The immunomodulatory potential of phage therapy to treat acne: a review on bacterial lysis and immunomodulation

Introduction

Acne vulgaris, or acne, is considered a chronic inflammatory skin disease, which affects many people around the world, principally adolescents (Zouboulis & Bettoli, 2015). The types of acne are classified by severity, and number, and localization of lesions (Table S1) (Moradi Tuchayi et al., 2015). This skin condition compromises the pilosebaceous unit, in which the first inflammatory events occur (Dréno, 2017). During the first stages of the disease, lesions may not present redness and inflammation of local tissue. Nevertheless, there is production of inflammatory mediators in these early lesions (Antiga et al., 2015).

Acne pathogenesis is markedly influenced by four pathogenic determinants: excess sebum production, abnormal keratinization within the follicle, Cutibacterium acnes (formerly, Propionibacterium acnes) colonization in the pilosebaceous duct, and release of inflammatory molecules in the skin (Gollnick, 2015). The precise order in which these pathogenic determinants trigger the inflammatory response is not clear. In fact, acne can be triggered by an alteration of the sebaceous lipid profile (dysseborrhea), stress, irritation, cosmetics, and diet (Dréno, 2017). At molecular level, acne has several factors associated with its pathogenesis, including the action of androgens, pro-inflammatory lipids, and Pathogen-Associated Molecular Patterns (PAMPs) derived from C. acnes acting at different levels of the immune system (Cong et al., 2019). Keratinocytes, sebocytes, and skin-resident immune cells have receptors which are activated by these molecules. This results in the secretion of cytokines, chemokines, and other pro-inflammatory agents (Bhat, Latief & Hassan, 2017).

C. acnes is an anaerobic, Gram-positive bacteria, and a typical colonizer of the skin. This bacterium can interact with other cutaneous skin commensal or pathogenic microbiota (Platsidaki & Dessinioti, 2018). Resident microbiota coexists with skin immunological components and reinforce barrier functions. It is now believed that microorganisms play an important role in skin immunity, maintaining the homeostasis of immune responses, including inhibition of pathogen colonization, inflammatory reactions, and immune cell-derived responses (O’Neill & Gallo, 2018). Dysregulation of the immune response is associated with various skin diseases, including acne, in which there is a disturbance in the relationship skin microbiota and skin immune sentinels (Wang et al., 2020; Abdallah, Mijouin & Pichon, 2017). In the pathogenesis of acne, it has been suggested that the loss of the skin’s microbial diversity activates innate immunity and lead to chronic inflammation (Dréno et al., 2018). C. acnes can interact with other cutaneous skin commensal or pathogenic microbiota (Platsidaki & Dessinioti, 2018). Although this bacterium can be found in healthy skin (Omer, McDowell & Alexeyev, 2017), dysregulation of its growth is associated with acne. C. acnes strains are classified into six phylogenetic groups: IA₁, IA₂, IB, IC, II, and III (Dagnelie et al., 2018). It has been hypothesized that some phylotypes are commensals and contribute to skin health, while others such as phylotype IA₁ can be associated with disease (Dagnelie et al., 2018; Dréno et al., 2018). C. acnes IA₁ strains had been linked to acne given the presence of virulence factors (Cong et al., 2019; Beylot et al., 2014; Dréno et al., 2018). The effect of enriched IA₁ isolates on skin is markedly pro-inflammatory. In contrast, a combination of different phylotypes of C. acnes is what leads to healthy skin (Dagnelie et al., 2019).

