Contact hypersensitivity (CHS) is a representative type of T-cell-mediated skin inflammation and also a murine model of human allergic contact dermatitis (1, 2). As in other T-cell-mediated inflammatory models, CHS is divided into two distinct stages: sensitization and elicitation. During sensitization, haptens applied to abdominal skin are taken up by antigen-presenting cells, such as Langerhans cells and dermal dendritic cells, and bind to and modify self-antigens; then the modified self-antigens sensitize CD4+ and CD8+ T cells. After 5 to 7 days, we induced an elicitation response by application of the same haptens to the ear, to induce skin swelling; the extent of swelling was correlated with the severity (1). Although T-cell responses are predominant in the pathology of CHS, other immune cells, such as neutrophils (3), innate lymphoid cells (4), γδ T cells (5, 6), NKT cells (7) and even B cells (8), can be involved.
LPC is a bioactive lysolipid produced by the cell-membrane metabolism by hydrolyzing phosphatidylcholine by phospholipase A2 (PLA2) in the Lands cycle (9). It can be divided into short-chain LPC or saturated/unsaturated LPC according to the acyl-chain length and degree of saturation (10). The levels of LPC can also be increased by lipid metabolism by means of the secretory PLA2 or the oxidative modification of lipoprotein phospholipids under inflammatory conditions (11, 12). In asthmatic patients (13) and animal models with lung injury (14), the concentrations of LPC were elevated in the lung fluids, accompanied by the increased activity of PLA2. Further study showed that the cell population responsible for the increase of LPC was the bronchial epithelial cells (15) and that LPC played a critical role in airway inflammation (16). LPC has various biological functions, including inflammatory responses, oxidative stress, and apoptosis (10), and the effects of LPC on immune cells are diverse, including increased bactericidal activity of neutrophils (17, 18), phagosome maturation of macrophages (19), integrin–mediated adhesion of eosinophils (20), activation of NKT cells (21, 22), and immune regulation (23). Altogether, these reports suggest that LPC can be upregulated under inflammatory conditions and play diverse roles, depending on environments.
Recently, it was reported that the proportion of short-chain LPC (C16 or C18) was increased significantly in atopic dermatitis, which is accompanied by downregulation of fatty-acid elongation enzymes, ELOVL3/ELOVL6 (24). Although these findings provide insight about how the expression of short-chain LPC is regulated, they did not show whether the ‘increase of short-chain LPC’ contributes to the development of skin inflammation.
Here, we examined the role of LPC in skin allergic inflammation in the DNFB-induced CHS model. We directly injected the short-chain LPC (C18:0) into the CHS mice and monitored the severity of the skin inflammation. Interestingly, LPC aggravated CHS by upregulating IL17 and CXCL1/2 and recruiting more neutrophils in a G2A-dependent manner.
To investigate the roles of LPC in skin inflammation, we decided to use the DNFB-induced CHS model mimicking human allergic dermatitis. We applied a high dose of DNFB to the abdominal skin two times at d0 and d1 for sensitization and challenged the ear with a low dose of DNFB at d5 (Fig. 1A). On the next day, we measured the ear thickness every day as a readout of the skin inflammation. During the period of the experiment, we injected LPC into the test-group mice every day. We also prepared three different control groups: the first one we treated with acetone (vehicle of DNFB) only; the second we sensitized with acetone and challenged it with DNFB to assess the ear swelling induced by nonspecific irritation. Last, we sensitized the third group, challenged it with DNFB, but treated it with BSA (vehicle of LPC). Ears treated with DNFB swelled significantly at d7-8 and the swelling was aggravated by LPC (Fig. 1B and 1C).
