Topical application of zwitterionic chitosan suppresses neutrophil-mediated acute skin inflammation

Abstract

Zwitterionic chitosan (ZWC), a water-soluble succinylated chitosan derivative, has anti-inflammatory activities with therapeutic effects on sepsis and colitis. However, it remains unknown whether ZWC has any influence on skin inflammation. Here, we investigated the role of ZWC in the tape-stripping-induced acute skin inflammation model. Topical application of ZWC to the wounded area significantly reduced skin lesion compared with PBS controls. Since tape-stripping-induced skin inflammation is mediated by neutrophils, we examined if ZWC has any suppressive effects on neutrophil’s function. ZWC treatment downregulated the skin recruitment of neutrophils, subsequently reducing inflammatory responses by keratinocytes. ZWC also suppressed LPS-induced inflammatory responses of neutrophils in vitro, indicating the direct effect of ZWC on neutrophils. Moreover, such anti-inflammatory effects of ZWC extended to other immune cells such as basophils in the spleen. Overall, our results support that ZWC may be used as a therapeutic material to control acute skin inflammation.

1. 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 NK and NKT cells, which play significant roles in the inflammatory response [6–9]. Previous studies show that the depletion of neutrophils downregulated the effector functions of NK and NKT cells such as their cytotoxicity and cytokine production [10–12]. 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 towards 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 downregulating LPS-induced inflammatory responses of macrophages [23–25]. 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.

2. Materials and methods

2.1. Mice and reagents

WT 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 sterile diet and provided with autoclaved tap water. In this study, age- and sex-matched mice were used for all experiments. The animal experiments were approved by the Institutional Animal Care and Use Committee at Sejong University (SJ-20161103). LPS derived from Escherichia coli (serotype 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitosan (15 kDa) was purchased from Polysciences, Inc. (Warrington, PA). Succinic anhydride was purchased from Sigma-Aldrich (St. Louis, MO).

2.2. Synthesis and characterization of zwitterionic chitosan (ZWC)

ZWC was synthesized by the previously established method with minor modifications [26]. In brief, chitosan was dissolved in 1% acetic solution and centrifuged at 3,724 rcf to separate a supernatant that contained an acetate salt form of chitosan. Four hundred mg of chitosan acetate was dissolved in 60 ml deionized (DI) water, and the pH was adjusted to pH 6.0 by 0.1N NaHCO₃. Subsequently, 140 mg succinic anhydride was added to the chitosan solution at room temperature (RT) under continuous stirring. The reaction was maintained at pH 6.0–6.5 for one hour and then adjusted to pH 7.5 overnight using 0.1N NaHCO₃. The product was purified using dialysis membrane (MWCO: 3.5 kDa) against DI water and lyophilized. For quality control, the zeta potential of ZWC was measured at different pHs with a Malvern Zetasizer Nano ZS90 (Worcestershire, UK). ZWC was dissolved in 10 mM NaCl with a concentration of 0.3 mg/ml, and the solution pH was adjusted with 0.1N HCl. The degree of substitution was determined by ¹H-NMR as described previously [22, 24].

2.3. Isolation of peritoneal neutrophils

WT B6 mice were i.p. injected with 2 ml of 4% thioglycollate broth (Sigma, St. Louis, MO, USA) in deionized water. Four hrs later, the peritoneal cavity was flushed with 10 ml ice-cold PBS, and peritoneal cells were harvested from peritoneal lavage fluid. Total cell suspension was then separated with 63% Percoll (GE Healthcare) gradients, and mononuclear cells were collected from the layer below 63% gradient. After the removal of red blood cells (RBCs) using ACK lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, and 2 mM EDTA), the peritoneal cells were washed with PBS. Cells were centrifuged and resuspended in complete RPMI 1640 medium. Neutrophil population was >87% pure among all purified populations.