To treat acne several drugs has been developed and are currently prescribed. These drugs include different retinoid-derived products, antibiotics, and hormonal antiandrogens for female patients (Zouboulis & Bettoli, 2015; Latter et al., 2019; Xu & Li, 2019). These therapeutic agents are commonly prescribed because of their anti-inflammatory and anti-comedogenic activity, but they present several side effects (Aslam, Fleischer, and Feldman 2015). Oral antibiotics are also prescribed to treat acne, especially because of their anti-inflammatory effects (Farrah & Tan, 2016). However, antibiotic administration has been associated with both adverse events and bacterial resistance (Oudenhoven et al., 2015). Despite their antibacterial effect in C. acnes virulent strains, antibiotics are not species-specific, hence they likely promote bacterial resistance, disturbance of skin microbiota, and a growth of opportunistic pathogens (Liu et al., 2015; Karadag et al., 2020). Given the importance of skin microbiota in the regulation of immunity, an enhancement of beneficial microbial communities should be considered as part of successful acne therapy. Microbial balance has also impact in the resolution of disease flare-ups and comeback of inflammatory lesions (Woo & Sibley, 2020).

In this review, we explore the potential use of bacteriophages as a therapeutic alternative to treat acne, from the interplay between their influence on the skin’s inflammatory response and the pathogenic determinants of acne. To this end, we first examine the inflammatory nature of acne, in particular the immune response against C. acnes. Next, we present the available acne treatments, focusing on the anti-inflammatory properties of these drugs. Finally, we discuss the available information along with the potential of phage therapy in the treatment of acne based on its anti-inflammatory properties. Most published articles about C. acnes phages and the intended use of these phages for phage therapy are focused on bacterial elimination. However, the potential of immunomodulation for these phages has not been completely addressed. Here, we emphasize the inflammatory nature of the disease and the available anti-inflammatory treatment of acne, to approach phage therapy from the immunomodulation perspective. With this article, we expect to point out important gaps in the field and encourage further research directed to investigate immunomodulatory properties of C. acnes phages. Moreover, we intend to raise interest in researchers of other scientific disciplines, including immunologists, dermatologists, and physicians interested in new therapeutic alternatives for inflammatory and infectious diseases.

Survey Methodology

We performed a literature search in four different databases: ePMC, Google Scholar, PubMed, and ScienceDirect, using combinations of the following keywords: “Acne”, “Acne pathology”, “Acne Therapy or Treatment”, “Antibiotic resistance”, “Propionibacterium acnes or Cutibacterium acnes”, “Inflammation or Inflammatory response”, “Immunity or Immune Response”, “Immunomodulation”, “Bacteriophage or Phage”, and “Phage therapy”. Keyword combinations were arranged according to the discussion object for each section. All relevant articles were included and summarized in a database. Following each search, a total of 372 manuscripts were screened. Articles to be included in this review were selected based on the following exclusion criteria: year of publication (2010–2020 for literature reviews, and 2015–2020 for original research articles), number of citations per year compared to other articles found, and quartile of the publishing journal (Quartiles 1 and 2). Applying selection criteria, 212 manuscripts were retrieved and assessed to be included in this review. Articles citing the literature found in our search were also explored. We included for analysis all reported outcomes of phage immunomodulation: anti- and pro-inflammatory results in phage-therapy contexts and in vitro assays of interactions between phages and immune system components.

Inflammatory Response in Acne

Inflammation is the fingerprint of acne pathogenesis, driven by Insulin-like Growth Factor (IGF-1), altered sebum production, and C. acnes. These are the main factors involved in the beginning of the disease, though it is not clear which of them appears first. Then, hyperkeratinization and altered sebum production appear as typical signs of the disease. In addition, hyperkeratinization produces anoxic conditions that confers an optimal environment for C. acnes proliferation (Cong et al., 2019). It has been proposed that early production of inflammatory cytokines is present in sub-clinical lesions, followed by disturbed lipid production and consequent C. acnes propagation (Quanico et al., 2017). Various pro-inflammatory cytokines and chemokines promote chronic inflammation and disease evolution. Later, chronic inflammation in acne can include innate immune cells and CD4+ helper T cells (Th cells) (Mattii et al., 2018; Kistowska et al., 2015). Figure 1 provides an overview of the acne pathogenesis, focusing on the inflammatory nature of the disease, and its details are covered in the following lines.

