Research ArticleAdaptations of Physiologic Systems that Promote Cancers

SLAMF1 is expressed and secreted by hepatocytes and the liver in nonalcoholic fatty liver disease

Published Online:https://doi.org/10.1152/ajpgi.00289.2021

Abstract

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disease in the United States and worldwide. Nonalcoholic steatohepatitis (NASH), the most advanced form of NAFLD, is characterized by hepatic steatosis associated with inflammation and hepatocyte death. No treatments are currently available for NASH other than lifestyle changes, and the disease lacks specific biomarkers. The signaling lymphocytic activation molecule family 1 (SLAMF1) protein is a self-ligand receptor that plays a role in orchestrating an immune response to some pathogens and cancers. We found that livers from humans and mice with NASH showed a more prominent immunohistochemistry staining for SLAMF1 than non-NASH controls. Furthermore, SLAMF1 levels are significantly increased in NASH plasma samples from mice and humans compared with their respective controls. In mice, the levels of SLAMF1 correlated significantly with the severity of the NASH phenotype. To test whether SLAMF 1 is expressed by hepatocytes, HepG2 cells and primary murine hepatocytes were treated with palmitic acid (PA) to induce a state of lipotoxicity mimicking NASH. We found that PA treatments of HepG2 cells and primary hepatocytes lead to significant increases in SLAMF1 levels. The downregulation of SLAMF1 in HepG2 cells improved the cell viability and reduced cytotoxicity. The in vivo data using mouse and human NASH samples suggests a potential role for this protein as a noninvasive biomarker for NASH. The in vitro data suggest a role for SLAMF1 as a potential therapeutic target to prevent hepatocyte death in response to lipotoxicity.

NEW & NOTEWORTHY This study identified for the first time SLAMF1 as a mediator of hepatocyte death in nonalcoholic fatty liver disease (NASH) and as a marker of NASH in humans. There are no pharmacological treatments available for NASH, and diagnostic tools are limited to invasive liver biopsies. Therefore, since SLAMF1 levels correlate with disease progression and SLAMF1 mediates cytotoxic effects, this protein can be used as a therapeutic target and a clinical biomarker of NASH.

INTRODUCTION

Hepatic steatosis is a hallmark feature of nonalcohol fatty liver disease (NAFLD). Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD that can rapidly progress to liver fibrosis and hepatocellular carcinoma (1). Although the molecular mechanisms underlying the progression from NAFLD to NASH are not completely understood, triglyceride accumulation in hepatocytes, inflammation, and apoptosis are critical processes in the disease progression (26). Since there is a close association among NASH, obesity, and metabolic syndrome (7), the treatment for NASH is limited to changes in the patient’s lifestyle, body weight management, use of antidiabetic drugs, or even bariatric surgery (8).

One of the predominant features of NASH is a robust activation of the innate immune response involving resident liver cells, including Kupffer cells, hepatic stellate cells, monocytes, and lymphocytes (9). Macrophages, neutrophils, monocytes, and T-lymphocytes infiltrate the liver, which contributes to and exacerbates inflammatory and fibrotic processes. This process eventually leads to hepatocyte death and fibrosis (10). The signaling lymphocytic activation molecule family 1 (SLAMF1) is a type I glycoprotein that belongs to the SLAM subfamily of the CD2-like group of proteins. SLAMF1 is highly expressed by activated macrophages, T, and B cells (11, 12). Moreover, SLAMF1 is a self-ligand receptor, and its activation is associated with signal-transduction events involved in the modulation of innate and adaptive immune responses and in the entry of certain viruses (e.g., measles virus; 13). Despite the role of SLAMF1 in modulating immune responses, no studies have reported a role for SLAMF1 in the liver in health or disease states. The present study found that SLAMF1 is detected in the liver, and its levels are more prominent in NASH livers. Moreover, SLAMF1 levels are significantly increased in plasma NASH samples from both mice and humans. These findings suggest a role for SLAMF1 as a biomarker of the disease. We also found that lipid accumulation triggered by palmitic acid (PA) treatment of HepG2 cells and primary murine hepatocytes increases SLAMF1 mRNA and protein levels and the secretion of SLAMF1 to the media. Meanwhile, the downregulation of SLAMF1 by siRNA improved cell viability, reduced cytotoxicity, and lessened the expression of proapoptotic markers in HepG2 cells. These results suggest that SLAMF1 may also play a role in modulating hepatocyte death in steatosis.

MATERIALS AND METHODS

Reagents

Palmitic acid (PA) was purchased from Sigma Aldrich (St. Louis, MO). The antibodies used in this study are commercially available, and information regarding their specificity is provided by the vendor on their respective website. All selected antibodies are specific for mice or humans. Monoclonal antibodies for SLAMF1 (Santa Cruz Biotechnology Cat. No. sc-166939, RRID:AB_10611179) were purchased from Santa Cruz Biotechnology (scbt. Heidelberg, Germany). Rabbit antibodies for SLAMF1 (Sigma-Aldrich Cat. No. SAB3500996-100UG, Anti-SLAMF1 antibody produced in rabbit) and mouse antibodies for α-tubulin (Sigma-Aldrich Cat. No. T9026, monoclonal anti-α-tubulin antibody produced in mouse) were purchased from Sigma. The peroxidase-conjugated anti-mouse [Bio-Rad Cat. No. 1706516, goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) conjugate] or anti-rabbit antibodies (Bio-Rad Cat. No. 1662408EDU, secondary antibody goat anti-rabbit antibody conjugated to horseradish peroxidase) were purchased from Bio-Rad (Hercules, CA). The secondary antibodies for immunofluorescence were purchased from Invitrogen [Thermo Fisher Scientific Cat. No. A-11008, goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody RRID AB_143165, Alexa Fluor 488]. Cell culture supplies were obtained from Lonza (Basel, Switzerland). siRNA for SLAMF1 (smart pool ON-TARGETplus Human SLAMF1 siRNA L-019583-00-0005) was purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO).

