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The Anti-Inflammatory Activities of Propionibacterium acnes CAMP Factor-Targeted Acne Vaccines

Yanhan Wang
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Yanhan Wang
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  • Department of Dermatology, School of Medicine, University of California, San Diego, California, USA
,
Tissa R. Hata
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Tissa R. Hata
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Affiliations

  • Department of Dermatology, School of Medicine, University of California, San Diego, California, USA
,
Yun Larry Tong
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Yun Larry Tong
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  • Department of Dermatology, School of Medicine, University of California, San Diego, California, USA
,
Ming-Shan Kao
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Ming-Shan Kao
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Affiliations

  • Department of Biomedical Sciences and Engineering, National Central University, Jhongli, Taiwan
,
Christos C. Zouboulis
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Christos C. Zouboulis
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  • Brandenburg Medical School Theodore Fontane, Dermatology, Venereology, Allergology and Immunology, Dessau, Germany
,
Richard L. Gallo
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Richard L. Gallo
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  • Department of Dermatology, School of Medicine, University of California, San Diego, California, USA
,
Chun-Ming Huang
x
Chun-Ming Huang
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  • Department of Dermatology, School of Medicine, University of California, San Diego, California, USA
  • Department of Biomedical Sciences and Engineering, National Central University, Jhongli, Taiwan
  • Moores Cancer Center; University of California, San Diego, California, USA

Correspondence

  • Correspondence: Chun-Ming Huang, Department of Dermatology, University of California, San Diego, 9452 Medical Center Drive, La Jolla, California 92037, USA
  • Chun-Ming Huang, Department of Biomedical Sciences and Engineering, National Central University, Jhongli, 320, Taiwan
'Correspondence information about the author Chun-Ming Huang
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Chun-Ming Huang
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Affiliations

  • Department of Dermatology, School of Medicine, University of California, San Diego, California, USA
  • Department of Biomedical Sciences and Engineering, National Central University, Jhongli, Taiwan
  • Moores Cancer Center; University of California, San Diego, California, USA

Correspondence

  • Correspondence: Chun-Ming Huang, Department of Dermatology, University of California, San Diego, 9452 Medical Center Drive, La Jolla, California 92037, USA
  • Chun-Ming Huang, Department of Biomedical Sciences and Engineering, National Central University, Jhongli, 320, Taiwan
'Correspondence information about the author Chun-Ming Huang
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Chun-Ming Huang
Search for articles by this author

Affiliations

  • Department of Dermatology, School of Medicine, University of California, San Diego, California, USA
  • Department of Biomedical Sciences and Engineering, National Central University, Jhongli, Taiwan
  • Moores Cancer Center; University of California, San Diego, California, USA

Correspondence

  • Correspondence: Chun-Ming Huang, Department of Dermatology, University of California, San Diego, 9452 Medical Center Drive, La Jolla, California 92037, USA
  • Chun-Ming Huang, Department of Biomedical Sciences and Engineering, National Central University, Jhongli, 320, Taiwan
accepted manuscript published online 30 June 2018; corrected proof published online XXX
The Anti-Inflammatory Activities of Propionibacterium acnes CAMP Factor-Targeted Acne Vaccines
DOI: https://doi.org/10.1016/j.jid.2018.05.032

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Figure 1

Up-regulation of Propionibacterium acnes CAMP factor under anaerobic conditions and the essential role of CAMP factor in P. acnes-induced proinflammatory MIP-2 cytokine and bacterial colonization. (a) P. acnes ATCC 6919 was grown under aerobic and anaerobic conditions. Lysates (1 mg) of P. acnes (ATCC 6919) from aerobic and anaerobic growth were labeled with 12C6-Nic-NHS and 13C6-Nic-NHS, respectively. After mixing 12C6-Nic-NHS– and 13C6-Nic-NHS–labeled samples, the mixture was subjected to an LC-LTQ mass spectrometer. CAMP factor with a mass difference of 3 Da per labeled site in mass spectra is shown. (b) A peptide, DLLKAAFDLR, was sequenced and assigned to the internal peptide of CAMP factor. (c) The right ears of Institute of Cancer Research (London, UK) mice were injected intradermally with a wild-type or CAMP factor-mutant P. acnes strain (Δcamp2) (107 CFU in 20 μl PBS). Injection of 20 μl PBS into left ears served as a control. (d) Three days after injection, the proinflammatory MIP-2 cytokines were quantified by ELISA. (e, f) The bacterial colonies (CFUs) were enumerated by plating serial dilutions (1:101–1:105) of the ear homogenate on an agar plate. Error bars represent mean ± standard deviation of five mice. ∗P < 0.05, ∗∗∗P < 0.001 by Student t test. CAMP, Christie-Atkins-Munch-Petersen; CFU, colony-forming unit; LC-LTQ, liquid chromatography linear ion trap quadrupole; Nic-NHS, N-nicotinoyloxy-succinimide; PBS, phosphate buffered saline; WT, wild type.

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Figure 2

The detectable antibodies to CAMP factor in acne patients and reduction of cytotoxicity of Propionibacterium acnes to macrophages by high titers of antibodies generated in the Escherichia coli overexpressing CAMP factor-vaccinated mice. (a) Sera from individuals without acne (normal control) and acne patients (acne) were assayed by ELISA for antibody to CAMP factor. Antibody (IgG) titers of were determined by using recombinant CAMP factor or GFP as a capture antigen for coating onto a 96-well ELISA plate. The endpoint was defined as the dilution of serum on CAMP factor-coated wells producing the same OD570–450 as a 1/100 dilution of serum on GFP-coated wells. Sera negative at the lowest dilution tested were assigned endpoint titers of 100. The data were presented as geometric mean endpoint ELISA titers. (b) For reduction of cytotoxicity of P. acnes, RAW264.7 macrophage cells were incubated with P. acnes ATCC 6919 along with different titers (as indicated) of inactivated sera obtained from individuals without acne, acne patients, or mice vaccinated with E. coli overexpressing CAMP factor or GFP for 18 hours. The cytotoxicity of P. acnes was determined by an ACP assay and calculated as the percentage of cell death. ∗P < 0.05, ∗∗P < 0.01 by Student t test. ACP, acid phosphatase; CAMP, Christie-Atkins-Munch-Petersen; GFP, green fluorescent protein; OD, optical density.

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Figure 3

The protective immunity in mice vaccinated with recombinant CAMP factor. Institute of Cancer Research mice were subcutaneously vaccinated with 10, 20, or 50 μg recombinant CAMP factor or GFP with or without 2% aluminum (alum) adjuvant in a 2-week interval. (a) Three weeks after vaccination, the titer of antibodies to CAMP factor was measured by ELISA. (b) Propionibacterium acnes (107 CFU/20 μl in PBS) bacteria or PBS (20 μl) was intradermally injected into the right or left ears of GFP- or CAMP factor-vaccinated mice, respectively, 3 weeks after the second booster. Ear thickness (mm) was measured 1 day after bacterial injection. (c) The levels of proinflammatory MIP-2 cytokines in the homogenates of P. acnes- or PBS-injected ears were measured by ELISA. (d) Bacterial colonization (CFUs) in the ears injected with P. acnes or PBS were enumerated by plating serial dilutions (1:101–1:105) of ear homogenate on an agar plate. Three separate experiments with five mice per group were performed. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by Student t test. Alum, aluminum; CAMP, Christie-Atkins-Munch-Petersen; CFU, colony forming unit; GFP, green fluorescent protein; PBS, phosphate buffered saline.

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Figure 4

The presence of Propionibacterium acnes CAMP factor in acne lesions. (a) Images of the biopsy sample (4 × 4 × 8 mm) of lesional back skin of patients with acne vulgaris within 2–14 days of onset of inflammatory papules. An open acne lesion, a hair shaft, and epidermal and dermal layers were indicated. The lesion was illustrated in a top-view picture of an acne biopsy sample with the epidermis facing up. Scale bar = 4 mm. Hematoxylin and eosin staining showed that the epidermis (b) in acne lesional skin was thicker than that (c) in nonlesional skin. Scale bar = 4 mm. The mRNA expression normalized to (d) 16S rRNA of P. acnes and (e) protein levels of CAMP factor detected by reverse transcription quantitative PCR and ELISA, respectively, in acne lesional skin (AL) were higher than those in nonlesional skin (NAL). ∗P < 0.05 by Student t test. High-magnification photos of selected areas in (f) nonlesional skin and (k) acne lesional skin are shown in g–j and l–o, respectively. (g, l) CAMP factor (red stains) was detectable in the hair follicle and was expressed at a higher abundance in (h, m) sebaceous glands, (i, n) epidermis, and (j, o) dermis in acne lesional skin than in nonlesional skin. Scale bars = 500 μm in f and k and 100 μm in g and l. CAMP, Christie-Atkins-Munch-Petersen.

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Figure 5

Reduction of Propionibacterium acnes-induced inflammation by monoclonal antibody to CAMP factor. Human skin was obtained by taking 4-mm punch biopsy samples from healthy individuals (NC), nonlesional skin (NAL), and acne lesional skin in patients with acne vulgaris(AL). (a) The mRNA expressions of 11 Th1/Th2/Th17-related proinflammatory cytokines normalized to GAPDH in nonlesional and acne lesional skin were examined by reverse transcription quantitative PCR. (b, c) The protein levels of IL-8 and IL-1β in the skin of healthy individuals and nonlesional and acne lesional skin of acne patients were measured by ELISA. (d, e) The nonlesional and acne lesional skin were cut in half, and half was incubated with monoclonal antibody (IgG1) to P. acnes CAMP factor for 24 hours in antibiotic-free the EpiLife keratinocyte medium (Thermo Fisher Scientific, Waltham, MA). The other half was incubated with HBsAg monoclonal antibody as a control (C). The protein levels of IL-8 and IL-1β in skins after monoclonal antibody incubation were quantified by ELISA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 by Student t test. CAMP, Christie-Atkins-Munch-Petersen; HBsAg, hepatitis B surface antigen; n.s., not significant; TGF, transforming growth factor; Th, T helper.

