Bone Marrow-derived MΦs Research Articles

Abstract We066: Macropinocytosis is essential for macrophage activation in acute ischemia.

Macrophages (Mφ) play central roles in inducing the robust inflammatory response to acute ischemia and promoting anti-inflammatory actions necessary for myocardial repair. The broad range of functional repertoire stems from Mφ plasticity dictated by their interaction with the dynamic ischemic milieu. The limited understanding of such interactions contributes to the lack of means to mitigate inflammatory injury in MI patients. We hypothesize that the dynamic Mφ plasma membrane-mediated process macropinocytosis (MP) is essential for Mφ’s activation and function evolvement in ischemia. By MP, Mφs engulf large amounts of soluble contents from the ischemic environment, and the macropinosomes formed from MP become signaling hubs of DAMP (Damage-Associated Molecular Pattern)-PRR (Pattern Recognition Receptor). We aim to determine if ischemia activates MP in Mφs, the signaling pathways that MP promotes, and the role of this process in post-ischemia repair. The rationale is based on our data demonstrating only soluble DAMP (sDAMP), not tissue debris, activates Mφs . We tested our hypothesis using the murine MI model and bone marrow-derived Mφs (BMDM) and used unpaired t-tests for statistical analyses. We uncover that ischemia markedly increases MP in Mφs; blocking MP by the inhibitors or siRNA-mediated knock-down of the Na+H+ exchanger abolished the production of potent cytokines such as IL1β from Mφs in vitro and in vivo . Furthermore, MP activates NF-κB and MAP kinase triggered by sDAMP and ischemia, critical pathways to produce inflammatory cytokine and chemokine. Intriguingly, mTORC1 activation in BMDM and Mφs in the ischemic myocardium depends on MP. These data suggest MP organizes the activation signal and the pathway required for metabolic rewiring in inflammatory Mφs. Suppressing MP at the earlier stage of ischemia (72h) when sDAMP is abundant in the ischemic tissue significantly reduces mortality and improves cardiac function. In conclusion, we identify MP functionalizes the danger signals in bulk and integrates activation signals with metabolic rewiring to promote and sustain the inflammatory function. Targeting MP is better equipped to reduce inflammatory injury as it modifies the dynamic interaction of Mφs with the ischemic environment.

Abstract 148: Loss Of Tlr4 Signaling Inhibits Thrombus Resolution In A Non-monocyte Dependent Manner

Background: Loss of TLR4 signaling reduces Venous Thrombosis (VT) resolution. Leukocyte expression of TLR4 is elevated in humans undergoing acute to chronic DVT transformation. We hypothesized that TLR4 signaling in monocytes directs thrombus clearance. Methods: Wild type, Tlr4 -/- , LysM cre+/- Tlr4 fl/fl mice underwent VT via IVC stasis at 4/7/14d timepoints. Thrombi subtypes were identified by flow cytometry. Thrombus size was assessed, and fibrinolytic mediators were assayed in splenic/bone marrow Mφs. Whole blood from mice was stimulated with TLR2/4 and assayed for thrombolytic potential via HALO assay. Immunohistochemistry was performed for cellular infiltrate. Results: We observed elevated TLR4 cell surface expression on CD11b+/Ly6C- Mo/Mφ from wild type thrombi. Thrombus weight in Tlr4 -/ mice was elevated at 4d stasis, confirming impaired thrombus clearance. In basal conditions, circulating Mo/Mφ saw decreased Urokinase & MMP9 mRNA levels in LysM cre+/- mice. In post thrombotic conditions, we saw decreased protein levels of Urokinase and MMP9 in Cre+ bone marrow-derived Mφs. Whole blood thrombolysis efficiency increased with TLR4 agonism in Cre- mice compared to Cre+. Efficiency was restored with LPS-mediated dual TLR2/4 agonism. Total thrombus weight was unchanged. No difference was identified in monocyte infiltrate into the thrombus (Fig. 1) Conclusions: Genetic deficiency of Mo/Mφ TLR4 signaling alters induction of fibrinolytic/matrix remodeling gene expression. Ex vivo TLR4 agonism increased whole blood thrombolysis in Cre- but not Cre+ mice. Additional TLR2 agonism of Cre+ mice restored thrombolytic activity, suggesting TLR2 as a compensatory pathway that may account for the unchanged in vivo phenotype in LysMcreTlr4fl/fl mice. Further clarifying these cellular pathways may provide an alternate pathway to target VT resolution.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our invitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Interleukin-10 increases macrophage-mediated chemotherapy resistance via FABP5 signaling in multiple myeloma

