DCLK1 is upregulated in macrophages of mouse atherosclerotic lesion

To determine whether DCLK1 is involved in atherosclerosis, we first examined the levels of DCLK1 in experimental atherosclerotic mouse models using publicly available transcriptome data. As shown in Fig 1A, DCLK1 was upregulated in atherosclerotic aortas of mice compared to that in normal mouse aortas. We built atherosclerotic mice using ApoE^−/− mice fed with a high‐fat diet (HFD) for 4 months. Consistently, our data validated that DCLK1 was upregulated in the aortas of HFD‐fed ApoE^−/− mice at both protein (Fig 1B and C) and mRNA (Fig 1D) levels. We then examined the cellular distribution of DCLK1 in aortas. Immunofluorescence staining showed that DCLK1 colocalized with F4/80, a macrophage marker, rather than with CD31, an endothelial cell marker, and α‐SMA, a smooth muscle cell marker in mouse aortas (Fig 1E–G). Figure 1E–G also showed that the expression of DCLK1 was markedly increased in macrophages in atherosclerotic plaques of HFD‐fed ApoE^−/− mice. We then identified that DCLK1 expression indeed increased in MPMs challenged with oxLDL in a time‐dependent manner (Fig 1H and I). These data suggest that DCLK1 is upregulated in macrophages of atherosclerotic lesions and may be involved in the atherosclerotic progression.

Macrophage‐specific DCLK1 deletion reduces atherosclerotic plaques in HFD‐fed ApoE ^−/− mice

To investigate the role of macrophage DCLK1 in atherosclerosis, macrophage‐specific DCLK1 knockout mice with an ApoE knockout background (ApoE^−/−DCLK1^MCKO) and the control DCLK1‐flox mice with an ApoE knockout background (ApoE^−/−DCLK1^f/f) were generated and used (Appendix Fig S1A and B). The DCLK1 deletion in macrophages was confirmed by Western blot analysis (Appendix Fig S1C). Feeding mice with a HFD for 16 weeks significantly increased the body weight and induced hyperlipidemia in ApoE^−/−DCLK1^f/f mice compared to the low‐fat diet (LFD) group, while macrophage‐specific DCLK1 deletion did not affect the increased body weight and serum lipid profile in HFD‐fed ApoE^−/− mice (Appendix Fig S2A–D). Interestingly, HFD feeding resulted in atherosclerotic plaques in the aortas of ApoE^−/−DCLK1^f/f mice, while macrophage‐specific DCLK1 deletion significantly reduced plaque size in the aortas of HFD‐fed ApoE^−/−DCLK1^MCKO mice (Fig 2A–D). Similarly, Oil Red O staining of aortic roots showed that DCLK1 deletion attenuated atherosclerotic lesions in HFD‐fed ApoE^−/− mice (Fig 2E and F). Masson's trichome staining also revealed reduced collagen deposition in atherosclerotic plaques in the ApoE^−/−DCLK1^MCKO mice with HFD compared to the ApoE^−/−DCLK1^f/f + HFD group (Fig 2G and H). Collectively, these results indicate that macrophage‐specific DCLK1 deletion alleviates HFD‐induced atherosclerotic development without affecting serum lipid profile.

Macrophage‐specific DCLK1 deletion suppresses inflammatory cell infiltration and alleviates inflammatory response in the aorta

