Abstract

   FcgRIIB dysfunction is commonly found in patients with lupus,
   especially in Asia. LPS-tolerance is prominent in FcgRIIB–/– lupus
   mice. LPS-tolerant macrophages demonstrate cell energy depletion, which
   might affect lipid metabolism. Therefore, to explore lipid metabolism,
   LPS-tolerance was induced twice by LPS administration in macrophages
   and in mice. LPS-tolerant FcgRIIB–/– macrophages demonstrated lesser
   mitochondrial DNA (mtDNA), more severe ATP depletion, lower cytokine
   production, and higher lipid accumulation (oil red O staining) compared
   to LPS-tolerant WT cells. Mass-spectrometry-based lipidomic analysis
   demonstrated a higher abundance of phosphatidylethanolamine (PE)
   phospholipid in LPS-tolerant FcgRIIB–/– macrophages than WT cells. This
   was at least in part due to the lower expression of
   phosphatidylethanolamine N-methyltransferase (pemt), an enzyme that
   converts PE to phosphatidylcholine (PC). Aminoimidazole-4-carboxamide
   ribonucleotide (AICAR), a pemt inhibitor, worsens LPS-tolerance in WT
   macrophages and supports the impact of pemt upon LPS-tolerant
   FcgRIIB–/– macrophages. Additionally, phosphorylated AMP-activated
   protein kinase (AMPK-p), a molecule for ATP-restoration associated with
   pemt, and phosphorylated acetyl CoA carboxylase, a downstream signaling
   of AMPK-p, were higher in LPS-tolerant FcgRIIB–/– macrophages than WT.
   Furthermore, Compound C, an AMPK inhibitor, attenuated LPS-tolerance in
   both FcgRIIB–/– macrophages and mice. Taken together, the intense
   decrease in cytokine production after the second LPS stimulation
   (LPS-tolerance) in FcgRIIB–/– macrophages was possibly due to the
   impact of an immense cytokine synthesis after the first dose of LPS.
   This includes using up PEMT, an enzyme of phospholipid synthesis during
   cytokine production, and AMPK-p induction in response to profound
   ATP-depletion. Therefore, the manipulation of the AMPK/PEMT axis
   provides a novel therapeutic candidate for the treatment of severe
   LPS-tolerance in lupus.

   Keywords: endotoxin tolerance, Fc gamma receptor IIb, macrophage,
   lupus, AMPK, phosphatidylethanolamine

Introduction

   One cause of systemic lupus erythematosus (SLE) is the functional
   defect in Fc gamma receptor IIB (FcgRIIB). This is the only inhibitory
   receptor among members of the FcgR family, especially in Asian
   population ([33]1–[34]3). In a mouse model of lupus, FcgRIIB–/– mice
   exhibited effective microbial control against several micro-organisms
   due to enhanced immune responses from inhibitory signaling loss ([35]2,
   [36]4, [37]5). However, immune responses in these mice showed an
   extremely exhausted phenotype after repeated stimulation, as
   demonstrated by the intense reduction of cytokine production after
   repeated LPS stimulation, often referred to as “LPS-tolerance” ([38]6,
   [39]7). Inadequate cytokine production due to prominent LPS-tolerance
   in FcgRIIB–/– lupus mice enhances sepsis susceptibility ([40]6, [41]7).
   This is similar to a secondary infection after immune exhaustion or
   immune paralysis ([42]8–[43]10). As such, LPS-tolerance might be
   responsible for the increased infection susceptibility in lupus
   patients ([44]11, [45]12).

   Interestingly, it has been reported that LPS-tolerance is possible in
   active lupus because of spontaneous endotoxemia both in patients and in
   mice ([46]13, [47]14). This could possibly lead to persistent LPS
   stimulation and LPS-tolerance ([48]15–[49]18). While LPS-tolerance
   protects the host from lethal doses of LPS by dampening cytokine
   responses, cytokine levels in LPS-tolerance are too low for organism
   control ([50]6). Although TLR4 signaling, microRNA, epigenetic
   alteration ([51]16), and cellular metabolism ([52]18) have been
   proposed as the mechanisms of LPS-tolerance, evidence is still
   inconclusive, and studies of LPS-tolerance in lupus are still lacking.
   Due to the hyper-responsiveness to LPS stimulation in FcgRIIB–/–
   macrophages ([53]6, [54]7), prominent LPS-tolerance of FcgRIIB–/– cells
   might result from profound energy insufficiency ([55]18–[56]20) or
   post-translational modification ([57]7, [58]21). Fatty acids are the
   source of β-oxidation, which is a catabolic process that converts fatty
   acids into acetyl-CoA for Krebs cycle in mitochondria of eukaryotic
   cells ([59]22). Therefore, further lipid exploration in LPS-tolerant
   macrophages is interesting because: (1) β-oxidation is important for
   mitochondria ([60]22); (2) intracellular lipids are responsible for
   diverse cell functions, such as phagocytosis, cytokine production, and
   mitochondrial function ([61]23, [62]24); and (3) increased lipid
   accumulation in activated macrophages has been documented ([63]25). The
   comparison between bone marrow-derived macrophages from FcgRIIB–/– and
   WT with either single or sequential LPS stimulation has led to the
   identification of lipid metabolism as one of the pathways that
   regulates LPS-tolerance and depresses cytokine production in
   LPS-tolerant FcgRIIB–/– macrophages.

Materials and Methods

Animal and Endotoxin-Tolerance Mouse Model

   Animal study protocols were approved by the Faculty of Medicine,
   Chulalongkorn University, following the National Institutes of Health
   (NIH) criteria. FcgRIIB–/– mice (on C57BL/6 background) were provided
   by Dr. Sylvia M. Bolland (NIAID, NIH, Maryland, USA). Other mice were
   purchased from the National Laboratory Animal Center, Nakornpathom,
   Thailand. Eight-week-old female C57BL/6J mice were used in all
   experiments. The endotoxin-tolerance model was performed by
   intra-peritoneal administration of endotoxin (LPS) from Escherichia
   coli 026:B6 (Sigma-Aldrich, St. Louis, MO, USA) in two separate doses
   at 0.8 and 4 mg/kg with 5-day rest between doses ([64]6). Then, blood
   was collected through tail vein nicking at specific time-points after
   the second dose of LPS to measure serum cytokines (TNF-α, IL-6, and
   IL-10) by ELISA (Bioplex, Bio-RAD, CA, USA). In addition, Compound C,
   an inhibitor against AMP activated protein kinase (AMPK), was tested in
   mice. Accordingly, 1 mg/kg of Compound C (Dorsomorphin, Sigma-Aldrich)
   was intra-peritoneally administered following a previous publication
   ([65]26) together with the second dose of LPS in the mouse model.

Bone Marrow-Derived Macrophages, Endotoxin Stimulation Protocol, and
Manipulation

   Activation of bone marrow (BM)-derived macrophages from progenitor
   cells in mice femur with L929-conditioned media was supported by flow
   cytometry by anti-F4/80 and anti-CD11c antibody (BioLegend, San Diego,
   CA, USA) before use ([66]6, [67]7). Then, endotoxin (LPS) Escherichia
   coli 026:B6 (Sigma-Aldrich) at 100 ng/ml in 100 μl/well was used to
   activate macrophages (1 × 10^5 cells/well) with two protocols,
   including single incubation and 2-sequential LPS stimulation ([68]6,
   [69]7). For single LPS stimulation (N/100), culture media without
   endotoxin was used for 24h then washed with phosphate buffer solution
   (PBS) and filled with LPS. For sequential LPS stimulations (100/100),
   LPS was incubated for 24 h, washed, and refilled with the same dose of
   LPS. In the control group (N/N), culture media without endotoxin was
   used before and after washing. Culture supernatant was collected at
   indicated time-points after the second incubation and stored at −80°C
   until cytokine analysis by ELISA (Thermo-Fisher Scientific, Waltham,
   MS, USA). In addition, aminoimidazole-4-carboxamide ribonucleotide
   (AICAR), an inhibitor of phosphatidylethanolamine N-methyltransferase
   (pemt), and Compound C, an AMPK inhibitor, were used to test the impact
   on LPS-tolerance. AICAR (50, 100, or 200 μM) or Compound C (5 μM)
   (Sigma-Aldrich) was incubated in macrophages together with the second
   dose of LPS in sequential LPS protocol (100/100) before supernatant
   cytokine determination.

Macrophage Phagocytosis

   Macrophage phagocytosis was measured by incubation of zymosan
   conjugated with 40 kDa fluorescein isothiocyanate dextran
   (FITC-dextran) (Sigma-Aldrich) at 200 μg/ml in 1 x 10^5 cells/ well for
   1 h at 37°C in 5% CO2 following a previous protocol ([70]27). Then, the
   extracellular fluorescence was washed out by PBS and quenched by Trypan
   blue solution. After that, residual adherent macrophages were fixed
   with 4% paraformaldehyde and stained with Hoechst 33342 nuclear stain
   (Molecular Probe, Eugene, OR, USA). Cells were explored by an Olympus
   IX81 inverted fluorescence microscope. The ratio of fluorescence
   intensity of FITC-dextran, normalized by the number of nuclear staining
   with a Varioskan Flash microplate reader, was used to represent
   phagocytosis activity.