Animals and Ethical Approval

All experimental procedures conformed to the regulations for animal experiments of Louisiana State University Health Sciences Center-Shreveport. All animal studies were approved by the Louisiana State University Health Sciences Center’s Animal Care and Use Committee (Protocol Approval 20-005). The animals were maintained in the Animal Facility of LSU Health Sciences Center-Shreveport, following their standard procedures for husbandry. Temperature and humidity in the animal room were maintained at 23°C and 40%–60%, respectively, with a 12:12 h light-dark cycle. Only male mice were used in these studies. Four-week-old male C57bl/6J (RRID: IMSR_JAX:000664) mice were fed a standard (control diet) or high-fat diet as specified in the section Experimental Procedures: Human and Mouse Liver Samples Processing. The sample size was determined using the software nQuary by Dotmatics (https://www.statsols.com/clinical-trial-design-solutions?hsLang=en-us). Animals were randomly assigned into control or high-fat diet groups. An equal number of animals (10/group) was assigned to each dietary treatment. Investigators were blinded regarding the treatments (control or high-fat diet) when performing the downstream analysis (imaging and biochemical assays). The number of biological replicates in each experimental group was at least three, which was the minimal number to check the statistical significance. The statistical methods used to analyze the data are described in Statistical Analysis. No inclusion/exclusion criteria were used. After 24 wk of control or high-fat feeding, mice were euthanized to collect blood and liver samples as described (see Experimental Procedures: Human and Mouse Liver Samples Processing). The data obtained using the blood and liver samples from our high-fat diet experiments are described in results. All procedures involving animals were confirmed by the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines checklist (https://arriveguidelines.org/).

Experimental Procedures: Human and Mouse Liver Samples Processing

Human histology slides were provided as a courtesy by the Pathology Department at LSU Health Sciences Shreveport. Samples were de-identified and coded as NASH or No-NASH by pathological evaluation. All samples were part of the Pathology Department Biorepository. Mouse livers from male C57BL/6J (RRID:IMSR_JAX:000664) fed a normal diet (10% fat; Stock No.: 000664) and a high-fat diet (60% fat; Stock No.: 380050) were purchased from The Jackson Laboratory (Bar Harbor, ME). SLAMF1 levels in plasma were determined with the mouse SLAMF1 ELISA (RAB1862, Millipore Sigma, St. Louis, MO) in samples from C57BL/6J (RRID:IMSR_JAX:000664) mice that were fed a high-fat (40%), high-fructose (22%), and high-cholesterol (2%) NASH diet (Research Diets, D17010103) or a low-fat (10%) control diet (Research Diets, D17072805) for 24 wk. This long-term dietary model features the coexistence of steatohepatitis and fibrosis, better mimicking advanced NAFLD in humans (14, 15). Human SLAMF1 levels in plasma were measured using the human SLAMF1 ELISA (RAB1083-1KT, Millipore Sigma, St. Louis, MO).

For both human and mouse samples, NASH was defined by using histological and biochemical indices as previously described (1417). Briefly, formalin-fixed liver tissues were sectioned at 4 μm thickness and stained for hematoxylin and eosin (H&E, Thermo Fisher Scientific) and Sirius red (Rowley Biochemical, Inc.). Frozen section processing was used for oil red O (ORO) staining (Rowley Biochemical, Inc., H-503-1B). NAFLD activity score (NAS) was determined based on H&E staining. Steatosis was scored from 0 to 3 (0: <5% steatosis; 1: 5%–33%; 2: 34%–66%; 3: >67%). Hepatocyte ballooning was scored from 0 to 2 (0: normal hepatocytes, 1: normal-sized with pale cytoplasm, 2: pale and enlarged hepatocytes, at least twofold). Lobular inflammation was scored from 0 to 2 based on foci of inflammation counted at 20× (0: none, 1: <2 foci; 2: ≥2 foci). NAS was calculated as the sum of steatosis, hepatocyte ballooning, and lobular inflammation scores (18, 19). Concentrations of SLAMF1 in the plasma were correlated with NAS.