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Inflammatory acne vulgaris afflicts hundreds of millions of people globally. Propionibacterium acnes, an opportunistic skin bacterium, has been linked to the pathogenesis of acne vulgaris. Our results show that a secretory Christie-Atkins-Munch-Petersen (CAMP) factor of P. acnes is up-regulated in anaerobic cultures. Mutation of CAMP factor significantly diminishes P. acnes colonization and inflammation in mice, demonstrating the essential role of CAMP factor in the cytotoxicity of P. acnes. Vaccination of mice with CAMP factor considerably reduced the growth of P. acnes and production of MIP-2, a murine counterpart of human IL-8. Acne lesions were collected from patients to establish an ex vivo acne model for validation of the efficacy of CAMP factor antibodies in the neutralization of the acne inflammatory response. The P. acnes CAMP factor and two proinflammatory cytokines (IL-8 and IL-1β) were expressed at higher levels in acne lesions than those in nonlesional skin. Incubation of ex vivo acne explants with monoclonal antibodies to CAMP factor markedly attenuated the amounts of IL-8 and IL-1β. Our work using an ex vivo acne model shows that P. acnes CAMP factor is an essential source of inflammation in acne vulgaris.

Abbreviations:

CAMP (Christie-Atkins-Munch-Petersen), GFP (green fluorescent protein), mAb (monoclonal antibody), TGF (transforming growth factor), Th (T helper)

Introduction

Propionibacterium acnes, now referred as Cutibacterium acnes based on a new taxonomic classification (Boisrenoult, 2018xBoisrenoult, 2018Boisrenoult, P. Cutibacterium acnes prosthetic joint infection: diagnosis and treatment. Orthop Traumatol Surg Res. 2018; 104: S19–S24

Abstract | Full Text | Full Text PDF | PubMed | Scopus (3) | Google ScholarSee all References), is a Gram-positive skin commensal bacterium and predominates (>60% of total bacteria) the facial skin in humans (Grice et al., 2009xGrice et al., 2009Grice, E.A., Kong, H.H., Conlan, S., Deming, C.B., Davis, J., Young, A.C. et al. Topographical and temporal diversity of the human skin microbiome. Science. 2009; 324: 1190–1192

Crossref | PubMed | Scopus (939) | Google ScholarSee all References). Nearly everyone hosts P. acnes (Ahn et al., 1996xAhn et al., 1996Ahn, C.Y., Ko, C.Y., Wagar, E.A., Wong, R.S., and Shaw, W.W. Microbial evaluation: 139 implants removed from symptomatic patients. Plast Reconstr Surg. 1996; 98: 1225–1229

Crossref | PubMed | Scopus (69) | Google ScholarSee all References, Brook and Frazier, 1991xBrook and Frazier, 1991Brook, I. and Frazier, E.H. Infections caused by Propionibacterium species. Rev Infect Dis. 1991; 13: 819–822

Crossref | PubMed | Google ScholarSee all References), which makes up almost half of the total skin microbiome (Tancrede, 1992xTancrede, 1992Tancrede, C. Role of human microflora in health and disease. Eur J Clin Microbiol Infect Dis. 1992; 11: 1012–1015

Crossref | PubMed | Scopus (119) | Google ScholarSee all References), with an estimated density of 102 to 105–6 cm2 (Leyden et al., 1998xLeyden et al., 1998Leyden, J.J., McGinley, K.J., and Vowels, B. Propionibacterium acnes colonization in acne and nonacne. Dermatology. 1998; 196: 55–58

Crossref | PubMed | Scopus (80) | Google ScholarSee all References, McGinley et al., 1978xMcGinley et al., 1978McGinley, K.J., Webster, G.F., and Leyden, J.J. Regional variations of cutaneous Propionibacteria. Appl Environ Microbiol. 1978; 35: 62–66

PubMed | Google ScholarSee all References). Overgrowth of P. acnes has been linked to acne vulgaris, a skin disease afflicting more than 85% of teenagers and over 40 million people in the United States (Fried and Wechsler, 2006xFried and Wechsler, 2006Fried, R.G. and Wechsler, A. Psychological problems in the acne patient. Dermatol Ther. 2006; 19: 237–240

Crossref | PubMed | Scopus (75) | Google ScholarSee all References, Taglietti et al., 2008xTaglietti et al., 2008Taglietti, M., Hawkins, C.N., and Rao, J. Novel topical drug delivery systems and their potential use in acne vulgaris. Skin Therapy Lett. 2008; 13: 6–8

PubMed | Google ScholarSee all References, White, 1998xWhite, 1998White, G.M. Recent findings in the epidemiologic evidence, classification, and subtypes of acne vulgaris. J Am Acad Dermatol. 1998; 39: S34–S37

Abstract | Full Text | Full Text PDF | PubMed | Google ScholarSee all References). Current treatments for acne are often inadequate or difficult to tolerate, but many therapies target the P. acnes organism, which has been implicated in the genesis of inflammation in acne (Dessinioti and Katsambas, 2010xDessinioti and Katsambas, 2010Dessinioti, C. and Katsambas, A.D. The role of Propionibacterium acnes in acne pathogenesis: facts and controversies. Clin Dermatol. 2010; 28: 2–7

Abstract | Full Text | Full Text PDF | PubMed | Scopus (99) | Google ScholarSee all References, Leyden, 2001xLeyden, 2001Leyden, J.J. The evolving role of Propionibacterium acnes in acne. Semin Cutan Med Surg. 2001; 20: 139–143

Crossref | PubMed | Scopus (88) | Google ScholarSee all References). Here, we used a vaccination approach to test whether P. acnes Christie-Atkins-Munch-Petersen (CAMP) factor, a secretory virulence factor (Valanne et al., 2005xValanne et al., 2005Valanne, S., McDowell, A., Ramage, G., Tunney, M.M., Einarsson, G.G., O’Hagan, S. et al. CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II. Microbiology. 2005; 151: 1369–1379

Crossref | PubMed | Scopus (78) | Google ScholarSee all References), is a main source of inflammation in acne vulgaris.

Five genes with approximately 32% sequence homology to the co-hemolytic CAMP factor of Streptococcus agalactiae were detected in the P. acnes genome (Bruggemann, 2005xBruggemann, 2005Bruggemann, H. Insights in the pathogenic potential of Propionibacterium acnes from its complete genome. Semin Cutan Med Surg. 2005; 24: 67–72

Crossref | PubMed | Scopus (78) | Google ScholarSee all References). P. acnes CAMP factor is able to bind to immunoglobulin G and M classes and acts as a pore-forming toxin (Valanne et al., 2005xValanne et al., 2005Valanne, S., McDowell, A., Ramage, G., Tunney, M.M., Einarsson, G.G., O’Hagan, S. et al. CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II. Microbiology. 2005; 151: 1369–1379

Crossref | PubMed | Scopus (78) | Google ScholarSee all References). Previous data from our laboratory showed that CAMP factor combined with sphingomyelinase exerts the co-hemolytic activity that can confer cytotoxicity to both keratinocytes and macrophages (Nakatsuji et al., 2011xNakatsuji et al., 2011Nakatsuji, T., Tang, D.C., Zhang, L., Gallo, R.L., and Huang, C.M. Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment. PloS One. 2011; 6: e14797

Crossref | PubMed | Scopus (50) | Google ScholarSee all References), enhancing virulence by degrading and invading host cells. Furthermore, P. acnes CAMP factor can induce cell death of sebocytes in sebaceous glands and trigger inflammation (Liu et al., 2011xLiu et al., 2011Liu, P.F., Nakatsuji, T., Zhu, W., Gallo, R.L., and Huang, C.M. Passive immunoprotection targeting a secreted CAMP factor of Propionibacterium acnes as a novel immunotherapeutic for acne vulgaris. Vaccine. 2011; 29: 3230–3238

Crossref | PubMed | Scopus (29) | Google ScholarSee all References). In addition, it has been shown that P. acnes targets skin cells, namely, keratinocytes and phagocytic cells like macrophages, stimulating the cells to produce proinflammatory cytokines, including IL-8, IL-1β, IL-12, and tumor necrosis factor-α, leading to the inflammation in acne vulgaris (Contassot and French, 2014xContassot and French, 2014Contassot, E. and French, L.E. New insights into acne pathogenesis: Propionibacterium acnes activates the inflammasome. J Invest Dermatol. 2014; 134: 310–313

Abstract | Full Text | Full Text PDF | PubMed | Scopus (27) | Google ScholarSee all References, Kurokawa et al., 2009xKurokawa et al., 2009Kurokawa, I., Danby, F.W., Ju, Q., Wang, X., Xiang, L.F., Xia, L. et al. New developments in our understanding of acne pathogenesis and treatment. Exp Dermatol. 2009; 18: 821–832

Crossref | PubMed | Scopus (295) | Google ScholarSee all References, Nagy et al., 2006xNagy et al., 2006Nagy, I., Pivarcsi, A., Kis, K., Koreck, A., Bodai, L., McDowell, A. et al. Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes. Microbes Infect. 2006; 8: 2195–2205

Crossref | PubMed | Scopus (195) | Google ScholarSee all References, Nakatsuji et al., 2011xNakatsuji et al., 2011Nakatsuji, T., Tang, D.C., Zhang, L., Gallo, R.L., and Huang, C.M. Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment. PloS One. 2011; 6: e14797

Crossref | PubMed | Scopus (50) | Google ScholarSee all References, Qin et al., 2014xQin et al., 2014Qin, M., Pirouz, A., Kim, M.H., Krutzik, S.R., Garban, H.J., and Kim, J. Propionibacterium acnes induces IL-1beta secretion via the NLRP3 inflammasome in human monocytes. J Invest Dermatol. 2014; 134: 381–388

Abstract | Full Text | Full Text PDF | PubMed | Scopus (71) | Google ScholarSee all References).

