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Prolonged Triglyceride Storage in Macrophages: pH o Trumps pO2 and TLR4 Mingfang Lu, Terry Kho and Robert S. Munford This information is current as of June 18, 2017. Subscription Permissions Email Alerts This article cites 46 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/193/3/1392.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References J Immunol 2014; 193:1392-1397; Prepublished online 27 June 2014; doi: 10.4049/jimmunol.1400886 http://www.jimmunol.org/content/193/3/1392 The Journal of Immunology Prolonged Triglyceride Storage in Macrophages: pHo Trumps pO2 and TLR4 Mingfang Lu,1 Terry Kho, and Robert S. Munford C ontaining mainly cholesteryl esters and triglycerides, cytosolic lipid droplets (also called lipid bodies) produce the foamy appearance often seen in macrophages residing in inflammatory lesions such as granulomas, xanthogranulomatous kidneys, and atherosclerotic plaques. Although cholesteryl esters typically contribute a larger fraction of the stored lipid, triacylglycerol (TAG) may comprise a substantial component (1), provide a critical energy source for phagocytosis (2), and be used by intracellular pathogens as a source of fatty acids (FAs) (3). The common stimuli known to promote TAG storage in macrophages include low oxygen tension (pO2) (3–5) and TLR agonists such as bacterial LPS (6), bacterial lipopeptides, or polyinosinic:polycytidylic acid (7). Hypoxiainduced triglyceride synthesis has been attributed to increases in lipid droplet proteins, FA synthesis, and TAG synthesis from glucose (8, 9), whereas changes in key enzymes (acyl-CoA synthetase long 1 [ACSL1], diacylglycerol acyltransferase-2 [DGAT-2], and adipose triglyceride lipase [ATGL]) have been proposed to promote prolonged TAG retention in response to TLR ligands (10). As noted by Mackenzie et al. in 1961 (11), another stimulus to lipid accumulation is low extracellular pH (pHo) (12, 13). Because both low pO2 and many inflammatory stimuli induce cells to release small carboxylic acids, tissues that are hypoxic and/or contain microbial agonists are often acidic (4, 14, 15). Measurements in human patients found that pH was often ,6.5 in abscesses (16), which typically are both anaerobic and microbe laden. In other studies, the median pH of pus, infected peritoneal fluid, or drainage fluid was 6.75, and the median pO2 was 28 mm Hg (14). In this study, we used a load-chase Antibacterial Host Defense Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 1 Current address: Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China. Received for publication April 7, 2014. Accepted for publication May 27, 2014. This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. strategy to study how extracellular acidity (pHo) influences the effects of ambient pO2 and LPS stimulation on the retention of TAG by cultured peritoneal macrophages. We found that low pHo strongly favors TAG retention in both low and high oxygen environments, and in the presence and absence of LPS. Macrophages that adapted to a low pHo environment decreased catabolism of both glucose and FAs, whereas they increased FA uptake and incorporation into TAG, promoting TAG retention throughout a 72-h chase period. Materials and Methods Reagents Oleic and palmitic acids were from NuChek. [1-14C]-palmitate and [9,10-3H]oleate were from Moravek, and 2-deoxy-3H-glucose was from PerkinElmer. Buffers, media, and other reagents were from Sigma-Aldrich. Macrophage cultures The animal protocol (LCID 11E) was approved by the National Institute of Allergy and Infectious Diseases Institutional Animal Care and Use Committee. Harvesting and culture of JAX C57BL/6 peritoneal macrophages were as described previously (10). Thioglycollate-elicited peritoneal macrophages were harvested 5 d after injecting 1.0 ml 3% thioglycollate i.p.; they were allowed to adhere to plastic wells for 3–6 h, washed, and incubated overnight in DMEM that contained 0.5% FBS (Hyclone), 5.5 mM glucose, 50 mM palmitic acid, 100 mM oleic acid, and 1 mCi/ml radiolabeled oleate (Fig. 