Macrophages (MΦs) protect multiple myeloma (MM) cells from chemotherapy-induced apoptosis, and interleukin-10 (IL-10) is frequently elevated in the MM microenvironment. However, the role of IL-10 in MΦ-induced tumor chemotherapy resistance has not yet been clarified. In the present study, bone marrow-derived MΦs were treated with IL-10 (IL10-MΦs), and IL10-MΦ-induced MM chemotherapy resistance was evaluated. IL-10 promoted MΦ-mediated resistance to MM chemotherapy. In addition, IL-10 treatment increased lipid accumulation and fatty acid β-oxidation in MΦs. Mechanistically, IL-10 increased fatty acid binding protein 5 (FABP5) expression in MΦs, and targeting FABP5 decreased MM chemotherapy resistance induced by IL10-MΦs in vitro and enhanced chemotherapeutic efficacy in vivo. Inhibition of FABP5 decreased the expression of Carnitine Palmitoyltransferase 1A (CPT1A) in IL10-MΦs. In addition, inhibition of CPT1A in IL10-MΦs decreased IL10-MΦ-mediated MM chemotherapy resistance. Peroxisome proliferator-activated receptor γ (PPARγ) is upstream of FABP5 signaling. Inhibition of PPARγ in IL10-MΦs decreased IL10-MΦ-mediated MM chemotherapy resistance in vitro. Collectively, our work indicates that IL-10 enhances MΦ-mediated MM chemotherapy resistance via FABP5 signaling and targeting FABP5 has potentially important clinical implications.

Abstract 419: Liberation Of Soluble Urokinase Plasminogen Receptor (supar) Occurs In Response To Coronavirus Infection But Not To Influenza Virus Infection

Objectives: Urokinase (uPA), and its receptor, uPAR, mediate fibrinolysis and matrix remodeling. Liberation of monocyte/macrophage (MO/Mϕ) bound uPAR to yield soluble uPAR (suPAR) occurs after infection with coronaviruses (CoVs) such as SARS-CoV-2, and suPAR levels correlate with systemic inflammation and degree of end organ injury. How suPAR liberation from MO/Mϕs occurs after CoV infection, and whether suPAR induction occurs after pneumotropic viral infections such as influenza A (IAV) is unclear. Methods: Murine bone marrow derived Mϕs (BMDMs) and immortalized Mϕs (RAW264.7) were infected with CoV [MHVA59 (murine model for SARS-CoV-2 infection) or MHV1] or with IAV (A/PR/8/34) and levels of mRNA and protein were measured using qRT-PCR and ELISA, respectively. uPA activity of cell supernatants was quantitated using a commercially available assay. uPA knockdown of Mϕs was performed using siRNA transfection. C57BL6/J mice underwent intranasal inoculation of either 2x10 5 pfu of MHVA59, 25 pfu of IAV, or with PBS (sham). Plasma levels of suPAR and splenic Mϕ (surrogate for circulating MOs) levels of mRNA and protein were analyzed at post-infection day 3. Results: Though both CoV or IAV infection resulted in induction of mRNA and protein levels of cellular uPAR and uPA, and uPA activity, only CoV infection yielded liberation of suPAR from Mϕs (Fig. 1A-C). Indicating a functional role in suPAR liberation, infection of uPA-silenced Mϕs yielded a blunted induction of suPAR release despite not affecting mRNA levels of uPAR (Fig 1D). Finally, inoculation of mice with MHVA59, but not with IAV, resulted in elevated plasma levels of suPAR, despite inducing a robust induction of uPAR in splenic Mϕs (Fig. 1E). Conclusions: suPAR induction results after CoV infection, but not IAV infection, and occurs through a uPA-dependent pathway. Antagonism of co-regulators of uPAR and uPA may prove to be a therapeutic strategy to combat post-CoV tissue injury mediated by Mϕs.

Abstract 15731: Rpl13a Small Nucleolar RNAs Promote Atherosclerosis and Oxidative Stress

