Eight-week-old male C57BL/6 mice (weighing 20g-25g) were purchased from HFK Laboratory (Beijing, China). After being allowed to adapt for 5 days, the mice were randomized assigned to different groups using the random number table. The numbers of mice used in each group were shown in figure legends. For the establishment of psoriasis model, mice were shaved and treated with a topical dose of 62.5 mg of imiquimod (IMQ) cream (3 M, USA) or vehicle cream (Ctrl) on their back for six consecutive days. At Day 7, mice were sacrificed. Their skin was fixed in 4% paraformaldehyde and then embedded in paraffin for HE staining. To evaluate the impact of D-mannose on IMQ-induced psoriasis, mice were administered 200 μl of 20% (w/v) D-mannose (1.1M) (Sigma, USA) by gavage twice a day while given drinking water with 20% D-mannose (1.1M) ad libitum for one week. Then, the IMQ-induced model was established, while D-mannose was still administered orally. After 6 consecutive days of IMQ treatment, the mice were harvested. The dosage of D-mannose used in our research was previously reported to have no adverse effect on the weight and health of the animals (29–32). All mice were housed in a pathogen-free, comfortable temperature environment with a 12 h light/dark cycle. All animal studies were performed in compliance with the ethical guidelines for animal studies and approved by the Animal Ethics Committee of Beijing Friendship Hospital, Capital Medical University (approval no. 20-2018).

The PASI score was used to assess the severity of the lesion upon IMQ exposure. The score was graded from 0 to 4 according to the erythema, scale and thickness of the skin. A score of 0 means normal, and a score of 4 means very severely altered. Every mouse was graded according to PASI daily, and the assessment was performed blinded.

Antibodies against TCRγδ (GL3), TCRβ (H57-597), CD3 (17A2), Foxp3 (MF23), CD25 (PC61), PD1 (J43), Annexin V, IFN-γ (XMG1.2), NK1.1 (S17016D), CD27 (LG.3A10), IL-23R (12B2B64), IL-17A (TC11-18H10), CD45 (A20), CCR2 (SA203G11), CCR5 (HM-CCR5), CCR6 (29-2L17), CXCR6 (SA051D1), ICOS (C398.4A), CD62L (MEL-14), Ki-67 (SoIA15), CD44 (IM7), IRF4 (IRF4.3E4), and Blimp1 (5E7) were purchased from Biolegend. Antibodies against p-AKT (Ser473) (SDRNR), p-HIF-1α (Mgc3), and p-mTOR (Ser2448) (MRRBY) were purchased from Thermo Fisher. Before cell staining, TruStain FcX™ PLUS (anti-mouse CD16/32) antibodies (Biolegend) were used to block nonspecific binding. For intracellular staining of cytokines, the cells were stimulated with Cell Activation Cocktail (with Brefeldin A) from Biolegend. Six hours later, the cells were stained with antibodies against surface molecules and prepared according to the instructions of the Cyto-Fast™ Fix/Perm Buffer Set (Biolegend). For intracellular staining of transcription factors and signaling molecules, the cells were prepared with a Foxp3/Transcription Factor Buffer Set (Thermal Fisher).

γδ T cells sorted from the spleens and peripheral LNs of 15 healthy mice were pooled together and cultured (2×10⁵ per well) for 72 hours with IL-2 (2 ng/ml) in a round-bottom 96-well plate. Antibodies against CD3 (10 μg/ml) and CD28 (1 μg/ml) were added. To evaluate the impact of D-mannose on γδ T cells, cells were also treated with D-mannose (50 mmol/L).

To determine the impact of D-mannose on γδ T cell glycolytic capabilities, γδ T cells were pretreated with or without D-mannose in vitro as described above for three days. Subsequently, these cells were harvested and seeded on a poly-D-lysine-coated 96-well XF microplate (2-4×10⁵ cells/well) and cultured in XF RPMI 1640 medium with glucose (25 mmol/L), pyruvate (1 mmol/L) and glutamine (4 mmol/L). In the D-mannose assay group, D-mannose (25 mmol/L) was added to the culture medium. Finally, to measure the extracellular acidification rate (ECAR) of these γδ T cells, an Agilent Seahorse XFe-96 metabolic analyzer and glycolytic rate assay kit (Agilent) were used according to the instructions. The level of ECAR may reflect the capabilities of glycolysis in γδ T cells.

RNA samples from skin-draining LN and splenic γδ T cells (from healthy and psoriatic mice) were sequenced using a standard Illumina protocol (Annoroad Gene Technology, Beijing). Each sample represented γδ T cells obtained from 15 mice. Reads were mapped to a mouse genome (Mm9) by using HISAT2. Gene counts were estimated by HTSeq. The R package DESeq2 was applied to determine differentially expressed genes (DEGs). Genes with a fold-change >2 and an adjusted P value < 0.05 were defined as DEGs. The R package clusterprofiler was applied to perform GO enrichment and GSEA of the DEGs. The data reported in this work have been uploaded to the Gene Expression Omnibus (GEO) database under accession number GSE188905.

