Intestinal Intervention Strategy Targeting Myeloid Cells to Improve Hepatic Immunity during Hepatocarcinoma Development

Biomedicines 2021, 9, 1633. https://doi.org/10.3390/biomedicines9111633 www.mdpi.com/journal/biomedicines Article Intestinal Intervention Strategy Targeting Myeloid Cells to Improve Hepatic Immunity during Hepatocarcinoma Development Adrián Bouzas Muñoz 1,2,3, Juan Antonio Giménez-Bastida 4, Aurora García Tejedor 5, Claudia Monika Haros 6, Marta Gómez de Cedrón 2, Ana Ramírez de Molina 2 and José Moisés Laparra Llopis 1,* 1 Molecular Immunonutrition Group, Madrid Institute for Advanced Studies in Food (IMDEA Food), Ctra Cantoblanco 8, 28049 Madrid, Spain; [email protected] 2 Molecular Oncology Group, Madrid Institute for Advanced Studies in Food (IMDEA Food), 28049 Madrid, Spain; [email protected] (M.G.d.C.); [email protected] (A.R.d.M.) 3 Canaan Research and Investment, 28003 Madrid, Spain 4 Laboratory of Food and Health, Research Group on Quality, Safety and Bioactivity of Plant Foods, Department Food Science and Technology, CEBAS-CSIC, Campus de Espinardo, 30100 Murcia, Spain; [email protected] 5 Faculty of Health Sciences, Valencian International University (VIU), Pintor Sorolla 21, 46002 Valencia, Spain; [email protected] 6 Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Av. Agustín Escardino 7, Parque Científico, Paterna, 46980 Valencia, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-(0)9-1787-8100 Abstract: Innate immunity in the tumor microenvironment plays a pivotal role in hepatocarcinoma (HCC) progression. Plant seeds provide serine-type protease inhibitors (SETIs), which can have a significant influence on liver inflammation and macrophage function. To elucidate the influence of SETIs to counter pro-tumorigenic conditions, at the early stages of HCC development, it was used as an established model of diethylnitrosamine/thioacetamide-injured liver fed with a standard diet (STD) or high-fat diet (42%) (HFD). The administration of SETIs improved survival and ameliorated tumor burden via modulation of monocyte-derived macrophages as key effectors involved in dietinduced HCC development. RT-qPCR analyses of hepatic tissue evidenced a diet-independent downregulatory effect of SETIs on the transcripts of CD36, FASN, ALOX15, and SREBP1c; however, animals fed with an STD showed opposing effects for PPAR and NRLP3 levels. These effects were accompanied by a decreased production of IL-6 and IL-17 but increased that of TNF in animals receiving SETIs. Moreover, only animals fed an HFD displayed increased concentrations of the stem cell factor. Overall, SETIs administration decreased the hepatic contents of lysophosphatydilcholine, phosphatidylinositol, phosphatidylcholine, and phosphatidyl ethanolamine. Notably, animals that received SETIs exhibited increased hepatic proportions of CD68+CX3CR1+CD74+ cells and at a higher rate in those animals fed an HFD. Altogether, the data evidence that oral administration of SETIs modulates the tumor microenvironment, improving hepatic innate immune response(s) and favoring a better antitumoral environment. It represents a path forward in developing coadjutant strategies to pharmacological therapies, with either a preventive or therapeutic character, to counter physiopathological conditions at early stages of HCC development. Keywords: macrophages; lipid homeostasis; immunonutrition; hepatocarcinoma 1. Introduction Hepatocellular carcinoma (HCC) is a major health problem with high morbidity and mortality, which often implies immune system disorders and local myeloid infiltration in Citation: Bouaz Muñoz, A.; Giménez Bastida, J.A.; Tejedor, A.G.; Haros, C.M.; Cedrón, M.G.d.; Molina, A.R.d.; Laparra Llopis, J.M. Intestinal Intervention Strategy Targeting Myeloid Cells to Improve Hepatic Immunity during Hepatocarcinoma Development. Biomedicines 2021, 9, 1633. https://doi.org/10.3390/biomedicines9111633 Academic Editor: Alexei Gratchev Received: 10 October 2021 Accepted: 3 November 2021 Published: 6 