Haemagglutinin and neuraminidase characterization of low pathogenic H5 and H7 avian influenza viruses isolated from Northern pintails (<Emphasis Type="Italic">Anas</Emphasis> <Emphasis Type="Italic">acuta</Emphasis>) in Japan,with special reference to genomic and biogeographical aspects

Pintails constitute an important host of avian influenza viruses (AIVs). Genetic, molecular, and antigenic characteristics of H5 and H7 AIVs, which we isolated from northern pintails (Anas acuta) wintering in Japan, were analyzed and found to be linked to various ecological features, chiefly in terms of gene geography, as shaped by various migratory aquatic host species. Although all the isolates were found to be of low pathogenicity (LP), we explored gene predispositions that may potentially underlie tentative transition to high pathogenicity (HP). Evolutionarily, the HA and NA genes of the isolates affiliated mostly with Eurasian lineage. The viruses closely related to ours were derived from China, Korea, Mongolia, Japan, and Australia. Comprehensive ecophylogenetic evaluations revealed that the pintail populations we sampled might have given rise to or been involved in the emergence of a LPAI H7N6 subtype that caused outbreaks in quail (Coturnix japonica) farms in Japan, as well as of the first H5N9 subtype ever isolated in Asia. The latter strain isolated by us showed, yet, notable affinity to certain North American and Australian strains, thereby signifying apparent intercontinental interfaces accounted for by extensive water-bird flyways. Noticeable conservation of certain antigenic sites within both Eurasian and North American H7 HAs is apparently an outcome of their advantageous survival value, in terms of restricted immunogenicity. Besides, the Japanese–Korean–Siberian regional axis seems to be particularly important for ongoing generation of novel viral strains due to conveyance of certain genes and genomes by migratory ducks, including such that circulate among pigs and human.     

Steroid and Xenobiotic Receptor (SXR) as a possible prognostic marker in epithelial ovarian cancer

We examined the expression of the steroid and xenobiotic receptor (SXR) and evaluated its clinical significance in human epithelial ovarian carcinoma. One hundred forty‐one cases were examined using immunohistochemistry for SXR with archival specimens. All cases were scored using a semi‐quantitative histological scoring (HSCORE) method. Specimens with an HSCORE > 60 were regarded as SXR‐positive. Various clinicopathologic variables were examined. SXR showed significant differences in age, histology, grade, ERα and PR. SXR was detected in 35 of 141 (24.8%) ovarian cancer tissues. There was a statistically significant negative correlation between SXR‐positive status and both disease‐free survival and overall survival (P= 0.0415 and 0.0316, respectively), independent of stage (P= 0.0167 and 0.021, respectively). In multivariate analysis, SXR was a statistically independent risk factor for both disease‐free survival and overall survival (P= 0.049 and 0.0354). Our results support an association of SXR between ERα and PR in epithelial ovarian cancers. Our data suggest that SXR is a prognostic factor in epithelial ovarian cancer and may represent a useful marker to identify patients at risk of recurrence or death.     

Fetal myocardial thickening measured by ultrasonic-based technique called 'phased-tracking method'

OBJECTIVE: To evaluate fetal myocardial movement by using newly developed ultrasonic technique. METHODS: We analyzed 50 normal fetuses between 25 and 41 weeks' gestation for changes in thickness of fetal myocardium using the phased-tracking method, a technique with high vertical distance resolution and the potential to evaluate fine ventricular wall movements. We analyzed differences in the rate of change in ventricular wall thickness and in changes in the inner and outer wall layers with advancing gestation. We also analyzed myocardial thickening period and evaluated the ratio of increasing thickness period to stroke interval. RESULTS: Mean thickness changing rate was significantly higher in the right (1.18 +/- 0.34 m/s/m) than in the left ventricular wall (0.86 +/-0.31 m/s/m) (p < 0.001). Mean ratio of increasing thickness period to stroke interval was significantly higher in the right (0.57 +/- 0.064) than in the left ventricle (0.46 +/- 0.075) (p < 0.001), indicating that myocardial contraction in the fetal right ventricle predominates. The thickness-changing rate of the bilateral ventricular walls was positively and linearly correlated with gestational age. The myocardial-wall thickness-changing rate was higher in the outer layer than in the inner layer in late gestation. CONCLUSIONS: We conclude that measurement of the thickness-changing rate of fetal ventricular walls using the phased-tracking method might be useful for evaluation of fetal cardiac function.     

