Early effects of Staphylococcus aureus biofilm secreted products on inflammatory responses of human epithelial keratinocytes

Each year millions of people are stricken with chronic wounds such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers that contribute to the morbidity and mortality in the U.S. annually[1‐3]. Recent evidence indicates that bacterial biofilms are present in chronic wounds more often than in acute wounds and are related to the wounds inability to heal[4‐6]. Bacterial biofilms consist of bacterial communities embedded in a self-made extracellular polysaccharide matrix that are often resistant to a variety of antibiotics[7]. Chronic wounds provide an excellent environment for this matrix due to the tissue surface for growth, poor blood flow, and poor oxygen flow that inhibit host cell defenses against the bacteria[8]. The surface of chronic wounds allow bacteria to readily adhere to a surface and relinquish their planktonic state to form an aggregate of bacteria that enable the newly formed biofilms to better survive in their environment[9]. The inhibitions of the host cell defenses allow the bacteria to cooperate in the distribution of nutrients, removal of wastes, and to resist host defense mechanisms[6].

The establishment of S. aureus biofilms in chronic wounds relies on the bacterial community’s ability to adhere to tissue. After attachment, the bacteria rapidly secrete cell signaling molecules that coordinate activities of the bacteria through a process known as quorum sensing[10]. Biofilm formation by Staphylococcus aureus has been shown to be highly dependent on the staphylococcal accessory regulator (sarA)[11] and to a certain degree on the (icaABCD) operon[12‐15], and the walRK operon[16]. Mutations in either sarA or the ica operon have been associated with reduced capacity of S. aureus to form biofilms. There is significant evidence that the molecules in the matrix of biofilms lead to impermeability to antibiotics and host defenses[17]. While the idea that biofilms affect wound healing has been established, the exact role of biofilms in chronic wounds is not well understood[7]. Biofilm’s resistance to antibiotics make the infections difficult to resolve and more likely to become chronic or recurrent infections in part due to the inhibition of the re-epithelialization process[18].

Wound healing is a complex series of pathogen and host cell interactions. Most chronic skin wound healing is mediated by keratinocytes[19, 20]. The ability of keratinocytes to migrate to the site of the wound and respond to inflammatory signals to eliminate infection and complete the process of reepithelialization is key to the ability of chronic wounds to heal[20]. Keratinocytes have been shown to express toll-like receptors 1–6 and 9, which can allow them to act as a first responder against pathogenic microorganisms. For example, S. aureus can activate nuclear transcription factor kB (NF-κB) in keratinocytes. Activated NF-κB then translocates into the nucleus and induces the transcription of NF-κB controlled genes such as interleukin 8 (IL-8) and nitric oxide synthase (iNOS)[21, 22]. The role that keratinocytes play in wound pathogenesis makes them an excellent model to investigate the pathogenesis of wound healing in vitro.

Inflammatory response of the host is important to the ability of wounds to heal. Biofilms have been shown to contribute to the failure of wounds to reepithelialize through the activation of the β-catenin/c-myc pathways which is in part attributed to the unresponsiveness of cells at the wounds edge[23‐25]. A part of that inflammatory response is the production of inflammatory cytokines that serve to mediate host immune responses. Recent evidence has revealed that S. aureus biofilms affect the gene regulation and cytokine production of keratinocytes and thus may affect the way that wounds heal[26]. The goal of this project was to further investigate and compare the effects of S. aureus biofilm secreted products and S. aureus planktonic secreted products on gene expression profiles and inflammatory responses of human epithelial keratinocytes (HEKa). The hypothesis is that products secreted by S. aureus growing as a biofilm actively impair wound healing by promoting inflammatory responses in keratinocytes. To test this hypothesis, we investigated and compared inflammatory responses of HEKa cells exposed to BCM and PCM at both transcriptional and translational levels.

The data collected in this study reveal a clearer picture of the role that S. aureus biofilms play on cultured keratinocytes. Keratinocytes serve as the primary cell type in the epidermis and primarily function in providing a barrier between the external and internal environment. When breaks in this barrier occur, basal keratinocytes migrate to the site and reepithelization ensues. Chronic wounds are characterized by prolonged inflammation and the failure of wound reepithelization[8]. Chronic wounds activate a number of inflammatory pathways that lead to the prevention of keratinocyte migration, growth, and differentiation and thus failure of wounds to heal[25]. Due to their importance in chronic wound pathogenesis HEKa cells were chosen for experiments on the effect of biofilms on early chronic wound pathogenesis. Kirker et al.[8] demonstrated that the viability of HEKa cells was significantly reduced when exposed to BCM or PCM for 24 hours. Results from our HEKa cell viability assays showed that viability was significantly reduced by BCM or PCM in as little as 8 hours of exposure (Figure 1), indicating that our findings are in general agreement with previous findings of Kirker et al.[8]. Visual inspection of the cells showed morphological differences between HEKa cells exposed to the two treatment conditions. Evidence of cellular stress in the form of rounding of cell membranes and decreased culture confluency were observed in HEKa cells exposed to BCM after 8 hours of exposure that was not seen in HEKa cells exposed to PCM or the control media (data not shown). Based on the viability assay results and these morphological changes, transcriptional changes were measured at two hours after treatment to detect early transcriptional changes in cell populations with high viabilities. For inflammatory cytokine response and NO production 4 and 8 hour time intervals were used. These time points were selected in an effort to measure downstream effects of transcription and inflammatory responses.

Increased apoptotic effects in HEKa cells exposed to S. aureus biofilm secreted products provides greater understanding of the pathogenesis of wound healing. Decreased cell viability and the inability of wounds to heal have been linked to chronic wounds associated with biofilms[8]. Figure 2C shows alterations in transcription of genes associated with apoptosis in BCM- and PCM-exposed HEKa cells. This correlates with our cell viability data (Figure 1) which reveals a statistically significant loss of HEKa cell viability in BCM and PCM after 8 hours. There are several definitions of what an actual chronic wound is, but some common themes are a prolonged inflammatory phase to the wounds and the failure of the wound to respond to standard treatments[38]. These wounds fail to reepithelialize which is due in part to the decrease of cell production and the increase in cell death. Our data indicate that there is an increase in apoptotic effect in HEKa cells exposed to BCM over PCM or the control conditions.

Microarray analysis was performed on RNA gathered after 2 hours exposed to BCM, PCM, or the media control the samples of HEKa cells exposed for 0 hours were used as a control. The data were then filtered in order to identify differentially expressed genes that were associated with inflammatory responses, apoptosis and nitric oxide production in HEKa cells. There was an increase in transcriptional activity in HEKa cells exposed to BCM for genes associated with inflammation, apoptosis and NO production (Figure 2). In HEKa cells exposed to BCM some of the largest upregulation of transcriptional activity came from the genes CXCL2, IL-8, DUSP1, and ATF3. The DUSP1 gene is important for the regulation of p38 activation of LPS-activated macrophages. The p38 pathway plays a central role in multiple pathways associated with inflammatory response in many cell types[39, 40]. The p38 MAPK pathway has been found to play a role in the production of inflammatory cytokines namely IL-1 and TNF-α but has also been found to contribute to the production of IL-8 in response to IL-1 osmotic shock and IL-6 in response to the production of TNF-α[41]. NFKBIA like DUSP1 is an important transcriptional regulator that induces innate and adaptive immune responses. NFKBIA is one of the genes that assist in the regulation of the magnitude and duration of inflammatory responses. One of its key roles is to prevent the inflammatory response from destroying excessive amounts of tissue[41]. Some of the other genes up regulated are associated with the production of inflammatory cytokines or chemokines. CXCL2, IL-6 and IL-8 are well known cytokines or chemokines which play important roles in mediating inflammatory responses to pathogen. CXCL2 and IL-8 are pro-inflammatory chemokines that assist in the mediation of neutrophil migration as well as the migration of other cellular and humeral factor components to the site of an infection. TNF-α, IL-6 and IL-8 were also upregulated but the fold change in expression over the control was much lower and the increase in expression was only seen in HEKa cells exposed to BCM. TNF-α, IL-6 and IL-1 are all multifunctional cytokines with a wide variety of functions. These three cytokines are interrelated with IL-1 and TNF-α inducing IL-6 and IL-6 in turn playing a role in the regulation of TNF-α[42]. TNF-α plays a role in tissue repair, inflammation and regulation of apoptosis as well as the activation of transcriptional factors such as NFΚB that are important to several processes including growth, death, and inflammation and stress responses[43]. While NF-κB is not regulated at the RNA level and is not be expected to be differentially expressed, Ingenuity Pathways Analysis software predicted its increased activation based on the expression patterns of 28 genes in the microarray results from BCM-treated but not PCM-treated HEKa cells relative to media controls (p = 1.06 x 10^-11). DUSP1 is produced in human skin cells and specifies a protein with structural features similar to members of non-receptor-type protein-tyrosine phosphatase family which inactivates MAPKs. MAPKs play an important role in the human cellular response to environmental stress as well in the negative regulation of cellular proliferation[44]. This can lead to an increase in cell death and thus contribute to S. aureus biofilms’ negative effects on the ability of HEKa cells ability to respond to bacterial challenges and prevent apoptosis and promote reepithelialization.

Seven cytokines or chemokines commonly produced by keratinocytes in response to pathogens were tested using sandwich ELISAs[45‐47]. After 4 hours, cells exposed to BCM showed statistically significant increases in concentrations of IL-6, TNF-α, and CXCL2 in HEKa cells exposed to BCM over HEKa cells exposed to PCM and the control media. The increase in inflammatory cytokine and chemokine responses after 4 hours of exposure to PCM, BCM, and the media control were in general agreement with earlier research by Secor et al.[6] who showed increases in inflammatory responses by HaCaT cells exposed to S. aureus biofilm products. HEKa cells exposed to PCM, BCM and the control media were also tested by ELISA’s for the same seven cytokines and chemokines. After 8 hours of exposure, levels of IL-8, along with previously elevated IL-6, TNF- α, and CXCL2 significantly increased in HEKa cells exposed to BCM over HEKa cells exposed to PCM and the media control. At the 8 hour time point HEKa cells treated with BCM showed a statistically significant increase in cytokines versus HEKa cells treated with PCM. These findings support the current hypothesis that biofilm-secreted products differ and have more dramatic effects than secreted products from planktonically grown bacteria which may contribute to difference between chronic and acute wounds[5]. The creation of biofilms enables bacteria to increase their interaction with one another and thus resist antibacterial and environmental pressures[6]. These biofilms are not only a congregate of bacterial cells but are also held together by a polysaccharide matrix. Understanding the components of the exopolysaccharide matrix produced by bacterial biofilms along with the difference in morphology of the cells may provide clues to the mechanisms of biofilms and why they produce the differential inflammatory responses that we have observed.

A somewhat unexpected finding from the microarray analysis was the upregulation of genes associated with nitric oxide production in HEKa cells exposed to BCM. Nine genes were up regulated in HEKa that were not found in control samples. Eight of those genes were unique to HEKa cells exposed to BCM and were not found in HEKa cells exposed to planktonically secreted products. Despite the genes associated with nitric oxide production there was not a significant upregulation of iNOS or NOS2 which are most often associated with nitrite production in cells. The lack of significant iNOS an NOS2 upregulation may have been due to the short time between the exposure of the HEKa cells to the PCM and BCM and the time of mRNA collections. Schnorr et al. reported that iNOS expression in keratinocytes was not detected until 4–8 hours after exposure to inflammatory cytokines and maximal expression does not occur until 24 hours post exposure[48]. Nitric oxide plays a key role in acute wound repair. Nitric oxide synthesis increases wound healing by enhancing collagen deposition within the wound and in dermal fibroblasts to enhance the mechanical strength of the tissue[29, 30]. However, high concentrations NO may actually inhibit healing[31]. In this study, we observed upregulation of genes associated with NO production in cells treated with BCM, and tested the downstream effects of such genes by performing nitrite assays on culture supernatants. These nitrite assays were done in order to measure nitrite as an index of NO formation. Statistically significant increases in the amount of nitrite produced by HEKa cells exposed to BCM compared to HEKa cells exposed to PCM or just Epilife media were detected. These results add to the understanding of NO role in chronic wounds as opposed to acute wounds. The role of NO in biofilm production and dispersal is controversial. Falsetta et al.[49] found that in terms of Neisseria gonorrhea biofilms there was a high level of NO which seemed to aid the biofilm’s growth. Other studies have found that NO has been linked to the dispersal of Pseudomonas aeruginosa biofilms as well the prevention of biofilm formation in S.aureus and Escherichia coli[50, 51]. It has been suggested that the disparate results are due to concentration of NO that is induced in the system. NO in large concentrations can be toxic to eukaryotic cells and in fact eukaryotic cells have developed defenses like metallothionein in response to oxidative stress[32]. Our results show that NO production by HEKa cells increased over time (Figure 4) as inflammatory effects of biofilm secreted products increased (Figure 3). Overproduction of reactive nitrogen species can impair cellular migration, proliferation, and synthesis of extracellular matrix that is important to wound healing by keratinocytes[35]. This lack of agreement between studies coupled with our results makes NO and its role in biofilm formation, dispersal, and/or chronic wound pathogenesis a topic that requires further investigation.

The majority of studies on immune responses to bacteria have been carried out with planktonically growing bacteria or cell components from planktonically growing bacteria. Infections due to S. aureus are characterized by strong inflammatory responses[52, 53]. Several molecules such as Panton-Valentine leukocidin[54] sphingomyelinase[55], peptidoglycan, lipoteichoic acid[56, 57], superantigens[58] as well as others have been shown to play a role in the inflammatory response directed towards S. aureus. It is possible that some of these same molecules may be responsible for the differences in inflammatory and nitric oxide responses we have measured; however, these molecules have not been specifically linked to S. aureus biofilms and chronic wound inflammation.

Studies presented here further our understanding of the role that S. aureus biofilms may play in chronic wound pathogenesis. We found greater transcriptional activity in HEKa cells exposed to BCM compared to HEKa cells exposed to PCM or the media controls. The increased transcriptional activity was corroborated by increases in inflammatory cytokine production, nitric oxide production, and reduced cell viability. The increase in nitrite production in HEKa cells exposed to BCM is an intriguing finding that could be an important biofilm-mediated factor that contributes to the failure of chronic wounds to heal and warrants further investigation. Experiments to further understand the role of nitric oxide in chronic wounds and studies to identify S. aureus biofilm secreted factors responsible for the increased inflammatory responses are planned.