2.4. Flow cytometry

The following monoclonal antibodies (mAbs) were obtained from BD Biosciences (San Jose, USA): FITC- or allophycocyanin (APC)-conjugated anti-CD11c (clone HL3); PE-Cy7-, or APC-conjugated anti-CD3ε (clone 145–2C11); phycoerythrin (PE)-, or APC-conjugated anti-NK1.1 (clone PK136); PE-conjugated anti-MHC II (clone M5/114.15.2); APC-, or biotin-conjugated anti-CD45 (clone 30-F11); PE-conjugated anti-CD40 (clone 3/23); PE-conjugated anti-CD86 (clone GL1); PE-, or APC-conjugated anti-CD25 (clone PC61); PE-Cy7-conjugated anti-CD11b (clone M1/70); PE-conjugated anti-caspase-3 (clone C92–605); PE-conjugated anti-TNFα (clone MP6-XT22); and PE-conjugated anti-IgG1 (κ isotype control).

The following mAbs were purchased from eBioscience: FITC-, or APC-conjugated anti-Ly-6G (Gr-1) (clone RB6–8C5); APC-conjugated anti-F4/80 (clone BM8); FITC- or APC-conjugated anti-FcεRI (clone MAR-1); PE-conjugated anti-Phospho-mTOR (Ser2448) (clone MRRBY); PE-conjugated anti-IL4 (clone BVD6–24G2); PE-conjugated anti-IL1β (clone NJTEN3); PE-conjugated anti-iNOS (clone CXNFT). The following mAbs were obtained from BioLegend: APC-conjugated anti-CD200R3 (clone Ba13). PE-conjugated anti-HIF1α (clone 241812) was purchased from R&D Systems (Minneapolis, MN, USA). Flow cytometric data were acquired with a FACSCalibur system (Becton Dickinson, USA) and analyzed with FlowJo software (Tree Star, USA).

For surface antibody staining, cells were harvested and washed twice with cold 0.5% BSA-containing PBS (FACS buffer). For blocking non-specific binding to Fc receptors, the cells were incubated with anti-CD16/CD32 mAbs on ice for 10 min and subsequently stained with fluorescence-labeled mAbs. Flow cytometric data were acquired using a FACSCalibur flow cytometer (Becton Dickson, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA) [27].

2.5. Preparation of skin cell suspensions.

The dorsal skin was dissected, and dermal fat was removed with scissors. The tissue was cut into small pieces with a scalpel and digested with 2.5 mg/ml collagenase type IV (Sigma, St. Louis, MO) and 1 mg/ml DNase I (Promega, USA) for 2 hrs at 37°C. At the end of the incubation, the digested tissue was dissociated into single-cell suspension using gentleMACS Dissociator (Miltenyi, Germany) in combination with C Tubes. The single-cell suspension was passed through a 70 μm-pore cell strainer to get rid of tissue debris, and cells were collected in a 50 ml Falcon tube and washed once with PBS + 10% FBS (1400 rpm, 10 min, 4°C). Total cell suspension was then separated with 37%/70% Percoll (GE Healthcare) gradients, and mononuclear cells were collected from the 37/70% Percoll interphase. After washing with PBS, the total mononuclear cell number was determined using a hemacytometer with 0.4% trypan blue (Welgene, Korea) before staining.

2.6. Determination of intracellular ROS.

ZWC-treated neutrophils were washed with PBS and then cultured with LPS for an additional 6 hrs, followed by incubation with 2 μM 2′−7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma, St Louis, MO) at 37°C for 30 min. ROS fluorescence intensity was determined by flow cytometry.

2.7. Intracellular cytokine staining

To perform intracellular staining, splenocytes were incubated with brefeldin A, an intracellular protein transport inhibitor (10 μg/ml), in RPMI medium for 2 hrs at 37°C. The cells were stained for cell surface markers, fixed with 4% PFA, washed once with cold FACS buffer, and permeabilized with 0.5% saponin. The permeabilized cells were then stained for an additional 30 min at room temperature with the indicated mAbs (PE-conjugated anti-TNFα, anti-IL6, anti-IL1β, anti-caspase-3, and anti-iNOS; PE-conjugated isotype control rat IgG mAbs). More than 5,000 cells per sample were acquired using a FACSCalibur and analyzed with the FlowJo software package.