For the primary hepatocyte experiments, 10-wk old male C57bl/6J (RRID:IMSR_JAX:000664) mice were used (only 1–2 mice/isolation). After the mouse was under anesthesia (30 mg/kg ketamine and 10 mg/kg xylazine), a midline laparotomy was performed, the inferior vena cava was identified, and a 22-gauge cannula was introduced distal to the renal bifurcation to perfuse retrogradely. The perfusate was allowed to outflow throw the transected hepatic portal vein. The flow rate was 5 mL/min, first with 25 mL of buffer 1 (d-glucose 22 mM, NaCl 122 mM, KCl 5 mM, NaHCO3 29 mM. KH2PO4 1 mM. MgSO4 1.2 mM, and EGTA 0.3 mM), second with buffer 2 (with the same composition than buffer 1 but without EGTA) and finally, with buffer 3, which consists of buffer 2 plus 5 mM CaCl2 (Sigma-Aldrich) and 0.05% of collagenase Type IV (Gibco), all of them warmed at 37°C. Lobes of the liver were collected, discarded the gall bladder, and transferred into a Petri dish containing RPMI 1640 medium (Lonza) with 2% FCS at 4°C and where hepatocytes were dispersed with agitation. Then cells were filtered through a 100-mm mesh and centrifuged twice at 400 rpm for 2 min at 4°C, and the pellet was resuspended in 15 mL RPMI 1640 medium with 2% FCS. Viable hepatocytes were counted on a hemocytometer, and 30,000 cells/cm2 were plated in DMEM (Lonza) containing 2 mM of sodium pyruvate, 1 mM of dexamethasone (Sigma), and 0.1 mm of insulin (Sigma), 10% of fetal bovine serum (FBS), 2 mM of Gln, and 1% of penicillin-streptomycin. The next day, cells were treated with palmitic acid (0.4 mM) for 24 h. Cells were then fixed with 4% of paraformaldehyde for 20 min, washed with PBS, and subjected to SLAMF1 immunocytochemistry using rabbit SLAMF1 (SAB3500996) antibody and a secondary antibody from Thermo Fisher Scientific [Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP from Thermo Fisher Scientific, Cat. No. 31460, RRID AB_228341].

Cell Culture

The HepG2, a human hepatocarcinoma cell line, was obtained from the American Type Culture Collection (CLS Cat. No. 300198/p2277_Hep-G2, RRID:CVCL_0027, Manassas, VA). HepG2 was cultured in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, and 1% antibiotic (penicillin-streptomycin), and incubated in a humidified incubator at 37°C and 5% CO2. When cells reached ∼80% confluency, they were treated with 0.4 mM or 0.8 mM palmitic acid (PA) to induce steatosis, lipotoxicity, and cell death. PA was dissolved in 100% isopropyl alcohol at a stock concentration of 40 mM. Control cells were treated with isopropyl alcohol. Palmitic acid was then added to Dulbecco’s modified Eagle’s medium containing 2% fetal calf serum, 1% fatty acid-free bovine albumin, 2 mM l-glutamine, and 1% antibiotic (penicillin-streptomycin). The extracellular medium (conditioned medium) was removed for lactate dehydrogenase (LDH) activity measurements and the immunoprecipitation assays. Adherent cells were used for oil-red-O staining, 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay, cytometry, protein was obtained for Western blot, and mRNA was extracted for qRT-PCR. Cells were trypsinized, and lipids were extracted following the Folch method. The samples were homogenized with chloroform-methanol (2/1) to a final volume 20 times the volume of the cell pellet. The solvent was washed with 0.2 volume of water, vortexed, and centrifuged for 10 min at 2,000 rpm. Fifty milliliters of the organic phase in duplicate were taken, and 1 μL of X-100 was added and evaporated at 50°C. Triglyceride levels were measured using the BioSystems kit (11528. Biosystems S.A., Spain) following the manufacturer’s instructions.

Protein Extraction and Quantification

Protein from adherent cells were collected on ice in 100 μL of lysis buffer, pH 7.6, containing 50 mmol/L HEPES, 10 mM EDTA, 50 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 10 mmol/L sodium orthovanadate, 1% Triton X-100, 2 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, and 10 μg/mL aprotinin. Samples were homogenized and centrifuged at 14,000 rpm for 20 min at 4°C to remove cellular debris. Clear supernatants were transferred to new tubes to determine the total protein concentration by bicinchoninic acid [BCA; Pierce, Thermo Fisher Scientific (Barcelona, Spain)].

Immunoprecipitation of Conditioned Medium

About 1 mL of conditioned medium was collected and spun at 1,700 rpm and 4°C for 10 min to remove cellular debris. Leupeptin and apoprotein (final concentration of 10 μg/mL) and 2 mL of anti-SLAMF1 monoclonal antibody (200 µg/mL) per sample were added and incubated in continuous agitation for 14 h at 4°C. Samples were incubated with 30 μL of protein A agarose (Roche, Mannheim, Germany) for 2 h at 4°C. Beads were sedimented by centrifugation at 3,500 g for 10 min, and the supernatant was removed. Immunoprecipitated proteins were released from the agarose A-antibody complex by heating at 95°C in a 2× sample buffer, and then electrophoresed on 10% polyacrylamide tris-tricine gels, and Western blot was performed as described below.

Western Blot Analysis

Total protein (30 μg) was resolved on a 10% SDS PAGE gel, then transferred to nitrocellulose membranes, and then blocked with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TTBS) and 5% (wt/vol) milk powder for 1 h at 25°C. Blots were incubated with primary antibodies diluted in TTBS at 4°C overnight. Antibodies included anti-SLAMF1 (SAB3500996, 1:1,000, dilution) anti-cleaved caspase 3 p17 (1:1,000 dilution), β-actin (1:1,000 dilution), or α-tubulin (1:1,000 dilution). The membranes were subsequently washed and incubated with the corresponding secondary antibody conjugated with horseradish peroxidase. Proteins were detected by chemiluminescence with a West Pico substrate (Applied Biosystem). Bands were quantified by densitometry using ImageJ software (RRID:SCR_003070, NIH, Bethesda, MD).

