The endocannabinoid system comprises cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol, and metabolic enzymes of these ligands. The endocannabinoid system has recently been implicated in the regulation of various pathophysiological processes of the skin that include immune competence and/or tolerance of keratinocytes, the disruption of which might promote the development of skin diseases. Recent evidence showed that CB1 in keratinocytes limits the secretion of proinflammatory chemokines, suggesting that this receptor might also regulate T cell dependent inflammatory diseases of the skin. In this article, we sought to investigate the cytokine profile of IFN-γ-activated keratinocytes, and found that CB1 activation by AEA suppressed production and release of signature TH1- and TH17-polarizing cytokines, IL-12 and IL-23, respectively. We also set up cocultures between a conditioned medium of treated keratinocytes and naive T cells to disclose the molecular details that regulate the activation of highly proinflammatory TH1 and TH17 cells. AEA-treated keratinocytes showed reduced an induction of IFN-γ-producing TH1 and IL-17-producing TH17 cells, and these effects were reverted by pharmacological inhibition of CB1. Further analyses identified mammalian target of rapamycin as a proinflammatory signaling pathway regulated by CB1, able to promote either IL-12 and IL-23 release from keratinocytes or TH1 and TH17 polarization. Taken together, these findings demonstrate that AEA suppresses highly pathogenic T cell subsets through CB1-mediated mammalian target of rapamycin inhibition in human keratinocytes. Thus, it can be speculated that the latter pathway might be beneficial to the physiological function of the skin, and can be targeted toward inflammation-related skin diseases.
Besides giving structure to the epidermis and functioning as a physical barrier, keratinocytes also play a key role in initiating cell-mediated immune responses in the skin through cytokine and chemokine release, as well as by expressing adhesion molecules. Pathogen- or stress-activated keratinocytes synthesize and secrete an array of cytokines to augment and shape the adaptive immunologic response (1). The main activators of keratinocytes are physical or chemical damage, UV radiation, and inflammatory signals (proinflammatory IFN-γ, IL-17 and IL-22, or anti-inflammatory IL-4 and IL-13), to which keratinocytes respond by releasing many pro- or anti-inflammatory cytokines. The latter substances trigger downstream TH1 and TH17 responses, typical of psoriasis, induce immunoregulatory TH2 or trigger regulatory T cell responses (1, 2). The endocannabinoid system (ECS) has been shown to regulate the homeostasis and function of different tissues including the brain, gut, blood, and skin (3), and cannabinoid-based therapies are under investigation for the treatment of many disorders, including chronic inflammation (4). The ECS consists mainly of two receptors (cannabinoid receptors 1 [CB1] and 2 [CB2]), their endogenous ligands termed endocannabinoids (primarily anandamide [AEA] and 2-arachidonoylglycerol), and different metabolic enzymes that are responsible for endogenous tone and biological activity of endocannabinoids (5, 6). CB2 is mainly expressed by immune cells, and is the key regulator of endocannabinoid-dependent suppression of various inflammatory responses (7). Instead, CB1 is more common in the central and peripheral nervous system, and acts mainly as a neuromodulator or neurotransmitter; yet it is also involved in the modulation of inflammation at the periphery. For instance, CB1 activation has been shown to protect against experimental colitis (8), liver fibrosis (9), and cutaneous inflammation (10). In the skin, ECS signaling is extensively implicated in the homeostasis of several cutaneous processes, including proliferation, differentiation and survival, as well as in immune competence and/or tolerance, whose disruption may lead to the development of skin diseases such as psoriasis, allergic dermatitis and skin cancer (11, 12). In particular, recent work by Tüting’s group has elegantly demonstrated that CB1 is functionally expressed by keratinocytes in vivo and attenuates the release of proinflammatory CXCL10 and CCL8 chemokines upon IFN-γ activation, which might regulate the downstream T cell dependent responses (13). In the current study, we further analyzed the cytokine profile expressed by IFN-γ-activated human keratinocytes (immortalized human keratinocytes [HaCaT] and normal human epidermal keratinocytes [NHEK] cells), and found that CB1 suppresses production and release of typical TH1- and TH17-polarizing cytokines. Furthermore, to evaluate T cell dependent responses, cocultures between HaCaT cell-conditioned medium and naive T cells or monocyte-derived dendritic cells (Mo-DC) were set up, showing that pharmacological CB1 activation in HaCaT cells prevented TH1 and TH17 induction. To further unravel the molecular mechanisms by which CB1 affects proinflammatory T helper differentiation, we also investigated the possible involvement of the mammalian target of rapamycin (mTOR) pathway, which profoundly influences skin biology including cell proliferation and survival, pigmentation and maintenance of barrier function (14, 15). These findings were reproduced in primary NHEK cells.