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Biomedicines 2021, 9, 1633 2 of 15 hepatic tissue [1]. Macrophages, as a main type of innate immune effector, can play opposing roles in regulating the tumor microenvironment, cell progression, and severity of the disease according to its different phenotype subtypes. Various risk factors for HCC have been well-defined, including liver damage caused by inflammation and metabolic syndrome [2]. Other cofactors such as innate immune ‘Toll-like’ receptor (TLR)-4 activation and gut microbiota have also been involved in the control and promotion of HCC [3]. Over the last decade, there has been an improvement in the understanding of the genetic pathogenesis of HCC; however, only a few of the main drivers responsible for tumor initiation and progression have been identified as druggable targets [4]. Of note, recent data suggest that environmental inflammation-associated conditions such as non-alcoholic fatty liver disease (NAFLD) or the severe variant non-alcoholic steatohepatitis (NASH) precedes the development of HCC [5]. Improved knowledge of the relative contribution of environmental immunometabolic factors to the overall inflamed hepatic network could help to develop and/or establish more effective intervention strategies coadjutant to classical pharmacological approaches with a preventive or therapeutic character. While the host’s endogenous factors influencing the risk of suffering HCC are difficult to influence, the environmental factors are predominant and addressable in a preventive or therapeutic intention. In this sense, recent discoveries have pointed to the close relationship between lipid homeostasis and metabolic derangements in liver cancer [6]. Thus, strategies to manipulate the tumor microenvironment in chemically injured livers of animals developing HCC are actively being investigated [7,8]. Our previous research has shown that Chenopodium quinoa seeds constitute a good source of serine-type protease inhibitors (SETIs) found in the globulin fraction, which display innate immune and metabolic effects ameliorating the HCC severity in mice [7–10]. The elucidation of functional and structural features showed that bioactive SETIs from C. quinoa appear as glycoproteins with a glucoside prosthetic group in complexes formed by homomeric subunits [9]. These compounds exhibited a significant partial resistance to gastrointestinal enzymes and, therefore, low bio-accessibility rates [10]. These compounds were mostly responsible for the effects showing biological correlation with TLR4 signaling [9,10]. SETIs appear to function primarily in the induction of a delayed wave downstream of TLR4 signaling [10]. Particularly, studies on high-fat diet (HFD) fed mice have shown beneficial innate immune and metabolic effects within the gut–liver axis targeting hepatocarcinogenesis derived from the administration of extracts enriched in serine-type protease inhibitors (SETIs) [8]. In this context, it was possible to target the hepatic lipid distribution of monoand poly-unsaturated (PUFA) fatty acids that improved the hepatocellular susceptibility to ferroptosis associated with increased PUFA levels [8]. To the best of our knowledge, only studies on microglial priming by fatty acids have shown that HFD consumption increased the gene expression of markers such as CX3CR1, MHC-II, and NLRP3 [11]. Interestingly, these studies concluded the participation of an additional mediator, contributing to the exaggerated inflammatory response observed. When considering HCC development, it could be hypothesized that environmental modulation of innate immune responses via TLR4 activation enable disease modulation [3]. Additionally, the chemokine receptor CX3CR1 is expressed on various cells of the macrophage lineage [12]. This receptor modulates liver inflammation by upregulating pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and by favoring the infiltration of inflammatory monocytes [13]. The important roles of the NFκB-mediated