2.8. Phosflow analysis of mTOR-protein phosphorylation levels

The cells were fixed in pre-warmed Fix Buffer I (BD Phosflow™ Cat. No. 557870) for 10 min at 37°C. Immediately after washing with cold-PBS, permeabilization was performed with cold Phosflow Perm Buffer II (BD Phosflow™ Cat. No. 558050) for 30 min on ice. The cells were washed twice with staining buffer in 1x PBS and 2% FBS for 10 min, and stained with PE anti–Phospho-mTOR (Ser2448) mAb in staining buffer for 30 min at room temperature. More than 5,000 cells per sample were acquired using the FACSCalibur and analyzed with the FlowJo software package.

2.9. Induction of acute skin inflammation by tape-stripping

The dorsal skin of anesthetized mice was shaven and tape-stripped six times with Transpore surgical tape (3M, St Paul, MN, USA). Either 20 mg of ZWC in 1 ml PBS or PBS only was applied on a patch of sterile gauze (2.5 × 2.5 cm) and attached to the tape-stripped area. Skin samples were collected 24 hrs after the treatment.

2.10. Statistical analysis

Statistical significance was determined using Excel (Microsoft, USA). Student’s t-test was performed for the comparison of two groups. *P<0.05, **P<0.01, and ***P<0.001 were considered significant in the Student’s t-test. Two-way ANOVA analysis was carried out using the VassarStats (http://vassarstats.net/anova2u.html).)^#P<0.05, ^##P<0.01, and ^###P<0.001 were considered to be significant in the two-way ANOVA.

3. Results

3.1. 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 the batch production. Both the succinylation degree and the transition pH were consistent with our previous reports of ZWCs prepared with the anhydride/amine ratio of 0.7 (ZWC_0.7) [24, 26, 28].

Fig. 1.

Characterization of ZWC.

(A) The structure of ZWC. (B) ¹H-NMR spectra of parent chitosan and ZWC (Solvent: 2% CD₃COOD in D₂O; at 70 °C). (C) pH-dependent zeta-potential profiles of ZWC. Data are expressed as averages with standard deviations of 3 repeated measurements.

3.2. Topical treatment of ZWC prevents acute skin inflammation elicited by tape-stripping

Upon cutaneous injury, neutrophils start to infiltrate the skin rapidly and contribute to the skin inflammation by releasing TNFα and IL1β at early time point [2, 4]. Moreover, as previously described, neutrophil depletion before skin injury reduces skin inflammation significantly, indicating that neutrophils are a critical initiator for skin inflammation [29]. To investigate the effect of ZWC on skin inflammatory immune responses, we prepared ZWC as described previously [26, 28]. We confirmed that the ZWC synthesized and used in this study displays similar pH-dependent zeta-potential profiles as those previously reported [26, 28]. We initially observed that ZWC suppresses inflammatory responses of neutrophils in vivo through TLR4-independent mechanisms with no additional stimulation in steady-state conditions (Fig. S1). Therefore, we asked whether ZWC treatment could block the progression of neutrophil-mediated acute skin inflammation. To test this, we employed the skin inflammation model induced by tape-stripping. The shaved dorsal skin was tape-stripped six times with surgical tapes. Subsequently, ZWC was topically applied to tape-stripped skin lesion area in wild-type (WT) C57BL/6 (B6) mice. After 24 hrs of topical treatment with ZWC, the severity of skin lesion area was examined (Fig. 2A). ZWC-treated mice exhibited significant reduction in tape-stripped skin lesions compared with those of vehicle-treated controls (Fig. 2B). Since IL33 and IL1β secreted by keratinocytes have pathogenic roles in acute skin injury [30, 31], we next examined whether such anti-inflammatory effects of ZWC on skin injury were associated with decreased levels of these cytokines and found that ZWC treatment significantly reduced IL33 and IL1β production from keratinocytes in the skin compared with the control group (Fig. 2C).

Fig. 2.

