Kilsun Myoung,Eun-Jeong Choi,Hanbyul Kim,Hung Su Baek,Hyoung-June Kim,Won-Seok Park

Research and Development Center,Amorepacific,Korea

Hyunhee Kim,Jaeho Yeon

Amorepacific(Shanghai)R&I Center Co.,Ltd.,China

Abstract Psychological stress can impair epidermal barrier function by inhibiting the proliferation and differentiation of keratinocytes.In this study,the effect of stress hormone on skin microorganisms was confirmed through an in vitro experiment.Cortisol,a typical stress hormone,inhibited the growth of skin microbes,especially Staphylococcus epidermidis,which is a commensal skin microbe.And cortisol enhanced the adhesion of the pathogenic bacterium Staphylococcus aureus to keratinocytes.The fermented lysate of Lactiplantibacillus isolated from green tea leaves(LFL)affected the growth of skin microbes in the opposite manner to cortisol,and increased the expression of a keratinocyte differentiation marker that was suppressed by cortisol and S.aureus.Moreover,LFL inhibited the adhesion of S.aureus to keratinocytes.The modulating effect of LFL on the growth and adhesion of skin microbes was unaffected by the presence of cortisol.LFL also alleviated cell damage in reconstructed human epidermis caused by S.aureus.These results suggest that LFL may be useful as a cosmetic ingredient capable of controlling skin microbiome balance and protecting skin health against psychological stress.

Key words stress hormone;skin microbiome;lactobacillus ferment lysate

Psychological stress negatively affects epidermal barrier function,which is mediated by increasing levels of cortisol.[1]Cortisol is a steroid hormone produced in the adrenal cortex,is regulated by the hypothalamic-pituitary-adrenal axis(HPA),and exerts systemic and adaptive effects;it regulates blood pressure,causes inflammation,and reduces immune responses.[2,3]Many studies have demonstrated the negative effects of stress hormones,such as cortisol,on skin.Cortisol regulates gene expression and the structures of tight junctions,thereby impairing skinbarrier function and wound healing,which increases susceptibility to skin infections.[4,5]On the other hand,the microbiome barrier is the outermost skinbarrier layer,[6]where microbes in the skin contribute to the immune defense of the host through various mechanisms;[7]they inhibit the growth of pathogenic bacteria in a manner that consumes space,produces antimicrobial compounds,and modulate the local cytokine production of lymphocytes in the epidermis.Recent studies have suggested that the skin microbiome is also affected by psychological stress,as the skin microbiomes of stressed and unstressed individuals differ in terms of richness and diversity.[8]Psychological stress can modulate communication between the host and the microbiome,which impairs wound healing and/or promotes pathologic infection.[9]However,the relationships between cortisol(a stress hormones)and skin microbes are still not well understood.

The fermented lysate(cell debris whose outer membranes have been ruptured through chemical or physical processes)of Lactiplantibacillus plantarum(formerly known as Lactobacillus plantarum)APsulloc 331261 isolated from green tea leaves in the Dolsongi tea field on Jeju Island by the AMOREPACIFIC(Figure 1)was found to affect microbial growth and biofilm formation by inhibiting quorum sensing and interfering with bacterial communication systems.[10]Extracellular vesicles of L.plantarum APsulloc 331261 were shown to promote the expression of macrophage-characteristic cytokines and representative anti-inflammatory cytokines in human skin organ culture.[11]In this study,we investigated the effect of cortisol on skin microorganisms through in vitro experiments and examined whether or not the effects of cortisol can be alleviated through the use of the fermented lysate of L.plantarum APsulloc 331261(LFL).

