Human Mφ Research Articles

Abstract 1365: INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers

Abstract Trastuzumab resistance is an important clinical issue in HER2+ breast cancer (BC), and few actionable targets are available. We demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect, and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response based on antitumoral macrophage-NK cell collaboration. Because immune checkpoint molecules (ICP) and T cell exhaustion foster tumor immune escape, we addressed whether INB03 could modulate macrophage polarization, T cell exhaustion, and ICP expression in both populations to boost an antitumor immune response. For in vitro assays, human monocytes and T cells were isolated from buffy coats and enriched with specific RosetteSep. Macrophages (Mϕ) were differentiated for 6 days with M-CSF (10 ng/ml) to M0, and polarized for 48h to the M1-like subtype with LPS (50 ng/ml)+IFNγ (20 ng/ml), or to the M2-like subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml). Then, INB03 was added (10 μg/ml) for 96h with corresponding cytokines to already-polarized Mϕ to test its ability to revert polarization. Also, Mϕ antibody-dependent cellular phagocytosis (ADCP) against human HER2+ breast cancer cell line JIMT-1 was assessed. T cells were activated with CD3/CD28 beads and cultured for 7 or 14 days. INB03 was added at those times for 48h to test its ability to modulate ICP and exhaustion markers. For in vivo assays, tumor-free mice or bearing the HER2+ C4HD tumor were treated with vehicle or INB03 (10 mg/kg, i.p.) for 21 days. Polarization, exhaustion and ICP markers were studied in Mϕ and T cells from spleen and tumor. Adding INB03 to already-polarized human Mϕ enhanced polarization towards M1-like (CD86+CD206−). Polarized Mϕ exposed to INB03 showed decreased PD1 and PDL-1 expression. Also, the expression of ADCP-inhibitory markers B7H4 and SIRPα was diminished when INB03 was present. These Mϕ exhibited increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Murine Mϕ from tumor-bearing mice treated with INB03 showed increased polarization to the M1-like phenotype. Human CD8+ T cells showed decreased expression of PD1, PD-L1, LAG3, TIM3 and TIGIT. However, only TIM3 and PD1 were downregulated in murine CD8+ T cells of tumor-bearing mice treated with INB03. In all, sTNF blockade skews Mϕ to the M1-like phenotype and reprograms already-polarized pro-tumoral M2-like Mϕ to antitumoral ones. INB03 can tailor the tumor microenvironment by promoting Mϕ ADCP against tumor cells and by acting as an ICP inhibitor for CD8+ T cells, possibly relieving their exhaustion. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted therapy efficacy and overcome tumor-induced macrophage and T-cell immune escape. Citation Format: Sofia Bruni, Maria F. Mercogliano, Roxana Schillaci. INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers [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 1365.

Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36, and TIM-3, whereas TIM-1, αVβ3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

Soluble CD83 modulates human-monocyte-derived macrophages toward alternative phenotype, function, and metabolism.

Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG, ABCA1, ABCG1, CD36) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions.

CD47 Blockade Enhances Phagocytic Activity of the TCR-Mimic Antibody Hu8F4 Against HLA-A2+ AML

