Abstract
Inflammatory skin diseases, such as atopic dermatitis, acne vulgaris, and psoriasis affect millions of individuals worldwide, and place an unprecedented cost on the medical system. Combined studies conducted in 2013 approximated the costs of treating inflammatory skin disease at $1.3 billion dollars (1,2). In atopic dermatitis (AD), the epidermidis of patients is disrupted, leading to a decrease in the expression of many anti-microbial peptides. These decreases, in turn, increases in the occurrence of microbial infections and colonization (3). Acne infections are characterized by blockage and inflammation of the pilosebaceous unit, and includes inflammation caused by activation of Toll-like receptor 2 (TLR2), the formation of microcomedones, hyperkeratization, and the excess production of sebum (seborrhea) from the sebaceous gland (4). In all inflammatory skin diseases, there are connecting underlying themes of tissue dysbiosis and overaction of the innate immune response. The sebaceous gland is an immunocompetent holocrine gland found in the skin, and its role is to produce and secrete a lipid mixture known as sebum. Sebum contains several distinct lipid components that provide structure and protection for the skin, as well as compounds that provide transport for fat soluble compounds to and from the skin (5). Short-chain fatty acids (SCFA) found in the sebum have been shown to have antimicrobial properties through the release of pro-inflammatory compounds to combat bacterial challenge (6). Interestingly however, SCFA also have the ability to modulate production of inflammatory precursors in a number of immune cells (7), whether to increase or decrease inflammation in order to maintain tissue homeostasis. Along with the host response of sebocytes, bacteria of the normal flora also have the ability to modulate their activity. By elucidating the interaction products between bacteria and cells of the skin, we can add depth to our knowledge and investigate possible therapeutic targets for inflammatory skin disorders. Bacteria of the microbiota reside in close contact with host cells and have developed methods of modulating sebocytes. It has been shown that the metabolic products of the microbiota can augment not only lipogenesis in sebocytes, but the composition of the sebum as well (8). Bacteria have also shown to have the ability to form biofilms when infecting the skin. Biofilms are multicellular groups of sessile bacteria that adhere first to a surface, then to one another by the secretion of an extracellular matrix consisting of polysaccharides, as well as extracellular DNA. Biofilms can provide antibiotic resistance through both steric hindrance to antibiotic treatment, as well as the passing of antibiotic resistance genes through horizontal gene transfer once together in close proximity within the biofilm (9). Cells also develop increased virulence through the passage of genetic material when part of the biofilm (10). These factors make biofilms difficult to treat, whether they are growing on industrial and medical equipment, or as part of an infection on a human host. Research into potential treatments against biofilm formation is paramount. Because of their multitude of functions, SCFA produced by the sebocytes may be a viable treatment option. In this study, we hope to further characterize interactions involving lipid production between live bacteria and the human sebocyte. We also seek to investigate whether SCFA produced during lipogenesis have the ability to modulate the development of early bacterial biofilms or disperse fully formed mature biofilms. This work seeks to answer the following questions: · Can we establish a living co-culture model to increase our understanding of the interplay between bacteria and sebocytes? · How does bacterial challenge affect sebocytes lipogenesis rates, and inflammatory marker production? · Can we quantify the changes we observe in lipogenesis and inflammation? · Does the presence of short chain fatty acids (SCFA) affect the development and growth of bacterial biofilms, or have modulation characteristics on mature biofilms? To answer these questions, we have developed living eukaryote and prokaryote co-culture assays for monitoring lipogenesis rates as well as inflammatory marker production. Using spectrophotometry and staining protocols, lipogenesis was monitored during co-culture. Inflammation and activation of the proposed lipogenesis pathway mTORC-1 was assed via Western Blot. To analyze biofilm formation, assays have been developed utilizing spectrophotometry, crystal violet staining, along with qRT-PCR. We investigated these questions by the following specific aims: A. Establish living co-culture models and determine possible cytotoxic effects of bacteria on eukaryotic cells • Co-Culture experiments conducted with immortalized SEB-1 sebocytes grown in the presence of Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), and Cutibacterium acnes (CA) show that in close proximity, these bacteria do not induce increased levels of eukaryotic cell death as analyzed by lactate dehydrogenase (LDH) cytotoxicity assay. B. Characterize lipogenesis and inflammation states produced by sebocytes during bacterial challenge. • Adipo red lipogenesis staining protocols demonstrate species specific lipogenesis modulation during co-culture, with SA significantly (p < 0.01) increasing lipogenesis while SE significantly (p < 0.01) decreasing lipogenesis at the 4-hour time point. Due to potential differences in growth dynamics, CA co-culture did not provide statistically significant results. • Western Blot analysis demonstrate that neither SA or SE significantly modulate lipogenesis through the mTORC-1 pathway, nor do these bacteria significantly alter the production of inflammatory markers 5-LOX and COX-2. C. Determine the effects of short chain fatty acids on biofilm growth and development. • Early biofilm modulation experiments show significant differential effects of SCFA on early biofilm formation in the bacterial species. The effects of SCFA are species specific, and dose dependent. Statistical significance was observed across all concentrations, with the greatest significance being observed at 500µm concentration. Mature biofilm experiments also showed differential species-specific effects, with fewer statistically significant findings. • Addition of SCFA modulated the gene expression of biofilm forming genes in both Staphylococcus species, as well as in Pseudomonas. Fold changes in relative gene expression of ica (Staphylococcus) and psl (Pseudomonas) were observed in accordance with the additions of acetate, butyrate, as well as propionate at the concentration of 500µM. Significant changes in expression (p < 0.01) were also observed between planktonic and biofilm cells in SA and PA.