Topical treatment of ZWC prevents the severity of neutrophil-mediated acute skin inflammation. (A-F) The dorsal skin of B6 mice was tape-stripped, and then mice were topically applied with either vehicle or ZWC (20 mg). (A) Experimental scheme to examine the effect of ZWC in skin inflammation. (B) Lesion areas of shaved mouse back skin 24 hrs after tape-stripping were measured by ImageJ software. Representative image of shaved mouse back skin (left panel) and a summary figure are shown in the right panels. (C-F) Twenty-four hrs later, skin excised for flow cytometric analysis from each group of mice. (C) The frequency of keratinocytes was measured by gating on Keratin-14⁺ population in CD45⁻ skin single cell suspension (left panels). Intracellular cytokine productions of both IL33 and IL1β by keratinocytes were determined by flow cytometry (right panels). The absolute numbers of (D) CD45⁺ cells and (E) neutrophils in skin-infiltrating leukocytes were assessed by flow cytometry. (F) Intracellular TNFα and IL1β productions by skin neutrophils (CD45⁺CD11b⁺Gr1⁺) were measured by flow cytometry. The mean values ± SD are shown (n = 4 per group in the experiment; Student’s t-test; *P<0.05). Two-way ANOVA (treatment × cytokine) showed an interaction between these two factors.

Since neutrophil infiltration is a pathological hallmark of the tape-stripping-induced skin inflammation [2], we examined whether the amelioration of skin injury is attributable to reduction of pro-inflammatory neutrophil functions by ZWC. Consistent with our expectation, ZWC efficiently inhibited the recruitment of neutrophils to an injured skin lesion (Fig. 2D-E). Moreover, ZWC-treated mice displayed a significant decrease in the frequencies of neutrophils producing pro-inflammatory cytokines such as TNFα and IL1β compared to vehicle-treated mice (Fig. 2F). The inhibitory effects of ZWC on skin neutrophil activation in the tape-stripped skin was dose-dependent (Fig. S2). Taken together, these findings suggest that topical ZWC application can downregulate the inflammatory function of neutrophils, which are the primary pathogenic immune cells in the tape-stripped skin injury.

3.3. Topical treatment of ZWC can affect splenic neutrophils to attenuate pro-inflammatory cytokine production elicited by tape-stripping

Emerging evidence has shown that skin-derived cytokine such as IL33 can reach the systemic circulation and activate peripheral immune cells [30]. Thus, we examined whether topical treatment of ZWC could affect the activation of splenic neutrophils triggered by tape-stripping. In vivo ZWC treatment did not alter the number of splenic neutrophils in the animals receiving tape-stripped skin injury (Fig. 3A-B). However, it significantly suppressed the production of TNFα, IL1β, and iNOS by splenic neutrophils (Fig. 3C). Topical application of ZWC to the skin lesion also exerted an inhibitory effect on splenic neutrophils in a dose-dependent manner (Fig. S2). Since it has been reported that mTOR-HIF1α signaling axis is necessary for neutrophil activation [32], we examined the effect of ZWC treatment on mTOR and HIF1α expression of splenic neutrophils upon tape-stripping skin injury. In ZWC-treated mice, mTOR and HIF1α expression on splenic neutrophils were significantly reduced compared with those from vehicle-treated mice (Fig. 3C), which indicates that topical treatment of ZWC could suppress peripheral neutrophil activation.

Fig. 3.

ZWC suppresses pro-inflammatory cytokine production of splenic neutrophils elicited by skin inflammation. (A-C) The dorsal skins of B6 mice were tape-stripped, and then mice were topically applied with either vehicle or ZWC (20 mg). Twenty-four hrs later, total splenocytes were prepared from each group of mice. (A) Splenocyte number were evaluated. (B) The absolute numbers and (C) intracellular productions of TNFα, IL1β, iNOS, HIF1α, and P-mTOR by splenic neutrophils (CD11b⁺Gr1⁺) were measured by flow cytometry. (A-B) Representative data (left panel) and their summary (right panel) are shown. (C) Representative data (upper panel) and their summary (lower panel) are shown. The mean values ± SD are shown (n = 4 per group in the experiment; Student’s t-test; **P<0.01, ***P<0.001).

