Cell-intrinsic Wnt4 ligand regulates mitochondrial oxidative phosphorylation in macrophages
2022; Elsevier BV; Volume: 298; Issue: 8 Linguagem: Inglês
10.1016/j.jbc.2022.102193
ISSN1083-351X
AutoresMouna Tlili, Hamlet Acevedo Ospina, Albert Descoteaux, Marc Germain, Krista M. Heinonen,
Tópico(s)Reproductive System and Pregnancy
ResumoMacrophages respond to their environment by adopting a predominantly inflammatory or anti-inflammatory profile, depending on the context. The polarization of the subsequent response is regulated by a combination of intrinsic and extrinsic signals and is associated with alterations in macrophage metabolism. Although macrophages are important producers of Wnt ligands, the role of Wnt signaling in regulating metabolic changes associated with macrophage polarization remains unclear. Wnt4 upregulation has been shown to be associated with tissue repair and suppression of age-associated inflammation, which led us to generate Wnt4-deficient bone marrow–derived macrophages to investigate its role in metabolism. We show that loss of Wnt4 led to modified mitochondrial structure, enhanced oxidative phosphorylation, and depleted intracellular lipid reserves, as the cells depended on fatty acid oxidation to fuel their mitochondria. Further we found that enhanced lipolysis was dependent on protein kinase C–mediated activation of lysosomal acid lipase in Wnt4-deficient bone marrow–derived macrophages. Although not irreversible, these metabolic changes promoted parasite survival during infection with Leishmania donovani. In conclusion, our results indicate that enhanced macrophage fatty acid oxidation impairs the control of intracellular pathogens, such as Leishmania. We further suggest that Wnt4 may represent a potential target in atherosclerosis, which is characterized by lipid storage in macrophages leading to them becoming foam cells. Macrophages respond to their environment by adopting a predominantly inflammatory or anti-inflammatory profile, depending on the context. The polarization of the subsequent response is regulated by a combination of intrinsic and extrinsic signals and is associated with alterations in macrophage metabolism. Although macrophages are important producers of Wnt ligands, the role of Wnt signaling in regulating metabolic changes associated with macrophage polarization remains unclear. Wnt4 upregulation has been shown to be associated with tissue repair and suppression of age-associated inflammation, which led us to generate Wnt4-deficient bone marrow–derived macrophages to investigate its role in metabolism. We show that loss of Wnt4 led to modified mitochondrial structure, enhanced oxidative phosphorylation, and depleted intracellular lipid reserves, as the cells depended on fatty acid oxidation to fuel their mitochondria. Further we found that enhanced lipolysis was dependent on protein kinase C–mediated activation of lysosomal acid lipase in Wnt4-deficient bone marrow–derived macrophages. Although not irreversible, these metabolic changes promoted parasite survival during infection with Leishmania donovani. In conclusion, our results indicate that enhanced macrophage fatty acid oxidation impairs the control of intracellular pathogens, such as Leishmania. We further suggest that Wnt4 may represent a potential target in atherosclerosis, which is characterized by lipid storage in macrophages leading to them becoming foam cells. Macrophages possess multiple functions, ranging from pathogen clearance and antigen presentation to T lymphocytes to tissue remodeling and immune suppression (1Hirayama D. Iida T. Nakase H. The phagocytic function of macrophage-enforcing innate immunity and tissue homeostasis.Int. J. Mol. Sci. 2017; 19: 92Crossref PubMed Scopus (279) Google Scholar, 2Gordon S. Pluddemann A. Tissue macrophages: heterogeneity and functions.BMC Biol. 2017; 15: 53Crossref PubMed Scopus (308) Google Scholar, 3Wang C. Yu X. Cao Q. Wang Y. Zheng G. 