Reactive oxygen species (ROS) exacerbate atherosclerosis (athero). ROS levels are elevated by specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene. We therefore tested the hypothesis that these snoRNAs promote athero, using “snoKO” mice deficient in Rpl13a snoRNAs, but not in Rpl13a itself. ROS levels assessed by CellROX Orange were 35% lower in snoKO than snoRNA +/+ aorta frozen sections ( p <0.01). After 14 wk on Western diet, female snoKO/ Apoe -/- unexpectedly showed total cholesterol levels 20% higher than Apoe -/- mice in (1,046 vs 869 mg/dl, p <0.05). Despite this, neointimal lesions in brachiocephalic artery (BCA) cross-sections were 50% smaller in snoKO/ Apoe -/- than in Apoe -/- mice, and lumen size was 45% larger (both p <0.01, n=8/group). Similar data were obtained in males: snoKO/ Apoe -/- mice had 40% smaller BCA lesion areas ( p <0.02, n=8/group). After being stained for cholesteryl ester with BODIPY, for ACTA2 by immunofluorescence and for DNA (Hoechst), BCAs from female snoKO/ Apoe -/- mice (n=9) demonstrated 50% less foam cell-positive and 95% more ACTA2 + area, 40% less necrotic core area, and a 60% lower prevalence of ACTA2 + foam cells ( p <0.05 for each). Thus, Rpl13a snoRNAs promote vascular ROS and athero. Assessed by MitoSOX Red, ROS levels were 25% lower in snoKO than WT M1-polarized bone marrow-derived Mϕs in vitro (n=3, p<0.05). To identify mechanisms linking the Rpl13a snoRNAs to ROS and athero, we performed LC-MS/MS on WT and snoKO aortic SMCs. COX4I2 was expressed 5.7-fold higher in snoKO than WT SMCs by MS/MS, and 2.5-fold higher in snoKO SMCs by immunoblot (n=3/group, p <0.05). As part of mitochondrial complex IV, COX4I2 lowers cellular ROS levels. We used CRISPR/Cas9 to create 293T cells lacking either the RPL13a -snoRNA U34A or the irrelevant snoRNA U25 . With mRNA from these cells we performed reverse transcription at low [dNTP] followed by qPCR (RTL-P) for COX4I2 . Inversely proportional to the degree of mRNA 2’- O -methylation (mediated by snoRNAs), the RTL-P efficiency was 4-fold higher in U34A -knockout than control cells (3 clones/genotype, p <0.01). Thus, COX4I2 mRNA appears to be regulated by RPL13A -snoRNA-guided 2’- O -methylation in a manner that could link Rpl13a snoRNAs, vascular ROS, and athero.

425.6: Recognition of Donor MHCI Disrupts Recipient Reparative Macrophage Differentiation

Introduction: After tissue injury, infiltrating monocytes differentiate into CD86hiLy6chi pro-inflammatory and CD206+CD301+ reparative macrophages (MΦs) that complete repair. MΦs differentiation is coordinated by tissue injury signals, often referred to as damage-associated molecular patterns (DAMPs), and local cytokines. For example, after heart transplant (HTx) IL-33 is released from graft fibroblasts and acts as a reparative/regulatory DAMP that metabolically reprograms MΦs to differentiate into reparative subsets that protect against chronic HTx rejection. Transplanting allogeneic materials is a novel situation where innate allorecognition of mismatched MHCI may disrupt normal MΦ responses to damaged tissues. For instance, paired immunoglobulin-like receptor (PIR)-A3 on C57BL/6 (B6) monocytes recognition of BALB/c H2-Dd triggers their differentiation into pro-inflammatory dendritic cells that promotes HTx rejection. Monocyte PIR-B binding to self MHCI negatively regulates PIR-A signals. How PIRs shapes DAMP-mediated MΦ differentiation after Tx is not understood, so we hypothesize that in addition to DAMPs, recognition of self versus allogeneic MHCI by PIRs modulates reparative or inflammatory MΦ differentiation. Methods: Monocyte-derived MΦ differentiation in response to donor materials was tested by administration of 20x106 irradiated BALB/c allogeneic (H-2d; Allo) or (B6) syngeneic (H-2b; Syn) splenocytes intraperitoneally alone or with recombinant IL-33. Recipients of donor cellular materials were B6 wildtype (WT), Rag2-/-γc-/-, Pira-/- and Pirb-/- mice. The precise impact of H-2Dd (Allo) or H-2Db (Syn) recombinant MHCI on MΦ differentiation was determined using B6 WT and Pira-/- bone marrow-derived MΦs (BMDMs). Flow cytometry was utilized to assess MΦ differentiation. Results: Administration of Syn cells predominantly stimulated monocyte differentiation into CD206+CD301+ reparative MΦs. Allo cells instead drove monocyte differentiation to pro-inflammatory Ly6chiCD86hi MΦs. A similar outcome was observed in Rag2-/-γc-/- recipients, suggesting these changes are not driven by adaptive immunity. Incubation of BMDMs with recombinant allo MHCI, but not syn MHCI, triggered inflammatory Ly6chiCD86hi MΦs in both WT and Pira-/- BMDMs. Co-administration of allo cells with recombinant IL-33 supported differentiation into CD206+CD301+ reparative MΦs. Interestingly, Pirb-/- mice display reduced C206+CD301+ MΦs, which correlated with decreased IL-33 receptor expression. Conclusions: These data indicate allorecognition by monocytes or MΦs disrupts the typical generation of reparative MΦs in response to injury. We show that monocyte-derived MΦs PIR-B recognition of self MHCI may trigger expression of the receptor needed for IL-33-mediated reparative MΦ differentiation. These new data provide important insights into how innate allorecognition and repair pathways intersect to direct the differentiation of reparative or inflammatory MΦs that shape HTx outcomes. #R01AR073527 (HRT) #5T32AI074490-14 (JPW).