Materials and Methods
Keratinocyte culture and treatments
HaCaT and primary NHEK were grown at 37°C in a humidified 5% CO2 atmosphere in D-MEM-F12 growth medium or KGM-Gold growth medium, respectively, as reported (16). Various doses of AEA (Tocris Bioscience), 500 nM SR141716A (SR1), 500 nM SR144528 (SR2) (Tocris Bioscience), 10 nM rapamycin (Sigma Aldrich) or 200 nM LY2584702 (Selleckchem) were added to fresh culture medium together with 100 ng/ml IFN-γ (Miltenyi Biotec), with vehicles alone added to controls. After each treatment, cell viability was measured by Trypan blue dye exclusion.
Preparation and sorting of peripheral blood cells
PBMCs were isolated after venous puncture from healthy donors, and were separated by density gradient over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), as reported (17). Naive T cells and monocytes were obtained from PBMCs using cell isolation kits (Miltenyi Biotec), and were purified by means of negative or positive selection through an autoMACS Pro Separator (Miltenyi Biotec), respectively. Briefly, non-naive T cells (effector and memory T cells, NK cells, B cells, dendritic cells, and granulocytes) were indirectly labeled using a mixture of biotin-conjugated Abs and anti-biotin magnetic microbeads. Highly purified unlabeled naive T cells were obtained by depletion of magnetically labeled cells. Monocytes were isolated by means of a CD14+ isolation kit (Miltenyi Biotec). The purity of both naive T cells and monocytes was evaluated by flow cytometry (17), and was consistently found to be >90%.
T cell polarization
For TH1 and TH17 polarization of T cells, isolated naive CD4+ T cells were activated with anti-CD3/CD28 beads (T cell per bead ratio of 5:1), and cultured for 6 d into 96-well round-bottom plates at 37°C in 200 μl final volumes of X-VIVO 15 medium, under TH1- and TH17-polarizing conditions. The following human recombinant cytokines were used: for TH1 polarization, 10 ng/ml IL-12 and IFN-γ; for TH17 polarization, 10 ng/ml IL-1β, 20 ng/ml IL-6, 100 ng/ml IL-23, and 1 ng/ml TGF-β.
Monocyte-derived dendritic cells
To obtain Mo-DC, highly purified CD14+ monocytes were left in adherence and cultured with 50 ng/ml GM-CSF and 20 ng/ml IL-4 for 5 d. Cultures were fed with fresh medium and cytokines every 2 d; at day 5, adherent cells were then activated with 100 ng/ml LPS (Sigma Aldrich) for two additional days.
To measure the intracellular levels of cytokines, their release from HaCaT cells was inhibited by adding 1 μg/ml brefeldin A (Sigma-Aldrich), 5 h before the end of stimulation (17). At the end of the incubation, cells were fixed with 4% formaldehyde for 10 min at room temperature, and then stained intracellularly with fluorescent Abs against IL-6, IL-8, TNF-α and IL12 p40 (see Table I) in 0.5% saponin, at room temperature for 30 min. For surface staining of activation markers, Mo-DC were stained with fluorescent Abs against CD80, CD86, and CD83 (Table I) for 10 min at 4°C. Intracellular cytokines and surface activation markers were analyzed by flow cytometry in a FACS-Cyan ADP (Beckman Coulter), as reported (17). For each analysis, at least 100,000 events were acquired.