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The prototypical canonical ligand that promotes β-catenin translocation into the nucleus, Wnt3a, increases arginase expression in primary macrophages after bacterial infection and inhibits the secretion of proinflammatory cytokines (15Neumann J. Schaale K. Farhat K. Endermann T. Ulmer A.J. Ehlers S. et al.Frizzled1 is a marker of inflammatory macrophages, and its ligand Wnt3a is involved in reprogramming Mycobacterium tuberculosis-infected macrophages.FASEB J. 2010; 24: 4599-4612Crossref PubMed Scopus (99) Google Scholar, 16Schaale K. Neumann J. Schneider D. Ehlers S. Reiling N. Wnt signaling in macrophages: augmenting and inhibiting mycobacteria-induced inflammatory responses.Eur. J. Cel. Biol. 2011; 90: 553-559Crossref PubMed Scopus (125) Google Scholar). Conversely, Wnt5a, which is usually associated with β-catenin–independent noncanonical Wnt signaling, promotes inflammatory responses via the transcription factor NF-κB to ensure immune surveillance (17Naskar D. Maiti G. 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Pastore C.F. et al.Cell-intrinsic Wnt4 influences conventional dendritic cell fate determination to suppress type 2 immunity.J. Immunol. 2019; 203: 511-519Crossref PubMed Scopus (5) Google Scholar), whose expression is upregulated in lung macrophages upon injury to promote tissue repair (20Hung L.Y. Sen D. Oniskey T.K. Katzen J. Cohen N.A. Vaughan A.E. et al.Macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a Trefoil factor 2-dependent mechanism.Mucosal Immunol. 2019; 12: 64-76Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Wnt4 overexpression in bone marrow was shown to inhibit age-associated inflammation (21Yu B. Chang J. Liu Y. Li J. Kevork K. Al-Hezaimi K. et al.Wnt4 signaling prevents skeletal aging and inflammation by inhibiting nuclear factor-kappaB.Nat. Med. 2014; 20: 1009-1017Crossref PubMed Scopus (164) Google Scholar), while its deletion from dendritic cells impacts their differentiation and promotes the development of type 2 immunity in response to the hookworm parasite Nippostrongylus brasiliensis (19Hung L.Y. Johnson J.L. Ji Y. Christian D.A. Herbine K.R. Pastore C.F. et al.Cell-intrinsic Wnt4 influences conventional dendritic cell fate determination to suppress type 2 immunity.J. Immunol. 2019; 203: 511-519Crossref PubMed Scopus (5) Google Scholar). We thus hypothesized that Wnt4 could also contribute to the metabolic reprogramming of bone marrow–derived macrophages (BMDMs). We show that Wnt4-deficient BMDMs display reduced AKT (Thr308) and ERK1/2 phosphorylation but increased ATP levels, which can be attributed to an enhanced mitochondrial OXPHOS activity. Furthermore, we identify FAO as a principal mechanism involved in the increase in mitochondrial activity. However, while Wnt4-deficient macrophages respond more strongly to lipopolysaccharide (LPS)/M1-type stimulation, their altered FA metabolism favors replication of the protozoan parasite Leishmania donovani. Wnt4-mediated regulation of macrophage metabolism and mitochondrial activity thus appear important for the control of intracellular pathogens. To elucidate the importance of Wnt4 in macrophages, we generated conditional knock-out mice in which Wnt4 is deleted from most macrophages and granulocytes by LysM-Cre–mediated excision (Wnt4Δ/Δ mice) (22Cross M. Mangelsdorf I. Wedel A. Renkawitz R. Mouse lysozyme M gene: isolation, characterization, and expression studies.Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6232-6236Crossref PubMed Google Scholar, 23Clausen B.E. Burkhardt C. Reith W. Renkawitz R. Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1550) Google Scholar). These mice present no overt alterations in myeloid differentiation in vivo (24Hetu-Arbour R. Tlili M. Bandeira Ferreira F.L. Abidin B.M. Kwarteng E.O. Heinonen K.M. Cell-intrinsic Wnt4 promotes hematopoietic stem and progenitor cell self-renewal.Stem Cells. 