Circadian control of macrophages in the tumor microenvironment.

Abstract Introduction All leukocytes tested to date have functional circadian clocks, and nearly every arm of the immune response is subject to circadian regulation. Circadian clocks instruct the time-of-day-dependent, rhythmic expression of genes in a tissue- and cell-specific manner. In macrophages (mΦs), the circadian clock regulates several factors that are critical to executing effective immune responses. Tumor-associated mΦs are major contributors to immune suppression in the tumor microenvironment (TME). Evidence suggests that metabolically stressful factors in the TME such as acidic pH and nutrient limitation promote mΦ-mediated immune suppression, and recent data point to dysregulation of the circadian clock downstream of metabolic stress. Methods We study the effect of TME-associated metabolic stress on the circadian clock of mΦs in vitro by culturing bone marrow-derived mΦs in conditions mimicking acidic pH and nutrient limitations that have been observed in the TME. To study the impact of mΦ-intrinsic circadian rhythms on tumorigenesis in vivo, we use mice genetically engineered to have a myeloid cell-specific disruption of the circadian clock via deletion of the key clock protein BMAL1. Results Oscillation of core clock proteins is altered in mΦs subjected to TME-associated metabolic stress. Additionally, we observe increased tumor growth in mice co-injected with mΦs whose circadian clocks were disrupted compared to mice co-injected with mΦs whose circadian clocks were functional. Conclusion Our data suggests that stressful conditions associated with the TME can alter the mΦ circadian clock, and that a functional circadian clock in mΦs can suppress tumor growth in a syngeneic murine tumor model of pancreatic cancer. This research has been supported by the following fellowships and grants: 2021-Current: Wilmot Predoctoral Cancer Research Fellowship, Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY 2020-2021: NIH T32 Training Grant in Cellular, Biochemical & Molecular Sciences, University of Rochester Medical Center, Rochester, NY

Post-Thrombotic loss of the epigenetic enzyme MLL1/KMT2a in macrophages suppresses urokinase expression and may contribute to an antifibrinolytic phenotype

Abstract Objectives: Macrophages (Mϕs) are critical in the process of subacute and chronic venous thrombus (VT) resolution. Epigenetic alterations can reprogram Mϕ function in chronic inflammatory states. We hypothesized that the chromatin modifying enzyme MLL1/KMT2a, which increases H3K4 trimethylation at NF-kβ targeted promoters during inflammatory processes, influences Mϕ-mediated post-thrombotic fibrinolysis. Methods: Bone marrow derived Mϕs (BMDMs) from C57bl6 mice and immortalized Mϕs (RAW264.7) were used for in vitro experiments. Small interfering RNAs (siRNAs) were transfected to achieve MLL1 silencing, and qPCR, immunoblotting, and chromatin immunoprecipitation (ChIP) assays were performed. In vivo, VT formation was induced by IVC ligation, after which BMDMs were harvested. Results: Analysis of procoagulant and antifibrinolytic transcripts in MLL1 silenced BMDMs and RAW264.7 cells revealed suppression of urokinase (Plau) mRNA and protein levels by >80% and >50% respectively (p<0.05) relative to controls. ChIP analysis of the Plau promoter in MLL1 knockdown cells showed decreased enrichment (~4.6 fold, p=0.0216) of H3K4me3 and suggested a functional role for MLL1 to promote Plau expression. BMDMs harvested from mice 7 days post-thrombosis showed suppressed mRNA and protein levels of MLL1 (by >65% and >55% respectively, p<0.05), PLAU (by >85% and >55% respectively, p<0.05) levels and decreased H3K4me3 enrichment on the Plau promoter (~7.2 fold, p=0.0031) relative to controls. Conclusions: The post-thrombotic inflammatory state induces MLL1-mediated epigenetic modifications in the bone marrow, resulting in suppression of Mϕ urokinase expression. These changes may contribute to an antibrinolytic Mϕ phenotype.

Malassezia spp. induce inflammatory cytokines and activate NLRP3 inflammasomes in phagocytes.

Malassezia spp. are common eukaryotic yeasts that colonize mammalian skin. Recently, the authors and others have observed that Malassezia globosa and Malassezia restricta can be found in the intestines in the context of certain diseases, including Crohn's disease and pancreatic cancer. In order to better understand the nature of innate inflammatory responses to these yeasts, inflammatory responses induced by M. restricta and M. globosa in mouse bone marrow-derived Mϕs (BMDM) and dendritic cells (BMDC) are evaluated. While Malassezia yeasts induce proinflammatory cytokine production from both Mϕs and dendritic cells, the levels of production from BMDC were more pronounced. Both M. restricta and M. globosa activated inflammatory cytokine production from BMDC in large part through Dectin2 and CARD9 signaling, although additional receptors appear to be involved in phagocytosis and activation of reactive oxygen production in response to the yeasts. Both M. restricta and M. globosa stimulate production of pro-IL-1β as well as activation of the NLRP3 inflammasome. NLRP3 inflammasome activation by Malassezia fungi requires SYK signaling, potassium efflux and actin rearrangement. Together, the data further the understanding of the coordinated involvement of multiple innate immune receptors in recognizing Malassezia globosa and Malassezia restricta and orchestrating phagocyte inflammatory and antimicrobial responses.