CX3CL1/CX3CR1 axis and NLR family in liver injury are known [14]. In this context, recent research associated CD74+ (major histocompatibility complex II-associated invariant chain, MHC-II) macrophages with favorable prognosis and immune contexture in hepatocellular carcinoma [15]. Besides the recognized role of CD74 in antigen presentation, it also plays key roles in inflammatory diseases such as liver fibrosis [16,17], and macrophage inflammation and function [16,18]. Additionally, the activation of TLRs on macrophages results in the production of multiple soluble mediators including the key inflammatory cytokines TNFα and IL-6, which display opposing regulatory functions on the Biomedicines 2021, 9, 1633 3 of 15 immune response via TLR4; whereas TNFα associates with TLR4 hypo-reactivity, IL-6 increases the responsiveness to activate NFκB [19]. Thus, as a consequence of environmental innate immune stimulatory conditions, subsets of monocyte-derived cells can generate a broad and diverse spectrum of innate immunometabolic responses, either worsening or improving HCC development and severity. In view of the pivotal role that tumor microenvironment plays in the natural history of HCC, this study focuses on the potential immunomodulatory role of naturally occurring SETIs in modulating the pro-tumorigenic microenvironment at early stages of HCC development in metabolic models of diethylnitrosamine/thioacetamide (DEN/TAA)-injured livers. 2. Materials and Methods 2.1. Isolation of Protease Inhibitors (SETIs) Innate immune and metabolically active extracts were obtained as described elsewhere [8]. In brief, commercial samples of Chenopodium quinoa seeds were purchased from local supermarkets (Madrid, Spain) to be finely grounded. The aliquots (0.5 g) were weighed into a centrifuge tube (50 mL) and extracted (×2) with 5 mL of phosphate-buffered saline solution (PBS) at 37 °C with gentle agitation during 2 h. The samples were thermal treated (60 °C) for 30 min, followed by sequential centrifugation: first (8000× g), to remove the precipitate and, second (10,000× g), to remove the supernatants using a 30 kDa membrane (Amicon®, Merck KGaA, Darmstadt, Germany). Clear filtrates were filtered through 0.45 μm and subjected to reverse-phase liquid chromatography–ESI–MS analyses to confirm (i) the presence of glucoside-carrying proteins with adequate molecular weights and (ii) the absence of bacterial lipopolysaccharide (LPS) contamination [10]. Only samples fulfilling these conditions were lyophilized and kept frozen until use. 2.2. Induction of the Hepatocarcinoma (HCC) C57Bl/6 male mice were obtained from the Centro de Investigaciones Biológicas (CIBCSIC) (Madrid, Spain). To induce hepatocarcinoma (Ethic code, Proex 220/17), the animals were randomly distributed into four different groups (n = 6 per group) under a standard (groups 1 and 2) or a high-fat diet (groups 3 and 4) (AIN93G mod. HF 43 kcal% fat, irradiated, Ssniff spezialdiäten gmbh): groups 1 and 3 received a saline solution as the ‘vehicle’ (control); groups 2 and 4 received SETIs (reconstituted lyophilisates). HCC was induced by a combined treatment with diethynitrosamine (DEN)/thioacetamide (TAA) [3,7,8]: DEN (20 mg/kg, i.p.), given 3 times per week (for 2 weeks), and TAA (saturated solution—0.5 mL/kg i.g., dissolved in PBS—dose equivalent to 80 mg/kg) administered 3 times per week (for 8 weeks). The mice were sacrificed 7 weeks after the initial DEN injection (Figure 1). Biomedicines 2021, 9, 1633 4 of 15 Figure 1. Liver tumor development in mice administered diethylnitrosamine (DEN) and the hepatotoxin thioacetamide (TAA). (A) Schematic representation of the procedure to induce the hepatocarcinoma in mice fed with a standard diet (STD) or a high-fat diet (HFD). (B) Survival curve (days) of serine-type protease inhibitors (SETIs) administered to mice fed with a standard diet (STD) or a high-fat diet (HFD). (C) Body weight gain of mice fed an STD or HFD, and those fed an HFD and administered SETIs were measured over 