Mitochondria Staining and Extracellular Flux Analysis

   LPS-tolerance might be associated with cell-energy adaptation.
   Therefore, several parameters of mitochondria were explored. As such,
   200 nM of Mitotracker Red CMxROS (Molecular probe) was added to each
   well and incubated at 37°C for 15 min before removal. Then, cells were
   fixed with cold methanol at −20°C for 15 min, washed twice with PBS,
   and photographed by an IX81 inverted microscope (Olympus, Tokyo,
   Japan). Energy metabolism profiles with estimation of glycolysis were
   performed and assessments of mitochondrial oxidative phosphorylation
   with extracellular acidification rate (ECAR) and oxygen consumption
   rate (OCR) were carried out by Seahorse XFp Analyzers (Agilent, Santa
   Clara, CA, USA) on macrophages at 1 × 10^5 cells/ well by Seahorse Wave
   2.6 software as previously described ([71]21).

Quantitative Real Time PCR (qRT-PCR) for Mitochondrial Genome (mtDNA),
Cytokines, and Lipid Metabolism Enzymes

   For mitochondrial DNA (mtDNA) detection, total DNA was extracted by
   FavorPrep™ Tissue Genomic DNA Extraction assay (Favorgen, Ping-Tung,
   Taiwan) before analysis by real time RT-PCR with the following
   sequences encoded for mtDNA (mmito-1); forward:
   5′-CGTACACCCTCTAACCTAGAGAAGG-3′, reverse:
   5′-GGTTTTAAGTCTTACGCAATTTCC-3′; compared to the following house-keeping
   sequences of β2 microglobulin (β2M); and forward:
   5′-GGACAGTGGGTAGGGAACTG-3′, reverse: 5′-GGACAGTGGGTAGGGAACTG-3′
   ([72]28). Then, mtDNA relative to β2M was analyzed by the comparative
   threshold cycle (ΔCt) method.

   For cytokines and enzymes in lipid metabolism, total RNA was extracted
   by an RNeasy mini kit (Qiagen, Albertslund, Denmark). Then, RNA (200
   ng) was converted into cDNA in 20 μl of reaction mix by RevertAid First
   Strand cDNA synthesis Kit (ReproTech, Oldwick, NJ, USA). Primers used
   for these experiments are demonstrated in [73]Table 1. Gene expression
   relative to β2M were analyzed by 2−ΔΔCT method, and fold change between
   the interested conditions and untreated WT macrophage control (N/N)
   were demonstrated. Measurements of transcript levels were performed
   with mastermix 1xKAPA fast SYBR Green (Kapa Biosystems, Wilmington, MA,
   USA). Real time RT-PCR was performed by a QuantStudio® 6 Real-Time PCR
   system (Applied Biosystems, Life Technology Corporation, CA, USA) with
   a final volume reaction of 10 μL containing 0.3 μmol/L of each forward
   and reverse primer. Mastermix 1xKAPA fast SYBR Green (Kapa Biosystems,
   Wilmington, MA, USA) and 2 μL of DNA template were used.

Table 1.

   A list of primers.
   Primers Forward Reverse
   Tumor necrosis factor α (TNF-α) 5′-CCTCACACTCAGATCATCTTCTC-3′
   5′-AGATCCATGCCG TTGGCCAG-3′
   Interleukin-10 (IL-10) 5′-GCTCTTACTGACTGGCATGAG-3′
   5′-CGCAGCTCTAGGAGCATGTG-3′
   Phosphatidylethanolamine N-methyltransferase (pemt)
   5′-TGGCTGCTGGGTTACATGG-3′ 5′-GCTTCCGAGTTCTCTGCTCC-3′
   Choline/ethanolamine phosphotransferase (cept1)
   5′-GCTCACTCTAATCATCACTA-3′ 5′-CCTGTTGTCCTTAATATGTTC-3′
   Ethanolamine kinase (ek2) 5′-AGCATCCTCTTCCACTTCTC-3′
   5′-TTCCGCCATTCAGTTCCA-3′
   Phosphatidylserine decarboxylase (psd) 5′-TGAGGACAATGACTAATGATG-3′ 5′-
   ACCAGACAAGCCAGTAAT-3′
   Phosphocholine cytidylyltransferase (pct1) 5′-CTTCTATCAGATTGACAGT-3′
   5′-CTAATTCCTTGGCTTCTT-3′
   β2 microglobulin (β2M) 5′-TTCTGGTGCTTGTCTCACTGA-3′
   5′-CAGTATGTTCGGCTTCCCATTC-3′
   [74]Open in a new tab

Reactive Oxygen Species (ROS), Adenosine Triphosphate (ATP) Measurement, and
Cell Viability Test

   Cellular total ROS production was determined by fluorescent dye,
   Dihydroethidium (DHE), according to the manufacturer's protocol.
   Briefly, 20 μM of DHE (Sigma-Aldrich) was incubated for 20 min at 37°C
   before DHE measurement at the indicated time-points. Fluorescence
   readings were analyzed at 520 nm by a Varioskan Flash microplate reader
   as presented by fluorescence arbitrary unit (a.u.). Cellular ATP
   content was identified by incubation with the substrate from
   Luminescent ATP Detection Assay (Abcam, San Francisco, CA, USA) for 15
   min in room temperature before analysis with a Varioskan Flash
   microplate reader (Thermo-Fisher Scientific) following the
   manufacturer's instruction. In addition, cell viability was analyzed by
   tetrazolium dye 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
   (MTT) solution (Thermo Fisher Scientific) according to the
   manufacturer's protocol. In short, macrophages at 1 × 10^5 cells/well
   were incubated with 0.5 mg/mL of MTT solution for 2 h at 37°C in the
   dark. Then, MTT was removed and diluted with Dimethyl sulfoxide (DMSO;
   Thermo Fisher Scientific) before measurement with a Varioskan Flash
   microplate reader with absorbance at 570 nm.

Total Lipid Staining by Oil Red O Dye and Fluorescent-Labeled Phospholipid
Uptake

   Cells were washed twice with PBS before staining with 0.3% Oil Red O
   solution (Sigma-Aldrich) for 10 min, washed with PBS, fixed with 4%
   paraformaldehyde for 15 min at room temperature, and washed again with
   PBS. Stained cells were evaluated under a microscope with 10 random
   fields from each well. Intensity was evaluated by ImageJ (NIH,
   Bethesda, MD, USA). Although cellular lipid accumulation might be due
   to increased lipid synthesis or enhanced uptake, and only lipid uptake
   was tested due to technical limitations of lipid synthesis ([75]29,
   [76]30). Fluorescent phospholipid analogs (Avanti Polar Lipids, Inc.,
   AL, USA), including lipid analogs
   1-acyl-2-[12-(7-nitro-2,1,3-benzoxadiazol-4-yl)
   amino]dodecanoyl]-sn-glycero-3- phosphocholine (NBD-PC) and
   1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine
   B sulfonyl) (Rhodamine liss PE), were prepared in 100% ethanol for the
   uptake assay following a published protocol ([77]31). Briefly, 20 μM of
   phospholipid analogs were added to macrophages in a 96-well plate. At
   indicated time-points, cells were washed twice, and PBS (50 μl) was
   added to each well. Fluorescence of NBD-PC and Rhodamine-liss PE taken
   up by cells was measured with Varioskan Flash microplate reader
   (Thermo-Fisher Scientific). Phospholipid uptake was calculated by ratio
   between intensity of phospholipid reporter and number of nuclei as
   stained by Hoechst 33342 (Molecular Probe).

Lipid Extraction and LC-MS Data Analysis

   A metabolomics protocol for sample preparation for adherent cells was
   followed ([78]32). In short, culture media of 2 × 10^6 cells/ well of
   macrophages were removed and placed on dry ice before 4 ml of 80%
   (vol/vol) methanol (cooled to −80°C) was added. Plates were then
   incubated in −80°C for 20 min before scraping to separate the cells.
   Samples were transferred to 15 ml conical tube and centrifuged at
   14,000 g for 10 min at 4°C before removal of the metabolite-containing
   supernatant to a new conical tube on dry ice. Subsequently, lipid
   components in the pellet were dissolved again with 80% (vol/vol)
   methanol according to previously mentioned procedures. The
   metabolite-containing supernatant from the pellet was combined with the
   previous supernatant before being concentrated with the total lipids
   from cell lysate by speed vacuum at −56°C for 16h to evaporate MeOH and
   then stored at −80°C before further analysis. Total lipids were
   reconstituted in 100 μl of 50% MeOH in water, MeOH:H2O 1:1 (v/v), and
   analyzed by an untargeted approach based on liquid chromatography
   coupled with an electrospray-ionization LC ESI-MS using micrOTOF-Q II
   (Bruker Daltonics, USA) with a chromatography system following the
   previous condition ([79]33, [80]34). In short, LC/MS/MS Q-TOF with LC
   separation on a C18 column (Agilent, Santa Clara, CA, USA) and mobile
   phases consisting of ultrapure water and acetonitrile containing 0.1%
   (v/v) formic acid were used. Data analysis was performed by Profile
   analysis software (Bruker Daltonics, USA) for LC/MS data. The
   difference between data sets was determined by using unpaired
   two-tailed Student's t-tests. The Lipidomics Gateway
   ([81]http://www.lipidmaps.org) was used to identify lipids based on
   major fragment ions of the MS spectrum after manual monoisotope
   selection. LC/MS data was subjected to Metaboanalyse 3.0
   ([82]https://www.metaboanalyst.ca) for principal cluster analysis
   (PCA), Volcano plots, and enrichment pathway analysis. The enrichment
   pathway analysis was performed as a Metabolite Set Enrichment Analysis
   by Metaboanalyse 3.0. This method was used to identify biologically
   meaningful patterns that were significantly enriched in quantitative
   metabolomic data. The intensity between data sets was compared using
   unpaired two-tailed Student's t-tests with profile analysis software.
   Only unknown metabolites with p < 0.01 were considered for further
   analysis. Out of 1,765 lipids, there were 187 selected unknown
   metabolites that were then identified by lipidomic map database with
   search parameters including Ion adducts: “M+H”, “M+H-H2O”, “M+Na”,
   “M+NH4”, “M+K”, and Mass Tolerance (m/z): +/– 0.05.

