Oral Mucosal Epithelial Cells

Abstract

Cellular Phenotype and Apoptosis: The function of epithelial tissues is the protection of the organism from chemical, microbial, and physical challenges which is indispensable for viability. To fulfill this task, oral epithelial cells follow a strongly regulated scheme of differentiation that results in the formation of structural proteins that manage the integrity of epithelial tissues and operate as a barrier. Oral epithelial cells are connected by various transmembrane proteins with specialized structures and functions. Keratin filaments adhere to the plasma membrane by desmosomes building a three-dimensional matrix. Cell-Cell Contacts and Bacterial Influence: It is known that pathogenic oral bacteria are able to affect the expression and configuration of cell-cell junctions. Human keratinocytes up-regulate immune-modulatory receptors upon stimulation with bacterial components. Periodontal pathogens including P. gingivalis are able to inhibit oral epithelial innate immune responses through various mechanisms and to escape from host immune reaction, which supports the persistence of periodontitis and furthermore is able to affect the epithelial barrier function by altering expression and distribution of cell-cell interactions including tight junctions (TJs) and adherens junctions (AJs). In the pathogenesis of periodontitis a highly organized biofilm community shifts from symbiosis to dysbiosis which results in destructive local inflammatory reactions. Cellular Receptors: Cell-surface located toll like receptors (TLRs) and cytoplasmatic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) belong to the pattern recognition receptors (PRRs). PRRs recognize microbial parts that represent pathogen-associated molecular patterns (PAMPs). A multimeric complex of proteins known as inflammasome, which is a subset of NLRs, assembles after activation and proceeds to pro-inflammatory cytokine release. Cytokine Production and Release: Cytokines and bacterial products may lead to host cell mediated tissue destruction. Keratinocytes are able to produce diverse pro-inflammatory cytokines and chemokines, including interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-α. Infection by pathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) can induce a differentiated production of these cytokines. Immuno-modulation, Bacterial Infection, and Cancer Cells: There is a known association between bacterial infection and cancer. Bacterial components are able to up-regulate immune-modulatory receptors on cancer cells. Interactions of bacteria with tumor cells could support malignant transformation an environment with deficient immune regulation. The aim of this review is to present a set of molecular mechanisms of oral epithelial cells and their reactions to a number of toxic influences.

Keywords: cancer; cytokines; differentiation; immuno-modulation; infection; oral epithelial cells; receptors.

Figure 1

Cytokeratin distribution patterns. Cytokeratin (CK) distribution patterns in oral epithelia. Modified according to Pöllänen et al. (6).

Figure 2

Structure of epithelial cell-cell contacts. Model of cellular junctions, modified according to Metha and Malik (24). Arrangement of junctions comprising tight junctions, adherens junctions, gap junctions, and integrin. Occludin, claudins, and junctional adhesion molecules (JAMs) required for tight junctions, whereas vascular endothelial (VE)-cadherin forms adherens junctions. Connexins are part of gap junctions. The extracellular domains of occludin, claudins, and VE-cadherin maintain cell-cell contacts. Intracellular domains provide junctional stability linking to the actin cytoskeleton via catenins (β,β-catenin, α,α-catenin; γ,γ-catenin; p120, p120-catenin) or zonula occludens-1 protein (ZO-1). Gap junctions are responsible for fast exchange of information by low-molecular-mass second messengers such as Ca²⁺ and IP₃ between contiguous cells. Integrin receptors link endothelial cells with the extracellular matrix (ECM) through matrix proteins like fibronectin (FN) or vitronectin (VN). The cytosolic domains of integrins are linked to the actin cytoskeleton through the proteins talin and vinculin (Vin), involved in integrin-mediated signaling.

Figure 3

Tight junction proteins of primary keratinocytes after infection with P. gingivalis. Immunostaining of the tight junction proteins in primary human gingival keratinocytes, claudin 1 (a,d) claudin 2 (b,e) and occludin (c,f); (a–c) cells in culture medium; (d–f) cells infected apically plus basolaterally with Pophyromonas gingivalis (P. gingivalis) W83 (MOI 10⁴) for 4 h. Arrows (e,f) show curved occludin strains in the walls of non-infected cells, in infected cells the arrows point to occludin aggregations, scale bar = 20 μm (71).