Hu8F4, a humanized monoclonal antibody that recognizes PR1/HLA-A2 complex, is a first-in-class T cell receptor (TCR) - mimic antibody is currently being tested in a phase 1 trial for AML. We previously reported that activity of Hu8F4 primarily depends on its binding to Fcγ receptors (FcγR) on effector cells. Additionally, we identified antibody-dependent cellular phagocytosis (ADCP) as a principal mechanism of Hu8F4 anti-AML activity in vivo. Leukemia cells overexpress the “don't eat me” membrane protein CD47. CD47 on AML binds SIRPα on macrophages and neutrophils to inhibit phagocytosis. Blocking this interaction can allow antibodies to exert powerful phagocytic activity. Importantly, the role CD47 may play in the anti-tumor activity of TCR-mimic antibodies has not yet been investigated. To examine the relationship between CD47 and Hu8F4 activity, we used both in vitro ADCP assays and in vivo NSG mouse models. CD47 blockade was studied as a potential strategy to enhance Hu8F4 activity. To characterize the in vitro phagocytosis, we used an HLA-A2 transfected leukemia cell line, U937 (U937-A2), and an endogenously expressing HLA-A2 cell line THP1, as targets. NSG mouse bone marrow derived macrophages (BMDM) were used as effectors. At an initial E:T=1, Hu8F4 alone (10 mg/ml) was insufficient to prevent growth of U937-A2 and THP1. However, addition of neutralizing anti-CD47 F(ab') 2 antibody significantly slowed leukemia growth and eliminated both target cells by day 5. To further confirm the critical role of CD47 in Hu8F4-mediated ADCP, we created CRISPR/Cas9 CD47 KO U937-A2 and THP1 cells lines. Complete phagocytosis of both CD47KO cell lines was only observed in the presence, but not the absence of Hu8F4. To test if CD47 blockade plays a role in Hu8F4 anti-AML activity in vivo, we injected NGS mice with U937-A2 (5x10^3). Mice were treated with Hu8F4 +/- anti-CD47 F(ab') 2 antibody at 1mg/kg (three times per week for a total of 10 doses starting on day 5, n=5 per group). Control mice, treated with PBS, succumbed to leukemia by day 28. Both Hu8F4 and anti-CD47 F(ab') 2 single-agent treatment slowed leukemia growth, while the combination of Hu8F4 and anti-CD47 F(ab') 2 completely prevented outgrowth of the aggressive U937-A2 cells ( Figure 1), indicating that blocking CD47 significantly increased the activity of Hu8F4 in vivo. Interestingly, CD47 KO U937-A2 did not engraft in NSG mice as expected, (n=5), confirming that CD47 expression is critically important for cell line engraftment in NSG. HuCD47 is known to bind to NSG mouse SIRPα with high affinity providing protection to the human cells from phagocytosis by mouse macrophages. Growth of CD47 KO THP1 in NSG mice was delayed compared to parental THP1, median survival of untreated mice increased from 58 days for THP1 to 99 days for CD47 KO THP1 (p=0.0017, n=5). To facilitate THP1 cell growth in vivo, we used NSGS mice which allows for faster growth of human myeloid cells. Treatment with Hu8F4 for 7 weeks (1 mg/kg three times per week; started on day 2), significantly reduced THP1 leukemia growth and fully eliminated CD47 KO THP1 cells, confirming the role of CD47 in the activity of Hu8F4 ( Figure 2). Finally, to test whether CD47 is also important in Hu8F4-mediated phagocytosis by human effector cells, we generated human macrophages (Mφ) from CD14+ monocytes from healthy donors. We incubated fluorescently labeled human Mφ with CFSE-labelled target cells (U937-A2 or THP1), that had been pre-treated with Hu8F4 or isotype control antibody (Herceptin), at E:T=10, with or without anti-CD47 F(ab') 2. After 3 hours, Hu8F4-mediated phagocytosis, defined as the mean percentage of double-positive target cells, increased from mean 9.7% to 12.3% (n=4, p=0.199) for U937-A2, and from 12.25% to 17.58% (n=4, p=0.005) for THP1 when anti-CD47 F(ab') 2 blocking antibody was added. Anti-CD47 F(ab') 2 alone and in the presence of control Herceptin did not induce phagocytosis. Our data shows that the presence of CD47 on leukemia cells can significantly limit the activity of Hu8F4 both in vitro and in vivo. Importantly, combining Hu8F4 with CD47 blockade significantly increased the Hu8F4 activity and together eliminated AML. The role of other phagocytosis checkpoints, such as SIRPα and LILRB-1 also warrant further investigation.

PACAP regulates VPAC1 expression, inflammatory processes and lipid homeostasis in M1- and M2-macrophages.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as an anti-atherogenic neuropeptide and plays an important role in cytoprotective, as well as inflammatory processes, and cardiovascular regulation. Therefore, the aim of this study is to investigate the regulatory effects of PACAP and its receptor VPAC1 in relation to inflammatory processes and lipid homeostasis in different macrophage (MΦ) subtypes. To investigate the role of PACAP deficiency in the pathogenesis of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Lumen stenosis in the aortic arch and different MΦ-subtypes were analyzed in atherosclerotic plaques by quantitative immunohistochemistry. Undifferentiated bone marrow-derived cells (BMDC) from 30-weeks-old ApoE-/- and PACAP-/-/ApoE-/- mice were isolated, differentiated into BMDM1- and BMDM2-MΦ, and incubated with oxidized low-density lipoprotein (oxLDL). In addition, PMA-differentiated human THP-1 MΦ were further differentiated into M1-/M2-MΦ and subsequently treated with PACAP38, the VPAC1 agonist [(Ala11,22,28)VIP], the antagonist (PG 97-269), and/or oxLDL. Uptake/accumulation of oxLDL was analyzed by oxLDL-DyLight™488 and Bodipy™ 493/503. The mRNA expression was analyzed by qRT-PCR, protein levels by Western blot, and cytokine release by ELISA. In vivo, after 30 weeks of SC, PACAP-/-/ApoE-/- mice showed increased lumen stenosis compared with ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice under CED, immunoreactive areas of VPAC1, CD86, and CD163 were increased compared with ApoE-/- mice. In vitro, VPAC1 protein levels were increased in PACAP-/-/ApoE-/- BMDM compared with ApoE-/- BMDM, resulting in increased TNF-α mRNA expression in BMDM1-MΦ and decreased TNF-α release in BMDM2-MΦ. Concerning lipid homeostasis, PACAP deficiency decreased the area of lipid droplets in BMDM1-/M2-MΦ with concomitant increasing adipose differentiation-related protein level. In THP-1 M1-/M2-MΦ, the VPAC1 antagonist increased the uptake of oxLDL, whereas the VPAC1 agonist decreased the oxLDL-induced intracellular triglyceride content. Our data suggest that PACAP via VPAC1 signaling plays an important regulatory role in inflammatory processes in atherosclerotic plaques and in lipid homeostasis in different MΦ-subtypes, thereby affecting foam cell formation. Therefore, VPAC1 agonists or PACAP may represent a new class of anti-atherogenic therapeutics.

Lead exposure suppresses the Wnt3a/β-catenin signaling to increase the quiescence of hematopoietic stem cells via reducing the expression of CD70 on bone marrow-resident macrophages.

Lead (Pb) is a heavy metal highly toxic to human health in the environment. The aim of this study was to investigate the mechanism of Pb impact on the quiescence of hematopoietic stem cells (HSC). WT C57BL/6 (B6) mice treated with 1250 ppm Pb via drinking water for 8 wk had increased the quiescence of HSC in the bone marrow (BM), which was caused by the suppressed activation of the Wnt3a/β-catenin signaling. Mechanically, a synergistic action of Pb and IFNγ on BM-resident macrophages (BM-Mφ) reduced their surface expression of CD70, which thereby dampened the Wnt3a/β-catenin signaling to suppress the proliferation of HSC in mice. In addition, a joint action of Pb and IFNγ also suppressed the expression of CD70 on human Mφ to impair the Wnt3a/β-catenin signaling and reduce the proliferation of human HSC purified from umbilical cord blood (UCB) of healthy donors. Moreover, correlation analyses showed that the blood Pb concentration was or tended to be positively associated with the quiescence of HSC, and was or tended to be negatively associated with the activation of the Wnt3a/β-catenin signaling in HSC in human subjects occupationally exposed to Pb. Collectively, these data indicate that an occupationally relevant level of Pb exposure suppresses the Wnt3a/β-catenin signaling to increase the quiescence of HSC via reducing the expression of CD70 on BM-Mφ in both mice and humans.

Culture density influences the functional phenotype of human macrophages.

Macrophages (MΦ) are commonly cultured in vitro as a model of their biology and functions in tissues. Recent evidence suggests MΦ to engage in quorum sensing, adapting their functions in response to cues about the proximity of neighboring cells. However, culture density is frequently overlooked in the standardization of culture protocols as well as the interpretation of results obtained in vitro. In this study, we investigated how the functional phenotype of MΦ was influenced by culture density. We assessed 10 core functions of human MΦ derived from the THP-1 cell line as well as primary monocyte-derived MΦ. THP-1 MΦ showed increasing phagocytic activity and proliferation with increasing density but decreasing lipid uptake, inflammasome activation, mitochondrial stress, and secretion of cytokines IL-10, IL-6, IL-1β, IL-8, and TNF-α. For THP-1 MΦ, the functional profile displayed a consistent trajectory with increasing density when exceeding a threshold (of 0.2 x 103 cells/mm2), as visualized by principal component analysis. Culture density was also found to affect monocyte-derived MΦ, with functional implications that were distinct from those observed in THP-1 MΦ, suggesting particular relevance of density effects for cell lines. With increasing density, monocyte-derived MΦ exhibited progressively increased phagocytosis, increased inflammasome activation, and decreased mitochondrial stress, whereas lipid uptake was unaffected. These different findings in THP-1 MΦ and monocyte-derived MΦ could be attributed to the colony-forming growth pattern of THP-1 MΦ. At the lowest density, the distance to the closest neighboring cells showed greater influence on THP-1 MΦ than monocyte-derived MΦ. In addition, functional differences between monocyte-derived MΦ from different donors could at least partly be attributed to differences in culture density. Our findings demonstrate the importance of culture density for MΦ function and demand for awareness of culture density when conducting and interpreting in vitro experiments.

CXCL13 chemokine is a novel player in multiple myeloma osteolytic microenvironment, M2 macrophage polarization, and tumor progression

BackgroundWe assessed the mechanism by which multiple myeloma (MM) shapes the bone marrow (BM) microenvironment and affects MΦ polarization.MethodsIn vivo xenograft model of BM-disseminated human myeloma, as well as analysis of MM cell lines, stromal components, and primary samples from patients with MM, was utilized.ResultsAnalysis of the BM from MM-bearing mice inoculated with human CXCR4-expressing RPMI8226 cells revealed a significant increase in M2 MΦ cell numbers (p < 0.01). CXCL13 was one of the most profoundly increased factors upon MM growth with increased levels in the blood of MM-bearing animals. Myeloid cells were the main source of the increased murine CXCL13 detected in MM-infiltrated BM. MM cell lines induced CXCL13 and concurrent expression of M2 markers (MERTK, CD206, CD163) in co-cultured human MΦ in vitro. Interaction with MΦ reciprocally induced CXCL13 expression in MM cell lines. Mechanistically, TGFβ signaling was involved in CXCL13 induction in MM cells, while BTK signaling was implicated in MM-stimulated increase of CXCL13 in MΦ. Recombinant CXCL13 increased RANKL expression and induced TRAP+ osteoclast (OC) formation in vitro, while CXCL13 neutralization blocked these activities. Moreover, mice inoculated with CXCL13-silenced MM cells developed significantly lower BM disease. Reduced tumor load correlated with decreased numbers of M2 MΦ in BM, decreased bone disease, and lower expression of OC-associated genes. Finally, higher levels of CXCL13 were detected in the blood and BM samples of MM patients in comparison with healthy individuals.ConclusionsAltogether, our findings suggest that bidirectional interactions of MΦ with MM tumor cells result in M2 MΦ polarization, CXCL13 induction, and subsequent OC activation, enhancing their ability to support bone resorption and MM progression. CXCL13 may thus serve as a potential novel target in MM.

Expression of O-glycosylated oncofetal fibronectin in alternatively activated human macrophages.

Macrophage (Mϕ) polarization is an essential phenomenon for the maintenance of homeostasis and tissue repair, and represents the event by which Mϕ reach divergent functional phenotypes as a result to specific stimuli and/or microenvironmental signals. Mϕ can be polarized into two main phenotypes, M1 or classically activated and M2 or alternatively activated. These two categories diverge in many aspects, such as secreted cytokines, markers of cell surface, and biological functions. Over the last 10years, many potential markers have been proposed for both M1 and M2 human Mϕ. However, there is scarce information regarding the glycophenotype adopted by these cells. Here, we show that M2- but not M1-polarized Mϕ expresses high levels of an unusual glycoform of fibronectin (FN), named O-glycosylated oncofetal FN (onf-FN), found in fetal/cancer cells, but not in healthy tissues. The onf-FN expression was confirmed in vitro by Western blot and real-time RT-qPCR in primary and cell line monocyte-derived Mϕ. onf-FN was induced by IL-4 and IL-13, but not by pro-inflammatory stimuli (LPS and INF-γ). RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization. In conclusion, we show by the first time that O-glycosylated onf-FN is expressed by M2-polarized Mϕ.

CD14 Is Involved in the Interferon Response of Human Macrophages to Rubella Virus Infection.

Macrophages (MΦ) as specialized immune cells are involved in rubella virus (RuV) pathogenesis and enable the study of its interaction with the innate immune system. A similar replication kinetics of RuV in the two human MΦ types, the pro-inflammatory M1-like (or GM-MΦ) and anti-inflammatory M2-like (M-MΦ), was especially in M-MΦ accompanied by a reduction in the expression of the innate immune receptor CD14. Similar to RuV infection, exogenous interferon (IFN) β induced a loss of glycolytic reserve in M-MΦ, but in contrast to RuV no noticeable influence on CD14 expression was detected. We next tested the contribution of CD14 to the generation of cytokines/chemokines during RuV infection of M-MΦ through the application of anti-CD14 blocking antibodies. Blockage of CD14 prior to RuV infection enhanced generation of virus progeny. In agreement with this observation, the expression of IFNs was significantly reduced in comparison to the isotype control. Additionally, the expression of TNF-α was slightly reduced, whereas the chemokine CXCL10 was not altered. In conclusion, the observed downmodulation of CD14 during RuV infection of M-MΦ appears to contribute to virus-host-adaptation through a reduction of the IFN response.

Quantitative Proteomics of Polarised Macrophages Derived from Induced Pluripotent Stem Cells.

Macrophages (MΦ) are highly heterogenous and versatile innate immune cells involved in homeostatic and immune responses. Activated MΦ can exist in two extreme phenotypes: pro-inflammatory (M1) MΦ and anti-inflammatory (M2) MΦ. These phenotypes can be recapitulated in vitro by using ligands of toll-like receptors (TLRs) and cytokines such as IFNγ and IL-4. In recent years, human induced pluripotent stem cells (iPSC)-derived MΦ have gained major attention, as they are functionally similar to human monocyte-derived MΦ and are receptive to genome editing. In this study, we polarised iPSC-derived MΦ to M1 or M2 and analysed their proteome and secretome profiles using quantitative proteomics. These comprehensive proteomic data sets provide new insights into functions of polarised MΦ.

SARS-CoV-2 Spike protein is not pro-inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders

IntroductionThe inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (MΦ). Different recombinant Spikes might display different effects in vitro, according to protein length and glycosylation, and endotoxin (lipopolysaccharide, LPS) contamination.ObjectivesTo assess (1) the effects of different Spikes on human primary MΦ inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased MΦ inflammation.MethodsHuman primary MΦ were incubated in the presence/absence of several different Spikes (10 nM) or graded concentrations of LPS. Pro-inflammatory marker expression (qPCR and ELISA) and supernatant endotoxin contamination (LAL test) were the main readouts.ResultsLPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary MΦ. Two (out of five) Spikes were contaminated with endotoxins ≥ 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced MΦ inflammation.ConclusionsGlycosylated Spike per se is not pro-inflammatory for human MΦ, a feature which may be crucial to evade the host innate immunity. In vitro studies with commercially available Spike should be conducted with excruciating attention to potential LPS contamination.Graphical abstract

Pro-inflammatory polarization primes Macrophages to transition into a distinct M2-like phenotype in response to IL-4.

Tissue repair is largely regulated by diverse Mϕ populations whose functions are timing- and context-dependent. The early phase of healing is dominated by pro-inflammatory Mϕs, also known as M1, followed by the emergence of a distinct and diverse population that is collectively referred to as M2. The extent of the diversity of the M2 population is unknown. M2 Mϕs may originate directly from circulating monocytes or from phenotypic switching of pre-existing M1 Mϕs within the site of injury. The differences between these groups are poorly understood, but have major implications for understanding and treating pathologies characterized by deficient M2 activation, such as chronic wounds, which also exhibit diminished M1 Mϕ behavior. This study investigated the influence of prior M1 activation on human Mϕ polarization to an M2 phenotype in response to IL-4 treatment in vitro. Compared to unactivated (M0) Mϕs, M1 Mϕs up-regulated several receptors that promote the M2 phenotype, including the primary receptor for IL-4. M1 Mϕs also up-regulated M2 markers in response to lower doses of IL-4, including doses as low as 10 pg/mL, and accelerated STAT6 phosphorylation. However, M1 activation appeared to also change the Mϕ response to treatment with IL-4, generating an M2-like phenotype with a distinct gene and protein expression signature compared to M2 Mϕs prepared directly from M0 Mϕs. Functionally, compared to M0-derived M2 Mϕs, M1-derived M2 Mϕs demonstrated increased migratory response to SDF-1α, and conditioned media from these Mϕs promoted increased migration of endothelial cells in transwell assays, although other common Mϕ-associated functions such as phagocytosis were not affected by prior polarization state. In summary, M1 polarization appears to prime Mϕs to transition into a distinct M2 phenotype in response to IL4, which leads to increased expression of some genes and proteins and decreased expression of others, as well as functional differences. Together, these findings indicate the importance of prior M1 activation in regulating subsequent M2 behavior, and suggest that correcting M1 behavior may be a therapeutic target in dysfunctional M2 activation.

MNSFβ Regulates TNFα Production by Interacting with RC3H1 in Human Macrophages, and Dysfunction of MNSFβ in Decidual Macrophages Is Associated With Recurrent Pregnancy Loss.

Decidual macrophages (dMϕ) are the second largest population of leukocytes at the maternal–fetal interface and play critical roles in maintaining pregnancy. Our previous studies demonstrated the active involvement of monoclonal nonspecific suppressor factor-β (MNSFβ) in embryonic implantation and pregnancy success. MNSFβ is a ubiquitously expressed ubiquitin-like protein that also exhibits immune regulatory potential, but its function in human dMϕ remains unknown. Here, we observed that the proportion of CD11chigh (CD11cHI) dMϕ was significantly increased in dMϕ derived from patients with recurrent pregnancy loss (RPL dMϕ) compared to those derived from normal pregnant women (Control dMϕ). The production of MNSFβ and TNFα by RPL dMϕ was also significantly increased compared to that by Control dMϕ. Conditioned medium from RPL dMϕ exerted an inhibitory effect on the invasiveness of human trophoblastic HTR8/SVneo cells, and this effect could be partially reversed by a neutralizing antibody against TNFα. Bioinformatics analysis indicated a potential interaction between MNSFβ and RC3H1, a suppressor of TNFα transcription. Immunoprecipitation experiments with human Mϕ differentiated from the human monocyte cell line Thp1 (Thp1-derived Mϕ) proved the binding of MNSFβ to RC3H1. Specific knockdown of MNSFβ in Thp1-derived Mϕ led to a marked decrease in TNFα production, which could be reversed by inhibiting RC3H1 expression. Interestingly, a significant decrease in the protein level of RC3H1 was observed in RPL dMϕ. Together, our findings indicate that aberrantly increased MNSFβ expression in dMϕ may promote TNFα production via its interaction with RC3H1, and these phenomena could result in the disruption of the immune balance at the maternal–fetal interface and thus pregnancy loss.

Macrophages Derived From Human Induced Pluripotent Stem Cells: The Diversity of Protocols, Future Prospects, and Outstanding Questions.

Macrophages (Mφ) derived from induced pluripotent stem cells (iMphs) represent a novel and promising model for studying human Mφ function and differentiation and developing new therapeutic strategies based on or oriented at Mφs. iMphs have several advantages over the traditionally used human Mφ models, such as immortalized cell lines and monocyte-derived Mφs. The advantages include the possibility of obtaining genetically identical and editable cells in a potentially scalable way. Various applications of iMphs are being developed, and their number is rapidly growing. However, the protocols of iMph differentiation that are currently used vary substantially, which may lead to differences in iMph differentiation trajectories and properties. Standardization of the protocols and identification of minimum required conditions that would allow obtaining iMphs in a large-scale, inexpensive, and clinically suitable mode are needed for future iMph applications. As a first step in this direction, the current review discusses the fundamental basis for the generation of human iMphs, performs a detailed analysis of the generalities and the differences between iMph differentiation protocols currently employed, and discusses the prospects of iMph applications.

804-P: Empagliflozin Reduces Inflammation and Bioenergetics in Proinflammatory Human Macrophages: Clues to Mechanisms of Blood Vessel Protection?

SGLT-2 inhibitors (SGLT2i) reduce cardiovascular (CV) risk in people with type 2 diabetes and atherosclerotic CV diseases. The inflammatory function of macrophages (Mφ) - which play a pivotal role in atherosclerosis - is strictly dependent on their metabolism. Aim: To determine whether empagliflozin (EMPA) reduces inflammation and modifies bioenergetic metabolism in proinflammatory human Mφ (M1). Methods: M1 were differentiated from healthy donors’ monocytes and incubated with/without EMPA 100 µM for 16h. Biomarkers of inflammation and oxidative stress were quantified by qPCR. Steady-state energetic metabolism (fluxes: pmol/min for 5x104 cells) was measured by indirect microcalorimetry [oxidations of lipids (LipOX,) glucose and amino acids; anaerobic glycolysis (AnaerGlyc); tricarboxylic acid cycle (TCA); energy expenditure (EPR); maximum estimate of ATP production (ATPflux)] which is based on the integration of measured O2 and H+ (Agilent Seahorse), lactate and NH4 (enzymatic method) fluxes with the stoichiometric equations of metabolic pathways. Results: In M1, EMPA reduces (≈50%) inflammation (IL1β, IL8, TNFα and MCP1; p&amp;lt;0.05-0.001) and oxidative stress (GPX and HO-1; p &amp;lt;0.05). EMPA lowers OCR (133±9.7 vs. 153±10.3, p&amp;lt;0.001), LipOX (1.15±0.1 vs. 1.33±0.1; p&amp;lt;0.005), TCA (45±3 vs. 52±4; p&amp;lt;0.001), AnaerGlyc (210±23 vs. 268±24; p&amp;lt;0.01), ATPflux (1097±94 vs. 1313±94; p&amp;lt;0.005) and EPR (83±7 vs. 98±7 µJ/min; p&amp;lt;0.001). After dissipating mitochondrial membrane potential and forcing maximal OCR with FCCP, TCA (77±11 vs. 97±12, p=0.001), LipOX (1.7±0.3 vs. 2.2±0.4, p&amp;lt;0.005) and EPR (125±17 vs. 153±17; p&amp;lt;0.005), but not AnaerGlyc, remain inhibited by EMPA. Conclusions: EMPA alters M1 inflammation along with cytosolic (AnaerGlyc) and - to a greater extent - mitochondrial (TCA) bioenergetics. EMPA-induced metabolic dysfunction of M1 in vivo might result into reduced metaflammation and contribute to SGLT2i-dependent CV protection. Disclosure V. Spigoni: None. G. Cinquegrani: None. F. Fantuzzi: None. S. Lorusso: None. A. Dei cas: Other Relationship; Self; Novo Nordisk. R. C. Bonadonna: Advisory Panel; Self; Sanofi, Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. Funding Italian Diabete Ricerca Foundation; Eli Lilly Italy

Selective killing of human M1 macrophages by Smac mimetics alone and M2 macrophages by Smac mimetics and caspase inhibition.

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma

Macrophages (Mϕ) represent a major component of tumor tissues and play an important role in both tumor progression and therapeutic response. Although tumor Mϕ are generally considered to be derived from circulating monocytes, emerging evidence indicates that tissue Mϕ pools can be maintained by self-renewal. We aimed to elucidate the contribution, phenotype, and regulatory mechanisms of proliferating Mϕ in human hepatocellular carcinoma (HCC). Flow cytometry analyses were performed to examine the presence and phenotype of proliferating Mϕ in fresh HCC tissues. Dual immunofluorescence staining was applied to analyze the prognostic value of proliferating Mϕ. The underlying regulatory mechanisms were examined using human monocyte-derived Mϕ. Tumor-infiltrating Mϕ exhibited a significantly higher proliferative capacity than Mϕ in non-tumor tissues. A higher level of Mϕ proliferation was positively correlated with Mϕ density in the tumor and a poor prognosis in patients with HCC. Proliferating Mϕ were less differentiated (with increased CD206 expression) and were induced by the tumor cell-derived soluble small molecule, adenosine, but not proteins, lipids, or large peptides. Mechanistic studies demonstrated that autocrine granulocyte-macrophage colony-stimulating factor (GM-CSF) released by tumor-stimulated Mϕ could enhance A2A receptor expression on Mϕ and function synergistically with adenosine to elicit Mϕ proliferation in HCC. Local Mϕ proliferation is an important mechanism for Mϕ accumulation in HCC tissues. Tumor-derived adenosine functions synergistically with autocrine GM-CSF released from activated Mϕ, which promotes Mϕ proliferation. Thus, selective modulation of Mϕ accumulation at the source may provide a novel strategy for cancer therapy. Tumor-associated macrophages (TAMs) have been reported to play an essential role in both tumor progression and therapeutic response. A fundamental understanding of the mechanisms that regulate macrophage accumulation in tumors will undoubtedly lead to the development of strategies to target macrophages with high specificity and efficiency. The current study unveils a novel mechanism by which local proliferation is linked to macrophage accumulation in the tumor milieu, identifying potential targets for future immune-based anticancer therapies.

Cytotrophoblasts suppress macrophage-mediated inflammation through a contact-dependent mechanism.

Gestational membrane (GM) infection provokes inflammation and can result in preterm prelabor rupture of membranes (PPROM). The choriodecidual layer of the GM includes decidual stromal cells (DSC), cytotrophoblasts (CTB), and macrophages (Mφ). Our laboratory has previously shown that DSCs suppress Mφ TNF-α production through secreted prostaglandin E2 . We hypothesized that CTBs would also inhibit Mφ cytokine expression through secreted mediators. THP.1 Mφ-like cells with an NF-κB reporter construct or human blood monocyte-derived Mφ were co-cultured with the Jeg3 CTB cell line or primary human CTBs and challenged with group B streptococcus (GBS) or Toll-like receptor (TLR) agonists. Conditioned medium generated from CTB cultures was applied to Mφ cultures before infection or treatment. Alternatively, CTBs were co-incubated with, but physically separated from, Mφ and GBS or TLR-stimulated. NF-κB was assessed via alkaline phosphatase assay, and proinflammatory mediators were assessed by qRT-PCR and ELISA. CTBs suppressed GBS- or TLR-stimulated Mφ NF-κB activity, and TNF-α and MMP9 production. Direct physical contact between CTBs and Mφ was required for full immunosuppression. Immunosuppression could be overcome by increasing the ratio of Mφ to CTB. CTBs limit Mφ NF-κB activation and production of TNF-α and MMP9 through an as-yet unknown, cell-to-cell contact-mediated mechanism. This suppression is distinct from the PGE2 -mediated Mφ TNF-α suppression by DSC, suggesting that DSCs and CTBs regulate Mφ inflammation through distinct mechanisms. How Mφ integrates these signals in an intact GM will be paramount to determining causes and prevention of PPROM.

Pseudomonas aeruginosa Planktonic- and Biofilm-Conditioned Media Elicit Discrete Metabolic Responses in Human Macrophages

Macrophages (MΦs) are prevalent innate immune cells, present throughout human bodily tissues where they orchestrate innate and adaptive immune responses to maintain cellular homeostasis. MΦs have the capacity to display a wide array of functional phenotypes due to different microenvironmental cues, particularly soluble bacterial secretory products. Recent evidence has emerged demonstrating that metabolism supports MΦ function and plasticity, in addition to energy and biomolecular precursor production. In this study, 1D 1H-NMR-based metabolomics was used to identify the metabolic pathways that are differentially altered following primary human monocyte-derived MΦ exposure to P. aeruginosa planktonic- and biofilm-conditioned media (PCM and BCM). Metabolic profiling of PCM- and BCM-exposed MΦs indicated a significant increase in glycolytic metabolism, purine biosynthesis, and inositol phosphate metabolism. In addition, these metabolic patterns suggested that BCM-exposed MΦs exhibit a hyperinflammatory metabolic profile with reduced glycerol metabolism and elevated catabolism of lactate and amino acids, relative to PCM-exposed MΦs. Altogether, our study reveals novel findings concerning the metabolic modulation of human MΦs after exposure to secretory microbial products and contributes additional knowledge to the field of immunometabolism in MΦs.