Western Blot

   Western blot analysis was used to explore the abundance of AMPK and
   phosphorylated-AMPK (AMPK-p) and phosphorylated-acetyl-CoA carboxylase
   (ACC-p), a downstream signaling of AMPK-p, in LPS-tolerant macrophages
   following the standardized procedure ([83]7, [84]21). In brief,
   stimulated macrophages were pelleted, washed with PBS, and lysed in
   RIPA lysis buffer with a protease/phosphatase inhibitor
   (Thermo-Scientific, Rockford, IL, USA). The samples were homogenized
   and protein quantification was performed by Bicinchoninic acid assay
   (BCA) (Pierce BCA Protein Assay, Thermo Scientific) and then separated
   in 10% sodium dodecyl sulfate (SDS) polyacrylamide gel before
   transferring into a nitrocellulose membrane. Subsequently, the
   preparation was incubated with primary antibodies for AMPK, AMPK-p, and
   ACC-p (Abcam, Cambridge, MA, USA), probed with the proper anti-IgG
   horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz
   Biotechnology), and detected by an enhanced chemiluminescence rapid
   step chemiluminescence detection system (Thermo-Scientific). Rabbit
   monoclonal antibody of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
   (Abcam) was used as a housekeeping control.

Statistical Analysis

   All statistical analyses were performed with GraphPad prism 5.0
   (GraphPad Software, Inc., San Diego, CA). In vitro data were based on
   triplicate independent experiments and represented by mean ± standard
   error (SE). A p value < 0.05 was considered statistically significant.
   Student's t-test or one-way analysis of variance (ANOVA) with Tukey's
   comparison test was used for the analysis of experiments with two and
   more than two groups, respectively.

Results

Characteristics of FcgRIIB–/– Macrophages After Single or Sequential LPS
Stimulation

   Both single (N/100) and sequential (100/100) LPS stimulation induced
   TNF-α and IL-6 ([85]Figures 1A,B) in WT and in FcgRIIB–/– macrophages.
   However, cytokine levels in FcgRIIB–/– cells with single LPS
   stimulation were significantly higher than those in WT macrophages. In
   contrast, cytokine levels in FcgRIIB–/– cells with sequential LPS
   stimulation (LPS-tolerance) were lower than WT cells ([86]Figures
   1A–C). Higher responses against single LPS stimulation and more
   depressed cytokine production during LPS-tolerance of FcgRIIB–/– cells
   were also demonstrated by the difference in cytokine levels between
   single LPS and control (N/100-N/N) and between sequential LPS vs.
   single LPS [(100/100)–(N/100)] ([87]Figures 1D–F). In parallel, gene
   expression of a pro-inflammatory cytokine (TNF-α) and anti-inflammatory
   cytokine (IL-10) was higher in single LPS-stimulated FcgRIIB–/–
   macrophages than WT cells ([88]Figures 1G,H). In LPS-tolerant (100/100)
   WT macrophages, TNF-α expression, but not IL-10 expression, was lower
   than single LPS stimulation. The expression of both genes in
   LPS-tolerant FcgRIIB–/– cells was lower than single LPS activation
   ([89]Figures 1G,H). These data support that the inhibitory signaling
   loss of FcgRIIB–/– macrophages induces hyper-responsiveness after
   single LPS stimulation and is followed by intense unresponsiveness to
   the second dose of LPS (LPS-tolerance) ([90]6). In addition,
   LPS-tolerance was also accompanied by enhanced phagocytosis activity in
   both WT and FcgRIIB–/– macrophages. There was higher activity in
   FcgRIIB–/– cells ([91]Figures 1I,J) at 24 h after the second LPS
   stimulation, which supports previous findings ([92]35).

Figure 1.

   [93]Figure 1
   [94]Open in a new tab

   Characteristics of wild-type (FcgRIIB+/+) macrophages and FcgRIIB–/–
   cells after single (N/100) or sequential (100/100) LPS stimulation vs.
   control (N/N) as determined by time-course of supernatant cytokines
   (TNF-α, IL-6, and IL-10) (A–C), the difference of cytokine level
   between single LPS stimulation vs. baseline [(N/100)–N/N)] and between
   single LPS stimulation vs. LPS tolerance [(100/100)–(N/100)] (D–F) and
   gene expression of TNF-α and IL-10 (G,H) are demonstrated. Phagocytosis
   function of these experimental groups evaluated by the uptake of
   FITC-Zymoxan beads in the representative figures (I) and phagocytosis
   score (J) are also shown (Independent triplicate experiments were
   performed; *p < 0.05; **p < 0.01).

Immense Reduction of Mitochondria Quantity and ATP Production in LPS-Tolerant
FcgRIIB–/– Macrophages

   Profound cytokine defect observed in LPS-tolerant macrophages is
   partially caused by cell-energy depletion and is often referred to as
   “inflammatory bioenergetics responses” ([95]36). Therefore, cell energy
   was explored in FcgRIIB–/– macrophages. When comparing FcgRIIB–/–
   macrophages with WT cells, LPS-tolerance (100/100) reduced
   mitochondrial quantification and, as evaluated by MitoTracker,
   mitochondrial DNA (mtDNA). It also reduced ATP production and total
   cellular reactive oxygen species (ROS) ([96]Figures 2A–E), especially
   at 24 h post-stimulation. In addition, cell energy evaluation by
   extra-cellular flux analysis demonstrated lower respiratory capacity
   ([97]Figure 3A) and a tendency of lower glycolysis capacity ([98]Figure
   3B) in LPS-tolerant FcgRIIB–/– macrophages compared with control
   FcgRIIB–/– cells with the non-different cell viability ([99]Figure 3C).
   Meanwhile, LPS-tolerance in WT macrophages showed a tendency of low
   mitochondria and glycolysis capacity compared with WT control but did
   not reach a significant value ([100]Figures 3A,B). This implies severe
   energy insufficiency in LPS-tolerant FcgRIIB–/– cells and the depletion
   in cell-energy might be associated with low cytokine production
   ([101]Figures 1A–D) in LPS-tolerant FcgRIIB–/– macrophages. The number
   of mitochondria, mtDNA, ATP production, and total cellular ROS in
   single LPS-stimulated FcgRIIB–/– macrophages (N/100) at 24 h
   ([102]Figure 2) and extracellular flux analysis ([103]Figures 3A,B)
   were not in the highest levels among all groups despite demonstrating
   the highest cytokine levels ([104]Figures 1A–C). This may suggest less
   correlation between cell-energy status and cytokine production in
   single LPS stimulation.

Figure 2.

   [105]Figure 2
   [106]Open in a new tab

   The evaluation of cell energy of wild-type (FcgRIIB+/+) macrophages and
   FcgRIIB–/– cells after single (N/100) or sequential (100/100) LPS
   stimulation vs. control (N/N) as determined by (1) mitochondrial
   assessment with Mitotracker Red as presented in representative figures
   from 6 h post-stimulation (A) and quantitative score (B), (2)
   semi-quantitative expression of mitochondrial DNA content (mtDNA) in
   relative to β2 microglobulin (β2M) gene by qRT-PCR (C), and (3)
   luminescence intensity of cellular ATP production (D) are demonstrated.
   Total cellular reactive oxygen species (ROS) as evaluated by
   Dihydroethidium (DHE) assay (E) are also shown (Independent triplicate
   experiments were performed; *p < 0.05).

Figure 3.

   [107]Figure 3
   [108]Open in a new tab

   Extracellular flux analysis pattern of macrophages with LPS-tolerance
   (100/100) or culture media control (N/N) in FcgRIIB–/– and wild-type
   (FcgRIIB+/+) groups as evaluated by oxygen consumption rate of
   mitochondrial stress test for mitochondrial pathway analysis (A) and
   extracellular acidification rate of glucose stress test for glycolysis
   pathway analysis (B) at 6 h after the second dose of LPS or control
   culture media are demonstrated. The evaluation on macrophages of
   wild-type (FcgRIIB+/+) and FcgRIIB–/– cells after the single (N/100) or
   the sequential (100/100) LPS stimulation vs. control (N/N) in the
   viability assay analyzed by tetrazolium dye 3-(4,
   5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) solution (C), the
   number of lipid droplets intensity determined by ImageJ software from
   Oil Red O staining (D), and the representative Oil Red O staining from
   macrophages in each condition (E) are demonstrated (Independent
   triplicate experiments were performed for all figures; FCCP, Carbonyl
   cyanide-4-(trifluoromethoxy)-phenylhydrazone; 2-DG, 2-Deoxy-d-glucose).