Considering that immune cell recruitment and inflammation are major promoters of atherosclerotic progression (Hansson et al, 2006), we examined the inflammatory indexes in these mice. Immunofluorescence staining showed that macrophage‐specific DCLK1 deletion significantly diminished the infiltration of F4/80‐positive macrophages into atherosclerotic lesions in HFD‐fed ApoE^−/− mice (Fig 3A and B). We also found that macrophage‐specific DCLK1 deletion reduces the level of F4/80‐ and iNOS‐positive proinflammatory macrophages in atherosclerotic lesions of HFD‐fed ApoE^−/− mice (Appendix Fig S3). Meanwhile, immunohistochemical results revealed that the recruitment of Ly6G‐positive neutrophils (Fig 3C and D) and Ly6C‐positive monocytes (Fig 3E and F) into atherosclerotic plaques was also significantly restrained by DCLK1 deletion, which was consistent with the quantitative analysis of neutrophils and monocytes in the plasma (Fig 3G–I). Macrophage‐specific DCLK1 deletion also reduced HFD‐increased serum proinflammatory cytokines (TNF‐α and IL‐6) at both the mRNA and protein levels in ApoE^−/− mice (Fig 3J and K). Similar changes were observed when we examined the mRNA levels of proinflammatory cytokines (Il1β, Il18), chemokines (Cxcl1, Ccl2), and adhesion molecules (Icam1, Vcam1) in mouse atherosclerotic aortas (Fig 3L and Appendix Fig S4). Together, these results indicate that macrophage‐specific DCLK1 deletion inhibits inflammatory cell infiltration and moderates the inflammatory response in the aorta of HFD‐fed ApoE^−/− mice.

DCLK1 deletion attenuates inflammatory response in macrophages via inhibiting NF‐κB activation

To validate the regulation of macrophage DCLK1 on inflammation, mouse primary peritoneal macrophages (MPMs) were isolated from DCLK1^f/f and DCLK1^MCKO mice and challenged with oxLDL. As expected, DCLK1 deficiency inhibited oxLDL‐induced upregulation of inflammatory cytokines TNF‐α and IL‐6 at both mRNA and protein levels in MPMs (Fig 4A–D). Subsequently, we found that DCLK1 deletion substantially impeded oxLDL uptake in oxLDL‐challenged macrophages (Fig 4E and F), leading to less foam cell formation. Next, to explore the mechanism by which DCLK1 regulates inflammation in macrophages, we performed RNA sequencing analysis using oxLDL‐stimulated MPMs from DCLK1^f/f and DCLK1^MCKO mice (Fig 4G). A GSEA enrichment analysis of the RNA‐sequencing data indicated that the anti‐inflammatory effect of DCLK1 deficiency may be related to the NF‐κB signaling pathway (Fig 4H and I), a canonical transcriptional factor regulating inflammation and involved in inflammatory atherosclerosis (Monaco et al, 2004). We confirmed the effects of DCLK1 on NF‐κB through examining IκB degradation and NF‐κB p65 subunit phosphorylation and nuclear translocation. The data in Fig 4J–M show that DCLK1 knockout suppressed oxLDL‐induced IκB degradation and p65 phosphorylation and nuclear transclocation in MPMs. Immunofluorescence staining assay also showed significantly reduced nuclear p65 level in oxLDL‐challenged DCLK1‐deficient MPMs (Fig 4N and O). In addition, the immunofluorescence staining for phosphorylated p65 (p‐p65) in aortic roots further verified that DCLK1 deletion suppressed NF‐kB p65 activation in atherosclerotic mice (Fig 4P and Q). Taken together, these data demonstrate that DCLK1 deletion attenuates inflammatory response by inhibiting NF‐κB activation in macrophages.

DCLK1 directly interacts with IKKβ to promote NF‐κB activation

To further decipher how DCLK1 activates NF‐κB, we performed LC–MS/MS analysis using 293T cells transfected with Flag‐DCLK1 plasmid (Fig 5A and B). Interestingly, the mass spectrometry data identified IKKβ, a canonical and direct upstream kinase of NF‐κB (Hayden & Ghosh, 2008), as a binding protein of DCLK1 (Fig 5C). As a upstream of NF‐κB, IKKβ has also been reported to be involved in atherosclerosis (Strnad & Burke, 2007). Therefore, we hypothesize that DCLK1 regulates NF‐κB activation and inflammatory atherosclerosis by directly binding to IKKβ.

