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Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation Yi-Fan Hong a,c,1 , Hea young Lee b,1 , Bong Jun Jung a , Soojin Jang e , Dae Kyun Chung a,c,d,∗∗ , Hangeun Kim d,∗ a
Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea Cancer Research Institute, Seoul National University, College of Medicine, Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea c Skin Biotechnology Center, Gyeonggi Biocenter, Suwon, Gyeonggi-do 443-766, Republic of Korea d RNA Inc., #308 College of Life Science, Kyung Hee University, Yongin 449-701, Republic of Korea e Institute Pasteur Korea, Sampyeong-dong, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea b
a r t i c l e
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Article history: Received 15 January 2015 Received in revised form 30 April 2015 Accepted 22 May 2015 Available online xxx Keywords: Matrix metalloproteinases-1 Lipoteichoic acid Reactive oxygen species Photo-aging Lactobacillus plantarum Human dermal fibroblasts
a b s t r a c t Background: Ultraviolet (UV) irradiation from the sun is the primary environmental factor that causes human skin aging. UV irradiation induces the expressions of matrix metalloproteinases (MMPs) and extracellular matrix degrading enzymes. Among the members of MMP family, MMP-1 is an interstitial collagenase that degrades the collagen triple helix. We investigated the effect of Lactobacillus plantarum, well known as useful microorganism, on UV-induced-MMP-1 expression in human dermal fibroblasts. Methods: Human dermal fibroblasts (HDF) was pre-stimulated with lipoteichoic acid isolated from L. plantarum followed by UV irradiation. Secreted protein level of MMP-1 was evaluated by Western blot analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-B) from the cell lysates was also examined by western blotting. Electrophoretic mobility-shift assay (EMSA) was used to detect the activated transcription factor, AP-1 and NF-B. The detection of type 1 procollagen was carried with Procollagen type 1 C-peptide (PIP) EIA kit. The generation of reactive oxygen species (ROS) by LTA and UV irradiation was examined by Griess reagent assay and fluorescence microscope. Results: We found that lipoteichoic acid (LTA), a cell-wall component of Gram-positive bacteria, isolated from L. plantarum, inhibited MMP-1 expression. Pretreatment with LTA from L. plantarum (pLTA) reduced MMP-1 expression in a dose-dependent manner and inhibited activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). It also led to the inhibition of DNA binding activity of activator protein-1 (AP-1) and of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-B). Furthermore, LTA promoted type 1 procollagen synthesis and reduced the generation of ROS induced by UV irradiation. Conclusion: Our study demonstrates that pLTA inhibits degradation of collagen and promotes its synthesis and that pLTA contributes to a decrease in ROS production. Therefore, pLTA from L. plantarum has potential abilities to prevent and treat skin photo-aging. © 2015 Published by Elsevier Ltd.
Abbreviations: UV, ultraviolet; MMP, matrix metalloproteinase; LTA, lipoteichoic acid; pLTA, Lactobacillus plantarum lipoteichoic acid; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinases; AP-1, activator protein-1; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cell; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +82 31 201 2487; fax: +82 31 202 3461. ∗∗ Corresponding author at: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea. Tel.: +82 31 201 2465; fax: +82 31 202 8333. E-mail addresses: [email protected] (D.K. Chung), [email protected] (H. Kim). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2015.05.019 0161-5890/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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1. Introduction Human skin is often directly exposed to the environment, especially to sun. Therefore, skin undergoes not only chronological aging, but also photo-aging (Fisher et al., 2002). Both are cutaneous and cause laxity, wrinkles and a decrease in elasticity. However, their molecular mechanisms and morphological changes are distinct (Helfrich et al., 2008; Rittie and Fisher, 2002). Photo-aging is also called extrinsic aging and is caused by external factors such as ultraviolet (UV) radiation. UV exposure can cause pigmented and wrinkled skin. UV irradiation induces the expression of MMPs and triggers cleavage of cell-surface receptors and protein kinase signal transduction pathways and activation of transcription factors (Fisher et al., 2002). MMPs degrade all kinds of extracellular matrix proteins and damage connective tissues (Kahari and SaarialhoKere, 1997) and induce the release of cytokines and chemokines and cleave cell surface receptors (Van and Libert, 2007). They also play a role in tissue remodeling and disease processes such as arthritis (Burrage et al., 2006). MMP-1, interstitial collagenase, is a member of the MMP family and is responsible for the degradation of collagen fibers (Moon et al., 2004). An increase in MMP-1 expression causes skin connective tissue damage. Collagen is the richest protein of the dermal connective tissue and is broken down by MMP-1. Among the 29 types of collagen, type 1 collagen is one of the most abundant proteins in the human body (Di Lullo et al., 2002). UV irradiation impairs synthesis of new type 1 collagen. Degradation and damage of collagen is the major feature of photo-aging (Brennan et al., 2003). Mitogen-activated protein kinase (MAPK) pathways are activated in response to extracellular stimuli including UV irradiation (Fisher et al., 1998), leading to the activation of activator protein 1 (AP-1) (Fisher and Voorhees, 1998; Kim et al., 2005; Whitmarsh and Davis, 1996). AP-1 is a transcription factor that regulates MMP1 gene expression in response to various stimuli (Karin, 1995; Wenk et al., 1999). Down-regulation of type 1 collagen and procollagen is mediated by UV-induced AP-1 (Fisher et al., 2002). NF-B activation occurs in response to various stimuli such as stress, free radicals and ultraviolet irradiation. UV-induced NF-B activation leads to the induction of various genes including MMP-1 and the production of MMP-1 in skin fibroblasts (Brenneisen et al., 2002; Tanaka et al., 2005). Reactive oxygen species (ROS) are free radicals and are naturally produced during cellular metabolism. The production of ROS is significantly increased by environmental stresses including UV irradiation, leading to the induction of MMPs (Bond et al., 1999). ROS causes oxidative damage and are a major contributor to aging (Muller et al., 2007). Lipoteichoic acid (LTA) is a major cell-wall component of Gram-positive bacteria and has antigenic properties that stimulate specific immune responses. LTA is known to increase the activity of MMP-9, play an active role in otitis media, inhibit melanogenesis in B16F10 mouse melanoma cells, protect UV-induced carcinogenesis and increases skin mast cell antimicrobial activity against vaccinia viruses (Park et al., 2012; Wang et al., 2012; Weill et al., 2013; Kim et al., 2015). In addition, tolerance using LTA has been used to introduce to protect against pro-inflammation and other diseases (Kim et al., 2008b; Ellingsen et al., 2002). Thus, to identify the role of LTA (pLTA) isolated from Lactobacillus plantarum on UV-mediated photo-aging, MMP-1 expression and ROS generation by pLTA was examined.
