Activation of TLR2 Enhances Tight Junction Barrier in Epidermal Keratinocytes

Abstract

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.

Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

“…In agreement with a single previous study (23), peptidoglycan also induced increased TEER in keratinocytes. However, the changes observed were strikingly different from those seen with whole lysates.…”

“…Rather, they demonstrated activation of protein kinase C alpha (PKC␣), an enzyme involved in TJ assembly (22). However, their study used ligands rather than whole organism lysates, and they also measured TEER over shorter times than in the present study (23). We also show that use of ligands such as peptidoglycan increases TEER.…”

Epidermal tight junctions in health and disease

“…For example, stimulation of primary human keratinocytes with TLR2 ligands enhances the barrier function of TJs. 54,55 An increase in cldn-1, cldn-23, occludin, and ZO-1 expression was observed commensurate with changes in barrier function 55 while, Yuki et al 54 demonstrated occludin-phosphorylation by atypical protein kinase C (aPKC). Similarly, treatment of human epidermis wounded by repeated tape stripping (e.g., model of mechanical injury) with TLR2 agonists enhanced skin barrier recovery (as measured by transepidermal water loss [TEWL]).…”

Molecular Genetic of Atopic Dermatitis : An Update

“…(26) In addition, it is also reported that activation of toll like receptor (TLR)-2 plays a role on tight junction barrier via the protein kinase in the tight junction biogenesis. (27) Moreover, IL-4 and IL-13 also promote keratinocyte CLDN-1 expression. (27) Thus TLR-2 activation enhanced expression of CLDN-1 in keratinocytes of AD patients.…”

“…(27) Moreover, IL-4 and IL-13 also promote keratinocyte CLDN-1 expression. (27) Thus TLR-2 activation enhanced expression of CLDN-1 in keratinocytes of AD patients. In addition, TLR-2 lacking mice showed delayed or incomplete barrier recovery.…”