Tissue-resident Macrophages Research Articles

Single-nucleus RNA sequencing of TMEM43 induced arrhythmogenic cardiomyopathy reveals altered cell compositions and transcriptional states in mice

Abstract Introduction and purpose TMEM43 induced Arrhythmogenic cardiomyopathy, also referred to as arrhythmogenic cardiomyopathy type 5 or ACM5, is caused by a point mutation in TMEM43 (p.S358L). The disease is characterised by chamber dilation and a progressive dystrophy of the ventricular myocardium causing life-threatening ventricular arrhythmias and sudden cardiac death. Loss of cardiomyocytes and fibrofatty replacement of the myocardium is clearly observed in our transgenic mouse model of ACM5 overexpressing TMEM43-S358L in postnatal cardiomyocytes. However, the mechanisms underlying cardiomyocyte death and fibroblast activation remain elusive, precluding the development of effective therapeutic options. Here we applied the state-of-the-art analysis on single-nucleus RNA sequencing (snRNA-seq) to characterise the cellular compositions and molecular states in ACM5 mouse hearts. Methods Left ventricular heart tissues of severely affected ACM5 mice and their corresponding transgenic controls overexpressing wild-type TMEM43 were used for snRNA-seq analysis. We identified 10 major cell types, which aggregated into 23 distinct clusters on the basis of transcriptional similarity. Results ACM5 nuclei showed significant depletion of cardiomyocytes and expanded populations of fibroblasts and myeloid cells. Fibroblasts from mutant mice acquired an activated phenotype related to remodelling of the extracellular matrix with a large number of genes like LOX, POSTN, NOX4 or COL1A1 significantly upregulated. In addition, the mutant cardiomyocytes showed altered transcriptional states characterized by cardiac stress and heart failure markers such as NPPB or MYH7. Interestingly, we were able to identify a cluster of mutant cardiomyocytes with a highly apoptotic profile. This cluster is also enriched in genes related to the unfolding protein response (UPR) stress sensors and the DNA damage response (DDR), elucidating a new potential mechanism of myocyte death in ACM5. Furthermore, heterogeneity of the myeloid populations is also observed. The number of tissue resident macrophages is increased in mutant samples and myeloid cells show additional disease-associated transcriptional states that are almost entirely absent in control samples. Conclusion Together these data illuminated shared and distinct cell and molecular architectures of ACM5 in mouse hearts. Further confirmation on diverse gene expression profiles like the UPR and DDR pathways related to cardiomyocyte death will establish a valuable resource to explore novel therapeutic targets and personalized therapies for ACM5.

Recruited macrophages incorporate into the resident cardiac macrophage pool and converge into a common phenotype in the healing infarct, but not in the failing heart exposed to pressure overload

Abstract Introduction Resident macrophages account for 5% of the cells in the healthy heart. In response to cardiac injury, monocytes infiltrate and differentiate into recruited macrophages which function distinctly from the resident subset of primarily embryonic origin. In this work, we aim to identify the extent to which macrophage origin, tissue location and type of cardiac injury determine macrophage phenotypes in ischemic and non-ischemic cardiac injuries over time. Results and methods We use a tamoxifen inducible CX3CR1 Yfp CreER/+ :R26tdT/+ mouse line to visualize and quantify fluxes of resident and recruited macrophages, and their respective localizations within the heart following ischemia and reperfusion (I/R) injury and pressure overload after transversal aortic constriction (TAC), respectively. Interestingly, macrophage numbers peaked during the first week post-surgery in both the local/acute and the global/continuous injury models. Initially, recruited macrophages outnumbered the resident macrophage pool within the infarct area but ultimately reached a 1:1 equilibrium at 4 weeks post I/R injury and at 8 weeks post TAC. In I/R injury, the 1:1 equilibrium established already during the first week in the remote myocardium. Gene expression profiles changed alongside shifts in macrophage ratios. For instance, in the infarct, recruited macrophages showed upregulation of cytokine production and cell chemotaxis pathways at early stage. By 4 weeks post MI their gene expression profiles have converged with those of resident macrophages. Likewise, in the remote myocardium, recruited and resident macrophages assimilated post MI, overexpressing genes associated adaptative immune response activation and cellular homeostasis. Similar to the I/R injury setting, a 1:1 equilibrium of resident and recruited macrophages established in the hypertrophied and dilated hearts within 4 to 8 weeks post TAC surgery. However, transcriptional profiles remained differentially regulated between recruited and resident cardiac macrophages at all time points tested, and distinct from those observed post I/R injury: recruited macrophages behavior changed from a profibrotic into a proliferative phenotype, meanwhile resident macrophages featured prominently in tissue homeostasis. Conclusion Ischemic and non-ischemic, focal and global, acute and chronic cardiac injuries induce a transient peak of monocyte recruitment and macrophage differentiation in and around cardiac lesions which lead to a permanent integration of recruited monocyte-derived macrophages into the pool of tissue resident macrophages – albeit at different paces. The type of injury and macrophage localization within the remodeling tissue determine partial or complete override of ontogenic cell programs.