Increased Lipid Droplet Accumulation and a Global Shift in
Glycerophospholipids Profile in FcgRIIB–/– Macrophages With LPS-Tolerance

   Severely reduced mitochondria biogenesis and decreased ATP in
   LPS-tolerant FcgRIIB–/– macrophages ([109]Figure 2) may affect lipid
   metabolism. Accordingly, lipid droplet count, as stained by Oil red O
   color in LPS-tolerant FcgRIIB–/– macrophage at 24 h after stimulation,
   was higher than other groups ([110]Figures 3D,E). This is in contrast
   to mitochondrial biogenesis, ATP production ([111]Figures 2B–D), and
   extra cellular flux analysis ([112]Figures 3A,B). Of note, lipid
   droplets were not increased in single LPS-stimulated FcgRIIB–/–
   macrophages ([113]Figures 3D,E). Furthermore, untargeted LC-MS-based
   lipidomic analysis was performed. The schematic diagram of the analysis
   is detailed in [114]Figure 4A. Increased expression of glycerolipid
   (GL), glycerophospholipid (GP), sphingosine (SP), and sterol (ST) in
   LPS-tolerant FcgRIIB–/– macrophages (100/100) compared with
   LPS-tolerant WT cells was visualized by clustering heat map analysis
   ([115]Figure 4B, right side). In parallel, reduced GP and increased SP
   and ST in single LPS stimulated FcgRIIB–/– cells (N/100) were observed
   when compared with WT ([116]Figure 4B, left side). A Venn diagram of
   lipid derivatives from LPS-tolerant FcgRIIB–/– macrophages (100/100)
   vs. single LPS stimulation (N/100) revealed 47 and 49 lipids,
   respectively. There were only eight lipids that shared similarity
   between groups ([117]Figure 4C). This implies different lipid
   metabolism between these groups. A list of lipid derivatives (lipids)
   in single and sequential LPS-stimulated macrophages identified by the
   Lipidomic Gateway database are shown in [118]Tables 2, [119]3. Among
   the significantly different lipid derivatives between groups, 20 and 29
   lipids were up- and downregulated, respectively, in FcgRIIB–/–
   macrophages with single LPS stimulation ([120]Table 2). While 39 and 11
   lipids in FcgRIIB–/– macrophages with LPS-tolerance were up- and down-
   regulated, respectively ([121]Table 3). These alterations mostly occur
   in glycerophospholipids. Raw data of lipidomic analysis in LPS-tolerant
   macrophages is included in [122]Supplemental Data.

Figure 4.

   [123]Figure 4
   [124]Open in a new tab

   Schematic of experimental design for mass-spectrometry (LC-MS) based
   lipidomic analysis from bone marrow derived macrophages (BMDM) of
   FcgRIIB–/– and wild-type (FcgRIIB+/+) with single (N/100) and
   sequential (100/100) LPS stimulation (A) is demonstrated. Lipidomic
   analysis presented by hierarchical clustering heat-map profiling groups
   of lipid derivatives identified as glycerolipid (GL),
   glycerophospholipid (GP), sphingosine (SP), and sterol (ST) between
   wild-type (FcgRIIB+/+) and FcgRIIB–/– macrophages after LPS stimulation
   with the green and red color indicated up and downregulated lipid
   derivatives, respectively, (B) are shown. Venn diagram indicating the
   number of lipid derivatives in FcgRIIB–/– macrophages with N/100 or
   100/100 LPS stimulation (C), principal cluster analysis (PCA) in
   principle component 1 (PC1) identifying variance between individual
   groups from wild-type (WT1-3) vs. FcgRIIB–/– (K1-3) and in principle
   component 2 (PC2) indicating variance between replicate experiments
   (D), Volcano plot of lipid derivatives quantification from sequential
   LPS stimulation (100/100) in wild-type vs. FcgRIIB–/– cells summarizing
   up- and downregulated lipid derivatives based on the adjusted log2 fold
   change (E), and enrichment pathway analysis (F) are also indicated.

Table 2.

   Summary of lipid derivatives based on lipidomic gateway analysis with
   mass spectrometric measurement identification: comparison of lipid
   profiles of FcgRIIB–/– and FcgRIIB+/+ macrophages with single LPS
   stimulation (N/100).
   Intensity (m/z)
   Compound Formula Ion Category FcgRIIB–/– FcgRIIB+/+ p-value[125]^*
   Regulation#
   Halaminol A C14H30NO [M+H]+ Sterolipids 155,650 ± 1,258 218,881 ± 2,045
   0.00006 Up
   C19 Sphingosine-1-phosphate C19H39NO4P [M+H-H2O]+ Sphingolipids 5,413 ±
   231 10,700 ± 108 0.00035 Up
   PI(O-16:0/14:0) C39H76O11P [M+H-H2O]+ Glycerophospholipids 1,362 ± 56
   2,210 ± 54 0.0004 Up
   PI(P-16:0/0:0) C25H49O11PNa [M+Na]+ Glycerophospholipids 8,278 ± 242
   14,536 ± 398 0.00054 Up
   PE(22:0/0:0) C27H57NO7P [M+H]+ Glycerophospholipids 881 ± 41 1,417 ± 40
   0.00071 Up
   PE(19:0/0:0) C24H50NO7P [M+H]+ Glycerophospholipids 16,526 ± 127 29,995
   ± 289 0.00135 Up
   PC(O-16:0/0:0) C24H53NO6P [M+H]+ Glycerophospholipids 1,034,750 ± 9,895
   1,293,654 ± 22,371 0.00261 Up
   PG(18:4(6Z,9Z,12Z,15Z)/13:0) C37H64O9P [M+H-H2O]+ Glycerophospholipids
   1,211 ± 68 3,239 ± 256 0.00613 Up
   PC(0:0/18:1(9E)) C26H52NO7P [M+H]+ Glycerophospholipids 6,502 ± 152
   9,787 ± 380 0.00649 Up
   PC(18:0/0:0) C26H55NO7P [M+H]+ Glycerophospholipids 1,099 ± 47 1,721 ±
   90 0.00869 Up
   PE(20:0/0:0) C25H53NO7P [M+H]+ Glycerophospholipids 20,662 ± 1,014
   29,342 ± 1,380 0.00893 Up
   PC(P-18:0/0:0) C26H55NO6P [M+H]+ Glycerophospholipids 4,206 ± 128 6,898
   ± 218 0.00917 Up
   PC(0:0/18:1(6Z)) C26H52NO7P [M+Na]+ Glycerophospholipids 2,172 ± 235
   4,161 ± 691 0.00965 Up
   PE(18:0/0:0) C23H48NO7PNa [M+Na]+ Glycerophospholipids 62,669 ± 263
   114,196 ± 2,124 0.00995 Up
   1-O-(2-methoxy-hexadecyl)-sn-glycerol C20H42O4Na [M+Na]+ Glycerolipids
   9,065 ± 150 13,224 ± 519 0.01045 Up
   PC(P-16:0/17:1(9Z)) C41H80NO7PK [M+K]+ Glycerophospholipids 69,884 ±
   1,489 78,782 ± 1,328 0.01149 Up
   PC(16:0/0:0) C24H50NO7PNa [M+Na]+ Glycerophospholipids 1,609 ± 91 2,857
   ± 199 0.013 Up
   PC(P-16:0/0:0) C24H51NO6P [M+H]+ Glycerophospholipids 998 ± 9 1,472 ±
   94 0.03591 Up
   Taccalonolide A C36H45O13 [M+H-H2O]+ Sterolipids 13,424 ± 237 15,225 ±
   443 0.03597 Up
   PC(18:1(9E)/2:0) C28H54NO8P [M+H-H2O]+ Glycerophospholipids 962 ± 76
   1,572 ± 155 0.04055 Up
   PA(16:0/14:0) C33H65O8PK [M+K]+ Glycerophospholipids 12,147 ± 125 8,148
   ± 191 0.00017 Down
   2-linoleoyl-sn-glycerol C21H38O4 [M+NH4]+ Glycerolipids 2,689 ± 52 999
   ± 21 0.00021 Down
   PE(16:0/0:0) C21H44NO7PNa [M+Na]+ Glycerophospholipids 5,832 ± 163
   3,124 ± 141 0.00026 Down
   16,17-didehydropregnenolone C21H30O2K [M+K]+ Sterolipids 9,000 ± 168
   5,804 ± 193 0.00026 Down
   3a,17a-Dihydroxy-5b-androstane C19H32O2K [M+K]+ Sterolipids 11,000 ±
   261 4,087 ± 114 0.00028 Down
   PE(0:0/20:5(5Z,8Z,11Z,14Z,17Z)) C25H42NO7P [M+H]+ Glycerophospholipids
   14,400 ± 45 6,040 ± 192 0.00028 Down
   PE(24:6(6Z,9Z,12Z,15Z,18Z,
   21Z)/0:0) C29H49NO7P [M+H]+ Glycerophospholipids 1,566 ± 58 604 ± 39
   0.00035 Down
   MG(18:2(9Z,12Z)/0:0/0:0)[rac] C21H38O4 [M+H-H2O]+ Glycerolipids 9,473 ±
   269 5,786 ± 226 0.00054 Down
   1-O-(2R-hydroxy-pentadecyl)-sn-glycerol C18H38O4Na [M+Na]+
   Glycerolipids 3,051 92 1,802 ± 78 0.00056 Down
   Dinorlithocholic acid C22H40NO3 [M+NH4]+ Sterolipids 66,119 ± 11,380
   19,952 ± 1,954 0.0006 Down
   PE(22:4(7Z,10Z,13Z,16Z)/0:0) C27H49NO7P [M+H]+ Glycerophospholipids
   1,737 ± 65 928 ± 47 0.00084 Down
   PE(20:5(5Z,8Z,11Z,14Z,17Z)/0:0) C25H42NO7PNa [M+Na]+
   Glycerophospholipids 1,819 ± 35 779 ± 12 0.00113 Down
   1alpha,25-dihydroxy-21-nor-20-oxavitam C25H44NO4 [M+NH4]+ Sterolipids
   1,425 ± 344 789 ± 44 0.00149 Down
   Cer(d18:1/2:0) C20H39NO3Na [M+Na]+ Sphingolipids 12,387 ± 456 5,162 ±
   129 0.00224 Down
   PE(18:2(9Z,12Z)/0:0) C23H44NO7P [M+H]+ Glycerophospholipids 9,532 ± 321
   3,359 ± 43 0.00234 Down
   3-ketosphinganine C18H36NO [M+H-H2O]+ Sphingolipids 64,307 ± 423 41,913
   ± 1,468 0.0024 Down
   1alpha,25-dihydroxy-19-nor-22-oxavitam C25H46NO4 [M+NH4]+ Sterolipids
   3,109 ± 55 2,244 ± 53 0.0032 Down
   12-Oxo-5alpha-cholan-24-oic Acid C24H38O3 [M+NH4]+ Sterolipids 10,217 ±
   451 5,319 ± 171 0.00388 Down
   PE(20:4(5Z,8Z,11Z,14Z)/0:0) C25H45NO7P [M+H]+ Glycerophospholipids
   5,535 ± 148 3,689 ± 40 0.00405 Down
   PE(22:5(4Z,7Z,10Z,13Z,16Z)/0:0) C27H46NO7PNa [M+Na]+ Sphingolipids
   32,509 ± 259 25,847 ± 889 0.01209 Down
   POV-PA C24H49NO9P [M+NH4]+ Glycerophospholipids 28,518 ± 833 23,737 ±
   543 0.01242 Down
   MG(0:0/20:4(5Z,8Z,11Z,14Z)/0:0) C23H37O3 [M+H-H2O]+ Glycerolipids 6,099
   ± 230 4,592 ± 96 0.01248 Down
   PS(16:1(9Z)/22:2(13Z,16Z)) C44H80NO10PN [M+Na]+ Glycerophospholipids
   11,108 ± 888 4,814 ± 173 0.01648 Down
   PC(16:0/2:0) C26H51NO7P [M+H-H2O]+ Glycerophospholipids 1,320 ± 72 798
   ± 107 0.02021 Down
   Sphingosine C18H38NO2 [M+H]+ Sphingolipids 1,177 ± 42 962 ± 32 0.02255
   Down
   PC(20:2(11Z,14Z)/0:0) C28H54NO7PNa [M+Na]+ Glycerophospholipids 3,152 ±
   164 2,146 ± 12 0.02515 Down
   Sphinganine C18H38NO [M+H-H2O]+ Sphingolipids 1,581 ± 148 730 ± 43
   0.02521 Down
   1-(2-methoxy-6Z-heptadecenyl)-sn-glyce C23H49NO7P [M+H]+
   Glycerophospholipids 1,014 ± 35 858 ± 21 0.0274 Down
   Sphingosine-1-phosphate C18H37NO4P [M+H-H2O]+ Sphingolipids 1,234 ± 46
   964 ± 39 0.0308 Down
   [126]Open in a new tab
   ^*