3.4. Direct suppressive effects of ZWC on pro-inflammatory cytokine production of neutrophils

Since our results showed that in vivo treatment of ZWC induces the inhibition of neutrophil activation, we wondered whether ZWC could directly suppress cytokine production from neutrophils. We first investigated whether ZWC can influence the survival of neutrophils, which have a short life span (less than 48 hrs after release from the bone marrow into the circulation), under in vitro culture conditions. There was no significant difference in the percentage of apoptotic neutrophils between vehicle- and ZWC-treated cells at 10 hrs after treatment (Fig. 4A-B). We also measured the expression level of anti-apoptotic BCL2 protein, known to influence neutrophil longevity, on neutrophils at 6 hrs after ZWC treatment. ZWC-treated neutrophils displayed similar levels of BCL2 protein expression to the vehicle-treated ones (Fig. 4C). Moreover, ZWC was not significantly cytotoxic to neutrophils at concentrations lower than 4 mg/ml (Fig. S3). Taken together, these findings suggest that in vitro ZWC treatment did not affect the survival of neutrophils.

Fig. 4.

ZWC directly suppresses pro-inflammatory cytokine production of neutrophils. (A-F) Peritoneal neutrophils purified from B6 mice were cultured with either vehicle or ZWC (0.5 and 2 mg/ml). (B) Ten hrs later, the apoptotic (annexin-V⁺7AAD⁻) frequencies among neutrophils were assessed by flow cytometric analysis. (C-D) Six hrs later, the intracellular expression of BCL2 and TNFα on neutrophils were estimated by flow cytometry. (E) Experimental scheme to examine the effect of ZWC on neutrophils in steady-state immune responses. (F-G) Neutrophils purified from B6 mice were stimulated with either vehicle or ZWC (0.5 and 2 mg/ml) for 6 hrs. Subsequently, these cells were thoroughly washed with PBS to remove any remaining ZWC and then cultured with LPS for an additional 6 hrs. LPS-stimulated intracellular productions of (F) TNFα and (G) ROS in neutrophils (CD11b⁺Gr1⁺) were assessed via flow cytometry. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01, ***P<0.001).

Next, to test the direct inhibitory effect of ZWC on neutrophils, we examined the production of pro-inflammatory cytokine TNFα by peritoneal neutrophils after in vitro ZWC treatment. ZWC significantly decreased the secretion of TNFα by peritoneal neutrophils, suggesting that ZWC is an effective suppressor of neutrophils (Fig. 4D). Next, to determine the effect of pre-treatment of ZWC seems redundant to the following sentence, we examined immune responses of ZWC-treated neutrophils to subsequent LPS challenge. Neutrophils were treated with ZWC for 6 hrs, thoroughly washed with PBS to remove extracellular ZWC (Fig. 4E), and then stimulated further with LPS for 6 hrs. We observed that pre-treatment with ZWC significantly reduced the TNFα production by neutrophils upon LPS challenge (Fig. 4F). A burst of reactive oxygen species (ROS) released from stimulated neutrophils negatively regulates inflammatory responses [33]; therefore, we explored whether there is any change in ROS production by neutrophils pre-treated with ZWC upon LPS challenge. ZWC attenuated ROS levels in neutrophils when exposed to LPS (Fig. 4G). Overall, these results provide strong evidence that ZWC directly acts as a neutrophil suppressor.

3.5. Topical treatment of ZWC can alleviate pro-inflammatory innate immune cell activation induced by tape-stripping

It has been reported that neutrophils induce the activation of NK cells via IL1β and IL18 [34]. Moreover, depletion of neutrophils diminished the effector function of NK and NKT cells [10–12]. Given that ZWC suppresses inflammatory response of neutrophils elicited by skin injury (Fig. 2), we examined whether the inhibition of skin neutrophils by ZWC can affect the functional phenotype of splenic NK and NKT cells. ZWC treatment had little impact on the absolute cell number of NK and NKT cells (Fig. 5A), but significantly decreased their production of inflammatory cytokines including IL4, IFNγ, TNFα, and IL1β (Fig. 5B-C).