Human T-cell leukemia virus type-I (HTLV-I)-specific T-cell responses detected using three-divided glutathione-S-transferase (GST)-Tax fusion proteins

Insufficient T-cell response to human T-cell leukemia virus type-I (HTLV-I) is a potential risk factor in adult T-cell leukemia (ATL). We established an assay system for detecting HTLV-I-specific T-cell response by using recombinant glutathione-S-transferase (GST) proteins fused with HTLV-I Tax protein that was divided into three portions, Tax-A, -B, and -C, corresponding to the N-terminal, central and C-terminal regions, respectively. When splenocytes from rats immunized with plasmids encoding Tax cDNA were incubated with these recombinant proteins, strong interferon gamma (IFN-gamma-producing responses occurred against GST-Tax proteins but not against control GST proteins. No such Tax-specific responses were observed in splenocytes from naive rats. Cocktails of oligopeptides corresponding to the Tax-A, -B, and -C regions also induced IFN-gamma-producing responses when incubated with splenocytes from immunized rats, but required higher amounts of antigens and there were a shorter periods of sustained T-cell responses than with GST-Tax protein-based assay. Although splenocytes from immunized rats predominantly reacted against GST-Tax-B protein, they failed to react with peptide cocktails corresponding to the Tax-B region, likely because the major epitope was interrupted in the initially prepared series of peptides. Using a newly prepared peptide series we found that splenocytes predominantly reacted with a peptide located in the Tax-B region that overlaps with a previously identified cytotoxic T lymphocytes (CTL) epitope of this rat strain. Using this system, we examined peripheral blood mononuclear cells (PBMC) from an ATL patient who underwent complete remission following hematopoietic stem cell transplantation (HSCT). PBMC from this patient produced a significant Tax-specific T-cell response predominantly against GST-Tax-A protein. This is consistent with the previous finding that this patient exhibited a strong HLA-A2-restricted CTL response to Tax 11-19 epitope, which is located in the Tax-A region. This study provides a diagnostic tool, useful for monitoring HTLV-I-specific T-cell immunity in patients and for surveying HTLV-I-carriers to identify an immunological group at high risk for ATL development, regardless of their human leukocyte antigen (HLA) types. It is also useful for predicting the location of T-cell epitopes, which may be applicable in future vaccine strategies.     

SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage’s respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells’ ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

Innate immune cells, including monocytes and dendritic cells, infiltrate inflamed tissues and mediate immune responses despite drastic changes in nutrient availability, low partial oxygen pressure, and other environmental stresses. Immune cells adapt to these environmental stresses by changing their metabolic state, and this adaptation is vitally important for these cells to fulfill their function in the immune system (1, 2). Solute carrier family 15 member 4 (SLC15A4) is a proton-coupled amino acid/oligopeptide transporter that is preferentially expressed in hematopoietic lineage cells such as macrophages, B cells, and plasmacytoid dendritic cells (3–5). This transporter, which localizes mainly in LAMP1⁺ vesicular compartments such as late endosomes and lysosomes, is required for Toll-like receptor (TLR)7- and TLR9-dependent production of cytokines such as type I interferon (IFN-I) and interleukin (IL)-6 (4, 6–8) and plays a critical role in autoimmune and other inflammatory diseases (3, 6, 9). SLC15A4 colocalizes with the mechanistic target of rapamycin (mTOR) (3, 10), and SLC15A4’s effects on TLR7 and TLR9 signaling are partly mediated through the regulation of mTORC1 activity (3).To understand SLC15A4’s mode of action in detail, we used BioID to identify molecules that are in close proximity to SLC15A4 (11, 12) using 293T transfectants expressing SLC15A4 proteins fused with BirA biotin ligase at the N terminus and used membrane raft-targeting methionine-glycine-cysteine (MGC) peptides (13) fused with BirA as a control (SI Appendix, Fig. S1 A–D). A large number of biotinylated proteins were precipitated from cells expressing BirA-SLC15A4 but not from parental 293T or cells expressing control-BirA (Fig. 1A). Mass spectrometry identified 424 precipitated proteins that increased more than twofold in cells expressing BirA-SLC15A4 