Anti-inflammatory Acne Treatment

Acne treatment includes the prescription of one or more drugs that can be applied topically or systemic. The different approaches, agents, and emerging treatments have been extensively reviewed elsewhere (Thiboutot et al., 2018; Fox et al., 2016; James, Burkhart & Morrell, 2009), and are summarized in Table 1. Alternative agents have been proposed, including antimicrobial peptides, probiotics, plant extracts, and minerals (Fox et al., 2016), and more research is being undertaken to study the potential of molecules such as sarecycline, tazarotene, minocycline, clascoterone, and cannabidiol (Kircik, 2019). Therapeutic targets are diverse and the use of one or a combination of different types of therapeutic agents depend on acne severity, a patient’s medical record and gender, propensity to scarring, and the physician’s criteria (Thiboutot et al., 2018). More affected individuals are usually treated with a combination of anti-acne agents; for instance, nodular or conglobate acne is treated with a combination of oral isotretinoin (a retinoid) and antibiotics. This treatment can be coupled with anti-androgen drugs in female patients (Gollnick et al., 2016).

Retinoids are a class of drugs derived from vitamin A and one of the most widely used treatments in acne therapy; given their properties as anti-comedogenic, reducers of abnormal keratinization, and anti-inflammatory drugs (Leyden, Stein-Gold & Weiss, 2017). These molecules act by binding on two different receptors (Retinoic acid receptors; Retinoid X Receptors), located in the nucleus, exerting retinoid-inducible gene activation and repression that derives in immunomodulation by decreasing the expression of TLR-2 receptors, reduction of abnormal follicular cell differentiation, prevention of pilosebaceous obstruction, and reduction of sebum production (Chien, 2018). There are four generations of retinoid molecules developed as acne treatments (Table S1) (Chien, 2018). Oral isotretinoin belongs to the first-generation retinoid, and is the most frequently prescribed to treat severe cases of acne (Costa et al., 2018). Isotretinoin has effects on hormone regulation, immunomodulation, and even reduction of C. acnes colonization (Tan et al., 2016; Ryan-Kewley et al., 2017). Nevertheless, the mechanism by which isotretinoin exerts anti-microbial properties on this bacterium or other organisms of the skin microbiota is not clear (Ryan-Kewley et al., 2017).

Treatment effectiveness depends upon isotretinoin concentration, delivery vehicle, combination with other drugs, exposure time, and patient condition (Landis, 2020). However, acne treatment with oral isotretinoin and other retinoids has been associated with several side effects. Isotretinoin most frequently causes xerosis, and facial erythema. Other less frequent adverse events include psychiatric symptoms, ocular lesions, increased cholesterol, and serum triglyceride levels (Brzezinski et al., 2017). Its prescription is not recommended in pregnant women (Costa et al., 2018). One way of reducing isotretinoin-related adverse events is to diminish its therapeutic concentration and combine it with another agent or drug. However, side effects can also appear for other treatments combined with isotretinoin (Xia et al., 2018). Acne relapses can occur following isotretinoin treatment, meaning that longer periods of treatment are needed to clear inflammatory lesions. Indeed, it is recommended to prolong the treatment once the acne has cleared up, to prevent relapses. However, higher doses can increase the probability of the occurrence of adverse events (Landis, 2020).

Other oral therapies with direct or indirect anti-inflammatory properties are anti-hormonal therapies, specially prescribed for women. These include contraceptives, spironolactone, cyproterone acetate, and flutamide. These molecules impact androgen availability, androgen binding, and sebaceous gland impairment. However, side effects also may appear and can be severe, including gastric disorders, depression, menstrual irregularities, and hematological disorders (Elsaie, 2016). Other chemical agents such as azelaic and pyruvic acid interfere with C. acnes metabolism, reducing free fatty acids, and therefore reducing inflammation derived from abnormal lipid profiles (Chilicka et al., 2020). Besides chemical treatments, light-based therapies and microneedle radiofrequency are physical alternatives that have recorded anti-inflammatory activities reducing IL-1α and controlling inflammatory reactions from sebaceous glands, respectively (Kim et al., 2020; Li et al., 2018). However, these therapeutic agents have been related to local adverse events, in particular irritation, pain, erythema, and edema.