Since the DNFB-induced skin inflammation is carried out by immune cells, we analyzed the phenotypes of immune cells in the skins by using flow cytometry. The percentage of total CD45+ cells in the ear skins was increased significantly by DNFB, but was not changed further by LPC treatment (Fig. 1D, left). Next, we analyzed the adaptive immune-cell populations, such as CD4+ helper, CD8+ cytotoxic, and Foxp3+ regulatory T cells, but did not find any significant difference between BSA and LPC (data not shown). Instead, the percentage of an innate cell population, like neutrophils (CD11β+Ly6G+; Fig. 1D, right), but not macrophages (CD11β+F4/80+; Fig. 1D, middle), were increased by LPC. Since LPC had been reported to promote the activity of neutrophils, as in H2O2 production (17), we speculated that LPC could aggravate skin inflammations by increasing neutrophil infiltration. However, it was reported that LPC was not chemotactic for neutrophils
Although G2A (G2 Accumulation, an orphan G protein coupled receptor) is not a
Next, we sought to investigate the expressions of IFN-γ and IL17 as signature cytokines of T-cell-mediated inflammations. Interestingly, the percentage of IL17-expressing cells increased in LPC-treated ear skins (Fig. 3A). We also observed similar results in RT-qPCR analysis (Fig. 3B). The subsequent FACS analysis revealed that the majority of IL17-expressing cells were TCRγδ cells. In contrast, TCRβ+ cellswere the major cellular sources of IFN-γ (Supplementary Fig. 2A). To check whether LPC is directly involved in the regulation of IL17 expression in T cells, we sought to culture naïve CD4+ T and TCRγδ+ cells in the presence of LPC under T helper type 17 (TH17) conditions and checked the expression of IL17. Unexpectedly, IL17 was not upregulated by LPC (data not shown), which caused concern that there might be another type of immune cell, such as innate lymphoid cells, not T cells, that produce IL17 in respond to LPC. Therefore, we repeated the CHS experiments in lymphocyte-deficient RAG-1 KO mice to confirm the role of T cells. RAG-1 KO mice developed the much-milder CHS, as shown previously (27), and the ear swelling of RAG-1 KO mice was not increased significantly by LPC (Supplementary Fig. 2B). Moreover, the percentage of IL17-expressing cells was pretty low in RAG-1 KO mice treated with DNFB plus BSA andwas not increased by LPC (Supplementary Fig. 2C), indicating that LPC upregulated IL17 indirectly in T cells.
IL17 is a signature cytokine in type 3 inflammation where neutrophils play an important role. We realized in this study that LPC upregulated IL17 (Fig. 3A), recruited neutrophils more efficiently (Fig. 1D), and exacerbated DNFB-induced skin inflammation (Fig. 1C). These findings led us to assume that LPC could recruit neutrophils by using IL17, which aggravated skin inflammation. To confirm the above hypothesis, we neutralized IL17 cytokine by using anti-IL17 mAb and did the CHS experiments. In mice treated with DNFB plus BSA, IL17 neutralization reduced the ear swelling slightly (Fig. 4A) but did not prevent the neutrophil accumulation (Fig. 4B and 4C). However, the effects of LPC on skin inflammation were dramatically reduced by IL17 neutralization. The ears treated with anti-IL17 mAb did not swell as much as did those treated with isotype control mAb in the presence of LPC (Fig. 4A). Furthermore, IL17 neutralization abolished the neutrophil recruitment (Fig 4B and 4C) and the upregulation of CXCL1/2 (Fig. 4D) induced by LPC, implying that the inflammatory effects of LPC depend on IL17.
Last, we investigated whether the upregulation of IL17 induced by LPC also depended on G2A and found that the expression of IL17 did not increase in G2A KO mice as much as in WT in the presence of LPC stimulation (Fig. 4E).
In this study, we investigated the effect of LPC in a gain-of-function approach and found that LPC upregulated CXCL1 and CXCL2 (Fig. 1E), recruited neutrophils (Fig. 1D), and aggravated DNFB-induced CHS (Fig. 1C). Moreover, once neutrophils were depleted, LPC did not cause the ear swelling anymore (Fig. 1F). These findings clearly indicated that neutrophils recruited by LPC and CXCL1/2 exacerbated CHS. Then, how was CXCL1/2 upregulated by LPC? Based on our finding that IL17was upregulated by LPC (Fig. 3) and the previous reports that IL17 drives neutrophil infiltration by inducing the expression of neutrophil-attracting chemokines such as CXCL1/2 (28-31), we hypothesized that LPC upregulated IL17, which subsequently increased the CXCL1/2 expressions and neutrophil infiltration. This hypothesiswas supported by the findings of our IL17 neutralization experiments (Fig. 4). However, we failed to identify the detailed mechanisms by which LPC upregulated IL17, which need further study.