2. Materials and methods 2.1. Cell culture Human dermal fibroblasts (HDF) were kindly provided by Dr. Y. S. Son (Kyung Hee University, Korea). NDFs were grown in FBM
with FGM-2 (Lonza, Basel, Switzerland) in humidified incubator of 5% CO2 at 37 ◦ C. 2.2. Preparation and treatment of LTA Highly purified LTA was isolated from L. plantarum by n-butanol extraction. The purity of the purified LTA was determined by measuring the protein and endotoxin contents through the conventional silver staining after polyacrylamide gel electrophoresis and through the Limulus amebocyte lysate (LAL) assay (BioWhittaker, Walkersville, MD), respectively. DNA or RNA contamination was assessed by measuring UV absorption at 260 and 280 nm, and no nucleic acids were detected. To treat the cells, LTA was diluted with non-pyrogenic water at the indicated concentrations. 2.3. UV irradiation Vilber Lourmat Bio-sun lamps (Marine, France) were used as a UV source. The emission spectrum was between 275 and 380 nm (peak spectrum, 312 nm) and UV strength was measured by UV meter (Waldmann, Germany). Before UV irradiation, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and medium was replaced by DPBS. After UV irradiation, the cells were washed with DPBS and cultured for 24 h in the serum-free media. 2.4. Western blot analysis For detection of MMP-1, cultured supernatants were harvested and for detection of phospho-ERK1/ERK2 and phospho-JNK, cells were lysed with RIPA buffer containing proteinase inhibitors. Insoluble debris was removed by centrifugation at 12,000 rpm for 10 min. For detection of NF-B, cells were lysed using nuclear extraction kit (Marligen Biosciences, Rockville, MD) and nuclear protein was extracted. Protein concentration was quantified using Bradford assay reagent (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved on SDS–polyacrylamide gels (10–15%) and then electrophoretically transferred onto nitrocellulose membrane (100 V, 2 h). Membranes were subsequently blocked with 5% skim milk in TBS-T (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, and 0.05% Tween 20) and incubated with the indicated antibodies. Commercially available anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK, anti-phospho-c-Jun, anti-phospho-p65, and anti--actin were purchased from Cell Signaling Technology (Boston, MA). Blotting proteins were visualized by enhanced chemiluminescence (ECL) reagents (GE Healthcare). 2.5. Electrophoretic mobility shift assay (EMSA) Fibroblasts were pretreated with the indicated concentrations of pLTA for 20 h, washed with DPBS and irradiated with 75 mJ/cm2 of UV in DPBS. To prepare nuclear fractions, cells were washed with DPBS and nucleus was extracted using nuclear extraction kit (Marligen Biosciences, Rockville, MD). The cytoplasmic extracts were removed by centrifugation and nuclear pellet were washed and solubilized with extraction buffer. AP-1 and NF-B consensus oligonucleotides (Jany et al., 1995; Husain et al., 2007) were labeled with 10 Ci/ml [␥-32 P] ATP by T4 polynucleotide kinase (Fermentas, Burlington, ON) at 37 ◦ C for 30 min. The sequence of the AP-1 and NF-B oligonucleotide is as follows: AP-1 sense 5 -CGCTTGATGAGTCAGCCGGAA3 , AP-1 antisense 5 -TTCCGGCTGACTCATCAAGCG-3 , NF-B sense 5 -AGTTGAGGGGACTTTCCCAGGC-3 , NF-B antisense 5 GCCTGGGAAAGTCCCCTCAACT-3 . Because unincorporated labeled nucleotides cause non-specific binding, purification was performed to remove them using PROBER Probe DNA purifying kit (Intron Biotechnology, Korea). After determination of nuclear
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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protein concentration, the reaction was performed according to the manufacturer’s instructions (Gel Shift Assay System, Promega, Madison, WI). The equal amounts of nuclear protein were added to the gel shift binding reaction mixtures and incubated for 10 min at room temperature. Then isotope-labeled oligonucleotides were added and incubated for 20 min at room temperature. This DNA–protein complex was loaded onto 6% non-denature gels. Gels were dried and labeled complexes were visualized by autoradiography.
2.6. Measurement of type 1 procollagen synthesis HDFs were pretreated with the indicated concentrations of pLTA for 72 h and supernatants were harvested. The detection of type 1 procollagen was performed using Procollagen type 1 C-peptide (PIP) EIA kit (TAKARA, Kyoto, Japan). Collagens are synthesized as precursor molecules called procollagens. These contain additional peptide, propeptide, at the amino-terminal and the carboxy-terminal ends. The amount of the free propeptides reflects the amount of collagen molecules synthesized. Quantitative detection of collagen synthesis was using polyclonal antibodies by recognizing procollagen type 1 carboxy-terminal peptide (PIP). TGF- was used as a positive control.
2.7. Cell viability The cytoxicity of pLTA on the cell was determined by WST1 assay (TAKARA BIO, Japan) which is based on the cleavage of tetrazolium salts by mitochondrial dehydrogenases in viable cells. The numbers of viable cells are measured by the detection of cleaved tetrazolium salts that added into a medium. HDFs were washed with DPBS and replaced with serum free media. Then pLTA was pretreated with indicated concentrations for 20 h. Then 10 l of premix WST-1 was added in 100 l of culture medium. After incubating for 2 h, absorbance was measured using ELISA reader. The formazen dye formed by metabolically active cell can be quantitated by measuring its absorbance. The measuring wavelength is 420 nm and the reference wavelength is 600 nm.
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3. Results 3.1. Ultraviolet (UV) radiation-induced MMP-1 expression and lipoteichoic acid from L. plantarum (pLTA) inhibited UV-induced MMP-1 expression in a dose-dependent manner MMP-1 expression was induced by UV irradiation with increasing intensities (0–100 mJ/cm2 ). After incubation for 24 h, culture media was harvested to determine MMP-1 protein levels by western blot analysis. As a result, UV irradiation-induced MMP-1 expression compared with non-irradiated cells, and this induction was dose-dependent (Fig. 1A). To elucidate the effect of pLTA on UV-induced MMP-1 expression, HDFs were pretreated with various concentrations of pLTA for 20 h and then irradiated with 75 mJ/cm2 of UV. After incubation for 24 h, culture media was harvested to determine MMP-1 protein levels by western blot analysis. As we expected, pretreatment with pLTA inhibited UV-induced MMP-1 expression in a dose-dependent manner (Fig. 1B). These results suggest that pLTA has an inhibitory effect on collagen degradation. 3.2. UV-induced MMP-1 expression was mediated by ERK, JNK and NF-B activation, and pLTA inhibited their activation To investigate the factors which are mediated on UV-induced MMP-1 expression, some specific inhibitors were pretreated, and then 75 mJ/cm2 of UV was irradiated. ERK, JNK and NF-B specific inhibitors decreased the expression of UV-induced MMP-1 (Fig. 2A). Therefore, this data suggests that ERK, JNK and NF-B mediated UV-induced MMP-1 expression. Two MAPKs, ERK and JNK were activated by UV irradiation and the activation was high at 60 and 30 min, respectively, after UV irradiation (Fig. 2B and C). We investigated the effects of pLTA on UV-induced ERK and JNK activation. HDFs were pretreated with pLTA for 20 h and then irradiated with 75 mJ/cm2 of UV. Levels of phospho and total ERK and JNK proteins were determined by western blot analysis at 60 and 30 min after UV treatment, respectively. Pretreatment with pLTA inhibited phospho-ERK and phospho-JNK level without affecting
2.8. Detection of reactive oxygen species generation HDFs were pretreated with NAC or pLTA for 20 h in serum-free media and then washed with DPBS. 1 M of H2 O2 or 75 mJ/cm2 of UV was treated to induce ROS generation. After washing the cells, 20 M of DCF-DA in HBSS was added and incubated at 37 ◦ C for 30 min. The cells were washed three times with DPBS and warm HBSS was added. The level of ROS was immediately analyzed using microplate readers (TECAN, Switzerland) and imaged using fluorescence microscope (Leica, Germany). The detection wavelength is 485 nm and reference wavelength is 535 nm. All experiments were performed in triplicate, and reactive oxygen species (ROS) levels are described as fold increase versus the non-treated control.
2.9. Statistical analysis All experiments were performed at least three times using triplicates. The data shown are representative results of the means ± standard deviation of triplicate experiments. Differences were judged to be statistically significant when the p-value was less than 0.05.
Fig. 1. Ultraviolet (UV) radiation induces MMP-1 expression and lipoteichoic acid from L. plantarum (pLTA) inhibits UV-induced MMP-1 expression in a dosedependent manner. Human dermal fibroblasts (HDFs) were cultured in FGM-2 until 80% confluency and washed with DPBS. (A) Cells were irradiated with increasing intensities (0–100 mJ/cm2 ) of UV. After UV irradiation, cells were cultured for 24 h in serum-free media. Supernatant was harvested and MMP-1 expression in the supernatant was measured by western blotting. (B) Cells were replaced with serum-free media and pLTA was pretreated with the indicated concentrations for 20 h. Then cells were washed with DPBS and 75 mJ/cm2 of UV was irradiated in DPBS. After 24 h incubation, supernatant was harvested MMP-1 expression in the supernatant was measured by western blotting. To verify the total number of cells used in the western blot analysis, -actin level was examined from cell lysates. All experiments were performed in triplicate.
