## 4 Discussion
### Non-Thermal Plasma Flux Promoted Healing Process of Burn Wounds, Regardless of Infection  
Amongst the proposed plasma flux applications, wound healing enhancement is one of the most attractive treatment indications among the potential plasma flux applications [61], owing to the anti-inflammation action on epithelial cells [38; 62; 63]. However, other cell types, including immune cells and fibroblasts, are also important in the wound healing process [64]. Unfortunately, data on the effects of plasma flux on non-epithelial cells are still limited. Because burn wounds are associated with a high mortality rate [65], partly through immune dysregulation-induced opportunistic infection [16; 66], burn wound models with and without _S. aureus_ infection were used [67]. Although MRSA-infected burn wounds are the most important problem in clinical practice, a standard ATCC strain of _S. aureus_, but not clinical isolated MRSA, was used due to the concern of model reproducibility at this level of proof-of-concept experimentation. Indeed, the plasma flux treatment attenuated burn wounds with and without infection as evaluated by the wound area and wound inflammatory score. Plasma flux might promote wound healing differently between burn wounds and infected burn wounds. There were no differences in cutaneous inflammatory cytokines between the plasma-treated and control groups in non-infected burn wounds. Plasma, on the other hand, improved wound healing in non-infected burn wounds, probably by increasing fibroblast migration and collagen formation. As such, the ability of fibroblasts to make collagen was enhanced by efficient cell migration [60]. On the other hand, plasma flux lowered pro-inflammatory cytokines in dermal tissue without affecting collagen formation in the infected burn wounds. These data imply promotion of the wound healing process through an anti-inflammatory effect in the infected burn wounds. Due to the activation by pathogen molecules, pro-inflammatory macrophages may be over-activated in infected burn wounds [68], and plasma flux adjusts the balance of the response. However, bacterial burdens in the total dermal tissue of the wound were not different between the plasma-treated and control groups. Perhaps the bactericidal impact may be restricted primarily to the surface of the lesions due to the limited depth of penetration by plasma flux. A more proper method to determine bacterial burdens on the surface of lesions is needed. Nevertheless, our experiments support the utilization of plasma flux in burn wounds, which could be easily used in real clinical practice. More studies on patients would be interesting. In conclusion, non-thermal atmospheric pressure argon-based plasma induced anti-inflammatory macrophages, possibly through (i) reduced NF-kB by ROS and (ii) enhanced fibroblast migration, which were responsible for wound healing promotion in murine burn wound models. [PERSON] and [PERSON] contributed equally to this work. [PERSON] designed plasma device. Conceptualization, C.P.D., S.W. and A.L. ; methodology, C.P.D., S.W., A.C., K.S.-K.; investigation, C.P.D., D.T.-A., A.L. ; data analysis and writing--original draft preparation, C.P.D. and S.W. ; writing--review and editing, A.L. ; funding acquisition, C.P.D., S.W., A.L. ; supervision, A.L. All authors have read and agreed to the published version of the manuscript. This work is supported by the Asahi Glass Foundation Grant (2020-2021) for Chulalongkorn University, the Program Management Unit for Human Resources & Institutional Development Research and Innovation-CU (Global Partnership B16F630071 and Flagship B05F630073) and National Research Council of Thailand. The study was conducted according to the approved animal protocol (029/2563) from the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Not applicable. All data analyzed during this study are included in this published article.