 15 mg of Vaseline served as a control. For histological observation, the ear was cross-sectioned, stained with hematoxylin and eosin (Sigma), and viewed on a Zeiss Axioskop2 plus microscope (Carl Zeiss). The increase in ear thickness was measured using a micro caliper (Mitutoyo, Kanagawa, Japan) 24 hours after the bacterial injection. The increase in ear thickness of the P. acnes-injected ear was calculated as percentage of a PBS-injected control.
To determine P. acnes number in the ear, the ear was cut off and punched with an 8 mm biopsy punch 24 hours after P. acnes injection. Wet kimwipes were used to remove the Vaseline remaining on the ear surfaces. The punch biopsy was homogenized in 200 μl of sterile PBS with a hand tissue grinder. CFUs of P. acnes in the ear were enumerated by plating serial dilutions (1:102-1:108) of the homogenate on a Brucella agar plate. To count colonies, the plate was anaerobically incubated for 72 hours at 37 °C. All experiments using mice were conducted according to institutional guidelines for animal experiments.
Statistical analysis
Data are presented as mean±SE. The Student’s t-test was used to assess the significance of independent experiments. The criterion P<0.05 was used to determine the statistical significance.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants (R01-AI067395-01, R21-R022754-01 and R21-I58002-01). We thank Dr Y.-T. Liu for providing valuable comments and Corbin Clawson for proofreading.
Abbreviations
BPObenzoyl peroxide
CFUcolony forming unit
FFAfree fatty acids
ICRInstitute of Cancer Research
MBCminimal bactericidal concentration
P. acnesPropionibacterium acnes

Footnotes
CONFLICT OF INTEREST
The authors state no conflict of interest.
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