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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Fig. 2. UV-induced MMP-1 expression is mediated by ERK, JNK and NF-B activation and pLTA inhibits their activation. HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. (A) Cells were pretreated with 10 M of specific inhibitors for 30 min in serum-free media. Cells were washed with DPBS and 75 mJ/cm2 of UV was irradiated in DPBS. After 24 h incubation, supernatant was harvested and MMP-1 expression in the supernatant was measured by western blotting. Cells were irradiated with 75 mJ/cm2 of UV and harvested at the indicated times. Cells were lysed and subjected to western blotting using phospho-specific ERK antibody (B) and phosphor-specific JNK antibody (C). Cells were pretreated with indicated dose of LTA and irradiated with 75 mJ/cm2 of UV. Levels of phospho-ERK at 60 min after irradiation (D) and phospho-JNK at 30 min after irradiation (E) were examined by western blotting. Total ERK and JNK were used as loading control. (F) Cells were irradiated with 75 mJ/cm2 of UV and harvested at the indicated times. Cells were lysed and nuclear extracts were prepared. Activation of NF-B was measured by western blotting using NF-B phospho-p65 specific antibody. Levels of -actin were used as loading control. All experiments were performed in triplicate.
total-ERK (Fig. 2D) or JNK-2 levels (Fig. 2E). Since NF-B activation was required for UV-induced MMP-1 expression, we examined the effect of pLTA on UV-induced NF-B activation. NF-B was activated within 2 h after UV irradiation and pretreatment of pLTA inhibited its activation (Fig. 2F). 3.3. Pretreatment of pLTA suppressed UV-induced AP-1 and NF-B activation As AP-1 and NF-B is activated by UV and play critical roles in UV-induced MMP-1 expression (Kim et al., 2005; Tanaka et al., 2005; Wenk et al., 1999), we investigated the effects of pLTA on UV-induced AP-1 and NF-B activity. HDFs were pretreated with pLTA for 20 h and then 75 mJ/cm2 of UV was irradiated. Pretreatment of pLTA inhibited UV-induced DNA binding activity of AP-1 and NF-B in nucleus (Fig. 3A). Variation in DNA-AP-1 or DNANF-B was analyzed by densitometry scanning (Fig. 3B). These results demonstrate that pLTA suppresses UV-induced AP-1 and NF-B activation. The translocation of AP-1 and NF-B into nucleus was also decreased by pLTA treatment followed by UV irradiation (Fig. 3C). The decreased nuclear protein level may also contribute to reduced shifting in EMSA assay, apart from decreased binding affinity of transcription factors. 3.4. pLTA promoted type 1 procollagen synthesis As shown in Fig. 1B, pLTA inhibited the expression of collagen degrading enzyme, MMP-1. Therefore, we could expect that pLTA promotes collagen synthesis. Procollagen is a synthesized form and precursor of collagen. Thus, by quantifying the amount of synthesized procollagen, we could measure the amount of collagen which is going to be synthesized. The type of procollagen we choose is type 1 because it is the most abundant collagen. pLTA increased the amount of type 1 procollagen by 37.2% versus the non-treated
control. TGF-, the positive control, increased it by 52.9% versus the control (Fig. 4A). Cell proliferation was measured by WST-1 assay and pLTA did not affect cell viability (Fig. 4B), indicating that pLTA does not have any cytotoxicity. Next, we examined whether LTA increases type I procollagen through protecting TGF- signaling, which is responsible for collagen synthesis. When the interaction between LTA and TLR2 was blocked by anti-TLR2 neutralization antibody, type 1 procollagen synthesis was significantly reduced as compared to LTA treated only. On the other hand, anti-TGF- neutralization antibody did not inhibit type 1 procollagen synthesis, suggesting that LTA itself, but not TGF-, has an ability to increase type 1 collagen synthesis (Fig. 4C).
3.5. pLTA inhibited UV- or H2 O2 -induced ROS generation To investigate whether pLTA inhibits UV-induced MMP-1 expression via an antioxidant effect, ROS levels were measured in two ways. UV irradiation significantly increased ROS generation and pLTA reduced this level to the similar level of positive control, NAC (Fig. 5A). Confocal microscope showed that UV irradiation dramatically increased ROS level, but it was decreased by pLTA pretreatment (Fig. 5B). This result suggests that the inhibition of UV-induced MMP-1 expression by pLTA is associated with the antioxidant effect of pLTA. Next, we examined whether LTA can protect the cells from other oxidative reagents such as H2 O2 . Hydrogen peroxide increased ROS generation in the fibroblasts but it was inhibited by pLTA in a dose-dependent manner (Fig. 5C). Hydrogen peroxide mediates the activation of mitogen-activated protein kinases (MAPKs) such as JNK, ERK and p38 kinase (Ho et al., 2011). Thus, pLTA-mediated inhibition of ROS generation in hydrogen peroxide-stimulated cells may be occurred by the inhibition of JNK and ERK pathway. 100 M hydrogen peroxide caused HDF cell death (Fig. 5D).
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Fig. 3. Pretreatment of pLTA suppresses UV-induced AP-1 and NF-B activation. (A) HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. pLTA was pretreated with the indicated concentrations for 20 h in serum-free media. After washing the cells with DPBS, 75 mJ/cm2 of UV was irradiated in DPBS and cultured in serum free media for 10 h. After incubation, cells were lysed and nuclear extracts were prepared. Electrophoretic mobility shift assay (EMSA) was performed with ␥-32 P-labeled AP-1 and NF-B DNA oligonucleotides. (+) and (−) on the figure indicate positive and negative control, respectively. (B) Intensity of shifted AP-1 and NF-B bands from (A) normalized to unbound probe. (C) Cells were pretreated with 100 g/ml pLTA and irradiated 75 mJ/cm2 of UV. After preparation of nuclear extracts, translocation of NF-B and AP-1 was examined by western blotting using phospho-p65 and phospho-c-Jun antibodies. -Actin from cytosol fraction was used as a loading control.
Fig. 4. pLTA promotes type 1 procollagen synthesis. HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. (A) pLTA was treated with the indicated concentrations for 72 h in serum-free media, and type 1 procollagen was measured from the culture supernatant. (B) Cell viability was measured by WST-1 assay. *P < 0.05 versus non treated cells. (C) HDFs were pretreated with 10 g/ml anti-TLR2 or anti-TGF- neutralization antibody for 30 min, and then cells were incubated with 50 g/ml pLTA for 72 h. type 1 procollagen was measured from the culture supernatant. **P < 0.05. TGF- was used as a positive control.
3.6. Inhibitory effect of pLTA mimics NAC function Based on the results, our observations may be explained by that LTA works as an anti-oxidative reagent to reduce UV triggered ROS. Thus, we examined whether all the beneficial effects of LTA can be mimicked by addition of NAC. NAC is a ROS inhibitor and commonly used to identify and test ROS inducers. When cells were stimulated with pLTA and NAC, UV-mediated ROS generation was significantly inhibited by both pLTA and NAC as compared to UV only treated cells (Fig. 6A). Next, we examined the effect of NAC on MMP-1 expression. NAC significantly inhibited UV-mediated MMP1 expression from fibroblasts (Fig. 6B). Very similar to NAC, pLTA effectively inhibited MMP-1 expression, suggesting that pLTA can be considered as an anti-oxidative reagent. 4. Discussion Photo-aging, unlike intrinsic aging, is strongly influenced by various external factors including repeated sun exposure (Chung et al., 2002; Gilchrest, 1996; Uitto, 1986). UV from sun exposure causes skin changes and triggers skin aging. UV-induced MMP1 degrades collagen and extracellular matrix (Chung et al., 2001; Fisher et al., 1996) and our data showed that LTA from L. plantarum inhibited UV-induced MMP-1 expression. MMP-1 gene expression is regulated by MAPK signal pathways which are involved in intracellular enzyme regulation in response to extracellular stresses including UV (Davis, 1994; Robinson and Cobb, 1997; Waskiewicz
and Cooper, 1995). We found that pretreatment of pLTA before UV irradiation inhibited UV-induced activation of ERK and JNK. Therefore, our results demonstrate that pLTA inhibits UV-induced MMP-1 expression and this inhibitory effect of pLTA may be mediated by the inhibition of the ERK and JNK dependent pathways. Also, because the MEK1/2 contributes to ERK1/2 activation and MEK4 contributes to JNK/SAPK activation (Robinson and Cobb, 1997), pLTA might effect on MEK 1/2 and MEK 4 inhibition prior to ERK and JNK inhibition. In certain cases, phosphorylation of p38, JNK and ERK was significantly enhanced by the low dose of LTA pretreatment, which increases IL-6 production. Low dose of LTA also increased phospho-ERK and phospho-JNK under UV irradiation. On the other hand, high dose of LTA decreased NF-B and MAPK signaling activation while phosphorylation of Akt and PI3K was increased. Decreased activation of NF-B and MAPK signaling affect the inhibition of LPS-mediated TNF-␣ production (Kim et al., 2014). Inhibition of ERK and JNK exerts influence on AP-1 and NF-B in nucleus. They are activated by UV and expression of MMP-1 is consistent with induction of AP-1 by UV irradiation (Rittie and Fisher, 2002). We found that pLTA inhibited DNA binding of AP-1 and NFB. Because they are proto-oncogene products and involved in the tumorigenesis (Cooper and Bowden, 2007), this result suggests the potential ability of pLTA as an agent for prevention of skin cancer. AP-1 up-regulates MMP-1 expression and down-regulates type 1 collagen synthesis. Because pLTA inhibits the activation of AP1, we can expect that pLTA is associated with collagen synthesis. Consistent with this mechanism, we found that pLTA promotes
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Fig. 5. pLTA inhibits UV-induced ROS generation. HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. pLTA was pretreated with the indicated concentrations for 20 h in serum-free media and washed with DPBS. Then cells were irradiated with 75 mJ/cm2 of UV in DPBS and 20 M of DCF-DA was treated in HBSS. After incubation for 30 min, the cells were washed triple times with DPBS. (A) ROS levels were analyzed using microplate readers. (B) Cells were imaged immediately. (C) HDFs were pretreated with the indicated dose of pLTA for 20 h, and then cells were re-stimulated with 50 M H2 O2 for 24 h. ROS generation was examined by DCF-DA assay as described above. (D) HDFs were incubated with the indicated dose of H2 O2 for 24 h, and then cell viability was examined by WST-1 assay. *P < 0.05 versus non-treated cells; **P < 0.05 versus UV-irradiated cells.