compared with those expressing control-BirA (project accession: PXD020370) (14). Among SLC15A4-dependent biotinylated proteins, we identified nine proteins involved in the mTOR signaling pathway, including RagA, RagB, RagC, and Lamtor1/2 (Fig. 1B and SI Appendix, Fig. S1 E and F). SLC15A4 itself was among the molecules with the highest scores, indicating that we had successfully collected proteins biotinylated by BirA-SLC15A4 (Fig. 1B). Functional annotation analyses of mass spectrometry datasets identified 15 biological processes associated with the enriched proteins, including glucose metabolism/glycolysis in SLC15A4’s profile (Fig. 1C). We then examined SLC15A4’s role in glycolysis using human plasmacytoid dendritic cell line CAL-1 (15). SLC15A4 knockdown CAL-1 cells (CAL-1-shA4) showed the significant reduction of SLC15A4 expression (SI Appendix, Fig. S2 A and B), and TLR9-mediated IFNB1 expressions was almost completely absent in CAL-1-shA4 (SI Appendix, Fig. S2C), consistent with previous observations (3, 6, 8, 16). Although we detected the decreased gene expression of glucose transporter 1 (GLUT1) in CAL-1-shA4 compared with CAL-1-shCont cells (SI Appendix, Fig. S2D), the protein expression levels of GLUT 1, GLUT 3, and HK2 were not affected by the presence or absence of SLC15A4 (Fig. 1D). We further assessed cellular glycolytic activities by measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) with a Seahorse XF Extracellular Flux Analyzer (17). In Glyco Stress Tests, the addition of glucose increased ECAR in both CAL-1-shCont and CAL-1-shA4 cells (Fig. 1E). The addition of oligomycin further increased ECAR in CAL-1-shCont cells irrespective of the presence or absence of TLR stimulation (Fig. 1E), suggesting that pyruvate, the end product of glycolysis, enters the mitochondria for oxidative phosphorylation (OXPHOS), and oligomycin treatment causes an elevated pyruvate flux toward lactate. In contrast, oligomycin had little effect on ECAR in CAL-1-shA4 cells (Fig. 1E). These results indicated that CAL-1-shA4 had a lower glycolytic reserve than CAL-1-shCont. Basal OCR and ECAR were lower in CAL-1-shA4 cells than in CAL-1-shCont cells in both the presence and absence of pyruvate (Fig. 1F). Similar trends were observed when only glucose was used as a carbon source (Fig. 1F). Oligomycin and (trifluoromethoxy) phenylhydrazone (FCCP) decreased and increased OCR, respectively, in CAL-1-shCont, with a concomitant change in ECAR (Fig. 1F). However, neither oligomycin nor FCCP affected ECAR in CAL-1-shA4 cells when glucose was used as a carbon source (Fig. 1F). These results suggested dysregulation of the coupling between OXPHOS and glycolysis in CAL-1-shA4 cells. When pyruvate was used as a carbon source, OCR and ECAR were concomitantly changed by oligomycin or FCCP, although ECAR values were lower in both CAL-1-shA4 and CAL-1-shCont cells compared with when glucose was used (Fig. 1F). Glucose uptake in CAL-1-shA4 cells was lower compared with CAL-1-shCont cells in a steady state but was comparable to CAL-1-shCont cells after CpG stimulation (Fig. 1G). Thus, although the decreased glycolytic activity in CAL-1-shA4 cells was partly explained by the decreased uptake of glucose, it was also possible that SLC15A4 contributed to the coordinated regulation between glycolysis and OXPHOS.Open in a separate windowFig. 1.Identification of SLC15A4-associated molecules by BioID. (A) Precipitation of SLC15A4-interacting proteins. Biotinylated precipitates were visualized by silver staining or by Western blot. (B) The enrichment of SLC15A4-interacting proteins involved in the mTOR pathway. Abundance ratio (AR) score: relative enrichment value over the control. (C) Pathway analysis of SLC15A4-interacting proteins by g:Profiler (https://biit.cs.ut.ee/gprofiler/gost). (D) Expression of glycolysis-related proteins in CAL-1-shCont or CAL-1-shA4 cells was analyzed by Western blotting. (E and F) Seahorse XF analysis of OCR and ECAR. (E) OCR and ECAR in CAL-1 cells were compared between the presence or absence of CpG stimulation in Seahorse XF basic RPMI medium containing 2 mM glutamine. Reagents were added at the indicated time points (arrows). (F) OCR (Upper) and ECAR (Bottom) were compared between CAL-1-shCont and CAL-1-shA cells in the presence of 10 mM glucose (Left) or 2 mM pyruvate (Right) by Mito Stress Test using Seahorse XF basic RPMI medium. (G) Glucose uptake assay was performed for CAL-1-shCont or CAL-1-shA4 cells treated with CpG-B (ODN2006) for the indicated periods. Uptake of 2-NBDG (a fluorescent D-glucose analog) was evaluated by