Fig. 5.

ZWC treatment attenuates splenic NK and NKT cell activation resulting from neutrophil-mediated inflammation. (A-C) The dorsal skin of B6 mice was tape-stripped, and these mice were topically applied with either vehicle or ZWC (20 mg). Twenty-four hrs later, total splenocytes were prepared from each group of mice. (A) The absolute cell numbers of NK and NKT cells were measured by gating on NK1.1⁺CD3ε⁻ and NK1.1⁺CD3ε⁺ populations, respectively. (B-C) Intracellular productions of IL4, IFNγ, TNFα, and IL1β from NK and NKT cells were measured by flow cytometry. The mean values ± SD are shown (n = 4 per group in the experiment; Student’s t-test; *P<0.05, **P<0.01, ***P<0.001). (D) Yeti B6 mice were i.p. injected with either vehicle or ZWC (20 mg). Six days later, total splenocytes were prepared from each group of mice. (E) The level of YFP(IFNγ) was assessed in NK cells (NK1.1⁺CD3ε⁻) and NKT cells (NK1.1⁺CD3ε⁺) using flow cytometry. The mean values ± SD are shown (n = 4 per group in the experiment; Student’s t-test; ***P<0.001). Two-way ANOVA (treatment × cell type) showed an interaction between these two factors (^#P<0.05, ##P<0.01, ^###P<0.001).

Previous studies have reported that a relatively large population of neutrophils accounts for inflammation in Yeti mice, which display an autoinflammatory syndrome due to their dysregulated IFNγ expression [7, 35]. Thus, we employed the Yeti mice to investigate whether ZWC exerts a suppressive effect on hyperactive NK and NKT cells. As expected, ZWC treatment restricted in vivo IFNγ production of NK and NKT cells in Yeti mice (Fig. 5D-E). Collectively, our findings suggest that ZWC can effectively limit initial activation of neutrophils responsible for skin inflammation, resulting in subsequent blockade of NK and NKT cell activation.

Innate allergic effector cells such as basophils, mast cells, and group 2 innate lymphoid cells (ILC2) are the main IL4-producing cell population [36]. Moreover, the release of pro-Th2 cytokine IL33 after skin injury stimulates basophils, mast cells, and ILC2 to produce IL4 [37]. Since ZWC treatment inhibited the tape-stripping-induced IL33 production by keratinocytes (Fig. 2C), we asked whether ZWC can suppress IL4 production from Th2-type innate immune cells (i.e., basophils, mast cells, and ILC2) in response to IL33. For this purpose, we first analyzed the absolute cell number and IL4 secretion of these cells at 24 hrs after skin injury. ZWC treatment did not alter the total cell number of these cells (Fig. 6A-B), but it significantly downregulated IL4 production by basophils. Although statistical difference was not reached, ZWC treatment showed a tendency toward decreased IL4 production in mast cells and ILC2 (Fig. 6C). Taken together, these results demonstrate that topical treatment of ZWC to the skin lesion affects the reduction of IL4 production by innate allergic effector cells in the spleen.

Fig. 6.

ZWC treatment attenuates IL4 production of Th2-type innate immune cells resulting from neutrophil-mediated inflammation. (A-C) The dorsal skin of B6 mice was tape-stripped, and these mice were topically applied with either vehicle or ZWC (20 mg). Twenty-four hrs later, total splenocytes were prepared from each group of mice. (A) The frequency and (B) absolute cell number of basophils, mast cells, and ILC2 were measured by gating on FcεRI⁺CD200R3⁺CD3⁻CD19⁻, FcεRI⁺CD200R3⁻CD3⁻CD19⁻, and CD25⁺ST2⁺Lin⁻(CD3, CD19, CD11b, CD11c, NK1.1, FcεRI, F4/80) populations, respectively. (C) Intracellular IL4 production by splenic basophils, mast cells, and ILC2 was measured by flow cytometry. The mean values ± SD are shown (n = 4 per group in the experiment; Student’s t-test; *P<0.05). Two-way ANOVA (treatment × cell type) showed an interaction between these two factors.