synthesis of type 1 procollagen. Thus, pLTA acts in both inhibition of collagen degradation and promotion of collagen synthesis. Reactive oxygen species (ROS) are normal product of metabolism but significantly triggered by UV irradiation. ROS is involved in the MAPK-mediated signal pathway (Kang et al., 2003; Lo and Cruz, 1995; Whisler et al., 1995) and implicated in cellular activity such as inflammatory responses as well as aging. ROS increases MMP1 expression in HDFs (Brenneisen et al., 1997) and ROS scavengers inhibit UV-induced AP-1 activation and MMP-1 expression (Ahmed et al., 2004; Grimm et al., 2004). Glutathione is a strong antioxidant which scavenges free radicals and it is synthesized from cysteine. N-acetyl cysteine (NAC), the precursor of glutathione, promotes the synthesis of glutathione within the cell (Gross et al., 1993). In this research, pLTA significantly reduced generation of ROS induced by
UV irradiation. Thus, we can say that pLTA has an antioxidant effect, although more specific studies are needed to define the mechanism. Lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, has an ability to protect against the proinflammation. Unlike other bacterial cell-wall component, such as LTA from Staphylococcus aureus, lipoprotein and lipopolysaccharide (LPS), pLTA was isolated from L. plantarum contributes to the health. pLTA can attenuate the proinflammatory signaling. In our previous study, pLTA inhibited LPS-induced TNF-␣ production and increased the survival rate of the endotoxin shock mice (Kim et al., 2008b). Moreover, it is reported that TLR ligands modulate the inflammation and immune response through TLR activation and LTA, a ligand of TLR2, inhibits allergenic Th2 responses (Hisbergues et al., 2007). The function of pLTA in this study on skin aging might be mediated
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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Fig. 6. The inhibitory effect of pLTA mimics NAC function. (A) ROS generation was examined from HDFs pretreated with 10 g/ml pLTA or 10 ng/ml NAC for 20 h followed by 75 mJ/cm2 of UV irradiation. (B) MMP-1 expression was examined by ELISA method using culture supernatants of HDFs pretreated with pLTA or NAC followed by 75 mJ/cm2 of UV irradiation. *P < 0.05 versus non-treated cells; **P < 0.05 versus UV-irradiated cells.
through TLR2 though further study is needed. Because we previously reported that cellular recognition of pLTA is associated with the TLR2 signal pathway (Kim et al., 2008b). Some reports demonstrated that LTA contributes beneficial effects for skin problems. For examples, The oral administration of purified LTA from Lactobacillus rhamnosus GG can modulate the immune-suppressive effect of UV and skin tumor development in female Crl:SKH-1-hrBR mice (Weill et al., 2013). Commensal bacteria lipoteichoic acid increases skin mast cell antimicrobial activity against vaccinia viruses (Wang et al., 2012). In the previous study we have shown that sLTA isolated from Lactobacillus sakei inhibited UVA-induced MMP-1 through the inhibition of MAPK/JNK signaling in human dermal fibroblasts (You et al., 2013). In addition, LTA from S. aureus (aLTA) reduced iNOS expression and NO production in the mouse model of renal ischemia reperfusion injury (Chatterjee et al., 2002). However, LTA from different species contributes different biological effects. For example, pLTA moderately induced TNF-␣, IL-10 and NO production, while sLTA and aLTA significantly increased these inflammatory cytokines in RAW 264.7 cells (Jeong et al., 2015). Similar results were observed in the mRNA and protein expression levels of iNOS. sLTA is one of the most strong TLR2 agonist, which may induce excessive inflammation and S. aureus is a major cause of bacteremia. Thus, these LTAs are not suitable for the development of therapies. On the other hand, pLTA moderately induces cytokine and NO production, while it strongly inhibits excessive inflammation induced by LPS, LTA, or cytokines (Kim et al., 2008a, 2008b; Jeong et al., 2015). In addition, L. plantarum have been used as a probiotic for a long time in human history. Therefore, pLTA has many merits as compared to other LTAs. Lactic acid bacteria is widely known microorganism but the study is usually focused on anti-inflammation and intestinal immune system and mainly used as a probiotic organism (Gaudana et al., 2010; Riqaux et al., 2009; Verdenelli et al., 2009). In the present study, we found new function of lactic acid bacteria on human skin photo-aging. We demonstrate that LTA isolated from L. plantarum inhibits that UV-induced MMP-1 expression by mediating inhibition of UV-induced ERK and JNK phosphorylation. It leads to inhibition of AP-1 and NF-B activation. Also, pLTA has an antioxidant effect by reducing ROS generation. Above all, pLTA did not influence on the cell viability, nor show any cytotoxicity. Therefore, we conclude that the LTA from L. plantarum has an anti-photoaging effect by contributing on the collagen contents and could be a potential agent for the prevention and treatment of skin aging without any toxicity. To investigate pLTA is effective in human
skin in vivo as well as in vitro, we are planning to further experiment by oral and dermal administration in vivo. In conclusion, L. plantarum, as lactic acid bacteria, is useful microorganism with no side effect and harmfulness. Moreover, pLTA inhibits degradation of collagen and promotes its synthesis, and that pLTA contributes to a decrease in UV-induced MMP-1 secretion and ROS generation. Thus, we could expect that pLTA and L. plantarum itself could be a new and natural ingredient for prevention and treatment of human skin aging. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment This work was supported by a grant from Kyung Hee University in 2011 (KHU-20110263). References Ahmed, S., Wang, N., Lalonde, M., Goldberg, V.M., Haqqi, T.M., 2004. Green tea polyphenol epigallocatechin-3-gallate (EGCG) differentially inhibits interleukin-1 beta-induced expression of matrix metalloproteinase-1 and -13 in human chondrocytes. J. Pharmacol. Exp. Ther. 308, 767–773. Bond, M., Baker, A.H., Newby, A.C., 1999. Nuclear factor B activity is essential for matrix metalloproteinase-1 and -3 upregulation in rabbit dermal fibroblasts. Biochem. Biophys. Res. Commun. 264, 561–567. Brennan, M., Bhatti, H., Nerusu, K.C., Bhagavathula, N., Kang, S., Fisher, G.J., Varani, J., Voorhees, J.J., 2003. Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem. Photobiol. 78, 43–48. Brenneisen, P., Briviba, K., Wlaschek, M., Wenk, J., Scharffetter-Kochanek, K., 1997. Hydrogen peroxide (H2 O2 ) increases the steady state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts. Free Radic. Biol. Med. 2, 515–524. Brenneisen, P., Sies, H., Scharffetter-Kochanek, K., 2002. Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events. Ann. NY Acad. Sci. 973, 31–43. Burrage, P.S., Mix, K.S., Brinckerhoff, C.E., 2006. Matrix metalloproteinases: role in arthritis. Front. Biosci. 1, 529–543. Chatterjee, P.K., Zacharowski, K., Cuzzocrea, S., Brown, P.A., Stewart, K.N., MotaFilipe, H., Thiemermann, C., 2002. Lipoteichoic acid from Staphylococcus aureus reduces renal ischemia/reperfusion injury. Kidney Int. 62, 1249–1263. Chung, J.H., Seo, J.Y., Choi, H.R., Lee, M.K., Youn, C.S., Rhie, G., Cho, K.H., Kim, K.H., Park, K.C., Eun, H.C., 2001. Modulation of skin collagen metabolism in aged and photoaged human skin in vivo. J. Invest. Dermatol. 117, 1218–1224. Chung, J.H., Seo, J.Y., Lee, M.K., Eun, H.C., Lee, J.H., Kang, S., Fisher, G.J., Voorhees, J.J., 2002. Ultraviolet modulation of human macrophage metalloelastase in human skin in vivo. J. Invest. Dermatol. 119, 507–512.