Figure 1.Scanning Electron Microscope Image of L.plantarum APsulloc331261

1 Experiment

1.1 Bacterial strains and growth conditions

S.aureus(ATCC6538)and S.epidermidis(ATCC12228)were purchased from ATCC(American Type Culture Collection)and grown in BDTM Tryptic Soy Broth medium(Franklin Lakes,NJ,USA)for 18～24 h at 32 °C and then sub-cultured under the same conditions after dilution by a factor of 100.L.plantarum APsulloc 331261(deposit number:KCCM11179P)was grown in de Man Rogosa and Sharp(MRS)broth at 37 °C for 24 h,and then subcultured under the same conditions following 100-fold dilution.The fermented lysates of APsulloc,cells,and the culture supernatants were ruptured under high pressure(>1,000 bar).The fractions containing nano-sized particles were enriched by continuous filtration through a 0.22 μm membrane(Merck Millipore,MA,USA)and ultrafiltration using a membrane module rated to a 10 kDa nominal molecular weight limit(Merck Millipore).

1.2 Cell culture

Human primary keratinocytes(NHEKs;Lonza,Basel,Switzerland)within two or three passages were cultured in KBM-Gold medium supplemented with a KGM-Gold Bullet Kit(Lonza,Basel,Switzerland)at 37 °C and 5% CO2.Hydrocortisone,a component of the KGM-Gold Bullet kit,was not added to the medium for this assay.HaCaT cells(300493;Cell Line Service,Eppelheim,Germany)were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum(FBS)at 37 °C and 5% CO2.To coculture HaCaT cells withS.aureus,HaCaT cells were plated onto a 12-well plate,andS.aureuswas added to transwell inserts(Corning Incorporated,NY,USA)and cultured for 24 h.

1.3 Evaluating skin microbe growth

To evaluate bacterial growth,each strain was inoculated into the medium at a concentration of 1 × 106CFU/mL and incubated at 35 °C for 24 h.Growth rates(percentages)were then determined by measuring absorbance at 600 nm and comparing them against the experimental group to which the same amount of PBS had been added.For the cocultured system,S.aureuswas inoculated with 1 mL of the medium at a concentration of 1 × 106CFU/mL,after which a 0.4 μm cell culture insert(Falcon,USA)was placed on top,andS.epidermidiswas inoculated at a concentration of 1 × 106CFU/mL in 0.5 mL of medium on the upper layer.Growth rates were compared by measuring the absorbance at 600 nm after incubation for 24 h at 35 °C.

1.4 Adhesion to skin-cell assay

Confluent HaCaT cells were exposed toS.aureus(1 × 109CFU/mL)for 1 h.After incubation,the cells were washed twice with PBS to remove nonadherent bacteria.The cells were trypsinized,and serial dilution plate counts were used to assess the number of adherent bacteria.

1.5 Quantitative real-time PCR

To determine the relative mRNA expressions of selected genes,total RNA was prepared using TRIzol(Thermo Fisher Scientific,Waltham,MA,USA),while cDNA was synthesized using a Revertaid RT kit(Thermo Fisher Scientific).Quantitative real-time PCR was performed using TaqMan probes(Thermo Fisher Scientific)and a 7500 Fast Real-Time PCR system(Thermo Fisher Scientific).

1.6 Reconstructed human epidermis

RHE was purchased from Episkin(Skinethic,Lyon,France)and maintained according to the manufacturer’s instructions.The RHE was systemically treated with LFL(0.6%)for 48 h followed by culturing with S.aureus(107 CFU/0.5-cm2)for 48 h.The viabilities of cells in the RHE were determined by measuring the release of lactate dehydrogenase(LDH)in the RHE-conditioned medium using an LDH Cytotoxicity Assay Kit(Thermo Fisher Scientific).For histological purposes,replicate RHE sections were stained with H&E.

1.7 Statistical Analysis

Data are expressed as means ± standard deviations(SDs),with statistical significance determined using Student’s t-test.Statistical significance was set top<0.05.

2 Results

2.1 Cortisol affects the growth and adhesion of skin microbes

In this study,Staphylococcus aureusandStaphylococcus epidermidiswere u sed a s microorganisms found in skin.S.aureus is found in atopic skin lesions and can harm skin by secreting proteases and toxins.S.epidermidisinhibits the colonization and formation ofS.aureusbiofilms by secreting serine proteases;[12,13]it also induces the expression of antimicrobial peptides(AMPs)in human keratinocytes.[14]Therefore,S.epidermidiscan be considered as beneficial bacteria for the skin.