Regulation of RNA degradation pathways during the lipopolysaccharide response in Macrophages.

The innate immune response to LPS is highly dynamic yet tightly regulated. The majority of studies of gene expression have focussed on transcription. However, it is also important to understand how post-transcriptional pathways are regulated in response to inflammatory stimuli as the rate of RNA degradation relative to new transcription is important for overall expression. RNA decay pathways include nonsense-mediated decay, the RNA decay exosome, P-body localized deadenylation, decapping and degradation, and AU-rich element targeted decay mediated by tristetraprolin. Here, bone marrow-derived Mϕs were treated with LPS over a time course of 0, 2, 6, and 24h and the transcriptional profiles were analyzed by RNA sequencing. The data show that components of RNA degradation pathways are regulated during an LPS response. Processing body associated decapping enzyme DCP2 and regulatory subunit DCP1A, and 5' exonuclease XRN1 and sequence specific RNA decay pathways were upregulated. Nonsense mediated decay was also increased in response to LPS induced signaling, initially by increased activation and at later timepoints at the mRNA and protein levels. This leads to increased nonsense mediated decay efficiency across the 24h following LPS treatment. These findings suggest that LPS activation of Mϕs results in targeted regulation of RNA degradation pathways in order to change how subsets of mRNAs are degraded during an inflammatory response.

JMJD3 Influences Macrophage-Mediated Inflammation in Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAAs) are characterized by inflammatory macrophage (Mφ) infiltration and pathologic vascular remodeling. The mechanisms regulating Mφ polarization during AAA development remain unknown. There is increasing evidence that epigenetic enzymes, specifically the histone demethylase JMJD3, direct Mφ polarization. The purpose of this study was to investigate whether JMJD3-mediated epigenetic modifications regulate Mφ inflammation and drive AAA formation. Single-cell RNA sequencing was conducted on human AAA and age-matched atherosclerotic control tissue samples. In addition, the angiotensin (Ang) II-induced AAA model was used to gain mechanistic insight. Briefly, after 4 weeks of high-fat diet, mice were infused with AngII or saline to induce AAAs. AAA maximum diameters were quantified and Mφs were sorted. Messenger RNA abundance of inflammatory cytokines and Jmjd3 were determined by quantitative polymerase chain reaction; levels of histone 3 lysine 27 trimethylation (H3K27me3) on gene promoters were determined by chromatin immunoprecipitation. Statistical significance was determined using Student t-test or analysis of variance. Single-cell RNA sequencing analysis of human AAAs and age-matched atherosclerotic controls demonstrated a significant upregulation of Jmjd3 and inflammatory cytokine expression in monocyte and Mφ subsets within AAA tissue. Congruently, AAA tissue- and bone marrow-derived Mφs from AngII-induced AAAs displayed marked upregulation of Jmjd3 and increased messenger RNA abundance of inflammatory genes (interleukins 1β, 12, and 23) during AAA development (P < .05). To determine the mechanistic impact of JMJD3 on Mφ polarization, histone methylation was evaluated on monocyte-macrophages and demonstrated decreased levels of the repressive histone methylation mark H3K27me3 on inflammatory gene promoters in AngII-induced AAAs compared with controls. In vitro inhibition of JMJD3 using short interfering RNA significantly reduced Mφ inflammatory cytokine expression (P < .05). These Results suggest an important role for JMJD3 in regulating macrophage-mediated inflammation and identify a potential target for the treatment of chronic inflammation in human AAAs.

Controlling the Phenotype of Tumor-Infiltrating Macrophages via the PHD-HIF Axis Inhibits Tumor Growth in a Mouse Model.

SummaryThe tumor microenvironment (TME) polarizes tumor-infiltrating macrophages toward tumor support. Macrophage-abundant tumors are highly malignant and are the cause of poor prognosis and therapeutic resistance. In this study, we show that the prolyl hydroxylase (PHD) inhibitor FG-4592 (FG) inhibits tumor growth of macrophage-abundant tumors and prolongs mouse survival. FG not only normalizes tumor vessels and improves tumor oxygenation but also directly affects macrophages and activates phagocytosis through the PHD-hypoxia-inducible factor (HIF) axis. Remarkably, FG can promote phagocytic ability of the Ly6Clo subset of tumor-infiltrating macrophages, leading to tumor growth inhibition. Moreover, Ly6Cneg macrophages contributed to blood vessel normalization. Using a malignant tumor mouse model, we characterized macrophage function and subsets. Altogether, our findings suggest that the PHD inhibitor can promote the anti-tumor potential of macrophages to improve cancer therapy.