Immunofluorescence Staining

Cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT) and then were washed three times with PBS. Unspecific unions were blocked using 0.5% BSA in continuous agitation for 30 min at RT. Thereafter, SLAMF1 antibody (SAB3500996, dilution 1:1,000) in 0.5% BSA 1× PBS was added and incubated in continuous agitation for 14 h at 4°C. The samples were washed three times in PBS and then incubated in 0.5% BSA PBS containing a 1:1,000 dilution of a rabbit Alexa-488 labeled secondary antibody. Both cells and tissue sections were mounted with Immu-Mount (Thermo Fisher Scientific, Pittsburg, PA). Images were acquired in a LEICA SP5 confocal microscope.

RNA Extraction and Quantitative Real-Time PCR Analysis

Total RNA from cells was obtained using the TriPure isolation reagent (Sigma-Aldrich, St. Louis, Missouri, MO), following the manufacturer’s instructions. RNAs were quantified using the NanoDrop 2000 (Take3, BioTek). Complementary DNA (cDNA) was synthesized from 1 μg of DNase-treated RNA. Quantitative real-time PCR (qRT-PCR) was performed by using ABI PRISM 7500 Fast Sequence Detection System instrument and software (Applied Biosystem, Foster City, CA). Relative quantification of target cDNA in each sample was performed from 10 ng of cDNA in SYBR Green Real-Time PCR Master Mix with the following primers purchased from Applied Biosystem: SLAMF1-specific forward primer 5′- GAGCATGCGCATGATGAACTGCCCAAAG-3′; antisense primer 5′- CCAGATCTTGAGGGGTCTGTCCTGGATCC-3′; and rRNA 18S (sense primer 5′- GCAATTATTCCCCATGAACG-3′; antisense primer 5′- GGGACTTAATCAACGAAGC-3′). For IFNg, we used the Hs00989291_m1 FAMMGB probe from Thermo Fisher Scientific. The relative gene expression for all genes was analyzed using the ΔΔCT method by normalizing with rRNA 18s gene expression in all experiments.

siRNA Silencing of SLAMF1

One day before the transfection, HepG2 cells were cultured for 24 h in a 1% BSA fatty acid free and 2% FBS medium with 0.4 mM palmitic acid; then they were transfected using the siIMPORTER siRNA reagent from Dharmacon (Thermo Fisher Scientific, Lafayette, CO). Smart siRNA targeting SLAMF1 was purchased from Dharmacon (smart pool ON-TARGETplus Human SLAMF1 siRNA L-019583-00-0005). The control nontargeting siRNA pool (D001810-10-05) was purchased from Dharmacon. Following the manufacturer’s protocol, cells were placed in 50% of the culture volume, and siRNAs were mixed with transfection reagent to a final concentration of 50 nM. Lipid complexes were allowed to form for 20 min at room temperature. The transfection mix was added to the HepG2 cells. Accuracy of the transfection was evaluated by flow cytometry using control siRNA FITC conjugated (sc-36869) as a scramble and transfected at a final concentration of 50 nM. To estimate the percentage of transfection efficacy, cells were transfected with a fluorescent scramble RNAi (ScF-36869, Santa Cruz BioTechnology). The percentage of the transfected cells was 83%, with a negligible effect on cell death, assayed by 7-amino-actinomycin D (7AAD) staining and analyzed by flow cytometry.

Apoptosis Assay by Flow Cytometry

Cell apoptosis was evaluated using an Annexin V-FITC Apoptosis Detection Kit with 7-AAD BD Biosciences (San Jose, CA) according to the manufacturer’s manual. First, cells were washed with cold cell staining buffer and resuspended in a binding buffer. Subsequently, cells were incubated with 5 μL annexin V/FITC and 5 μL 7-AAD for 15 min at room temperature in the dark. Then, 400 µL of annexin V binding buffer was added to each tube containing the cell. Apoptosis analysis was performed using a FACSCanto II flow cytometer, and the data were analyzed using FACSDiva v 6.1.3. Software (BD FACSDiva Software, RRID:SCR_001456).

MTT Colorimetric Assay

Cell viability was determined by using the MTT colorimetric assay. Cells were plated in 48-well plates and treated as described in Cell Culture. After 24 h, the cells were washed with PBS and incubated with 0.5 mg/mL MTT dye (Sigma) in DMEM medium without phenol red for 30 min at 37°C. Blue formazan crystals were solubilized by dimethyl sulfoxide (DMSO), and colorimetric evaluation was performed using a spectrophotometer NanoDrop 2000 (BioTek) at 555 nm and 690 nm. A reading was also performed at 690 nm to subtract background.

Lactate Dehydrogenase Assay

The lactate dehydrogenase (LDH) assay was used to evaluate cytotoxicity. This assay is based on the procedure published by Wroblewski and LaDue (20) with minor modifications. Conditioned medium LDH was quantified by measuring the oxidation of the reduced form of nicotinamide adenine dinucleotide (Sigma-Aldrich 3.5 mM) in the presence of pyruvate (Sigma-Aldrich 21 mM), measuring absorbance at 340 nm 20 min after the mixture was made.

