Patients with NAFLD come from different centers of Buenos Aires whereas samples processing was entirely performed at the Clinical Hospital “José de San Martín”. NAFLD patients group 1 comprised 68 individuals with biopsy-proven diagnosis of SS (n=34) or NASH (n=34). Its diagnosis was based on a daily alcohol consumption <20 g (females) or 30 g (males), absence of other causes of liver disease and abdominal ultrasound [16]. The histological diagnosis was based on the NAFLD Activity Score [17] and fibrosis staging [18] with score values ≥5 as the cut-off for NASH. Liver biopsies and concomitant blood samples were used to evaluate TLR9 protein expression. NAFLD patients from group 2 were eight individuals who met the general criteria for NAFLD diagnosis but lacked histological diagnosis, hereafter called “NAFLD patients”, used to evaluate TLR9 mRNA expression. Control group 1 included 11 metabolically healthy individuals who underwent antireflux surgery or cholecystectomy and did not have fatty liver which was confirmed by biopsy. Their biopsy samples were used to evaluate TLR9 protein expression. Control group 2 included 40 individuals recruited among the staff members of the Institute of Immunology, Genetics and Metabolism. All of the control individuals were given information of the study and those who intended to participate gave their written informed consent. Patients in the control groups did not received any medications within 6 months before the study, had low daily alcohol consumption, a body mass index (BMI) <25 kg/m² and a waist circumference <80 cm (females) or 94 cm (males). Their blood samples were used in different experiments as indicated. The BMI and waist circumference were evaluated according to the World Health Organization criteria [19]. Homeostasis Assessment Model for insulin resistance was calculated as glucose (mg/dL) × insulin (µU/mL) / 405 and measures were performed using commercial kits. Plasma levels of total cholesterol and triglycerides, aspartate and alanine aminotransferases were determined in a Cobas C-501 autoanalyzer (Roche Diagnostics, Mannheim, Germany). As transaminases normal levels slightly vary depending on the individual laboratory´s reference values, data were normalized before use. The experimental protocols and sample studies were approved by the local Ethics Committee (Clinic Hospital Ethics Committee, “Immunopathogenic factors that determine the progression of diseases associated with metabolic syndrome”, March 6, 2017) which uses the internationally endorsed standards for the application of the Helsinki Declaration.

We used Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden), TRIZOL^® reagent and Roswell Park Memorial Institute 1640 medium (Life Technologies, Gaithersburg, MD, USA) and gentamicin, glutamine, ionomycin, fetal bovine serum, phorbol 12-myristate 13-acetate and brefeldin A (Sigma Chemical Co, Saint Louis, MO, USA). Phorbol 12-myristate 13-acetate was prepared in dimethyl sulfoxide at 25 µg/mL. We used BD™ IMag anti-human CD3 magnetic particles, PermWash™, Cytofix/Cytoperm, anti-human CD3 monoclonal antibody (UCHT1) for cell stimulation, anti-TLR9-phycoerythrin (PE) (eB72-1665) and anti-CD4-Peridinin-Chlorophyll-protein (PerCP) (SK3) from Becton-Dickinson (San Diego, CA, USA), anti-CD8-Fluorescein isothiocyanate (FITC) (UCHT-4), anti-CD69-PE (FN50) and anti-IFN-γ-PE (B27) from ImmunoTools GmbH (Friesoythe, Germany). The synthetic cytidine-phosphateguanosine oligodeoxynucleotide (CpG-ODN) 2395 5’-tcgtcgttttcggcgcgcgccg-3’ (Thermo Fisher Scientific, Waltham, MA, USA) used as TLR9 agonist was solubilized in sterile endotoxin-free water at 100 µM and stored at 20X until use.

Peripheral blood mononuclear cells were obtained using FicollHypaque gradients and resuspended in Roswell Park Memorial Institute 1640 medium 10% fetal bovine serum, 2 mmol/L L-glutamine, and 50 µg/mL gentamicin. Twice-enriched fractions of CD3⁺ T cells were isolated from peripheral blood mononuclear cells (PBMC) by negative selection using magnetic particles. Recovered cells were at least 98% CD3⁺. We handled liver tissues at 4ºC using a hands-on homogenization method, washed liver cell suspensions twice in phosphate buffered saline and harvested cells by centrifugation.