Abstract 17622: CCN3 Deficiency in Myeloid Cells Exacerbates Aortic Dissection by Governing a Proinflammatory Program in Macrophages

Introduction: Aortic dissection (AD) is a fatal disease. Macrophages are known central regulators of aortic inflammation and elastic fiber disruption, two major hallmarks of AD. Cellular communication network factor 3 (CCN3/NOV) is a matricellular protein associated with various biological cellular functions in cardiovascular disease and cancers. Emerging evidence strongly implicates a protective role of CCN3 against several types of vascular pathologies, however its role in AD remains unknown. Hypothesis: CCN3 deficiency in myeloid cells promotes AD development in murine models through the modulation of macrophage phenotype. Aims: We investigated the causal role of CCN3 loss in AD pathogenesis, focusing on its role in macrophages. Methods: 3-4-week-old male myeloid-specific CCN3 knockout mice (CCN3F/F;LysMCre) and control mice (LysMCre) were given BAPN (β-aminopropionitrile monofumarate; 0.2%) in their drinking water for 4 weeks for AD modeling. Mouse survival rate and histological changes of the aorta were assessed. Single cell RNA sequencing analysis was performed to recapitulate cellular transcriptome profile changes. Finally, gain and loss of function studies in bone marrow-derived macrophages (BMDMs) were performed to decipher the underlying mechanism by which CCN3 controls macrophage plasticity. Results: Following BAPN administration, myeloid-specific CCN3 knockout mice had a higher mortality rate due to AD formation and aortic rupture. In accordance with this phenotype, vascular media thickness was markedly reduced, and elastic fiber fragmentation and macrophage infiltration were elevated in myeloid-specific CCN3 knockout mice. Using single-cell RNA-seq, we found that myeloid-deficiency of CCN3 was associated with a pathogenic pro-inflammatory phenotype of perivascular tissue-resident macrophages, which promoted deleterious aortic wall remodeling at early stages. Furthermore, CCN3 deficient BMDMs are prone to M1 macrophage polarization, an effect dependent on the activation of the JAK/STAT1 signaling pathway. Conclusions: Myeloid-deficiency of CCN3 drives vascular macrophages toward an M1-like, pathogenic phenotype and promotes dissecting aortic aneurysm.