First, the interaction between DCLK1 and IKKβ was confirmed in 293T cells transfected with Flag‐DCLK1 by coimmunoprecipitation (Fig 5D). The DCLK1‐IKKβ complex was further found in oxLDL‐treated MPMs using coimmunoprecipitation assay (Fig 5E). Considering that DCLK1 is a protein kinase (Nevi et al, 2021), we hypothesize that the DCLK1‐IKKβ interaction may phosphorylate IKKβ. Two phosphorylating sites, serine 177 and serine 181 (S177/181), are critical for IKKβ phosphorylation (Delhase et al, 1999; Scheidereit, 2006). As shown in Fig 5F, oxLDL challenging induced IKKβ phosphorylation at S177/181, while DCLK1 deletion significantly reduced this change in MPMs. We then explored whether IKKβ is indispensable for DCLK1‐mediated NF‐κB activation and inflammatory response. We knocked IKKβ expression down in 293T cells using IKKβ siRNA (Fig 5G). These cells were simultaneously transfected with Flag‐DCLK1 plasmid to induce DCLK1 overexpression. As shown in Fig 5H–J, DCLK1 overexpression in 293T cells could increase the phosphorylation level of IKKβ at S177/181 and induce NF‐κB activation and inflammatory cytokine production. However, knocking down IKKβ completely reversed the NF‐κB activation and inflammatory cytokine production induced by DCLK1 overexpression in 293T cells (Fig 5H–J). These results show that DCLK1 directly interacts with IKKβ to promote the phosphorylation of IKKβ at S177/181 and NF‐κB‐mediated inflammation.

Pharmacological inhibitor of DCLK1 mitigates NF‐κB activation and inflammatory response in oxLDL‐challenged macrophages

To strengthen our findings, a selective small‐molecule inhibitor of DCLK1, DCLK1‐IN‐1 (Fig 6A; Ferguson et al, 2020) was used. The dose of DCLK1‐IN‐1 (5 and 10 μM) was selected based on previous studies (Ding et al, 2021; Patel et al, 2021). As expected, DCLK1‐IN‐1 treatment dose‐dependently inhibited oxLDL‐induced upregulation of pro‐inflammatory cytokines at both mRNA and protein levels in MPMs (Fig 6B and C). We also found that DCLK1‐IN‐1 significantly inhibited IKKβ phosphorylation at S177/181, p65 phosphorylation, IkBa degradation, and p65 nuclear translocation in oxLDL‐challenged macrophages (Fig 6D–I). These results demonstrate that the pharmacological inhibition of DCLK1 mitigates NF‐κB activation and inflammatory response in macrophages.

Pharmacological inhibitor of DCLK1 prevents atherosclerotic progression and inflammation in mice

DCLK1‐IN‐1 was further used in in vivo experiments to enhance a translational significance. The dose of DCLK1‐IN‐1 (10 mg/kg/day) was selected based on a previous study (Ferguson et al, 2020). As shown in Appendix Fig S5A–D, DCLK1‐IN‐1 treatment did not affect the increased body weight and serum lipid profile in HFD‐fed ApoE^−/− mice. However, DCLK1‐IN‐1 substantially decreased the plaque area in the aorta of HFD‐fed ApoE^−/− mice (Fig 7A–D). Similarly, Oil Red O staining of the aortic roots showed that DCLK1‐IN‐1 attenuated atherosclerotic lesions (Fig 7E; Appendix Fig S6A). Masson's trichome staining also revealed reduced collagen deposition in atherosclerotic plaques in the DCLK1‐IN‐1‐treated group (Fig 7F; Appendix Fig S6B). Immunofluorescence and immunohistochemical staining showed that DCLK1‐IN‐1 significantly reduced the infiltration of inflammatory cells (macrophages, neutrophils, and monocytes) into atherosclerotic lesions in HFD‐fed ApoE^−/− mice (Fig 7G–I; Appendix Fig S6C–E). The staining of p‐p65 in aortic roots showed that DCLK1‐IN‐1 constrained NF‐kB activation in the aortic roots of atherosclerotic mice (Fig 7J; Appendix Fig S6F), which was consistent with the in vitro results. Finally, DCLK1‐IN‐1 reduced the levels of proinflammatory cytokines (TNF‐α, IL‐6) upregulated in HFD‐fed ApoE^−/− mice (Fig 7K–N). Collectively, these results suggest that pharmacological inhibitor of DCLK1 significantly prevent atherosclerotic progression and inflammation in mice.