|Ab .||Manufacturer .||Dilution .|
|TNF-α FITC||Miltenyi Biotec||1:100|
|IL-6 FITC||BD Pharmingen||1:60|
|IL-8 FITC||Becton Dickinson||1:60|
|IL-12 p40 APC||Miltenyi Biotec||1:100|
|CD80 PE-Cy5||BD Pharmingen||1:100|
|CD86 FITC||BD Pharmingen||1:100|
|CD83 PE-Cy7||BD Pharmingen||1:80|
|Ab .||Manufacturer .||Dilution .|
|TNF-α FITC||Miltenyi Biotec||1:100|
|IL-6 FITC||BD Pharmingen||1:60|
|IL-8 FITC||Becton Dickinson||1:60|
|IL-12 p40 APC||Miltenyi Biotec||1:100|
|CD80 PE-Cy5||BD Pharmingen||1:100|
|CD86 FITC||BD Pharmingen||1:100|
|CD83 PE-Cy7||BD Pharmingen||1:80|
Keratinocyte/immune cell cocultures
Supernatants of untreated or treated HaCaT cells were tested for their ability to polarize allogenic naive T cells into TH1 or TH17 cells, or to activate Mo-DC, both obtained from healthy controls and seeded at a density of 5 × 104/well. HaCaT cells were challenged with IFN-γ for 24 h in the presence or absence of AEA, SR1 or SR2, and then their conditioned media were used to culture naive T cells for 6 d into 96-well round-bottom plates at 37°C, in 200 μl final volumes of X-VIVO 15 medium (Lonza, Walkersville, MD). Supernatants from polarized Th cells or mature dendritic cells were then collected and kept at −20°C until evaluation of cytokine levels through ELISA assay.
Cytokine content was determined by standard two-site sandwich ELISAs, using available commercial kits for IL-12 p40, IL-23, IFN-γ, and IL-17 (eBioscience), as previously reported (18).
Detection of mTOR pathway
HaCaT or NHEK cells were disrupted with a lysis buffer, and cell homogenates were subjected to 10% SDS-PAGE (50 μg/lane) under reducing conditions. Gels were then electroblotted on to 0.45 μm nitrocellulose filters (Bio-Rad, Hercules, CA) and were immunoreacted with anti-phospho-mTOR (Ser2448, 1:1000, Cat. No. #5536; Cell Signaling), anti-mTOR (1:1000, Cat. No. #2983; Cell Signaling), anti-phospho-p70S6k (Thr389, 1:1000, Cat. No. #9232; Cell Signaling), anti-phospho-p70S6k (Ser371, 1:1000, Cat. No. #9208; Cell Signaling), anti-phospho-4EBP1 (Thr37/46, 1:1000, Cat. No. #2855; Cell Signaling), and anti-FKBP1 (1:500, Cat. No. #H00002280-M01; Abnova) Abs, or with anti-α-actin mAb (1:5000; Bio-Rad), and goat-anti-rabbit polyclonal Ab (1:2000; Santa Cruz Biotechnologies, Santa Cruz, CA) as a secondary Ab. The phosphorylated forms of these proteins were also measured by applying cell lysates to the human AKT pathway phosphorylation array (RayBiotech, Norcross, GA), and were processed according to the manufacturer’s protocol. In both cases, proteins were detected by ECL (ECL, Amersham Pharmacia Biotech) and by CDiGit Chemiluminescent Western Blot Scanner (LI-COR Biosciences, Lincoln).
Quantitative real-time PCR
Total RNA was extracted with an RNeasy Micro kit (Qiagen). A mixture containing random hexamers, oligo(dT)15 (Promega) and SuperScript II Reverse Transcriptase (Invitrogen) was used for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7900 sequence detector (Applied Biosystems) with Applied Biosystems’ predesigned TaqMan Gene Expression Assays and ABsolute qPCR, ROX mix (Thermo Fisher Scientific). The FKBP12 (Hs04192058_gH) probe was used (assay identification numbers in parentheses; Applied Biosystems). For each sample, mRNA abundance was normalized to the amount of β-actin (Hs01060665_g1).
Data were expressed as mean ± SD of at least three independent experiments, each performed in duplicate or triplicate, and were analyzed by means of the Prism 5 program (GraphPad Software, San Diego, CA). Comparisons between groups were calculated by one-way ANOVA followed by Kruskal-Wallis’s test. A p value <0.05 was considered significant. FACS analysis was performed using the Flowjo analysis program (Treestar, Ashland, OR).