fluorescence intensity within cells using flow cytometry. The results are representative of three independent experiments. *P < 0.05, as determined by one-way ANOVA followed by Tukey’s post hoc test.The dysregulation of glucose use in CAL-1-shA4 cells compared with CAL-1-shCont cells was consistent with our recent report, whereby we found that CAL-1-shA4 promptly decreased mitochondria membrane potential in amino acid–starved conditions in which glucose was included as the carbon source in Earle’s Balanced Salt Solution medium (16). Therefore, we further examined the role of SLC15A4 by focusing on the coupling between glycolysis and the tricarboxylic acid (TCA) cycle in additional cell types. Macrophages change the metabolic state in response to environmental cues such as infectious stimuli and the availability of nutrients, and this dynamic metabolic reprograming is vitally important in macrophages’ functional polarization (2, 18). In Glyco Stress Tests using bone marrow–derived macrophages (BMMϕ), Slc15a4^−/− BMMϕ showed larger ECAR and OCR than Slc15a4^+/+ BMMϕ in the steady state (Fig. 2A). TLR9 or TLR4 stimulation increased ECAR in both Slc15a4^+/+ and Slc15a4^−/− BMMϕ (Fig. 2A), consistent with previous observations that macrophages shift to glycolysis after stimulation (2, 18). These results indicated that SLC15A4 is not required for a metabolic switch toward glycolysis after stimulation. It should be noted that Slc15a4^+/+ BMMϕ showed increased OCR with a concomitant increase in ECAR after TLR9 or TLR4 stimulation (Fig. 2A). In contrast, OCR in Slc15a4^−/− BMMϕ were nearly unchanged after stimulation even though ECAR increased (Fig. 2A). The protein expression of enzymes involved in pyruvate metabolism, such as lactate dehydrogenase A (LDHA), pyruvate dehydrogenase (PDH), and succinate dehydrogenase complex flavoprotein subunit A (SDHA), were not largely affected by SLC15A4 loss, although a slight decrease in LDHA was detectable at 18 h after stimulation in Slc15a4^−/− BMMϕ (SI Appendix, Fig. S3A). These results suggested that Slc15a4^+/+ BMMϕ increased the mitochondrial pyruvate flux of OXPHOS after inflammatory stimulation, while in Slc15a4^−/− BMMϕ, mitochondrial pyruvate flux was not changed before and after stimulation even though it increased glycolysis.Open in a separate windowFig. 2.Metabolic analyses in BMMϕ. (A) Energy map of Seahorse XF Glyco stress test. BMMϕ were stimulated with the indicated TLR agonists for 2 h in complete RPMI containing 10% fetal calf serum. Data showed OCR and ECAR 15 min after the addition of glucose. (B) Principal component analysis of metabolites in the steady state (0 h) 1 and 18 h after CpG stimulation (n = 3). (C and D) Summary of metabolic flow analyses using ¹³C₆-glucose and ¹³C₅-glutamine. BMMϕ stimulated with CpG for 1 or 18 h were incubated for 1 h in the presence of ¹³C₆-glucose (C) and ¹³C₅-glutamine (D), and metabolic intermediates in glycolysis and the TCA cycle were quantified by MS. Values in bar graphs represent the ratio between ¹²C and ¹³C, and the reference of graphs was shown in Bottom Left. Red and closed circles in individual metabolites denote the numbers of ¹³C and ¹²C in the metabolites. References of graphs were shown in the area surrounded by dotted lines. (E) Bar graphs of statistical differences in the indicated metabolites (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s two-tailed t test. (F) Measurement of acetyl-CoA. The ratio of ¹³C₆-glucose–derived acetyl-CoA was calculated and shown as bar graphs of statistical differences (n = 3). **P < 0.01. (G) Phosphorylation of PDHA at Ser293 was compared between wild-type (WT) and Slc15a4^−/− BMMϕ. WT or Slc15a4^−/− BMMϕ were cultured and stimulated with CpG1668 for the indicated periods. (H) The ratio of Itaconic acid and transaconitate derived from ¹³C₆-glucose and ¹³C₅-glutamine (n = 3). Data are representative of two independent experiments.We further examined SLC15A4’s effect on glucose metabolism more directly by metabolic flux analysis using ¹³C₆-glucose and ¹³C₅-glutamine. Principal component analyses of metabolites showed wide differences in metabolite intermediates produced by Slc15a4^+/+ and Slc15a4^−/− BMMϕ, particularly after CpG stimulation (Fig. 2B). Most glycolysis intermediates were elevated 1 h after CpG treatment in both Slc15a4^+/+ and Slc15a4^−/− BMMϕ (Fig. 2C). Pyruvate and lactate levels were higher in Slc15a4^−/− BMMϕ than in Slc15a4^+/+ BMMϕ 18 h after treatment (Fig. 2 C and E). Glucose uptake was not strikingly different between Slc15a4^+/+ and Slc15a4^−/− BMMϕ, although the latter