TNF-α promotes CXCL-1/8 production in keratinocytes by downregulating galectin-3 through NF-κB and hsa-miR-27a-3p pathway to contribute psoriasis development

Abstract Objective Treatment with TNF-α inhibitors improve psoriasis with minimize/minor neutrophils infiltration and CXCL-1/8 expression in psoriatic lesions. However, the fine mechanism of TNF-α initiating psoriatic inflammation by tuning keratinocytes is unclear. Our previous research identified the deficiency of intracellular galectin-3 was sufficient to promote psoriasis inflammation characterized by neutrophil accumulation. This study aims to investigate whether TNF-α participated in psoriasis development through dysregulating galectin-3 expression. Methods mRNA levels were assessed through quantitative real-time PCR. Flow cytometry was used to detect cell cycle/apoptosis. Western blot was used to evaluate the activation of the NF-κB signaling pathway. HE staining and immunochemistry were used to detect epidermal thickness and MPO expression, respectively. Specific small interfering RNA (siRNA) was used to knock down hsa-miR-27a-3p while plasmids transfection was used to overexpress galectin-3. Further, the multiMiR R package was utilized to predict microRNA-target interaction. Results and discussion We found that TNF-α stimulation altered cell proliferation and differentiation and promoted the production of psoriasis-related inflammatory mediators along with the inhibition of galectin-3 expression in keratinocytes. Supplement of galectin-3 could counteract the rise of CXCL-1/8 but not the other phenotypes of keratinocytes induced by TNF-α. Mechanistically, inhibition of the NF-κB signaling pathway could counteract the decrease of galectin-3 and the increase of hsa-miR-27a-3p expression whereas silence of hsa-miR-27a-3p could counteract the decrease of galectin-3 expression induced by TNF-α treatment in keratinocytes. Intradermal injection of murine anti-CXCL-2 antibody greatly alleviated imiquimod-induced psoriasis-like dermatitis. Conclusion TNF-α initiates psoriatic inflammation by increasing CXCL-1/8 in keratinocytes mediated by the axis of NF-κB-hsa-miR-27a-3p-galectin-3 pathway.


Introduction
Psoriasis is an immune-mediated, chronic inflammatory skin disease characterized by keratinocyte hyperproliferation, infiltration of immune cells, and expression of the diversity and abundance of pro-inflammatory mediators, among which TNF-α is highly expressed in lesional skin and plays a pivotal role in the mechanism of psoriasis [1].TNF-a was reported to be highly expressed in both skin lesions and serum of psoriasis patients [2].Knockout TNF can improve dermatitis and inflammation on the imiquimod cream-induced psoriasis murine model, indicating the critical role of TNF-a in the pathogenesis of psoriasis [3].It is currently believed that the pro-inflammatory function of TNF-a is due to the activation of NF-kB and MAPK signaling pathways and the transcription of their downstream pro-inflammatory genes, including a wide range of cytokines, chemokines, related receptors, and adhesion molecules [4].However, the underlying pathogenetic mechanisms of TNF-a in psoriasis are complex and remain elusive.Besides, the efficacy of TNF-α inhibitors in psoriasis treatment only reaches about 46-66% [5].Thus, it is necessary to improve the therapeutic efficacy and initiate precision medicine by thoroughly exploring the mechanism of TNF-α involved in psoriasis development.The improvement of psoriasis treated with TNF-α inhibitor (Etanercept) was characterized by a decrease of neutrophils infiltration and a reduction of CXCL-1/8 expression in psoriatic lesions [6], indicative of a critical role of TNF-α in regulating chemokines and neutrophils recruitment.
Galectin-3 is a member of the β-galactoside-binding lectin family and is a ubiquitously present protein with a unique chimera structure and a variety of biological roles [7,8].In skin, galectin-3 is expressed in keratinocytes, hair follicles, sebaceous, sweat glands, and other resident cells in the epidermis and dermis, including melanocytes, dendritic cells, fibroblasts, and monocytes [9].Galectin-3 was reported to be associated with many functions of keratinocytes, including differentiation, migration, apoptosis, and inflammation.Our previous study has demonstrated that galectin-3 was greatly reduced in psoriatic lesions and the absence of galectin-3 in the skin was sufficient to induce psoriasis-like dermatitis that was characterized by neutrophil accumulation [10].However, the fine mechanism of galectin-3 reduction and involvement in psoriasis remains unexplored.
In the present study, we showed that TNF-α promoted the production of proinflammatory mediators and meanwhile downregulated galectin-3 expression in keratinocytes.The enhanced expression of CXCL-1 and CXCL-8 was mediated by galectin-3 downregulation.The activation of the NF-κB pathway and the consequent production of hsa-miR-27a-3p resulted in the inhibition of galectin-3 expression in keratinocytes in response to TNF-α stimulation.The blockade of murine CXCL-2 (murine homolog of human CXCL-8) with antibodies alleviated imiquimod-induced psoriasis-like dermatitis.Overall, our study suggests that TNF-α participates in psoriasis development by promoting the production of neutrophil chemoattractants through the downregulation of galectin-3 expression with the involvement of NF-κB and hsa-miR-27a-3p pathway.