AEA suppresses proinflammatory cytokines from IFN-γ-activated HaCaT cells
To analyze the cytokine profile expressed by inflamed keratinocytes and the immunomodulatory effects of ECS, preliminary experiments were performed on HaCaT cells activated with 100 ng/ml IFN-γ, in the presence or absence of several concentrations of AEA (up to 10 μM), and TNF-α production was evaluated by flow cytometry. We showed that AEA significantly inhibited TNF-α production from 2.5 μM up to 10 μM, being ineffective at lower concentrations (Supplemental Fig. 1A). Furthermore, 2.5 μM AEA did not affect cell viability, which decreased to ∼70% at 10 μM (data not shown). Thus, all subsequent experiments were performed using AEA at the lowest effective concentration of 2.5 μM. Upon activation with IFN-γ, HaCaT cells also produced significantly higher levels of several proinflammatory cytokines (IL-6, IL-8, TNF-α, IL-12 p40), as compared with untreated controls. When cells were pretreated with 2.5 μM AEA, the production of TNF-α and IL-12 p40 was significantly suppressed, whereas no significant effect was observed on IL-6 and IL-8 levels (Fig. 1A). Moreover, AEA alone or in combination with IFN-γ did not exert any cytotoxic effect when used at 2.5 μM. Because IL-12 p40 is the common subunit of IL-12 and IL-23, which both have a key role in skin inflammation and the induction of skin autoimmunity (e.g., in psoriasis) (1), we confirmed the actual release of these cytokines in cell supernatants of both HaCaT and NHEK cells. We found that indeed AEA strongly inhibits release from HaCaT cells of both IL-12p70 (3.0 ± 1.0 pg/ml versus 62.0 ± 3.0 pg/ml) and IL-23 (93.0 ± 18.0 pg/ml versus 245.0 ± 55.0 pg/ml) (Fig. 1B), and in primary NHEK cells (IL-12p70, 7.0 ± 3.0 pg/ml versus 16.0 ± 0.2 pg/ml; IL-23, 2.0 ± 2.0 pg/ml versus 97.0 ± 17.0 pg/ml) (Fig. 1C). Incidentally, net levels of cytokine production were 2.5- to 3.5-fold lower in NHEK cells than in HaCaT cells. Furthermore, AEA also significantly reduced TNF-α release (160 ± 42 pg/ml versus 408 ± 43 pg/ml) (Fig. 1B), whereas no effect could be observed on the release of IL-6 and IL-8 (data not shown). In additional experiments, aimed at disclosing the possible involvement of cannabinoid receptors in AEA-mediated anti-inflammatory effects, CB1 and CB2 were blocked by using their selective antagonists (SR1 and SR2, respectively). Treatment with SR1 but not SR2 was able to counteract the immunosuppressive effects of AEA on TNF-α, IL-12p70 and IL-23 release from HaCaT cells (Fig. 1B), as well as IL-12p70 and IL-23 release from NHEK cells (Fig. 1C). As expected, treatment with SR1 and SR2, alone or in combination with IFN-γ, in the absence of AEA, did not significantly change cytokine release compared with controls or IFN-γ-activated cells, respectively (Supplemental Fig. 1B, Table I).
AEA inhibits keratinocyte-dependent induction of TH1 and TH17 responses via CB1
IL-12 and IL-23 play a critical role in the downstream activation of adaptive T helper responses (1). Thus, cocultures were set up between the conditioned medium of untreated or AEA-treated IFN-γ-activated keratinocytes with highly purified CD4+CD45RA+CD27+ naive T cells to evaluate whether keratinocytes were able to affect naive T cell polarization into pathogenic IFN-γ-producing TH1 or IL-17-producing TH17 cells. To this aim, a standard naive CD4 T cell differentiation assay was performed in the presence of polyclonal stimulation with anti-CD3 and anti-CD28, and specific polarizing cytokines or keratinocyte-conditioned medium. As shown in Fig. 2A, in the presence of their specific polarizing conditions, naive CD4 T cells released high levels of IFN-γ (625 ± 125 pg/ml) and IL-17 (180 ± 59 pg/ml), as compared with non-polarized cells (TH0). Although less potent, supernatants of IFN-γ-activated HaCaT cells also significantly stimulated the release of IFN-γ (345 ± 105 pg/ml) and IL-17 (93 ± 8 pg/ml) from CD4 T cells, suggesting that the culture media of IFN-γ-activated keratinocytes contained appreciable levels of TH1- and TH17-polarizing cytokines. Interestingly, pretreatment of activated keratinocytes with AEA abrogated both TH1 and TH17 polarization, inducing a 5-fold inhibition of IFN-γ and a 2-fold inhibition of IL-17 release, respectively. Furthermore, coincubation of HaCaT cells with SR1 or SR2 revealed that only SR1 was able to significantly revert the effects of AEA on TH1 and TH17 polarization, suggesting that the T cell-dependent AEA-mediated immunosuppressive effect was mediated by CB1 only. These findings were also reproduced in IFN-γ-activated NHEK cells (Fig. 2A), although the culture media of the