showed a slight decrease in its uptake (SI Appendix, Fig. S3B). Notably, acetyl-CoA levels 18 h after stimulation were significantly decreased in Slc15a4^−/− BMMϕ compared with Slc15a4^+/+ BMMϕ (Fig. 2F). The observation that glucose-derived citrate decreased and pyruvate accumulated in the absence of SLC15A4 was consistent with this observation (Fig. 2 C and E). Succinate was increased 18 h after CpG stimulation in both Slc15a4^+/+ and Slc15a4^−/− BMMϕ, although glucose-derived succinate was decreased in Slc15a4^−/− BMMϕ compared with Slc15a4^+/+ BMMϕ (Fig. 2 C and E). However, succinate from glutamine was more increased in Slc15a4^−/− BMMϕ than in Slc15a4^+/+ BMMϕ (Fig. 2 D and E), suggesting that SLC15A4 loss prevented the smooth integration of glycolysis with the TCA cycle, causing Slc15a4^−/− BMMϕ to draw more glutamine into the TCA cycle. Importantly, phosphorylation of the PDH E1 component subunit alpha PDHA at S293 was increased in Slc15a4^−/− BMMϕ (Fig. 2G). Since PDHA activity is suppressed by the phosphorylation of S293 by PDHK, this observation was consistent with the decreased acetyl-CoA in Slc15a4^−/− BMMϕ. The same trend of PDHA S293 phosphorylation was observed in alveolar Mϕs (SI Appendix, Fig. S3C), strongly suggesting that SLC15A4 mediates the conversion of pyruvate to acetyl-CoA not only in BMMϕ but also in a certain type of tissue-resident macrophage. Taken together, these results indicated that SLC15A4 increased the efficiency of acetyl-CoA formation by controlling PDH activity. More interestingly, Slc15a4^−/− BMMϕ produced significantly low levels of itaconate and transaconitate (Fig. 2H), metabolites that are produced in response to the stimulation-dependent interruption of the TCA cycle in M1 macrophages (19–21). Particularly, itaconate not only has antibacterial activity but also regulates a panel of gene expression associated with immune regulation (21). Thus, these observations revealed that SLC15A4 loss impaired M1-prone metabolic changes and probably the acquisition of M1-associated effector functions after stimulation.We next investigated the significance of SLC15A4-mediated metabolic regulation in Mϕ inflammatory responses and found that CpG-stimulated Slc15a4^−/− BMMϕ showed the severe decrease of Il12b and Il27 expression (Fig. 3A). IL-12 cytokine family uses two different β chains, IL-12/23 p40 (Il12b) and Ebi3, and covers a very broad range of immune responses including proinflammatory to anti-inflammatory responses (22). The expression of Ebi3 was less influenced, and Il12a and Il23a expressions were rather increased in Slc15a4^−/− BMMϕ (Fig. 3A). Tnf was not affected by the presence or absence of SLC15A4, although Il6 was increased (Fig. 3B). These results in BMMϕ were inconsistent with previous studies of B cells and dendritic cells (3, 4). Because BMMϕ were stimulated with CpG in the presence of macrophage colony-stimulating factor (M-CSF), by which PI3K–AKT-mediated signaling events are continuously transmitted, it was likely that the effect of SLC15A4 loss on inflammatory responses differed between cell types depending on the crosstalk with other signaling pathways. The decreased production of IL-12/23 p40 was also confirmed at the protein level (Fig. 3C). These results indicated the selective requirement of SLC15A4 in the expression of IL-12 family members. We further investigated the interrelation between the suppression of Il12b and the metabolic change caused by SLC15A4 loss. Since Slc15a4^−/− BMMϕ showed the increased use of glutamine for TCA cycle, we used Compound 968 (C968), an inhibitor of glutaminase, to inhibit the conversion from glutamine to glutamate, which can be further metabolized to α-ketoglutarate, resulting in blocking the replenishment of glutamine-derived α-ketoglutarate. In the presence of C968, Il12b expression augmented in both Slc15a4^+/+ and Slc15a4^−/− BMMϕ (Fig. 3D). Although Il12b expression induced by C968 in Slc15a4^−/− BMMϕ was lower than that in Slc15a4^+/+ BMMϕ, C968 increased Il12b expression in Slc15a4^−/− BMMϕ to similar levels of Il12b in C968-untreated Slc15a4^+/+ BMMϕ, suggesting the contribution of glutaminolysis to the decreased Il12b in Slc15a4^−/− BMMϕ. At the same time, these results implied a multifaceted contribution of glutaminolysis to Il12b expression because Il12b expression in Slc15a4^+/+ BMMϕ was also inhibited by C968. In contrast, Ebi3 expression was almost not changed by the C968 treatment (Fig. 3D). Conversely, the addition of dimethyl-oxoglutarate (dimethyl-α-ketoglutarate, DMoG) suppressed Il12b expression (Fig. 3E). The effects of C968 and DMoG on IL-12/23 p40 protein expression showed similar trends to Il12b gene expression, although the influence on protein expression became smaller than those on gene expression (Fig. 3F). We did not observe a severe defect in TLR9-mediated canonical signaling events in Slc15a4^−/− BMMϕ (Fig. 3G), consistent with the normal ranges of the TNF-α production by Slc15a4^−/− BMMϕ (Fig. 3 B and C). These results strongly suggested that the SLC15A4-dependent restriction of glutamine replenishment is critical for the selectivity of IL12 family cytokine expressions.Open in a separate windowFig. 