RNA was extracted from γδ T cells using the RNeasy Plus Micro Kit (Qiagen), and cDNA was obtained using PrimeScript™ RT Master Mix (TaKaRa). Quantitative PCR was performed using Hieff qPCR SYBR Green Master mix (YEASEN, Beijing) on a QuantStudio™ 3 Real-Time PCR Instrument (Thermal Fisher), with each sample in triplicate. The quantification was based on 2^-ΔΔCt calculations and was normalized to β-actin. The primers used in the article were listed in Table S1 .

The protein of γδ T cells cultured in vitro was extracted by RIPA lysis buffer (Roche). Each sample represented γδ T cells obtained from 5 mice. After quantified the concentration of protein by bicinchoninic acid assay (Thermal Fisher), the expressions of p-AKT (Ser473), p-mTOR (Ser2448), and β-acitn (8H10D10) were evaluated using a capillary western blot analyzer (ProteinSimple). The 25-lane plates were used according to the instructions. All the antibodies for western blot were purchased from Cell Signaling Technology.

GraphPad Prism 8 software was applied to perform the statistical analysis. An unpaired Student’s t test was used to evaluate the significance of the difference between two groups. For comparisons between multiple groups, the two-way ANOVA with Sidak’s multiple comparison test was performed. Data were presented as the mean ± SEM, and a P value < 0.05 was considered significant (*p < 0.05, **p < 0.002, ***p < 0.0002, ****p < 0.0001). The t values and F values of each comparison were also shown in the figure legends.

To investigate the alteration of γδ T cells, we first treated the mice with IMQ on their backs for six consecutive days. As shown in Figures 1A–C , IMQ treatment resulted in signs of scurf and thickened skin ( Figure 1A ). Histological analysis of skin sections showed that IMQ-treated mice had parakeratosis and thickened dermal and inflammatory cell infiltrations ( Figures 1B, C ). These results indicated that the psoriasis model was successfully established. We then compared the ratio of γδ T cells to CD3⁺ T cells in skin-draining LNs (DLNs) and spleens between IMQ-treated and control mice ( Figure 1D ). The gating strategy of γδ T cells for flow cytometry analysis was shown in Figure S1 . Upon IMQ treatment, the frequency of γδ T cells was increased in both DLN and spleen; however, the frequency of γδ T cells in the DLN was higher, and the fold-change of the γδ T cell ratio in the DLN was more remarkable than that in the spleen (by 2.28 in the DLN and 1.30 in the spleen) ( Figure 1D ). These results were consistent with previous reports (16, 18) and showed that IMQ stimulation expanded γδ T cells in the DLNs and spleens.

We next characterized the γδ T cells from the DLN and spleen by RNA-seq analysis. Both principal component analysis (PCA) and a dendrogram provided a clear demonstration that γδ T cells from different groups were distinct from each other ( Figure 2A and Figure S2A ). Compared to control mice, the γδ T cells from IMQ-treated mice had a total of 2091 upregulated genes and 3563 downregulated genes in the DLN, as well as 1469 and 3353, respectively, in the spleen (absolute fold change>2, P.adj<0.05) ( Figure 2B ). The transcriptional differences between DLN and splenic γδ T cells were also significantly amplified upon IMQ stimulation, from 552 upregulated genes and 2673 downregulated genes (Ctrl LN vs. Ctrl Spl) to 2230 upregulated genes and 4849 downregulated genes (IMQ LN vs. IMQ Spl) ( Figure 2B ). Comparing the differentially expressed genes (DEGs) from different groups by Gene ontology (GO) analysis, we found that the upregulated DEGs in the DLN were enriched in lymphocyte proliferation, leukocyte migration, positive regulation of the cell cycle and cytokine production; however, the upregulated DEGs in the spleen were primarily enriched in small GTPase-mediated signal transduction, leukocyte migration, and cell chemotaxis ( Figure 2C ). Further comparison of DEGs between DLN and splenic γδ T cells from IMQ-treated mice also showed that the γδ T cells in DLN attained a higher level of proliferation, while the splenic γδ T cells got enhanced in the capabilities such as cell chemotaxis and adhesion ( Figure S2B ). Consistent with the RNA-seq results, our flow cytometry analysis showed that the DLN γδ T cells from IMQ-treated mice had the highest level of Ki-67 expression among different γδ T populations, supporting the augmented proliferation of these cells ( Figure 2D and Figure S2C ).

