Blockade of Tigit on AML-Derived M2 Macrophages Results in Reprograming into the M1 Phenotype and Enhances CD47-Mediated Phagocytosis

Background: Bidirectional interactions between the tumor microenvironment (TME) and AML cells lead to disease progression through induction of angiogenesis, migration, cancer stemness and local immunosuppression. Leukemia-associated macrophages (LAM) constitute an important cell population within the TME, but little is known about the phenotype, function, and plasticity of these cells. In the present study we provide an extensive characterization of the macrophage population in patients with AML. Methods: The phenotype and expression of co-regulatory receptors was assessed on different bone marrow-derived CD68 +CD14 + LAM populations, in comparison to corresponding CD3 + T-cells and CD117 +CD34 + AML cells (n=35), as well as peripheral blood monocytes from healthy donors (HD, n=16) using multi-parameter flow cytometry. The expression of surface markers and the distribution of LAM subpopulations was correlated with clinical parameters. The effect of a blocking anti-TIGIT antibody on the in vitro plasticity on primary LAMs and monocyte-derived macrophages from healthy donors was investigated. Furthermore, we analyzed if the treatment with blocking anti-TIGIT and anti-CD47 antibodies could increase the anti-leukemic phagocytosis of AML cell lines and in vitro polarized monocyte-derived M2 macrophages. Results: Phenotypic analysis of M1 and M2 macrophages in AML and HD revealed that the predominant macrophage population in patients with AML is made up of immunosuppressive alternatively activated M2 LAMs defined by expression of CD163 and CD86 (M1 AML vs. HD p<0.01 and M2 AML vs. HD p=0.02). These M2 LAMs contained significantly higher frequencies of cells expressing the immune checkpoint receptors TIGIT and TIM-3 than M1 LAMs (TIGIT + M2 vs. M1 p<0.01 and TIM-3 + M2 vs. M1 p<0.01, respectively). Regarding co-expression of multiple co-inhibitory receptors, the frequency of macrophages co-expressing TIM-3 or LAG-3 with TIGIT was higher in samples from AML patients in comparison to HDs (p=0.01 and p<0.01, respectively). This difference was caused by the significant up-regulation of TIM-3 and LAG-3 on TIGIT + M2 LAMs in comparison to their corresponding M1 LAMs (p<0.01and p<0.01, respectively). Importantly, in vitro blockade of TIGIT in primary LAMs of AML patients or differentiated PB-derived M2 macrophages of HDs resulted in a change in polarization from the M2 towards the M1 phenotype after 24 hours (AML: anti-TIGIT vs. IgG2a p<0.01, n=7 and HD: anti-TIGIT vs. IgG2a p=0.02, n=3). Moreover, the additional blockade of TIGIT on PB-derived M2 macrophages augmented the anti-CD47-mediated phagocytosis of the AML cell lines MOLM-13 and MV4-11 after 4 hours (MOLM-13: anti-CD47 vs. IgG1a 31% vs. 10.9%, p=0.04; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 31% vs. 46.4%, p<0.01 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 46.4% vs. 13.6%, p<0.01, n=3 and for MV4-11: anti-CD47 vs. IgG1a 14.4% vs. 7.345%, p=0.03; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 14.4% vs. 28.6%, p=0.03 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 28.6% vs. 12.85%, p=0.04, n=2). Next, we correlated the phenotypic data with clinical parameters. AML patients of the intermediate risk group according to ELN criteria exhibited a significantly higher frequency of M2 LAMs co-expressing TIGIT and LAG-3 than those in the favorable group (p=0.04 and p=0.01). Moreover, the frequency of TIM-3 + M2 LAMs was significantly increased in patients with adverse and intermediate risk in comparison to those with a favorable risk (p=0.01, p=0.0053). Furthermore, TIGIT + M2 LAMs were significantly more frequent in patients with the FLT3 ITD mutation in comparison with the wilde type (p=0.03). Conclusions: Our findings suggest that the proven clinical effect of monoclonal antibodies against TIGIT and TIM-3 in cancer may be due in part to their action on macrophages and depend on macrophage polarization. Our study identifies TIGIT + M2 LAMs co-expressing TIM-3 and LAG-3 as a promising effector population in AML. Further experiments should be conducted to investigate macrophage-mediated cytotoxicity in AML. Disclosures Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: meeting attendance; Servier: Consultancy, Honoraria, Other: meeting attendance; Jazz Pharmaceuticals: Other: meeting attendance; Novartis: Other: meeting attendance. Bokemeyer: BMS: Honoraria, Other: Travel accomodation, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel accomodation; Merck Serono: Consultancy, Other: Travel accomodation ; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicine: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Gylcotope GmbH: Research Funding; GlaxoSmithKline: Research Funding; Inside: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding; Janssen-Cilag: Research Funding; Karyopharm Therapeutics: Research Funding; Lilly: Research Funding; Millenium: Research Funding; MSD: Research Funding; Merck KGaA: Honoraria; Bayer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Merck Sharp Dohme: Consultancy, Honoraria; AstraZeneca: Honoraria, Research Funding; Lilly/ImClone: Consultancy; Nektar: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other. Fiedler: Celgene: Consultancy; Servier: Consultancy, Other: support for meeting attendance; Abbvie: Consultancy, Honoraria; Morphosys: Consultancy; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: support for meeting attendance; Jazz Pharmaceuticals: Consultancy, Other: support for meeting attendance; Stemline: Consultancy; Novartis: Consultancy; ARIAD/Incyte: Consultancy; Amgen: Consultancy, Other: support for meeting attendance, Patents & Royalties, Research Funding.

