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To more accurately assess the activity and role of epithelial-cell derived antimicrobial peptides in their native settings, it is essential to perform assays at the surfaces under relevant conditions. In order to carry this out, we utilize 3-dimensional cultures of airway and gingival epithelium, which are grown at an air-liquid interface. Under these conditions, the cultures can be subjected to challenge with a variety of factors known to cause an increase in antimicrobial peptide gene expression. The functional relevance of this induction can then be assessed by quantifying antibacterial activity either directly on the surface of the cells or using the fluid secreted onto the apical surface of the cultures. The relative contribution of the peptides can also be measured by pre-incubation of the secreted fluid with specific inhibitory antibodies. Thus, a relatively inexpensive in vitro model can be used to evaluate the role of antimicrobial peptides in mucosal epithelium.
In general, most antimicrobial peptides have been identified by protein purification methods from tissues, followed by in vitro assays to identify the active fraction (see (1-3), for example). Alternatively, DNA sequences coding for homologous peptides are identified through computational or experimental methods (e.g., (4,5)). Subsequently, the purified natural peptides or synthetic peptides based on the DNA sequences are characterized by in vitro antimicrobial assays against target organisms, which provides important information regarding the potential for the peptide's use as a therapeutic, or give clues to its role in innate defense. Furthermore, the expression of many host defense peptide genes is induced by a variety of factors both in cell culture (e.g., (6,7)) and in vivo (8,9), supporting their role in innate host defense of mucosal epithelia such as the airway, the oral cavity and the intestinal tract. However, to accurately assess the relationship between the induction of a peptide gene (as measured by mRNA levels) and an increase in antibacterial activity in a tissue is difficult.
Recently, novel methods for cell culture which provide three-dimensional model systems that recapitulate the mucosal epithelium for different tissues have been described (10,11). The systems rely on culturing epithelial cells in an air-liquid interface (ALI) in serum-free medium, leading to well-differentiated cultures, often covered with a cell-derived secretion. This provides an environment that allows for quantification of antimicrobial activity in mucosal secretions under conditions that are more representative of the original tissue, and allows the investigator to modulate antimicrobial peptide gene expression with exogenous factors. Assay conditions can then be developed to determine the role of endogenous antimicrobial peptides on mucosal surfaces.
Beta-defensins and cathelicidins are antimicrobial peptides expressed in mucosal epithelial cells (reviewed in (13,14)). Their expression is induced in response to a variety of agents including bacterial Lipopolysaccharide (LPS), Interleukin (IL)-1β and the active form of vitamin D, 1,25(OH)2 D3 (reviewed in (15,16)).
To assess the activity of these peptides in airway epithelial cells, primary cultures of bronchial epithelial cells are grown in an air-liquid interface and are allowed to mature and differentiate for 20 days before any experiments are performed. The bronchial epithelial cells are then basolaterally treated with an inducing agent, such as IL-1β (100ng/ml) or vitamin D at a concentration of 10-8M. The airway surface fluid (ASF) is then collected by washing the cells with 50 μl of filter-sterilized 1× PBS. As a control for the poorly water soluble 1,25(OH)2 D3, control cells are treated with an equal volume of ethanol. The effect of the inducing agents on the bactericidal activity of ASF is studied using airway pathogens such as B. bronchiseptica or P. aeruginosa. To determine whether specific peptides are responsible for all or part of the activity, the ASF can be pre-treated with inhibitory antibodies prior to incubation with bacteria. A relative increase in the number of colonies relative to control (pre-treated with non-specific IgG) suggests a role for that peptide in killing.
To quantify the activity of peptides on the surface of gingival epithelial cells, a 3-D culture system is used whereby epithelial cells are cultured on transwells coated with a feeder layer of cells in collagen (10). Once the top layer is confluent, surface medium is removed and the epithelial cells are cultured similar to airway cells in an air-liquid interface. Activity against bacteria is measured using challenge with the periodontal pathogen A. actinomycetemcomitans.
The authors would like to thank Drs. Leslie Fulcher and Scott Randell (University of North Carolina, Chapel Hill) and Helena Kashleva and Anna Dongari-Bagtzoglou (University of Connecticut) for assistance in growing the ALI cultures of airway and gingival cells, respectively. GD is supported by grants from the Cystic Fibrosis Foundation and the NIH (DE18781).