Figure 4

Cellular location of toll like receptors (TLRs) and the identity of their ligands/agonists. The stimulation of surface TLRs (TLR-2, TLR-4, and TLR-5) with appropriate ligands results in the activation of nuclear factor (NF)-κB. The ensuing increase in levels of pro-inflammatory cytokines and the influx of inflammatory cells then provides an environment, which protects against both virus and bacterial challenge. Activation of intracellular TLRs (TLR-3, TLR-7, TLR-8, and TLR-9) leads to interferon regulating factor (IRF) activation and the production of Type 1 interferons (IFNs) and pro-inflammatory cytokines, again providing an environment not conducive for pathogens (100).

Figure 5

Toll like receptor (TLR)-signaling pathways. TLR-4, TLR-5, and the heterodimers TLR-1/TLR-2 and TLR-2/TLR-6 are located on the cell surface where they are activated by the appropriate ligand. Conversely, TLR-3, TLR-7, TLR-8, and TLR-9 are located within endosomal compartments of the cell and recognize microbial and viral nucleic acids. Stimulation of TLR-1/TLR-2, TLR-2/TLR-6, TLR-4, and TLR-5 leads to the engagement of myeloid differentiation primary response protein (MyD88) and MYD88-adapter-like protein (MAL) with the toll/interleukin-1 receptor (TIR) domain-containing adapter proteins. This stimulates downstream signaling pathways that involve the interactions between IL-1R-associated kinases (IRAKs) and the adapter molecules tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and activates mitogen-activated protein kinases (MAPKs) JUN N-terminal kinases (JNK) and p38. Activation of these kinases leads to the activation of transcriptional factors such as nuclear factor-κB (NF-κB), cyclic adenosine mono phosphate (AMP)-responsive element binding protein (CREB), and activator protein-1 (AP-1). A major consequence of activation of surface TLRs is the induction of pro-inflammatory cytokines. Activation of TLR-7, TLR-8, and TLR-9 also leads to the engagement of MyD88, MAL, IRAKs, and NF-κB inhibitor kinase (IKK)α, however, interferon-regulatory factors (IRFs) are activated, which leads to the production of type 1 interferons (IFN). Stimulation of TLR-3 results in the association of TIR domain-containing adapter protein inducing IFNβ (TRIF). This leads to the downstream signaling of TNF receptor-associated factors (TRAFs) and IKK leading to the activation of IRF3 and the production of type 1 IFNs (100).

Figure 6

Model of Nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 signaling cascades. NOD1 and NOD2 recognize bacterial peptidoglycans (PGNs), (iE-DAP), and muramyl dipeptide (MDP), respectively. Following ligand sensing the NODs recruit their common adaptor receptor-interacting serine/threonine-protein kinase (RIP)2 by caspase activation and recruitment domains (CARD)–CARD interactions and induce RIP2 to undergo phosphorylation. The members of the tumor necrosis factor receptor-associated factor (TRAF) family (TRAF2, TRAF5, and TRAF6), the inhibitor of apoptosis (IAP) family (XIAP, cIAP1, and cIAP2), and the B-cell lymphoma (BCL)2 family (BID) bind to RIP2 and facilitate its ubiquitination allowing the recruitment of transforming growth factor-β-activated kinase (TAK)1 and ubiquitinated nuclear factor (NF-κB) essential modulator (NEMO) to the nodosome. On one hand, NEMO instigates activation of the canonical NF-κB pathway by phosphorylating NF-κB inhibitor kinase (IKK)α and IKKβ, by inducing nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) phosphorylation and proteasomal degradation, and by freeing p50 and p65 NF-κB subunits. On the other hand, transforming growth factor-β-activated kinase 1 (TAK1) recruits, transforming growth factor-β-activated kinase binding protein (TAB)1 and TAB2/3 inducing both (p38, extracellular-signal Regulated Kinases = ERK, and JUN N-terminal kinases = JNK) mitogen activated protein kinases (MAPK) and NF-κB activation. Stimulation of both arms culminates in the induction of anti-microbial peptides (AMPs), cytokines, and chemokines. The formation of the nodosome promotes autophagy and conversely, a fully functional autophagy machinery helps in signal transduction through the nodosome. Autophagy-related protein (ATG)16L1 along with ATG5 and ATG12 is required for autophagosome formation, however, independently of its autophagy functions, ATG16L1 negatively regulates NOD/RIP2 signaling (136).