7 weeks. (D) Food intake in STDor HFD-fed mice, receiving or not receiving SETIs, was measured every 48 h during the study period. (E) Liver masses in STDor HFD-fed mice, receiving or not receiving SETIs, measured at week 7. (F) Liver to body weight (BW) ratio of DEN/TAA-treated mice fed with STPIs. (G) The number of infiltrated F4/80+ macrophages in hepatic tissues of STDand HFD-fed mice receiving or not receiving SETIs was determined at week 7 after first DEN/TAA administration. Results are expressed as mean ± mean standard error (SEM) (n = 6). Changes in body weight (BW) and food consumption were monitored every two days. After treatment, the mice were sacrificed by cervical luxation. Whole blood samples were preserved in EDTA-treated tubes (at room temperature) for analyses. Different sections (30–50 mg) of the liver were immersed in RNA later buffer (Qiagen, USA), Krebs’s buffer, and RIPA buffer and kept at −80 °C until analysis. 2.3. Quantitative Reverse Transcription Real-Time Polymerase Chain Reaction (qRT-PCR) Validated Gene Expression Assays for murine (Mus musculus) CD36 (forward 5′- AAA AGC CAA GCC AGT GAC AAG-3′, reverse 5′-GGA CGC TTT TTC CTC AAG TCA3′), fatty acid synthase (FASN) (forward 5′- TTC CCA CCA AGT GTG GGT AT -3′, reverse 5′- TGG GAC CTT CAG CTT GCT TC -3′), sterol regulatory element-binding protein 1 (SREBP1c) (forward 5′- TCA AAA CCG CTG TGT CCA GT -3′, reverse 5′- GAC GTC TCA ACC CGC TAG G -3′), arachidonate 15-lipoxygenase (ALOX15) (forward 5′- TCC CAT TCT AGG GGA GAG GG -3′, reverse 5′- CCT TGA CCA GCT CAG TAG GC -3′), peroxisome proliferator activated receptor (PPAR)-γ (forward 5′- TTG GTG GGA TTG TGT CTC GG -3′, reverse 5′- GGC CAA GAT CTC ACA GTG CT -3′), NRLP3 inflammasome (forward 5′- GGT GAC CAC GGG ACA AAA CT -3′, reverse 5′- ATT TGG TCC CAC ACA Biomedicines 2021, 9, 1633 5 of 15 AGC -3′), and β-Actin (forward 5′-TGC ACC AAC TGC TTA-3′, reverse 5′-GGA TGC AGG GAT GAT GTT-3′) were purchased from Applied Biosystems (Foqter City, CA, USA). PCR reactions were performed with 50 ng of cDNA from liver sections, using the Universal PCR Master Mix (Applied Biosystems). The cycling conditions were 10 min of denaturation at 95 °C and 40 cycles at 95 °C for 15 s and at 65 °C for 30 s and 72 °C for 20 s. Quantitative values were calculated by using the 2−ΔCt method, wherein the ΔCt value of each sample was calculated by subtracting the average Ct value of the target gene from the average Ct value of the β-Actin gene. 2.4. Cytokine Production Liver aliquots (25 mg) kept in Krebs’s buffer (0.3 mL) containing the Complete Protease Inhibitor Cocktail (Sigma) were thawed up and homogenized using a TissueRuptor (Qiagen) freshly before use. Then, the samples were centrifuged (10,000× g/15 min) to obtain clear supernatants for cytokine determination. Interleukin (IL)-6 (Cat. n° 32670069), IL-17 (Cat. n° 32670179), Tumor necrosis factor (TNF)-α (Cat. n° 32673019), and stem cell factor (SCF) (Cat. n° 32673329) were determined by ELISAs according to the manufacturer’s instructions (ImmunoTools, Friesoythe, Germany). 2.5. Analysis of the Hepatic Phospholipid Profile Liver aliquots (50 mg) kept in Krebs’s buffer (0.2 mL) were homogenized. Whole homogenates were added (0.4 mL) with a 20:80 methanol/acetonitrile solution and extracted at 37 °C with gentle agitation during 1 h. The analyses were carried out on an Agilent HPLC system using a HILIC (75 × 3.2 mm) (Agilent) column. The elution phases consisted of mobile phase A (acetonitrile, 0.1% formic acid) and mobile phase B (deionized 18.2 MΩ cm water, 0.1% formic acid). Aliquots (30 μL) of the precipitation supernatants were injected in each cycle, and the analysis was performed with the following gradient (min, %B): 0, 5; 3, 5; 7, 50; 10, 50; 15, 100; 18, 100; 18.1, 5; and 20, 5. Phospholipids were identified by matching the retention times and by co-injecting the samples with the phospholipid mixture for HPLC used as standards (P3817, MerckSigma). 