1A, FA load). The cells were then washed and reincubated (Fig. 1A, chase) in medium that contained half the original concentrations of nonradioactive FAs and no bicarbonate. The chase medium was buffered by adding 25 mM Mops, Hepes, or Tris to achieve starting pHo of 6.95–7.1, 7.3–7.5, or 7.6–7.7, respectively, and cells were then cultured either in a humidified incubator in 21% O2 or in a sealed, humidified chamber that contained a mixture of 4% O2 and 96% N2. The cells were harvested after a chase period of 48 or 72 h, and the final pHo was measured using a Mettler Seven Compact S220 pH/ion reader. In experiments to study the effect of lactate production on LPS-induced TAG retention, cells were loaded with [3H]oleate and nonradioactive FA as described earlier, washed, and cultured in a CO2 incubator in cDMEM that contained 44 mM NaHCO3, 5% FBS (Hyclone), 20 mM Tris, pH 7.5, 50 mM oleate, 25 mm palmitate, and either 5.5 mM glucose or 4 mM glutamine (17, 18). LPS (2.5–5.0 ng/ml) was added to the chase medium as indicated. Address correspondence and reprint requests to Dr. Robert S. Munford, 9000 Rockville Pike, Bethesda, MD 20892-3206. E-mail address: [email protected] Lipid analysis Abbreviations used in this article: ACSL-1, acyl-CoA synthetase long 1; ATGL, adipose triglyceride lipase; DGAT-2, diacylglycerol acyltransferase-2; FA, fatty acid; pHo, extracellular pH; pO2, oxygen tension; TAG, triacylglycerol. Preparation of albumin-bound FAs was as described previously (10). The amount of the radiolabel that remained in TAG ([3H]TAG retention) (10) was determined by extracting the cellular lipids into isopropanol, thin- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400886 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Lipid-laden macrophages contribute to pathologies as diverse as atherosclerosis and tuberculosis. Three common stimuli are known to promote macrophage lipid storage: low tissue oxygen tension (pO2), low extracellular pH (pHo), and exposure to agonists such as bacterial LPS. Noting that cells responding to low pO2 or agonistic bacterial molecules often decrease pHo by secreting lactic and other carboxylic acids, we studied how pHo influences the stimulation of triacylglycerol (TAG) storage by low pO2 and LPS. We found that TAG retention after incubation for 48–72 h was inversely related to pHo when primary macrophages were cultured in 21% oxygen, 4% oxygen, or with LPS at either oxygen concentration. Maintaining pHo at ∼7.4 was sufficient to prevent the increase in prolonged TAG storage induced by either low pO2 or LPS. The strong influence of pHo on TAG retention may explain why lipid-laden macrophages are found in some tissue environments and not in others. It is also possible that other long-term cellular changes currently attributed to low pO2 or bacterial agonists may be promoted, at least in part, by the decrease in pHo that these stimuli induce. The Journal of Immunology, 2014, 193: 1392–1397. The Journal of Immunology layer chromatography on silica gel G to isolate TAG, and b-scintillation counting of TAG-containing silica (10). The recovered [3H]TAG correlated strongly (r2 = 0.90) with measurements of TAG mass in the same cells (10). To estimate the lipolytic rate in intact cells, we loaded macrophages with [3H]oleate, incubated them in buffered media for 48 h, then washed and reincubated them for 48 h in the presence of 10 mM triacsin C (ENZO Life Sciences) before measuring the extent to which [3H]TAG was depleted (10, 19). Triacsin C inhibits macrophage acyl-CoA synthetases (20, 21), blocking FA thioesterification, and thereby reducing the reincorporation of released FA into TAG. TAG depletion is then attributable to lipolysis (19). Assays Results TAG retention increases at low pHo We studied TAG retention by loading cells with 3H-FA and following the retention of labeled TAG over 48 or 72 h (10) (Fig. 1A). We first noticed that retention of radiolabeled TAG increased when the chase medium was buffered at low pHo. The relationship between pHo and log10[3H dpm in TAG] after a 48- or 72-h chase in 21% O2 