Antibiotics are also frequently prescribed to manage acne, namely tetracyclines, macrolides, clindamycin, and trimethoprim/sulfamethoxazole. Their use in acne therapy may be justified because of their antimicrobial properties, controlling C. acnes proliferation. Nevertheless, beyond bacterial elimination, these antimicrobials have anti-inflammatory properties (Bienenfeld, Nagler & Orlow, 2017). Oral tetracyclines reduce initial inflammatory responses, targeting neutrophil chemotaxis, inflammatory cytokines, metalloproteinases (MMPS), and ROS (Farrah & Tan, 2016). As a second option, macrolides can be prescribed when tetracyclines are contraindicated (Marson & Baldwin, 2019). The macrolide-class antibiotic erythromycin was one of the most frequently used antibiotics to treat acne. Nevertheless, prescription patterns of topical erythromycin changed to other antimicrobials, such as clindamycin. This change can be attributed to the emergence and spread of antibiotic resistance, since erythromycin was first introduced to the market. A similar pattern is to be expected for clindamycin and other antibiotics (Austin & Fleischer, 2017). New antibiotic molecules are arising, including sarecycline (Deeks, 2019), topical minocycline (Gold et al., 2019), the sulfone dapsone (Al-Salama & Deeks, 2017), and the quinolone-like antimicrobial VCD-004 (Ghosh et al., 2018). Although promising, these new alternatives have drawbacks: sarecycline has high activity against tetracycline-resistant strains, but less so against isolates bearing tetracycline-resistance mutations, tet(K) and tet(M) (Zhanel et al., 2019); long-term efficacy and safety studies are still lacking for minocycline foam (Gold et al., 2019); dapsone may lead to local erythema, pruritus, dryness, exfoliation, and pain (Al-Salama & Deeks, 2017); and there is already a report of C. acnes isolates with mutations in DNA gyrase, which is the target of VCD-004 (Nakase et al., 2016).

Due to the increasing acquisition of antibiotic-resistance mechanisms, it is important to reduce antibiotic use. As a matter of fact, no antibiotic is recommended as a monotherapy or as a long-lasting therapy (Marson & Baldwin, 2019). The first-line therapeutic option to antimicrobial resistance in acne therapy is benzoyl peroxide (BPO), which is an antimicrobial with a potent oxidative effect (Thiboutot et al., 2018). However, BPO is also considerably irritant for most patients, diminishing patient adherence to treatment (Otlewska, Baran & Batycka-Baran, 2020).

New therapeutic alternatives for acne are emerging. These new drugs are sebo-suppressive, anti-inflammatory, or comedolytic, interfering in different inflammatory pathways including Melanocortin 5 Receptor (MC5R), interleukin signaling, and leukotriene inhibitors (Trivedi et al., 2018). Superoxide dismutase 3 (SOD3) can suppress TLR-2 expression in sebocytes and keratinocytes exposed to C. acnes or bacterial lipopolysaccharide (LPS), resulting in NF-κB inhibition. SOD3 also reduces NLRP3 inflammasome activation. Similar results were observed in vivo, in a murine model (Nguyen, Sah & Kim, 2018). Plant-derived products, such as Myrtus communis leaf extract have anti-microbial activity against IA₁ and clindamycin-resistant C. acnes strains, presumably by targeting bacterial virulence (Pécastaings et al., 2018). Designed peptides based on naturally-occurring peptides can display antimicrobial properties, that may be efficient at relatively low concentrations (Woodburn, Jaynes & Clemens, 2020). Nonetheless, large clinical studies are required to reveal its exact therapeutic mechanism and subsequent development.

Other emerging alternatives target microbiota homeostasis, an interesting approach for acne treatment, since its pathogenesis has been related to microbial imbalance (Trivedi et al., 2018). Among these, phage therapy emerges as an alternative to anti-microbial resistance without disturbing the host microbiota (Kutter et al., 2010).