Antibiotics for acne treatment may run a risk of developing resistant bacteria and have no capability of neutralizing the secretory toxins. Isotretinoin, 13-cis-retinoic acid, has been extensively prescribed for the systemic treatment of severe acne (Layton et al., 2006xLayton et al., 2006Layton, A.M., Dreno, B., Gollnick, H.P., and Zouboulis, C.C. A review of the European Directive for prescribing systemic isotretinoin for acne vulgaris. J Eur Acad Dermatol Venereol. 2006; 20: 773–776

Crossref | PubMed | Scopus (71) | Google ScholarSee all References). However, isotretinoin can cause depression and an increased rate of birth defects if taken during pregnancy (Mondal et al., 2017xMondal et al., 2017Mondal, D., Shenoy, R.S., and Mishra, S. Retinoic acid embryopathy. Int J Appl Basic Med Res. 2017; 7: 264–265

Crossref | PubMed | Google ScholarSee all References). Thus, the drug is highly regulated by the US Food and Drug Administration. Despite the success of isotretinoin in the treatment of acne vulgaris, there remains no effective modality that can prevent the occurrence of acne vulgaris. We have previously shown that the vaccination approach prevents the inflammation caused by P. acnes CAMP factor (Huang et al., 2008xHuang et al., 2008Huang, C.P., Liu, Y.T., Nakatsuji, T., Shi, Y., Gallo, R.R., Lin, S.B. et al. Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes. Proteomics Clin Appl. 2008; 2: 1234–1245

Crossref | PubMed | Scopus (4) | Google ScholarSee all References, Kao and Huang, 2009xKao and Huang, 2009Kao, M. and Huang, C.M. Acne vaccines targeting Propionibacterium acnes. G Ital Dermatol Venereol. 2009; 144: 639–643

PubMed | Google ScholarSee all References, Liu et al., 2011xLiu et al., 2011Liu, P.F., Nakatsuji, T., Zhu, W., Gallo, R.L., and Huang, C.M. Passive immunoprotection targeting a secreted CAMP factor of Propionibacterium acnes as a novel immunotherapeutic for acne vulgaris. Vaccine. 2011; 29: 3230–3238

Crossref | PubMed | Scopus (29) | Google ScholarSee all References, Lo et al., 2011xLo et al., 2011Lo, C.W., Lai, Y.K., Liu, Y.T., Gallo, R.L., and Huang, C.M. Staphylococcus aureus hijacks a skin commensal to intensify its virulence: immunization targeting beta-hemolysin and CAMP factor. J Invest Dermatol. 2011; 131: 401–409

Abstract | Full Text | Full Text PDF | PubMed | Scopus (30) | Google ScholarSee all References, Nakatsuji et al., 2008axNakatsuji et al., 2008aNakatsuji, T., Liu, Y.T., Huang, C.P., Zoubouis, C.C., Gallo, R.L., and Huang, C.M. Antibodies elicited by inactivated Propionibacterium acnes-based vaccines exert protective immunity and attenuate the IL-8 production in human sebocytes: relevance to therapy for acne vulgaris. J Invest Dermatol. 2008; 128: 2451–2457

Abstract | Full Text | Full Text PDF | PubMed | Scopus (46) | Google ScholarSee all References, Nakatsuji et al., 2008bxNakatsuji et al., 2008bNakatsuji, T., Liu, Y.T., Huang, C.P., Zouboulis, C.C., Gallo, R.L., and Huang, C.M. Vaccination targeting a surface sialidase of P. acnes: implication for new treatment of acne vulgaris. PloS One. 2008; 3: e1551

Crossref | PubMed | Scopus (53) | Google ScholarSee all References, Nakatsuji et al., 2008cxNakatsuji et al., 2008cNakatsuji, T., Rasochova, L., and Huang, C.M. Vaccine therapy for P. acnes-associated diseases. Infect Disord Drug Targets. 2008; 8: 160–165

Crossref | PubMed | Scopus (15) | Google ScholarSee all References, Nakatsuji et al., 2008dxNakatsuji et al., 2008dNakatsuji, T., Shi, Y., Zhu, W., Huang, C.P., Chen, Y.R., Lee, D.Y. et al. Bioengineering a humanized acne microenvironment model: proteomics analysis of host responses to Propionibacterium acnes infection in vivo. Proteomics. 2008; 8: 3406–3415

Crossref | PubMed | Scopus (25) | Google ScholarSee all References, Nakatsuji et al., 2011xNakatsuji et al., 2011Nakatsuji, T., Tang, D.C., Zhang, L., Gallo, R.L., and Huang, C.M. Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment. PloS One. 2011; 6: e14797

Crossref | PubMed | Scopus (50) | Google ScholarSee all References). Results from our previous publications illustrated that vaccination using surface sialidase (Nakatsuji et al., 2008bxNakatsuji et al., 2008bNakatsuji, T., Liu, Y.T., Huang, C.P., Zouboulis, C.C., Gallo, R.L., and Huang, C.M. Vaccination targeting a surface sialidase of P. acnes: implication for new treatment of acne vulgaris. PloS One. 2008; 3: e1551

Crossref | PubMed | Scopus (53) | Google ScholarSee all References) or heat-killed P. acnes (Nakatsuji et al., 2008axNakatsuji et al., 2008aNakatsuji, T., Liu, Y.T., Huang, C.P., Zoubouis, C.C., Gallo, R.L., and Huang, C.M. Antibodies elicited by inactivated Propionibacterium acnes-based vaccines exert protective immunity and attenuate the IL-8 production in human sebocytes: relevance to therapy for acne vulgaris. J Invest Dermatol. 2008; 128: 2451–2457

Abstract | Full Text | Full Text PDF | PubMed | Scopus (46) | Google ScholarSee all References) as an antigen significantly suppressed P. acnes-induced inflammation. Specifically, immunized mice had a reduction in ear swelling and production of MIP-2. Here, we explored the use of a specific secretory virulence factor as a vaccine because it has been reported that specific inhibition of secretory virulence factors presents less selective pressure for the development of resistant bacteria (Rasko et al., 2008xRasko et al., 2008Rasko, D.A., Moreira, C.G., Li de, R., Reading, N.C., Ritchie, J.M., Waldor, M.K. et al. Targeting QseC signaling and virulence for antibiotic development. Science. 2008; 321: 1078–1080

Crossref | PubMed | Scopus (288) | Google ScholarSee all References). We have previously shown that CAMP factor of P. acnes is immunogenic and a secretory toxin (Nakatsuji et al., 2011xNakatsuji et al., 2011Nakatsuji, T., Tang, D.C., Zhang, L., Gallo, R.L., and Huang, C.M. Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment. PloS One. 2011; 6: e14797

Crossref | PubMed | Scopus (50) | Google ScholarSee all References) and that vaccination of mice with Escherichia coli overexpressing CAMP factor provides therapeutic protection against P. acnes (Liu et al., 2011xLiu et al., 2011Liu, P.F., Nakatsuji, T., Zhu, W., Gallo, R.L., and Huang, C.M. Passive immunoprotection targeting a secreted CAMP factor of Propionibacterium acnes as a novel immunotherapeutic for acne vulgaris. Vaccine. 2011; 29: 3230–3238

Crossref | PubMed | Scopus (29) | Google ScholarSee all References, Lo et al., 2011xLo et al., 2011Lo, C.W., Lai, Y.K., Liu, Y.T., Gallo, R.L., and Huang, C.M. Staphylococcus aureus hijacks a skin commensal to intensify its virulence: immunization targeting beta-hemolysin and CAMP factor. J Invest Dermatol. 2011; 131: 401–409

Abstract | Full Text | Full Text PDF | PubMed | Scopus (30) | Google ScholarSee all References, Nakatsuji et al., 2011xNakatsuji et al., 2011Nakatsuji, T., Tang, D.C., Zhang, L., Gallo, R.L., and Huang, C.M. Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment. PloS One. 2011; 6: e14797

Crossref | PubMed | Scopus (50) | Google ScholarSee all References). With the goal of vaccination approaches for acne treatments in the future, we decided to further assess the relationship of P. acnes in humans. These experiments included vaccination with a clinical aluminum adjuvant, consideration of the status of P. acnes as commensal bacteria in humans, and evaluation of the effectiveness of antibodies to CAMP factor using human ex vivo acne explants.