2.6. Immunohistochemistry for Hepatic F4/80+ Cells The liver (4 μm) sections, prepared from tissue samples kept in O.C.T. [7], were incubated with a blocking solution (1X PBS/5% bovine serum/0.3% Triton™ X-100) for 60 min. After blocking, samples were washed with PBS. Fluorescent-tagged antibodies (F4/80) (Biolegend) were diluted in the dilution buffer (1X PBS/1% BSA/0.3% Triton™ X100) and pipetted onto the slides. The samples were incubated overnight. Subsequently, after 3 washes of 10 min each in PBS-Triton X-100 0.05% (v/v), the liver sections were analyzed using an inverted fluorescence microscope Leica DM IL LED. 2.7. Immunophenotyping of Hepatic Infiltrated Cells Liver aliquots (150 mg) kept in Krebs’s buffer (0.5 mL) were immersed in 0.5 mL of a trypsin/EDTA mixture (0.25%, sterile-filtered, 2.5 g porcine trypsin, and 0.2 g EDTA) (T4049, Merck-Sigma, Darmstadt, Germany). The samples were incubated at 37 °C with gentle shaking. Afterwards, large particles from tissue sections were completely disaggregated using pre-separation cell strainers (70 μm) (130-041-407 Miltenyi Biotech, Bergisch Gladbach, Germany). After centrifugation (1200 rpm/5 min), the cells were resuspended in PBS and mixed with the following fluorochrome-conjugated antibodies: Anti-mouse CD68 (Cat # F-2761, Thermo-Fisher), CX3CR1 (Cat No: 567805, BD Biosciences), CD74 (Cat No: 740939, BD Biosciences), and TLR4 (Cat No: 558294, BD Biosciences). Then, the samples were prepared for flow cytometry analysis with the Immunoprep kit (Beckman Coulter, USA) Biomedicines 2021, 9, 1633 6 of 15 according to the manufacturer’s instructions and further analyzed on a SORP Flow Cytometer (BD Biosciences, New Jersey, NJ, USA). 2.8. Statistical Analyses Statistical analyses were performed using SPSS v.15 software (SPSS Inc., Chicago, IL, USA). For normally distributed data, ANOVA with the post hoc Tukey test were applied, and for nonnormally distributed data from microbial analyses, the Mann–Whitney U test was used. Statistical significance was established at p < 0.05 for all comparisons. 3. Results 3.1. Amelioration of Tumor Burden in Hepatocarcinoma-Developing Mice Major features in the development of HCC in mice fed a standard diet (STD) or highfat diet (HFD) were measured (Figure 1). Cohorts of HCC-developing mice and age-match animals receiving SETIs were assessed for survival (Figure 1B). Animals administered SETIs displayed a significantly lower mortality in comparison with those only subjected to the concurrent administration of the DEN/TAA solutions. Herein, ≥80% of chemically treated mice completed the 7 weeks of treatment. This value represents a 20% higher survival percentage in comparison with the groups not receiving SETIs, regardless of the feeding diet. The sharp losses in body weight gain (∆BW) (Figure 1C) between the initial 10 days suggests severe inflammatory hepatitis as the main cause of liver failure and death. Notably, the administration of SETIs was able to ameliorate the curve at this early stage of treatment and could explain the higher survival percentages quantified. This effect may be interpreted as a preservation of the liver function as it was accompanied of an increased food intake in HFD-fed mice (Figure 1D), while STD-fed mice only showed an upward trend in food intake. None of the mice administered SETIs that completed the study period displayed significant changes in liver masses compared with those receiving the STD or HFD alone (Figure 1E). In addition, of the mice that reached the treatment, SETI-administered mice did not display a significantly higher liver/body weight ratio than their respective counterparts (Figure 1F). Both STDor HFD-fed mice receiving SETIs displayed significantly a higher number of intrahepatic F4/80+ macrophages (Figure 1G), reflecting resistance to HCC development and sustained metabolism. These findings point to monocyte-derived macrophages as key effectors involved in the diet-induced control of HCC development. 