Reagents

Oxidized low‐density lipoproteins (oxLDL) and DiI‐labeled oxLDL (DiI‐oxLDL) were purchased from Peking Union‐Biology (Beijing, China). Antibodies against GAPDH (#5174, 1:1,000), β‐actin (#3700, 1:1,000), p‐IKKβ (#2697, 1:1,000), IKKβ (#8943, 1:1,000), p‐p65 (#3033, 1:1,000 for western blotting and 1:200 for immunofluorescence staining), NF‐κB p65 (#8242, 1:1,000), and IκBα (#9242, 1:1,000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against DCLK1 (#ab31704, 1:1,000 for western blotting and 1:200 for immunofluorescence staining), F4/80 (#ab6640, 1:200), CD31 (#ab9498, 1:200), α‐SMA (#ab7817, 1:200), iNOS (#ab178945, 1:200), and Lamin B1 (#ab133741, 1:1,000) were purchased from Abcam (Cambridge, UK). Antibodies against Ly6G (#sc‐53515, 1:100) and Ly6C (#sc‐271811, 1:100) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against Flag (#20543‐1‐AP, 1:1,000) was purchased from Proteintech (Wuhan, China). DCLK1‐IN‐1 (BD01203503) was purchased from Bidepharm (Shanghai, China).

Animal experiments

All animal care and experimental procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (approval ID: wydw2021‐0057). All animal experiments followed the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA). Wildtype C57BL/6 (Strain NO. N000013), ApoE knockout mice on a C57BL/6 background, B6/JGpt‐Dclk1em1Cflox/Gpt (DCLK1‐flox) mice, and B6/JGpt‐Lyz2em1Cin(iCre)/Gpt (lysozyme 2 (Lyz2)‐driven Cre) mice were obtained from GemPharmatech. Myeloid cell‐specific DCLK1 knockout mice (DCLK1‐Lyz^Cre, DCLK1^MCKO) were generated with technical expertise from GemPharmatech Co. Ltd (Nanjing, China). Then, the DCLK1‐flox (DCLK1^f/f), DCLK1^MCKO, and ApoE‐knockout (ApoE^−/−) mice on a C57BL/6 background were further generated in Gempharmatech (Nanjing, China). ApoE^−/− mice were crossed with DCLK1^MCKO mice to generate ApoE^−/−DCLK1^f/f and ApoE^−/−DCLK1^MCKO mice, respectively. Validation of genotype of ApoE^−/−DCLK1^f/f and ApoE^−/−DCLK1^MCKO mice was performed by PCR using the gene primers in Appendix Table S1. Animals were housed in a 12:12 h light–dark cycle at a constant room temperature and fed a standard rodent diet. The animals were acclimatized to the laboratory for at least 2 weeks before initiating the studies. All animal experiments were performed and analyzed by blinded researchers.

For studies using DCLK1 knockout mice, male eight‐week‐old ApoE^−/−DCLK1^f/f and ApoE^−/−DCLK1^MCKO mice were fed an LFD containing 10 kcal.% fat, 20 kcal.% protein and 70 kcal.% carbohydrate (H10010, HFK Bioscience, Beijing, China) or a HFD containing 40 kcal.% fat, 20 kcal.% protein, 40 kcal.% carbohydrate and 1.25% cholesterol (H10540, HFK Bioscience) for 16 weeks, respectively. LFD and HFD groups were assigned in a randomized fashion. Body weight was recorded weekly. All mice were sacrificed under sodium pentobarbital anesthesia, and blood and aortas samples were collected.

For studies involving DCLK1 inhibitor DCLK1‐IN‐1, eight‐week‐old ApoE^−/− mice were randomly divided into three groups: (i) LFD group: mice fed with a LFD and intragastrically treated with 1% CMC‐Na vehicle control; (ii) HFD group: mice fed with a HFD and intragastrically treated with 1% CMC‐Na vehicle control; and (iii) HFD + DCLK1‐IN‐1 group: mice fed with a HFD and intragastrically treated with 10 mg/kg/2 days DCLK1‐IN‐1 reconstituted in 1% CMC‐Na solution. All mice were fed with a LFD or HFD for total 16 weeks, while mice were treated with the vehicle or DCLK1‐IN‐1 only for the final 8 weeks. Body weight was recorded weekly. All mice were sacrificed under sodium pentobarbital anesthesia at week 16, and blood samples were collected. Aortas were fixed in 4% paraformaldehyde or snap‐frozen in liquid nitrogen.

Atherosclerotic lesion analysis

For en face lesion analysis of the aorta, the whole aorta and aortic sinus were dissected, opened longitudinally from the heart to the iliac arteries, and stained with Oil Red O (G1260, Solarbio, Beijing, China). The heart and proximal aorta were collected and embedded in optimum cutting temperature compound for quantification of plaque lesions. Serial 5 μm‐thick cryosections of the aortic sinus were obtained from each mouse. The sections were stained with Oil Red O and hematoxylin for plaque size analysis.

Paraffin‐embedded sections were used for Ly6G and Ly6C immunohistochemistry and Masson's trichrome staining (G1340, Solarbio). For immunohistochemistry, sections were deparaffinized and rehydrated, followed by heat‐induced antigen retrieval using 10 mM sodium citrate buffer (pH 6.5). Sections were blocked with 3% H₂O₂ and then with 5% bovine serum albumin for 30 min. Primary Ly6G (1:200) and Ly6C (1:200) antibodies were added. Sections were incubated at 4°C overnight. Horseradish peroxidase‐conjugated secondary antibodies and DAB were used for detection.

Frozen sections were used for immunofluorescence staining. Slides were fixed in cold methanol and permeabilized using 0.5% Triton‐X. Then, slides were blocked using 5% bovine serum albumin for 30 min and incubated overnight with primary antibodies. Alexa‐488 and Alexa‐647 conjugated secondary antibodies (Abcam, 1:200) were used for detection. Images were captured using a fluorescence microscope (Nikon, Tokyo, Japan).

Analysis of leukocytes in plasma

Plasma neutrophils and monocytes were analyzed using an automated blood cell analyzer (XN‐1000, Sysmex, Kobe, Japan).

Cytokine measurements

The levels of TNF‐α and IL‐6 in the serum and cell culture media were determined using ELISA kits (Cat#. 88‐7324‐76, 88‐7064‐76, 88‐7346‐76 and 88‐7066‐76, Thermo Fisher, Carlsbad, CA, USA).

Cell culture

Mouse primary peritoneal macrophages (MPMs) were isolated as described previously (Chen et al, 2020; Huang et al, 2022). Briefly, mice received a single intraperitoneal injection of 6% thioglycolate solution. Two days later, the mice were euthanized, and the peritoneal cavity was flushed with RPMI‐1640 medium (Gibco, Eggenstein, Germany). Samples were centrifuged, and the cell suspension was plated in RPMI‐1640 medium containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Invitrogen, Waltham, MA, USA). Nonadherent cells were removed 2 h after seeding the cell suspension. 293T cells (GNHu17) were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in high‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) with 10% FBS and 1% penicillin/streptomycin. All cells were cultured in a humidified incubator maintained at 37°C and 5% CO₂.

Gene knockdown and overexpression

Gene silencing and overexpression in cells were achieved by transfecting specific siRNAs and plasmids. The custom siRNA synthesized for human IKKβ (5′‐ GGAUUACAUUAGUGGACAATT‐3′) was purchased from RiboBio (Guangzhou, China). Plasmid encoding Flag‐DCLK1 was constructed by Genechem (Shanghai, China). Transfection of 293T cells with siRNA and plasmid was performed using Lipofectamine 2000 (Thermo Fisher Scientific, Carlsbad, CA, USA).

oxLDL uptake assay

For uptake detection, MPMs were incubated 50 μg/ml DiI‐oxLDL for 3 h at 37°C. Cells were washed with PBS and imaged using a Leica TCS SP8 confocal laser‐ scanning microscope (Buffalo Grove, IL, USA). Cells under identical conditions were dislodged and analyzed using flow cytometry. The results of flow cytometry were expressed as mean fluorescence intensity after subtracting the auto‐fluorescence of cells (absence of DiI‐oxLDL).