4. 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 cell such as basophils in the spleen.

IL1β secretion can be triggered by activated NLRP3 inflammasome upon inflammatory skin diseases [38]. Moreover, skin inflammation with excessive production of IL1β is strongly associated with neutrophilic infiltration [38]. Our data showed that ZWC treatment strongly attenuated the release of IL1β by neutrophils, suggesting that ZWC would be useful for alleviating the symptoms of inflammatory skin diseases. Since TNFα induces keratinocytes to produce IL33 [39], which can aggravate skin inflammation [30, 40], neutrophil-derived TNFα might indirectly contribute to skin inflammation. Indeed, our data showed that topical application of ZWC downregulated the cytokine production of neutrophils (i.e., IL1β and TNFα), consequently reducing the IL33 output by keratinocyte in the inflamed skin lesion. Thus, the regulation of TNFα-IL33 axis by ZWC might be the primary mechanism of preventing skin inflammation.

Peripheral neutrophils rapidly undergo a process called spontaneous apoptosis, which largely depends on the levels of BCL2 expression [41]. The induction of spontaneous neutrophil apoptosis is a resolution mechanism of neutrophil-mediated inflammation [42]. We observed that ZWC had little effect on spontaneous apoptosis and BCL2 expression of neutrophils in vitro. This result indicates that ZWC mediates anti-inflammatory effects on neutrophils in an apoptosis-independent manner.

Moreover, acute skin inflammation initiated by neutrophils is implicated in etiology of chronic allergic skin inflammation such as atopic dermatitis (AD) [2] as well as the atopic march, which is an orderly progression from AD to asthma [43]. Based on these reports, it will be of interest to further investigate the preventive effects of ZWC on the pathogenesis of other allergic diseases such as asthma. Moreover, although neutrophils are critical initiators of skin inflammation, it has been shown that CD4⁺ T cells are also essential for inducing an optimal Th2 response in response to repeated tape-stripping [2]. ZWC-mediated downregulation of IL4-producing immune cells (i.e., NKT cells and basophils) might contribute to inhibiting the progression of skin inflammation to other types of allergic diseases.

A recent study has demonstrated that oral administration of ZWC diminished local inflammation in the colon by increasing intestinal Treg cell population, consequently protecting against TNBS (2,4,6-Trinitrobenzenesulfonic acid)-induced colitis [44]. IL1β-mediated neutrophil accumulation is associated with Helicobacter hepaticus-driven colitis [45]. Moreover, it has been reported that TNBS-induced colitis could be suppressed by regulating neutrophil infiltration into the lamina propria [46]. Our results favor the scenario that ZWC-mediated suppression of neutrophil activation at early immune responses may contribute to the protection against TNBS- or bacteria-induced intestinal inflammation.

ZWC, unlike unmodified chitosan, has low pro-inflammatory activities, probably because of its high solubility and low levels of amine content [24]. It is currently unknown which receptors recognize the succinylated chitosan. However, several studies have provided some clue. For example, succinylated proteins are specifically taken up by scavenger receptor SR-BI [47, 48] and also SR-BI-mediated uptake of lipids attenuates neutrophil activation [49]. Therefore, SR-BI may be one of the receptors responsible for ZWC’s suppression of neutrophil activation. Accordingly, it would be exciting to investigate the role of SR-BI on neutrophils in the context of the immunomodulatory effects of ZWC on neutrophils.

5. 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 resulting in a reduction of 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. These findings support that ZWC is a promising biomaterial for the treatment of acute skin inflammation.

Highlights.

• Zwitterionic chitosan (ZWC) has shown anti-inflammatory activity.

• However, the effect of ZWC on skin inflammation is unknown.

• Topical ZWC application attenuated acute skin inflammation induced by tape-stripping.

• ZWC treatment suppressed neutrophils responsible for acute skin inflammation.

• Thus, ZWC is a promising biomaterial to control neutrophil-mediated skin injury.