Phage Therapy and Immunomodulation

The increasing phenomenon of antimicrobial resistance had led to propose new therapeutic alternatives (Tacconelli et al., 2017; World Health Organization, 2015). One of these alternatives is phage therapy, which consists in the use of bacteriophages (phages for short), viruses that infect bacteria, to control bacterial infections and contamination (Kutter et al., 2010). Although phage therapy is intended to kill bacteria, in recent years alternative applications have been suggested for phages due to their potential interactions with the eukaryotic cells (Górski et al., 2018). Scientific reports have showed that phages interact direct or indirectly with the mammal immune system; by infecting and eliminating bacterial hosts, phages can indirectly impact immunity, helping or improving immune responses to bacterial pathogens (Barr, Youle & Rohwer, 2013; Roach et al., 2017). However, direct interactions between phages and the immune system have also been reported (Fig. 2) (Van Belleghem et al., 2019; Sweere et al., 2019; Khan Mirzaei et al., 2016). These induced immune responses to phages trigger innate and adaptative mechanisms.

Perspectives for Immunomodulatory Acne Phage Therapy

To date, studies on C. acnes phages and acne phage therapy are scarce. The available information about C. acnes phages has been reviewed previously (Castillo, Nanda & Keri, 2019; Brüggemann & Lood, 2013; Jończyk-Matysiak et al., 2017) in studies that outline the history of C. acnes phages, phage characteristics, and prior in vitro studies. Below, we present a summary of their main findings.

The first isolated and characterized Cutibacterium phages were used to identify the Corynebacterium species, then classified into the Propionibacterium genus. Later, these early isolated phages were used to divide the genus into two morphologically related but different genera: Corynebacterium and Propionibacterium. Moreover, Corynebacterium acnes strains were further divided using phage’s ability to differentially infect various strains; resulting in the classification of these strains into new Corynebacterium species (Brüggemann & Lood, 2013). Like its host bacteria, C. acnes phages are usually found in the skin, in the pilosebaceous unit, and their titers are usually lower than C. acnes cell densities (Castillo, Nanda & Keri, 2019). The principal sources of C. acnes phages are sebum-rich areas, but they are isolated from other skin areas, including nostrils, oral cavity, and the gastrointestinal tract (Brüggemann & Lood, 2013). Due to the restricted C. acnes niche, it is thought that C. acnes phages are derived from a single clone that has been conserved given the low possibility of interaction with other bacterial species in this anaerobic and lipid-rich environment. This theory is supported by high nucleotide identity among C. acnes phage genomes, genome organization, and almost identical viral morphology, even between phages isolated from different countries (Castillo, Nanda & Keri, 2019; Brüggemann & Lood, 2013, Vives et al., 2018). Although lytic C. acnes phages have been described, most of those characterized are pseudolysogenic and have Siphoviridae morphology (Castillo, Nanda & Keri, 2019). Pseudolysogeny is an unstable life cycle presented by some phages, in which a phage exists inside the cell as an episome, outside bacterial chromosome. This state does not allow direct bacterial lysis and may conduce to bacterial resistance to other phages, by means of superinfection establishment to secure pseudolysogenic phage lifestyle (Jończyk-Matysiak et al., 2017).

Early studies on C. acnes phages as potential phage therapy agents were conducted in vitro, testing their ability to kill their host embedded in different types of formulations: ionic creams and water-oil nanoemulsions. In these preparations, phages were able to lyse bacteria and maintain their stability over several days (Jończyk-Matysiak et al., 2017). However, to date there are no in vivo studies in humans that explore C. acnes phages’ efficiency in killing this bacterium, even though various phages have been isolated and characterized from acne-affected and healthy individuals. Thus, data on phage therapy to treat acne in humans is still lacking (Castillo, Nanda & Keri, 2019).