   p < 0.05 FcgRIIB–/– vs. FcgRIIB+/+; #, direction of change in
   FcgRIIB–/– vs. FcgRIIB+/+.

Table 3.

   Summary of lipid derivatives based on lipidomic gateway analysis with
   mass spectrometric measurement identification: comparison of lipid
   profile of FcgRIIB–/– and FcgRIIB+/+ macrophages with sequential LPS
   stimulation (100/100).
   Intensity (m/z)
   Compound Formula Ion Category FcgRIIB–/– FcgRIIB+/+ p-value[127]^*
   Regulation#
   PI(18:1(9Z)/0:0) C27H51O12P [M+H]+ Glycerophospholipids 10,111 ± 418
   4,037 ± 356 0.00044 Up
   PE(18:0/0:0) C23H47NO6P [M+H-H2O]+ Glycerophospholipids 11,731 ± 528
   4,683 ± 327 0.00088 Up
   PA(12:0/14:0) C29H61NO8P [M+NH4]+ Glycerophospholipids 4,970 ± 206
   1,769 ± 266 0.0009 Up
   POV-PA C24H49NO9P [M+NH4]+ Glycerophospholipids 67,817 ± 2,890 18,364 ±
   1,193 0.00104 Up
   PE(18:1(11Z)/0:0) C23H47NO7P [M+H]+ Glycerophospholipids 10,818 ± 567
   4,489 ± 396 0.00132 Up
   PC(0:0/18:1(9Z)) C26H53NO7P [M+H]+ Glycerophospholipids 12,620 ± 574
   6,270 ± 565 0.0014 Up
   CPA(18:0) C21H45NO6P [M+NH4]+ Glycerophospholipids 42,165 ± 1,515
   24,175 ± 1,674 0.0014 Up
   PE(16:0/0:0) C21H45NO7P [M+H]+ Glycerophospholipids 27,108 ± 3,887
   6,325 ± 454 0.00164 Up
   PI(20:4(5Z,8Z,11Z,14Z)/0:0) C29H49O12PNa [M+Na]+ Glycerophospholipids
   3,039 ± 158 1,690 ± 124 0.0031 Up
   1-(2-methoxy-6Z-heptadecenyl)-sn-g C23H49NO7P [M+H]+
   Glycerophospholipids 165,910 ± 8,158 65,591 ± 2,816 0.00323 Up
   1-(2-methoxy-6Z-octadecenyl)-sn-gly C24H51NO7P [M+H]+
   Glycerophospholipids 31,731 ± 1,377 18,167 ± 1,601 0.00326 Up
   PE(18:2(9Z,12Z)/0:0) C23H45NO7P [M+H]+ Glycerophospholipids 6,852 ± 419
   2,183 ± 165 0.00345 Up
   PE(0:0/20:3(11Z,14Z,17Z)) C25H47NO7P [M+H]+ Glycerophospholipids 9,856
   ± 490 4,819 ± 221 0.00346 Up
   MG(18:2(9Z,12Z)/0:0/0:0)[rac] C21H42NO4 [M+NH4]+ Glycerolipids 3,843 ±
   168 2,371 ± 94 0.00395 Up
   C17 Sphinganine C17H38NO2 [M+H]+ Sphingolipids 48,051 ± 3,025 6,969 ±
   444 0.00465 Up
   PC(16:1(9Z)/0:0) C24H49NO7P [M+H]+ Glycerophospholipids 3,700 ± 198
   2,262 ± 171 0.00572 Up
   PC(20:1(11Z)/0:0) C28H57NO7P [M+H]+ Glycerophospholipids 3,947 ± 224
   2,396 ± 171 0.00646 Up
   1-(2-methoxy-5Z-hexadecenyl)-sn-gly C22H47NO7P [M+H]+
   Glycerophospholipids 5,112 ± 303 2,600 ± 130 0.00668 Up
   PG(P-18:0/0:0) C24H53NO8P [M+NH4]+ Glycerophospholipids 5,074 ± 383
   2,186 ± 95 0.00849 Up
   PI(P-18:0/0:0) C27H54O11P [M+H]+ Glycerophospholipids 3,420 ± 228 1,394
   ± 49 0.00984 Up
   PG-PG C27H51O12PNa [M+Na]+ Glycerophospholipids 96,567 ± 662 5,593 ±
   656 0.0101 Up
   POV-PG C27H52O11P [M+H]+ Glycerophospholipids 25,827 ± 2,012 12,884 ±
   1,066 0.01041 Up
   1-(2-methoxy-13-methyl-6Z-tetradec C22H45NO9P [M+H]+
   Glycerophospholipids 8,387 ± 771 2,493 ± 180 0.01309 Up
   PC(P-18:0/0:0) C26H55NO6P [M+H]+ Glycerophospholipids 88,434 ± 734
   4,488 ± 754 0.01441 Up
   PI(O-18:0/0:0) C27H56O11P [M+H]+ Glycerophospholipids 2,241 ± 195 1,097
   ± 81 0.01669 Up
   PE(P-18:0/0:0) C23H49NO6P [M+H]+ Glycerophospholipids 12,713 ± 1,050
   7,280 ± 513 0.02018 Up
   PE(0:0/22:5(4Z,7Z,10Z,13Z,16Z)) C27H47NO7P [M+H]+ Glycerophospholipids
   49,295 ± 3,189 31,897 ± 1,223 0.02097 Up
   3-ketosphinganine C18H36NO [M+H-H2O]+ Sphingolipids 25,083 ± 1,925
   13,497 ± 430 0.02217 Up
   PI(O-16:0/0:0) C25H51O11PNa [M+Na]+ Glycerophospholipids 8,319 ± 886
   3,302 ± 164 0.02651 Up
   PC(18:1(11Z)/0:0) C26H52NO7PNa [M+Na]+ Glycerophospholipids 2,725 ± 147
   1,957 ± 173 0.02885 Up
   1-(2-methoxy-nonadecanyl)-sn-glyce C25H53NO6P [M+H-H2O]+
   Glycerophospholipids 1,505 ± 88 1,071 ± 98 0.03033 Up
   Sphinganine-1-phosphocholine C23H55N3O5P [M+NH4]+ Sphingolipids 1,028 ±
   30 899 ± 18 0.03139 Up
   PI(18:0/0:0) C27H54O12P [M+H]+ Glycerophospholipids 30,517 ± 3,132
   15,545 ± 797 0.03438 Up
   PE(22:2(13Z,16Z)/0:0) C27H52NO7PNa [M+Na]+ Glycerophospholipids 5,334 ±
   677 1,894 ± 94 0.03444 Up
   Sphingosine C18H38NO2 [M+H]+ Sphingolipids 24,517 ± 1,557 13,190 ± 247
   0.03854 Up
   Homochenodeoxycholic acid C25H42O4 [M+NH4]+ Sterol lipids 2,662 ± 315
   1,439 ± 167 0.04069 Up
   1-O-(2-methoxy-4Z-hexadecenyl)-sn- C25H53NO7P [M+H]+
   Glycerophospholipids 3,239 ± 227 2,354 ± 184 0.04107 Up
   MG(0:0/20:4(5Z,8Z,11Z,14Z)/0:0) C23H37O3 [M+H-H2O]+ Glycerolipids 6,632
   ± 825 1,951 ± 222 0.04491 Up
   DG(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,1 C41H70NO5 [M+NH4]+ Glycerolipids
   3,015 ± 70 5,661 ± 40 0.00004 Down
   PE(12:0/0:0) C42H67O8PNa [M+Na]+ Glycerophospholipids 1,268 ± 36 2,191
   ± 61 0.00067 Down
   MG(0:0/20:5(5Z,8Z,11Z,14Z,17Z)/0:0) C43H71O5 [M+H]+ Glycerolipids 7,846
   ± 441 14,896 ± 694 0.00205 Down
   16,17-didehydroprogesterone C25H44NO7P [M+H]+ Glycerophospholipids
   13,306 ± 472 22,673 ± 944 0.00327 Down
   ST 25:4;O5;T C18H28O2K [M+K]+ Sterol lipids 30,089 ± 1,980 71,772 ±
   4,231 0.00369 Down
   Estrone C27H41NO7S [M+H]+ Sterol lipids 33,834 ± 2.081 53,084 ± 3,034
   0.00886 Down
   PE(20:4(5Z,8Z,11Z,14Z)/0:0) C23H36O4Na [M+Na]+ Glycerolipids 2,034 ±
   132 2,709 ± 110 0.0183 Down
   DG(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,1 C17H37NO7P [M+H]+
   Glycerophospholipids 1,041 ± 37 1,293 ± 53 0.0219 Down
   PA(17:2(9Z,12Z)/22:6(4Z,7Z,10Z,13Z,1 C21H32NO2 [M+NH4]+ Sterol lipids
   4,140 ± 380 6,764 ± 595 0.02732 Down
   19-norandrosterone C27H49NO7P [M+H]+ Glycerophospholipids 13,207 ± 302
   15,902 ± 649 0.03638 Down
   PE(22:4(7Z,10Z,13Z,16Z)/0:0) C18H22O2K [M+K]+ Sterol lipids 5,097 ± 185
   10,229 ± 1,200 0.04762 Down
   [128]Open in a new tab
   ^*

