Edward Putnins

Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria. Enzyme monoamine oxidase B (MAO B) gene and protein expression was upregulated in diseased tissue of LPS-induced periodontal mucosal chronic inflammation in rat; MAO inhibition with phenelzine reduced signs of inflammation (Ekuni et al., 2009). Current MAO inhibitors are brain permeable and clinically used to treat CNS-associated diseases. Interestingly, some of them showed promising peripheral effects. To manage chronic non-CNS inflammation, Dr. Putnins and collaborators redeveloped novel MAO B inhibitors with reduced blood-brain barrier permeability. These novel inhibitors have significantly reduced LPS-induced cytokine gene and protein expression in various epithelial and endothelial cell models via unknown mechanisms. We investigated the possible signaling pathways mediating LPS-induced interleukin-8 (IL-8) protein expression, and the mechanism by which novel MAO B inhibitor Compound B reduced such effects in intestinal epithelial cell line Caco-2 cells. Caco-2 cell differentiation was induced by culturing in Transwell-inserts for extended culture times. MAO B protein expression, levels of IL-8 secretion, total NF-kB p65 nuclear translocation, phosphorylation of NF-kB p65 at Ser536 and phosphorylation of ERK1/2, p38 and JNK MAPKs were assessed following stimulation by LPS with or without Compound B in differentiated and undifferentiated Caco-2 cultures via immunoprecipitation, ELISA, immunofluorescence and Western blotting. We showed that LPS-induced IL-8 secretion in Caco-2 cultures was independent of the common LPS-induced signaling pathways including NF-kB p65 nuclear translocation and phosphorylation of NF-kB p65 and MAPKs. MAO B protein expression was only detected in differentiated Caco-2 cultures. Compound B exhibited equal efficacy at downregulating LPS-induced IL-8 secretion in both undifferentiated and differentiated Caco-2 cultures. In summary, IL-8 downregulation by Compound B appears to be independent of the presence of MAO B protein, suggesting a possible off-target effect.

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Epithelial tissues play a critical role in maintaining systemic health by establishing a functional barrier that separates the external environment from the host to provide an innate defense against environmental insult. Epithelial barrier disruption is suspected to play a central role in the onset of chronic inflammatory disease, although, fundamental knowledge of the underlying pathogenesis remains poorly understood. Thus, identifying factors that mediate epithelial barrier loss is clinically relevant as it will open the possibility that novel interventional strategies may be developed to mitigate early disease-associated signaling events. Lipopolysaccharide (LPS) is a Gram-negative bacterial virulence factor implicated in periodontal disease onset. Amphiregulin (AR) is a ligand for the epidermal growth factor receptor (EGFR) and downstream mediator of tumor necrosis factor-alpha (TNF-α) (Chokki et al., 2006) that is normally sequestered at cell-cell contacts in stable epithelial barriers. AR and corresponding signaling components modulating the EGFR pathway are altered in a rat model of periodontal disease that exhibited concomitant altered barrier architecture (Fujita et al., 2011; Firth et al., 2011). Treatment of this model with monoamine oxidase (MAO) inhibitors ameliorated disease indices (Ekuni et al., 2009). This study employs an in vitro histiotypic model of epithelium to provide evidence that LPS-reduced epithelial barrier function associated with chronic inflammatory disease may be mediated by altered AR and TNF-α secretion. MAO-B inhibition by (−)-deprenyl enhanced barrier model transepithelial electrical resistance (TER), prevented LPS-, AR- and H₂O₂-induced reduction in TER and attenuated LPS-induced AR and TNF-α secretion and H₂O₂-induced AR secretion. Furthermore, immunostaining of barrier model cultures showed that markers of cell-cell junctions were altered by LPS challenge and treatment of the model with (−)-deprenyl protects against this disruption. This study addresses the underlying mechanism by which (−)-deprenyl protects against bacterial virulence factor-induced epithelial barrier disruption and points to a significant role for AR as a central mediator of barrier integrity. Ultimately, this project aims to provide in vitro evidence for the efficacy of (−)-deprenyl treatment of LPS-induced epithelial barrier disruption, which may promote development of enhanced MAO-B inhibitors and lead to an effective clinical treatment for disease-associated epithelial barrier loss.

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Epithelial tissues play a critical role in maintaining systemic health by establishing a functional barrier that separates the external environment from the host to provide an innate defense against environmental insult. Epithelial barrier disruption is suspected to play a central role in the onset of chronic inflammatory disease, although, fundamental knowledge of the underlying pathogenesis remains poorly understood. Thus, identifying factors that mediate epithelial barrier loss is clinically relevant as it will open the possibility that novel interventional strategies may be developed to mitigate early disease-associated signaling events. Lipopolysaccharide (LPS) is a Gram-negative bacterial virulence factor implicated in periodontal disease onset. Amphiregulin (AR) is a ligand for the epidermal growth factor receptor (EGFR) and downstream mediator of tumor necrosis factor-alpha (TNF-α) (Chokki et al., 2006) that is normally sequestered at cell-cell contacts in stable epithelial barriers. AR and corresponding signaling components modulating the EGFR pathway are altered in a rat model of periodontal disease that exhibited concomitant altered barrier architecture (Fujita et al., 2011; Firth et al., 2011). Treatment of this model with monoamine oxidase (MAO) inhibitors ameliorated disease indices (Ekuni et al., 2009). This study employs an in vitro histiotypic model of epithelium to provide evidence that LPS-reduced epithelial barrier function associated with chronic inflammatory disease may be mediated by altered AR and TNF-α secretion. MAO-B inhibition by (−)-deprenyl enhanced barrier model transepithelial electrical resistance (TER), prevented LPS-, AR- and H₂O₂-induced reduction in TER and attenuated LPS-induced AR and TNF-α secretion and H₂O₂-induced AR secretion. Furthermore, immunostaining of barrier model cultures showed that markers of cell-cell junctions were altered by LPS challenge and treatment of the model with (−)-deprenyl protects against this disruption. This study addresses the underlying mechanism by which (−)-deprenyl protects against bacterial virulence factor-induced epithelial barrier disruption and points to a significant role for AR as a central mediator of barrier integrity. Ultimately, this project aims to provide in vitro evidence for the efficacy of (−)-deprenyl treatment of LPS-induced epithelial barrier disruption, which may promote development of enhanced MAO-B inhibitors and lead to an effective clinical treatment for disease-associated epithelial barrier loss.

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