Characterisation of macrophage infiltration and polarisation based on integrated transcriptomic and histological analyses in Primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a progressive inflammatory autoimmune disease. Immune cell infiltration into glandular lobules and ducts and glandular destruction are the pathophysiological hallmarks of pSS. Macrophages are one of the most important cells involved in the induction and regulation of an inflammatory microenvironment. Although studies have reported that an abnormal tissue microenvironment alters the metabolic reprogramming and polarisation status of macrophages, the mechanisms driving macrophage infiltration and polarisation in pSS remain unclear. Immune cell subsets were characterised using the single-cell RNA sequencing (scRNA-seq) data of peripheral blood mononuclear cells (PBMCs) from patients with pSS (n = 5) and healthy individuals (n = 5) in a public dataset. To evaluate macrophage infiltration and polarisation in target tissues, labial salivary gland biopsy tissues were subjected to histological staining and bulk RNA-seq (pSS samples, n = 24; non-pSS samples, n = 12). RNA-seq data were analysed for the construction of macrophage co-expression modules, enrichment of biological processes and deconvolution-based screening of immune cell types. Detailed mapping of PBMCs using scRNA-seq revealed five major immune cell subsets in pSS, namely, T cells, B cells, natural killer (NK) cells, dendritic cells (DCs) and monocyte-macrophages. The monocyte-macrophage subset was large and had strong inflammatory gene signatures. This subset was found to play an important role in the generation of reactive oxygen species and communicate with other innate and adaptive immune cells. Histological staining revealed that the number of tissue-resident macrophages was high in damaged glandular tissues, with the cells persistently surrounding the tissues. Analysis of RNA-seq data using multiple algorithms demonstrated that the high abundance of pro-inflammatory M1 macrophages was accompanied by the high abundance of other infiltrating immune cells, senescence-associated secretory phenotype and evident metabolic reprogramming. Macrophages are among the most abundant innate immune cells in PBMCs and glandular tissues in patients with pSS. A bidirectional relationship exists between macrophage polarisation and the inflammatory microenvironment, which may serve as a therapeutic target for pSS.

Single-cell RNA sequencing-guided fate-mapping toolkit delineates the contribution of yolk sac erythro-myeloid progenitors

Erythro-myeloid progenitors of the yolk sac that originates during early embryo development has been suggested to generate tissue-resident macrophage, mast cell, and even endothelial cell populations from fetal to adult stages. However, the heterogeneity of erythro-myeloid progenitors (EMPs) is not well characterized. Here, we adapt single-cell RNA sequencing to dissect the heterogeneity of EMPs and establish several fate-mapping tools for each EMP subset to trace the contributions of different EMP subsets. We identify two primitive and one definitive EMP subsets from the yolk sac. In addition, we find that primitive EMPs are decoupled from definitive EMPs. Furthermore, we confirm that primitive and definitive EMPs give rise to microglia and other tissue-resident macrophages, respectively. In contrast, only Kit+ Csf1r- primitive EMPs generate endothelial cells transiently during early embryo development. Overall, our results delineate the contribution of yolk sac EMPs more clearly based on the single-cell RNA sequencing (scRNA-seq)-guided fate-mapping toolkit.

Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability

BackgroundMyeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. ObjectivesWe aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. MethodsWe used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. ResultsGlycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. ConclusionCollectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.

Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages.

Even though gammaherpesvirus and parasitic infections are endemic in parts of the world, there is a lack of understanding about the outcome of coinfection. In humans, coinfections usually occur sequentially, with fluctuating order and timing in different hosts. However, experimental studies in mice generally do not address the variables of order and timing of coinfections. We sought to examine the variable of coinfection order in a system of gammaherpesvirus-helminth coinfection. Our previous work demonstrated that infection with the intestinal parasite, Heligmosomoides polygyrus, induced transient reactivation from latency of murine gammaherpesvirus-68 (MHV68). In this report, we reverse the order of coinfection, infecting with H. polygyrus first, followed by MHV68, and examined the effects of preexisting parasite infection on MHV68 acute and latent infection. We found that preexisting parasite infection increased the propensity of MHV68 to reactivate from latency. However, when we examined the mechanism for reactivation, we found that preexisting parasite infection increased the ability of MHV68 to reactivate in a vitamin A dependent manner, a distinct mechanism to what we found previously with parasite-induced reactivation after latency establishment. We determined that H. polygyrus infection increased both acute and latent MHV68 infection in a population of tissue resident macrophages, called large peritoneal macrophages. We demonstrate that this population of macrophages and vitamin A are required for increased acute and latent infection during parasite coinfection.

Vancomycin-induced gut microbial dysbiosis alters enteric neuron-macrophage interactions during a critical period of postnatal development.