Inhibition of NF-κB signaling pathway
JSH-23 (Selleck, S7351) was dissolved in DMSO and then diluted with a complete medium.Cells were pretreated with 30 µM of JSH-23 for 3 h before TNF-α stimulation.An equal volume of DMSO served as control.Cells were harvested at 15 min/72 h thereafter for protein/RNA analysis, respectively.

MicroRNA-LGALS3 mRNA interaction prediction
The multiMiR R package was utilized to obtain predicted or validated microRNA-target interaction from 11 databases [11].In total, 86 microRNA were found to target LGALS3 mRNA.The interaction between hsa-miR-27a-3p and LGALS3 mRNA was predicted by 3 databases (miRanda, microcosm, and targetscan).

Keratinocytes transfected with plasmids or siRNAs
The plasmids were purchased from BGI Tech Solutions (Beijing Liuhe) CO.HaCaT cells were transiently transfected with indicated plasmids (pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro used as backbone plasmid, plasmid sequence of galectin-3 is shown in the supplemental material) using Lipofectamine 3000 reagent (Invitrogen, L3000008) according to the manufacturer's instructions.The transfected cells were harvested and analyzed using RT-qPCR 96 h after transfection.

mRNA extraction, reverse transcription, and qPCR
Total RNA was extracted using EZ-press RNA Purification Kit for cultured keratinocytes (EZBioscience, B0004DP) or RNeasy Fibrous Tissue Mini Kit for animal tissue (QIAGEN, 74704).Complementary DNA of mRNA was synthesized using Color Reverse Transcript Kit (EZBioscience, A0010CGQ) according to the manufacturer's instructions, and reverse transcription for microRNA was performed using EZ-press microRNA Reverse Transcription Kit (EZBioscience, EZB-miRT4-L).Quantitative PCR (qPCR) was performed on Roche LightCycler TM 480 using 2 × SYBR Green Color qPCR Mix (EZBioscience, A0012-R2) and EZ-Probe qPCR Master Mix (EZBioscience, EZB-Probe-L).The primer sequences were shown in Table S1-S3.The formula for calculating the relative expression of target genes was: 2 −ΔCt (2^housekeeping gene/2^gene of interest).The primer sequences are listed in the supplemental material.

Cell cycle and apoptosis assay of keratinocytes
For cell cycle analysis, floating cells were discarded, and adherent cells were digested using trypsin (Gibco, 25200056 for HaCaT cells, ATCC, PCS-999-003 for NHEK), washed with PBS and DPBS, and fixed with ice-cold 70% ethanol followed by centrifugation.Cell pellets were resuspended with 2 mL PBS/D-PBS containing 50 µl RNAse A and 450 µL propidium iodide (Invitrogen) and analyzed by flow cytometry.
For apoptosis assay, floating and adherent cells were collected, resuspended by binding buffer (eBioscience) containing annexin V/FITC(eBioscience), incubated in darkness for 15 min, added propidium iodine (Invitrogen) and analyzed using flow cytometry immediately.

