Topical application of zwitterionic chitosan suppresses neutrophil-mediated acute skin inflammation
Introduction
Neutrophils play an essential role in many pathological processes including infection, inflammation, wound healing, and tumor [1]. Neutrophils are the first cells recruited to the injury site. Particularly in the inflamed skin, these cells induce dermal infiltration of eosinophils and CD4+ T cells, which are the primary source of Th2-type cytokines, leading to allergic skin inflammation [2]. Suppression of neutrophil infiltration and IL1β production may be a reasonable therapeutic strategy for the treatment of tissue inflammation [3,4]. Moreover, tissue-infiltrating neutrophils mediate skin tissue damage through the release of cytokines such as TNFα and IL1β. Subsequently, they undergo apoptosis and are taken up by macrophages, enhancing the macrophage efferocytosis [5]. Neutrophils also interact with innate immune cells such as natural killer (NK) and natural killer T (NKT) cells, which play significant roles in the inflammatory response [[6], [7], [8], [9]]. Previous studies show that the depletion of neutrophils downregulated the effector functions of NK and NKT cells such as their cytotoxicity and cytokine production [[10], [11], [12]]. Conversely, NK and NKT cells can enhance cytokine production and recruitment of neutrophils into the inflamed tissue [13,14]. These studies suggest that there is a positive crosstalk between neutrophils and NK/NKT cells.
Chitosan is a deacetylated polysaccharide derived from chitin, which is one of the main components of the shrimp shell. Chitosan is a biocompatible material with applications in drug delivery and various immunological activities. In particular, chitosan induces type I IFN response and maturation of dendritic cells (DCs) via STING-cGAS-dependent pathway, resulting in a Th1 polarized response [15]. Chitosan enhances NK cell activity and promotes both Th1 and Th2 immune responses in OVA-treated mice [16]. The immunostimulatory effects of chitosan on macrophages are dependent on its molecular weight and concentration [17]. On the other hand, anti-inflammatory effects of chitosan have also been reported. For example, chitosan reduces LPS-induced inflammation in the intestinal epithelial cell line [18] and also attenuates the progression of inflammatory bowel disease by inhibiting the NFκB pathway [19]. The immunomodulatory effect of chitosan varies with cell types. While chitosan up-regulates pro-inflammatory responses of DCs, it skews macrophage polarization toward an M2-like phenotype with anti-inflammatory activities [20].
Chitosan is poorly soluble in water in physiological conditions with near neutral pH and has in vivo toxicity such as thrombogenic action and tissue inflammation [21]. A chitosan derivative with partial succinylation of amine groups (named zwitterionic chitosan, ZWC) is water-soluble at physiological pH [22]. ZWC with hydrophilic property is considered safe and biocompatible because it does not show pro-inflammatory activities [23]. In addition, ZWC has shown a protective effect on sepsis and intestinal inflammation by down-regulating LPS-induced inflammatory responses of macrophages [[23], [24], [25]]. Nevertheless, previous studies on immunological functions of ZWC are limited to macrophages. Thus, it will be of interest to explore the effects of ZWC on neutrophils, the initiator of acute inflammation in injured tissues.
Here, we hypothesized that ZWC, a water-soluble succinylated chitosan derivative, can attenuate acute skin inflammation by inhibiting inflammatory neutrophils. To test our hypothesis, we investigated the role of ZWC in acute skin inflammation induced by tape-stripping. We found that topical application of ZWC can attenuate the severity of skin lesion elicited by tape-stripping. In vivo ZWC treatment effectively inhibited both skin recruitment and pro-inflammatory cytokine production of neutrophils. Moreover, ZWC had a direct suppressive effect on neutrophil activity. These results find ZWC to be a promising biomaterial for treating acute skin inflammation.
Section snippets
Mice and reagents
Wild-type (WT) C57BL/6 (B6) mice were purchased from Jung Ang Lab Animal Inc. (Seoul, Korea). IFNγ/YFP (Yeti) cytokine reporter mice were kindly provided by Dr. R. Locksley (University of California at San Francisco, CA, USA). All mice were maintained at Sejong University and used at 6–12 weeks of age for experiments. They were maintained on a 12-hour light/12-hour dark cycle in a temperature-controlled barrier facility with free access to food and water. These mice were fed a γ-irradiated
Characterization of ZWC
ZWC was synthesized by partial succinylation of primary amines of chitosan (Fig. 1A). The degree of succinylation, as determined by 1H NMR, was 54.7% (Fig. 1B). The ZWC showed negative charges at relatively basic pH and positive charges at acidic pH, unlike unmodified chitosan that assumes positive charges at pH <7.2. The transition pH for the charge conversion was in the range of 4.5–4.8 (Fig. 1C). Three representative batches showed similar charge profiles, supporting the reproducibility of
Discussion
We demonstrate that topical application of ZWC to the skin lesion effectively attenuates skin inflammation elicited by tape-stripping. Such effects of ZWC on tape-stripping-induced skin inflammation are strongly correlated with direct suppression of the pro-inflammatory activity of neutrophils. Furthermore, ZWC treatment inhibited the activation of NK and NKT cells as well as innate allergic effector cells such as basophils in the spleen.
IL1β secretion can be triggered by activated NLRP3
Conclusions
In this study, we report for the first time that topical application of ZWC on the skin damaged by tape-stripping prevents the infiltration of neutrophils into the skin lesion and suppresses the release of pro-inflammatory cytokines from the skin and splenic neutrophils, consequently reducing skin lesion area. Moreover, these effects are closely related to direct suppression of neutrophil activation by ZWC. Topical treatment of ZWC can decrease the activation status of splenic immune cells.
CrediT author statement
Sung Won Lee: Conceptualization, Methodology, Software, Validation, Formal analysis, Data curation, Writing - Original draft preparation, Writing - Reviewing and Editing, Funding acquisition. Hyun Jung Park: Conceptualization, Methodology, Software, Validation, Formal analysis, Data curation, Writing - Original draft preparation, Writing - Reviewing and Editing. Yihua Pei: Formal analysis, Data curation, Writing - Original draft preparation. Yoon Yeo: Resources, Writing - Reviewing and Editing,
Declaration of competing interest
The authors have no potential conflicts of interest to declare.
Acknowledgments
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1A09919293 to S.H.; NRF-2018R1D1A1A02086057 to S.W.L.) and NIH R21 AI119479 to Y.Y.
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These authors contributed equally to this work.