Myeloid Slc2a1-Deficient Murine Model Revealed Macrophage Activation and Metabolic Phenotype Are Fueled by GLUT1.

Macrophages (MΦs) are heterogeneous and metabolically flexible, with metabolism strongly affecting immune activation. A classic response to proinflammatory activation is increased flux through glycolysis with a downregulation of oxidative metabolism, whereas alternative activation is primarily oxidative, which begs the question of whether targeting glucose metabolism is a viable approach to control MΦ activation. We created a murine model of myeloid-specific glucose transporter GLUT1 (Slc2a1) deletion. Bone marrow-derived MΦs (BMDM) from Slc2a1M-/- mice failed to uptake glucose and demonstrated reduced glycolysis and pentose phosphate pathway activity. Activated BMDMs displayed elevated metabolism of oleate and glutamine, yet maximal respiratory capacity was blunted in MΦ lacking GLUT1, demonstrating an incomplete metabolic reprogramming. Slc2a1M-/- BMDMs displayed a mixed inflammatory phenotype with reductions of the classically activated pro- and anti-inflammatory markers, yet less oxidative stress. Slc2a1M-/- BMDMs had reduced proinflammatory metabolites, whereas metabolites indicative of alternative activation-such as ornithine and polyamines-were greatly elevated in the absence of GLUT1. Adipose tissue MΦs of lean Slc2a1M-/- mice had increased alternative M2-like activation marker mannose receptor CD206, yet lack of GLUT1 was not a critical mediator in the development of obesity-associated metabolic dysregulation. However, Ldlr-/- mice lacking myeloid GLUT1 developed unstable atherosclerotic lesions. Defective phagocytic capacity in Slc2a1M-/- BMDMs may have contributed to unstable atheroma formation. Together, our findings suggest that although lack of GLUT1 blunted glycolysis and the pentose phosphate pathway, MΦ were metabolically flexible enough that inflammatory cytokine release was not dramatically regulated, yet phagocytic defects hindered MΦ function in chronic diseases.

Pentose Phosphate Shunt Modulates Reactive Oxygen Species and Nitric Oxide Production Controlling Trypanosoma cruzi in Macrophages

Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (Mφ) anti-microbial response. Mφs infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classical Mφs. How Mφs fail to elicit a potent ROS/NO response during infection and its link to glycolysis is unknown. Herein, we evaluated for ROS, NO, and cytokine production in the presence of metabolic modulators of glycolysis and the Krebs cycle. Metabolic status was analyzed by Seahorse Flux Analyzer and mass spectrometry and validated by RNAi. Tc infection of RAW264.7 or bone marrow-derived Mφs elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-α expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO by 18 h. Interferon (IFN)-γ addition enhanced the Tc-induced ROS/NO release and shut down mitochondrial respiration to the levels noted in classical Mφs. Inhibition of PPAR-α attenuated the ROS/NO response and was insufficient for complete metabolic shift. Deprivation of glucose and inhibition of pyruvate transport showed that Krebs cycle and glycolysis support ROS/NO generation in Tc + IFN-γ stimulated Mφs. Metabolic profiling and RNAi studies showed that glycolysis-pentose phosphate pathway (PPP) at 6-phosphogluconate dehydrogenase was essential for ROS/NO response and control of parasite replication in Mφ. We conclude that IFN-γ, but not inhibition of PPAR-α, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/NO response in Tc-infected Mφs. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by Mφs.

Myeloid-derived NF-κB negative regulation of PU.1 and c/EBP-β-driven pro-inflammatory cytokine production restrains LPS-induced shock.

Sepsis is a life-threatening event predominantly caused by Gram-negative bacteria. Bacterial infection causes a pronounced macrophage (MΦ) and dendritic cell activation that leads to excessive pro-inflammatory cytokine IL-1β, IL-6 and TNF-α production (cytokine storm), resulting in endotoxic shock. Previous experimental studies have revealed that inhibiting NF-κB signaling ameliorates disease symptoms; however, the contribution of myeloid p65 in endotoxic shock remains elusive. In this study, we demonstrate increased mortality in mice lacking p65 in the myeloid lineage (p65Δmye) compared with wild type mice upon ultra-pure LPS challenge. We show that increased susceptibility to LPS-induced shock was associated with elevated serum level of IL-1β and IL-6. Mechanistic analyses revealed that LPS-induced pro-inflammatory cytokine production was ameliorated in p65-deficient bone marrow-derived MΦs; however, p65-deficient 'activated' peritoneal MΦs exhibited elevated IL-1β and IL-6. We show that the elevated pro-inflammatory cytokine secretion was due, in part, to increased accumulation of IL-1β mRNA and protein in activated inflammatory MΦs. The increased IL-1β was linked with heightened binding of PU.1 and CCAAT/enhancer binding protein-β to Il1b and Il6 promoters in activated inflammatory MΦs. Our data provide insight into a role for NF-κB in the negative regulation of pro-inflammatory cytokines in myeloid cells.