As shown in Figure 2E , γδ T cells from IMQ DLNs demonstrated a higher level of activation/effector gene expression, including Icos, Pdcd1, and Itgae, and lower levels of Sell and Ccr7 expression. Hence, we examined the cells by flow cytometry and found that the DLN γδ T cells from IMQ-treated mice included more activated T cells (CD44^hiCD62L^lo) with higher expression of ICOS and PD1 than other γδ T populations ( Figure 2F and Figure S2C ). Together, these results indicated that the inflammatory γδ T cells in the DLN of psoriatic mice are an activated population with highly proliferative features.

To address the inflammatory function of γδ T cells in the psoriasis model, we first investigated the cytokine and cytokine receptor signatures of γδ T cells from control and IMQ-treated mice. As shown in Figure 3A , the expression of cytokines from the IL-17 superfamily (Il17a, Il17f, Il22), cytokine receptors (Il23r, Il2ra, Il1r1) and Gzmb was exclusively higher in DLN γδ T cells from the IMQ-treated mice than in other γδ T populations ( Figure 3A ). Flow cytometry analysis also confirmed that they included more cells with positive expression of IL-23R and CD25 [molecules associated with IL-17 production (33)], but fewer cells expressing CD27 and NK1.1 ( Figure 3B add Figure S3A ). Indeed, upon PMA and ionomycin stimulation, the DLN γδ T cells from psoriatic mice had a significantly elevated percentage of IL-17A⁺ cells and decreased IFN-γ production ( Figure 3C and Figure S3B ).

We also compared the expression of transcription factors within γδ T cells of healthy and psoriatic mice. Consistent with the augmented capability of IL-17 production, RNA-seq and flow cytometry analysis both confirmed that more RORγt⁺ cells were found in γδ T cells from IMQ-treated DLNs than in γδ T cells from other groups ( Figures S4A, B and Figure 3D ). In addition, these pathogenic γδ T cells from the DLN of IMQ-treated mice showed higher level expression of Prdm1, Maf, Irf4, Runx3, Stat5, and Batf, and flow cytometry analysis further confirmed the exclusive upregulation of Blimp1 and IRF4 ( Figure S4A and Figure 3E ). These transcriptional data again demonstrated that the γδ T cells in DLN were imprinted by the destiny of γδ17 T cells after IMQ treatment.

Following dermatitis, both γδ T cells in the DLN and spleen upregulated the genes associated with migration ( Figure 2C ). Therefore, we also determined the expression of chemokine receptors. Notably, the transcription of Ccr1, Ccr2, Ccr4, Ccr5, Ccr6, and Cxcr6 was higher in DLN γδ T cells from IMQ-treated mice than in other γδ T populations ( Figure S4A ). The increased ratios of CCR2⁺, CCR5⁺, CCR6⁺, and CXCR6⁺ cells were confirmed by flow cytometry ( Figure 3F and Figure S4B ). Interestingly, compared to control splenic γδ T cells, the splenic γδ T cells from IMQ-treated mice also showed increased percentages of CCR6⁺ and CXCR6⁺ cells, and their CCR6 expression was the highest between each population ( Figure 3F and Figure S4B ). CCR2 and CCR6 were reported to direct pathogenic γδ17 T cells to migrate into the dermis and cause inflammatory injury (18, 34). Hence, these results suggested that IMQ stimulation augmented the chemotaxis of γδ T cells from peripheral tissues to inflamed sites.

We next characterized the metabolic pattern of γδ T cells from IMQ-treated mice by gene set enrichment analysis (GSEA). After the psoriatic model was built, enrichments of genes associated with mTOR1 signaling and the metabolic processes of carbohydrates, fatty acids, glutamine family amino acids, glucose 6-phosphate, and glycolysis were observed in skin DLN γδ T cells ( Figure 4A and Figure S5A ). In spleen, the enriched pathways associated with metabolism were the metabolic processes for carbohydrates, glucose 6-phosphate, and cholesterol ( Figure S5A ). To meet the large energy demands for activation or differentiation, T cells usually switch their metabolic pattern to glycolysis (35, 36). Previous works have reported that the mTOR1-regulated glycolysis is required for the development and function of γδ17 T cells (35). The enrichments of pathways associated with mTOR1 signaling and glycolytic process in psoriatic DLN γδ T cells were consistent with these reports and implied the enhanced capability of glycolysis ( Figure 4A ). Hence, we determined the glycolytic signatures of γδ T cells in psoriatic mice. Upon IMQ stimulation, genes upregulated in γδ T cells tied to glycolysis included Slc2a1, Hk2, Pfkm, Aldoc, Pgk1, Ldha, Tpi1, Eno1, Pkm, and Pfkl ( Figure 4B ). Among these highly expressed genes, DLN γδ T cells from psoriatic mice exhibited the most distinctive differences in the expression of Pgk1 and Ldha relative to other γδ T populations ( Figures 4B, C ). As expected, the DLN γδ T cells from psoriatic mice also expressed the highest level of p-HIF-1α, the key regulator of glycolysis (23), and our flow cytometry analysis confirmed this finding ( Figure S4A and Figure 4D ). Collectively, these data indicated that the skin-draining LN γδ T cells from psoriatic mice had a higher level of glycolysis.

D-mannose, as a C-2 epimer of glucose, has been reported to impair glycolysis and suppress the activation of HIF-1α (31); therefore, we hypothesized that it may also have a beneficial effect in the model of psoriasis. As expected, D-mannose alleviated the scaly erythematous epidermis and resulted in smaller spleens in psoriatic mice, while it didn’t affect the weight ( Figures S5B-D ). Compared to mice exposed only to IMQ, the PASI score was decreased in mannose-treated mice starting on the third day after IMQ treatment ( Figure 4E ). Histological analysis also showed that the mice given D-mannose orally had decreased inflammatory cell infiltration and hyperkeratosis ( Figure 4F ). The layers of keratinocytes in skin were decreased significantly because of D-mannose treatment ( Figure 4F ). Together, these data demonstrated that oral administration of D-mannose successfully alleviated the skin inflammation caused by IMQ exposure.

To determine whether the beneficial effect of D-mannose on psoriasis was achieved by the regulation of γδ T cells, we performed flow cytometry analysis. As shown in Figure 5A , mannose treatment selectively downregulated the frequency of γδ T cells in DLN from psoriatic mice. Compared to mice treated without D-mannose, mice given mannose also had a lower level of ICOS expression exclusively in DLN γδ T cells ( Figure 5B and Figure S6A ). Considering the decreased frequency and ICOS expression of DLN γδ T cells upon D-mannose treatment, we determined their capabilities of proliferation and cytokine production. Both spleens and DLNs from mice given mannose had reduced percentages of Ki-67⁺ and IL17A⁺ γδ T cells compared to mice treated with IMQ alone; however, the expression of IFN-γ in γδ T cells was not altered ( Figures 5C, D and Figure S6A ). Nevertheless, the γδ T cells from DLN contained more activated cells with positive expression of ICOS, Ki-67, and IL-17A than splenic γδ T cells after feeding with D-mannose ( Figures 5B–D and Figure S6A ). To examine the potential underlying machinery, we compared the transcription factors of these γδ T cells. As shown in Figure 5E , oral administration of D-mannose resulted in decreased expression of p-HIF-1α in the spleen and DLN compared to mice that received IMQ alone, which may imply the lowered glycolytic capabilities of γδ T cells ( Figure 5E ). However, a reduced frequency of RORγt⁺ cells was only found in the spleen of psoriatic mice treated with D-mannose ( Figure 5F and Figure S6A ). Together, these results suggested that D-mannose could attenuate psoriatic inflammation by suppressing the proliferation and IL-17 production of γδ T cells.

Previous findings by Dunfang et al. demonstrated that D-mannose could induce Treg cells and suppress autoimmune diabetes and airway inflammation (29). To verify whether the suppression of γδ T cells by D-mannose was achieved by the induction of Tregs, we examined the ratio of Treg cells in the spleen and DLN of psoriatic mice treated with or without D-mannose. Flow cytometry analysis showed that D-mannose treatment significantly increased the percentage of Treg cells in spleen; however, the ratio of Tregs in DLN was not altered ( Figure S6B ). This partially excluded the effect of Treg induction on the suppression of DLN γδ T cells by D-mannose.

We next investigated the direct effect of D-mannose on γδ T cells. γδ T cells sorted from spleens and LNs were pooled together and cultured in vitro. After three days of TCR stimulation, γδ T cells treated with D-mannose contained fewer cells with positive expression of Ki-67 and more apoptotic cells ( Figures 6A, B ). To reveal the underlying mechanism, we hypothesized that the suppression of γδ T cells may result from alterations in metabolism and performed a glycolytic rate assay. Indeed, the lower extracellular acidification rate (ECAR) indicated that D-mannose significantly suppressed the glycolytic capabilities of γδ T cells, while quantitative PCR showed that D-mannose reduced the expression of key glycolytic molecules in γδ T cells, such as Pgk1, Ldha, Pfkm, Aldoa and Slc2a1 ( Figures 6C, D ). Moreover, both flow cytometry analysis and capillary western blot illustrated the expression of phospho-AKT and phospho-mTOR was downregulated in γδ T cells after culture with D-mannose ( Figures 6E, F and Figure S7 ). Together, considering the crucial roles of AKT/mTOR signaling in glycolysis (35, 37), our results suggested that D-mannose treatment contributed to the lower proliferation and higher apoptosis of γδ T cells by impairing glycolysis via the AKT/mTOR axis.