was approximately linear from pHo ∼6.7 to pHo ∼7.6, with or without supplemental acids (each at 5 mM) to lower pHo (Fig. 1B). For each decrease of 0.1 U pHo between 7.4 and 7.0, TAG retention increased ∼25%. Low pHo increases FA uptake and FA incorporation into TAG while decreasing lipolysis Providing supplementary palmitate and oleate during the chase greatly increased the amount of cellular TAG mass at the 72-h time point (Fig. 1C). We therefore studied the effect of pHo on TAG synthesis from exogenous FA. As expected (11–13), FA uptake (Fig. 1D) and FA incorporation into TAG (Fig. 1E) were enhanced at low pHo. In addition, [14C]palmitate added to the medium during the 72-h chase was incorporated into TAG with the same relationship to pHo that [3H]TAG was retained from the 3H-oleate load (Fig. 1F). Incubation at low pHo was also associated with decreased lipolytic activity in cell lysates (Fig. 1G) and there was less loss of [3H]TAG at low pHo when FA thioesterification was inhibited with 10 mM triacsin C to provide a measure of the lipolytic rate (10, 19) (Fig. 1H). These results suggest that macrophages that adapt to low pHo increase TAG storage mainly by increasing the uptake and incorporation of FA into TAG; a decrease in lipolysis may also contribute. pHo can trump pO2 Because cells living at low pO2 lower their pHo by secreting lactate (anaerobic glycolysis), a role for pHo in hypoxia-induced TAG retention seemed plausible. We cultured cells in 4% O2 (pO2 ∼28 mm Hg), a level measured in tissues such as lymph nodes and spleen (22, 23). As expected, cells cultured in 4% O2 retained more of the FA load in TAG than did cells cultured in 21% O2 (pO2 ∼150 mm Hg), but again TAG retention was strongly related to the final pHo (Fig. 2A, 2B). TAG retention was greatly reduced by preventing the decline in pHo that occurred as cells adapted to living at a moderate level of hypoxia, suggesting that pHo may play a more important role in promoting TAG retention than does oxygen at this concentration. pHo-dependent changes in cell metabolism Although glycolysis increases when macrophages become hypoxic, we found that glucose uptake decreased when cells were cultured for 72 h at acidic pHo (Fig. 3A), as did both glucose consumption (Fig. 3B) and the amount of lactate that remained in the culture medium at the end of the chase period (Fig. 3C). The relationship between glucose consumption and the medium lactate level after a 72-h chase was similar in both 4 and 21% O2 (Fig. 3D), and in the presence and absence of supplemental FA (Fig. 3E), suggesting that neither low pO2 nor providing FA as an alternative energy source influenced the effect of pHo on glucose catabolism. FA b-oxidation also decreased at low pHo (Fig. 3F). Both glucose and FA catabolism thus decreased as the cells adapted to low pHo, whereas FA uptake and storage increased. Although a role for extracellular L-lactic acid (or its uptake and utilization) (24) in promoting TAG retention at low pH o seemed likely, providing supplemental L-lactic acid during the chase did not increase TAG retention more than would be predicted by its effect on pHo (Fig. 1B). Extracellular acidity may have dramatic effects on cells, altering their mobility, secretory ability, and other properties (25–27). The adherent cells studied in this article were impermeable to trypan blue after the 72-h chase, yet we found 10% less DNA in wells with final pHo ,7.0 than in wells cultured at final pHo 7.3–7.5 (210 6 3% [SD], 3 experiments, each with n = 4/condition), and there was a commensurate decrease in cell protein (212 6 11%, 6 experiments with n = 4/condition). Cellular ATP levels were also lower in cells carried at low pHo (215 6 5% relative to HEPEScultured cells, 3 experiments with n = 4, p , 0.05; Fig. 3G, 3Ia), yet the AMP/ATP ratio was higher (Fig. 3H), suggesting that the low ATP levels were produced, at least in part, by greater ATP utilization. Switching cells from low pHo (MOPS buffer) to pHo 7.4 (HEPES buffer) after the 72-h chase period allowed restoration of cellular ATP to control levels 72 h later (total 144 h of chase; Fig. 3I). These results are consistent with prior reports that macrophages cultured at low pHo expend ATP to defend intracellular pH, enabling recovery if pHo increases (26, 28). pHo versus TLR4 LPS, a potent TLR4 agonist, induces acute macrophage FA uptake and TAG synthesis (6). We recently reported that LPS stimulated TAG storage for as long as 72–96 h in media buffered with 44 mM NaHCO3, 5% CO2 (initial pHo 7.7) (10). LPS also stimulated glucose consumption (data not shown) and lactate accumulation (Fig. 4A) during the chase period. When we noted the effect of pHo on prolonged TAG storage, we stimulated macrophages with LPS while controlling pHo. We again found that TAG storage was maintained above control (unstimulated) levels for 72 h, but now the importance of final pHo became apparent in cells cultured in either 4% O2 or 21% O2 (Fig. 4B–D). Low pHo did not account for the acute effects of LPS on TAG storage, however, because LPSinduced TAG retention was evident 24 h after the stimulus was added, before sufficient lactate had accumulated to lower pHo (10), and because macrophages stimulated with LPS in media that contained glutamine instead of glucose (17) retained TAG after chasing for 24 or 72 h in the absence of lactate accumulation (Fig. 4E) and without a low final pHo. In macrophages exposed to LPS and cultured for prolonged periods, the accumulation of extracellular lactate induced a decline in pHo that likely extended and enhanced the acute stimulatory effects of LPS on TAG storage Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Quantitative PCR was performed as previously described (10). Glucose and L-lactate were measured using kits from Sigma and Eton Biosciences, respectively. Lipolytic activity (cleavage of [3H]oleate from [3H]triolein [Amersham]) was measured as described previously (10) in lysates of cells cultured without FA loading. Cell protein was measured by dissolving the isopropanol-insoluble residue from each culture well in 200 ml 1N NaOH before assay using the Pierce BCA kit (Thermo Scientific). DNA was measured using the Invitrogen NF cell proliferation assay kit (C35007). For ATP and AMP analysis, cells were lysed in buffer from the Promega AMP kit before assay using the ATP Determination Kit from Molecular Probes and the AMP kit from Promega. Tests of significance and linear regression analysis were performed using GraphPad Prism software. 1393 1394 LOW pH PROLONGS TRIGLYCERIDE STORAGE IN MACROPHAGES (Fig. 5A, 5B). Decreasing pHo may also have prevented the switch from glycolysis to FA catabolism that was found by Liu et al. (29) in a human macrophage cell line that was stimulated with LPS for shorter periods. As expected (30), LPS stimulation boosted glucose uptake (Fig. 3A), glucose consumption (Fig. 4F), lactate accumulation in the medium (Fig. 4A), and ATP stores (Fig. 3G). As was also FIGURE 2. pHo trumps pO2. (A) Cells cultured in 4% O2 after the FA load retain more [3H]TAG than do cells cultured in 21% O2. H, Hepes, initial pH 7.3; M, Mops, initial pH 6.95; T, Tris, initial pH 7.5. (Inset) The same data plotted to show the relationship between final pHo and log10 (cellular [ 3H]TAG). (B) pHo influences TAG retention when cells are cultured in either low or high O2. Added to the data for 21% O2 shown in Fig. 1B (closed circles) are data for cells cultured in 4% O2 (open boxes) from seven experiments (total n = 70) in which cells were chased at both 4 and 21% O2. Linear regression lines: solid = 21% O2, dashed = 4% O2. Four percent O2 regression: r2 = 0.76, n = 70. *p , 0.05, **p , 0.01. found in cultures without LPS, however, glucose consumption (Fig. 4F) and lactate accumulation (data not shown) decreased when the cells were cultured at low pHo. LPS also induced increases in mRNA abundance for genes associated previously with FA uptake and TAG synthesis (ACSL-1 and DGAT-2) (31) whereas decreasing ATGL, the enzyme that carries out the first step in TAG lipolysis (32) (Fig. 4G). At low pHo the LPS-induced Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 1. pHo influences