Possible limitations to acne phage therapy have also been discussed and reviewed (Castillo, Nanda & Keri, 2019). Previous works have found that different C. acnes strains are sensitive to various phages, and these phages can infect diverse C. acnes strains. In fact, initial studies on C. acnes phage typing (the use of phages to identify new bacterial species based on phage ability to differentially infect certain species) become useless because of the broad host range that these phages have within the C. acnes species (Brüggemann & Lood, 2013). In principle, this can represent an advantage in terms of finding lytic phages for phage therapy purposes. Nevertheless, previous studies indicate that some C. acnes isolates can develop resistance to individual C. acnes phages. The use of phage cocktails is the first answer to tackle phage resistance, but it is important to further explore this approach in C. acnes isolates considering the phages’ limited genetic diversity (Castillo, Nanda & Keri, 2019). Lysogeny also represents a drawback since this life cycle type does not allow the rapid lysis of the bacterium (Jończyk-Matysiak et al., 2017). Thus, it is important to refine the search for C. acnes phages for those that only have lytic life cycles.

Besides phage therapy, phage-derived products offer additional alternatives. Endolysins are phage-encoded enzymes involved in the lysis of bacteria required for the viral progeny liberation. C. acnes phages encode endolysins, which are highly conserved. These enzymes catalyze the bacterial peptidoglycan destruction, and there is low reported resistance to these anti-bacterial agents (Castillo, Nanda & Keri, 2019). Another option is the concomitant use of phages and antibiotics. Their combined use can restore sensitivity to antibiotic-resistant C. acnes strains. In this scenario, phages establish selection pressure on resistant bacteria that present multidrug efflux pumps, which are receptors for certain phages. Consequently, phages select bacterial populations sensitive to antimicrobials (Saha & Mukherjee, 2019). Antibiotic effectivity is also achieved by using phages as sensitivity genes vectors or as antimicrobial vehicles to deliver antibiotic molecules in the site of infection. However, special care must be taken to avoid double-resistant phage bacterial isolates, by using phage cocktails or gradual use of phages and antibiotics (Jończyk-Matysiak et al., 2017). Phage cocktails should be explored while new C. acnes phages are found. Nevertheless, if genomes of new phages still demonstrate low genetic diversity, gradual use of single phages, custom individual therapies, and phage genetic modification would be studied.

These previous studies considered phage therapy from the most classical approach: by testing the lysis efficacy of phages. However, phages can interact with eukaryotic organisms and have a more systemic effect by modulating bacterial populations, hence impacting human microbiota, which in turn influences host health. As discussed in this review, C. acnes colonization-related acne pathogenesis goes beyond its abundance in affected skin, to be more closely related to a microbial dysbiosis characterized by the loss of C. acnes diversity. Production of short-chain fatty acids and differential immune response to C. acnes phylotypes can increment inflammatory responses, which is a hallmark for acne pathogenesis (O’Neill & Gallo, 2018). In fact, a high abundance of Cutibacterium spp. and C. acnes phages are related to healthy skin. In contrast, acne affected skin has lower richness of C. acnes phylotypes and C. acnes phages which has been associated to the augmented presence of virulence factors (Barnard et al., 2016). Interestingly, the presence of complete bacterial “immune” CRISPR systems has been reported in most type II C. acnes strains; isolates characterized into the phylotype II are usually related to healthy skin. In contrast, disease-related type I and III isolates apparently do not have entire CRISPR systems, which may have allowed a more expeditious acquisition of virulence traits to these strains (McLaughlin et al., 2019). Additionally, the absence of a competent CRISPR system in isolates belonging to phylotype I can be related to their susceptibility to lytic phages, as has been reported (Webster & Cummins, 1978). According to these findings, strains that belong to phylotype I are usually more susceptible to lytic phage infection (McLaughlin et al., 2019). This would represent a change in the classic viewpoint of selecting phages for phage therapy with a broad host range (Fernández et al., 2019), since healthy skin is characterized by a high diversity of phylotypes, in particular abundance of II and III phylotypes (Barnard et al., 2016). Therefore, selection of C. acnes phages should focus on those specific for strains belonging to phylotype I, aiming to restore skin microbial homeostasis and diminish exposure to virulence-bearing isolates that promote inflammation (Spittaels et al., 2021). Nonetheless, this approach must be further explored due to the limited genetic diversity of C. acnes phages.