2021; 39: 1207-1220Crossref PubMed Scopus (5) Google Scholar). We isolated BM cells from Wnt4Δ/Δ and Cre- littermate control mice, and we obtained comparable numbers of Wnt4Δ/Δ and control BMDM after 1 week in culture (Fig. 1, A and B). While Wnt4 deletion was highly efficient in culture (Fig. 1C), Wnt4Δ/Δ and control BMDM expressed similar levels of the macrophage surface marker F4/80 (Fig. 1D), suggesting that Wnt4 deficiency did not significantly alter BMDM differentiation from BM progenitors. There was no difference in β-catenin phosphorylation (Fig. 1E) or in the activation of c-Jun N-terminal protein kinase (JNK) (Fig. 1, F and G), suggesting that the deletion of Wnt4 did not alter the balance between canonical and JNK-dependent noncanonical signaling in the absence of other exogenous ligands. Macrophage function is regulated not only by JNK but also other members of the mitogen-activated protein kinase (MAPK) family, such as the extracellular signal–regulated kinases 1 and 2 (ERK1/2) (25Traves P.G. de Atauri P. Marin S. Pimentel-Santillana M. Rodriguez-Prados J.C. Marin de Mas I. et al.Relevance of the MEK/ERK signaling pathway in the metabolism of activated macrophages: a metabolomic approach.J. Immunol. 2012; 188: 1402-1410Crossref PubMed Scopus (52) Google Scholar, 26Papa S. Choy P.M. Bubici C. The ERK and JNK pathways in the regulation of metabolic reprogramming.Oncogene. 2019; 38: 2223-2240Crossref PubMed Scopus (148) Google Scholar). Unlike JNK, there was a notable decrease in ERK1/2 phosphorylation in Wnt4Δ/Δ BMDM (Fig. 1,H and I). Wnt4Δ/Δ BMDM also showed a decreased level of AKT phosphorylation on Thr308 (Fig. 1, J and K), suggesting that these signal transduction pathways are altered in these cells. Nevertheless, phosphorylation of the mTORC2-dependent site in AKT (Ser473) (Fig. 1L), as well as that of the mTORC1 downstream effectors S6 (Ser235/236) (Fig. 1M) and 4EBP1(Thr37/46) (Fig. 1N) was not affected in Wnt4Δ/Δ BMDM, indicating that Wnt4 regulates ERK1/2 and AKT signaling independently from the mTORC axis. Considering the dual roles of ERK1/2 and AKT in cell proliferation and metabolism (27Yu J.S. Cui W. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination.Development. 2016; 143: 3050-3060Crossref PubMed Scopus (589) Google Scholar, 28Altomare D.A. Khaled A.R. Homeostasis and the importance for a balance between AKT/mTOR activity and intracellular signaling.Curr. Med. Chem. 2012; 19: 3748-3762Crossref PubMed Scopus (86) Google Scholar, 29Cargnello M. Roux P.P. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases.Microbiol. Mol. Biol. Rev. 2011; 75: 50-83Crossref PubMed Scopus (1819) Google Scholar), we further evaluated the proliferative state of Wnt4Δ/Δ BMDM using Ki-67. Ki-67 expression was not significantly changed in Wnt4Δ/Δ BMDM compared to control (Fig. 1O), which together with the comparable cell counts (Fig. 1B) suggests that Wnt4 does not affect BMDM proliferation. However, impaired ERK1/2 and AKT (Thr308) activation prompted us to further investigate the functional consequences of Wnt4 deletion on BMDM metabolism. To address the impact of Wnt4 on macrophage metabolism, we first investigated their ATP levels. Wnt4Δ/Δ BMDM displayed higher intracellular ATP levels (Fig. 2A) but a comparable ADP/ATP ratio relative to control cells (Fig. 2B), suggesting that this increase in ATP is not the consequence of alterations in ATP consumption. In addition, intracellular ATP was decreased to similar levels in both genotypes upon inhibition of the ATP synthase with oligomycin (Fig. 2A). As these data suggest that OXPHOS is increased in Wnt4Δ/Δ BMDM, we measured oxygen consumption rates (OCRs) in control and Wnt4Δ/Δ BMDM. Consistent with Wnt4Δ/Δ BMDM having increased OXPHOS activity, basal OCR was significantly increased in these cells (Fig. 2, C and D). ATP-linked respiration was also increased in Wnt4Δ/Δ BMDM (Fig. 2E), further supporting a role for OXPHOS in the elevated cellular ATP levels observed in Wnt4Δ/Δ cells. On the other hand, the spare respiratory capacity (a measure of the ability of mitochondria to respond to an increased energy demand) (Fig. 2F) was similar between the two genotypes while proton leak (a measure of proton diffusion across the inner