TAG storage. (A) Experimental approach: load-chase. The retention of [3H]TAG is a measure of net TAG synthesis and degradation during the chase (10). (B) Relationship between final pHo and cellular [3H]TAG for cells chased 72 h in Tris, Hepes, or Mops buffers, with or without the addition of 5 mM acetic acid (HAc), b-hydroxybutyrate (BHB), L-lactic acid, or sodium L-lactate to Hepes-buffered medium. Compiled from 12 independent experiments; the values from each experiment were normalized so that log10[3H]TAG dpm at pHo 7.25 was ∼3.7. Linear regression was performed on the values obtained without added acid (blue circles). (C) Providing supplemental FA increases TAG retention at low pHo. Macrophages were loaded with FA for 18 h, washed, and chased for 72 h in buffered media in the presence (+ FA) or absence (2 FA) of 25 mM palmitate and 50 mM oleate. Cellular TAG mass was much greater at 72 h if FAs were added to the medium during the chase. (D) FA uptake is pHo dependent. Cells were incubated overnight in Hepes buffer, pH 7.4, before their ability to take up [3H]oleate (in 20 mM nonradiolabeled oleate) in 5 h was measured at different pHo. n = 3; symbols show mean 6 1 SD. Representative of two independent experiments. (E) Incorporation of FA into TAG was also pHo dependent. After overnight culture in buffered media, cells were provided [3H]oleate and [14C]palmitate (in 20 mM oleate and 20 mM palmitate) in media at different pHo for 5 h before lipid analysis. Results are expressed as nanomoles FA in TAG per milligram cell protein. Representative of two independent experiments. (F) Cells were loaded overnight with FA containing [3H]oleate, washed, and incubated for 72 h in media that contained [14C]palmitate (with 25 mM nonradiolabeled palmitate and 50 mM nonradiolabeled oleate). The amounts of [3H]TAG (acquired during load) and [14C]TAG (acquired during chase) were both higher at low pHo. Representative of three independent experiments. (G) Cellular lipolytic activity decreased at low pHo. [3H]oleate was released from [3H]triolein by lysates of cells that had been cultured in media at different pHo for 72 h. A similar trend was observed at 24 and 96 h. (H) Lipolysis decreased at low pHo. Cells loaded with [3H]oleate were incubated with or without 10 mM triacsin C (Tc) for 48 h before measuring the percentage of the pretreatment [3H]TAG that was lost from the cells. Tc blocks thioesterification so that TAG loss may be attributed to lipolysis (19). More TAG was lost in the presence of Tc at high pHo. Data combined from two independent experiments, each with n = 3. **p , 0.01. H, Hepes; M, Mops; T, Tris. The Journal of Immunology 1395 changes in mRNA abundance were significantly smaller (Fig. 4G), yet TAG retention increased, suggesting that low pHo had a greater influence on TAG storage than did the observed changes in mRNA or protein abundance. None of these changes in mRNA abundance was noted in cells cultured without LPS. Cells cultured in 4% oxygen had increased levels of mRNAs for HIF-1a target genes (GLUT1, LDHa, VEGF), but these changes did not differ substantially in cells cultured at different pHo (Fig. 4H–J). Discussion The retention of TAG stores for 48–72 h in vitro was closely related to the final pHo in both low and high oxygen environments, and in the presence and absence of a potent microbial stimulus. Maintaining pHo at ∼7.3–7.5 prevented the increase in TAG storage induced by either low pO2 or LPS. Although low pHo influenced both TAG synthesis and lipolysis, its major effect was to increase the uptake and incorporation of FA into TAG, an activity first described by Spector (12). Low pHo may promote FA uptake by decreasing the affinity of albumin for FA (12, 33) or by allowing greater protonation of the carboxyl moiety, increasing transmembrane diffusion or transport (34). It seems likely that many of the other effects of pHo on cell metabolism are mediated by changes in intracellular pH that alter protein function (26–28). In fibroblasts and a glioma cell line, for example, decreased lactate production at low pHo was