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Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation Yi-Fan Hong a,c,1 , Hea young Lee b,1 , Bong Jun Jung a , Soojin Jang e , Dae Kyun Chung a,c,d,∗∗ , Hangeun Kim d,∗ a
Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea Cancer Research Institute, Seoul National University, College of Medicine, Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea c Skin Biotechnology Center, Gyeonggi Biocenter, Suwon, Gyeonggi-do 443-766, Republic of Korea d RNA Inc., #308 College of Life Science, Kyung Hee University, Yongin 449-701, Republic of Korea e Institute Pasteur Korea, Sampyeong-dong, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 15 January 2015 Received in revised form 30 April 2015 Accepted 22 May 2015 Available online xxx Keywords: Matrix metalloproteinases-1 Lipoteichoic acid Reactive oxygen species Photo-aging Lactobacillus plantarum Human dermal fibroblasts
a b s t r a c t Background: Ultraviolet (UV) irradiation from the sun is the primary environmental factor that causes human skin aging. UV irradiation induces the expressions of matrix metalloproteinases (MMPs) and extracellular matrix degrading enzymes. Among the members of MMP family, MMP-1 is an interstitial collagenase that degrades the collagen triple helix. We investigated the effect of Lactobacillus plantarum, well known as useful microorganism, on UV-induced-MMP-1 expression in human dermal fibroblasts. Methods: Human dermal fibroblasts (HDF) was pre-stimulated with lipoteichoic acid isolated from L. plantarum followed by UV irradiation. Secreted protein level of MMP-1 was evaluated by Western blot analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-B) from the cell lysates was also examined by western blotting. Electrophoretic mobility-shift assay (EMSA) was used to detect the activated transcription factor, AP-1 and NF-B. The detection of type 1 procollagen was carried with Procollagen type 1 C-peptide (PIP) EIA kit. The generation of reactive oxygen species (ROS) by LTA and UV irradiation was examined by Griess reagent assay and fluorescence microscope. Results: We found that lipoteichoic acid (LTA), a cell-wall component of Gram-positive bacteria, isolated from L. plantarum, inhibited MMP-1 expression. Pretreatment with LTA from L. plantarum (pLTA) reduced MMP-1 expression in a dose-dependent manner and inhibited activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). It also led to the inhibition of DNA binding activity of activator protein-1 (AP-1) and of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-B). Furthermore, LTA promoted type 1 procollagen synthesis and reduced the generation of ROS induced by UV irradiation. Conclusion: Our study demonstrates that pLTA inhibits degradation of collagen and promotes its synthesis and that pLTA contributes to a decrease in ROS production. Therefore, pLTA from L. plantarum has potential abilities to prevent and treat skin photo-aging. © 2015 Published by Elsevier Ltd.
Abbreviations: UV, ultraviolet; MMP, matrix metalloproteinase; LTA, lipoteichoic acid; pLTA, Lactobacillus plantarum lipoteichoic acid; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinases; AP-1, activator protein-1; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cell; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +82 31 201 2487; fax: +82 31 202 3461. ∗∗ Corresponding author at: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea. Tel.: +82 31 201 2465; fax: +82 31 202 8333. E-mail addresses: [email protected] (D.K. Chung), [email protected] (H. Kim). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2015.05.019 0161-5890/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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2
1. Introduction Human skin is often directly exposed to the environment, especially to sun. Therefore, skin undergoes not only chronological aging, but also photo-aging (Fisher et al., 2002). Both are cutaneous and cause laxity, wrinkles and a decrease in elasticity. However, their molecular mechanisms and morphological changes are distinct (Helfrich et al., 2008; Rittie and Fisher, 2002). Photo-aging is also called extrinsic aging and is caused by external factors such as ultraviolet (UV) radiation. UV exposure can cause pigmented and wrinkled skin. UV irradiation induces the expression of MMPs and triggers cleavage of cell-surface receptors and protein kinase signal transduction pathways and activation of transcription factors (Fisher et al., 2002). MMPs degrade all kinds of extracellular matrix proteins and damage connective tissues (Kahari and SaarialhoKere, 1997) and induce the release of cytokines and chemokines and cleave cell surface receptors (Van and Libert, 2007). They also play a role in tissue remodeling and disease processes such as arthritis (Burrage et al., 2006). MMP-1, interstitial collagenase, is a member of the MMP family and is responsible for the degradation of collagen fibers (Moon et al., 2004). An increase in MMP-1 expression causes skin connective tissue damage. Collagen is the richest protein of the dermal connective tissue and is broken down by MMP-1. Among the 29 types of collagen, type 1 collagen is one of the most abundant proteins in the human body (Di Lullo et al., 2002). UV irradiation impairs synthesis of new type 1 collagen. Degradation and damage of collagen is the major feature of photo-aging (Brennan et al., 2003). Mitogen-activated protein kinase (MAPK) pathways are activated in response to extracellular stimuli including UV irradiation (Fisher et al., 1998), leading to the activation of activator protein 1 (AP-1) (Fisher and Voorhees, 1998; Kim et al., 2005; Whitmarsh and Davis, 1996). AP-1 is a transcription factor that regulates MMP1 gene expression in response to various stimuli (Karin, 1995; Wenk et al., 1999). Down-regulation of type 1 collagen and procollagen is mediated by UV-induced AP-1 (Fisher et al., 2002). NF-B activation occurs in response to various stimuli such as stress, free radicals and ultraviolet irradiation. UV-induced NF-B activation leads to the induction of various genes including MMP-1 and the production of MMP-1 in skin fibroblasts (Brenneisen et al., 2002; Tanaka et al., 2005). Reactive oxygen species (ROS) are free radicals and are naturally produced during cellular metabolism. The production of ROS is significantly increased by environmental stresses including UV irradiation, leading to the induction of MMPs (Bond et al., 1999). ROS causes oxidative damage and are a major contributor to aging (Muller et al., 2007). Lipoteichoic acid (LTA) is a major cell-wall component of Gram-positive bacteria and has antigenic properties that stimulate specific immune responses. LTA is known to increase the activity of MMP-9, play an active role in otitis media, inhibit melanogenesis in B16F10 mouse melanoma cells, protect UV-induced carcinogenesis and increases skin mast cell antimicrobial activity against vaccinia viruses (Park et al., 2012; Wang et al., 2012; Weill et al., 2013; Kim et al., 2015). In addition, tolerance using LTA has been used to introduce to protect against pro-inflammation and other diseases (Kim et al., 2008b; Ellingsen et al., 2002). Thus, to identify the role of LTA (pLTA) isolated from Lactobacillus plantarum on UV-mediated photo-aging, MMP-1 expression and ROS generation by pLTA was examined.