3.Effect of glutaminolysis on the production of IL-12 family cytokines in Mϕ. BMMϕ stimulated with CpG1668 in 10% fetal calf serum–containing medium were analyzed. (A) Gene expressions of IL-12 family members after CpG1668 stimulation in BMMϕ were analyzed by RT-PCR. (B) Gene expressions of Tnf or Il6 after CpG1668 stimulation in BMMϕ were analyzed by RT-PCR. (C) TNF-α and IL-12/23p40 productions by BMMϕ. Wild-type (WT) or Slc15a4^−/− BMMϕ were cultured and stimulated with CpG1668 for 18 h, and the culture supernatant was subjected to ELISA. (D and E) Gene expressions of Il12b or Ebi3 after CpG1668 stimulation for the indicated periods in the presence of Glutaminase inhibitor 968 (appeared as C968 in D) or Dimethyl-α-oxo-glutarate (appeared as DMoG in E) in BMMϕ were analyzed by RT-qPCR. (F) Effect of glutaminolysis modulation on IL-12/23p40 production by BMMϕ. WT or Slc15a4^−/− BMMϕ were stimulated with CpG1668 for 18 h in the presence or absence of C968 glutaminase inhibitor or DMoG, and the culture supernatant was analyzed by ELISA. (G) Western blot analysis of the CpG-triggered signaling in BMMϕ. *P < 0.05, **P < 0.01, ns, not significant, as determined by Student’s two-tailed t test. Data are representative of at least two independent experiments.During metabolic analyses, we noticed that respiratory functions in Slc15a4^−/− BMMϕ were easily influenced by serum concentration in culture medium. While Slc15a4^+/+ BMMϕ maintained the OCR and ECAR rates at a certain level regardless of the concentration of serum in the culture medium, Slc15a4^−/− BMMϕ changed OCR and ECAR more sensitively depending on serum concentration, and Slc15a4^−/− BMMϕ possessed higher respiratory reservation than Slc15a4^+/+ BMMϕ in low serum concentration (Fig. 4 A and B). These observations implied that SLC15A4 is critical for the macrophage’s respiratory homeostasis by adapting their metabolic state to different nutrient conditions. Since a macrophage’s nutrient environment changes drastically upon inflammation, the ability of Slc15a4^+/+ BMMϕ to maintain respiratory homeostasis is likely important to enhance their competence for fulfilling inflammatory responses. In addition, OXPHOS generates reactive oxygen species through mitochondrial activity, which has to occur in the controlled manner to avoid host cell damage. We therefore examined the mechanism of respiratory fragility in Slc15a4^−/− BMMϕ by focusing on glutaminolysis and observed that the fragility in respiratory potential was abrogated in the absence of glutamine (Fig. 4C). These results indicated that SLC15A4 is required for macrophage metabolic homeostasis by limiting the availability of glutamine for the TCA cycle.Open in a separate windowFig. 4.SLC15A4-mediated resistance to nutrient stress. (A) Energy maps of Seahorse XF Mito stress test. OCR and ECAR of BMMϕ in different nutrient conditions were measured. The assay medium used was Seahorse XF basic RPMI containing 10% or 1% fetal calf serum (FCS). (B) Data showed values of ECAR and OCR 6 h after CpG stimulation measured by Mito Stress Test. BMMϕ were treated with CpG1668 (open circles) or control medium (closed circles) for 6 h in the presence of 10% (red squares) or 1% (black symbols) FCS. Before starting assay, media were changed to the assay medium (Seahorse XF basic RPMI medium containing 10% or 1% FCS with 10 mM glucose and 2 mM glutamine). (C) Data showed values of ECAR and OCR of BMMϕ in the steady state. BMMϕ were cultured for 6 h in the presence of 10% (red squares) or 1% (black symbols) FCS without glutamine. Assay medium used was Seahorse XF basic RPMI containing 1% or 10% FCS and 10 mM glucose without glutamine. (D) Immunoblotting of BMMϕ stimulated with CpG1668 for the indicated periods in different nutrient conditions. (E and F) Association between SLC15A4 and AMPK. (E) Myc-tagged SLC15A4 was precipitated from WEHI231-SLC15A4 transfectants using anti-myc or isotype-matched antibodies, and precipitates were analyzed by Western blot