PROPIONIBACTERIUM ACNES AND CHRONIC DISEASES
Ajay Bhatia, Ph.D., Jean-Francoise Maisonneuve, Ph.D., and David H. Persing, M.D., Ph.D.
Corixa Corporation, Seattle, WA
Propionibacterium acnes is a gram-positive human skin commensal that prefers anaerobic growth conditions and is involved in the pathogenesis of acne (Kirschbaum and Kligman, 1963). Acne is one of the most common skin diseases, affecting more than 45 million individuals in the United States. It is estimated that nearly 20 percent of all visits to dermatologists are related to the treatment of acne. Acne often debuts during changes in hormonal levels in pre-teens; however, it is also very common as an adult-onset condition, often associated with hormonal fluctuation during the menstrual cycle and pregnancy. While not life-threatening, acne can persist for years and is known to have serious psychosocial effects such as decreased self-esteem, depression, frustration, and social withdrawal. In addition to dermatological pathology, P. acnes is also suspected to be discreetly involved in post-operative infections, prostheses failure, and more recently, in inflammation of lumbar nerve roots leading to sciatica.
P. acnes, previously known by the name Corynebacterium parvum, has been studied extensively by immunologists for its ability to stimulate the reticuloendothelial system (Adlam and Scott, 1973). Not too long ago, an important cytokine, interleukin (IL)-18 was cloned from the liver of mice primed with P. acnes followed by challenge with LPS (Okamura et al., 1995). In the early eighties, certain bacteria, including BCG and P. acnes, were commonly used to stimulate the innate immune response against cancer in mice and human cells (Cantrell and Wheat, 1979; Davies, 1982). One of the great ironies of this organism is that it is a powerful nonspecific immune stimulant that resides naturally in the skin; its role as an immunostimulant in humans is appreciated when cases of severe acne also develop adjuvant-type arthritis.
Some investigators have gone so far as to suggest that severe acne, by virtue of the nonspecific immunostimulatory effects of P. acnes, might have played a role in natural protection against life-threatening diseases such as malaria and plague. In contrast, the acquired immune response to P. acnes has received little attention in humans.
Pathogenesis of Acne
Chronic inflammatory acne cannot be defined as an infectious disease, since the bacteria are normally present on the skin of a vast majority of individuals, irrespective of the presence of acne lesions. P. acnes apparently only triggers the disease when it meets favorable dermatophysiological terrain; P. acnes colonization of the skin is therefore necessary but not sufficient for the establishment of the pathology. The 4 major recognized pathophysiological features of acne include androgen stimulated seborrhea, hyperkeratinization and obstruction of the follicular epithelium, proliferation of P. acnes, and then inflammation.
Comedogenesis, the transformation of the pilosebaceous follicle into the primary acne lesion, the comedone, is the product of abnormal follicular keratinization related to excessive sebum secretion. During this process, P. acnes often gets trapped in layers of corneocytes and sebum and rapidly colonizes the comedonal kernel, resulting in a microcomedone, a structure invisible to the naked eye (Plewig and Kligman, 2000). A microcomedone can develop into larger structures, called comedones. Comedones can be a closed structure (whitehead) that appears like a colored bump on the skin or an open structure (blackhead). Unlike open comedones, closed comedones cannot evacuate the thread-looking conglomerate of cell debris, sebum, P. acnes and its products to the skin surface, and this makes them more prone to inflammation and rupture. In inflammatory acne, comedones rupture and the follicular material becomes dispersed in the dermis rather than on the skin surface. Depending on the extent of the damage to the comedone wall, various types of inflammatory lesions are produced and these are classified as papules, pustules, or nodules. Nodules are the most severe types of acne lesions and scarring may be associated with any form of severe inflammatory acne.