Interplay between myofibers and pro-inflammatory macrophages controls muscle damage in mdx mice

ABSTRACT Duchenne muscular dystrophy is a genetic muscle disease characterized by chronic inflammation and fibrosis mediated by a pro-fibrotic macrophage population expressing pro-inflammatory markers. Our aim was to characterize cellular events leading to the alteration of macrophage properties and to modulate macrophage inflammatory status using the gaseous mediator hydrogen sulfide (H2S). Using co-culture experiments, we first showed that myofibers derived from mdx mice strongly skewed the polarization of resting macrophages towards a pro-inflammatory phenotype. Treatment of mdx mice with NaHS, an H2S donor, reduced the number of pro-inflammatory macrophages in skeletal muscle, which was associated with a decreased number of nuclei per fiber, as well as reduced myofiber branching and fibrosis. Finally, we established the metabolic sensor AMP-activated protein kinase (AMPK) as a critical NaHS target in muscle macrophages. These results identify an interplay between myofibers and macrophages where dystrophic myofibers contribute to the maintenance of a highly inflammatory environment sustaining a pro-inflammatory macrophage status, which in turn favors myofiber damage, myofiber branching and establishment of fibrosis. Our results also highlight the use of H2S donors as a potential therapeutic strategy to improve the dystrophic muscle phenotype by dampening chronic inflammation. This article has an associated First Person interview with the first author of the paper.

Predictive potential of macrophage population phenotyping in malignization of H. pylori-associated chronic gastritis

Tumor-associated macrophages are able to regulate the tumor cell proliferation and to affect the tumor cell dissemination. The study was aimed to assess the predictive potential of the macrophage population immunohistochemical phenotyping in early malignization of H. pylori-associated chronic gastritis. Gastic biopsy samples of male and female patients aged 48 ± 7.2 infected with Helicobacter pylori were used as the research material. The patients were divided into three groups: non-atrophic chronic gastritis (NACG, n = 10), atrophic chronic gastritis (ACG, n = 10), G1/G2 gastric adenocarcinoma (GAC, n = 10). The macrophage population was visualized using the CD68 pan-macrophage marker and the type 2 monocyte/macrophage marker CD163. Intensity of neoangiogenesis was defined using the CD31 endothelial marker by assessing the total cross sectional area of blood vessels. It was found that chronic gastritis was accompanied by the dynamic increase in the size of the general macrophage population with the progression of atrophic and metaplastic processes. According to immunohistochemical study of biopsies obtained from patients with NCG, the CD163 : CD68 ratio was 0.67 ± 0.02, and the total cross sectional area of blood vessels was 3590.92 ± 356.27 µm2. Atrophic gastritis and adenocarcinoma were characterized by vector redistribution of monocytes/macrophages into the 2nd functional phenotype. The CD163 : CD68 expression index in the group with ACG was 0.81 ± 0.04, and in the group with GAC it was 0.88 ± 0.03. Microvascular area was significantly increased in the groups with ACG and GAC, which reflected tumor neoangiogenesis intensification under the influence of М2 monocytes/macrophages. The increased expression of CD163 can serve as a predictor of chronic gastritis malignization together with evaluation of the glandular epithelium atrophy and metaplasia degree.

A Novel Treatment for Glomerular Disease: Targeting the Activated Macrophage Folate Receptor with a Trojan Horse Therapy in Rats

Since activated macrophages express a functional folate receptor β (FRβ), targeting this macrophage population with folate-linked drugs could increase selectivity to treat inflammatory diseases. Using a macrophage-mediated anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN) in WKY rats, we investigated the effect of a novel folic acid-aminopterin (AMT) conjugate (EC2319) designed to intracellularly deliver AMT via the FR. We found that treatment with EC2319 significantly attenuated kidney injury and preserved renal function. Kidney protection with EC2319 was blocked by a folate competitor, indicating that its mechanism of action was specifically FRβ-mediated. Notably, treatment with methotrexate (MTX), another folic acid antagonist related to AMT, did not protect from kidney damage. EC2319 reduced glomerular and interstitial macrophage infiltration and decreased M1 macrophage recruitment but not M2 macrophages. The expression of CCL2 and the pro-fibrotic cytokine TGF-β were also reduced in nephritic glomeruli with EC2319 treatment. In EC2319-treated rats, there was a significant decrease in the deposition of collagens. In nephritic kidneys, FRβ was expressed on periglomerular macrophages and macrophages present in the crescents, but its expression was not observed in normal kidneys. These data indicate that selectively targeting the activated macrophage population could represent a novel means for treating anti-GBM GN and other acute crescentic glomerulonephritis.