M1/M2-macrophage phenotypes regulate renal calcium oxalate crystal development.

In our previous report, M2-macrophage (Mφs) deficient mice showed increased renal calcium oxalate (CaOx) crystal formation; however, the role of Mφs-related-cytokines and chemokines that affect kidney stone formation remains unknown. Here, we investigated the role of M1/M2s in crystal development by using in vitro and in vivo approaches. The crystal phagocytic rate of bone marrow-derived M2Mφs was higher than that of bone marrow-derived Mφs and M1Mφs and increased on co-culture with renal tubular cells (RTCs). However, the amount of crystal attachment on RTCs reduced on co-culture with M2Mφs. In six hyperoxaluric C57BL/6J mice, M1Mφ transfusion and induction by LPS and IFN-γ facilitated renal crystal formation, whereas M2Mφ transfusion and induction by IL-4 and IL-13 suppressed renal crystal formation compared with the control. These M2Mφ treatments reduced the expression of crystal-related genes, such as osteopontin and CD44, whereas M1Mφ treatment increased the expression of pro-inflammatory and adhesion-related genes such as IL-6, inducible NOS, TNF-α, C3, and VCAM-1. The expression of M2Mφ-related genes was lower whereas that of M1Mφ-related genes was higher in papillary tissue of CaOx stone formers. Overall, our results suggest that renal crystal development is facilitated by M1Mφs, but suppressed by M2Mφs.

Abstract 417: Control of Adipose Tissue Macrophage Metabolism by Oxidized Phospholipids

Our group has previously shown that oxidized phospholipids (OxPL) induce a unique macrophage (MΦ) phenotype known as “Mox,” distinct from pro- and anti-inflammatory M1 and M2 phenotypes. We have also shown that Mox make up 30% of the MΦs found in atherosclerotic lesions. OxPL induce TLR2-dependent inflammatory gene expression in MΦs. TLR2 activation by peptidoglycan was shown to induce accumulation of ceramides, which can alter bioenergetics by inhibiting the mitochondria. Recently, it has been shown that spleen tyrosine kinase (Syk) is phosphorylated upon TLR2 agonist Pam3CSK stimulation in MΦs. The effect of OxPL on bioenergetics has never before been studied. Here we test the hypothesis that OxPL change MΦ bioenergetics and inflammatory capacity via a TLR2-Syk-Ceramide pathway. Using flow cytometry, we found that in mice fed a high-fat diet, more than 20% of all adipose tissue MΦs can be described as Mox. Concomitantly, using immunohistochemistry and liquid-chromatography mass spectrometry, we measured increased levels of OxPL in the stromal vascular fraction of obese murine adipose tissue, as compared to lean controls. We treated bone marrow-derived MΦs (BMDMs) from WT, TLR2-KO, and Syk-KO mice with OxPL and measured bioenergetics using a Seahorse Flux Analyzer. Our results show that OxPL decrease oxygen consumption rate (OCR), a measure of oxidative phosphorylation, and decrease extracellular acidification rate (ECAR), a measure of glycolytic capacity. These MΦs can be described as quiescent, but they still engage in low-level cytokine production. Moreover, OxPL result in the accumulation of ceramides, as quantified by mass spectrometry. Finally, using Syk-KO MΦs and Syk inhibitors, we show that OxPL-induced inflammatory gene expression and ceramide accumulation are dependent on Syk. In summary, our results demonstrate that OxPL accumulate in obese adipose tissue and induce a change in the inflammatory and metabolic profiles of MΦs, involving a TLR2-ceramide-Syk dependent pathway. These results suggest that OxPL are triggers of adipose tissue inflammation and subsequent development of insulin resistance. Furthermore, we identify Syk as a therapeutic target for inhibiting diet-induced adipose tissue inflammation and insulin resistance.

Metabolic reprogramming through fatty acid transport protein 1 (FATP1) regulates macrophage inflammatory potential and adipose inflammation.