using the indicated antibodies. (F) HEK293T cells transiently expressing Flag-tagged mouse AMPK-α in combination with the indicated HA-tagged SLC15A4 proteins (Wild type [WT], EK, or mWT for human WT SLC15A4, human E465K, or mouse WT, respectively) were collected. After lysis, samples were subjected to HA or Flag immunoprecipitation and immunoblotting for the HA-tagged proteins. Data are representative of at least two independent experiments.We finally investigated how SLC15A4 affects glutamine metabolism for the TCA cycle. Previous studies reported that glucose limitation causes the AMPK-dependent augmentation of glutaminolysis in T cells (23). AMPK plays a key role in controlling catabolic responses and mitochondrial respiratory function by sensing ATP levels (24, 25). Recent studies demonstrating that AMPK functions at lysosomes (26, 27) prompted us to examine functional relationships between SLC15A4 and AMPK. We compared AMPK activity between the presence or absence of SLC15A4 and found that AMPK-α phosphorylation in the steady state appeared to be increased in Slc15a4^−/− BMMϕ irrespective of serum concentration (Fig. 4D). This might be explained by the decreased mTORC1 activity in Slc15a4-deficient cells (3, 10) and possibly contributes to the increase of glutamine replenishment. Unexpectedly, Slc15a4^−/− BMMϕ severely impaired TLR9-induced AMPK-α activation (Fig. 4D). The phosphorylation of acetyl-CoA carboxylase (ACC) 1/2, the major AMPK substrate, was not affected by the presence or absence of SLC15A4 (Fig. 4D). AMPK-α phosphorylation was entirely absent also in CAL-1-shA4 cells after TLR9 or TLR7 stimulation (SI Appendix, Fig. S4). TLR9-dependent AMPK phosphorylation is mediated by TAK1 and involved in actin remodeling through Rho/Rock phosphorylation (28). TLR9-induced inflammatory response is ATP-consuming processes and reduces energy substrate, leading to AMPK activation (29). AMPK activation switches off some ATP-consuming processes and therefore is important to increase the cellular tolerance against the metabolic stress (24). The activities of AMPK and mTORC1 after TLR9 stimulation were differently controlled by the presence or absence of SLC15A4 (Fig. 4D), indicating that the balance between anabolic and catabolic metabolism after receiving TLR9 stimulation was not correctly regulated in the absence of SLC15A4. We also found that AMPK-α was coprecipitated with SLC15A4 (Fig. 4E). SLC15A4 precipitates also included mTOR and phosphorylated TAK1, and TLR9 stimulation increased the amount of mTOR in the precipitate (Fig. 4E). The association of SLC15A4 and AMPK appeared to be constitutive since it did not differ between the CpG-stimulated and steady states (Fig. 4E). The reconstitution experiments using 293T cells showed that both human and murine SLC15A4 interacted with AMPK-α, although murine SLC15A4 interacted more efficiently than human SLC15A4 (Fig. 4F). SLC15A4’s transporter activity seemed not to be important for AMPKα interaction since the SLC15A4 E465K mutant, which exhibited a severe decrease in transporter activity (3, 16), also could interact with AMPK-α (Fig. 4F). Although TAK1 and AMPK were not identified in BioID, it is possible that SLC15A4 interacts with these molecules via interactions with mTORC1 (30, 31). Taken together, our observations revealed a regulation in M1 macrophage function in which SLC15A4 guards immune cells from metabolic stress by controlling both mTORC1 and AMPK.Our results demonstrated that SLC15A4 plays a vitally important role in the metabolic regulation of innate immune cells such as DCs and macrophages. SLC15A4 enables glycolysis to integrate efficiently with the TCA cycle by governing the acetyl-CoA supply. SLC15A4-deficient macrophages increase glutamine use probably to compensate for carbon in the TCA cycle. Glutamine is an important source of carbon and nitrogen, both of which are also important for massively proliferating cells such as growing tumor cells and clonally expanding T cells (32–34). Furthermore, glutamine metabolism is required for alternative activated macrophages (M2a) and for monocyte epigenetic reprogramming (35, 36), and a lack of glutamine and glutaminolysis augments M1-prone, proinflammatory metabolic changes (35). Therefore, the steady-state expression of SLC15A4 in these innate immune cells safeguards their ability to adapt their metabolic function to the proinflammatory state by connecting glycolysis and the TCA cycle concomitantly with the restriction of glutaminolysis (SI Appendix, Fig. S5). Intriguingly, the use of glutamine to fuel the TCA cycle is also