Results

Up-regulation of P. acnes CAMP factor under anaerobic conditions

P. acnes is an anaerobic microbe endemic in humans. The genome of P. acnes has revealed genes for many virulence factors involved in degrading host tissues and genes involved in the induction of inflammation (Bruggemann, 2005xBruggemann, 2005Bruggemann, H. Insights in the pathogenic potential of Propionibacterium acnes from its complete genome. Semin Cutan Med Surg. 2005; 24: 67–72

Crossref | PubMed | Scopus (78) | Google ScholarSee all References, Schaefer et al., 1980xSchaefer et al., 1980Schaefer, H., Schalla, W., Hagele, W., and Stuttgen, G. P. acnes and the chemistry of sebum. Acta Derm Venereol Suppl (Stockh). Suppl. 1980; 89: 23–26

Google ScholarSee all References). It has been shown that the production of virulence factors of P. acnes was markedly increased in the absence of oxygen (Cove et al., 1983xCove et al., 1983Cove, J.H., Holland, K.T., and Cunliffe, W.J. Effects of oxygen concentration on biomass production, maximum specific growth rate and extracellular enzyme production by three species of cutaneous Propionibacteria grown in continuous culture. J Gen Microbiol. 1983; 129: 3327–3334

PubMed | Google ScholarSee all References). These virulence factors, which are either secreted from P. acnes or anchored in the cell wall, stimulate adjacent host cells and trigger inflammation and cell damage. In an effort to identify the virulence factors that are highly expressed under anaerobic conditions, a quantitative proteomic analysis was performed on P. acnes in the presence or absence of oxygen. Changes in the abundances of individual protein species in P. acnes grown with/without oxygen were analyzed using the non–gel-based isotope-coded protein label method (Schmidt et al., 2005xSchmidt et al., 2005Schmidt, A., Kellermann, J., and Lottspeich, F. A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics. 2005; 5: 4–15

Crossref | PubMed | Scopus (388) | Google ScholarSee all References). A total of 342 proteins were sequenced by nano-liquid chromatography linear ion trap quadrupole tandem mass spectrometry. Of these, 23 proteins were identified as up- or down-regulated under anaerobic or aerobic conditions (see Supplementary Table S1 online). By quantification of an internal peptide, DLLKAAFDLR, of CAMP factor (accession number: WP_002518322), we found that the anaerobic culture led to an approximately 1.5-fold increase in the expression of CAMP factor (Figure 1a). The internal peptide was sequenced and is presented in Figure 1b.

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Figure 1

Up-regulation of Propionibacterium acnes CAMP factor under anaerobic conditions and the essential role of CAMP factor in P. acnes-induced proinflammatory MIP-2 cytokine and bacterial colonization. (a) P. acnes ATCC 6919 was grown under aerobic and anaerobic conditions. Lysates (1 mg) of P. acnes (ATCC 6919) from aerobic and anaerobic growth were labeled with 12C6-Nic-NHS and 13C6-Nic-NHS, respectively. After mixing 12C6-Nic-NHS– and 13C6-Nic-NHS–labeled samples, the mixture was subjected to an LC-LTQ mass spectrometer. CAMP factor with a mass difference of 3 Da per labeled site in mass spectra is shown. (b) A peptide, DLLKAAFDLR, was sequenced and assigned to the internal peptide of CAMP factor. (c) The right ears of Institute of Cancer Research (London, UK) mice were injected intradermally with a wild-type or CAMP factor-mutant P. acnes strain (Δcamp2) (107 CFU in 20 μl PBS). Injection of 20 μl PBS into left ears served as a control. (d) Three days after injection, the proinflammatory MIP-2 cytokines were quantified by ELISA. (e, f) The bacterial colonies (CFUs) were enumerated by plating serial dilutions (1:101–1:105) of the ear homogenate on an agar plate. Error bars represent mean ± standard deviation of five mice. ∗P < 0.05, ∗∗∗P < 0.001 by Student t test. CAMP, Christie-Atkins-Munch-Petersen; CFU, colony-forming unit; LC-LTQ, liquid chromatography linear ion trap quadrupole; Nic-NHS, N-nicotinoyloxy-succinimide; PBS, phosphate buffered saline; WT, wild type.

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The essential role of CAMP factor in P. acnes-induced inflammation

Although results in our previous publication (Huang et al., 2008xHuang et al., 2008Huang, C.P., Liu, Y.T., Nakatsuji, T., Shi, Y., Gallo, R.R., Lin, S.B. et al. Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes. Proteomics Clin Appl. 2008; 2: 1234–1245

Crossref | PubMed | Scopus (4) | Google ScholarSee all References) showed that sera from CAMP factor-immunized mice were able to neutralize the virulence of CAMP factor, a knock-out mutant (Δcamp) of P. acnes CAMP factor (Sorensen et al., 2010xSorensen et al., 2010Sorensen, M., Mak, T.N., Hurwitz, R., Ogilvie, L.A., Mollenkopf, H.J., Meyer, T.F. et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants. J Microbiol Methods. 2010; 83: 211–216

Crossref | PubMed | Scopus (17) | Google ScholarSee all References) was used to verify the essential role of CAMP factor in P. acnes-induced inflammation. We first examined the in vitro growth of Δcamp. The Δcamp and wild-type P. acnes (266; 1-1a, ST18) were grown in Reinforced Clostridium Medium (BD, Sparks, Baltimore, MD) in a 96-well microplate under anaerobic conditions. The optical density600 was read every day. We found that both Δcamp and wild-type P. acnes bacteria have the same growth rates, indicating that mutation of CAMP factor did not affect its growth in vitro (data not shown). To investigate the essential role of CAMP factor in P. acnes-induced inflammation, ears of Institute of Cancer Research (Harlan Labs, Placentia, CA) mice were injected intradermally with live Δcamp (107 colony-forming units in 20 μl phosphate buffered saline) or wild-type P. acnes. Injection of the same amount of phosphate buffered saline served as a control. Compared with injection with wild-type P. acnes, injection of mice with Δcamp caused less swelling (data not shown), redness (Figure 1c), and secretion of MIP-2 (Figure 1d). Furthermore, the mouse ear injected with Δcamp resulted in significantly less P. acnes growth than those injected with wild-type P. acnes (Figure 1e and f). These results show the requirement of CAMP factor for P. acnes-induced inflammation.

Detectable but low titers of antibodies to CAMP factor in acne patients

Previous findings have shown that acne patients produce a higher titer of antibody to P. acnes bacteria than individuals without acne (Basal et al., 2004xBasal et al., 2004Basal, E., Jain, A., and Kaushal, G.P. Antibody response to crude cell lysate of Propionibacterium acnes and induction of pro-inflammatory cytokines in patients with acne and normal healthy subjects. J Microbiol. 2004; 42: 117–125

PubMed | Google ScholarSee all References). Here, we conducted ELISA to determine the titer of antibody to P. acnes CAMP factor. As shown in Figure 2a, the titer (1:400) of antibody to CAMP factor in healthy individuals was low but still detectable and was also detected in acne patients at a titer of 1:800. To determine whether antibodies to CAMP factor in humans at these low titers have the ability to reduce the cytotoxicity of P. acnes, murine RAW264.7 macrophage cells were incubated with P. acnes ATCC 6919 overnight in the presence of different titers of inactivated sera 2.5% (volume/volume) obtained from healthy individuals and acne patients with titers of antibody to CAMP factor at 1:400 and 1:800. Cells were also incubated with P. acnes in the presence of inactivated sera collected from mice vaccinated with UV-inactivated E. coli overexpressing CAMP factor with titers of antibodies to CAMP factor ranging from 1:500 to 1:7,821,500. Cells incubated with P. acnes and inactivated sera obtained from mice vaccinated with UV-inactivated E. coli overexpressing green fluorescent protein (GFP) served as a negative control. As shown in Figure 2b, human sera with a titer of antibody to CAMP factor at 1:800 were unable to abrogate the P. acnes-induced cell death. Cell death was significantly inhibited (>24%) only when the anti-CAMP factor mouse sera with titers greater than 1:62,500 were used, suggesting that the low titers of antibodies to CAMP factor produced in humans are not sufficient to reduce the cytotoxicity of P. acnes.

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Figure 2

The detectable antibodies to CAMP factor in acne patients and reduction of cytotoxicity of Propionibacterium acnes to macrophages by high titers of antibodies generated in the Escherichia coli overexpressing CAMP factor-vaccinated mice. (a) Sera from individuals without acne (normal control) and acne patients (acne) were assayed by ELISA for antibody to CAMP factor. Antibody (IgG) titers of were determined by using recombinant CAMP factor or GFP as a capture antigen for coating onto a 96-well ELISA plate. The endpoint was defined as the dilution of serum on CAMP factor-coated wells producing the same OD570–450 as a 1/100 dilution of serum on GFP-coated wells. Sera negative at the lowest dilution tested were assigned endpoint titers of 100. The data were presented as geometric mean endpoint ELISA titers. (b) For reduction of cytotoxicity of P. acnes, RAW264.7 macrophage cells were incubated with P. acnes ATCC 6919 along with different titers (as indicated) of inactivated sera obtained from individuals without acne, acne patients, or mice vaccinated with E. coli overexpressing CAMP factor or GFP for 18 hours. The cytotoxicity of P. acnes was determined by an ACP assay and calculated as the percentage of cell death. ∗P < 0.05, ∗∗P < 0.01 by Student t test. ACP, acid phosphatase; CAMP, Christie-Atkins-Munch-Petersen; GFP, green fluorescent protein; OD, optical density.