2. Materials and methods 2.1. Cell culture Human dermal fibroblasts (HDF) were kindly provided by Dr. Y. S. Son (Kyung Hee University, Korea). NDFs were grown in FBM
with FGM-2 (Lonza, Basel, Switzerland) in humidified incubator of 5% CO2 at 37 ◦ C. 2.2. Preparation and treatment of LTA Highly purified LTA was isolated from L. plantarum by n-butanol extraction. The purity of the purified LTA was determined by measuring the protein and endotoxin contents through the conventional silver staining after polyacrylamide gel electrophoresis and through the Limulus amebocyte lysate (LAL) assay (BioWhittaker, Walkersville, MD), respectively. DNA or RNA contamination was assessed by measuring UV absorption at 260 and 280 nm, and no nucleic acids were detected. To treat the cells, LTA was diluted with non-pyrogenic water at the indicated concentrations. 2.3. UV irradiation Vilber Lourmat Bio-sun lamps (Marine, France) were used as a UV source. The emission spectrum was between 275 and 380 nm (peak spectrum, 312 nm) and UV strength was measured by UV meter (Waldmann, Germany). Before UV irradiation, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and medium was replaced by DPBS. After UV irradiation, the cells were washed with DPBS and cultured for 24 h in the serum-free media. 2.4. Western blot analysis For detection of MMP-1, cultured supernatants were harvested and for detection of phospho-ERK1/ERK2 and phospho-JNK, cells were lysed with RIPA buffer containing proteinase inhibitors. Insoluble debris was removed by centrifugation at 12,000 rpm for 10 min. For detection of NF-B, cells were lysed using nuclear extraction kit (Marligen Biosciences, Rockville, MD) and nuclear protein was extracted. Protein concentration was quantified using Bradford assay reagent (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved on SDS–polyacrylamide gels (10–15%) and then electrophoretically transferred onto nitrocellulose membrane (100 V, 2 h). Membranes were subsequently blocked with 5% skim milk in TBS-T (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, and 0.05% Tween 20) and incubated with the indicated antibodies. Commercially available anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK, anti-phospho-c-Jun, anti-phospho-p65, and anti--actin were purchased from Cell Signaling Technology (Boston, MA). Blotting proteins were visualized by enhanced chemiluminescence (ECL) reagents (GE Healthcare). 2.5. Electrophoretic mobility shift assay (EMSA) Fibroblasts were pretreated with the indicated concentrations of pLTA for 20 h, washed with DPBS and irradiated with 75 mJ/cm2 of UV in DPBS. To prepare nuclear fractions, cells were washed with DPBS and nucleus was extracted using nuclear extraction kit (Marligen Biosciences, Rockville, MD). The cytoplasmic extracts were removed by centrifugation and nuclear pellet were washed and solubilized with extraction buffer. AP-1 and NF-B consensus oligonucleotides (Jany et al., 1995; Husain et al., 2007) were labeled with 10 Ci/ml [␥-32 P] ATP by T4 polynucleotide kinase (Fermentas, Burlington, ON) at 37 ◦ C for 30 min. The sequence of the AP-1 and NF-B oligonucleotide is as follows: AP-1 sense 5 -CGCTTGATGAGTCAGCCGGAA3 , AP-1 antisense 5 -TTCCGGCTGACTCATCAAGCG-3 , NF-B sense 5 -AGTTGAGGGGACTTTCCCAGGC-3 , NF-B antisense 5 GCCTGGGAAAGTCCCCTCAACT-3 . Because unincorporated labeled nucleotides cause non-specific binding, purification was performed to remove them using PROBER Probe DNA purifying kit (Intron Biotechnology, Korea). After determination of nuclear
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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protein concentration, the reaction was performed according to the manufacturer’s instructions (Gel Shift Assay System, Promega, Madison, WI). The equal amounts of nuclear protein were added to the gel shift binding reaction mixtures and incubated for 10 min at room temperature. Then isotope-labeled oligonucleotides were added and incubated for 20 min at room temperature. This DNA–protein complex was loaded onto 6% non-denature gels. Gels were dried and labeled complexes were visualized by autoradiography.
2.6. Measurement of type 1 procollagen synthesis HDFs were pretreated with the indicated concentrations of pLTA for 72 h and supernatants were harvested. The detection of type 1 procollagen was performed using Procollagen type 1 C-peptide (PIP) EIA kit (TAKARA, Kyoto, Japan). Collagens are synthesized as precursor molecules called procollagens. These contain additional peptide, propeptide, at the amino-terminal and the carboxy-terminal ends. The amount of the free propeptides reflects the amount of collagen molecules synthesized. Quantitative detection of collagen synthesis was using polyclonal antibodies by recognizing procollagen type 1 carboxy-terminal peptide (PIP). TGF- was used as a positive control.
2.7. Cell viability The cytoxicity of pLTA on the cell was determined by WST1 assay (TAKARA BIO, Japan) which is based on the cleavage of tetrazolium salts by mitochondrial dehydrogenases in viable cells. The numbers of viable cells are measured by the detection of cleaved tetrazolium salts that added into a medium. HDFs were washed with DPBS and replaced with serum free media. Then pLTA was pretreated with indicated concentrations for 20 h. Then 10 l of premix WST-1 was added in 100 l of culture medium. After incubating for 2 h, absorbance was measured using ELISA reader. The formazen dye formed by metabolically active cell can be quantitated by measuring its absorbance. The measuring wavelength is 420 nm and the reference wavelength is 600 nm.
3
3. Results 3.1. Ultraviolet (UV) radiation-induced MMP-1 expression and lipoteichoic acid from L. plantarum (pLTA) inhibited UV-induced MMP-1 expression in a dose-dependent manner MMP-1 expression was induced by UV irradiation with increasing intensities (0–100 mJ/cm2 ). After incubation for 24 h, culture media was harvested to determine MMP-1 protein levels by western blot analysis. As a result, UV irradiation-induced MMP-1 expression compared with non-irradiated cells, and this induction was dose-dependent (Fig. 1A). To elucidate the effect of pLTA on UV-induced MMP-1 expression, HDFs were pretreated with various concentrations of pLTA for 20 h and then irradiated with 75 mJ/cm2 of UV. After incubation for 24 h, culture media was harvested to determine MMP-1 protein levels by western blot analysis. As we expected, pretreatment with pLTA inhibited UV-induced MMP-1 expression in a dose-dependent manner (Fig. 1B). These results suggest that pLTA has an inhibitory effect on collagen degradation. 3.2. UV-induced MMP-1 expression was mediated by ERK, JNK and NF-B activation, and pLTA inhibited their activation To investigate the factors which are mediated on UV-induced MMP-1 expression, some specific inhibitors were pretreated, and then 75 mJ/cm2 of UV was irradiated. ERK, JNK and NF-B specific inhibitors decreased the expression of UV-induced MMP-1 (Fig. 2A). Therefore, this data suggests that ERK, JNK and NF-B mediated UV-induced MMP-1 expression. Two MAPKs, ERK and JNK were activated by UV irradiation and the activation was high at 60 and 30 min, respectively, after UV irradiation (Fig. 2B and C). We investigated the effects of pLTA on UV-induced ERK and JNK activation. HDFs were pretreated with pLTA for 20 h and then irradiated with 75 mJ/cm2 of UV. Levels of phospho and total ERK and JNK proteins were determined by western blot analysis at 60 and 30 min after UV treatment, respectively. Pretreatment with pLTA inhibited phospho-ERK and phospho-JNK level without affecting
2.8. Detection of reactive oxygen species generation HDFs were pretreated with NAC or pLTA for 20 h in serum-free media and then washed with DPBS. 1 M of H2 O2 or 75 mJ/cm2 of UV was treated to induce ROS generation. After washing the cells, 20 M of DCF-DA in HBSS was added and incubated at 37 ◦ C for 30 min. The cells were washed three times with DPBS and warm HBSS was added. The level of ROS was immediately analyzed using microplate readers (TECAN, Switzerland) and imaged using fluorescence microscope (Leica, Germany). The detection wavelength is 485 nm and reference wavelength is 535 nm. All experiments were performed in triplicate, and reactive oxygen species (ROS) levels are described as fold increase versus the non-treated control.
2.9. Statistical analysis All experiments were performed at least three times using triplicates. The data shown are representative results of the means ± standard deviation of triplicate experiments. Differences were judged to be statistically significant when the p-value was less than 0.05.
Fig. 1. Ultraviolet (UV) radiation induces MMP-1 expression and lipoteichoic acid from L. plantarum (pLTA) inhibits UV-induced MMP-1 expression in a dosedependent manner. Human dermal fibroblasts (HDFs) were cultured in FGM-2 until 80% confluency and washed with DPBS. (A) Cells were irradiated with increasing intensities (0–100 mJ/cm2 ) of UV. After UV irradiation, cells were cultured for 24 h in serum-free media. Supernatant was harvested and MMP-1 expression in the supernatant was measured by western blotting. (B) Cells were replaced with serum-free media and pLTA was pretreated with the indicated concentrations for 20 h. Then cells were washed with DPBS and 75 mJ/cm2 of UV was irradiated in DPBS. After 24 h incubation, supernatant was harvested MMP-1 expression in the supernatant was measured by western blotting. To verify the total number of cells used in the western blot analysis, -actin level was examined from cell lysates. All experiments were performed in triplicate.