Statistical Analysis

Data are presented as the means ± SE of three to four independent experiments. All data were tested for normality and equal variance. If passed, the Student’s t test was used to compare two groups or one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc test for comparisons among >2 groups. Otherwise, nonparametric tests (the Mann–Whitney U test or Kruskal–Wallis test, followed by Dunn’s post hoc test) were used. Spearman’s correlation was used to determine the significance of the SLAMF1 plasma concentrations correlated with NASH indices. Graphs were generated using GraphPad Prism software version 5.00 for Windows (GraphPad Prism, RRID:SCR_002798, San Diego, CA). BD Accuri C6 software (version 1.0.264.21) was used for flow cytometry data analysis.

RESULTS

SLAMF1 Protein-Positive Staining Is Prominent in NASH Human Livers and High-Fat Diet Mouse Livers

The SLAMF1 protein was detected in the control and NASH liver samples by immunofluorescence microscopy (Fig. 1). Microscopic examination of the images suggested that SLAMF1 appears to be present in several cell types in the liver (Fig. 1). Liver samples classified as NASH showed a more pronounced positive staining for SLAMF1 as compared with No-NASH samples (controls; Fig. 1A). Liver samples from mice fed a high-fat diet for 16 wk (60% fat) also showed positive staining for SLAMF1 staining as compared with livers harvested from mice fed a normal diet (Fig. 1B).

Figure 1.

Figure 1.Representative immunofluorescence staining of liver sections from normal and NASH liver and high-fat diet samples with SLAMF-1 antibody (red) and the nuclear marker DAPI (blue). A: SLAMF1 staining is more prominent in human NASH liver samples as compared with healthy livers. B: liver samples were harvested from mice fed on a high-fat diet (60% fat) and mice fed on a regular diet (10% fat). Liver SLAMF1 staining was more pronounced in high-fat diet livers as compared with regular diet samples. Data represent n = 3 samples/group. All human samples were courtesy of the Pathology Department at LSU Health Sciences Center-Shreveport. All pictures were acquired on a Leica TCS SP5 spectral confocal microscope equipped with a with a ×40 (oil) objective. Scale bar: 100 µm. NASH, nonalcoholic steatohepatitis; SLAMF1, signaling lymphocytic activation molecule family 1.


SLAMF1 Is Detected in the Plasma of Mice and Humans, and Its Levels Correlate with NASH Severity

Studies showed that an activated lymphocyte T cell secretes SLAMF1 into circulation (12, 21). Therefore, we next tested the plasma concentrations of SLAMF1 in mice fed a NASH diet (40% fat, 22% fructose, and 2% cholesterol) for 24 wk featuring the coexistence of steatohepatitis and hepatic fibrosis (Fig. 2A). Plasma SLAMF1 concentrations were significantly increased in mice with NASH compared with control mice 3.1-fold (Fig. 2B). Circulating SLAMF1 concentrations were positively and significantly correlated with NASH severity determined by the nonalcoholic fatty liver disease activity score (NAS; Fig. 2B). To test if these results translate to humans, we measured the circulating levels of SLAMF1 in plasma from controls and patients with NASH. Our data show that SLAMF1 is increased in liver biopsies from patients with NASH, but also, plasma levels were significantly increased in NASH human samples (Fig. 2, C and D), recapitulating our findings in mice. Therefore, SLAMF1 seems to be secreted by the liver into circulation, and its levels in NASH increased threefold (Fig. 2D).

Figures 2.

Figures 2. Plasma SLAMF1 is increased in human and murine NASH and its levels positively correlated with nonalcoholic fatty liver disease activity score (NAS) in mice. Male C57BL6/J mice were fed a high-fat, high-fructose, and high-cholesterol NASH diet or low-fat control diet for 24 wk. A: histological confirmation of NASH using H&E, Sirius red (scale bar: 50 µm), and oil red O (ORO, scale bar: 100 µm) staining of liver sections. B: plasma concentrations of SLAMF1 in mice with NASH (n = 9) and control mice (n = 7). *P < 0.05 vs. control mice. Spearman’s correlation analyses between plasma SLAMF1 and NAFLD activity score (NAS), C: representative immunohistochemistry images of human liver sections from control (healthy liver) and NASH. Human NASH section showed a more pronounced positive staining for SLAMF1. D: plasma concentrations of SLAMF1 in human with NASH (n = 9) and controls (n = 8). ***P < 0.001 vs. controls. Values are shown as means ± SE. All r and P values are shown. H&E, hematoxylin and eosin; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SLAMF1, signaling lymphocytic activation molecule family 1.


Palmitic Acid Treatment of HepG2 Induces Cell Death

We tested the cytotoxic effect of palmitic acid (PA) in HepG2 cells using different methods. Cell viability was evaluated with MTT assay after 24 h of PA treatment at two different doses (0.4 and 0.8 mM). We found that HepG2 cell viability was significantly decreased in response to both doses of PA (Fig. 3A). Next, we tested the effects of PA in a time course of 0, 1, 3, 6, 12, and 24 h using a dose of 0.4 mM of PA. Lactate dehydrogenase (LDH) enzyme activity was measured in the medium as described in materials and methods. LDH activity was significantly increased in response to PA at 12 and 24 h of treatment (Fig. 3B). These results confirmed that treating HepG2 cells with PA significantly reduces cell viability and increases cytotoxicity in our model.