Total RNA was isolated from CD3⁺ cells using TRIZOL^® reagent and reverse transcribed using oligo (dT) primers and SuperScript™ II First-Strand Synthesis System (Invitrogen, Life Technologies, Carlsbad, CA, USA). Forward and reverse primers: TLR9, 5´-gaagggacctcgagtgtgaag-3´ and 5´-gtaggaaggcaggcaaggta-3´; glyceraldehyde 3-phosphate dehydrogenase, 5´-cgaccactttgtcaagctca-3´ and 5´-acatggcctccaaggagtaa-3´. Quantitative polymerase chain reaction was performed on a Stratagene Mx3005p RT-PCR Detection System (Agilent Technologies, La Jolla, CA, USA) using SYBR^® Green PCR Master Mix (applied Biosystems, Life Technologies). Relative gene expression and fold change expression were calculated using the comparative Ct method [20].

Suspension of 5×10⁵ isolated peripheral blood mononuclear cells or liver cells was centrifuged at 2,000 rpm for 5 minutes and incubated with anti-CD4-PerCP and anti-CD8-FITC in darkness at 4ºC for 20 minutes. Fixed and permeabilized cells were incubated with anti-TLR9-PE or the isotype control in darkness at 4ºC for 40 minutes, washed twice in PermWash™ 1X, resuspended in phosphate buffered saline 1X and assessed using flow cytometry. At least 100,000 events were acquired on a FACSCalibur flow cytometer (BD). The acquired data were analyzed using FlowJo software version 5.7.2 (Treestar, San Carlos, CA, USA). To analyze TLR9 expression, lymphocytes were gated on a forward and sidescatter plot and further gated based on CD4 or CD8 expression. TLR9 expression was expressed as a percent difference compared with isotype control using mean fluorescence intensity (MFI) values as follows: [(MFI of TLR9 / MFI of isotype control) - 1 / MFI of isotype control × 100] [9].

1×10⁶ isolated CD3⁺ cells were activated with soluble anti-CD3 (250 ng/mL) alone or in combination with 2 µM CpG-ODN in 24-well culture plates at 37ºC for 24 hours. The cells were harvested by centrifugation, incubated with anti-CD4-PerCP, anti-CD8-FITC and anti-CD69-PE monoclonal antibodies in darkness at 4ºC, washed twice and resuspended in phosphate buffered saline 1X. A frequency index for CD69⁺ cells was calculated within CD4⁺ and CD8⁺ cell compartments as the ratio of frequencies observed after treatments with anti-CD3+CpG-ODN and anti-CD3 alone.

To evaluate the frequency of IFN-γ⁺ cells at baseline, peripheral blood mononuclear cells were stimulated with 25 ng/mL phorbol 12-myristate 13-acetate and 1 mM Ionomycin in the presence of 1 mM Brefeldin A at 37°C for 4 hours. After stimulation, cell surface staining using anti-CD4-PerCP and anti-CD8-FITC monoclonal antibodies was performed as described above, and intracytoplasmic detection of IFN-γ was done as described for TLR9. Lymphocytes were gated on a forward versus size scatter plot, the lymphoid region was further gated based on CD4 or CD8 expression, and IFN-γ expression was measured in the selected populations. To evaluate the frequency of IFN-γ⁺ cells after co-stimulation, isolated PBMC were divided into 5×10⁵ cells per well each containing 500 µL of Roswell Park Memorial Institute medium and stimulated at 37ºC for 18 hours in the presence of 1 mM Brefeldin A with 250 ng/mL soluble anti-CD3 alone or anti-CD3⁺2 uM CpG-ODN in 24-well culture plates. A frequency index for IFN-γ⁺ cells was calculated as described for CD69.

We used GraphPad Prism software (GraphPad, San Diego, CA, USA). We performed the two-tailed Mann-Whitney U test (two groups), the Kruskal-Wallis with Dunn’s multiple comparison test (three or more groups), and Wilcoxon paired test (intragroup comparisons). Data were summarized using the median, first and third interquartiles and minimum and maximum values. We used the Spearman’s rank correlation analysis to measure the statistical relations between TLR9 expression and continuous variables. P<0.05 was accepted as a significant value.