Infusion of Phagocytic Macrophages Overexpressing CPT1a Ameliorates Kidney Fibrosis in the UUO Model

Phagocytosis is an inherent function of tissue macrophages for the removal of apoptotic cells and cellular debris during acute and chronic injury; however, the dynamics of this event during fibrosis development is unknown. We aim to prove that during the development of kidney fibrosis in the unilateral ureteral obstruction (UUO) model, there are some populations of macrophage with a reduced ability to phagocytose, and whether the infusion of a population of phagocytic macrophages could reduce fibrosis in the murine model UUO. For this purpose, we have identified the macrophage populations during the development of fibrosis and have characterized their phagocytic ability and their expression of CPT1a. Furthermore, we have evaluated the therapeutic effect of macrophages overexpressing CPT1a with high phagocytic skills. We evidenced that the macrophage population which exhibits high phagocytic ability (F4/80low-CD11b) in fibrotic animals decreases during the progression of fibrosis while the macrophage population with lower phagocytic ability (F4/80high-CD11b) in fibrotic conditions, conversely, increases and CPT1a macrophage cell therapy with a strengthening phagocytic ability is associated with a therapeutic effect on kidney fibrosis. We have developed a therapeutic approach to reduce fibrosis in the UUO model by enrichment of the kidney resident macrophage population with a higher proportion of exogenous phagocytic macrophages overexpressing CPT1a.

Probiotic Bifidobacterium bifidum G9-1 Has a Preventive Effect on the Acceleration of Colonic Permeability and M1 Macrophage Population in Maternally Separated Rats

Although probiotics may be useful for the treatment of irritable bowel syndrome (IBS), it is unclear how probiotics play a role in colonic mucosal integrity and immunity. Here, we aimed to investigate the effect of Bifidobacterium bifidum G9-1 (BBG9-1) on colonic mucosal integrity and macrophage behavior in rats subjected to maternal separation (MS) as a model of IBS. MS pups were individually separated from their mother rats, and a proportion of the MS rats were orally administered BBG9-1. The colonic mucosal permeability was evaluated by Ussing chamber assay. The expression of tight junction proteins and cytokines and the population of CD80-positive cells was examined in the colonic tissues by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Caco2 cells were stimulated with cytokines and the transepithelial electric resistance (TEER) was measured. MS rats showed significantly higher colonic permeability and lower claudin 4 expression in the colonic epithelium relative to controls. The number of CD80-positive macrophages was significantly increased in the colonic mucosa of MS rats, accompanied by the increase of IL-6 and IFN-γ expression. BBG9-1 treatment ameliorated the increase of M1 macrophage and IL-6/IFN-γ expression in the colonic tissue of MS rats. Simultaneously, BBG9-1 treatment improved the enhanced mucosal permeability and the decreased claudin 4 expression in the colon of MS rats. IL-6 and IFN-γ, whose expression is enhanced in the colon of MS rats, significantly decreased TEER in Caco2 cells in vitro. Probiotic BBG9-1 has a preventive effect on the acceleration of colonic permeability and M1 macrophage population in maternally separated rats.

Obesity-instructed TREM2high macrophages identified by comparative analysis of diabetic mouse and human kidney at single cell resolution

Mouse models are a tool for studying the mechanisms underlying complex diseases; however, differences between species pose a significant challenge for translating findings to patients. Here, we used single-cell transcriptomics and orthogonal validation approaches to provide cross-species taxonomies, identifying shared broad cell classes and unique granular cellular states, between mouse and human kidney. We generated cell atlases of the diabetic and obese kidney using two different mouse models, a high-fat diet (HFD) model and a genetic model (BTBR ob/ob), at multiple time points along disease progression. Importantly, we identified a previously unrecognized, expanding Trem2high macrophage population in kidneys of HFD mice that matched human TREM2high macrophages in obese patients. Taken together, our cross-species comparison highlights shared immune and metabolic cell-state changes.

Identification and characterization of a non-conventional CD45 negative perivascular macrophage population within the mouse brain.

Perivascular macrophages (pvM) are closely associated with cerebral vasculature and play an essential role in drainage of the brain and regulation of the immune response. Here, using reporter mouse models and immunofluorescence on sections and whole brain, flow cytometry and single cell sequencing, we identify a Lyve1+ brain perivascular population lacking classical macrophage markers such as CD45 and Cx3cr1. We named the new non-conventional CD45 negative perivascular macrophages pvM2. These cells have a similar location, morphology and phagocytic function as conventional pvM. The pvM2 are not derived from hematopoietic stem cells, as they are negative in the VavtdT lineage tracing model. They increase in number after photothrombotic induced stroke established by flow cytometry and 3D immunofluorescence analysis. Since CD45 negative cells were typically excluded from macrophage studies, the presence of pvM2 has been previously missed and their role is of importance to assess in the brain disease models.