membrane) (Fig. 2G) was decreased in Wnt4Δ/Δ BMDM. In addition, mitochondrial membrane potential and mitochondrial ROS levels in Wnt4Δ/Δ BMDM were comparable to controls (Fig. 2,H and I). Altogether, our results indicate that mitochondria in Wnt4Δ/Δ cells have increased flux through the electron transport chain and ATP synthase without major impairment in mitochondrial function. We then determined if the functional changes we observed in Wnt4Δ/Δ BMDM were associated with changes in mitochondrial structure or mass. We first stained mitochondria in control and Wnt4Δ/Δ BMDM macrophages for the mitochondrial outer membrane protein TOM20 and imaged them by confocal microscopy (Fig. 2J). While control BMDM had on average very short mitochondria, we observed a significant increase in intermediate mitochondria in Wnt4Δ/Δ BMDM (Fig. 2K). Mitochondrial elongation and increased OXPHOS can be associated with changes in cristae structure, the folds of the inner membrane where the electron transport chain resides. We thus used electron microscopy to evaluate cristae width in control and Wnt4Δ/Δ BMDM. Wnt4Δ/Δ BMDM had tighter cristae than their control counterparts (Fig. 2L), suggesting improved OXPHOS efficiency. On the other hand, there was no difference in mitochondrial mass as measured by citrate synthase activity (Fig. 2M) or TOM20 immunoblotting (Fig. 2N). In sum, these findings indicate that Wnt4 controls mitochondrial activity without altering mitochondrial mass. As our results suggest that the increased OCR and ATP levels observed in Wnt4Δ/Δ BMDM is the consequence of increased metabolic flow through OXPHOS rather than a major change in mitochondrial structure, we then analyzed the potential substrates supporting OXPHOS in these cells. Wnt4Δ/Δ BMDM showed a substantial decrease in lactate production (Fig. 3A), which was associated with a smaller but significant reduction in glucose consumption (Fig. 3B). While these results are consistent with the greater OXPHOS activity, we observed they also suggest that glucose is not the major source of metabolic intermediates supporting enhanced mitochondrial activity in Wnt4Δ/Δ BMDM. In contrast, there was a drastic reduction in the lipid droplets present in Wnt4Δ/Δ BMDM relative to control cells, as measured by Oil Red O staining (Fig. 3, C and D), suggesting that fatty acids (FAs) could be fueling the increased OXPHOS in these cells. Lipid droplets store triglycerides that must be hydrolyzed to liberate the FA used in mitochondrial ß-oxidation. As lysosomal acid lipase (LAL) is one of the key enzymes that cells use to liberate FA from lipid droplets (30Remmerie A. Scott C.L. Macrophages and lipid metabolism.Cell Immunol. 2018; 330: 27-42Crossref PubMed Scopus (178) Google Scholar, 31Singh R.K. Barbosa-Lorenzi V.C. Lund F.W. Grosheva I. Maxfield F.R. Haka A.S. Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse.J. Cel. Sci. 2016; 129: 1072-1082Crossref PubMed Scopus (24) Google Scholar), we measured its activity in control and Wnt4Δ/Δ BMDM. Consistent with the reduction in lipid droplets, LAL activity was increased 2-fold in Wnt4Δ/Δ BMDM as compared to controls (Fig. 4A). However, this increase is neither due to an increase in Lipa gene expression (Fig. S1A) nor to an increase in the overall lysosomal content as we did not observe any change in the activity of the lysosomal protease Cathepsin B (Fig. 4B) or the expression of the lysosomal membrane protein LAMP-1 (Fig. 4C). Moreover, LAL inhibition restored cytosolic lipid content in Wnt4Δ/Δ BMDM (Fig. 4, D and E). Consistent with a specific role for LAL, the cytosolic neutral lipase activity was similar between Wnt4Δ/Δ and control BMDM (Fig. 4F), and the genes for the enzymes responsible for this activity, Lipe and Pnpla2, were expressed at very low levels but similar levels in both genotypes (Fig. S1, B and C). Altogether these data indicate that enhanced LAL activity is responsible of FA generation in Wnt4Δ/Δ cells. As autophagy has been implicated in LAL-dependent lipid metabolism in macrophages (32Ouimet M. Franklin V. Mak E. Liao X. Tabas I. Marcel Y.L. Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase.Cell Metab. 2011; 13: 655-667Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), we assessed autophagy flux with the membrane autophagosome marker LC3. Although we observed a decrease in the number of LC3 puncta in Wnt4Δ/Δ BMDM that was partially rescued with bafilomycin treatment (Fig. 4,G and I), there was no significant increase in the ratio of LC3 puncta (treated/untreated) between Wnt4Δ/Δ BMDM and controls (Fig. 4H), indicating that the autophagic flux was not enhanced in Wnt4Δ/Δ BMDM. Put together, these data point toward increased lipid degradation by LAL in Wnt4Δ/Δ BMDM, irrespective of alterations in autophagy. Decreased glucose consumption and decreased lipid storage support the hypothesis of FAO as a major source of energy in Wnt4Δ/Δ BMDM. To more directly evaluate the relative importance of each carbon source, we inhibited glycolysis with 2-deoxy-d-glucose (2-DG) or FA transport into mitochondria using etomoxir. We then measured OCR in otherwise unstimulated and nonpolarized macrophages, similar to Figure 2. Control BMDM showed very little alteration in their basal OCR in response to blocking either one of the two pathways (Fig. 5, A–C), likely reflecting their relatively low level of metabolic activity in the absence of stimulation (33Van den Bossche J. Baardman J. de Winther M.P. Metabolic characterization of polarized M1 and M2 bone marrow-derived macrophages using real-time extracellular flux analysis.JoVE. 2015; 28: 53424Google Scholar). Consistent with ß-oxidation providing the extra carbon source to fuel OXPHOS in Wnt4Δ/Δ cells, etomoxir significantly decreased basal OXPHOS in these cells. Similarly, etomoxir significantly reduced ATP-linked OCR in Wnt4Δ/Δ but not in control BMDM (Fig. 5D), and it also increased the spare capacity of Wnt4Δ/Δ BMDM but not of control cells (Fig. 5E). Moreover, Wnt4Δ/Δ BMDM challenged with palmitate showed a significant decrease in OCR upon etomoxir treatment while control BMDM was not affected (Fig. 5F). This coincided with an increase in Oil Red O staining in etomoxir-treated Wnt4Δ/Δ BMDM, restoring their lipid droplets to control levels (Fig. 5, G and H) and further supports a role for ß-oxidation in the metabolic changes observed in Wnt4Δ/Δ BMDM. Treatment with 2-DG also somewhat decreased basal OCR in Wnt4Δ/Δ BMDM (Fig. 5, A–C), suggesting that glucose can also contribute to their enhanced OXPHOS. However, the expression of Pdh1, an enzyme required to commit pyruvate to the TCA cycle, was not enhanced in Wnt4Δ/Δ BMDM (Fig. S1D), suggesting no major changes in pyruvate handling by Wnt4Δ/Δ BMDM. Altogether, these results indicate that the loss of Wnt4 stimulates the usage of lipids as an important source of energy. To better establish how Wnt4 regulates lipid metabolism, we evaluated putative signaling pathways downstream of Wnt4. While JNK phosphorylation (Fig. 1, E and F) and AKT-dependent β-catenin phosphorylation were not altered in Wnt4Δ/Δ BMDM (Fig. 1D), the classical β-catenin-dependent target genes c-Myc and Ccnd1 (Axin2 was not expressed in BMDM) were downregulated in Wnt4Δ/Δ BMDM (Fig. S1, E and G). As the noncanonical protein kinase C (PKC)/Ca2+ pathway negatively regulates TCF/β-catenin-dependent gene expression without impacting intracellular β-catenin levels (34Ishitani T. Kishida S. Hyodo-Miura J. Ueno N. Yasuda J. Waterman M. et al.The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling.Mol. Cell. Biol. 2003; 23: 131-139Crossref PubMed Scopus (470) Google Scholar) and inhibits ERK1/2 (35Valledor A.F. Xaus J. Marques L. Celada A. Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.J. Immunol. 1999; 163: 2452-2462PubMed Google Scholar), our results suggest that the absence of Wnt4 promotes the activation of this PKC pathway. Since PKC activity has also been associated with LAL induction during monocyte differentiation into macrophage (36Ries S. Buchler C. Langmann T. Fehringer P. Aslanidis C. Schmitz G. Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2.J. Lipid Res. 1998; 39: 2125-2134Abstract Full Text Full Text PDF PubMed Google Scholar), we evaluated the impact of PKC inhibition on LAL activity in Wnt4Δ/Δ BMDM. While PKC inhibition had no impact on control BMDMs, LAL activity in Wnt4Δ/Δ BMDM was reduced to control levels upon PKC inhibition (Fig. 5I), suggesting that the enhanced lipolysis observed in the absence of Wnt4 is PKC-dependent. Considering the decrease in AKT (Thr308) phosphorylation observed in Wnt4Δ/Δ BMDM (Fig. 1I), we also evaluated the contribution of the PI3K/PTEN axis. To our surprise, LAL activity was further enhanced in Wnt4Δ/Δ BMDM upon PTEN inhibition (Fig. 5J). While we cannot exclude a potential contribution of AKT downstream of PI3K upon PTEN inhibition, it is possible that the increase in PI3K activity triggered by PTEN inhibition promotes PKC activation via PDK-1 (37Wolf A.M. Lyuksyutova A.I. Fenstermaker A.G. Shafer B. Lo C.G. Zou Y. Phosphatidylinositol-3-kinase-atypical protein kinase C signaling is required for Wnt attraction and anterior-posterior axon guidance.J. Neurosci. 2008; 28: 3456-3467Crossref PubMed Scopus (81) Google Scholar, 38Pearn L. Fisher J. 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However, flow cytometry analysis revealed no significant differences in the expression of M1 (CD86, MHCII) or M2 cell surface markers (CD206) between unstimulated Wnt4Δ/Δ and control BMDM (Fig. 6, A–C). There was also no difference in cathepsin B activity (Fig. 4B), the most abundant lysosomal protease (40Cavallo-Medved D. Moin K. Sloane B. Cathepsin B: basis sequence: mouse.The AFCS-Nature Mol. Pages. 2011; 2011A000508Google Scholar) whose activity has been shown to be increased in M2 macrophages (41Oelschlaegel D. Weiss Sadan T. Salpeter S. Krug S. Blum G. Schmitz W. et al.Cathepsin inhibition modulates metabolism and polarization of tumor-associated macrophages.Cancers. 2020; 12: 2579Crossref Scopus (13) Google Scholar). In summary, the metabolic differences in Wnt4Δ/Δ BMDM did not appear to result in an inherent bias in unstimulated cells. LPS is a toll-like receptor 4 agonist that is widely used to promote the secretion of proinflammatory cytokines by macrophages (42Park B.S. Lee J.O. Recognition of lipopolysaccharide pattern by TLR4 complexes.Exp. Mol. Med. 2013; 45: e66Crossref PubMed Scopus (600) Google Scholar). Moreover, LPS-treated macrophages reduce their oxygen consumption and adopt a strongly glycolytic profile (10Liu L. Lu Y. Martinez J. Bi Y. Lian G. Wang T. et al.Proinflammatory signal suppresses proliferation and shifts macrophage metabolism from Myc-dependent to HIF1alpha-dependent.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 1564-1569Crossref PubMed Google Scholar). We thus stimulated Wnt4Δ/Δ and control BMDM with LPS to determine if the metabolic differences in Wnt4Δ/Δ BMDM were reversible. There was a strong suppression of OCR in LPS-treated Wnt4Δ/Δ BMDM, with basal OCR decreasing even slightly below levels detected in LPS-treated controls (Fig. 6, D and E). Unsurprisingly, this also corresponded to a significant decrease in ATP-linked respiration (Fig. 6F), indicating that LPS inhibits mitochondrial activity in both Wnt4Δ/Δ and control BMDM. Similarly, extracellular acidification rate was increased to similar levels in LPS-treated Wnt4Δ/Δ and control BMDM, suggesting an increase in lactate production (Fig. 6G). These data indicate that the LPS-induced metabolic switch to glycolysis is not impaired in Wnt4Δ/Δ BMDM. To further evaluate the inflammatory potential of Wnt4Δ/Δ BMDM, we measured nitric oxide (NO) and TNFα production in culture supernatants with and without LPS stimulation. Wnt4Δ/Δ BMDM produced slightly more NO (Fig. 6H) and similar levels of TNFα (Fig. 6I) upon LPS stimulation as compared to their normal counterparts. Furthermore, Wnt4Δ/Δ and control BMDM showed a comparable expression of M1 (iNOS) and M2 (arginase-1) markers following LPS/IFN-γ and IL-4
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