Vancomycin is a broad-spectrum antibiotic widely used in cases of suspected sepsis in premature neonates. While appropriate and potentially lifesaving in this setting, early-life antibiotic exposure alters the developing microbiome and is associated with an increased risk of deadly complications, including late-onset sepsis (LOS) and necrotizing enterocolitis (NEC). Recent studies show that neonatal vancomycin treatment disrupts postnatal enteric nervous system (ENS) development in mouse pups, which is in part dependent upon neuroimmune interactions. This suggests that early-life antibiotic exposure could disrupt these interactions in the neonatal gut. Notably, a subset of tissue-resident intestinal macrophages, muscularis macrophages, has been identified as important contributors to the development of postnatal ENS. We hypothesized that vancomycin-induced neonatal dysbiosis impacts postnatal ENS development through its effects on macrophages. Using a mouse model, we found that exposure to vancomycin in the first 10 days of life, but not in adult mice, resulted in an expansion of pro-inflammatory colonic macrophages by increasing the recruitment of bone-marrow-derived macrophages. Single-cell RNA sequencing of neonatal colonic macrophages revealed that early-life vancomycin exposure was associated with an increase in immature and inflammatory macrophages, consistent with an influx of circulating monocytes differentiating into macrophages. Lineage tracing confirmed that vancomycin significantly increased the non-yolk-sac-derived macrophage population. Consistent with these results, early-life vancomycin exposure did not expand the colonic macrophage population nor decrease enteric neuron density in CCR2-deficient mice. Collectively, these findings demonstrate that early-life vancomycin exposure alters macrophage number and phenotypes in distinct ways compared with vancomycin exposure in adult mice and results in altered ENS development.

The ligation between ERMAP, galectin-9 and dectin-2 promotes Kupffer cell phagocytosis and antitumor immunity.

Kupffer cells, the liver tissue resident macrophages, are critical in the detection and clearance of cancer cells. However, the molecular mechanisms underlying their detection and phagocytosis of cancer cells are still unclear. Using in vivo genome-wide CRISPR-Cas9 knockout screening, we found that the cell-surface transmembrane protein ERMAP expressed on various cancer cells signaled to activate phagocytosis in Kupffer cells and to control of liver metastasis. ERMAP interacted with β-galactoside binding lectin galectin-9 expressed on the surface of Kupffer cells in a manner dependent on glycosylation. Galectin-9 formed a bridging complex with ERMAP and the transmembrane receptor dectin-2, expressed on Kupffer cells, to induce the detection and phagocytosis of cancer cells by Kupffer cells. Patients with low expression of ERMAP on tumors had more liver metastases. Thus, our study identified the ERMAP-galectin-9-dectin-2 axis as an 'eat me' signal for Kupffer cells.

Inhibitory effect of telocyte-induced M1 macrophages on endometriosis: Targeting angiogenesis and invasion

PurposeTelocytes (TCs), a novel type of stromal cells found in tissues, induce macrophage differentiation into classically activated macrophages (M1) types and enhance their phagocytic function. The purpose of this study was to investigate the inhibitory effects of TC-induced M1 macrophages on endometriosis (EMs). Methodsmouse uterine primary TCs and endometrial stromal cells (ESCs) were isolated and identified using double immunofluorescence staining. For the in vitro study, ESCs were treated with TC-induced M1 macrophages, and the vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP9), and nuclear factor kappa B (NF-κb) genes were identified by quantitative real-time PCR (qRT-PCR) or western blotting (WB). For the in vivo study, an EMs mouse model received TC-conditioned medium (TCM) via abdominal administration, and characterized the inhibitory effects on growth (lesion weight, volume, and pathology), tissue-resident macrophages differentiation by immunostaining, angiogenic capacity (CD31 and VEGF), invasive capacity (MMP9), and NF-κb expression within EMs lesions. Resultsimmunofluorescent staining showed that uterine TCs expressed CD34+ and vimentin+, whereas ESCs expressed vimentin+ and cytokeratin-. At the cellular level, TC-induced M1 macrophages can significantly inhibit the expression of VEGF and MMP9 in ESCs through WB or qRT-PCR, possibly by suppressing the NF-κb pathway. The in vivo study showed that macrophages switch from the alternatively activated macrophages (M2) in untreated EMs lesions to the M1 subtype after TCM exposure. Thereby, TC-induced M1 macrophages contributed to the inhibition of EMs lesions. More importantly, this effect may be achieved by suppressing the expression of NF-κb to inhibit angiogenesis (CD31 and VEGF) and invasion (MMP9) in the tissue. ConclusionTC-induced M1 macrophages play a prevailing role in suppressing EMs by inhibiting angiogenic and invasive capacity through the NF-κb pathway, which provides a promising therapeutic approach for EMs.