   p < 0.05 FcgRIIB–/– vs. FcgRIIB+/+; #, direction of change in
   FcgRIIB–/– vs. FcgRIIB+/+.

   Lipids in LPS-tolerant macrophages in FcgRIIB–/– and WT were further
   analyzed as a result of mitochondrial defect where energy depletion and
   increased cellular lipid content ([129]Figures 2, [130]3) were more
   dominant in LPS-tolerant macrophages compared with single
   LPS-stimulated cells. Firstly, principle component analysis (PCA) of
   lipid derivatives showed a clear separation between LPS-tolerant
   FcgRIIB–/– macrophages (100/100) compared with WT. PCA analysis also
   identified a 39% principle component 1 (PC1) measurement of variance,
   which suggests the variability of lipids in WT and FcgRIIB–/–
   macrophages ([131]Figure 4D). Secondly, a Volcano plot demonstrated the
   distribution of up- and downregulated lipids with a cut off p-value at
   0.05 and log[2]FC at 1.5 folds difference between LPS-tolerant
   FcgRIIB–/– macrophages vs. WT cells ([132]Figure 4E). Thirdly, among
   the upregulated lipid derivatives of FcgRIIB–/– vs. WT cells,
   biological significance from pathway enrichment analysis identified
   that phospholipid biosynthesis was the most significantly enriched
   lipid ([133]Figure 4F, red), followed by the derivatives in arachidonic
   acid metabolism ([134]Figure 4F, yellow). Fourthly, the lists of
   upregulated lipids were further explored and analyzed by map-gateway
   analysis. The top 20 abundant lipids among 39 upregulated lipid
   derivatives of LPS-tolerant FcgRIIB–/– macrophages over WT ([135]Table
   3 and [136]Supplemental Data) with p value differences of less than
   0.01, and ratios (FcgRIIB–/– divided by FcgRIIB+/+) of more than 1.5
   fold in FcgRIIB–/– macrophages over WT are demonstrated in [137]Figure
   5. Most of these lipids were in glycerophospholipid pathways. Although
   the abundance of 2-methoxy-6Z-heptadecenyl-sn-glycero-3-PE of
   LPS-tolerant FcgRIIB–/– macrophages was the highest (top column of
   [138]Figure 5), the increase was only 2.5 folds higher than WT. In
   addition, the second and third most abundant derivatives were C17
   Sphinganine at 6.9 folds and POV-PA at 3.7 folds higher than WT,
   respectively. However, C17 Sphinganine, as a sphingolipid derivative,
   and POV-PA, as an oxidized phospholipid, were not enriched in
   phospholipid biosynthesis or the arachidonic acid metabolism pathway
   that was derived from the pathway analysis ([139]Figure 4F). This
   implies biologically less meaningful patterns. PE (16:0/0:0) (PE) was
   the fourth most abundant lipid at 4.3 folds higher in FcgRIIB–/–
   macrophage compared with WT and became the focus for further study
   ([140]Figure 5, red dotted line). Indeed, phosphatidylcholines (PC) and
   phosphatidylyethanolamine (PE) are major lipid derivatives in the GP
   group ([141]37, [142]38). PE is also considered as one of the major
   phospholipid components of eukaryotic cell membrane ([143]39). Enriched
   PE component in LPS-tolerant FcgRIIB–/– macrophages over WT was
   possibly due to increased lipid uptake or enhanced lipid synthesis.

Figure 5.

   [144]Figure 5
   [145]Open in a new tab

   Top first 20 high intensity lipid derivatives in macrophages after
   LPS-tolerance (100/100) from FcgRIIB–/– vs. wild-type (FcgRIIB+/+)
   identified by lipids map gateway with a cut off p-value < 0.01 together
   with the difference of values with more than 1.5 fold-change is
   demonstrated (Independent triplicate experiments were performed to
   prepare cells for lipidomic analysis).

Involvement of Phosphatidylethinolamine Methyltransferease (PEMT) and AMPK in
LPS-Tolerant FcgRIIB–/– Macrophages

   Technical limitations in the evaluation of intracellular lipid
   synthesis ([146]29, [147]30) resulted in performing only the lipid
   uptake assay. Accordingly, uptake of PE analog (Rhodamine-liss PE) was
   higher in LPS-tolerant FcgRIIB–/– macrophages when compared with WT at
   24h of incubation, while the uptake of phosphatidylcholine (PC) analog
   (NBD-PC) was similar between WT and FcgRIIB–/– macrophages ([148]Figure
   6). Although mechanisms of enhanced-uptake of Rhodamine-liss PE in
   LPS-tolerant FcgRIIB–/– macrophages were not clear, these results
   suggested an association between PE and LPS-tolerance in macrophages of
   lupus mice.

Figure 6.

   [149]Figure 6
   [150]Open in a new tab

   Uptake of lipid analogs Rhodamine-liss phosphatidylethanolamine
   (Rhodamine-liss PE) (A,B) or fluorescent phosphatidylcholine (NBD-PC)
   (C,D) normalized by Hoechst 33342 nucleus staining, in FcgRIIB–/– and
   wild-type (FcgRIIB+/+) macrophages in control (N/N) and LPS-tolerance
   (100/100) with representative immunofluorescence images (E) are
   demonstrated (images of NBD-PC uptake are not shown). (Independent
   triplicate experiments were performed).

   Since several enzymes are needed for PE and PC lipogenesis ([151]Figure
   7, diagram), the expression of these enzymes between LPS-tolerant
   FcgRIIB–/– macrophages vs. WT cells were examined. Interestingly, there
   was high expression of phosphoethanolamine cytidylyltransferase (et),
   the encoding enzyme responsible for PE synthesis, at the baseline
   condition (N/N) of FcgRIIB–/– macrophages compared with WT ([152]Figure
   7A). LPS-tolerant FcgRIIB–/– macrophages demonstrated a decreased
   expression of phosphatidylethanolamine N-Methyltransferase (pemt)
   ([153]Figure 7A), which is the encoding enzyme responsible for the
   conversion of PE into PC. Indeed, the reduced pemt expression
   ([154]Figure 7A), and increased PE uptake ([155]Figure 6) in
   LPS-tolerant FcgRIIB–/– macrophages is consistent with the higher
   intracellular PE ([156]Figure 5) in LPS-tolerant FcgRIIB–/– macrophages
   compared with WT.