Western blotting and co‐immunoprecipitation

Total protein from cells and aortic tissues was extracted using lysis buffer (AR0103, Boster Biological Technology, Pleasanton, CA, USA). Proteins were separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride membranes. Before adding specific primary antibodies, the membranes were blocked in Tris‐buffered saline (pH 7.4, containing 0.1% Tween 20 and 5% nonfat milk) for 1.5 h at room temperature. Protein bands were detected by incubation with horseradish peroxidase‐conjugated secondary antibodies and an enhanced chemiluminescence reagent (Bio‐Rad, Hercules, CA, USA). Band densities were quantified using ImageJ software (version 1.38 e, NIH, Bethesda, MD, USA) and normalized to the loading controls.

For co‐immunoprecipitation assays, cell extracts prepared following treatments were incubated with indicated antibodies at 4°C overnight. Then the proteins were immunoprecipitated with Protein A + G Agarose (P2012, Beyotime, Shanghai, China) at 4°C for 2 h. Immunoprecipitation samples were immunoblotted for co‐precipitated protein detection. Total lysates were subjected to western blot analysis as input controls. Protein interactions were quantified using ImageJ software.

Real‐time quantitative PCR

Total RNA was extracted from cells or aortic tissues using RNAiso Plus (9109, Takara, Shiga, Japan). Reverse transcription was performed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (RR047A, Takara, Shiga, Japan). Quantitative PCR was performed using TB Green® Premix Ex Taq™ II (RR820A; Takara, Shiga, Japan) in a QuantStudio™ 3 Real‐Time PCR System (Thermo Fisher Scientific, Carlsbad, CA, USA). The efficiency of PCR amplification was required to be 90–110%. The CT values were normalized to Rn18s or β‐actin and the $2^{-\mathrm{\Delta \Delta }{C}_{\mathrm{T}}}$ method was used to calculate the relative amount of target mRNA. The primers were obtained from Thermo Fisher Scientific. The primer sequences used in this study are listed in Appendix Table S2.

Transcriptome sequencing

Total RNA from the cells was collected using RNAiso Plus and subjected to genome‐wide transcriptomic analysis using LC‐Bio (Hangzhou, China). Differentially expressed genes (DEGs) were selected with fold change > 2 or fold change < 0.5 and P‐value < 0.05. Gene‐set enrichment analysis (GSEA, https://www.gsea‐msigdb.org/gsea/index.jsp) of the signaling pathways was performed as described by LC‐Bio (https://www.lc‐bio.cn/). Publicly available transcriptome data GSE94044 (Mohanta et al, 2022b; Data ref: Mohanta et al, 2022a) and GSE137581 (Guo et al, 2020b; Data ref: Guo et al, 2020a) were acquired from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/). DEGs were selected with fold change > 2 or fold change < 0.5 and P‐value < 0.05, using GEO2R.

LC–MS/MS analysis

Anti‐Flag antibody was added to the lysate of 293T cells transfected with Flag‐DCLK1 for co‐immunoprecipitation and IgG was used as a negative control. LC–MS/MS analysis was performed by PTM Bio Co., Ltd (Zhejiang, China). We screened the substrate proteins that may bind to DCLK1 according to the score and Flag/IgG ratio of the detected proteins in the mass spectrometry data.

Statistical analysis

Sample sizes were defined by a priori power calculation with G‐Power 3.1.9 software (http://www.gpower.hhu.de/), considering a statistical power of 80% and α = 0.05. We employed a random number table to perform randomization. Briefly, all animal experiments in the present study were performed and analyzed in a blinded manner. Treatment groups were assigned in a randomized fashion. Every mouse was assigned a temporary random number within the weight range. Mice were given their permanent numerical designation in the cages after they were randomly divided into each group. For each group, a cage was selected randomly from the pool of all cages. All data were collected and analyzed by two observers who were not aware of the group assignment or treatment of the mice. Data represented at 10 biological replicates in animal experiments and three biological replicates in cell experiments and were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA). Comparisons between two groups were analyzed using two‐tailed unpaired Student's t‐test. One‐way ANOVA followed by Dunnett's post‐hoc test was used to compare more than two data groups. Statistical significance was set at P < 0.05. Post‐tests were run only if F achieved P < 0.05, and there was no significant variance in homogeneity.