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Protective immunity against P. acnes conferred by vaccination with recombinant CAMP factor

Institute of Cancer Research mice were subcutaneously vaccinated with recombinant CAMP factor or green fluorescent protein (50 μg) as antigens without addition of an exogenous adjuvant and boosted with the same amount antigen 2 weeks after first vaccination. The production of antibody against CAMP factor was detected by ELISA assay 5 weeks after first vaccination. Antibodies to CAMP factor were produced in mice vaccinated with recombinant CAMP factor alone at a titer of approximately 1:800,000 (Figure 3a). The result indicates that P. acnes CAMP factor is immunogenic by itself. However, because of the possibility of development of a human vaccine, aluminum, a common adjuvant for human use (Lindblad, 2004xLindblad, 2004Lindblad, E.B. Aluminium compounds for use in vaccines. Immunol Cell Biol. 2004; 82: 497–505

Crossref | PubMed | Scopus (315) | Google ScholarSee all References), was selected as an adjuvant. With the addition of aluminium, there was more than a 4-fold increase in the titers of antibodies to CAMP factor, when mice were vaccinated with recombinant CAMP factor (50 μg) along with 2% aluminum.

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Figure 3

The protective immunity in mice vaccinated with recombinant CAMP factor. Institute of Cancer Research mice were subcutaneously vaccinated with 10, 20, or 50 μg recombinant CAMP factor or GFP with or without 2% aluminum (alum) adjuvant in a 2-week interval. (a) Three weeks after vaccination, the titer of antibodies to CAMP factor was measured by ELISA. (b) Propionibacterium acnes (107 CFU/20 μl in PBS) bacteria or PBS (20 μl) was intradermally injected into the right or left ears of GFP- or CAMP factor-vaccinated mice, respectively, 3 weeks after the second booster. Ear thickness (mm) was measured 1 day after bacterial injection. (c) The levels of proinflammatory MIP-2 cytokines in the homogenates of P. acnes- or PBS-injected ears were measured by ELISA. (d) Bacterial colonization (CFUs) in the ears injected with P. acnes or PBS were enumerated by plating serial dilutions (1:101–1:105) of ear homogenate on an agar plate. Three separate experiments with five mice per group were performed. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by Student t test. Alum, aluminum; CAMP, Christie-Atkins-Munch-Petersen; CFU, colony forming unit; GFP, green fluorescent protein; PBS, phosphate buffered saline.

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To determine if CAMP factor vaccination can decrease P. acnes-induced inflammation, the right ear of each vaccinated mouse was intradermally injected with 20 μl of P. acnes (107 colony-forming units), and the left ear was injected with 20 μl of phosphate buffered saline as a control (Figure 3b) 5 weeks after first vaccination. Compared with mice immunized with green fluorescent protein, mice vaccinated with recombinant CAMP factor alone without aluminum decreased P. acnes-induced erythema and ear thickness (Figure 3b). In addition, vaccination with CAMP factor alone markedly reduced the production of proinflammatory MIP-2 cytokine (Figure 3c) and led to one log10 reduction in P. acnes colonization in ear skin (Figure 3d). When the titers of antibodies to CAMP factor were augmented by 2% aluminum, the reduction in ear thickness and MIP-2 production (5,109 ± 212.1 to 511 ± 5.9 pg/ml) was considerably enhanced (Figure 3b and c), and two log10 reduction in P. acnes colonization in ear skin was detected (Figure 3d). The titers of antibodies to CAMP factor in mice vaccinated with 10 or 20 μg recombinant CAMP factor plus 2% aluminum can reach the levels at 1:512,500 and 1:1,562,500, respectively (Figure 3a). In the presence of aluminum, vaccination of 10 or 20 μg CAMP factor versus green fluorescent protein conferred effective protection against P. acnes in terms of suppression of the production of the MIP-2 cytokine and colonization of P. acnes in ear skin (see Supplementary Figure S1 online). These results clearly show that vaccination with P. acnes CAMP factor elicited protective immunity against P. acnes.

The presence of P. acnes CAMP factor in acne lesions

The immunity in mouse ears may not completely recapitulate that in human acne lesions. Human acne explants provide the closest laboratory model attainable to the in vivo environment in terms of local immune response and fidelity to human physiology (Ng et al., 2009xNg et al., 2009Ng, K.W., Pearton, M., Coulman, S., Anstey, A., Gateley, C., Morrissey, A. et al. Development of an ex vivo human skin model for intradermal vaccination: tissue viability and Langerhans cell behaviour. Vaccine. 2009; 27: 5948–5955

Crossref | PubMed | Scopus (20) | Google ScholarSee all References). Punch biopsy samples of nonlesional and lesional (2–14 days after onset of inflammatory papules) back skin of patients with acne vulgaris were obtained (see Supplementary Figure S2 online). Acne lesions were used to establish the ex vivo acne explants, which develop open lesions with thickened and inflamed epidermal layers (Figure 4a–c ). We first examined whether CAMP factor is present within acne lesions. After homogenization, total cellular RNA was extracted for reverse transcription quantitative PCR analysis (Figure 4d), and the supernatants were used for ELISA assay (Figure 4e). Both the mRNA and protein expressions of P. acnes CAMP factor in acne lesions were substantially higher than those in nonlesional skin. Immunohistochemical staining using a monoclonal antibody (mAb) to the P. acnes CAMP factor was performed to determine the distribution of CAMP factor in nonlesional and acne lesional skin. The CAMP factor can be detected in hair follicles and sebaceous glands in nonlesional skin (Figure 4g–j), whereas it was distributed everywhere in an acne lesion (Figure 4l–o).

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Figure 4

The presence of Propionibacterium acnes CAMP factor in acne lesions. (a) Images of the biopsy sample (4 × 4 × 8 mm) of lesional back skin of patients with acne vulgaris within 2–14 days of onset of inflammatory papules. An open acne lesion, a hair shaft, and epidermal and dermal layers were indicated. The lesion was illustrated in a top-view picture of an acne biopsy sample with the epidermis facing up. Scale bar = 4 mm. Hematoxylin and eosin staining showed that the epidermis (b) in acne lesional skin was thicker than that (c) in nonlesional skin. Scale bar = 4 mm. The mRNA expression normalized to (d) 16S rRNA of P. acnes and (e) protein levels of CAMP factor detected by reverse transcription quantitative PCR and ELISA, respectively, in acne lesional skin (AL) were higher than those in nonlesional skin (NAL). ∗P < 0.05 by Student t test. High-magnification photos of selected areas in (f) nonlesional skin and (k) acne lesional skin are shown in g–j and l–o, respectively. (g, l) CAMP factor (red stains) was detectable in the hair follicle and was expressed at a higher abundance in (h, m) sebaceous glands, (i, n) epidermis, and (j, o) dermis in acne lesional skin than in nonlesional skin. Scale bars = 500 μm in f and k and 100 μm in g and l. CAMP, Christie-Atkins-Munch-Petersen.

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Evaluation of efficacy of CAMP factor mAb in ex vivo acne models

To measure the expression of proinflammatory cytokines in acne vulgaris, 11 cytokines related to T helper (Th) type 1 (IFN-γ, tumor necrosis factor-β, and IL-8 [a human counterpart of murine MIP-2]), Th2 (IL-4, IL-9, IL-10, and IL-13), and Th17 (IL-1α, IL-1β, IL-17A, and transforming growth factor [TGF]-β1) were quantified in nonlesional and acne lesional skin in acne patients. As shown in Figure 5a, the mRNA expression levels of eight cytokines (IFN-γ, tumor necrosis factor-β, IL-8, IL-4, IL-10, IL-1α, IL-1β, and IL-17A) in acne lesional skin were significantly higher than those in nonlesional skin. The mRNA expressions of IL-8 and IL-1β normalized to those of GAPDH were extremely high in acne lesional skin. The protein levels of IL-8 (4,474.0 ± 901.9 pg/mg) and IL-1β (361.2 ± 93.0 pg/mg) in acne lesional skin were noticeably higher than those in nonlesional skin in acne patients and normal skin in healthy individuals (Figure 5b and c).

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Figure 5

Reduction of Propionibacterium acnes-induced inflammation by monoclonal antibody to CAMP factor. Human skin was obtained by taking 4-mm punch biopsy samples from healthy individuals (NC), nonlesional skin (NAL), and acne lesional skin in patients with acne vulgaris(AL). (a) The mRNA expressions of 11 Th1/Th2/Th17-related proinflammatory cytokines normalized to GAPDH in nonlesional and acne lesional skin were examined by reverse transcription quantitative PCR. (b, c) The protein levels of IL-8 and IL-1β in the skin of healthy individuals and nonlesional and acne lesional skin of acne patients were measured by ELISA. (d, e) The nonlesional and acne lesional skin were cut in half, and half was incubated with monoclonal antibody (IgG1) to P. acnes CAMP factor for 24 hours in antibiotic-free the EpiLife keratinocyte medium (Thermo Fisher Scientific, Waltham, MA). The other half was incubated with HBsAg monoclonal antibody as a control (C). The protein levels of IL-8 and IL-1β in skins after monoclonal antibody incubation were quantified by ELISA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 by Student t test. CAMP, Christie-Atkins-Munch-Petersen; HBsAg, hepatitis B surface antigen; n.s., not significant; TGF, transforming growth factor; Th, T helper.

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Incubation of acne ex vivo explants with the mAb to CAMP factor permits the mAb direct access to secreted CAMP factor in acne lesions. The method also allows us to quantify the proinflammatory cytokines released from immune or skin cells in ex vivo explants, predicting the feasibility of immunotherapy using CAMP factor vaccination for the treatment of acne vulgaris in humans and confirming the role of P. acnes CAMP factor in the pathogenesis of acne vulgaris.