Although antibiotics have anti-bacterial and immunomodulatory effects, these benefits may be limited or short-termed. Besides, these benefits are not comparable to potential long-lasting perturbations to the microbiota and development and spread of antibiotic resistance (Knackstedt, Knackstedt & Gatherwright, 2020). Phages or phage lysins can be used as selective microbiome therapeutics and contribute to restore the microbial balance in the skin (Woo & Sibley, 2020). Microbiome-based therapies may have better benefits to acne patients in re-establishing a regulated microbiota, which in turn will influence the immune response function (Xu & Li, 2019). However, the relationship between C. acnes and other skin microbiota organisms must be explored in greater detail. Culture-based studies on acne-related microbiota are biased because they do not reveal the complete skin microbial community (Ramasamy et al., 2019). Furthermore, there are other potential microbial agents that should be considered in acne pathogenesis. Microbial dysbiosis can also include changes in Staphylococcus epidermidis richness, which is suggested to be related to C. acnes uncontrolled colonization of the skin (Claudel et al., 2019). Malassezia spp. are commensal inhabitants of the skin that are related to other pathological conditions. Like C. acnes, this yeast can also metabolize lipids and produce irritant and allergenic molecules that compromise skin immune homeostasis (Ramasamy et al., 2019). A summary of different studies of phage therapy targeting other skin bacteria different to C. acnes to treat various dermatosis, including acne, was published by Steele and coworkers (Steele et al., 2020)

To the best of our knowledge, there are only two studies that addresses the relationship between C. acnes phages and the immune system. Kim et al. (2019) used a murine model to examine the efficacy of C. acnes phages in alleviating inflammatory lesions caused by C. acnes inoculated in mice skin. Phage administration was associated to decreased nodule size, compared with control groups. Histological findings showed reduced epidermal thickness in the phage treated group, which was in turn related to decreased expression of Integrin α6, a molecule known to cause epidermal hyperplasia. Moreover, a reduction of inflammatory markers, such as neutrophils, IL-1β, MMP-3, MMP-9 and CD8+ T cells (Kim et al., 2019), related to acne pathogenesis was observed in phage treated animals. More recently, Lam et al. (2021) carried out similar experiments to assess the therapeutic effect of a C. acnes phage. In this study, C. acnes was also inoculated on mice skin and reduction of lesions was evaluated post-mortem. Reduced inflammation of the lesions was observed and confirmed by histological findings. IL-1β expression in inoculated skin was reduced by phage therapy, along with the apoptosis marker Caspase-3 (Lam et al., 2021). These studies show that C. acnes phages can have anti-inflammatory properties. However, the mechanisms by which phages can diminish inflammatory reactions associated with C. acnes colonization must be further explored, especially in human cells. As reviewed earlier, these effects can be derived from bacterial elimination and subsequent control of bacterial population, or phages directly interact with certain components of the immune system, as has been reported for other phages that were associated with inflammatory response reduction. Phages may exert different immune reactions in their interaction with different cells (Khan Mirzaei et al., 2016); even different capsid proteins can represent contrasting relationships with the immune system (Dabrowska et al., 2014). Thus, selection of phages for acne phage therapy should include assessment of the relationship between candidate phages and immune response.

The most remarkable difficulty in the search for lytic phages useful in acne phage therapy is the limited diversity of phages. Nevertheless, further studies on Cutibacterium phage diversity are worthy. For other Propionibacteriaceae species, phages have been characterized with different morphologies, including prophages (latent state of a phage consisting in its genome inserted into bacterial genome) and filamentous ssDNA phages infecting Propionibacterium freudenreichii (Brüggemann & Lood, 2013). For that matter, a prophage found in P. freudenreichii, named PFR1, can cause lysis in a C. acnes strain by inducing the lytic cycle of a pseudolysogenic phage found in its genome. Interestingly, another P. freudenreichii prophage (PFR2) was able to infect C. acnes cells without causing bacterial lysis, becoming a prophage (Brown et al., 2017; Łoś & Wegrzyn, 2012). These findings show that prophages or lysogenic phages can interact with each other and provoke lysis, but it also indicates that phages infecting other Propionibacteriaceae genera may infect C. acnes, broadening the limited diversity of phages. Nevertheless, more studies are needed given the scarce information about lysogenic phages infecting C. acnes.