latter cells showed a reduced ability to induce TH1 and TH17 lymphocytes, reflecting their low levels of IL-12 and IL-23 previously shown in Fig. 1C. These results demonstrate a direct activity of keratinocytes on T helper cells. Additionally, keratinocytes are known to regulate dendritic cells, which are the most abundant immune cells of the skin, and the major players in the control of TH1/TH17 balance, thus contributing to the specificity of the inflammatory responses (1). To evaluate whether the ability of AEA to prevent TH1 and TH17 polarization also engaged dendritic cell activation, cocultures were set up between conditioned media of untreated or AEA-treated IFN-γ-activated HaCaT cells and LPS-activated monocyte-derived dendritic cells (Mo-DC). As shown in Fig. 2B, LPS-activated Mo-DC released high levels of IL-12p70 (875 ± 75 pg/ml), and IL-23 (475 ± 125 pg/ml) as compared with resting Mo-DC. Furthermore, supernatants of IFN-γ-activated HaCaT cells also significantly stimulated IL-12p70 (525 ± 65 pg/ml) and IL-23 (380 ± 90 pg/ml) release from Mo-DC, and their levels were significantly higher than those released from keratinocytes in the form of their common subunit IL-12 p40. Overall, these data suggest that the majority of IL-12 and IL-23 in the skin derives from Mo-DC rather than from keratinocytes themselves. Interestingly, pretreatment of activated HaCaT cells with AEA abrogated both IL-12p70 and IL-23 release from Mo-DC, inducing a 2-fold inhibition of IL-12p70 and a 3-fold inhibition of IL-23 release, respectively. In addition, coincubation of HaCaT cells with SR1 or SR2 revealed that only SR1 was able to significantly revert the effects of AEA on both TH1- and TH17-polarizing cytokines, suggesting that the Mo-DC-dependent AEA-mediated immunosuppressive effect was once again only mediated by CB1. Moreover, the same AEA-mediated and CB1-dependent effect was also observed when analyzing the expression of CD80, CD86 and CD83 activation markers on Mo-DC. Indeed, AEA downregulated their cell surface expression via CB1 (Fig. 2C), especially in the case of CD86 and CD83, eventually causing Mo-DC to lose their ability to induce adaptive T helper responses (5).
Mechanism of AEA-mediated and CB1-dependent immunomodulatory effects on HaCaT cells
Next, we interrogated the potential signaling mechanism through which AEA exerts its immunomodulatory effects. Recent evidence has shown that mTOR is a novel pathway that modulates inflammatory responses in human keratinocytes (15, 19). Thus, we sought to assess its possible involvement in AEA-mediated and CB1-dependent anti-inflammatory effects on activated keratinocytes. To this aim, we first ascertained whether mTOR was able to modulate per se the release of proinflammatory cytokines upon IFN-γ stimulation of HaCaT cells. Thus, cells were pretreated with different concentrations of mTOR inhibitor rapamycin for 2 h, and then were further challenged with IFN-γ. Cells were evaluated for intracellular expression of TNF-α and IL-12 p40 24 h poststimulation, and supernatants were assayed for IL-12p70 and IL-23. We found that pharmacological inhibition of mTOR by 10 nM rapamycin significantly reduced both intracellular production and extracellular release of cytokines in IFN-γ-activated HaCaT cells (Fig. 3A). Because rapamycin did not exert any effect at 1 nM, and at 100 nM it was as effective as at 10 nM (Supplemental Fig. 1C), the latter dose was chosen for all subsequent experiments. Incidentally, rapamycin alone did not affect IL-12p70 and IL-23 release (data not shown). The effect of mTOR inhibition on IL-12 and IL-23 release was observed also in primary NHEK cells (Fig. 1C). In addition, TH1- or TH17-polarized naive T cells were cocultured in the presence of conditioned media of activated HaCaT cells, pretreated or not with rapamycin, and supernatants were assayed for IFN-γ and IL-17 levels. Fig. 3B shows that mTOR inhibition by rapamycin prevented both TH1 and TH17 polarization (IFN-γ, 98 ± 53 pg/ml versus 345 ± 105 pg/ml; IL-17, 46 ± 24 pg/ml versus 140 ± 10 pg/ml), supporting the engagement of mTOR signaling in keratinocyte-dependent T cell responses. Because rapamycin has many off-targets, we further confirmed our data by using LY2584702, a potent and selective inhibitor of the downstream kinase p70S6K activated by mTOR (20). Our results showed that indeed LY2584702 significantly suppressed cytokine production and release from activated HaCaT cells (Fig. 3A), as well as of IFN-γ and IL-17 from polarized TH1 and TH17 cells, suggesting that the mTOR pathway is likely to be involved in cytokine production from inflamed keratinocytes.