In a Western blot analysis, we found that the mAb to CAMP factor specifically recognized P. acnes CAMP factor 2 (data not shown). Punch biopsy samples of nonlesional and lesional skin from the backs of patients with acne vulgaris were collected as shown in Figures 4a–e. To establish the ex vivo skin explants (Ng et al., 2009xNg et al., 2009Ng, K.W., Pearton, M., Coulman, S., Anstey, A., Gateley, C., Morrissey, A. et al. Development of an ex vivo human skin model for intradermal vaccination: tissue viability and Langerhans cell behaviour. Vaccine. 2009; 27: 5948–5955

Crossref | PubMed | Scopus (20) | Google ScholarSee all References), punch skin biopsy samples were fully covered by antibiotic-free media with the epidermal layer side facing up. The ex vivo explants derived from acne lesional and nonlesional skin were incubated with the mAb to CAMP factor for 24 hours. Incubation of ex vivo explants with the mAb to hepatitis B surface antigen served as a control. As shown in Figure 5d and e , the mAb to CAMP factor exhibited the neutralizing capability to attenuate the production of both IL-8 and IL-1β in ex vivo explants obtained from acne lesional skins. Because sebocytes, key cells in a pilosebaceous unit, express high levels of IL-6, we measured IL-6 levels in ex vivo explants and a three-dimensional culture of SZ95 sebocyte cells. As shown in Supplementary Figures 3 and 4 online, the mAb to CAMP factor efficiently reduced the levels of IL-6 in ex vivo acne explants and P. acnes-induced IL-6 secretions from sebocyte grown in a three-dimensional culture. These results validate the efficacy of the mAb to P. acnes CAMP factor in the suppression of inflammation in acne vulgaris.

Discussion

Although P. acnes CAMP factor triggered cell death (Huang et al., 2008xHuang et al., 2008Huang, C.P., Liu, Y.T., Nakatsuji, T., Shi, Y., Gallo, R.R., Lin, S.B. et al. Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes. Proteomics Clin Appl. 2008; 2: 1234–1245

Crossref | PubMed | Scopus (4) | Google ScholarSee all References), it has been described that cell death is a key factor that induces the proinflammatory and anti-inflammatory responses in bacterial infection (Pinheiro da Silva and Nizet, 2009xPinheiro da Silva and Nizet, 2009Pinheiro da Silva, F. and Nizet, V. Cell death during sepsis: integration of disintegration in the inflammatory response to overwhelming infection. Apoptosis. 2009; 14: 509–521

Crossref | PubMed | Scopus (43) | Google ScholarSee all References). The CAMP factor acts as a pore-forming toxin that may induce cytolysis and cytokine secretion via activation of the inflammasome (Averette et al., 2009xAverette et al., 2009Averette, K.M., Pratt, M.R., Yang, Y., Bassilian, S., Whitelegge, J.P., Loo, J.A. et al. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent. PloS One. 2009; 4: e7913

Crossref | PubMed | Scopus (37) | Google ScholarSee all References). Toxin-induced membrane permeability results in a decrease in cytoplasmic potassium, which triggers the formation of the inflammasome, leading to caspase-1 activation (Averette et al., 2009xAverette et al., 2009Averette, K.M., Pratt, M.R., Yang, Y., Bassilian, S., Whitelegge, J.P., Loo, J.A. et al. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent. PloS One. 2009; 4: e7913

Crossref | PubMed | Scopus (37) | Google ScholarSee all References). These findings indicate that inflammation and cytolysis occur together in infected tissues. The granulomatous inflammation induced by P. acnes (Figures 1c and 3b) may result from a lesion of epithelioid macrophages surrounded by a lymphocyte cuff (Carpinteiro et al., 2008xCarpinteiro et al., 2008Carpinteiro, A., Dumitru, C., Schenck, M., and Gulbins, E. Ceramide-induced cell death in malignant cells. Cancer Lett. 2008; 264: 1–10

Abstract | Full Text | Full Text PDF | PubMed | Scopus (86) | Google ScholarSee all References).

Although ex vivo skin explants were used for incubation of mAb for 24 hours the same day they were collected (Figure 5), it has been documented that skin explants exhibit rapid sebaceous gland degradation and disruption of epidermal and dermal integrity after 6 days of ex vivo maintenance (Nikolakis et al., 2015xNikolakis et al., 2015Nikolakis, G., Seltmann, H., Hossini, A.M., Makrantonaki, E., Knolle, J., and Zouboulis, C.C. Ex vivo human skin and SZ95 sebocytes exhibit a homoeostatic interaction in a novel coculture contact model. Exp Dermatol. 2015; 24: 497–502

Crossref | PubMed | Scopus (7) | Google ScholarSee all References). Besides a three-dimensional culture of sebocyte cells (see Supplementary Figure S4) with a sebaceous-like phenotype (Barrault et al., 2012xBarrault et al., 2012Barrault, C., Dichamp, I., Garnier, J., Pedretti, N., Juchaux, F., Deguercy, A. et al. Immortalized sebocytes can spontaneously differentiate into a sebaceous-like phenotype when cultured as a 3D epithelium. Exp Dermatol. 2012; 21: 314–316

Crossref | PubMed | Scopus (7) | Google ScholarSee all References), other skin models with insertion of complete pilosebaceous unit (Michel et al., 1999xMichel et al., 1999Michel, M., L’Heureux, N., Pouliot, R., Xu, W., Auger, F.A., and Germain, L. Characterization of a new tissue-engineered human skin equivalent with hair. In Vitro Cell Dev Biol Anim. 1999; 35: 318–326

Crossref | PubMed | Scopus (150) | Google ScholarSee all References) will be used to examine the efficacy of mAb to CAMP factor for suppression of P. acnes-induced secretion of proinflammatory cytokines. CAMP factor is a protein named by its biological function of CAMP reaction. The sequence homology of P. acnes CAMP factor to other CAMP factors among different bacteria is low. It has been reported that P. acnes types IA, IB, or II bacteria encode five different CAMP factor (CAMP factors 1–5) genes (Valanne et al., 2005xValanne et al., 2005Valanne, S., McDowell, A., Ramage, G., Tunney, M.M., Einarsson, G.G., O’Hagan, S. et al. CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II. Microbiology. 2005; 151: 1369–1379

Crossref | PubMed | Scopus (78) | Google ScholarSee all References). The CAMP factor 2 of P. acnes in this study shares approximately 36% to 49% amino acid sequence identity with other CAMP factor homologues in P. acnes. Previous studies via proteomics have shown that CAMP factor 2 is secreted from all five human-isolated P. acnes strains (Sorensen et al., 2010xSorensen et al., 2010Sorensen, M., Mak, T.N., Hurwitz, R., Ogilvie, L.A., Mollenkopf, H.J., Meyer, T.F. et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants. J Microbiol Methods. 2010; 83: 211–216

Crossref | PubMed | Scopus (17) | Google ScholarSee all References). Only CAMP factor 2 and 4 are detectable in the secretion of the P. acnes (KPA171202) strain (Sorensen et al., 2010xSorensen et al., 2010Sorensen, M., Mak, T.N., Hurwitz, R., Ogilvie, L.A., Mollenkopf, H.J., Meyer, T.F. et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants. J Microbiol Methods. 2010; 83: 211–216

Crossref | PubMed | Scopus (17) | Google ScholarSee all References). Also, it has been shown that CAMP factor 2, but not 4, is the major active co-hemolytic factor of P. acnes (Sorensen et al., 2010xSorensen et al., 2010Sorensen, M., Mak, T.N., Hurwitz, R., Ogilvie, L.A., Mollenkopf, H.J., Meyer, T.F. et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants. J Microbiol Methods. 2010; 83: 211–216

Crossref | PubMed | Scopus (17) | Google ScholarSee all References). Here, we conclude that CAMP factor 2 is essential for P. acnes-induced inflammation because mutation of CAMP factor 2 in P. acnes (Figure 1c–f) abolished the inflammatory response caused by P. acnes. Antibodies produced by CAMP factor-vaccinated mice target secretory CAMP factor instead of bacterial particles. As shown in Figure 1e and f, the bacterial load in the mouse ear was significantly reduced, implying that that CAMP factor vaccination may disarm bacteria that could be eliminated locally and naturally by host immune systems. P. acnes CAMP factor was differentially expressed in distinct types of inflammatory acne vulgaris (Quanico et al., 2017xQuanico et al., 2017Quanico, J., Gimeno, J.P., Nadal-Wollbold, F., Casas, C., Alvarez-Georges, S., Redoules, D. et al. Proteomic and transcriptomic investigation of acne vulgaris microcystic and papular lesions: insights in the understanding of its pathophysiology. Biochim Biophys Acta. 2017; 1861: 652–663

Crossref | PubMed | Scopus (1) | Google ScholarSee all References). There is no conclusive evidence showing the correlation of CAMP factor expression with acne severity and the role of CAMP factor in the pathogenesis of acne vulgaris. Future studies will include (i) examining whether CAMP factor 2-targeted vaccines can diminish the inflammation caused by various P. acnes subtypes, (ii) determining if antibodies generated by CAMP factor 2 vaccination are cross-reactive with other CAMP factor homologues, and (iii) creating a multivalent vaccine composed of a mixture of various recombinant CAMP factor homologues because vaccination of mice with CAMP factor 2 does not fully suppress P. acnes-induced inflammation (Figure 3c).