attributed to a decrement in phosphofructokinase activity (35). pHo has also been reported to dominate pO2 in the regulation of glucose utilization by cultured chondrocytes (36). Peritoneal macrophages elicited by thioglycollate have been used for many of the foundational studies of macrophage metabolism (17, 18, 24, 37, 38), including the recent analysis of macrophage lipids by the Lipid MAPS consortium (39). By harvesting 5 d after thioglycollate injection and studying only adherent cells, we sought to reduce contamination by other cells, particularly neutrophils and eosinophils, and to obtain the “small peritoneal macrophages” described by Ghosn et al. (40). Although we know no evidence that the FA and glucose metabolism of thioglycollate-elicited peritoneal macrophage differs substantially from that of other primary macrophages (as distinct from RAW 264.7 cells and possibly other macrophage cell lines) (10, 39), it will be important to confirm our results in other macrophage types. Our findings suggest that local acidity can contribute prominently to the development of “lipid-laden” macrophages, even in an in vivo environment that is hypoxic and/or contains microbial agonists (such as an infected tissue, tuberculous granuloma, abscess, or atherosclerotic plaque) (4, 41) (Fig. 5). In addition to promoting FA uptake and incorporation into TAG (but not cholesteryl esters) (12), low pHo increases macrophage uptake of lipoproteins (42) and likely contributes to the acidic environment that facilitates lipoprotein processing and cholesterol uptake within surface-connected compartments (43). M1 or classically activated macrophages may be particularly likely to become lipid laden, because they carry out aerobic glycolysis and secrete much of the lactate they produce (30). In contrast, macrophages in uninflamed tissues, even ones with lower levels of pO2, such as the spleen and lymph nodes (23), rarely appear “lipid laden.” Our findings suggest that an acidic environment may be needed to sustain long-term TAG storage in macrophages. Low pHo promoted TAG retention, yet glucose uptake, lactate production, lipolysis, and FA b-oxidation all decreased, in keeping Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 3. Cell metabolism produces and responds to low pHo. (A) Glucose uptake decreased in acidic media. 2-[3H]deoxyglucose (3H-DOG) uptake during a 1-h incubation period was measured after 72-h chase at different pHo. n = 4 per condition. Similar results were found in two additional experiments. (B) Glucose consumption decreased as pHo decreased. 72-h chase. (C) L-lactate levels in the culture medium at the end of the chase were also lower at low pHo. (D) Lactate accumulation decreased with decreasing glucose consumption when cells were cultured in either 4 or 21% O2. Similar results to those shown in (B)–(D) were obtained in four additional experiments, each with n = 3 or 4 at each pHo and % O2. (E) Glucose consumption and lactate accumulation were not influenced by providing supplemental FA during the chase. Representative of two experiments. (F) b-Oxidation, producing 3H2O from [3H]oleate, decreased when FA-loaded cells were incubated at low pHo. A similar relationship between pHo and 3H2O release was observed in three additional experiments. (G–I) After the 72-h chase period, ATP levels were lower (G) and the AMP/ATP ratio was higher (H) in cells cultured low pHo. (I) Restoring physiological pHo (25 mM Hepes buffer, pH 7.3–7.5) allowed cellular ATP levels to increase. After 72-h chase at initial pH, FA-loaded cells were washed and reincubated either in media containing the same buffer (a) or Hepes buffer, pH 7.4 (b), for an additional 72 h before ATP was measured. (G–I) Data were combined from three or four independent experiments, each with n = 3 or 4. *p , 0.05, **p , 0.01, ***p , 0.001. 