Bacterial growth was compared after inoculating each bacterium in a medium treated with cortisol,a typical stress hormone,for 24 h,the results of which are shown in Figure 2-a.TheS.epidermidisandS.aureuscultures were slightly inhibited by cortisol;however,cortisol significantly inhibited the growth of beneficial bacteria(S.epidermidis)when the two species were co-cultured in a transwell system,while it did not affectS.aureus(Figure 2-b).In addition,HaCaT cells(human immortalized keratinocytes)were directly treated with a high concentration ofS.aureus(109CFU/mL)and adherent bacteria were examined after 1 h following washing with phosphate buffered saline(PBS).Cortisol was found to increase the adhesion of S.aureus to skin cells(Figure 2-c).

Figure 2.Effect of cortisol on the growth and adhesion of skin microbes.(a)Cortisol slightly inhibits the growth of S.epidermidis and S.aureus.(b)Cortisol inhibits the growth of S.epidermidis but not of S.aureus in a co-cultured system.(c)Cortisol increases adhesion between S.aureus and HaCaT cells(＊ P<0.05 vs control)

2.2 LFL modulates the growth of skin microbes and adhesion to skin cells

Bacterial growth was compared after inoculating each bacterium in a medium treated with LFL for 24 h,the results of which are shown in Figure 3-a.LFL was observed to induce the growth of S.epidermidis and inhibit the growth of S.aureus at 1.2% concentration.Beneficial bacteria grew more when treated with LFL,even when the two species were co-cultured,while the growth of harmful bacteria was inhibited(Figure 3-b).This trend was also observed when cortisol was included in the co-cultured system(Figure 3-c).

Figure 3.Effect of LFL on the growth of skin microbes.(a)LFL induces the growth of S.epidermidis and inhibits the growth of S.aureus in independent culture systems,(b)in a co-culture system,and(c)even in the presence of cortisol.(＊ P<0.05,＊＊P<0.005,＊＊＊P<0.001 vs.control,# P<0.05,##P<0.005 vs.cortisol 10 μM treated group)

HaCaT cells were then treated with a high concentration ofS.aureus(109CFU/mL)and bacterial adherence to the cells was examined after 1 h.LFL was found to inhibit the adhesion ofS.aureusto HaCaT cells in a dose-dependent manner(Fig.4-a).While the adhesion ofS.aureusincreased when HaCaT cells were treated with cortisol,1.2% LFL reduced the level of adhesion,even in the presence of cortisol(Figure 4-b).

Figure 4.(a)Effect of LFL on the adhesion of S.aureus to HaCaT cells.(b)Cortisol induces the adhesion of bacteria to skin cells,which was improved by LFL treatment.(＊ P<0.05 vs.control,# P<0.05 vs.cortisol 10 μM treated group)

2.3 LFL improves keratinocyte differentiation inhibited by cortisol and S.aureus

We examined whether or not LFL is able to improve keratinocyte differentiation inhibited by cortisol.Normal human epidermal keratinocytes(NHEKs)were treated with cortisol(0.1 μmol/L)in the presence or absence of LFL for 4 d.LFL was found to improve the mRNA expression of KRT1 that had been lowered by cortisol(Figure 5-a).In addition,we investigated the promoting effect of LFL on the reduced keratinocyte differentiation byS.aureus.HaCaT cells were co-cultured with S.aureus for 24 h in a transwell system in which the cells and bacteria were separated by a 0.4 μm membrane.The observed decrease in KRT1 expression due toS.aureuswas restored in a dose-dependent manner by LFL treatment(Figure 5-b).These results indicate that LFL is able to ameliorate cortisol- as well asS.aureus-inhibited keratinocyte differentiation.