Next, we investigated whether AEA could modulate p70S6K and 4-EBP1, downstream targets of the mTOR pathway. We found that activation of HaCaT cells with IFN-γ significantly stimulated phosphorylation of p70S6K (Thr389/Ser371) and 4-EBP1, and that pretreatment with AEA significantly inhibited phosphorylation of p70S6K, in particular at Ser371 and not at Thr389, but not of 4-EBP1 (Fig. 4A). Treatment with SR1 but not SR2 restored the phosphorylation of p70S6K, suggesting a CB1-dependent mechanism. Remarkably, also in primary NHEK cells AEA suppressed phosphorylation of p70S6K in a CB1-dependent manner (Fig. 4A). Such a CB1-mediated effect of AEA was also confirmed through a phosphorylation proteomic array (Fig. 4B), suggesting that AEA interferes with the mTOR pathway through p70S6K rather than 4-EBP1. To further dissect the signaling mechanism responsible for CB1-dependent inhibition of the mTOR pathway, we also analyzed the expression of FKBP12. Indeed, the latter substance belongs to the immunophilin family, which comprises the only endogenous inhibitors of mTOR as yet known (21). IFN-γ-activated HaCaT cells show reduced mRNA (Fig. 4C) and protein (Fig. 4D) levels of FKBP12, demonstrating that mTOR is also upregulated in activated keratinocytes because of a reduction of its physiological inhibitor. Lower doses of IFN-γ (10 and 50 ng/ml) could not significantly affect FKPB12 expression (Supplemental Fig. 1D). Interestingly, AEA restored FKBP12 mRNA and protein content back to that of untreated cells only in IFN-γ-activated cells (Fig. 4B, 4C) but not when given alone and at different concentrations (Supplemental Fig. 1D). Furthermore, antagonism of CB1 counteracted (yet not significantly) this AEA-induced effect on FKBP12 protein expression (Fig. 4B, 4C), suggesting that this physiological mTOR inhibitor might partly be involved in mediating AEA actions. Overall, it can be suggested that AEA exerts its anti-inflammatory effects on keratinocytes through a CB1-dependent inhibition of mTOR signaling, as schematically depicted in Fig. 5.
