Aggregatibacter Actinomycetemcomitans Induces Autophagy in Human Junctional Epithelium Keratinocytes.

Emiliano Vicencio,Esteban M Cordero,Sebastián Palominos,Paola Murgas,Pedro Parra,Bastián I Cortés,Gustavo Monasterio,Constanza Henrríquez,Emilio A Cafferata,Tania Mella,Rolando Vernal,Nelda Salazar,Cristian Cortez

doi:10.3390/cells9051221

Abstract

The adverse environmental conditions found in the periodontium during periodontitis pathogenesis stimulate local autophagy responses, mainly due to a continuous inflammatory response against the dysbiotic subgingival microbiome. The junctional epithelium represents the main site of the initial interaction between the host and the dysbiotic biofilm. Here, we investigated the role of autophagy in junctional epithelium keratinocytes (JEKs) in response to Aggregatibacter actinomycetemcomitans or its purified lipopolysaccharides (LPS). Immunofluorescence confocal analysis revealed an extensive nuclear translocation of transcription factor EB (TFEB) and consequently, an increase in autophagy markers and LC3-turnover assessed by immunoblotting and qRT-PCR. Correspondingly, challenged JEKs showed a punctuate cytosolic profile of LC3 protein contrasting with the diffuse distribution observed in untreated controls. Three-dimensional reconstructions of confocal images displayed a close association between intracellular bacteria and LC3-positive vesicles. Similarly, a close association between autophagic vesicles and the protein p62 was observed in challenged JEKs, indicating that p62 is the main adapter protein recruited during A. actinomycetemcomitans infection. Finally, the pharmacological inhibition of autophagy significantly increased the number of bacteria-infected cells as well as their death, similar to treatment with LPS. Our results indicate that A. actinomycetemcomitans infection induces autophagy in JEKs, and this homeostatic process has a cytoprotective effect on the host cells during the early stages of infection.

Highlights

Macroautophagy is a cellular homeostatic process that sequesters and delivers intracellular components to a lysosomal degradation/recycling pathway [1].This cellular process has been investigated in numerous pathologies with heterogeneous etiologies, including inflammatory diseases of the oral cavity such as periodontitis [1,2,3]
A. actinomycetemcomitans has been strongly implicated in the development of rapidly progressing periodontal disease due to its ability to adhere, invade, and damage the junctional epithelium, adjacent
1) and induces autophagy in this periodontal context, junctional epithelium keratinocytes (JEKs) were incubated with A. actinomycetemcomitans processed immunofluorescence to visualize

Summary

Introduction

Macroautophagy (hereafter referred to as autophagy) is a cellular homeostatic process that sequesters and delivers intracellular components to a lysosomal degradation/recycling pathway [1]. This cellular process has been investigated in numerous pathologies with heterogeneous etiologies, including inflammatory diseases of the oral cavity such as periodontitis [1,2,3]. Autophagy begins when an isolation membrane sequesters and encloses cytosolic targets into a double-membrane vesicle, which subsequently elongates to form the autophagosome. The autophagosome fuses with lysosomes to form the autolysosome, leading to the degradation of its cargo [5,6]. Autophagy is essential in (i) the recognition, degradation, and selective elimination of bacterial pathogens (a phenomenon termed xenophagy), including Streptococcus pyogenes, Mycobacterium tuberculosis, Salmonella sp., and Listeria monocytogenes; as well as, in (ii) protection of the host cell from exotoxins and endotoxins released by pathogens, such as lipopolysaccharides (LPS) [4,8]

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