Figure 5.Effect of LFL on keratinocyte differentiation inhibited by cortisol or S.aureus.(a)LFL overcomes the reduction in KRT1(a keratinocyte differentiation marker)expression in cortisol-treated NHEKs as well as(b)in HaCaT cells co-cultured with S.aureus(＊ P<0.05,＊＊P<0.005,＊＊＊ P<0.001 vs.control,# P<0.05,vs.cortisol or S.aureus treated group)

LFL protects reconstructed human epidermis againstS.aureus.To investigate whether or not LFL protects the epidermis fromS.aureus,reconstructed human epidermis(RHE)was co-cultured withS.aureusfor 2 d in the presence or absence of LFL.We determined the lactate dehydrogenase(LDH)in the conditioned medium to assess the toxicity ofS.aureustoward RHE.Since LDH is released from damaged cells,the amount of LDH in the conditioned medium provides an indication of the level of cell damage caused byS.aureus.LFL treatment was found to improve LDH levels(Figure 6-a).Furthermore,we subjected the RHE to hematoxylin and eosin(H&E)staining(Figure 6-b),which revealed fewer attachedS.aureusin LFLtreated RHE.These results suggest that LFL protects the epidermis againstS.aureus.

Figure 6.Effect of LFL on reconstructed human epidermis(RHE)co-cultured with S.aureus.(a)LFL inhibits the toxic effect of S.aureus on RHE.(b)Hematoxylin and eosin stained RHE.(Scale bars:100 μm.＊＊＊ P<0.001 vs.control,### P<0.001 vs.S.aureus treated group)

3 Discussion

Cortisol,a stress hormone,is secreted by various cells and produced by keratinocytes and melanocytes in the epidermis.[15,16]Cortisol is also known to impair skin barrier function by inhibiting gene expression and tight junction structures.Moreover,cortisol affected the growth of skin microorganisms,especially by reducing the growth of beneficial bacteria in the skin.As shown in this study,the effects of certain substances,including cortisol,on skin microorganisms depend on the individual growth and co-culture conditions of the skin microorganisms.Since various microorganisms live on skin,evaluating their individual effects is important,but understanding the influence of the substance on the interactions between beneficial and deleterious strains is also important.Cortisol interfered with the growth ofS.epidermidis(which is known to be normal and beneficial to skin)in a co-culture system containingS.aureusandS.epidermidis.Moreover,cortisol promoted the adhesion ofS.aureusto skin cells,and both cortisol andS.aureusdecreased the expression of a differentiation marker in skin cells.Taken together,these results indicate that psychological stress promotes skin fragility caused by pathogenic bacteria.

On the other hand,probiotics contain live microorganisms and are used to promote a variety of health benefits to the host.[17]Recent studies have shown that the extracts and fermented products from probiotics are beneficial to skin health.[18]The lysates of Lactobacillus and Bifidobacterium have been shown to increase tight-junction barrier resistance.[19]The lysates of six Lactobacillus strains were found to stimulate the proliferation of keratinocytes,and some strains of L.plantarum accelerated re-epithelization by promoting keratinocyte migration.[20]LFL,a fermented lysate of L.plantarum APsulloc331261(deposit number:KCCM11179P),was also found to improve the expression of the KRT1 differentiation marker in keratinocytes that had been inhibited by cortisol orS.aureus.KRT1 is crucial for maintaining skin integrity and it participates in an inflammatory network.[21]S.aureusis known to inhibit the expression of such proteins and the differentiation of keratinocytes by stimulating the secretion of interluekin-6.[22]The lysates of certain probiotics reduce the secretion of pro-inflammatory cytokines from keratinocytes,[20]and our previous study showed that the extracellular vesicles of L.plantarum APsulloc331261 promote the release of anti-inflammatory cytokines in human monocytes.[11]Furthermore,treatment with LFL was shown to significantly ameliorate the toxic effects of S.aureus on RHE.Moreover,we also confirmed that treatment with LFL rebalances skin microbes.LFL was found to increase the growth of S.epidermidis and decrease the growth ofS.aureus;this trend was also observed in the co-cultured system,even in the presence of cortisol.LFL also inhibited the adhesion ofS.aureusto keratinocytes,both in the presence and absence of cortisol.These results provide a promising care option for the amelioration of skin disorders associated with psychological stress or pathogenic bacteria.Future studies will determine which LFL components exhibit these effects.