To our knowledge, the present investigation demonstrates for the first time that in keratinocytes CB1 inhibits the activation of T cell responses, in particular of the highly proinflammatory TH1 and TH17 cells. The role of CB1 in skin homeostasis has been extensively investigated, and has been shown to favor maturation and activation of mast cells (19), to inhibit epidermal differentiation (22, 23), and to induce melanogenesis (24). The first evidence for a role of the ECS in skin inflammatory diseases was obtained in knockout mice (25): those lacking both CB1 and CB2 showed exacerbated allergic inflammation, whereas those deficient in fatty acid amide hydrolase showed higher AEA content and reduced cutaneous allergic responses. Overall, these data suggested a protective role of ECS in allergic contact dermatitis. In line with this, Tüting’s group has recently shown that CB1 in keratinocytes helps to limit the secretion of proinflammatory chemokines CXCL10 and CCL8 in a mouse model of allergic contact dermatitis. Both substances regulate T cell-dependent inflammation, and attenuate thymic stromal lymphopoietin- and CCL8-dependent TH2-type allergic inflammatory responses (13, 26). In this article, we provide additional evidence that AEA limits production and release of IL-12 and IL-23 (TH1- and TH17-inducing cytokines, respectively) from inflamed HaCaT cells in a CB1-dependent manner. We thus reasoned that CB1 could negatively drive the polarization of CD4 naive T cells into TH1 and TH17. This hypothesis was confirmed through TH1/TH17 polarization assays in the presence of conditioned supernatants from HaCaT cells treated with AEA, alone or in combination with the CB1 selective antagonist SR1. Indeed, the observed reduction in TH1 and TH17 lineages upon treatment with AEA and SR1 demonstrates a direct role of CB1 in driving proinflammatory Th cell responses in the skin. It should be recalled that the skin also hosts constituents of the innate immune system, whose dysfunction is critical in the development of pathological inflammatory conditions (1). The most important innate immune cells of the skin are Langerhans cells, dendritic cells of the skin and mucosa that derive from the differentiation of monocytes, and are the main regulators of TH1 and TH17 induction (1, 27). Treatment of Mo-DC with the conditioned medium of AEA- or SR1-treated inflamed HaCaT cells demonstrated that Mo-DC are indeed the main producers of the TH1-inducing IL-12 and of the TH17-inducing IL-23. The CB1-dependent inhibition by AEA of the release of these polarizing cytokines from dendritic cells, along with inhibition of their activation markers, suggests that CB1 might be truly involved in the control of TH1 and TH17 commitment. To determine the molecular mechanisms through which CB1 in keratinocytes may regulate these anti-inflammatory responses of the skin, the possible engagement of a new player in skin immunology like mTOR was investigated. Indeed, accumulated evidence points to a crucial role of mTOR signaling in the epidermal changes that lead to the psoriatic phenotype (15, 28, 29). In this context, recent reports suggest that overexpression of the PI3K/AKT/mTOR pathway may play an important role in the development of psoriasis, by mediating immunopathogenesis, epidermal hyperplasia and/or angiogenesis in the course of the disease (30). Furthermore, relevant cytokines known to be critical in psoriasis, psoriatic arthritis, and rheumatoid arthritis, such as IL-17 and IL-22, have been found to activate mTOR-dependent signal transduction (29, 31). Our results demonstrate not only that IFN-γ-induced IL-12 and IL-23 production in HaCaT cells is mediated by mTOR signaling, and that its inhibition with rapamycin limits TH1 and TH17 polarization, but also that activation of CB1 by AEA blocks the phosphorylation of the downstream executioners of mTOR pathway. Interestingly, the latter effect seemed to be partly mediated by upregulation of the immunophilin FKBP12, the only endogenous inhibitor of mTOR as yet known. Taken together, these findings account for an unprecedented transduction mechanism through which AEA exerts protective and anti-inflammatory effects in the skin. On a more general note, it should be recalled that the role of mTOR in immunity is somewhat controversial, because its activation in APCs and T cells has been reported to be both host protective and disease inductive. On the one hand, mTOR activation can upregulate IL-10 and inhibit IL-12, thus promoting TH2/Treg cells and enhancing the TH1/TH17 axis; on the other hand, inhibition of mTOR signaling in Treg and memory cells can augment expansion of Tregs with increased suppressive capacity, thus improving the quality of memory cells (32, 33). In this scenario, our data supports the view that mTOR plays a proinflammatory role in skin, thus advocating a pharmacological inhibition of its pathway to limit skin inflammation. However, although such effects may be the result of AEA/CB1-mediated mTOR inhibition, present data do not rule out the possibility of an off-target (i.e., other than mTOR-related) effect, particularly given the broad effects of mTOR activity on protein synthesis, lipid metabolism, and autophagy (34). Much like CB2 in the immune system, which is considered a panacea to control both innate and adaptive immune responses (17, 18), CB1 seems to be central in skin inflammation, inasmuch as its activation could limit TH2-driven allergic inflammatory disabilities (such as atopic dermatitis) and TH1/TH17-driven skin autoimmunity. Overall, our findings provide further mechanistic insights into the role of CB1 in the regulation of T cell immune responses, and pave the way for a better understanding of skin physiology as well as the development of novel therapeutic strategies that target ECS (and/or mTOR pathway). These new drugs hold promise for the treatment of skin inflammatory imperfections or TH-1/TH-17-mediated skin diseases, such as psoriasis or contact dermatitis.
This work was supported by L’Oréal Research and Innovation, Paris (Grant 2011-2013 to M.M.).
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.