Western blot analysis
Cytoplasmic and nuclear proteins of HaCaT cells were extracted using an extraction kit (Beyotime, P0027) together with protease and phosphatase inhibitors (Beyotime, P1045).Immunoblot analysis of nuclear protein was achieved using antibodies against phospho-NF-κB p65 (Cell Signaling Technology, 3033), NF-κB p65 (Cell Signaling Technology, 8242), and PCNA (Cell Signaling Technology, 13110) according to the manufacturer's protocols.The intensity of fluorescence of bands was quantified using Image J software.

Intradermal injection of neutralizing antibodies
Female C57BL/6J mice between 8 and 10 weeks of age were purchased from GemPharmatech Co, Ltd (Guangzhou, China).Mice received a daily topical application of imiquimod cream (5%) (3 M Pharmaceutical, Maplewood, Minnesota, uSA) on bilateral ears consecutively for 6 days to induce psoriasis-like dermatitis.Some mice received 3 µg of anti-CXCL-1(MAB453-100, R&D), anti-CXCL-2 antibodies (AF-452-NA, R&D), or both through intradermal injection into the unilateral ear.An equal volume of PBS was injected into the contralateral ear at the same time points as the control.

Measurement of dermatitis scores and epidermal thickness
The severity was evaluated on day 6 using a modified psoriasis severity index (PSI) scoring system.Erythema, scaling, and thickening was scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked.The cumulative score was summed up.Paraffin sections of mouse ear tissue were stained with hematoxylin and eosin.Photos were captured under a microscope (Nikon, Japan).Length from the stratum corneum to the basal layer was measured as epidermal thickness using Image pro-plus 6.0 software.

Statistical analysis
Data were presented as mean ± SEM.The student's t-test was used to compare two conditions and one-way analysis of variance (ANOVA) was used to compare more than two conditions.Paired T-test was used to compare the changes between two ears of the same mouse.
We then investigated whether TNF-α could regulate galectin-3 expression in keratinocytes by stimulating keratinocytes with TNF-α at a range of concentrations (0-300 ng/ mL).We found that galectin-3 expression was suppressed by TNF-α in a dose-dependent manner (Figure 1(E)).In addition, kinetic data (24-72 h) revealed the suppression started 72 h after stimulation with 100 ng/mL of TNF-α (Figure 1(F)).The concentration of 100 ng/mL and the time point of 72 h were thereafter applied to the following studies.Together, these findings suggested that TNF-α not only dysregulated the phenotypes of keratinocytes but also down-regulated galectin-3 expression.

