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Studies of the human skin microbiome suggest that Propionibacterium acnes strains may contribute differently to skin health and disease. However, the immune phenotype and functions of T helper type 17 (Th17) cells induced by healthy (P H ) versus acne (P A ) skin-associated P. acnes strains are currently unknown. We stimulated peripheral blood mononuclear cells from healthy donors and observed that P A strains induce higher IL-17 levels than P H strains. We next generated P H and P A strain-specific Th17 clones and show that P. acnes strains induce Th17 cells of varied phenotype and function that are stable in the presence of IL-2 and IL-23. Although P H - and P A -specific clones expressed similar levels of LL-37 and DEFB4, only P H -specific clones secreted molecules sufficient to kill P. acnes. Furthermore, electron microscopic studies showed that supernatants derived from activated P H and not P A -specific clones exhibited robust bactericidal activity against P. acnes, and complete breaches in the bacterial cell envelope were observed. This antimicrobial activity was independent of IL-26, because both natural IL-26 released by Th17 clones and rhIL-26 lacked antimicrobial potency against P. acnes. Overall, our data suggest that P. acnes strains may differentially modulate the CD4 + T-cell responses, leading to the generation of Th17 cells that may contribute to either homeostasis or acne pathogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Propionibacterium acnes is a skin commensal bacterium that contributes to the development of acne vulgaris and other infections. Recent work revealed that P. acnes clinical isolates can be classified into distinct phylotypes, several of which have associations with healthy skin or acne. We sought to determine if these phylotypes induce different immunological responses and express protein factors that may contribute to their disease associations. We found that acne-associated P. acnes phylotypes induced 2- to 3-fold higher levels of IFN-γ and IL-17 in peripheral blood mononuclear cells compared with healthy phylotypes. On the other hand, P. acnes phylotypes associated with healthy skin induced 2- to 4-fold higher levels of IL-10. Comparative proteomic analysis of P. acnes phylotypes revealed a differential expression of several proteins, including an adhesion protein that was expressed at least 10-fold higher in acne-associated phylotypes and a cell surface hydrolase expressed in all phylotypes except those associated with healthy skin. Taken together, our data provide insight into how specific P. acnes phylotypes influence immune responses and the pathogenesis of acne., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Propionibacterium acnes induction of IL-1 cytokines through the NLRP3 (NLR, nucleotide oligomerization domain-like receptor) inflammasome was recently highlighted as a dominant etiological factor for acne vulgaris. Therefore, therapeutics targeting both the stimulus and the cascade would be ideal. Nitric oxide (NO), a potent biological messenger, has documented broad-spectrum antimicrobial and immunomodulatory properties. To harness these characteristics to target acne, we used an established nanotechnology capable of generating/releasing NO over time (NO-np). P. acnes was found to be highly sensitive to all concentrations of NO-np tested, although human keratinocyte, monocyte, and embryonic zebra fish assays revealed no cytotoxicity. NO-np significantly suppressed IL-1β, tumor necrosis factor-α (TNF-α), IL-8, and IL-6 from human monocytes, and IL-8 and IL-6 from human keratinocytes, respectively. Importantly, silencing of NLRP3 expression by small interfering RNA did not limit NO-np inhibition of IL-1 β secretion from monocytes, and neither TNF-α nor IL-6 secretion, nor inhibition by NO-np was found to be dependent on this pathway. The observed mechanism by which NO-np impacts IL-1β secretion was through inhibition of caspase-1 and IL-1β gene expression. Together, these data suggest that NO-np can effectively prevent P. acnes-induced inflammation by both clearing the organism and inhibiting microbial stimulation of the innate immune response.

Although antibiotics are a common treatment for acne, the difficulties inherent to effective antimicrobial penetration in sebum and selective antimicrobial action in the skin are compounded by increasing resistance of Propionibacterium acnes clinical isolates. To address these problems, we engineered Pentobra, a peptide-aminoglycoside molecule that has multiple mechanisms of antibacterial action and investigated whether it can be a potential candidate for the treatment of acne. Pentobra combines the potent ribosomal activity of aminoglycosides with the bacteria-selective membrane-permeabilizing abilities of antimicrobial peptides. Pentobra demonstrated potent and selective killing of P. acnes but not against human skin cells in vitro. In direct comparison, Pentobra demonstrated bactericidal activity and drastically outperformed free tobramycin (by 5-7 logs) against multiple P. acnes clinical strains. Moreover, electron microscopic studies showed that Pentobra had robust membrane activity, as treatment with Pentobra killed P. acnes cells and caused leakage of intracellular contents. Pentobra may also have potential anti-inflammatory effects as demonstrated by suppression of some P. acnes-induced chemokines. Importantly, the killing activity was maintained in sebaceous environments as Pentobra was bactericidal against clinical isolates in comedones extracts isolated from human donors. Our work demonstrates that equipping aminoglycosides with selective membrane activity is a viable approach for developing antibiotics against P. acnes that are effective in cutaneous environments.