Independent prognostic biomarker FERMT3 associated with immune infiltration and immunotherapy response in glioma

Background Adult glioma progresses rapidly and has a poor clinical outcome. The focal adhesion protein Kindlin-3 (encoded by the FERMT3 gene) participates in tumor development, drug resistance, and progression. However, the relationship between Kindlin-3 and glioma prognosis or immune microenvironment is poorly understood. Methods We comprehensively analyzed the expression, prognostic value, mutation landscape, functional enrichment, immune infiltration, and therapeutic role of FERMT3 in glioma using multiple datasets and validated Kindlin-3 expression in clinical tissue specimens by immunohistochemistry and multiple immunofluorescence staining. Results FERMT3 is an independent predictor of glioma prognosis and is highly expressed in glioblastoma tissues. Functional enrichment analyses indicated that FERMT3 participates in multiple immune-related pathways such as immune response and cytokine production. Furthermore, FERMT3 expression was positively correlated with the infiltration of several immune cells, immune scores, and the expression of genes related to immune checkpoints. Further analyses revealed that overexpression of FERMT3 was linked to a better response to anti-PD1 therapy. Data from single-cell RNA-seq reveal that FERMT3 was largely expressed in microglial cells and tissue-resident macrophages. Multiple immunofluorescence staining confirmed the overexpression of Kindlin-3 in the glioma-associated microglia/macrophages (GAMs). Conclusion The findings of this study provide a new perspective on the role of Kindlin-3 in glioma and may have a significant impact on the discovery of novel biomarkers and targeting of GAMs in the future.

220 Ezrin is required to maintain the number and function of tissue resident alveolar macrophages in cystic fibrosis during infection

220 Ezrin is required to maintain the number and function of tissue resident alveolar macrophages in cystic fibrosis during infection

Distinct hypoxia-induced translational profiles of embryonic and adult-derived macrophages

Tissue resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Posttranscriptional regulation determining mRNA stability and translation rate may override transcriptional signals in response to hypoxia. We profiled differentially regulated BMDM and FLDM transcripts in response to hypoxia at the level of mRNA translation. Using a translating ribosome affinity purification (TRAP) assay and RNA-seq, we identified non-overlapping transcripts with increased translation rate in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2) after hypoxia. We further identified hypoxia-induced transcripts within these subsets that are regulated by the RNA-binding protein HuR. These findings define translational differences in macrophage subset gene expression programs, highlighting potential therapeutic targets in ischemic myocardium.

Metabolic heterogeneity of tissue-resident macrophages in homeostasis and during helminth infection

Tissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using high-dimensional spectral flow cytometry, we observe distinct metabolic profiles between different organs and functionally link acetyl CoA carboxylase activity to efferocytotic capacity. Additionally, differences in metabolism are evident within populations from a specific site, corresponding to relative stages of macrophage maturity. Immune perturbation with intestinal helminth infection increases alternative activation and metabolic rewiring of monocyte-derived macrophage populations, while resident TIM4+ intestinal macrophages remain immunologically and metabolically hyporesponsive. Similar metabolic signatures in alternatively-activated macrophages are seen from different tissues using additional helminth models, but to different magnitudes, indicating further tissue-specific contributions to metabolic states. Thus, our high-dimensional, flow-based metabolic analyses indicates complex metabolic heterogeneity and dynamics of tissue-resident macrophage populations at homeostasis and during helminth infection.