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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Fig. 2. UV-induced MMP-1 expression is mediated by ERK, JNK and NF-B activation and pLTA inhibits their activation. HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. (A) Cells were pretreated with 10 M of specific inhibitors for 30 min in serum-free media. Cells were washed with DPBS and 75 mJ/cm2 of UV was irradiated in DPBS. After 24 h incubation, supernatant was harvested and MMP-1 expression in the supernatant was measured by western blotting. Cells were irradiated with 75 mJ/cm2 of UV and harvested at the indicated times. Cells were lysed and subjected to western blotting using phospho-specific ERK antibody (B) and phosphor-specific JNK antibody (C). Cells were pretreated with indicated dose of LTA and irradiated with 75 mJ/cm2 of UV. Levels of phospho-ERK at 60 min after irradiation (D) and phospho-JNK at 30 min after irradiation (E) were examined by western blotting. Total ERK and JNK were used as loading control. (F) Cells were irradiated with 75 mJ/cm2 of UV and harvested at the indicated times. Cells were lysed and nuclear extracts were prepared. Activation of NF-B was measured by western blotting using NF-B phospho-p65 specific antibody. Levels of -actin were used as loading control. All experiments were performed in triplicate.
total-ERK (Fig. 2D) or JNK-2 levels (Fig. 2E). Since NF-B activation was required for UV-induced MMP-1 expression, we examined the effect of pLTA on UV-induced NF-B activation. NF-B was activated within 2 h after UV irradiation and pretreatment of pLTA inhibited its activation (Fig. 2F). 3.3. Pretreatment of pLTA suppressed UV-induced AP-1 and NF-B activation As AP-1 and NF-B is activated by UV and play critical roles in UV-induced MMP-1 expression (Kim et al., 2005; Tanaka et al., 2005; Wenk et al., 1999), we investigated the effects of pLTA on UV-induced AP-1 and NF-B activity. HDFs were pretreated with pLTA for 20 h and then 75 mJ/cm2 of UV was irradiated. Pretreatment of pLTA inhibited UV-induced DNA binding activity of AP-1 and NF-B in nucleus (Fig. 3A). Variation in DNA-AP-1 or DNANF-B was analyzed by densitometry scanning (Fig. 3B). These results demonstrate that pLTA suppresses UV-induced AP-1 and NF-B activation. The translocation of AP-1 and NF-B into nucleus was also decreased by pLTA treatment followed by UV irradiation (Fig. 3C). The decreased nuclear protein level may also contribute to reduced shifting in EMSA assay, apart from decreased binding affinity of transcription factors. 3.4. pLTA promoted type 1 procollagen synthesis As shown in Fig. 1B, pLTA inhibited the expression of collagen degrading enzyme, MMP-1. Therefore, we could expect that pLTA promotes collagen synthesis. Procollagen is a synthesized form and precursor of collagen. Thus, by quantifying the amount of synthesized procollagen, we could measure the amount of collagen which is going to be synthesized. The type of procollagen we choose is type 1 because it is the most abundant collagen. pLTA increased the amount of type 1 procollagen by 37.2% versus the non-treated
control. TGF-, the positive control, increased it by 52.9% versus the control (Fig. 4A). Cell proliferation was measured by WST-1 assay and pLTA did not affect cell viability (Fig. 4B), indicating that pLTA does not have any cytotoxicity. Next, we examined whether LTA increases type I procollagen through protecting TGF- signaling, which is responsible for collagen synthesis. When the interaction between LTA and TLR2 was blocked by anti-TLR2 neutralization antibody, type 1 procollagen synthesis was significantly reduced as compared to LTA treated only. On the other hand, anti-TGF- neutralization antibody did not inhibit type 1 procollagen synthesis, suggesting that LTA itself, but not TGF-, has an ability to increase type 1 collagen synthesis (Fig. 4C).
3.5. pLTA inhibited UV- or H2 O2 -induced ROS generation To investigate whether pLTA inhibits UV-induced MMP-1 expression via an antioxidant effect, ROS levels were measured in two ways. UV irradiation significantly increased ROS generation and pLTA reduced this level to the similar level of positive control, NAC (Fig. 5A). Confocal microscope showed that UV irradiation dramatically increased ROS level, but it was decreased by pLTA pretreatment (Fig. 5B). This result suggests that the inhibition of UV-induced MMP-1 expression by pLTA is associated with the antioxidant effect of pLTA. Next, we examined whether LTA can protect the cells from other oxidative reagents such as H2 O2 . Hydrogen peroxide increased ROS generation in the fibroblasts but it was inhibited by pLTA in a dose-dependent manner (Fig. 5C). Hydrogen peroxide mediates the activation of mitogen-activated protein kinases (MAPKs) such as JNK, ERK and p38 kinase (Ho et al., 2011). Thus, pLTA-mediated inhibition of ROS generation in hydrogen peroxide-stimulated cells may be occurred by the inhibition of JNK and ERK pathway. 100 M hydrogen peroxide caused HDF cell death (Fig. 5D).
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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Fig. 3. Pretreatment of pLTA suppresses UV-induced AP-1 and NF-B activation. (A) HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. pLTA was pretreated with the indicated concentrations for 20 h in serum-free media. After washing the cells with DPBS, 75 mJ/cm2 of UV was irradiated in DPBS and cultured in serum free media for 10 h. After incubation, cells were lysed and nuclear extracts were prepared. Electrophoretic mobility shift assay (EMSA) was performed with ␥-32 P-labeled AP-1 and NF-B DNA oligonucleotides. (+) and (−) on the figure indicate positive and negative control, respectively. (B) Intensity of shifted AP-1 and NF-B bands from (A) normalized to unbound probe. (C) Cells were pretreated with 100 g/ml pLTA and irradiated 75 mJ/cm2 of UV. After preparation of nuclear extracts, translocation of NF-B and AP-1 was examined by western blotting using phospho-p65 and phospho-c-Jun antibodies. -Actin from cytosol fraction was used as a loading control.
Fig. 4. pLTA promotes type 1 procollagen synthesis. HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. (A) pLTA was treated with the indicated concentrations for 72 h in serum-free media, and type 1 procollagen was measured from the culture supernatant. (B) Cell viability was measured by WST-1 assay. *P < 0.05 versus non treated cells. (C) HDFs were pretreated with 10 g/ml anti-TLR2 or anti-TGF- neutralization antibody for 30 min, and then cells were incubated with 50 g/ml pLTA for 72 h. type 1 procollagen was measured from the culture supernatant. **P < 0.05. TGF- was used as a positive control.
3.6. Inhibitory effect of pLTA mimics NAC function Based on the results, our observations may be explained by that LTA works as an anti-oxidative reagent to reduce UV triggered ROS. Thus, we examined whether all the beneficial effects of LTA can be mimicked by addition of NAC. NAC is a ROS inhibitor and commonly used to identify and test ROS inducers. When cells were stimulated with pLTA and NAC, UV-mediated ROS generation was significantly inhibited by both pLTA and NAC as compared to UV only treated cells (Fig. 6A). Next, we examined the effect of NAC on MMP-1 expression. NAC significantly inhibited UV-mediated MMP1 expression from fibroblasts (Fig. 6B). Very similar to NAC, pLTA effectively inhibited MMP-1 expression, suggesting that pLTA can be considered as an anti-oxidative reagent. 4. Discussion Photo-aging, unlike intrinsic aging, is strongly influenced by various external factors including repeated sun exposure (Chung et al., 2002; Gilchrest, 1996; Uitto, 1986). UV from sun exposure causes skin changes and triggers skin aging. UV-induced MMP1 degrades collagen and extracellular matrix (Chung et al., 2001; Fisher et al., 1996) and our data showed that LTA from L. plantarum inhibited UV-induced MMP-1 expression. MMP-1 gene expression is regulated by MAPK signal pathways which are involved in intracellular enzyme regulation in response to extracellular stresses including UV (Davis, 1994; Robinson and Cobb, 1997; Waskiewicz
and Cooper, 1995). We found that pretreatment of pLTA before UV irradiation inhibited UV-induced activation of ERK and JNK. Therefore, our results demonstrate that pLTA inhibits UV-induced MMP-1 expression and this inhibitory effect of pLTA may be mediated by the inhibition of the ERK and JNK dependent pathways. Also, because the MEK1/2 contributes to ERK1/2 activation and MEK4 contributes to JNK/SAPK activation (Robinson and Cobb, 1997), pLTA might effect on MEK 1/2 and MEK 4 inhibition prior to ERK and JNK inhibition. In certain cases, phosphorylation of p38, JNK and ERK was significantly enhanced by the low dose of LTA pretreatment, which increases IL-6 production. Low dose of LTA also increased phospho-ERK and phospho-JNK under UV irradiation. On the other hand, high dose of LTA decreased NF-B and MAPK signaling activation while phosphorylation of Akt and PI3K was increased. Decreased activation of NF-B and MAPK signaling affect the inhibition of LPS-mediated TNF-␣ production (Kim et al., 2014). Inhibition of ERK and JNK exerts influence on AP-1 and NF-B in nucleus. They are activated by UV and expression of MMP-1 is consistent with induction of AP-1 by UV irradiation (Rittie and Fisher, 2002). We found that pLTA inhibited DNA binding of AP-1 and NFB. Because they are proto-oncogene products and involved in the tumorigenesis (Cooper and Bowden, 2007), this result suggests the potential ability of pLTA as an agent for prevention of skin cancer. AP-1 up-regulates MMP-1 expression and down-regulates type 1 collagen synthesis. Because pLTA inhibits the activation of AP1, we can expect that pLTA is associated with collagen synthesis. Consistent with this mechanism, we found that pLTA promotes
Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
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Fig. 5. pLTA inhibits UV-induced ROS generation. HDFs were cultured in FGM-2 until 80% confluency and washed with DPBS. pLTA was pretreated with the indicated concentrations for 20 h in serum-free media and washed with DPBS. Then cells were irradiated with 75 mJ/cm2 of UV in DPBS and 20 M of DCF-DA was treated in HBSS. After incubation for 30 min, the cells were washed triple times with DPBS. (A) ROS levels were analyzed using microplate readers. (B) Cells were imaged immediately. (C) HDFs were pretreated with the indicated dose of pLTA for 20 h, and then cells were re-stimulated with 50 M H2 O2 for 24 h. ROS generation was examined by DCF-DA assay as described above. (D) HDFs were incubated with the indicated dose of H2 O2 for 24 h, and then cell viability was examined by WST-1 assay. *P < 0.05 versus non-treated cells; **P < 0.05 versus UV-irradiated cells.