The antibodies to CAMP factor are detectable, although low, in human blood (Figure 2a). It has been shown that pre-existing antibodies, acting in concert with complement, are endogenous adjuvants for the generation of protective CD8+ T cells after vaccination against visceral leishmaniasis (Stager et al., 2003xStager et al., 2003Stager, S., Alexander, J., Kirby, A.C., Botto, M., Rooijen, N.V., Smith, D.F. et al. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses. Nat Med. 2003; 9: 1287–1292

Crossref | PubMed | Scopus (143) | Google ScholarSee all References). The pre-existing antibodies to respiratory syncytial virus are present in low titers in human sera and are a short-lived antibody (Welliver et al., 1980xWelliver et al., 1980Welliver, R.C., Kaul, T.N., Putnam, T.I., Sun, M., Riddlesberger, K., and Ogra, P.L. The antibody response to primary and secondary infection with respiratory syncytial virus: kinetics of class-specific responses. J Pediatr. 1980; 96: 808–813

Abstract | Full Text PDF | PubMed | Scopus (89) | Google ScholarSee all References). Because of relatively rapid decay of the pre-existing antibodies after respiratory syncytial virus infection, respiratory syncytial virus vaccines may be best given more frequently before the expected exposure. Because vaccines that contain toxic antigens are less acceptable candidates for human application, future work will need to include engineering a nontoxic yet highly immunogenic vaccine by either a chemical or genetic approach (Sheng and Kong, 2012xSheng and Kong, 2012Sheng, S. and Kong, F. Separation of antigens and antibodies by immunoaffinity chromatography. Pharm Biol. 2012; 50: 1038–1044

Crossref | PubMed | Scopus (10) | Google ScholarSee all References) to achieve clinical utility.

Although aluminum is a known Th2 adjuvant, it has been shown to potentiate either the Th1 or Th2 immune response (Averette et al., 2009xAverette et al., 2009Averette, K.M., Pratt, M.R., Yang, Y., Bassilian, S., Whitelegge, J.P., Loo, J.A. et al. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent. PloS One. 2009; 4: e7913

Crossref | PubMed | Scopus (37) | Google ScholarSee all References). Thus, it is worthwhile to determine which T-cell populations (Th1, Th2, and/or Th17) are activated by vaccination with P. acnes CAMP factor in the presence or absence of aluminum. P. acnes bacteria stimulate skin cells to produce cytokines, leading to inflammatory acne disease (Contassot and French, 2014xContassot and French, 2014Contassot, E. and French, L.E. New insights into acne pathogenesis: Propionibacterium acnes activates the inflammasome. J Invest Dermatol. 2014; 134: 310–313

Abstract | Full Text | Full Text PDF | PubMed | Scopus (27) | Google ScholarSee all References, Kurokawa et al., 2009xKurokawa et al., 2009Kurokawa, I., Danby, F.W., Ju, Q., Wang, X., Xiang, L.F., Xia, L. et al. New developments in our understanding of acne pathogenesis and treatment. Exp Dermatol. 2009; 18: 821–832

Crossref | PubMed | Scopus (295) | Google ScholarSee all References, Nagy et al., 2006xNagy et al., 2006Nagy, I., Pivarcsi, A., Kis, K., Koreck, A., Bodai, L., McDowell, A. et al. Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes. Microbes Infect. 2006; 8: 2195–2205

Crossref | PubMed | Scopus (195) | Google ScholarSee all References, Nakatsuji et al., 2011xNakatsuji et al., 2011Nakatsuji, T., Tang, D.C., Zhang, L., Gallo, R.L., and Huang, C.M. Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment. PloS One. 2011; 6: e14797

Crossref | PubMed | Scopus (50) | Google ScholarSee all References, Qin et al., 2014xQin et al., 2014Qin, M., Pirouz, A., Kim, M.H., Krutzik, S.R., Garban, H.J., and Kim, J. Propionibacterium acnes induces IL-1beta secretion via the NLRP3 inflammasome in human monocytes. J Invest Dermatol. 2014; 134: 381–388

Abstract | Full Text | Full Text PDF | PubMed | Scopus (71) | Google ScholarSee all References). IL-17, expressed in acne lesions (Agak et al., 2014xAgak et al., 2014Agak, G.W., Qin, M., Nobe, J., Kim, M.H., Krutzik, S.R., Tristan, G.R. et al. Propionibacterium acnes induces an IL-17 response in acne vulgaris that is regulated by vitamin A and vitamin D. J Invest Dermatol. 2014; 134: 366–373

Abstract | Full Text | Full Text PDF | PubMed | Scopus (67) | Google ScholarSee all References), can be induced by P. acnes bacteria in peripheral blood mononuclear cells. Recently, Th1- (IFN-γ and IL-8), Th2- (IL-10), and Th17- (IL-1β and TGF-β) related cytokines were strongly up-regulated in acne lesions (Kelhala et al., 2014xKelhala et al., 2014Kelhala, H.L., Palatsi, R., Fyhrquist, N., Lehtimaki, S., Vayrynen, J.P., Kallioinen, M. et al. IL-17/Th17 pathway is activated in acne lesions. PloS One. 2014; 9: e105238

Crossref | PubMed | Scopus (34) | Google ScholarSee all References). This evidence supports our findings in Figure 5a–c showing that the levels of IL-8 and IL-1β mRNA and protein expression in acne lesions were higher than those in nonlesional skin. P. acnes bacteria play a key role in the development of inflammatory lesions via induction of secretion of IL-6 and IL-8 by follicular keratinocytes, IL-1β, tumor necrosis factor-α, IL-8, and IL-12 by monocytic cells in a toll-like receptor 2–dependent manner (Bojar and Holland, 2004xBojar and Holland, 2004Bojar, R.A. and Holland, K.T. Acne and Propionibacterium acnes. Clin Dermatol. 2004; 22: 375–379

Abstract | Full Text | Full Text PDF | PubMed | Scopus (130) | Google ScholarSee all References, Kurokawa et al., 2009xKurokawa et al., 2009Kurokawa, I., Danby, F.W., Ju, Q., Wang, X., Xiang, L.F., Xia, L. et al. New developments in our understanding of acne pathogenesis and treatment. Exp Dermatol. 2009; 18: 821–832

Crossref | PubMed | Scopus (295) | Google ScholarSee all References, Leeming et al., 1988xLeeming et al., 1988Leeming, J.P., Holland, K.T., and Cuncliffe, W.J. The microbial colonization of inflamed acne vulgaris lesions. Br J Dermatol. 1988; 118: 203–208

Crossref | PubMed | Scopus (86) | Google ScholarSee all References, Lheure et al., 2016xLheure et al., 2016Lheure, C., Grange, P.A., Ollagnier, G., Morand, P., Desire, N., Sayon, S. et al. TLR-2 recognizes Propionibacterium acnes CAMP factor 1 from highly inflammatory strains. PloS One. 2016; 11: e0167237

Crossref | PubMed | Scopus (10) | Google ScholarSee all References, Vowels et al., 1995xVowels et al., 1995Vowels, B.R., Yang, S., and Leyden, J.J. Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne. Infect Immun. 1995; 63: 3158–3165

PubMed | Google ScholarSee all References) and IL-1β, IL-12, and IL-23 by peripheral blood mononuclear cells (Ng et al., 2009xNg et al., 2009Ng, K.W., Pearton, M., Coulman, S., Anstey, A., Gateley, C., Morrissey, A. et al. Development of an ex vivo human skin model for intradermal vaccination: tissue viability and Langerhans cell behaviour. Vaccine. 2009; 27: 5948–5955

Crossref | PubMed | Scopus (20) | Google ScholarSee all References). In addition, sebocytes secreted IL-8 in response to P. acnes exposure (Nagy et al., 2006xNagy et al., 2006Nagy, I., Pivarcsi, A., Kis, K., Koreck, A., Bodai, L., McDowell, A. et al. Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes. Microbes Infect. 2006; 8: 2195–2205

Crossref | PubMed | Scopus (195) | Google ScholarSee all References). P. acnes bacteria triggered mixed Th17/Th1 responses by inducing the simultaneous secretion of IL-17A and IFN-γ from specific CD4+ T cells (Agak et al., 2014xAgak et al., 2014Agak, G.W., Qin, M., Nobe, J., Kim, M.H., Krutzik, S.R., Tristan, G.R. et al. Propionibacterium acnes induces an IL-17 response in acne vulgaris that is regulated by vitamin A and vitamin D. J Invest Dermatol. 2014; 134: 366–373

Abstract | Full Text | Full Text PDF | PubMed | Scopus (67) | Google ScholarSee all References, Kistowska et al., 2015xKistowska et al., 2015Kistowska, M., Meier, B., Proust, T., Feldmeyer, L., Cozzio, A., Kuendig, T. et al. Propionibacterium acnes promotes Th17 and Th17/Th1 responses in acne patients. J Invest Dermatol. 2015; 135: 110–118

Abstract | Full Text | Full Text PDF | PubMed | Scopus (30) | Google ScholarSee all References). As shown in Figure 5a, the mRNA expressions of eight cytokines were significantly higher in acne lesions compared with those in nonlesional skin in acne patients. Our results are in line with the prior observations showing up-regulated IFN-γ, IL-8, IL-10, IL-1β, and IL-17A in acne lesions examined by reverse transcription quantitative PCR (Bechara et al., 2012xBechara et al., 2012Bechara, F.G., Sand, M., Skrygan, M., Kreuter, A., Altmeyer, P., and Gambichler, T. Acne inversa: evaluating antimicrobial peptides and proteins. Ann Dermatol. 2012; 24: 393–397