critical for inducing trained immunity (innate immune memory) through the fumarate-dependent regulation of histone demethylases (37). Therefore, SLC15A4’s effect on glutaminolysis also may play a role in trained immunity. In this context, the observation that glutaminolysis largely affected the expression of IL-12 cytokine members might be important. Given that SLC15A4 is critical for IL-12/23 p40 expression partly by restricting glutaminolysis, it is likely that the Il12b gene locus in Slc15a^+/+ BMMϕ is released from glutaminolysis-dependent epigenetic suppression.Our findings also revealed the critical role of SLC15A4 in developing the tolerance against metabolic stress by controlling AMPK and mTORC1 activities. Both mTORC1 and AMPK probably exist in close proximity to SLC15A4, and SLC15A4 may support the crosstalk and/or cooperative regulation of these molecules by providing a scaffold. Thus, SLC15A4 might be one of key molecules for integrating the immune and metabolic circuit to induce appropriate adaptive responses in macrophages, and expressing SLC15A4 gives macrophages an important advantage when responding to inflammatory stimulation in any milieu.In summary, SLC15A4 plays novel and important roles in the link between glycolysis and the TCA cycle and in M1-prone metabolic reprogramming. SLC15A4 is critical for the expression of inflammatory IL-12 cytokine members by restricting glutamine replenishment to the TCA cycle. Furthermore, SLC15A4 maintains cellular respiratory homeostasis and increases cells’ resistance to metabolic stress. Thus, SLC15A4 may stand guard over metabolic resilience in both human and mouse innate immune cells. Our findings shed light on a mechanism of how macrophages exert their functions in the presence of metabolic stress during inflammation and, at the same time, raise a number of intriguing questions relating to the cooperative regulation of anabolic and catabolic responses during inflammation. Since tissue-resident Mϕs contain highly diverse populations in vivo, further careful and detailed analyses using various types of tissue-resident Mϕs are required to clarify the significance of SLC15A4-mediated metabolic regulations in unique functional properties of tissue-resident Mϕs. On the basis of our present findings, it will be an interesting future issue to examine Slc15a4^−/− in inflammatory/infection models in which Mϕs/monocytes play pivotal roles in pathological conditions.     

The RASSF3 candidate tumor suppressor induces apoptosis and G1-S cell-cycle arrest via p53

RASSF3 is the smallest member of the RASSF family of proteins that function as tumor suppressors. Unlike other members of this important family, the mechanisms through which RASSF3 suppresses tumor formation remain unknown. Here, we show that RASSF3 expression induces p53-dependent apoptosis and its depletion attenuates DNA damage-induced apoptosis. We found that RASSF3-induced apoptosis depended upon p53 expression. Exogenous expression of RASSF3 induced G(1)-S arrest, which was also p53 dependent. In contrast, loss of RASSF3 promoted cell-cycle progression, abrogated UVB- and VP-16-induced G(1)-S arrest, decreased p53 protein and target gene expression, and prevented DNA repair. RASSF3 was shown to directly interact with and facilitate the ubiquitination of MDM2, the E3 ligase that targets p53 for degradation, thereby increasing p53 stabilization. Together, our findings show the tumor suppressor activity of RASSF3, which occurs through p53 stabilization and regulation of apoptosis and the cell cycle.     

The root canal bonding of chemical-cured total-etch resin cements

Discovering a durable restorative method to reconstruct and reinforce pulpless teeth is a vital key to help prevent root fractures. Complete and firm adhesion of resin cement in root canal dentin using a post is critical to achieve it. The null hypothesis in the present study was that the bond strength of dual-cured and chemical-cured adhesive resin cements to root canal dentin is not affected by their vertical locations in the root canal. In the experiments, extracted human incisors restored with fiber-reinforced posts and adhesive resin cements were subjected to microtensile bond strength testing. Then, the failure modes and the dentin-bonding interfaces were observed. Self-etch and self-adhesive dual-cured resin cements showed frequent pretesting failure despite using a silane coupling agent. Chemical-cured total-etch adhesive material showed stable bonding performances throughout the entire post space and thus has an advantage in post-core restorations.