Figure 7

Microbial activation of the inflammasomes. Pathogenic microorganisms activate the inflammasomes through multiple agonists and pathways. Salmonells typhimurium (S. typhimurium), Legionells pneumophila (L. pneumophila), and Mycobacterium tuberculosis (M. tuberculosis) reside within the host cell phagosome and are capable of activating inflammasomes through secreted flagellin, effectors, or undefined NACHT, LRR, and PYD domains-containing protein (NLRP)3 agonists. NACHT = NAIP, neuronal apoptosis inhibitor protein; C2TA, class 2 transcription activator, of the MHC; HET-E, heterokaryon incompatibility; TP1, telomerase-associated protein 1; LRR, leucine-rich repeat; PYD, PYRIN domain. Francisella tularensis (F. tularensis) and Listeria monocytogenes (L. monocytogenes), which escape the phagosome activate absent in melanoma (AIM)2 that senses cytosolic deoxyribonucleic acid (DNA). Bacillus anthracis (B. anthracis) lethal toxin activates the NLRP1 inflammasome. Candida. albicans (C. albicans) and hemozoin activate NLRP3 through Spleen tyrosine kinase (SYK) signaling. Viral-mediated inflammasome activation is heavily dependent on the detection of nucleic acids by NLRP3, AIM2, and retinoic acid-inducible gene (RIG)-I. Dotted lines indicate signaling through an unknown mechanism (161).

Figure 8

Mechanism for canonical NACHT, LRR and PYD domains-containing protein (NLRP)3- inflammasome activation. NACHT = NAIP, neuronal apoptosis inhibitor protein; C2TA, class 2 transcription activator, of the MHC; HET-E, heterokaryon incompatibility; TP1, telomerase-associated protein 1; LRR, leucine-rich repeat; PYD, PYRIN domain. Various pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) provide the signal 2 required to assemble and activate the NLRP3 inflammasome comprised of NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1. Although the precise mechanism leading to NLRP3 activation is still controversial, it is speculated that potassium ion K⁺ efflux may be the common cellular response that triggers inflammasome activation. However, this notion has not been fully verified and it is possible that an unidentified or intermediate adaptor may be required for transmitting signals between K⁺ efflux and the NLRP3 inflammasome. Crystals and particulate DAMPs enter the cell via endocytosis directly inducing K⁺ efflux and NLRP3-inflammasome formation. In addition, the endo- lysosomes carrying these DAMPs undergo lysosomal rupture and release cathepsin B, which acts as an intracellular DAMP and can induce K⁺ efflux. However, contradicting studies indicate that lysosomal rupture may cause K⁺ efflux and inflammasome activation even in the absence of cathepsin B. Adenosin triphosphate (ATP) binds to the P2X purinoceptor 7 (P2X7) receptor on the cell membrane and causes opening of the annexin 1 (PANX1) channels allowing K⁺ efflux and influx of any PAMPs and DAMPs present in the extracellular space. PAMPs such as pore-forming toxins activate the NLRP3 inflammasome and facilitate K⁺ efflux. Liposomes instigate Ca²⁺ influx through opening of (TRPM2) channels. Accumulation of excessive Ca²⁺ in the cytosol causes mitochondrial dysfunction and release of mitochondrial reactive oxygen species (mtROS) and oxidized mitochondrial deoxyribonucleic acid (mtDNA), which may activate the NLRP3 inflammasome either directly or by inducing K⁺ efflux. Clearance of distressed mitochondria by mitophagy serves to evade such inflammasome activation. Mitochondrial cardiolipin binds to NLRP3 and is required for the NLRP3-inflammasome activation. Following NLRP3-inflammasome assembly, caspase-1 undergoes proximity driven proteolytic cleavage and further processes pro-interleukin (IL)-18 and pro-IL-1β into their mature active forms. Activation of the NLRP3-caspase-1 axis results in inflammation and pyroptotic cell death (136).