## 4 Discussion  
Keratinocytes, located in the outer layer of human skin, play an essential role in the skin defense against infections. In addition to creating a physical barrier between the environment and the inside of the body, keratinocytes exert a strong immune function against pathogenic infections. Skin keratinocytes express proinflammatory cytokines and antimicrobial peptides including hBD2, hBD3, and LL37 that affect skin immune system [20]. Because immune defenses of the skin can be increased by probiotics, the role of probiotics in disease management and immune regulation has recently received renewed attention from society. The skin immunoregulatory effect of _L. plantarum_ KG was improved by fermentation with ginseng extracts. KG lysates suppressed _S. aureus_ infection by increasing hBD3 expression in HaCaT cells. HBD3 expression in the skin affects skin inflammation, bacterial infections, and wound healing [21]. Indeed, hBD3 was significantly increased in HaCaT cells by KG lysates. Interestingly, hBD3 expression was more inducible in cells pretreated with KG lysates followed by infection of _S. aureus_. The KG lysates also showed a reduced _S. aureus_ infection rate compared to the K8 lysates. Treatment with only K8 lysates did not affect, suggesting that it is a characteristic of KG lysates. It is unclear whether the inhibition of _S. aureus_ intracellular infection was caused by antimicrobial activity of hBD3 or if it affects the mechanism used by _S. aureus_ for intracellular infection. However, there was a study stating that hBD3 kills _S. aureus_[1]. In our research, _S. aureus_ was cultured in CM prepared from HaCaT cells treated with KG lysates, but it did not significantly affect the viability of _S. aureus_ (data not shown). _S. aureus_ uses the Fibronectin \(\alpha\)5\(\beta\)1 integrin pathway to enter host cells [22]. The interaction between _S. aureus_ and its host cell receptors such as TLR2, CD36, and CD14 may also be important. Considering the anti-inflammatory and resistance-inducing efficacy of _L. plantarum_ lysates, intracellular infection may be suppressed by reducing the expression of genes related to _S. aureus_ infection. The mechanism of inducing the expression of hBD3 in skin keratinocytes is not well known, but EGFR may be involved [23]. EGFR induces interferon regulatory factor 1 (IRF-1) expression through STAT1 activation [24; 25]. We observed that KG lysates increased IRF-1 from 5 min treatment and peaked at 6 h (data not shown). IRF-1 acts as a transcriptional activator and regulates expression of type I interferon genes [26]. When cells were treated with EGFR inhibitor, IRF-1 expression was inhibited (data not shown). However, in this study, we have not yet confirmed directly that IRF-1 is involved in hBD3. Further studies on the relationship between IRF-1 and hBD3 expression are needed. The expression of hBD3 in HaCaT cells increased significantly at 72 h. This is thought to be because the activity of JAT-STAT1 occurs late. The expression of EGFR increased starting 5 min after treatment with KG lysates, but EGF increased from 1 h post-treatment and reached a peak at 24 h. In other words, we speculate that time is necessary for the signal initiated from EGF-EGFR to activate IFN-\(\gamma\) signaling to activate JAK-STAT1. In addition, the relationship between EGFR signaling and IFN-\(\gamma\)-mediated JAK/STAT activation will require further clarification. Figure 4: EGFR is involved in KG lysate-mediated hBD3 induction. HaCaT cells were treated with \(1\times 10^{9}\) cells/mL KG lysates for the indicated times and EGF mRNA (**A**) and EGFR mRNA (**B**) was examined by real-time PCR. (**C**) HaCaT cells were pretreated with afatinib and re-treated with KG lysates. hBD3 mRNA level was examined by real-time PCR. **_p_\(<\) 0.01 and ***_p_\(<\) 0.001.