The putative LPC receptor is the G protein coupled receptors, G2A. Although G2A KO mice developed a late-onset autoimmune disease that looked like Systemic Lupus Erythematosus (32), recent studies have shown evidence that G2A can work as both pro- and anti-inflammatory mediators (26, 33, 34). Here, we demonstrated that LPC exacerbated CHS in a G2A-dependent manner (Fig. 2D), suggesting the proinflammatory roles of LPC and G2A in skin inflammations. Particularly, it was intriguing that G2A KO mice developed less-severe CHS even in the absence of LPC treatment. Given that diverse G2A ligands including LPC and oxidized fatty acids (35) are available in skin, these findings imply the importance of G2A and its lipid ligands in skin homeostasis.
In conclusion, we demonstrated that the upregulation of LPC could exacerbate allergic skin inflammation by increasing IL17 expression and neutrophil recruitment via G2A receptor. Further study on LPC and G2A would help our understanding of the roles of lipid metabolites in skin immunology.
We purchased WT C57BL/6 mice from Koatech (Pyeongtaek, Korea). The G2A knockout (KO) mice (36) on the C57BL/6 background, as we described previously, we received from Dr. DK Song (Hallym University). We did all animal experiments in accordance with guidelines and approval of the International Animal Care and Use Committees of Hallym University (Hallym 2018-9, 2019-18).
We did the induction of CHS in mice as described previously (37, 38). The extent of CHS was shown as the increase of the ear thickness (ear swelling), which we calculated by subtracting the ear thickness of the treated mice from that of the control mice (mice challenged with DNFB (Sigma Korea, Seoul, Korea) without sensitization), which we measured every 24 h after challenging them using a micrometer (Mitutoyo, Kanagawa, Japan). We injected LPC (18:0 Lyso PC, 1-stearoyl-2-hydroxy-snglycero-3-phosphocholine, Avanti Polar Lipids, Alabaster, AL) or 2% BSA (vehicle of LPC, Sigma) subcutaneously for the whole period of the experiments.
We removed ears, split them in half, and cut them into small pieces. We treated skin tissues in RPMI media containing 0.1 mg/ml DNase I and 0.1 mg/ml Liberase TL (Sigma) for 2 h at 37°C. We filtered digested tissues with a 70-μm cell strainer (SPL, Seoul, Korea). For cytokine analysis, we cultured cells for 4 h in the presence of PMA/ionomycin plus monensin (BD Biosciences, San Jose, CA) before intracellular cytokine staining unless otherwise specified. We acquired data by means of FACS Canto-II (BD Biosciences) and analyzed the data with FlowJo software (BD Biosciences).
We isolated RNA using the RNeasy Mini kit (Qiagen, Germantown, MD) or Trizol (Thermo Fisher Scientific Korea, Seoul, Korea), and reverse-transcribed it into cDNA using QuantiTect Reverse Transcription kit (Qiagen). We normalized all data to actin. We checked non-specific amplification by the use of melting curves and agarose gel electrophoresis (39). The sequences of primers (Genotech, Daejon, Korea) are as follows. Il17a forward, 5’-AC TACCTCAACCGTTCCACGTC-3’; Il17a reverse, 5’-ATGTGGTGG TCCAGCTTTCC-3’; Ifng forward, 5’-GATGCATTCATGAGTATT GCCAAGT-3’; Ifng reverse, 5’-GTGGACCACTCGGATGAGCTC-3’; Cxcl1 forward, 5’-TGAGCTGCGCTGTCAGTGCCT-3’; Cxcl1 reverse, 5’-AGAAGCCAGCGTTCACCAGA-3’; Cxcl2 forward, 5’-GAGCTTGAGTGTGACGCCCCCAGG-3’; Cxcl2 reverse, 5’-GTT AGCCTTGCCTTTGTTCAGTATC-3’; G2a forward, 5’-AAGTGT CCAGAATCCACACAGGGT-3’; G2a reverse, 5’-AGTAAACCTA GCTTCGCTGGCTGT-3’; actin forward, 5’-CATCCGTAAAGACC TCTATGCCAAC-3’; actin reverse, 5’-ATGGAGCCACCGATCCA CA-3’.
We used a two-tailed, unpaired, student’s
This work was supported by NRF of Korea (2018R1D1A1B070 4642813), Hallym University (H20160646) and Korea Healthcare R&D project (HI17C0597).
The authors have no conflicting interests.