Paxillin mechanisms control epithelial tissue remodeling under inflammatory conditions

Introduction: Tissue remodeling via the dynamic equilibrium between cells and their environment plays an important role in maintaining epithelial tissue functionality. However, upon infection, epithelial tissue homeostasis is altered by the invasion of pathogens, disruption of tissue barriers, and inflammation. Although the role of pathogens in infectious diseases may have been extensively explored, the impact of those pathogens and other inflammatory cues in regulating epithelial tissue restoration via the dynamic interactions between cells and their environment is unclear. The current study aims to explore new cell-matrix-based mechanisms in the epithelial tissue during tissue repair and restoration in normal and disease states. Specifically, we explored paxillin-mediated mechanisms, which mediate the cross-talk between cell-matrix interactions and cell adhesion and influence cell migration, proliferation, and differentiation during epithelial remodeling. To recapitulate the inflammatory conditions and identify the wound healing mechanisms, keratinocytes were exposed to E. coli and P. gingivalis Lipopolysaccharide (LPS). We have also developed a microfluidic device to monitor single-cell migration under exposure to LPS conditions. Overall, the study delves into cellular dynamics, investigating how inflammatory cues control cell migration, proliferation, and differentiation during the healing phase. Ultimately, elucidating these intricate cellular dynamics aims to inform the development of targeted therapeutic strategies for enhancing tissue remodeling upon infectious states.

Materials and

Methods: 1) Cell culture: Keratinocytes were cultured in serum-free media. Subsequently, the cells were exposed to lipopolysaccharide (LPS) derived from E. coli and P. gingivalis, individually and in combination, at a concentration of 100 ng/ml.
2) Scratch assay: After achieving confluence in the 6-well plates, keratinocyte cells underwent a scratch assay. Over time, the closure of the scratch was monitored using a microscope, providing insights into the cell migration and wound healing process.
3) Immunofluorescence: Cells fixed with 4% PFA and treated with permeability solution and blocking solution. To visualize the paxillin primary antibody used Alexa Fluor™ 488 goat anti rabbit IgG. Furthermore, Phalloidin 455 and Dapi were used to see the cytoskeleton and nucleus respectively.
4) Western blot: After LPS treatment, cell lysis was carried out using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Subsequently, the lysates were analyzed via Western blotting to evaluate the expression levels of paxillin and phospho-paxillin. Additionally, Rac pulldown assays were conducted using PAK-PBD beads on the lysates to investigate Rac activation.
5) Microfluidic device: Polydimethylsiloxane (PDMS) was poured into the channel mold and cured under heat. Afterward, the cured PDMS and a glass slide underwent plasma treatment. The PDMS devices were then attached to the slide and kept at 80°C. Following this, the devices were treated with poly-lysine (PLL) and 0.5% glutaraldehyde, followed by UV sterilization, before cell loading. Live cell imaging was performed for phase analysis involved capturing time-lapse images before and after cell exposure to LPS

Results, Conclusions, and Discussions: Our study revealed the complex mechanisms involved in LPS-mediated effects on epithelial tissue restoration, offering a deeper understanding of the underlying cellular dynamics. Scratch assay revealed that exposure to LPS resulted in delayed wound closure by 3-fold (p < 0.05) after 18 hours compared to control conditions. Using a microfluidic device, we investigated epithelial migration at the single-cell level and observed increased protrusion formation and reduced migration by 2-fold (p < 0.05) in response to LPS treatment. Immunofluorescence analysis confirmed prominent paxillin expression by 2-fold (p < 0.05) was observed under the LPS conditions. Furthermore, paxillin was predominantly localized on the distal side of the cells, suggesting potential implications for cellular adhesion and migration processes under inflammatory conditions. Based on this, we investigated multiple phospho-paxillin sites to pinpoint the key phosphorylation site controlling migration-related signaling. We found that Ser273 was prominently phosphorylated, indicating its pivotal role in cellular migration pathways. Furthermore, the phosphorylation of Ser273 is linked to PAK enzyme activation, triggered by Rac activation induced by LPS exposure. Notably, the downregulation of both Rac expression and paxillin phosphorylation at Ser 273 by Rac inhibitors (NSC23766) underscores the critical role of Rac-mediated pathways in LPS-impaired migration. Based on this finding, in scratch assay experiments, we introduced a Rac inhibitor under LPS conditions and observed improved wound closure by 2-fold compared to LPS conditions alone (p < 0.05). Altogether, under LPS conditions, there was an upregulation of phosphorylation of Paxillin at Ser273 and Rac expression, and the introduction of the Rac inhibitor reduced both expressions. Ultimately, this study suggests that wound recovery under infection conditions is dependent on the phosphorylation of Paxillin at Ser273 and the expression of Rac. Overall, these findings underscore the complex interplay between inflammatory cues, cellular signaling pathways, and cytoskeletal dynamics in regulating epithelial function upon wound healing. Further validation in animal models will confirm our findings, providing valuable insights for developing targeted therapies to improve tissue remodeling under infectious conditions.

Acknowledgements (Optional): This work was supported by the National Institute of Dental and Craniofacial Research (NIDCR) of the National Institutes of Health (R01DE031046). This research was supported by the Microscopy and Imaging Shared Resource (MISR) and the Lombardi Comprehensive Cancer Center grant (P30-CA051008). Instrumentation used in this research included Upgrade existing multiwell Fluorescence Imaging (S10RR025661). We greatly appreciate Alimperti lab members for their help.