Figure 3.

Figure 3.Effects of palmitic acid (PA) on cell viability, cell apoptosis, and cytotoxicity in HepG2 cells. A: the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay confirmed that the viability of HepG2 cells was decreased by PA, and the effect was concentration-dependent. *P < 0.05, 0.4 mM of PA vs. control, #P < 0.05, 0.8 mM of PA vs. control. B: lactate dehydrogenase (LDH) activity in the extracellular medium. At 0.4 mM of PA-induced cytotoxicity in a time-dependent manner. Statistically significant differences were found at 12 and 24 h of PA treatment. *P < 0.05 vs. control at 12 h, #P < 0.05 vs. control at 24 h. C: the percentage of annexin V-positive cells was significantly increased in PA-treated cells. *P < 0.05 vs. control. D: cleaved caspase 3 p17 levels were significantly increased (normalized to α-tubulin) in HepG2 cells treated with PA in a concentration-dependent manner. *P < 0.05 vs. control, #P < 0.05 vs. 0.04 mM PA. Values are shown as means ± SE. n = 3–4 independent experiments.


To investigate whether the increase in cytotoxicity and reducing cellular viability leads to apoptosis, we performed an annexin V 7AAD assay using flow cytometry (Fig. 3C). Treatment of hepatocytes with 0.4 mM PA for 24 h led to a significant increase in the percentage of cells in apoptotic phase (annexin V-positive and 7AAD-negative cells; 26.0 ± 0.6%, P < 0.05) as compared with control cells (9.3 ± 1.2%; Fig. 3C). This effect was also confirmed by Western blot, where we found a significant increase in the levels of cleaved caspase 3 p17 in response to PA (Fig. 3D).

Palmitic Acid Treatment Increases SLAMF1 Levels in the HepG2 Cells and in Murine Primary Hepatocytes

Immunofluorescence imaging showed an increase in SLAMF1 levels in response to PA treatment in HepG2 cells (Fig. 4A). Our data show that SLAMF1 is strongly expressed on the surface of PA-treated cells compared with vehicle control cells in which SLAMF1 is barely detected (Fig. 4A). At the gene expression level, we found that SLAMF1 mRNA levels were undetectable in vehicle-treated cells. In contrast, the expression of SLAMF1 was significantly upregulated in PA-treated cells (Fig. 4B). Similar results were found at the protein level; Western blot analyses of HepG2 lysates showed that SLAMF1 levels are significantly increased in HepG2 cells in response to 0.4 mM and 0.8 mM of PA treatment (Fig. 4C). Since HepG2 cells are a cancer cell line, we isolate primary murine hepatocytes to test if SLAMF1 is expressed by these cells and if PA treatment increases SLAMF1 levels. As shown in Fig. 4D, SLAMF1 levels are almost undetectable at baseline conditions in primary hepatocytes; however, on PA treatment, the SLAMF1 levels increased on the surface of the cell. Also, in agreement with the results from HepG2 cells, SLAMF1 mRNA and protein levels increased in response to PA in primary hepatocytes (Fig. 4E). These results confirmed that hepatocytes express SLAMF1, and its levels are upregulated by PA treatment.

Figure 4.

Figure 4.Levels of SLAMF1 are significantly increased HepG2 and primary murine hepatocytes in response to PA treatment, and it is released into the medium by PA-treated HepG2 cells. HepG2 cells grown for 24 h in DMEM plus 1% fatty acid free BSA and 2% FBS with/out 0.4 mM of PA. A: PA treatment led to a more prominent positive immunofluorescence staining for SLAMF1 as compared with vehicle-treated cells (control). Cells were fixed with 4% of paraformaldehyde and incubated with anti-SLAMF1 and a secondary antibody as described in the materials and methods. The nucleus was stained with Hoechst 33342 staining solution to stain DNA. Scale bar: 25 µm. B: SLAMF1 mRNA levels increased in response to PA in HepG2 cells. Cells were treated with 0.4 mM of PA in a time course from 0 to 24 h. RNA was extracted, and qRT-PCR was performed to measure SLAMF1 and 18S expression. Values are normalized to the levels of 18S (2–ΔCT) and are represented in the Y-axis without decimals (2–ΔCT × 106). *P < 0.05 vs. control. C: representative Western blot showing the effect of 0.4 mM of PA in SLAMF1 levels. The protein values were determined by densitometry using ImageJ (NIH). The density o SLAMF1 was normalized to α-tubulin. D: PA treatment induces increases in SLAMF1 levels in primary hepatocytes as compared with vehicle-treated cells. The primary hepatocytes were treated with 0.4 mM of PA for 24 h. Cells were then fixed with 4% of paraformaldehyde and incubated with anti-SLAMF1 and the respective secondary antibody. Blue: Hoechst 33342 and red: SLAMF1. Scale bar: 10 µm. E: SLAMF1 mRNA levels increased in response to PA in primary hepatocytes. Cells were treated with 0.4 mM of PA in a time course for 12, 18, and 24 h, and RNA levels were measured by qRT-PCR as described for HepG2 cells. *P < 0.05 vs. control at 12, 18, and 24 h, #P < 0.001 vs. 12 and 24 h. F: SLAMF1 is released from HepG2 cells. Conditioned medium was harvested from PA-treated HepG2 cells and used to precipitate SLAMF1 with a monoclonal SLAMF1 antibody. Representative blot showing SLAMF1 levels from 0 to 24 h of PA treatment. The percentage (%) change in the levels of SLAMF1 was calculated relative to its levels at 0 h. *P < 0.05 vs. control (0 h PA). Values are shown as means ± SE. n = 3–4 independent experiments. PA, palmitic acid; SLAMF1, signaling lymphocytic activation molecule family 1.