Acne vulgaris is the most common skin disorder affecting millions of people worldwide and inflammation resulting from the immune response targeting Propionibacterium acnes has a significant role in its pathogenesis. In this study, we have demonstrated that P. acnes is a potent inducer of T helper 17 (Th17) and Th1, but not Th2 responses in human peripheral blood mononuclear cells (PBMCs). P. acnes stimulated expression of key Th17-related genes, including IL-17A, RORα, RORc, IL-17RA, and IL-17RC, and triggered IL-17 secretion from CD4(+), but not from CD8(+) T cells. Supernatants from P. acnes-stimulated PBMCs were sufficient to promote the differentiation of naive CD4(+)CD45RA T cells into Th17 cells. Furthermore, we found that the combination of IL-1β, IL-6, and transforming growth factor-β-neutralizing antibodies completely inhibited P. acnes-induced IL-17 production. Importantly, we showed that IL-17-expressing cells were present in skin biopsies from acne patients but not from normal donors. Finally, vitamin A (all-trans retinoic acid) and vitamin D (1,25-dihydroxyvitamin D3) inhibited P. acnes-induced Th17 differentiation. Together, our data demonstrate that IL-17 is induced by P. acnes and expressed in acne lesions and that both vitamin A and D could be effective tools to modulate Th17-mediated diseases such as acne.

Propionibacterium acnes induction of inflammatory responses is a major etiological factor contributing to the pathogenesis of acne vulgaris. In particular, the IL-1 family of cytokines has a critical role in both initiation of acne lesions and in the inflammatory response in acne. In this study, we demonstrated that human monocytes respond to P. acnes and secrete mature IL-1β partially via the NLRP3-mediated pathway. When monocytes were stimulated with live P. acnes, caspase-1 and caspase-5 gene expression was upregulated; however, IL-1β secretion required only caspase-1 activity. P. acnes induced key inflammasome genes including NLRP1 and NLPR3. Moreover, silencing of NLRP3, but not NLRP1, expression by small interfering RNA attenuated P. acnes-induced IL-1β secretion. The mechanism of P. acnes-induced NLRP3 activation and subsequent IL-1β secretion was found to involve potassium efflux. Finally, in acne lesions, mature caspase-1 and NLRP3 were detected around the pilosebaceous follicles and colocalized with tissue macrophages. Taken together, our results indicate that P. acnes triggers a key inflammatory mediator, IL-1β, via NLRP3 and caspase-1 activation, suggesting a role for inflammasome-mediated inflammation in acne pathogenesis.

The human skin microbiome has important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that although the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne, and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may have roles in determining virulence properties of P. acnes strains, and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain-level analysis of the human microbiome to define the role of commensals in health and disease.

Advances in nanotechnology have demonstrated potential application of nanoparticles (NPs) for effective and targeted drug delivery. Here we investigated the antimicrobial and immunological properties and the feasibility of using NPs to deliver antimicrobial agents to treat a cutaneous pathogen. NPs synthesized with chitosan and alginate demonstrated a direct antimicrobial activity in vitro against Propionibacterium acnes, the bacterium linked to the pathogenesis of acne. By electron microscopy (EM) imaging, chitosan-alginate NPs were found to induce the disruption of the P. acnes cell membrane, providing a mechanism for the bactericidal effect. The chitosan-alginate NPs also exhibited anti-inflammatory properties as they inhibited P. acnes-induced inflammatory cytokine production in human monocytes and keratinocytes. Furthermore, benzoyl peroxide (BP), a commonly used antiacne drug, was effectively encapsulated in the chitosan-alginate NPs and demonstrated superior antimicrobial activity against P. acnes compared with BP alone while demonstrating less toxicity to eukaryotic cells. Together, these data suggest the potential utility of topical delivery of chitosan-alginate NP-encapsulated drug therapy for the treatment of dermatologic conditions with infectious and inflammatory components.