Figure 7.

   [157]Figure 7
   [158]Open in a new tab

   Schematic diagram of important enzymes including ethanolamine kinase
   (ek), CTP: phosphoethanolamine cytidylyltransferase (et),
   choline/ethanolamine phosphotransferase (cept), phosphatidylserine
   decarboxylase (psd), phosphatidylethanolamine N-methyltransferase
   (pemt), and CTP-phosphocholine
   cytidylyltransferase/choline/ethanolamine phosphotransferase (cpt/cept)
   in lipogenesis pathway with phosphatidylserine (PS),
   phosphatidylyethanolamine (PE), and phosphatidylcholine (PC) (A, upper
   part) and gene expression from macrophages from FcgRIIB–/– and
   wild-type (FcgRIIB+/+) in control (N/N) or LPS-tolerance (100/100) (A,
   lower part) are demonstrated (DAG, diacyl glycerol). Protein abundance
   of macrophages in control (N/N) or LPS-tolerance (100/100) as
   determined by AMP-activated protein kinase (AMPK) in relative to
   Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (B), phosphorylated
   AMPK (AMPK-p) in relative to GAPDH (C), AMPK-p abundance in relative to
   AMPK (D), and phosphorylated acetyl-CoA carboxylase (ACC-p) (E) of
   LPS-tolerant macrophages with representative pictures of Western blot
   analysis are also demonstrated (Independent triplicate experiments were
   performed).

   Activation status of AMPK in LPS-tolerance was also explored because of
   the well-known association between PEMT pathway of lipid metabolism and
   the sensor of cellular energy status, AMP-activated protein kinase
   (AMPK) ([159]40, [160]41). Accordingly, higher protein burdens of AMPK,
   phosphorylated AMPK (AMPK-p), and phosphorylated acetyl-CoA carboxylase
   (ACC-p, a downstream signaling of AMPK) in LPS-tolerant FcgRIIB–/–
   macrophages compared with WT cells was observed ([161]Figures 7B–E). Of
   note, burdens of AMPK but not AMPK-p, was also increased in
   LPS-tolerant WT macrophages ([162]Figure 7B). Hence, prominent pemt
   reduction and increased AMPK-p in response to energy depletion of
   LPS-tolerant FcgRIIB–/– macrophages compared with WT might be
   responsible for the severe unresponsiveness against the second dose of
   LPS in LPS-tolerant FcgRIIB–/– cells. To address the role of PEMT,
   5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an
   inhibitor of pemt (in low dose; <500 μM) with the AMPK enhancer
   property (in high dose) ([163]42), was incubated in LPS-tolerant WT
   macrophages to see if AICAR could enhance severity of LPS exhaustion as
   seen in LPS-tolerant FcgRIIB–/– macrophages. As expected, AICAR
   worsened LPS-tolerance in WT macrophages in a dose-dependent manner as
   demonstrated by cytokine reduction with increased cellular lipid
   droplets, similar to LPS-tolerant FcgRIIB–/– cells ([164]Figures
   8A–E,H). However, AICAR did not alter cell energy as determined by
   mtDNA and ATP production ([165]Figures 8F,G). This is possibly due to
   the selected doses were not high enough to induce AMPK.

Figure 8.

   [166]Figure 8
   [167]Open in a new tab

   Schematic diagram (A) of the experiment with LPS-tolerance induction
   (LPS/LPS) in wild-type (FcgRIIB+/+) bone marrow-derived macrophages
   (BMDMs) with aminoimidazole-4-carboxamide ribonucleotide (AICAR; a pemt
   inhibitor) compared with LPS-tolerant FcgRIIB–/– macrophages as
   determined by supernatant cytokines (B–D), number of lipid droplets
   intensity from Oil Red O staining by ImageJ software evaluation (E),
   semi-quantitative expression of mitochondrial DNA content (mtDNA)
   relative to β2 microglobulin (β2M) gene by qRT-PCR (F), luminescence
   intensity of cellular ATP production (G), and the representative Oil
   Red O staining (H) from these cells after the stimulations are
   demonstrated. Independent triplicate experiments were performed.

   The known association between PEMT and AMPK ([168]40, [169]41) together
   with prominent AMPK-p in LPS-tolerant FcgRIIB–/– macrophages of lupus
   mice ([170]Figure 7C) makes AMPK an interesting target to harness
   LPS-tolerance in lupus. To test the role of AMPK in LPS-tolerance, more
   experiments were performed using Compound C, a specific AMPK inhibitor.
   As such, Compound C was given during LPS challenge and increased
   supernatant TNF-α in LPS-tolerant WT macrophages. It enhanced all
   cytokines in LPS-tolerant FcgRIIB–/– macrophages ([171]Figures 9A–C).
   Compound C reduced mtDNA and cellular ATP in LPS-tolerant WT
   macrophages but not in FcgRIIB–/– cells ([172]Figures 9D,E). In
   addition, Compound C also attenuated LPS-tolerance in FcgRIIB–/– mice
   as determined by serum cytokines at 1 h after the second LPS injection.
   However, it was not effective in LPS-tolerance on WT mice ([173]Figures
   9F–H).

Figure 9.

   [174]Figure 9
   [175]Open in a new tab

   Schematic diagram of the experiment with LPS-tolerance induction
   (LPS/LPS) in macrophages and LPS injection in mice are shown (upper
   part). Supernatant cytokines of LPS-tolerant FcgRIIB–/– macrophages and
   wild-type (FcgRIIB+/+) with or without compound C (AMPK inhibitor)
   (A–C), semi-quantitative expression of mitochondrial DNA content
   (mtDNA) relative to β2 microglobulin (β2M) gene by qRT-PCR (D), and
   luminescence intensity of cellular ATP production (E) are demonstrated
   (independent triplicate experiments were performed). Serum cytokine
   from FcgRIIB–/– and wild-type mice after LPS-tolerance induction with
   placebo control or compound C treatment (F–H) are shown (n =
   6–7/group). BMDMs, bone marrow-derived macrophages.

Discussion

   FcgRIIB–/– mice provide a good representative lupus model for Asian
   population due to the high prevalence of FcgRIIB
   dysfunction-polymorphisms ([176]43). Persistent LPS exposure due to
   active lupus induced spontaneous endotoxemia ([177]13, [178]14)
   possibly induces extreme LPS exhaustion with increased susceptibility
   to secondary infection ([179]6). Here, we demonstrated that prominent
   LPS-tolerance in FcgRIIB–/– macrophages is, at least in part, due to an
   alteration in lipid-derivative metabolism. In a translational
   implication, an AMPK inhibitor rescued LPS-tolerance in lupus mice and
   might be a candidate for treatment of frequent infections in patients
   with lupus.

Severe LPS-Tolerance, Mitochondrial Defects, and Lipid Accumulation in
FcgRIIB–/– Macrophages

   FcgRIIB–/– macrophages showed hyper-responsiveness after a single LPS
   stimulation but more severe depressed cytokine production after
   subsequent doses of LPS (referred to as “LPS-tolerance”) in comparison
   with WT cells ([180]6, [181]7). Immense exhaustion of cytokine
   production in LPS-tolerant FcgRIIB–/– macrophages compared with WT was
   possibly associated with decreased cell energy as determined by
   mitochondrial biogenesis, mtDNA, ATP production, and extracellular flux
   analysis. In contrast, energy status of single LPS-stimulated
   FcgRIIB–/– macrophages was not higher than LPS-stimulated WT cells.
   This indicates that LPS hyper-responsiveness in FcgRIIB–/– macrophages
   is more complicated than energy status alone. It could be that cell
   energy of hyper-responsive FcgRIIB–/– macrophages increases in a short
   period of time before robustly exhausting. This can be visualized by
   repetitive LPS-stimulations. Our data suggests that prominent energy
   exhaustion after the second dose of LPS in FcgRIIB–/– macrophages might
   be responsible from the hyper-responsive responses against first LPS
   challenge. Accordingly, defect in mitochondrial Krebs cycle (36 ATP
   production) and glycolysis (2 ATP production) in leukocytes with
   LPS-tolerance is reported ([182]18, [183]19).

   Lipid β-oxidation is a part of the cell energy process ([184]22) where
   energy depletion in LPS-tolerant macrophages might be associated with
   lipid metabolism. Indeed, increased lipid accumulation was demonstrated
   in LPS-tolerant FcgRIIB–/– macrophages as previously mentioned
   ([185]44) but not in WT cells. This is possibly due to the energy
   depletion in WT cells not being severe enough. Most of the lipid
   derivatives in LPS-tolerant FcgRIIB–/– macrophages were upregulated
   ([186]Table 3), while approximately half of the derivatives were
   upregulated in single LPS stimulated FcgRIIB–/– macrophages ([187]Table
   2) when compared with WT cells. Among intracellularly accumulated
   lipids, glycerophospholipid (GP), the lipid component of cell membrane
   and sphingosine (SP), the lipid of cellular energy ([188]45) were both
   predominant in LPS-tolerant FcgRIIB–/– macrophages when compared with
   WT cells by mass-spectrometry analysis (LC-MS). Within several
   derivatives in GP of LPS-tolerant macrophages, phosphatidylethanolamine
   (PE) is an important lipid component of cell membrane ([189]37,
   [190]38). PE alteration could interfere with membrane fluidity, block
   LPS trans-membrane signaling and deplete cytokine production ([191]46,
   [192]47). Additionally, high PE in LPS-tolerant FcgRIIB–/– macrophages
   might directly reduce cytokine production because PE is an
   anti-inflammatory lipid-derivative ([193]48–[194]50).