Our mouse and human data showed that SLAMF1 levels are significantly increased in plasma from NASH-positive samples, suggesting that SLAMF1 is secreted by hepatic cells into circulation. Therefore, we tested whether SLAMF1 was released by the cultured HepG2 cells and primary hepatocytes into the medium. No SLAMF1 was detected in the conditioned medium of nontreated cells, but there was an increase in SLAMF1 release as early as 1 h and peaking at 12 h after exposure to PA in HepG2 cells (Fig. 4F). Based on these results, we hypothesized that SLAMF1 expression increases in the cell membrane and is then released into the extracellular medium. The secretion of SLAMF1 may then be mediating the lipotoxicity associated with PA treatment. To test this hypothesis, we used siRNA to SLAMF1, a pool of four different siRNAs specific to SLAMF1, to reduce SLAMF1 protein synthesis.

Inhibition of SLAMF1 Synthesis Improves Cell Viability and Prevents Palmitic Acid Lipotoxicity in HepG2 Cells

Treatment of HepG2 cells with 50 nM SLAMF1 siRNA leads to an approximate 50% reduction in SLAMF1 mRNA and protein levels (Fig. 5A). After confirming the efficiency of the SLAMF1 knockdown, we tested the effects of knocking down SLAMF1 on cell viability and cytotoxicity. SLAMF1 knockdown by siRNA led to a significant reduction in cell death and cytotoxicity (Fig. 5B) and in the number of annexin V positive cells (Fig. 5C). To test if the protective effects of SLAMF1 knockdown resulted from SLAMF1 siRNA altering PA-dependent increase in triglycerides (TG), we measured TG levels in all of our treatments (Fig. 5B). We found no significant differences in the effects of PA treatment on TG levels among our group treatments. SLAMF1 knockdown by siRNA did not inhibit triglyceride increases in response to PA treatment of HepG2 cells. Therefore, downregulation of SLAMF1 improves cell viability in response to PA treatment not by decreasing the lipid accumulation but by an alternative mechanism. The activation of the SLAMF1 signaling pathway has been suggested to modulate apoptosis (20). To test if SLAMF1 knockdown protects HepG2 cells from apoptosis, we measured the levels of the apoptotic marker annexin V in response to PA treatment (0.4 nM). We found that the number of annexin V-positive cells was significantly reduced in SLAMF1 knockdown cells compared with controls (Fig. 5C). These results suggest that PA cytotoxic and apoptotic effects are partly mediated by SLAMF1 signaling in hepatocytes.

Figure 5.

Figure 5.Effects of SLAMF1 downregulation by siRNA on cytotoxicity and cell viability after palmitic acid (PA) treatment. HepG2 cells at 80% confluency were for 24 h with 0.4 mM of PA and transfected with control or SLAMF1 siRNA (siSLAMF1). A: RNA and protein levels were quantified by qRT-PCR and Western blots after PA treatment for 24 h. siSLAMF1 significantly decreased SLAMF1 upregulation in response to PA (*P < 0.05 vs. control). B: effects of SLAMF1 knockdown on PA-mediated cytotoxicity and viability. Cell viability was determined using the MTT assay. LDH activity was used as an indicator of increased cytotoxicity. SLAMF1 knockdown significantly improved cell viability (*P < 0.05 vs. control) and reduced cytotoxicity (*P < 0.05 vs. control). SLAMF1 knockdown did not block triglyceride levels increased in response to PA treatment. Therefore, downregulation of SLAMF1 by siRNA improves cell viability in response to PA treatment not by decreasing the lipid accumulation but by an alternative mechanism. C: SLAMF1 knockdown significantly decreases (*P < 0.05 vs. control) the percentage of Annexin V-7AAD-positive cells in PA treatment. Top: representative flow chart. Values are shown as means ± SE from 3 to 4 independent experiments. n = 3–4 independent experiments. LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PA, palmitic acid; SLAMF1, signaling lymphocytic activation molecule family 1; TG, triglycerides; 7AAD-A, 7-amino-actinomycin D-Annexin V.


DISCUSSION

NASH is one of the most common liver diseases in the United States, and its prevalence is increasing worldwide. NASH is characterized by liver steatosis, chronic inflammation, hepatocyte injury, death, and fibrosis (22). Despite the high incidence and its associated complications, the pathogenesis of NASH and the mechanisms of progression of hepatic steatosis to NASH remain unknown.