1396 LOW pH PROLONGS TRIGLYCERIDE STORAGE IN MACROPHAGES with the general decrease in cell metabolism noted by Taylor (25) and others. Glucose and FA catabolism did not cease, however, and the cells produced sufficient ATP to maintain a high level of FIGURE 5. Summary diagrams. (A) Experimental time course. After loading overnight with FA, macrophages were exposed to TLR agonists (LPS) and/or low pO2. In cells exposed to LPS, TAG retention was associated with enzyme changes that either promote FA uptake and FA incorporation/retention in TAG or decrease lipolysis. Both hypoxia and LPS stimulated glycolysis, producing extracellular acidosis that also promoted TAG retention while decreasing glucose and FA catabolism. (B) Proposed in vivo scenario. Blood monocytes enter an infected tissue and encounter TLR agonists that acutely induce FA uptake, TAG synthesis, and lactic acid production. As pHo decreases, lipoprotein and FA uptake both increase, as does TAG and cholesterol storage in lipid droplets. Cells become more “lipid laden” and their metabolism slows. Low tissue pO2 also promotes TAG retention as anaerobic glycolysis decreases pHo; the enzyme changes noted with TLR stimulation are not induced by low pHo. viability and allow recovery when pHo was restored to 7.4. These changes are similar to those described in human tissues during the chronic phase of severe sepsis (44), raising the possibility that Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 4. LPS versus pHo. (A) Lactate accumulated in the media of cells cultured in 44 mM NaHCO3, 5% CO2, 21% O2 for 72 h. Supplemental FAs were present during the chase (see Materials and Methods). Representative of three experiments with similar results. (B) LPS (2.5 ng/ml) increased [3H]TAG retention in cells cultured in either Mops or Hepes buffers for 72 h. (Inset) log10[3H]TAG versus pHo. (C) LPS-induced TAG retention after 72-h chase was pHo dependent. Data for cells treated with 2.5 ng/ml LPS for 72 h in 21% O2 (red boxes) are shown with results for cells incubated without LPS (blue circles; see Fig. 1B). Data from four independent experiments. Linear regression lines: blue = no LPS; red = + LPS. (D) pHo also influences the impact of LPS on TAG retention at low pO2. Data shown in (B) for 21% O2 + LPS are reproduced (red symbols and regression line) and data are added (green triangles and regression line) from 3 experiments in which cells were chased for 48–72 h at 4% O2 with LPS (2.5 ng/ml). (E) LPS-stimulated TAG retention in the absence of glucose and without increased lactate production. Adding LPS (2.5 ng/ml) increased retention of [3H]TAG for 72 h (bottom) whether or not L-lactate was produced (top). n = 4. LPS also stimulated TAG retention at 24 h and in two other experiments when glutamine was used instead of glucose (not shown). (F) LPS stimulation of glucose consumption was pHo dependent. Measurements after 72-h chase. (G) mRNA abundance in cells cultured 48 h at different pHo or with LPS (M+LPS, H+LPS). LPS-induced increases in mRNA abundance for ACSL-1 and DGAT-2 and decreases in ATGL (10); these changes were smaller at pHo 6.9 (M+LPS) than at pH 7.4 (H+LPS). Data (n = 3) are expressed as fold-change relative to the value obtained using HEPES buffer. Bars show mean + 1 SE. Similar results in two independent experiments. (H–J) mRNA abundance for hypoxia-responsive genes: VEGF, LDHa, and GLUT1. LPS induced increases in each of these mRNAs in both 21 and 4% oxygen; each increase was significantly higher in cells chased in 4% O2 (for each comparison, p , 0.01, unpaired t test.) Representative of two (VEGF, LDHa) or three (GLUT1) independent experiments, each with n = 2 or 3. *p , 0.05, **p , 0.01. H, Hepes; M, Mops; T, Tris. The Journal of Immunology local or systemic acidosis may contribute to the hibernation-like phenomena that are reported to occur in critically ill individuals. Spurred by observing that metabolic pathways play important roles in macrophage and lymphocyte function, the new field of “immunometabolism” promises to revise many ideas about immune cell biology. It is now appreciated that glycolysis, for example, is important not only for producing nucleotides, glycerolipids, and ATP, but also for regulating IFN-g production (45) and inflammasome activation (46). 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