Anthropometric measurements, blood analysis and ultrasonography examination were performed at the beginning of the study (Table 1). All patients showed overweight or obesity and altered values of waist circumference and homeostasis assessment model for insulin resistance. We observed higher levels of aspartate and alanine aminotransferases in NASH than in SS patients from group 1. Anthropometric and biochemical values from control subjects fell into the reference ranges indicated (not shown). We described a direct relationship between plasma triglycerides concentration and fat content in NAFLD patients after comparing triglyceride levels between patients with steatosis scores “≤1” and “≥2” (P=0.0342, not shown).

We assessed TLR9 protein expression in liver cell suspensions (Fig. 1A, B). TLR9 expression was lowest within the CD4⁺ cell compartment in SS patients (P<0.05, vs. controls and vs. NASH patients) (Fig. 1B). TLR9 expression on CD8⁺ T cells was similar among groups. To get further insight into the biological relevance of these findings, we evaluated the relationship between TLR9 expression and NAFLD histological features (Table 2) after stratification of NAFLD patients according to their histological scores within the categories indicated in Table 2. Our results showed a positive association between TLR9 expression in CD4⁺ cells and liver damage. Then, we assessed TLR9 protein expression in peripheral blood mononuclear cells from the individuals previously evaluated (Fig. 1C, D). SS patients showed the lowest TLR9 expression within CD4⁺ (P=0.0219) and CD8⁺ (P=0.0022) cell compartments (Fig. 1D). A quantitative polymerase chain reaction-based analysis of TLR9 mRNA in CD3⁺ T cells confirmed a decreased expression of TLR9 mRNA in about 50% of patients (not shown). To assess the relationship between TLR9 expression at peripheral blood and clinic-pathological variables relevant to NAFLD, we performed a correlation analysis (Table 3). NAFLD patients with a lower alteration of BMI and aminotransferases activity exhibited a lower TLR9 expression within both cell compartments. We also found a positive association between plasma triglycerides concentration and TLR9 expression in peripheral CD8⁺ cells.

Given that a dysfunctionality of Kupffer cells in NASH [21] might enhance the influx of TLR9 ligands into the systemic circulation and interact with T cells, we explored the consequences of TLR9-mediated signaling of circulating T cells. Because cell activation is a prerequisite for T cell differentiation, we assessed CD69 upregulation and IFN-γ production as phenotypic markers of these processes. We employed isolated CD3⁺ cells for the assessment of cell activation to avoid the indirect effects of non-T cells [22], and the higher affinity of non-T cells for TLR9 ligands [23] (Fig. 2). Regarding CD69 expression, the simultaneous addition of anti-CD3 and CpG-ODN only provided co-stimulation to CD8⁺ cells (intragroup comparisons: anti-CD3⁺CpG-ODN vs. anti-CD3 alone, not shown). Upregulation of CD69 was lowest in SS patients (P<0.01 vs. controls, P<0.05 vs. NASH patients). The addition of CpG-ODN did not affect CD69 expression (intragroup comparison: CpG-ODN treatment vs. medium, not shown). We employed peripheral blood mononuclear cells for cell differentiation assessment as a representative cellular platform that would allow the association of these results with the frequencies of IFN-γ⁺-producing cells at baseline (Fig. 3). Again, anti-CD3⁺CpG-ODN only provided co-stimulation for IFN-γ production to CD8⁺ cells (intragroup comparisons: anti-CD3⁺CpG-ODN vs. anti-CD3 alone, not shown). Up-regulation of IFN-γ⁺ cells was lowest in SS patients (P<0.05 vs. controls, P<0.01 vs. NASH patients). As for CD69, the addition of CpG-ODN did not alter intracellular IFN-γ production. Thus, our results demonstrated that T cells co-stimulation promoted the lowest activation and differentiation of circulating CD8⁺ cells in SS patients. The small size of our liver biopsies precluded its use for functional studies.

We measured the frequency of circulating IFN-γ⁺ T cells (Fig. 4). The frequency of IFN-γ⁺ cells within CD4⁺ (P<0.01) and CD8⁺ (P=0.0005) cell compartments was higher in NASH patients than control subjects. SS patients and control subjects showed similar frequencies of type 1 CD8⁺ cells and T helper 1 cells, respectively.