A break in the lining of the comedone was initially attributed to free fatty acids generated by P. acnes-mediated triglyceride hydrolysis, but for several reasons, it is now thought that substances produced by P. acnes are directly involved in the rupture the comedone epithelial lining (Holland et al., 1981). The bacteria secrete many polypeptides, among which are numerous extracellular enzymes such as proteases, hyaluronidases, neuraminidases, and others that could be involved in epithelium permeabilization and inflammatory infiltration (Noble, 1984). P. acnes is also known to produce chemotactic factors (Puhvel and Sakamoto, 1977), proinflammatory cytokine inducing-factors (Vowels et al., 1995), and to activate both the direct and indirect complement pathways (Webster et al., 1978). The infiltrate of an early inflamed lesion consists of polymorphonuclear cells that certainly contribute to the lining breakage, but eventually, as time goes by and infection becomes chronic, these cells attract and are replaced by mononuclear cells, predominantly T-cells of the CD4 phenotype (Norris and Cunliffe, 1988; Layton et al., 1994). As the inflammation propagates to the lining of adjacent sebaceous follicules, it can start a chain reaction that results in multiple lesions connected together and called a sinus. Studies by Hoffler et al. (1985) have revealed differences in the production of various enzymes by Propionibacterium isolates of acne lesions versus bacteria isolated from healthy controls. These studies are important for differentiating bacterial antigens that lead healthy controls to generate a protective immune response and those that might be involved in pathogenesis.
Antibody against P. acnes antigenic determinants are found in the blood of most adults, whether they have had acne or not (Ingham et al., 1987); amounts may vary between the two populations, and possibly the nature of the determinants the antibodies recognize (Holland et al., 1993). Recent investigations by our group suggest that differential recognition might involve surface molecules with physiological functions. P. acnes specific IgG and IgA are also found at the level of the follicular infudibulum (Knop et al., 1983); these antibodies might be of great importance in limiting or preventing P. acnes proliferation, and maybe more importantly, in preventing comedonal lining destruction by P. acnes-derived soluble factors. Our preliminary data suggests that a robust P. acnes specific T-cell response is also common in adult donors, but its specificity at the antigen level is currently under investigation. We like to think that there possibly exists a P. acnes-specific protective immunity against acne. This hypothesis is supported by the fact that some people never get acne, as well as by the observation that acne is mostly a disease of young people, (although there are numerous exceptions), and that even in countries where people are unable to afford sophisticated medications, chronic disease of adolescents eventually resolves with age. Finally, there have been successful human trials of therapeutic vaccination against P. acnes, and although the rate of success has not been high, some individuals refractory to conventional approaches experienced remission (Goldman et al., 1979; Vymola et al., 1970).
Role of P. acnes in Chronic Inflammation and Systemic Infections
The chronic inflammatory condition of the pilosebaceous follicle caused by P. acnes is generally considered non-pathogenic. However, there is a growing body of evidence that point to the bacterium as being low virulence pathogen in several types of postoperative infections and other chronic conditions. P. acnes have been associated with endocarditis of prosthetic (Lazar and Schulman, 1992) and native aortic valves (Mohsen et al., 2001), corneal infections (Underdahl et al., 2000) and postoperative endophthalmitis (Clark et al., 1999). It has also been recognized as a source of infection in focal intracranial infections (Chu et al., 2001) and various cerebrospinal fluid shunt infections (Thompson and Albright, 1998).
A recent study from Japan (Ishige et al., 1999) has shown that P. acnes DNA can be detected in lymph nodes of Japanese individuals with sarcoidosis. Sarcoidosis is a granulomatous disease that results in the inflammation of lymph nodes, lungs, eyes, liver, and other tissues. P. acnes have also been implicated in sciatica, a chronic inflammatory condition of the lower back. Stirling et al. (2001) have isolated P. acnes from intervertebral disc material of patients with severe sciatica and they hypothesize that low virulent organisms such as P. acnes can gain access to the injured spinal disc and initiate chronic inflammation. However, until confirmatory data is available, the proposed role of P. acnes in sarcoidosis and sciatica should be considered intriguing but preliminary.