   High PE in LPS-tolerant FcgRIIB–/– macrophages is possibly due to
   increased PE-uptake as demonstrated by PE intracellular-influx and/ or
   enhanced PE lipogenesis. This is a possible process from decreased
   expression of pemt, which is the enzyme responsible for converting PE
   into phosphatidylcholine (PC). Alternatively, prominent PE in
   LPS-tolerant FcgRIIB–/– cells might be due to an increase in PE uptake.
   Unfortunately, technical limitations on cellular lipogenesis preclude
   further investigation in this topic ([195]29, [196]30). On the other
   hand, low PC in LPS-tolerant FcgRIIB–/– macrophages is possibly from
   the shortage on PC because it is necessary for cytokine secretion
   ([197]23, [198]24). In this study, cytokine secretion of FcgRIIB–/–
   macrophages toward the first dose LPS was very prominent.
   Alternatively, a reduction of PC in LPS-tolerant FcgRIIB–/– macrophages
   might be due to increased PC degradation. Although mechanisms for the
   alteration of lipid derivatives in LPS-tolerant FcgRIIB–/– macrophages
   is inconclusive, prominent PE and/ or shortage on PC in LPS-tolerant
   FcgRIIB–/– macrophages are, at least in part, responsible for lower
   cytokine levels. AICAR, a pemt inhibitor, enhanced LPS-tolerant
   severity in WT macrophages by dampening cytokine levels and enhancing
   lipid accumulation into similar levels with LPS-tolerant FcgRIIB–/–
   macrophages. This suppression of macrophage cytokine production by
   AICAR has also been previously reported ([199]51, [200]52). However,
   pemt inhibition did not alter cell energy despite inducing some
   LPS-tolerant characteristics, including reduced cytokine production and
   enhanced phagocytosis activity, suggesting the diverse mechanisms of
   LPS-tolerance. Although direct exploration of PE and PC in
   AICAR-treated LPS-tolerant macrophages was not determined, our data
   supported that pemt inhibition enhanced the severity of LPS-tolerance
   in WT macrophages.

Inadequate PEMT and High AMPK, a Proposed Mechanism of LPS-Tolerance in
FcgRIIB–/– Macrophages

   While PEMT is encoded by pemt and associated with lipogenesis, pemt
   also co-operates with AMPK, a sensor of cellular energy status
   ([201]40, [202]41), because AMPK is upregulated in pemt-deficient mice
   ([203]41). Accidentally, AICAR is not only a potent pemt inhibitor, but
   it has also been reported to act as an AMPK activator in high doses
   ([204]42). Therefore, the cross talk between PEMT and AMPK after LPS
   activation is possible. As such, LPS induces cytokine production
   through PEMT-mediated lipogenesis ([205]23, [206]53, [207]54). From our
   data, the inhibition of PEMPT by a low dose AICAR reduced cytokine
   production ([208]42). In addition, LPS promotes fatty acid oxidation by
   AMPK-dependent-TLR4 activation ([209]23, [210]53, [211]54). On the
   other hand, AMPK activation that occurs during cell stress, including
   starvation, induces fatty acid translocation into mitochondria for
   enhancing energy production ([212]55) and directly inhibits cytokine
   production to possibly restore cell energy ([213]56). Here, we
   demonstrated increased AMPK in LPS-tolerant macrophages, which was
   partly responsible for low cytokine production, especially in
   LPS-tolerant FcgRIIB–/– macrophages. Compound C, an AMPK inhibitor,
   reduced ATP only in LPS-tolerant WT macrophages but not in FcgRIIB–/–
   macrophages. This is perhaps due to already minimized cell energy in
   LPS-tolerant FcgRIIB–/– macrophages where further energy-depletion is
   prohibited to maintain normal cell-homeostasis ([214]57). Nevertheless,
   AMPK inhibitor could attenuate LPS-tolerance in macrophages and mice in
   FcgRIIB–/– groups. This might be an interesting target for harnessing
   LPS-tolerance in lupus in the future. Indeed, the enhanced cytokine
   production by Compound C in several situations has also been mentioned
   ([215]58, [216]59). However, Compound C was not effective in rescuing
   LPS-tolerance in WT macrophages and in WT mice because the energy
   depletion in LPS-tolerant WT group was perhaps not severe enough to
   upregulate AMPK. Of note, the direct effect of Compound C on cell
   energy and lipid accumulation in macrophages, in vivo, was not
   evaluated because the cell sorting process might affect these
   parameters. Due to the possibility that extreme LPS-tolerance in lupus
   might be associated with increased susceptibility to infections
   ([217]6), AMPK inhibition is an interesting treatment in such
   condition. Further studies on Compound C and LPS-tolerance in lupus
   might provide novel therapeutic insight.

   LPS-tolerance seems to be a characteristic that is inducible by several
   mechanisms of either energy dependent or energy independent pathways.
   Our proposed hypothesis on LPS-tolerant FcgRIIB–/– macrophages
   ([218]Figure 10) initially shows that the first dose of LPS in
   FcgRIIB–/– macrophages produces higher cytokine production than WT due
   to inhibitory signaling loss ([219]6, [220]7). This results in
   enhancing PEMT utilization for the lipid-associated cytokine secretion
   process ([221]23). The second dose of LPS had inadequate PEMT to alter
   PE into PC, which leads to (1) shortage on PC, a lipid-derivative of
   cytokine secretion process ([222]23, [223]24), (2) accumulation of PE,
   an anti-inflammatory lipid-derivative ([224]48–[225]50), and (3)
   enhancement of AMPK, a signaling that reduces cytokine production to
   preserve cell energy ([226]53), according to the known association
   between PEMT and AMPK ([227]41, [228]60). In parallel, severity of cell
   energy depletion after the second dose of LPS in FcgRIIB–/– macrophages
   was enough to induce AMPK. As a metabolic sensor molecule of ATP
   depletion ([229]53), AMPK sets the anti-inflammatory state to restore
   cell energy partly by reducing cytokine production ([230]56).
   Therefore, prominent energy depletion, low PEMT, prominent PE
   ([231]48–[232]50), reduced PC, and enhanced AMPK ([233]56) are possibly
   responsible for severe LPS-tolerance in FcgRIIB–/– macrophages.
   Manipulation of these factors, alone or in combination, might be able
   to attenuate LPS-tolerance in lupus. As a proof of concept, attenuation
   of LPS-tolerance in FcgRIIB–/– mice by AMPK inhibitor was demonstrated.
   Because of the association between LPS-tolerance and infection
   susceptibility in lupus ([234]6, [235]7, [236]21), attenuation of
   LPS-tolerance might improve the outcome of infectious diseases in
   patients with lupus.

Figure 10.

   [237]Figure 10
   [238]Open in a new tab

   Diagram of the proposed hypothesis is shown. In wild-type macrophages,
   LPS-tolerance does not induce extreme energy depletion because no
   exaggerated cytokine production after the first LPS stimulation (left
   side of the figure). In contrast, in FcgRIIB–/– macrophages (right side
   of the figure), LPS-tolerance induces severe energy depletion
   (mitochondria and ATP) (1), which is possibly due to the extreme
   cytokine production after the first LPS stimulation from inhibitory
   signaling loss. Subsequently, lipid uptake (2) is increased in response
   to cell-energy depletion. Conversion from phosphatidylyethanolamine
   (PE) into phosphatidylcholine (PC) is reduced (3) from inadequate pemt
   (4), which is consumed during profound cytokine secretion process
   ([239]23, [240]24) after the first LPS stimulation. In parallel, AMPK
   (5), in response to ATP depletion, restores cell energy through reduced
   cytokine production ([241]56) leading to LPS-tolerance.

   In conclusion, our results demonstrated an impact of cell-energy
   depletion and lipid metabolisms in LPS-tolerant FcgRIIB–/– macrophages.
   We propose that alteration of lipid metabolism is another possible
   mechanism of LPS-tolerant macrophages, especially in FcgRIIB–/– cells.
   This finding is in addition to other well-described hypotheses
   ([242]61). Manipulations in cell energy and/ or lipid metabolisms are
   potential strategies for harnessing LPS-tolerance in lupus. Further
   studies are warranted.

Data Availability Statement

   The raw data supporting the conclusions of this article will be made
   available by the authors, without undue reservation, to any qualified
   researcher.

Ethics Statement

   The animal study was reviewed and approved by the Institutional Animal
   Care and Use Committee of the Faculty of Medicine, Chulalongkorn
   University, Bangkok, Thailand.

Author Contributions

   AL, PR, and TP carried out the conceptualization. TJ, PV, PD, and AL
   were responsible for the methodology and carried out the investigation.
   TJ and AL prepared the original draft. AL and TP wrote, reviewed, and
   edited the manuscript, supervised the study, and acquired the funding.

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgments