ENPP3 drives ccRCC progression by cGAMP hydrolysis and STING–IFN suppression

e21097 Background: Although anti-PD-1/PD-L1 monotherapy has achieved clinical success in non-small cell lung cancer (NSCLC), definitive predictive biomarkers remain to be elucidated. We assumed that by combining gene expression signatures with patient clinical data, we could identify a novel promising biomarker to predict response to anti-PD-1/PD-L1 monotherapy in NSCLC patients. Moreover, the characterization of these signatures will help us to decipher the complexity of tumor-immune interactions and better understand the tumor microenvironment (TME) that favors clinical response to nivolumab monotherapy. Methods: From clinically annotated NSCLC patients (n = 40) with nivolumab monotherapy in the second- or later-line settings, we prospectively collected tumor tissues and peripheral blood mononuclear cells (PBMCs) before first dose of nivolumab and PBMCs after first 4 or 5 doses of nivolumab. All tumor tissue and PBMC samples obtained were applied to whole-transcriptome sequencing (RNA-seq). We extracted transcriptomic datasets of lung adenocarcinoma (LUAD) (n = 20) and lung squamous cell carcinoma (LUSC) (n = 18) from the results, separately analyzed each histological subtype. To elucidate biological processes associated with clinical outcomes, we performed a supervised gene set enrichment analysis (GSEA) approach and an unsupervised single sample scoring approach. Results: In LUAD, we observed that gene sets related to interferon (type I and II) signaling (‘IFN signatures’) and antigen processing and presentation (‘APP signatures’) were significantly enriched in pre-treatment PBMCs of responders. IFN and APP signatures, which are closely related to each other, functionally cooperate to activate anti-tumor immune response. The enrichment of IFN and APP signatures provides the possibility that responders have a pre-existing anti-tumor immunity prior to nivolumab monotherapy. In LUSC, neither IFN nor APP signatures were enriched in pre-treatment tumor tissues and PBMCs of responders. Instead, gene sets related to the regulation of the TME (‘TME signatures’) are significantly enriched in pre-treatment tumor tissues of non-responders. The enrichment of TME signatures suggested that non-responders have an extremely immunosuppressive TME. These findings highlighted that responsive LUAD inherently have a high immunogenicity to elicit effective anti-tumor responses, whereas responsive LUSC have a similar level of immunogenicity as non-responsive LUSC but are free from an extremely immunosuppressive TME. Conclusions: We found that nivolumab enhanced anti-tumor immunity in patients with LUAD in a quite different way from patients with LUSC. Our study provides a blueprint for innovating combinational immunotherapy and supporting patient selection and treatment strategies on long-term clinical outcomes.

<h3>Background</h3> Immunotherapy for hepatocellular carcinoma (HCC) exhibits minimal clinical benefit due to immunosuppressive tumor microenvironment. The recently discovered cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling in tumor-associated macrophages (TAMs) plays an important role in stimulating the antitumor immune response. As2O3 has been demonstrated to inhibit HCC development, while the effect of immune-regulation in HCC remains elusive. Here, we aim to investigate the immune-regulatory mechanism of As2O3-induced HCC elimination. <h3>Methods</h3> Human Huh7 and murine Hepa1–6 HCC cells were treated with As2O3, followed by the co-culture with THP1 and RAW264.7 cells respectively. Comet assay and western blot of γ-H2AX expression were performed to assess DNA damage occurring in HCC cells. RT-qPCR and western blot were used to identify the As2O3-targeted signals in macrophages, and the signaling was confirmed in the loss-of-effect experiment of gene inhibition. Immunofluorescence, flow cytometry and RT-qPCR were utilized to assess the expression of phenotype-specific markers and cytokines secretion in macrophages. <h3>Results</h3> Low dose of As2O3 induced double-strand DNA (dsDNA) breaks in HCC cells, and the damaged dsDNA was released into the extracellular space. Macrophages absorbed the dsDNA, which was recognized and bound to cyclic GMP-AMP synthase (cGAS) for the activation of the stimulator of interferon genes (STING) signaling. Activated cGAS-STING signaling promoted the reprogramming of macrophage to M1 phenotype with the production of pro-inflammatory cytokine IFN-I, causing the suppression of tumor cell proliferation. Blockade of cGAS-STING signaling by STING inhibitor could effectively abrogate the antitumor effect of As2O3-activated macrophages. (IDDF2022-ABS-0038 Figure 1. Graphic abstract) <h3>Conclusions</h3> This study reveals that As2O3 functions as an antitumor immune modulator to promote the activation of cGAS-STING signaling and the conversion of TAM from M2 to M1, leading to the immune regression of HCC.

Objective: Cigarette smoking is the main cause of chronic obstructive pulmonary disease (COPD) and may modulate the immune response of exposed individuals. Mast cell function can be altered by cigarette smoking, but the role of smoking in COPD remains poorly understood. The current study aimed to explore the role of cigarette smoke extract (CSE)-treated mast cells in COPD pathogenesis.Methods: Cytokine and chemokine expression as well as degranulation of bone marrow-derived mast cells (BMMCs) were detected in cells exposed to immunoglobulin E (IgE) and various doses of CSE. Adoptive transfer of CSE-treated BMMCs into C57BL/6J mice was performed, and macrophage infiltration and polarization were evaluated by fluorescence-activated cell sorting (FACS). Furthermore, a coculture system of BMMCs and macrophages was established to examine macrophage phenotype transition. The role of protease serine member S31 (Prss31) was also investigated in the co-culture system and in COPD mice.Results: CSE exposure suppressed cytokine expression and degranulation in BMMCs, but promoted the expressions of chemokines and Prss31. Adoptive transfer of CSE-treated BMMCs induced macrophage infiltration and M2 polarization in the mouse lung. Moreover, CSE-treated BMMCs triggered macrophage M2 polarization via Prss31 secretion. Recombinant Prss31 was shown to activate interleukin (IL)-13/IL-13Rα/Signal transducers and activators of transcription (Stat) 6 signaling in macrophages. Additionally, a positive correlation was found between Prss31 expression and the number of M2 macrophages in COPD mice.Conclusion: In conclusion, CSE-treated mast cells may induce macrophage infiltration and M2 polarization via Prss31 expression, and potentially contribute to COPD progression.

Tumor associated macrophages, mainly represented by the M2-like phenotype, are a highly immunosuppressive macrophage subset that exert a pro-tumorigenic role in a variety of cancer types. Understanding the factors that promote M2 macrophage differentiation and activity is of critical importance to unlocking the potential of anti-tumor immune therapy. Thus, we investigated the role of the AhR pathway in M2 macrophages. We found the Aryl Hydrocarbon Receptor (AhR) highly expressed in in vitro polarized human M2 macrophages derived from peripheral blood mononuclear cells. Stimulation of AhR with small molecule agonists TCDD or kynurenine resulted in a significant enhancement of the suppressive activity of M2 macrophages on anti-CD3/CD28 stimulated CD4 T cell proliferation and IFNγ production. Inhibition of AhR using a novel, highly potent and selective AhR inhibitor (IDE-AhRi-1) fully inhibited the suppressive activity of M2 macrophages on T cells. Surprisingly, stimulated CD4 T cells co-cultured with IDE-AhRi-1 exposed M2 macrophages had an enhanced ability to proliferate and produce IFNγ compared with stimulated T cells in the absence of M2 macrophages. This suggests that IDE-AhRi-1 not only inhibits the suppressive mechanisms of M2 macrophages, but also boosts their ability to generate pro-inflammatory responses that enhance T-cell activity. Further studies to elucidate the AhR-driven molecular mechanisms responsible for this activity are underway. To find out whether AhR driven M2 polarization and activity is evident in human cancer, we analyzed TCGA-derived RNA sequencing data from an array of solid tumor indications. In a subset of cancer indications, high AhR activity correlated with key M2 macrophage and immuno-regulatory signature genes. Surprisingly, expression of CYP1B1 also correlated with expression of TDO2 mRNA, but not IDO1 mRNA. These finding establish AhR as a key modulator of M2 macrophages and demonstrate a correlation between AhR activity and tumor associated macrophages in human tumor samples. Furthermore, we demonstrate that IDE-AhRi-1 can potently suppress M2 macrophages and propose IDE-AhRi-1 as a novel approach for potentiating anti-tumor immunity. Citation Format: Candy Garcia, Hadia Lemar, Christina Galang, James Joseph, Marcos Gonzalez-Lopez, Jeffrey Hager, Michael P. Dillon, Fred J. Aswad. A novel small molecule inhibitor of AhR suppresses the polarization and activity of M2 macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3255.

BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer Background: Immune checkpoint inhibitors (ICIs) has received approval due to its encouraging clinical efficacy with improving the outcome of cancer patients. However, current immunotherapeutic regimens benefit merely a minority of populations. Thus, combining ICIs with other potential modalities is of great significance to enhance the response of single drug alone. Modifying tumor-associated macrophages (TAMs) strategies have attracted attention for ameliorating tumor immunosuppression and improving anti-tumor immunity. Here we exhibit APG2575, a proven BCL-2 inhibitor, can act as an antitumor immune modulator that resets TAMs from M2 to pro-tumoral M1 phenotype and further augment immunotherapeutic efficacy. Methods: By applying non-small cell lung cancer cell lines and murine syngeneic tumor xenograft, we investigated the effects of combined APG-2575 and PD-1 antibody on tumor growth. Furthermore, we explored the role of APG-2575 in regulating TAMs polarization through utilizing human and murine macrophages, involving immortalized and primary models. Besides, IL-10 secretion in cell culture supernatants was detected via ELISA assay. In addition, we also assessed the tumor immune microenvironment regulation of APG-2575 using flow cytometry. Results: In vitro, APG-2575, a BCL-2 inhibitor, augmented the tumor cell lysis induced by T cells when combined with ICIs. Moreover, mice treated with APG-2575 and ICIs showed remarkable tumor regression and prolonging survival which were associated with the increase of granzyme-B, IFN-γ and TNF-α release of CD8+ cytotoxic lymphocytes. Importantly, we found that APG-2575-mediated antitumor T cell immunity is dependent on macrophages. Furthermore, we discovered APG-2575 could induce M2 macrophage switching to M1 phenotype and suppress IL-10 secretion in vitro. Meanwhile, reduced M2 macrophages as well as increased M1 polarization were also observed in the tumor-infiltrating immune cells from mice treated with APG-2575. Additionally, APG-2575-mediated macrophage transition could improve tumor immunosuppression, with decreasing number of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells in tumor microenvironment. Conclusions: Our data demonstrate that APG-2575 can synergizes with ICIs in vitro and in vivo, which was involved in switching TAMs from M2 to M1 polarization and further improving tumor immunosuppression, finally enhancing T cell anti-tumor activity. These results warranted future clinical evaluation of combination therapy of APG-2575 and ICIs. Key words: BCL-2, APG-2575, ICIs, macrophages, non-small cell lung cancer Citation Format: Fan Luo, Fei Teng Lu, Miao Zhen Qiu, Wen Tao Pan, Lin Zhang, Hong Yun Zhao, Li Zhang, Da Jun yang. BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 439.

The interplay between tumor cells and immune cells within the tumor microenvironment (TME) dictates protumor or antitumor immune responses. Tumor-associated macrophages (TAMs) respond to signals of the TME and exhibit a spectrum of phenotypes ranging between M1 (antitumor) and M2 (protumor) macrophages. In most tumor types, including melanoma, the balance between M1 and M2 macrophages is critical with a higher M1/M2 ratio favoring antitumor immunity. Therefore, strategies enhancing the M1/M2 ratio can significantly alter the TME towards antitumor immunity. In this study, we administered luciferase and GFP-expressing M1 macrophages as an adoptive cell therapy (ACT) through intravenous (i.v) and intratumor routes respectively, into mice bearing SM1 murine melanoma tumors to determine the effective treatment modality. We reprogrammed antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma and humanized NSG-SGM3 melanoma models. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations to demonstrate the benefit of macrophage ACT. Tail vein injected, luciferase-expressing M1 macrophages localized in the lungs and spleen, failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v administration is ineffective for macrophage therapy. On the contrary, intratumor M1 macrophage ACT resulted in diminished tumor growth. Single-cell RNA-seq analysis of the CD45+ sorted tumor-associated immune cell population revealed distinct macrophage subsets and a significant M1/M2 macrophage ratio increase. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Trajectory analysis of infiltrated monocytes indicated differentiation toward inflammatory macrophages in ACT tumors. Flow cytometry analysis corroborated that ACT increased the M1/M2 macrophage ratio and an increase in CD8 effector T-cells. Furthermore, HDAC6i-treated macrophages increased antigen cross-presentation. Single-cell secretome analysis of F4/80+ TAMs by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages secreting inflammatory cytokine Tnfa and T-cell recruiting chemokine Cxcl10. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. We demonstrated the potential of reprogramming macrophages ex vivo with HDAC6 inhibitors as a feasible macrophage cell therapy to treat solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Nithya Gajendran, Manasa Suresh, Xintang Li, Maria D. Hernandez, Christian Zevallos Delgado, Nima Aghdam, Michael Berrigen, Tessa Knox, Karen Tan, Marie Durr, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman H. Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra. Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5247.

186 NRF2 regulation of the interferon signature in lupus macrophages

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis.

Objective To investigate the differences of glycoprotein non-metastatic melanoma b （Gpnmb） expression between M1 and M2 bone marrow-derived macrophages （BMMφs） in mouse. Meth- ods Primary BMMφs were cultured and then identified by immunofluorescenee staining for F4/80 and flow cytometry testing of CDllb. Interferon-γ and lipopolysaccharide were used to induce differentiation of BMMφ towards M1 macrophages and interleukin-4 was adopted to induce differentiation of M2 macropha- ges. Realtime PCR was performed to analyze mRNA expressions of tumor necrosis factor （TNF-α）, induc- ible NO synthase （ iNOS）, macrophage mannose receptor （MMR）, arginase-1 （ Arg-1 ） and Gpnmb. Pro- teins of Gpnmb and MMR were detected by double immunofluorescence staining, Western blot and flow cy- tometry. Results （1） Immunofluorescence staining showed high expression of F4/80 in BMMφs and flow cytometry results showed that CDllb was expressed in 92.7% ±6.1% of BMMφs, suggesting that primary BMMφs were successfully cultured. （2） Compared with M0 BMMφs, mRNAs of TNF-α and iNOS were highly up-regulated in M1 BMMφs （ both P〈0. 01 ）, and mRNAs of MMR and Arg-1 were highly up-regula- ted in M2 BMMφs （ both P〈0.01 ）, indicating that differentiation of BMMφs towards M1 and M2 BMMφs were successfully induced. （3） Expressions of Gpnmb mRNA and Gpnmb protein were predominantly up- regulated in M2 BMMφs in comparison with those in M0 and M1 BMMφs （ both P〈0. 01 ）. Gpnmb and MMR were co-expressed in M2 BMMφs and 83.2% ±9.7% of MMR positive BMMφs expressed Gpnmb. Conclusion Gpnmb expression is significantly increased in M2 macrophages than that in M1 macrophages in vitro, indicating that Gpnmb which takes part in the differentiation of macrophages might be used as a marker for identification of M1 and M2 macrophages. Key words: Gpnmb; Bone marrow-derived macrophages; M1 macrophages; M2 macrophages; MMR

42 Background: Chronic inflammation plays an important role in tumorigenesis and tumor progression in human cancers. We aim to investigate the role of NF-kB in cancer inflammation of esophageal squamous cell carcinoma (ESCC). Methods: To generate M2-polarized macrophages, cells of human U937 monocyte cell line were treated with phorbol myristate acetate (PMA, 50 ng/ml) for 6 hours, and then cultured with PMA plus Th2 cytokines, IL-4 (20 ng/ml) and IL-13 (20 ng/ml), for another 18 hours. M2 phenotype was verified by flow cytometry and by cytokine profiling using enzyme-linked immunosorbent assay (ELISA). After co-culture with M2 macrophages, transcription nuclear factor-kB (NF-kB) activity was measured using quantitative polymerase chain reaction (Q-PCR), followed by reconfirmation with Western blot analysis for IkBα in KYSE-170 and -510 ESCC cell lines (kindly provided by Dr. Yutaka Shimada at Kyoto University, Japan). A selective inhibitor to NF-kB, Bay11-7082, was used to treat ESCC cell lines co-cultured with M2 macrophages, followed by cell proliferation, migration, invasion assays and vascular endothelial growth factor (VEGF) secretion by ELISA. The effect of Bay11-7082 (5 mg/kg) against growth of ESCC tumor was tested in xenografted tumors. Results: PMA plus Th2 cytokines treatment promoted differentiation of U937 cells into M2 macrophages. When treated with Bay11-7082, proliferation, migration, invasion and induction of VEGF expression was significantly inhibited in M2 macrophage co-cultured ESCC cells with a down-regulation of IkBα expression. Tumor growth was significantly increased in M2 macrophage co-cultured ESCC cells compared to that of the non-co-cultured controls, which was significantly retarded by treatment with Bay11-7082. Conclusions: NF-kB pathway was activated in ESCC cell lines co-cultured with M2 macrophages with an increase in cell proliferation, cell motility and angiogenic factor in vitro and tumor growth in vivo, which were significantly suppressed by a NF-kB inhibitor, Bay11-7082. These results suggest a role of M2 macrophage in promoting aggressiveness of ESCC cells, possibly through an activation of NF-kB pathway that may serve as a potential therapeutic target for ESCC.

Vitamin D enhances the effect of Soufeng sanjie formula in alleviating joint inflammation in CIA mice through VDR-NOTCH3/DLL4 signaling in macrophages.

SPP1-mediated M2 macrophage polarization shapes the tumor microenvironment and enhances prognosis and immunotherapy guidance in nasopharyngeal carcinoma.

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

High intake of dietary sodium is a risk factor for cardiovascular disease. Previously, we have shown that an increase in extracellular hypertonic sodium (+40 mM Na + ; HS) inhibits alternatively-activated M2 macrophage (M(IL4+IL13)) gene signature and function, while boosting classic M1 macrophage polarization (M(LPS)) and bacterial killing capacity. M1 macrophages predominantly rely on glycolysis, whereas M2 macrophages use oxidative phosphorylation, fueled by the oxidation of fatty acids, at a late phase of activation (24 h). The aim of this study was to elucidate the role of HS on the metabolic signature of M1 and M2 macrophages. Therefore, we quantified 13 C incorporation into intermediates of central carbon metabolism via GC-MS based pulsed stable isotope resolved metabolomics (pSIRM), and performed Seahorse analyses at varying time points during M1/M2-activation. After 24 h, LPS-induced increase in basal ECAR (extracellular acidification as surrogate parameter for glycolysis) and lactate production were further boosted under HS compared to isotonic salt conditions (NS), whereas mitochondrial respiration (OCR) was not affected. On the other hand, IL4+IL13-induced increase in OCR was decreased under HS compared to NS, while ECAR was not affected at this stage. Regarding TCA-cycle, we have found an early (1 h and 3 h post-stimulation) increase in citrate levels in M1 and M2 macrophages independent of HS, which further increased after 24 h in M1 and M1+HS. Interestingly, both in early and late phase of activation, glucose-derived 13 C incorporation into citrate was lower in HS-treated compared to NS-treated M0, M1 and M2 macrophages, suggesting an alternative replenishing source. Consistently, other TCA-cycle intermediates increased after 24h (especially in M1 and even further in M1+HS), whereas glucose-derived 13 C incorporation was minimal. Our data suggests that under hypertonic sodium macrophages re-direct TCA-cycle intermediates into the production of pro-inflammatory mediators (as described for late-phase M1 macrophages), whereas TCA-cycle itself is replenished by other alternative metabolic substrates. These HS-induced metabolic modifications might play an important role for macrophage activation and function.