Galectin-3 supplement alleviates CXCL-1/8 expression promoted by TNF-α in keratinocytes
We next examined whether the function and phenotype of keratinocytes dysregulated by TNF-α were mediated by the reduction of galectin-3 expression.For this purpose, we transiently transfected keratinocytes with galectin-3 expressing plasmids followed by stimulation with TNF-α for 72 h.Galectin-3 overexpression was verified by RT-qPCR (Figure 2(A)).In the absence of TNF-α, galectin-3 overexpression did not affect mRNA expression of CXCL-1/8, IL-23, or IL-1β, but increased the transcripts of keratin10 and filaggrin, suggesting that galectin-3 had a minor effect on the expression of proinflammatory mediators but could alter the differentiation of keratinocytes under steady state (Figure 2(B,C)).In agreement with the results in Figure 1(A), the transcripts of CXCL-1/8, IL-23, and IL-1β were greatly promoted, whereas the expression of keratin 10 and filaggrin was suppressed by TNF-α stimulation.unexpectedly, CXCL-2 mRNA was not increased by TNF-α treatment (Figure 2(B)), which might result from the off-target effect of the transfection reagent.Supplementary galectin-3 ceased the overexpression of CXCL-1/8 but had little effect on the mRNA expression of IL-23, IL-1β, keratin 10, and filaggrin induced by TNF-α (Figure 2(B,C)).As for the role of galectin-3 on cell proliferation and apoptosis, keratinocytes were stimulated with TNF-α and galectin-3 protein at 10 µg/ml.However, neither the proliferation nor the apoptosis altered by TNF-α was reversed by adding exogenous galectin-3 (Figure 2(D,E)).Together, these results suggested that galectin-3 regulated the expression of CXCL-1/8, but barely affected the other pro-inflammatory mediators, cell proliferation, and apoptosis induced by TNF-α.
The activation of transcript factor NF-κB is more likely to upregulate the expression of downstream target genes [15], raising the possibility that the NF-κB pathway might regulate galectin-3 indirectly.MicroRNAs are well-known for suppressing gene expression by disrupting mRNA translation and degradation.A few aberrantly expressed microRNAs that participate in psoriasis development were identified [16].Thus, we explored the association between microRNA and galectin-3 expression in keratinocytes by database searching (miRanda, microcosm, and targetscan) and found 6 microRNAs with potential interaction with 3′ uTRs of mRNA of LGALS3 gene (gene for galectin-3) and examined their expression in keratinocytes stimulated by TNF-α.The regulation of these microR-NAs by TNF-α was then verified using RT-qPCR analysis and it was shown that only hsa-miR-27a-3p expression was increased by TNF-α stimulation (Figure 3(C), Fig S3) and decreased by inhibiting the NF-κB signaling pathway with JSH-23 in keratinocytes (Figure 3(D)).We then inhibited hsa-miR-27a-3p expression with siRNA (Figure 3(E)) and observed galectin-3 expression inhibited by TNF-α was rescued (Figure 3(F)).The matching of the nucleotide sequence of hsa-miR-27a-3p and 3′ uTR of LGALS3 gene was shown in Figure 3(G).Collectively, these results demonstrated that TNF-α inhibited galectin-3 expression by increasing hsa-miR-27a-3p through activation of NF-κB signaling pathway in keratinocytes.

Intradermal neutralizing murine CXCL-2 alleviates psoriasis-like dermatitis induced by imiquimod
One of the most significant observations of this study was the upregulation of CXCL-1/8 expression mediated by galectin-3 in response to TNF-α stimulation in vitro.CXCL-1/8 are important chemokines controlling neutrophil recruitment and are highly expressed in psoriatic lesions [17,18].In psoriasis-like dermatitis induced by imiquimod, murine CXCL-1 and CXCL-2 expression (murine CXCL-2 is the main homolog for human CXCL-8) were reported to be increased (Fig S4B, data from GSE110658) [19] and galectin-3 expression was also down-regulated in the epidermis (Fig S4A).To investigate the therapeutic potential of the blockade of murine CXCL-1/2, we adapted the imiquimod-induced psoriasis model and intradermally injected anti-CXCL-1 and/or anti-CXCL-2 antibodies into the unilateral ear with the Figure 2. galectin-3 mediates cxcl-1 and cxcl-8 production in keratinocytes stimulated by tnF-α.hacat keratinocytes were transfected with empty vector or galectin-3 overexpression plasmids followed by tnF-α stimulation.the mrna expression of (a) galectin-3, (B) proinflammatory mediators and (c) KRT10 and FLG were examined using rt-pcr.nhEK were co-cultured with 10µg/ml commercial galectin-3 (gal3) protein in the presence and absence of tnF-α.(d) cell cycles and (E) apoptosis after tnF-α stimulation for 72 h were evaluated by flow cytometry.the data are presented as the means ± SEms.Statistical analysis was carried out by one-way analysis of variance (anoVa) test with multiple comparisons (*p < 0.05, **p < 0.01 and ***p < 0.001).Each experiment was independently repeated 3 times.
injection of PBS into the other ear as a control (Figure 4(A)).Notably, anti-CXCL-2 antibodies alone or together with anti-CXCL-1 antibodies, but not anti-CXCL-1 antibodies alone, alleviated skin inflammation, which was evidenced by lower PSI scores, thinner ear thickness, and less epidermal hyperplasia as compared to the controls (Figure 4(B,C)).Less infiltration of neutrophils was also observed only when the anti-CXCL2 antibody was applied (Fig S5).In addition, the mRNA expression of psoriasis-related inflammatory mediators was also reduced with the treatment of the anti-CXCL-2 antibody (Figure 4(D)).Application of the anti-CXCL-2 antibodies resulted in the reduction of Ki67 (Figure 4(E)) and rescued the dysregulated cell differentiation by increasing Krt10 mRNA expression (Figure 4(F)).Together, these results demonstrated that neutralizing murine anti-CXCL-2 could alleviate psoriasis-like dermatitis induced by imiquimod.

Discussion
It has been experimentally demonstrated by our previous study that the absence of the galectin-3 in skin alone was sufficient to provoke psoriasis-like dermatitis [10].On the other hand, the induction of various inflammatory mediators by TNF-α, a widely reported psoriasis-related cytokine, has been extensively studied [20].However, studies on the specific role of galectin-3 under TNF-α-mediated psoriasis-related inflammatory conditions in keratinocytes are quite limited.
Our present study revealed that galectin-3 serves as a pivotal mediator in TNF-α-induced inflammation in keratinocytes via regulating the expression of neutrophil-associated chemokines CXCL-1 and CXCL-8 specifically among the various proinflammatory mediators provoked by TNF-α.These findings were consistent with what was reported in keratinocytes [10], spleens [21], and spinal tissue [22] of mice with galectin-3 absent where neutrophil recruitment was greatly increased.Interestingly, we found that the supplement of galectin-3 alone could promote the differentiation of keratinocytes but showed a minor effect on differentiation with TNF-α present.These results agree with existing studies where galectin-3 was found to promote differentiation of keratinocytes [10] and preadipocytes [23] but also suggested a rather disposable role of galectin-3 on differentiation in TNF-α induced phenomenon, which further emphasized the specificity of the regulatory function of galectin-3 on CXCL-1 and CXCL-8.
In the present data, galectin-3 exacerbated the effect of TNF-α on apoptosis.According to the previous reports, extracellular and intracellular galectin-3 functioned differently on apoptosis where apoptosis was induced by the extracellular but was inhibited by the intracellular galectin-3 [24,25].Due to the technical limitation, exogenous galectin-3 was added into the apoptosis system without measuring the endocytosed galectin-3 level, which may limit the information provided by the experiment and require further exploration.
Of greater interest is our findings that TNF-α regulates galectin-3 via hsa-miR-27a-3p, a microRNA downstream of NF-κB signaling pathway.Emerging evidence has shown that dysregulation of microRNAs participates in the differentiation, proliferation, and cytokine production of both keratinocytes and T cells and plays a central role in the onset and development of psoriasis [16].Our data showed that blocking hsa-miR-27a-3p could regulate the expression of galectin-3 exposed to TNF-α but had little effect under steady state, suggesting that hsa-miR-27a-3p may specifically target the transcription of galectin-3 under inflammatory conditions.Hsa-miR-27a-3p was reported to be associated with heart failure and a variety of tumors.It is reduced in coronary sinus samples of patients with congestive heart failure [26] and in the circulation of patients with acute heart failure as well [27].Besides, expression of hsa-miR-27a-3p was reduced in circulating vesicles after the removal of the colorectal tumor [28].It was also positively associated with a hypoxia gene signature in breast cancer and contributed to hepatocellular carcinoma growth [29].However, the role of hsa-miR-27a-3p in psoriasis was barely investigated.Our data revealed the regulating mechanism among TNF-α, hsa-miR-27a-3p, and galectin-3, suggesting a potential role of hsa-miR-27a-3p in mediating the dysregulation of keratinocytes in psoriasis.In future studies, we plan to both verify the expression level of hsa-miR-27a-3p in the human lesional epidermis or the epidermis of murine IMQ-induced dermatitis and to verify hsa-miR-27a-3p/galectin-3/CXCL-1,8 regulation in vivo and to prove the interaction between hsa-miR-27a-3p and the 3'uTR of galectin-3 directly.
CXCL-1 and CXCL-8 act as chemoattractant for immune cells, primarily for neutrophils [30].Both are secreted by various cells and tissues and are regulated by the NF-κB signaling pathway [31,32].In our study, we found that instead of IL-23A and IL-1β, galectin-3 regulated the expression of CXCL-1/8 especially in the inflammatory circumstance caused by TNF-α.Our previous research has revealed that keratinocytes transfected with galectin-3-siRNA exhibited higher expression of chemokines including CXCL1 and CXCL8 through up-regulation of c-Jun N-terminal kinase (JNK) pathway, resulting in enhanced leukocyte-attracting capacity [10].On the other hand, the association between IL-23A and JNK signaling pathway was rarely reported.As for IL-1β, however, JNK has been reported to be both up-stream [33] and down-stream [34] signaling pathway, therefore the lack of regulation to IL-1β by galectin-3 may be more complicated and need further investigation.
CXCL-1/8 are highly expressed in psoriatic lesions [35,36].Mouse does not express CXCL-8 but expresses CXCL-2 as a major homolog of human CXCL-8.Partly in line with these findings, our data showed that murine anti-CXCL-2 neutralizing antibodies could efficiently alleviate imiquimod-induced psoriatic inflammation in mice, with a marginal effect observed of anti-CXCL-1 antibodies.That might be explained by that although both murine CXCL-1 and CXCL-2 were high in psoriatic dermatitis induced by imiquimod, the elevation of murine CXCL-2 was much higher than the elevation of murine CXCL-1 (Fig S4B ), implicating a more important role of CXCL-2, which might explain a better efficacy of CXCL-2 neutralization.Further research is warranted to probe the therapeutic effect of local blocking CXCL-8 on patients with psoriasis.
Taken together, our findings suggested that TNF-α participates in psoriasis development by downregulating galectin-3 expression in keratinocytes through NF-κB and hsa-miR-27a-3p signaling pathway (Figure 5).These findings provided a more profound understanding of the involvement of TNF-α in the pathogenesis of psoriasis and implicated the galectin-3/ hsa-miR-27a-3p/CXCL-8 pathway being a potential option for treating or preventing relapse of psoriasis.

