Bifidobacterium longum extract exerts pro‐differentiating effects on human epidermal keratinocytes, in vitro (2016)

The term “probiotic” was popularised more than 20 years ago,1 and more recently defined as “living microorganisms which, when administered in adequate amounts, confer a health effect on the host.”2 Traditionally, probiotics are shown to be beneficial in numerous inflammatory and allergic, mostly gastrointestinal, diseases.3 However, orally consumed probiotics were also found to improve conditions of other organs, skin includeds1,s2,4 Importantly, recent intriguing data suggest that microorganism-based topical formulations are also effective in various skin conditions. Indeed, locally applied living Lactobacillus plantarum suppressed inflammation and accelerated wound healing in patients with chronic infected leg ulcers.5 Topical formulation containing non-pathogenic Gram-negative bacterium Vitreoscilla filiformis lysates improved symptoms and promoted the repair of the skin barrier in atopic dermatitis patients.6 Of further importance, a specific Bifidobacterium longum (B. longum) extract from CLR Richter laboratories (Berlin, Germany), when applied as a cream to volunteers with reactive skin, decreased cutaneous sensitivity, increased skin resistance, and decreased skin dryness.7 In addition, this B. longum extract inhibited neuropeptide-induced inflammatory cutaneous response in organ-cultured human skin and inhibited the release of neuropeptides from capsaicin-stimulated cultured sensory neurons.7 Epidermal keratinocytes are key players in establishing and maintaining the complex cutaneous barrier homoeostasiss3. However, very limited information is available on the direct actions of beneficial microorganisms on these cells. Therefore, in the current study, as a continuation of previously published work,7 the effects of various preparations of B. longum, one of the most widely used probiotics, were investigated on cellular functions of primary normal human epidermal keratinocytes (NHEKs). In this study, the following B. longum preparations (BL) were used to examine their effect on the viability, differentiation, antimicrobial peptide production and calcium homoeostasis: First, the effect of the various BL preparations on the growth of the keratinocytes was assessed. Treatment of preconfluent (i.e. proliferating) NHEKs with various concentrations of BL for up to 72 hours did not modify the proliferation (as assessed by MTT assay) of the cells (Fig. S1). In addition, as MTT assay determines the viable cell number, it can also be concluded that none of the BL extract affected the viability of the NHEKs. Therefore, throughout the experiments, the highest BL extract concentrations were employed (3 v/v %). Next, the effects of BL preparations on the epidermal differentiation process were investigated. For this, the expression of various differentiation markerss3 was compared in proliferating (pre-confluent) and differentiating NHEKs; the latter was achieved by culturing the cells for 2 days after reaching confluence (high cell density-induced differentiation model;s4. As expected (Fig. 1a), the expression of differentiation markers (KRT1, TGM1) was significantly higher in the postconfluent cultures (when compared with the pre-confluent ones). Importantly, treatment of preconfluent NHEK with BL/81 increased the expression of all differentiation markers (Fig. 1b–e) to much higher levels than seen in the control (untreated) postconfluent cells (Fig. 1a,c–e). BL/84 also up-regulated the levels of keratin (KRT) 1, KRT10, and transglutaminase 1 (TGM1). However, it did not induce an increase in filaggrin, loricrin and involucrin. Interestingly, BL/64 was ineffective in these assays. The effects of BL on the expression of certain antimicrobial peptides (AMP) (β defensin 1 –BDEF- and Cathepsin S) and molecules with pivotal role in wound healing (Cathepsins B, D and H)s5,s6,8 were also examined. Interestingly, levels of most of these markers (the only exception was Cathepsin S) were significantly elevated in postconfluent differentiating NHEKs (Fig. 2a,c,d). Therefore, in the subsequent experiments, only preconfluent NHEKs were treated by BL. As shown in Fig. 2, BL/81 and BL/84 (with BL/81 being more efficient) markedly elevated the levels of mRNA transcripts and released amount of all (BL/81) or most (BL/84) molecules, BL/64, again, proved to be almost ineffective significantly up-regulating BDEF-specific mRNAs albeit not causing the release of the AMP. Although the exact signalling pathway(s) that mediate the above effects remain to be determined, we have preliminarily attempted to assess the effect of BL extracts on the intracellular calcium levels, the elevation of which is one of the key factors that induce differentiation in NHEKss7. Importantly, only BL/81 (but not BL/84 or BL/64) was effective in elevating the calcium concentration of the cells. This effect was completely absent when calcium was removed from the medium suggesting that BL/81 induces a calcium influx from the extracellular medium (Fig. 2e,f). The intriguing findings of this study provide the first evidence that certain B. longum extract preparations exhibit significant pro-differentiating and potentially pro-regenerating (cathepsins’ expression) actions in NHEKs, together with an increased expression of BDEF, one key actor of cutaneous innate immune response. In the single available previous study, various probiotics (certain B. longum and Lactobacillus strains) were shown to increase the expression of multiple tight-junction proteins of NHEKs and enhanced barrier function (a decreased transepidermal water loss).9 These data, therefore, collectively suggest that the beneficial effects of topical microbial formulations derived from bacteria may be mediated, at least in part, by direct modulation of epidermal keratinocytes’ differentiation, most probably via inducing Ca2+ influx. The current work was supported by Hungarian research grants (LP2011-003/2015, OTKA 101761, OTKA 105369, and TÁMOP-4.2.2./A-11/1/KONV-2012-0025) and by L'Oréal Research and Innovation. AGS, JSP and NV performed the experiments, AG, OJ, CB, LB and TB participated in designed the research study and evaluation and interpretation of the obtained data. MA and IJ contributed human samples for the experiments. AGS, AG and TB also contributed to writing the manuscript. The authors have declared no conflicting interests. Figure S1 Effects of bacterial extract on proliferation and viability of NHEKs Data S1 Supplementary Materials and Methods Data S2 Supplementary References Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.