Crossref | PubMed | Scopus (30) | Google ScholarSee all References, Kang et al., 2005xKang et al., 2005Kang, S., Cho, S., Chung, J.H., Hammerberg, C., Fisher, G.J., and Voorhees, J.J. Inflammation and extracellular matrix degradation mediated by activated transcription factors nuclear factor-kappaB and activator protein-1 in inflammatory acne lesions in vivo. Am J Pathol. 2005; 166: 1691–1699

Abstract | Full Text | Full Text PDF | PubMed | Scopus (139) | Google ScholarSee all References, Kelhala et al., 2014xKelhala et al., 2014Kelhala, H.L., Palatsi, R., Fyhrquist, N., Lehtimaki, S., Vayrynen, J.P., Kallioinen, M. et al. IL-17/Th17 pathway is activated in acne lesions. PloS One. 2014; 9: e105238

Crossref | PubMed | Scopus (34) | Google ScholarSee all References, Kelhala et al., 2016xKelhala et al., 2016Kelhala, H.L., Fyhrquist, N., Palatsi, R., Lehtimaki, S., Vayrynen, J.P., Kubin, M.E. et al. Isotretinoin treatment reduces acne lesions but not directly lesional acne inflammation. Exp Dermatol. 2016; 25: 477–478

Crossref | PubMed | Scopus (5) | Google ScholarSee all References). However, our results showed no significant difference in the mRNA expression of TGF-β1 between acne lesional and nonlesional skin. TGF-β1 is a well-studied player in the pathogenesis of scarring (Klass et al., 2009xKlass et al., 2009Klass, B.R., Grobbelaar, A.O., and Rolfe, K.J. Transforming growth factor beta1 signalling, wound healing and repair: a multifunctional cytokine with clinical implications for wound repair, a delicate balance. Postgrad Med J. 2009; 85: 9–14

Crossref | PubMed | Scopus (74) | Google ScholarSee all References), an event in the late stages of acne vulgaris. It has been reported that overproduction of TGF-β1 can result in excessive deposition of scar tissue and fibrosis (Ignotz and Massague, 1986xIgnotz and Massague, 1986Ignotz, R.A. and Massague, J. Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem. 1986; 261: 4337–4345

PubMed | Google ScholarSee all References). The acne lesions in this study were isolated from lesions within 2–14 days of onset of inflammatory papules, not in the late stages. This may explain why no difference in TGF-β1 expression between acne lesional and nonlesional skin was noted.

Although effective in decreasing expression of TGF-β1 in skin cells (Carroll et al., 2002xCarroll et al., 2002Carroll, L.A., Hanasono, M.M., Mikulec, A.A., Kita, M., and Koch, R.J. Triamcinolone stimulates bFGF production and inhibits TGF-beta1 production by human dermal fibroblasts. Dermatol Surg. 2002; 28: 704–709

Crossref | PubMed | Scopus (41) | Google ScholarSee all References, Furuzawa-Carballeda et al., 2005xFuruzawa-Carballeda et al., 2005Furuzawa-Carballeda, J., Krotzsch, E., Barile-Fabris, L., Alcala, M., and Espinosa-Morales, R. Subcutaneous administration of collagen-polyvinylpyrrolidone down regulates IL-1beta, TNF-alpha, TGF-beta1, ELAM-1 and VCAM-1 expression in scleroderma skin lesions. Clin Exp Dermatol. 2005; 30: 83–86

Crossref | PubMed | Scopus (14) | Google ScholarSee all References), triamcinolone acetonide has known adverse effects for intralesional injection (Levine and Rasmussen, 1983xLevine and Rasmussen, 1983Levine, R.M. and Rasmussen, J.E. Intralesional corticosteroids in the treatment of nodulocystic acne. Arch Dermatol. 1983; 119: 480–481

Crossref | PubMed | Scopus (41) | Google ScholarSee all References). If the humanized mAb to CAMP factor can be developed, the injection of the mAb to CAMP factor directly into acne lesions potentially can replace triamcinolone acetonide for intralesional therapy against acne vulgaris. The vaccination approaches developed in this study may provide an effective means with protective immunity against acne vulgaris. The vaccination may benefit not only patients with acne vulgaris but also those with other P. acnes-associated diseases including prostate cancers, polymer (device)-associated diseases, sepsis, toxic shock syndrome, endocarditis, osteomyelitis, and various surgery infections (Fassi Fehri et al., 2011xFassi Fehri et al., 2011Fassi Fehri, L., Mak, T.N., Laube, B., Brinkmann, V., Ogilvie, L.A., Mollenkopf, H. et al. Prevalence of Propionibacterium acnes in diseased prostates and its inflammatory and transforming activity on prostate epithelial cells. Int J Med Microbiol. 2011; 301: 69–78

Crossref | PubMed | Scopus (63) | Google ScholarSee all References, Haidar and Najjar, 2010xHaidar and Najjar, 2010Haidar, R. and Najjar, M. Der Boghossian A, Tabbarah Z. Propionibacterium acnes causing delayed postoperative spine infection: review. Scand J Infect Dis. 2010; 42: 405–411

Crossref | PubMed | Scopus (33) | Google ScholarSee all References, Perry and Lambert, 2011xPerry and Lambert, 2011Perry, A. and Lambert, P. Propionibacterium acnes: infection beyond the skin. Expert Rev Anti Infect Ther. 2011; 9: 1149–1156

Crossref | PubMed | Scopus (91) | Google ScholarSee all References, Severi et al., 2010xSeveri et al., 2010Severi, G., Shannon, B.A., Hoang, H.N., Baglietto, L., English, D.R., Hopper, J.L. et al. Plasma concentration of Propionibacterium acnes antibodies and prostate cancer risk: results from an Australian population-based case-control study. Br J Cancer. 2010; 103: 411–415

Crossref | PubMed | Scopus (14) | Google ScholarSee all References).

Materials and Methods

Ethics

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and an approved Institutional Animal Care and Use Committee protocol (no. S10058) at University of California, San Diego. The institutional review board at UCSD approved the procedure of written informed patient consent and sampling of acne biopsy samples under an approved protocol (no. 121230).

Bacterial culture

P. acnes bacteria including ATCC 6919, wild-type P. acnes (266; 1-1a, ST18) strain (Bruggemann et al., 2004xBruggemann et al., 2004Bruggemann, H., Henne, A., Hoster, F., Liesegang, H., Wiezer, A., Strittmatter, A. et al. The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Science. 2004; 305: 671–673

Crossref | PubMed | Scopus (271) | Google ScholarSee all References), and a knock-out mutant of CAMP factor 2 (Δcamp2, [PPA0687]) (Nakatsuji et al., 2008bxNakatsuji et al., 2008bNakatsuji, T., Liu, Y.T., Huang, C.P., Zouboulis, C.C., Gallo, R.L., and Huang, C.M. Vaccination targeting a surface sialidase of P. acnes: implication for new treatment of acne vulgaris. PloS One. 2008; 3: e1551

Crossref | PubMed | Scopus (53) | Google ScholarSee all References, Sorensen et al., 2010xSorensen et al., 2010Sorensen, M., Mak, T.N., Hurwitz, R., Ogilvie, L.A., Mollenkopf, H.J., Meyer, T.F. et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants. J Microbiol Methods. 2010; 83: 211–216

Crossref | PubMed | Scopus (17) | Google ScholarSee all References) were used.

Mass spectrometry, vaccination, in vitro neutralization, and ex vivo acne model

All procedures for the identification of P. acnes CAMP factor by isotope-coded protein labeling (Schmidt et al., 2005xSchmidt et al., 2005Schmidt, A., Kellermann, J., and Lottspeich, F. A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics. 2005; 5: 4–15

Crossref | PubMed | Scopus (388) | Google ScholarSee all References) using light (12C6) and heavy (13C6) forms of N-nicotinoyloxy-succinimide for liquid chromatography linear ion trap quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA), the expression of recombinant CAMP factor, vaccination, quantification of antibody titers, injection of P. acnes into mouse ear, in vitro neutralization, ELISA, bacteria counts, and establishment of ex vivo acne model were performed according to the methods described in Nakatsuji et al. (2008a)xNakatsuji et al., 2008aNakatsuji, T., Liu, Y.T., Huang, C.P., Zoubouis, C.C., Gallo, R.L., and Huang, C.M. Antibodies elicited by inactivated Propionibacterium acnes-based vaccines exert protective immunity and attenuate the IL-8 production in human sebocytes: relevance to therapy for acne vulgaris. J Invest Dermatol. 2008; 128: 2451–2457

Abstract | Full Text | Full Text PDF | PubMed | Scopus (46) | Google ScholarSee all References) and are described in detail in the Supplementary Materials online.

Conflict of Interest

CCZ is owner of an international patent of the human immortalized sebaceous gland cell line SZ95 (WO0046353). The other authors state no conflict of interest.

Acknowledgments

This work was supported by National Institutes of Health (R21 RR022754-01 and 1R41AR056169-01), National Health Research Institutes (NHRI-EX106-10607SI), Ministry of Science and Technology (106-2622-B-008-001-CC1), and a research agreement from Sanofi Pasteur pharmaceutical company.

Author Contributions

CMH conceived the project, designed experiments, supervised the research, and assisted in analyzing data and writing the manuscript. YW performed the experiments and prepared the figures. TRH and YLT wrote the institutional review board protocol, assisted in writing the manuscript, and collected skin biopsy samples. CCZ provided the SZ95 sebocyte cell lines and protocols for cell culture. RLG provided clinical knowledge and designed experiments.

Supplementary Material

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Supplementary Data

References

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© 2018 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
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