Acne phage therapy would be likely addressed via topical application. As a general principle, local phage therapy is easy to implement since it does not have to overcome physical and chemical barriers that diminish phage titers (i.e., gastrointestinal tract). However, mechanisms of phage transcytosis through the skin require further exploration (Huh et al., 2019). C. acnes phage penetration should be evaluated, since most of the C. acnes population is inside pilosebaceous units, which can be considered a physical barrier (Christensen & Brüggemann, 2014). Moreover, hyperkeratinization in acne involves the process of comedogenesis due to abnormal keratinocyte differentiation and accumulation of lipid droplets (Kurokawa et al., 2009). Such conditions may hamper phage penetration into the skin, implying the need for excipients or pharmaceutical vehicles to improve phage absorption. Depending on the phage, conjugation with emulsions, liposomes or similar vehicles may represent a reduction of phage titers or limited phage stability (Jończyk-Matysiak et al., 2017). Moreover, phage penetration to biofilms generated by C. acnes IA₁ strains should be considered (Kuehnast et al., 2018). Future studies on acne phage therapy must emphasize phage interaction with skin barrier and control of biofilm-embedded C. acnes, of strains belonging to IA₁ phylotype.

Conclusions

The paradigm of phage therapy as an alternative to antibiotic therapy is broadening to consider its additional effects. By regulating bacterial population, phages may exert indirect mechanisms that help balance the microbiota, and therefore immunity. Moreover, direct impact on immune responses is recorded for several phages. The blockage of bacterial virulence factors by phages can reduce inflammatory reactions. Other phages have been correlated with the reduction of pro-inflammatory cytokines, or the expression of anti-inflammatory cytokines. Inflammatory markers such as IL-1, IL-6, IL-8, MMPs, and TNFα are the most relevant cytokines expressed in acne pathogenesis. Since there are isolated phages that have regulatory properties on these cytokines, phage therapy studies for acne therapy should emphasize in the activity of C. acnes phages regarding the control of these inflammatory cytokines. The mechanisms by which cytokine regulation related to phages or phage therapy is presented is not fully known; their regulation may be given by the expression control of NF-κB or inflammasomes.

The impact of phage therapy on lymphocytes should also be evaluated. Late acne immune responses involve various Th cell lineages, including Th17, Th1, and Treg. It would also be important to evaluate the effects of any emerging therapy to treat acne in relation to the modulation of the response of these families of T cells, given the impact that these cells may have on acne inflammatory persistence and abnormal keratinization of the skin.

Phage therapy for acne and C. acnes regulation should be evaluated from an immunomodulatory and microbiota restoration perspective. As acne is a multifactorial disease with a markedly inflammatory nature, emerging phage therapy to manage it should have different functionalities that help to diminish or control inflammation. This skin condition involves various mechanisms of immune responses, ranging from innate immunity to cellular responses. Successful phages to treat acne might be related to the regulation in the expression of receptors such as TLR-2, TLR-4, and CD36; reduction of the activity of inflammasomes (specially NLRP3) and NF-κB; and modulation of lymphocyte and other immune cells chemoattraction by reducing KC, CXCL2, and IL-8 chemokines. Other desired effects might also be expected in acne phage therapy, such as a reduction of MMPs expression and Th cells modulation and to avoid neutralizing antibodies, which may have the potential of reducing not only inflammatory lesions but also to lower abnormal scarring risk. These immunomodulatory events in combination with the reports that indicate that phages are generally innocuous to human cells position phage therapy as a promising alternative therapy to regulate acne pathogenesis.

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