The signaling lymphocyte-activation molecule SLAMF1 is an Ig-like receptor and a costimulatory molecule. SLAMF1 triggers signal transduction networks, leading to innate and adaptive immune responses in T cells, macrophages, and dendritic cells, natural killer-T cell development, and the production of proinflammatory cytokines and reactive oxygen species (14, 15). Regarding the role of SLAMF1 in the gastrointestinal system, studies have shown that SLAMF1 mediates macrophage infiltration into the intestinal lining in murine models of enterocolitis (14). The present study is the first to propose a role for SLAMF1 in NASH and hepatocytes. We found that SLAMF1 is detected at baseline levels in human and mouse livers by immunofluorescence and immunohistochemistry staining. Human and mouse NASH livers showed a more pronounced SLAMF1 positive staining. Plasma levels of SLAMF1 were significantly increased in NASH samples from mice, and its levels correlated with disease severity in mouse liver samples. These results translated to humans; SLAMF1 levels were also considerably increased in NASH samples compared with samples from healthy volunteers. Our findings showed that the liver secretes SLAMF1 in NASH, suggesting a clinical use for SLAMF1 as a biomarker of the disease. Future studies are needed to expand these results and validate SLAMF1 as a biomarker for NASH diagnosis or severity.

Our findings also showed that SLAMF1 is expressed by HepG2 cells and primary murine hepatocytes. Treatment with PA significantly increased SLAMF1 mRNA expression and protein levels in both the cell line and primary hepatocytes. Also, both cell culture systems secreted SLAMF1 into the medium. These findings support the hypothesis that hepatocytes in the NASH liver are producing SLAMF1 and secreting this protein into circulation.

Studies have shown that the expression of SLAMF1 in B cells leads to the activation of CD40 and Fas/CD95 signaling, which triggers cell apoptosis (19). There are no studies on the role of SLAMF1 in hepatocyte cell death or apoptotic markers’ expression. Our data showed that PA treatment of HepG2 cells led to a rapid increase in the proapoptosis markers cleaved caspase 3 and annexin V, which correlated with decreased cell viability and upregulation in the levels of SLAMF1. Increases in cleaved caspase 3 have been associated with the expression of biomarkers of disease severity in NASH (23). Lipotoxicity in NASH has been linked with the activation of proinflammatory and proapoptotic pathways (21), including caspase-3 and caspase-7, the apoptotic executioner caspases (24). Our results show that the knockdown of SLAMF1 by siRNA led to a decrease in the activation of annexin V and caspase 3. These results suggest that the upregulation of SLAMF1 correlates with the activation of proapoptotic pathways in PA-treated hepatocytes. Therefore, SLAMF1 inhibition may serve as a therapeutic target to reduce hepatocyte apoptosis and attenuate NASH pathogenesis. Future studies using animal models of NASH and inhibitors of SLAMF1 are needed to fully clarify the potential therapeutic use of SLAMF1.

In summary, our data showed that the levels of SLAMF1 are elevated in NASH in both mice and humans. Moreover, the increased circulating SLAMF1 positively and significantly correlated with NAS in mouse livers. Hepatocytes express SLAMF1, and its levels are exacerbated in response to PA treatment (in an in vitro model of NASH). Our data on HepG2 cells also showed that a reduction in the levels of SLAMF1 by siRNA protected the cells from PA lipotoxicity. Therefore, SLAMF1 is a novel mediator of hepatocyte lipotoxicity, and it has potential clinical use as a biomarker of the disease and perhaps as a therapeutic target. Future studies need to be performed to fully elucidate the role of this protein in the pathology and progression of NASH.

GRANTS

This work was supported by Ayuda a Grupos UCLM (FEDER) Grant 2020-GRIN-28737 (to O. Gomez-Torres and E. Burgos-Ramos); National Institutes of Health Grants HL098435, HL133497, and HL141155 (to W. A. Orr), NHLBI K99/R00 HL150233 and R01DK134011 (to O. Rom); R00HL145131 (to A. Yurdagul); Collaborative Intramural Research Program (to A. Yurdagul, O. Rom, P. Thevenot, and A. Cohen), and National Heart, Lung, and Blood Institute Grants K01 HL144882 and 3P20GM121307-03S1 (to D. Cruz-Topete).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

O.G.-T., H.D., O.R., W.A.O., K.N., P.T., A.C., H.S., J.S.A., E.B.-R., A.C.-R., and D.C.-T. conceived and designed research; O.G.-T., S.A., L.K., H.D., P.K., O.R., P.T., A.C., J.S.A., A.C.-R., and D.C.-T performed experiments; O.G.-T., H.D., O.R., A.Y., W.A.O., A.C., H.S., A.C.-R., and D.C.-T. analyzed data; O.G.-T., H.D., O.R., A.Y., W.A.O., K.N., A.C., H.S., J.S.A., A.C.-R., and D.C.-T. interpreted results of experiments; O.G.-T., H.D., O.R., W.A.O., H.S., and D.C.-T. prepared figures; O.G.-T., H.D., O.R., W.A.O., H.S., and D.C.-T. drafted manuscript; O.G.-T., S.A., H.D., O.R., W.A.O., A.C., H.S., A.C.-R., and D.C.-T. edited and revised manuscript; O.G.-T., S.A., L.K., H.D., P.K., O.R., W.A.O., K.N., P.T., A.C., H.S., J.S.A., E.B.-R., A.C.-R., and D.C.-T. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors thank Dr. Serrano del Arco Santamaria and Dr. Alberto Davalos for providing us with the reagents necessary for the realization of this work. We also thank the Microscopy Imaging Core Facility at LSU Health Sciences Shreveport.

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AUTHOR NOTES