3.2. Modulation of Neoplastic Markers Rt-qPCR analyses of hepatic tissue revealed significant differences in the control of the selected markers depending on the diet fed to the groups of treatment (Figure 2). The administration of SETIs downregulated the expression of CD36 to a higher extent in animals fed an HFD (Figure 2A). In animals fed an HFD, a diet-induced significant decrease in the transcripts of FASN was quantified. Thus, the administration of SETIs only caused significant changes in the expression of FASN in the STD-fed group (Figure 2B). The data for SREBP1c and ALOX15 showed similar trends. For both markers, the administration of SETIs seemed to not influence the expression levels in the groups fed an STD, but the transcripts for both markers were significantly decreased in animals receiving SETIs that were fed an HFD. When considering the effects in the expression of PPARγ and NRLP3, the administration of SETIs sharply increased the expression levels for both makers in animals fed an STD, while significant opposite patterns were quantified in HFD-fed mice. Collectively, these data highlight the interference of molecular pathways triggered by SETIs with the lipid management and homeostasis. Data from SREBP1c and PPARγ could suggest a potential increased phagocytic condition in animals administered SETIs, which may interpret the results in a way to obtain better control of the lipid accumulation influencing innate immune activation to ameliorate HCC severity under an HFD. Biomedicines 2021, 9, 1633 7 of 15 Figure 2. Interaction of SETIs with diet-induced lipid accumulation. Transcript levels (mRNA) of lipogenic markers: (A) CD36, fatty acid translocase; (B) FASN, fatty acid synthase; (C) SREBP1c sterol regulatory element-binding protein 1; (D) the coronary risk marker-arachidonate 15-lipoxygenase (ALOX15). Expression levels of the ‘double-edged’ neoplastic markers: (E) PPARγ, Peroxisome proliferator-activated receptor; (F) NRLP3 inflammasome. Results are expressed as mean ± mean standard error (SEM) (n = 4 for panel A and F; n = 6 for panels (B–D)). Untreated controls are represented by the dotted line. 3.3. Modulation of Inflammatory Mediators and Cellular Pattern Recognition Agonists The administration of SETIs led to a significant downregulation of IL-6 protein production (by 20%) in the groups fed an STD (Figure 3A), while this effect occurred to a higher extent (by 33%) in mice fed an HFD. However, an opposite trend was quantified for TNFα (Figure 3B), showing increased mean values of 57% and 34% in STDand HFDfed mice receiving SETIs, respectively. For both IL-6 and TNFα, variations in the same direction despite the diet fed to animals were quantified. Otherwise, contrasting patterns were quantified in the production of IL-17 and SCF depending on the diet (Figure 3C,D). Animals fed an STD receiving SETIs displayed increased levels of IL-17, associated with a decreased production of SCF, while feeding an HFD decreased the concentration of IL17 but increased that of SCF. Altogether, SETI administration favors a cytokine profile in line with better controlled development of HCC. Decreased levels of cytokines mediating immunosuppression (i.e., IL-6), inhibition of autophagy, and promotion of cell migration (i.e., IL-17) together with the development of enriched granulocyte-macrophage colonyforming cells (i.e., SCF) may be interpreted as the experimental data showing immunostimulatory effects, which ameliorate HCC progression. Biomedicines 2021, 9, 1633 8 of 15 Figure 3. Hepatic concentration of immunological mediators in diethylnitrosamine (DEN)/thioacetamide (TAA)-treated mouse livers at 7 weeks, receiving or not receiving serine-type protease inhibitors (SETIs). Tissue cytokine concentration: (A) interleukin 6; (B) interleukin 17; (C) tumor necrosis factor (TNF)-α; (D) stem cell factor (SCF); and phospholipids (E) lysophosphatydilcholine—LPC, (F) phosphatidylinositol—PI, (G) phosphatidylcholine—PC, and (H) phosphatidylethanolamine—PE. Results are expressed as mean ± mean standard error (SEM) (n = 4 for panel (A–D); n = 6 for