ObjectiveA novel approach to regulate obesity-associated adipose inflammation may be through metabolic reprogramming of macrophages (MΦs). Broadly speaking, MΦs dependent on glucose are pro-inflammatory, classically activated MΦs (CAM), which contribute to adipose inflammation and insulin resistance. In contrast, MΦs that primarily metabolize fatty acids are alternatively activated MΦs (AAM) and maintain tissue insulin sensitivity. In actuality, there is much flexibility and overlap in the CAM-AAM spectrum in vivo dependent upon various stimuli in the microenvironment. We hypothesized that specific lipid trafficking proteins, e.g. fatty acid transport protein 1 (FATP1), would direct MΦ fatty acid transport and metabolism to limit inflammation and contribute to the maintenance of adipose tissue homeostasis.MethodsBone marrow derived MΦs (BMDMs) from Fatp1−/− and Fatp1+/+ mice were used to investigate FATP1-dependent substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. We also generated C57BL/6J chimeric mice by bone marrow transplant specifically lacking hematopoetic FATP1 (Fatp1B−/−) and controls Fatp1B+/+. Mice were challenged by high fat diet (HFD) or low fat diet (LFD) and analyses including MRI, glucose and insulin tolerance tests, flow cytometric, histologic, and protein quantification assays were conducted. Finally, an FATP1-overexpressing RAW 264.7 MΦ cell line (FATP1-OE) and empty vector control (FATP1-EV) were developed as a gain of function model to test effects on substrate metabolism, bioenergetics, metabolomics, and inflammatory responses.ResultsFatp1 is downregulated with pro-inflammatory stimulation of MΦs. Fatp1−/− BMDMs and FATP1-OE RAW 264.7 MΦs demonstrated that FATP1 reciprocally controled metabolic flexibility, i.e. lipid and glucose metabolism, which was associated with inflammatory response. Supporting our previous work demonstrating the positive relationship between glucose metabolism and inflammation, loss of FATP1 enhanced glucose metabolism and exaggerated the pro-inflammatory CAM phenotype. Fatp1B−/− chimeras fed a HFD gained more epididymal white adipose mass, which was inflamed and oxidatively stressed, compared to HFD-fed Fatp1B+/+ controls. Adipose tissue macrophages displayed a CAM-like phenotype in the absence of Fatp1. Conversely, functional overexpression of FATP1 decreased many aspects of glucose metabolism and diminished CAM-stimulated inflammation in vitro. FATP1 displayed acyl-CoA synthetase activity for long chain fatty acids in MΦs and modulated lipid mediator metabolism in MΦs.ConclusionOur findings provide evidence that FATP1 is a novel regulator of MΦ activation through control of substrate metabolism. Absence of FATP1 exacerbated pro-inflammatory activation in vitro and increased local and systemic components of the metabolic syndrome in HFD-fed Fatp1B−/− mice. In contrast, gain of FATP1 activity in MΦs suggested that Fatp1-mediated activation of fatty acids, substrate switch to glucose, oxidative stress, and lipid mediator synthesis are potential mechanisms. We demonstrate for the first time that FATP1 provides a unique mechanism by which the inflammatory tone of adipose and systemic metabolism may be regulated.

Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice.

In inflammatory bowel diseases (IBDs), particularly ulcerative colitis, intestinal macrophages (MΦs), eosinophils, and the eosinophil-selective chemokine CCL11, have been associated with disease pathogenesis. MΦs, a source of CCL11, have been reported to be of a mixed classical (NF-κB-mediated) and alternatively activated (STAT-6-mediated) phenotype. The importance of NF-κB and STAT-6 pathways to the intestinal MΦ/CCL11 response and eosinophilic inflammation in the histopathology of experimental colitis is not yet understood. Our gene array analyses demonstrated elevated STAT-6- and NF-κB-dependent genes in pediatric ulcerative colitis colonic biopsies. Dextran sodium sulfate (DSS) exposure induced STAT-6 and NF-κB activation in mouse intestinal F4/80(+)CD11b(+)Ly6C(hi) (inflammatory) MΦs. DSS-induced CCL11 expression, eosinophilic inflammation, and histopathology were attenuated in RelA/p65(Δmye) mice, but not in the absence of STAT-6. Deletion of p65 in myeloid cells did not affect inflammatory MΦ recruitment or alter apoptosis, but did attenuate LPS-induced cytokine production (IL-6) and Ccl11 expression in purified F4/80(+)CD11b(+)Ly6C(hi) inflammatory MΦs. Molecular and cellular analyses revealed a link between expression of calprotectin (S100a8/S100a9), Ccl11 expression, and eosinophil numbers in the DSS-treated colon. In vitro studies of bone marrow-derived MΦs showed calprotectin-induced CCL11 production via a p65-dependent mechanism. Our results indicate that myeloid cell-specific NF-κB-dependent pathways play an unexpected role in CCL11 expression and maintenance of eosinophilic inflammation in experimental colitis. These data indicate that targeting myeloid cells and NF-κB-dependent pathways may be of therapeutic benefit for the treatment of eosinophilic inflammation and histopathology in IBD.