Staphylococcus aureus skin infections represent a significant public health threat because of the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA). As greater understanding of protective immune responses and more effective antimicrobial therapies are needed, a S. aureus skin wound infection model was developed in which full-thickness scalpel cuts on the backs of mice were infected with a bioluminescent S. aureus (methicillin sensitive) or USA300 community-acquired MRSA strain and in vivo imaging was used to noninvasively monitor the bacterial burden. In addition, the infection-induced inflammatory response was quantified using in vivo fluorescence imaging of LysEGFP mice. Using this model, we found that both IL-1α and IL-1β contributed to host defense during a wound infection, whereas IL-1β was more critical during an intradermal S. aureus infection. Furthermore, treatment of a USA300 MRSA skin infection with retapamulin ointment resulted in up to 85-fold reduction in bacterial burden and a 53% decrease in infection-induced inflammation. In contrast, mupirocin ointment had minimal clinical activity against this USA300 strain, resulting in only a 2-fold reduction in bacterial burden. Taken together, this S. aureus wound infection model provides a valuable preclinical screening method to investigate cutaneous immune responses and the efficacy of topical antimicrobial therapies.

Propionibacterium acnes is a critical component in the pathogenesis of acne vulgaris, stimulating the production of various inflammatory mediators, such as cytokines and chemokines, important in the local inflammatory response found in acne. This study explored the role of P. acnes and its ability to induce matrix metalloproteinases (MMPs) in primary human monocytes and how this induction is regulated by retinoids. MMP-1- and MMP-9-expressing cells were present in perifollicular and dermal inflammatory infiltrates within acne lesions, suggesting their role in acne pathogenesis. In vitro, we found that P. acnes induced MMP-9 and MMP-1 mRNA, and the expression of MMP-9, but not of MMP-1, was found to be Toll-like receptor 2-dependent. P. acnes induced the mRNA expression of tissue inhibitors of metalloproteinase (TIMP)-1, the main regulator of MMP-9 and MMP-1. Treatment of monocytes with all-trans retinoic acid (ATRA) significantly decreased baseline MMP-9 expression. Furthermore, co-treatment of monocytes with ATRA and P. acnes inhibited MMP-9 and MMP-1 induction, while augmenting TIMP-1 expression. These data indicate that P. acnes-induced MMPs and TIMPs may be involved in acne pathogenesis and that retinoic acid modulates MMP and TIMP expression, shifting from a matrix-degradative phenotype to a matrix-preserving phenotype.

Propionibacterium acnes is a key therapeutic target in acne, yet this bacterium has become resistant to standard antibiotic agents. We investigated whether the human antimicrobial protein granulysin is a potential candidate for the treatment of acne. Granulysin and synthetic granulysin-derived peptides possessing a helix-loop-helix motif killed P. acnes in vitro. Modification of a helix-loop-helix peptide, 31-50, by substitution of a tryptophan for the valine at amino acid 44 (peptide 31-50v44w) to increase its interaction with bacterial surfaces also increased its antimicrobial activity. Moreover, when synthesized with D- rather than L-type amino acids, this peptide (D-31-50v44w) became less susceptible to degradation by proteases and more effective in killing P. acnes. Granulysin peptides were bactericidal, demonstrating an advantage over standard bacteriostatic antibiotics in their control of P. acnes. Moreover, peptide D-31-50v44w killed P. acnes in isolated human microcomedone preparations. Importantly, peptides 31-50, 31-50v44w, and D-31-50v44w also have potential anti-inflammatory effects, as demonstrated by suppression of P. acnes-stimulated cytokine release. Taken together, these data suggest that granulysin peptides may be useful as topical therapeutic agents, providing alternatives to current acne therapies.

Toll-like receptors (TLR) are crucial players in the innate immune response to microbial invaders. These receptors are expressed on immune cells, such as monocytes, macrophages, dendritic cells, and granulocytes. Importantly, TLR are not only expressed by peripheral blood cells, but their expression has been demonstrated in airway epithelium and skin, important sites of host-pathogen interaction. Host cells expressing TLR are capable of recognizing conserved pathogen-associated molecular patterns, such as lipopolysaccharide and CpG DNA, and their activation triggers signaling pathways that result in the expression of immune response genes and cytokine production. As TLR are instrumental in both launching innate immune responses and influencing adaptive immunity, regulation of TLR expression at sites of disease such as in leprosy, acne, and psoriasis may be important in the pathophysiology of these diseases. Furthermore, since TLR are vital players in infectious and inflammatory diseases, they have been identified as potential therapeutic targets. Indeed, synthetic TLR agonists such as imiquimod have already established utility in treating viral pathogens and skin cancers. In the future, it seems possible there may also be drugs capable of blocking TLR activation and thus TLR-dependent inflammatory responses, providing new treatment options for inflammatory diseases.