synthesis of type 1 procollagen. Thus, pLTA acts in both inhibition of collagen degradation and promotion of collagen synthesis. Reactive oxygen species (ROS) are normal product of metabolism but significantly triggered by UV irradiation. ROS is involved in the MAPK-mediated signal pathway (Kang et al., 2003; Lo and Cruz, 1995; Whisler et al., 1995) and implicated in cellular activity such as inflammatory responses as well as aging. ROS increases MMP1 expression in HDFs (Brenneisen et al., 1997) and ROS scavengers inhibit UV-induced AP-1 activation and MMP-1 expression (Ahmed et al., 2004; Grimm et al., 2004). Glutathione is a strong antioxidant which scavenges free radicals and it is synthesized from cysteine. N-acetyl cysteine (NAC), the precursor of glutathione, promotes the synthesis of glutathione within the cell (Gross et al., 1993). In this research, pLTA significantly reduced generation of ROS induced by
UV irradiation. Thus, we can say that pLTA has an antioxidant effect, although more specific studies are needed to define the mechanism. Lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, has an ability to protect against the proinflammation. Unlike other bacterial cell-wall component, such as LTA from Staphylococcus aureus, lipoprotein and lipopolysaccharide (LPS), pLTA was isolated from L. plantarum contributes to the health. pLTA can attenuate the proinflammatory signaling. In our previous study, pLTA inhibited LPS-induced TNF-␣ production and increased the survival rate of the endotoxin shock mice (Kim et al., 2008b). Moreover, it is reported that TLR ligands modulate the inflammation and immune response through TLR activation and LTA, a ligand of TLR2, inhibits allergenic Th2 responses (Hisbergues et al., 2007). The function of pLTA in this study on skin aging might be mediated
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Fig. 6. The inhibitory effect of pLTA mimics NAC function. (A) ROS generation was examined from HDFs pretreated with 10 g/ml pLTA or 10 ng/ml NAC for 20 h followed by 75 mJ/cm2 of UV irradiation. (B) MMP-1 expression was examined by ELISA method using culture supernatants of HDFs pretreated with pLTA or NAC followed by 75 mJ/cm2 of UV irradiation. *P < 0.05 versus non-treated cells; **P < 0.05 versus UV-irradiated cells.
through TLR2 though further study is needed. Because we previously reported that cellular recognition of pLTA is associated with the TLR2 signal pathway (Kim et al., 2008b). Some reports demonstrated that LTA contributes beneficial effects for skin problems. For examples, The oral administration of purified LTA from Lactobacillus rhamnosus GG can modulate the immune-suppressive effect of UV and skin tumor development in female Crl:SKH-1-hrBR mice (Weill et al., 2013). Commensal bacteria lipoteichoic acid increases skin mast cell antimicrobial activity against vaccinia viruses (Wang et al., 2012). In the previous study we have shown that sLTA isolated from Lactobacillus sakei inhibited UVA-induced MMP-1 through the inhibition of MAPK/JNK signaling in human dermal fibroblasts (You et al., 2013). In addition, LTA from S. aureus (aLTA) reduced iNOS expression and NO production in the mouse model of renal ischemia reperfusion injury (Chatterjee et al., 2002). However, LTA from different species contributes different biological effects. For example, pLTA moderately induced TNF-␣, IL-10 and NO production, while sLTA and aLTA significantly increased these inflammatory cytokines in RAW 264.7 cells (Jeong et al., 2015). Similar results were observed in the mRNA and protein expression levels of iNOS. sLTA is one of the most strong TLR2 agonist, which may induce excessive inflammation and S. aureus is a major cause of bacteremia. Thus, these LTAs are not suitable for the development of therapies. On the other hand, pLTA moderately induces cytokine and NO production, while it strongly inhibits excessive inflammation induced by LPS, LTA, or cytokines (Kim et al., 2008a, 2008b; Jeong et al., 2015). In addition, L. plantarum have been used as a probiotic for a long time in human history. Therefore, pLTA has many merits as compared to other LTAs. Lactic acid bacteria is widely known microorganism but the study is usually focused on anti-inflammation and intestinal immune system and mainly used as a probiotic organism (Gaudana et al., 2010; Riqaux et al., 2009; Verdenelli et al., 2009). In the present study, we found new function of lactic acid bacteria on human skin photo-aging. We demonstrate that LTA isolated from L. plantarum inhibits that UV-induced MMP-1 expression by mediating inhibition of UV-induced ERK and JNK phosphorylation. It leads to inhibition of AP-1 and NF-B activation. Also, pLTA has an antioxidant effect by reducing ROS generation. Above all, pLTA did not influence on the cell viability, nor show any cytotoxicity. Therefore, we conclude that the LTA from L. plantarum has an anti-photoaging effect by contributing on the collagen contents and could be a potential agent for the prevention and treatment of skin aging without any toxicity. To investigate pLTA is effective in human
skin in vivo as well as in vitro, we are planning to further experiment by oral and dermal administration in vivo. In conclusion, L. plantarum, as lactic acid bacteria, is useful microorganism with no side effect and harmfulness. Moreover, pLTA inhibits degradation of collagen and promotes its synthesis, and that pLTA contributes to a decrease in UV-induced MMP-1 secretion and ROS generation. Thus, we could expect that pLTA and L. plantarum itself could be a new and natural ingredient for prevention and treatment of human skin aging. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment This work was supported by a grant from Kyung Hee University in 2011 (KHU-20110263). References Ahmed, S., Wang, N., Lalonde, M., Goldberg, V.M., Haqqi, T.M., 2004. Green tea polyphenol epigallocatechin-3-gallate (EGCG) differentially inhibits interleukin-1 beta-induced expression of matrix metalloproteinase-1 and -13 in human chondrocytes. J. Pharmacol. Exp. Ther. 308, 767–773. Bond, M., Baker, A.H., Newby, A.C., 1999. Nuclear factor B activity is essential for matrix metalloproteinase-1 and -3 upregulation in rabbit dermal fibroblasts. Biochem. Biophys. Res. Commun. 264, 561–567. Brennan, M., Bhatti, H., Nerusu, K.C., Bhagavathula, N., Kang, S., Fisher, G.J., Varani, J., Voorhees, J.J., 2003. Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem. Photobiol. 78, 43–48. Brenneisen, P., Briviba, K., Wlaschek, M., Wenk, J., Scharffetter-Kochanek, K., 1997. Hydrogen peroxide (H2 O2 ) increases the steady state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts. Free Radic. Biol. Med. 2, 515–524. Brenneisen, P., Sies, H., Scharffetter-Kochanek, K., 2002. Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events. Ann. NY Acad. Sci. 973, 31–43. Burrage, P.S., Mix, K.S., Brinckerhoff, C.E., 2006. Matrix metalloproteinases: role in arthritis. Front. Biosci. 1, 529–543. Chatterjee, P.K., Zacharowski, K., Cuzzocrea, S., Brown, P.A., Stewart, K.N., MotaFilipe, H., Thiemermann, C., 2002. Lipoteichoic acid from Staphylococcus aureus reduces renal ischemia/reperfusion injury. Kidney Int. 62, 1249–1263. Chung, J.H., Seo, J.Y., Choi, H.R., Lee, M.K., Youn, C.S., Rhie, G., Cho, K.H., Kim, K.H., Park, K.C., Eun, H.C., 2001. Modulation of skin collagen metabolism in aged and photoaged human skin in vivo. J. Invest. Dermatol. 117, 1218–1224. Chung, J.H., Seo, J.Y., Lee, M.K., Eun, H.C., Lee, J.H., Kang, S., Fisher, G.J., Voorhees, J.J., 2002. Ultraviolet modulation of human macrophage metalloelastase in human skin in vivo. J. Invest. Dermatol. 119, 507–512.
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Please cite this article in press as: Hong, Y.-F., et al., Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.05.019
