The role of commensal bacteria in the maintenance of the tight junction skin barrier

Abstract

The skin is a critical barrier that prevents water and heat loss from the body and prevents ingress of toxins and pathogens. This barrier comprises the physical (stratum corneum and tight junctions (TJs)), chemical and immune components. Recent studies have shown that the skin microbiota also plays a role in epidermal barrier function by preventing infection by pathogenic bacteria and enhancing skin immune responses. Some recent studies have also suggested that the physical barrier can be regulated by bacterial metabolites such as butyrate, from the fermentation of glycerol. However, to date most studies have been conducted in mouse models using consortia of bacteria and few studies have looked at individual bacteria and their effects on the barrier. The aim of this doctoral thesis was to identify human skin bacterial isolates that have the potential to regulate TJ barrier function possibly via production of butyrate and other metabolites. Skin swabs were collected from five healthy participants to create a biobank of skin commensal bacteria using culture-based methods. The isolated species were used in screening assays of TJ function in primary normal human epidermal keratinocytes (NHEKs). One of the enhancers of the intestinal barrier is butyrate, a bacterial metabolite from fermentation of carbohydrates. Similar mechanisms may also exist in the skin since some skin commensal bacteria have been shown to produce butyrate from the fermentation of glycerol. A significant increase in transepithelial electrical resistance (TEER) was observed in NHEKs treated with 4 mM butyrate for 24 h compared to untreated cells. However, treatment with lower concentrations (2 and 0.5 mM) induced no change in TEER. Staphylococcal isolates were able to ferment glycerol as a source of carbohydrate, but the concentrations of butyrate produced were low. In keeping with this, the glycerol-fermented supernatants of all of the tested species (S. epidermidis, S. hominis, S. capitis S. lugdunensis, and S. caprae) had no impact on TJ barrier function in NHEKs. The effect of cell-free supernatants of different skin commensal isolates grown in tryptic soy broth (to maximise the production of metabolites), on TJ integrity were investigated in NHEKs. The supernatants from S. hominis, S. aureus and M. luteus significantly increased the TJ barrier integrity as measured by TEER. However, the supernatants of S. epidermidis, S. capitis S. lugdunensis, and S. caprae had no impact on TJs. Metabolite identification by mass spectrometry showed that all the species produced multiple metabolites, with many in common between the different isolates. However, tryptophol, a known enhancer of the intestinal TJ barrier was detected only in the supernatants of the TJ barrier-enhancing species. There was almost zero production by the ineffective species. Further investigation sought to determine whether this was a general effect of a certain species, or whether there were any strain-specific effects. Therefore, other isolates of S. hominis were investigated for their ability to increase TJ function. Only specific isolates of S. hominis could regulate TJ function in NHEKs and this correlated with the production of tryptophol. Furthermore, an isolate that was unable to produce tryptophol or modify TJ function, was sequenced and found to contain mutations in a gene for tryptophol production. This study shows for the first time that specific bacteria within the skin microbiota can produce metabolites with the capacity to regulate human keratinocytes tight junction function. The identification of such activities could be utilised in the development of therapeutic approaches to different skin conditions where the barrier is known to be aberrant.