It also appears to be significant that P. acnes have been isolated from several orthopedic infections, silicone breast prosthesis, and prosthetic joint infections (Yu et al., 1997; Tunney et al., 1999). The infected prostheses have been shown to contain bacterial biofilms of P. acnes and/or Staphylococcus epidermidis. The adhesion of P. acnes to the surface of the prostheses has been postulated to be a result of binding of propionibacterial cell surface proteins or adhesion molecules to host plasma or connective tissue proteins such as fibronectin (Yu et al., 1997). Evidence for this hypothesis comes from the studies of Herrmann et al. (1988), who show that fibronectin, fibrinogen, and laminin are mediators of adherence of staphylococcal isolates to polymer surfaces in intravenous device infection.
Corixa Acne Vaccine Program
The gamut of acne treatments range from topical and systemic antibiotics to oral and topical isotretinoins, chemicals like benzoyl peroxide, oral contraceptives and corticosteroids. Antibiotics have been in use for several decades as one of the most common treatments for acne. Antibiotics, both topical and systemic, take a relatively long time to reduce the numbers of P. acnes bacteria in the skin and do not address other causative factors of acne. More recently, vitamin A derivatives called retinoids have been used effectively for acne treatment since these drugs help unclog pores, reduce sebum production and help normalize skin shedding and growth. However, oral isotretinoins are also known to cause severe side effects including elevated serum triglyceride levels, acute pancreatitis, hepatotoxicity, clinical depression, and birth defects in pregnant women.
To help identify components of P. acnes involved in pathogenesis or a protective immune response and develop a therapeutic vaccine for acne, we recently sequenced the genome of P. acnes. The genome is approximately 2.6 Mb and organized into 100 contigs. It shares similarity with the genomes of other bacteria, including Streptomyces coelicor, Mycobacterium tuberculosis, and other gram-positive cocci. Numerous homologues to virulence factors of other gram-positive pathogens have been found in the P. acnes genome, including homologues of known vaccine targets.
Whole genome sequencing of microbial pathogens has been used successfully to predict vaccine candidates in Streptococcus pneumoniae and Haemophilus influenzae (Adamou et al., 2001; Wizemann et al., 2001; Chakravarti et al., 2000). We are using a multifaceted approach that combines traditional immunological and biochemical antigen discovery strategies along with a genomics approach to identify antigens for use as vaccine targets. This approach includes serological expression cloning, proteomics, and CD4 T-cell expression cloning. We are further enhancing antigen discovery methods by using in-silico approaches to predict targets for antibody-based vaccines and antimicrobial agents. The products of these various research strategies provide attractive antigen candidates, i.e., a polypeptide that is detected by serum from adult individuals who never suffered acne, and predicted to be extracellular and involved in P. acnes metabolism, or an immunogenic extracellular enzyme potentially involved in epithelial destruction. Such antigens may prove to be valuable vaccine candidates for the other chronic diseases associated with P. acnes as well.
Knowing the physiological function of our targets allows us to tailor in-vitro and in-vivo assays to evaluate the potential of specific immune components to limit or abolish the events that lead to inflammatory acne. Since the antigens of choice will be delivered under a recombinant protein format, they will require a strong adjuvant that induces an adequate immune response at the correct site. Recent data indicates that Corixa's proprietary adjuvants, MPL® and AGPs (aminoalkyl glucosaminide phosphates), induce strong mucosal and systemic immunity when administered mucosally. Adjuvants such as these would be useful to prime a local immune system against P. acnes at the pilosebaceous level.
Lastly, the molecules discovered by immunological methods could be used in immunodiagnostic assays. For example, we might be able to develop serological markers to predict in early adolescence the likelihood of future acne flares. In addition, since many of the studies of the involvement of P. acnes outside of the skin have so far relied on culture-based and molecular techniques that are prone to false positive results, future studies of disease associations of P. acnes might be facilitated by the availability of a specific immunoassay comprising recombinant P. acnes proteins.
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