panels (E–H)). To investigate the effects of such cytokines on the levels of hepatic phospholipids (PLs), the levels of major PLs that play important roles in the development and progression of hepatocellular carcinoma were quantified (Figure 3). The chromatogram obtained for the methanol/ACN extract of DEN/TAA-treated mouse livers at 7 weeks, receiving or not receiving SETIs are shown in Figure 4. Feeding an HFD increased the hepatic content of PLs in animals not receiving SETIs. Moreover, mice administered SETIs, feeding either under an STD or HFD, exhibited downward trends or significant lower levels of the different PLs quantified. All PLs were significantly decreased in animals fed an STD. Otherwise, in animals fed an HFD, a slight negative variation of the LPC and PI concentration was quantified while PC and PE appeared to be significantly reduced. Overall, data demonstrate that PL metabolic pathways are coherently altered when animals are administered SETIs to impair hepatocyte switch to proliferation. Unfortunately, the PL speciation method does not allow us to identify oxidized PLs that could be recognized by soluble and cellular pattern recognition receptors (i.e., ‘Toll-like’ receptors) to modulate innate immunoreactivity. Biomedicines 2021, 9, 1633 9 of 15 Figure 4. Overlaid HPLC-RP-UV chromatograms for the methanol/ACN extract of DEN/TAA-treated mouse livers at 7 weeks, receiving or not receiving SETIs. (A,B) Chromatographic signals for the standard mixture composed of Lysophosphatydilcholine (LPC), Phosphatidylinositol (PI), Phosphatidylcholine (PC), and Phosphatidylethanolamine (PE). (C,D): Chromatographic signals for hepatic extracts from mice fed a standard diet (STD) without receiving SETIs (C) or administered STEIs (D). (E,F) Chromatographic signals for the hepatic extracts from mice fed a high-fat diet (HFD) without receiving SETIs (E) or administered STEIs (F). 3.4. Promotion of Infiltrated Macrophages with Antitumoral Activity Based on the above results, the promotion of hepatic infiltration by the monocytederived macrophage population (ihMθ) and its immunophenotyping in mice was directly examined (Figure 5). The ihMθ population was detected as CD68+CX3CR1+CD74+. Feeding animals with an HFD did not result in the infiltration of CD68+ macrophages into hepatic tissue, a condition linked to HCC development. When animals were administered SETIs, significantly increased proportions of CD68+ macrophages in the hepatic tissue of mice fed an HFD were observed, whereas STD-fed mice only display upward trends (Figure 5A). Given this finding and the association of M1 macrophages (Figure 1F) with antitumoral activity, CX3CR1 and CD74 identification in the infiltrated CD68+ population was performed. In HFD-fed animals, a significant effect directly caused by the diet was quantified. However, for both STDand HFD-fed animals, the administration of SETIs significantly increased the CX3CR1+ population (Figure 5B). When considering CD74, proportions of the CD68+CD74+ population were similar among the two diets (Figure 5C). Only mice fed an STD displayed significant increases in the CD68+CD74+ population. The combination of all these markers allowed us to identify increased proportions of CD68+CX3CR1+CD74+ cells under either STDor HFD-fed conditions (Figure 5D). Changes in the proportion of these populations are shown in the density plots (Figure 5E,F). Collectively, these data highlight that SETI administration is involved in the induction of antitumoral macrophage populations, allowing for a better control of the tumoral microenvironment. Increased levels of clusters of differentiation contributing to macrophage survival in tumor metastasis and their differentiation into pro-inflammatory F4/80+ macrophages in the liver (i.e., CX3CR1+CD74+) associated with a better survival rate allow for the results to be interpreted as supporting myeloid-derived antitumoral responses. All of these changes were associated with increased values for fluorescent staining of TLR4.