Figure 1 .
Figure 1.tnF-α dysregulates the function and phenotype of keratinocytes with downregulation of galectin-3 expression.(a) the expression of psoriasis-related cytokines in human epidermal keratinocytes (nhEK) after tnF-α stimulation for 72 h.(B) the cell cycles of nhEK evaluated by flow cytometry.(c) the mrna expression of keratin 10 (KRT10) and filaggrin (FLG) in nhEK.(d) the apoptotic cells analyzed by flow cytometry.the bar graph shows the percentage of apoptotic cells.(E) galetin-3 mrna expression in nhEK after co-cultured with tnF-α at indicated concentration for 72 h and (F) at 100 ng/ml for 24-72 h. the data are presented as the means ± SEms.Statistical analysis was carried out by Student's t-test (a, B, c, d) and one-way anoVa with multiple comparisons (E, F) (*p < 0.05, **p < 0.01 and ***p < 0.001).Each experiment was independently repeated 3 times.

Figure 4 .
Figure 4. Blockade of murine cxcl-2 by intradermal injection of anti-cxcl-2 antibody alleviates imiquimod-induced psoriasis-like inflammation.(a) mice with imiquimod-induced psoriasis-like dermatitis on both ears received intradermal injection of murine anti-cxcl-1 and/or anti-cxcl-2 antibodies unilaterally in one ear and pBS injection as control in the contralateral ear every other day according to the experimental scheme shown.(B) Skin manifestations, whole ear thickness and the severity of skin inflammation measured by pSI score (n = 4-5).(c) histologic examination of ear skin and measurement of epidermal thickness.(d) the mrna expression of psoriasis-related inflammatory mediators, (E) Ki67 and (F) Krt 10.Statistical analysis was carried out by anoVa with multiple comparisons (c), Student's t-test (D) or paired T test (B, E and F).(*p < 0.05, **p < 0.01 and ***p < 0.001).the experiment was independently repeated twice (n = 4-5 per group).