P12.15.A THE IMPACT OF MICROGLIA MODULATION ON THE IMMUNE TUMOR MICROENVIRONMENT IN SYNGENEIC GLIOBLASTOMA MOUSE MODELS

Abstract BACKGROUND Glioblastoma is exceptionally resistant to T-cell targeting immunotherapies for which the immunosuppressive myeloid compartment is held responsible. As influencer and instructor of antitumoral T-cell-immunity this compartment mainly consists of Monocyte-derived Cells (MdCs) and tissue-resident (TR) macrophages called microglia (MG). Since the response to immunotherapies depend on antigen presenting cells at the tumor site, we aim to investigate this compartment and its individual role in tumor progression by genetically depleting/activating the TR MG. METHODS We made use of two MG-specific and tamoxifen inducible knockout mouse models in which 1) we activate MG by excision of the activation-repressing transcription factor Sall1 (Sall1CreERT2/wt x Sall1fl/wt) and 2) deplete MG by deleting the “survival-receptor” Csf1r on MG (Sall1CreERT2/wt x Csf1rfl/fl). To investigate the effect of MG activation and depletion on the immune tumor microenvironment (iTME), immunocompetent mice were engrafted with syngeneic glioma cells (CT-2A, GL261) and the iTME subsequently characterized by scRNAseq, spectral flow cytometry and immunofluorescence imaging. RESULTS Upon Sall1-KO MG demonstrate a morphologically reactive phenotype reminiscent of activated MG. The MG-specific deletion of Csf1r effectively depletes the brain parenchyma of TR MG which rapidly gets repopulated by a morphologically distinct microglia-(like) population. Genetic activation of MG leads to a survival benefit in Gl261-engrafted mice compared to Cre-negative littermates. In contrast, depletion of MG significantly reduces the survival in Gl261 and CT-2A engrafted mice. ScRNAseq confirms the effective depletion of MG and the subsequent emergence of a microglia-like cell population that despite microscopical resemblance to MG, transcriptionally remains distinct. The iTME in MG-depleted mice is characterized by a decreased abundance of exhausted and naïve CD8-Tcells and an increased infiltration of MdCs. Gene set enrichment analysis demonstrate a dampened inflammatory immune profile of infiltrating MdCs highlighted by a downregulation of hallmark gene sets of “interferon-α and γ response” and “TNFα-signaling via NF-κB”. CONCLUSION The presence of MG orchestrate the development towards a more antitumorigenic iTME by shaping the antitumoral response of incoming myeloid cells and furthermore directly or indirectly influencing T-cell immunity. Understanding this interplay of MG and MdCs in cancer and its impact on instruction of antitumoral T-cell immunity could help guiding the development of novel immunotherapeutic strategies in GBM.

Optimizing a Novel Fate-Mapping Strategy to Assess Tissue Resident Macrophages in Muscle Regeneration

Tissue resident macrophages (TRMs) are heterogenous innate immune cells involved in tissue homeostasis and repair, yet their role in muscle regeneration is currently unknown. TRMs differ from classical monocyte-derived macrophages (MDMs) in both ontogeny and function. Our inability to differentiate TRMs from MDMs has limited the development of subset specific immunotherapies. The novel Cx3cr1CreER/+:RosaTd/+ fate-mapping mouse model employs tamoxifen inducible labeling of MDMs and TRMs with a TdTomato reporter (Td) that persists only in self-renewing TRMs, while MDMs are replaced by Td- monocyte progenitors over a 4-week period. This strategy has been successfully used in adult mice heart and brain tissue. However, in the skeletal muscle, expression level of the Cx3cr1 reporter gene is unknown. We assessed muscle TRM Cx3cr1 expression in neonate (P3), young (P21), and adult (10wk) mice to optimize a pulse/chase strategy to label muscle TRMs with the Td reporter. Like heart and brain tissue, we found muscle TRMs express high levels of Cx3cr1 throughout development and adulthood suggesting viability of this strategy. We performed a single injection of tamoxifen in adult Cx3cr1CreER/+:RosaTd/+ mice and showed ~20% induction in blood monocytes and muscle TRMs. Although Td induction was poor, levels were similar between the blood and muscle, suggesting accuracy of the model. The next step is to optimize tamoxifen administration to achieve >90% labelling in blood and muscle. To assess spatial localization of TRMs with muscle resident stem cells (satellite cells) we optimized immunofluorescence staining on tissue sections. We found a statistically significant increase in colocalization of macrophages with satellite cells in healthy and injured gastrocnemius tissue, consistent with their predicted role in satellite cell proliferation. Once complete, our fate-mapping approach will allow for the labelling of muscle TRM subsets and reveal their role in the regeneration process.

Fate-Mapping of Yolk Sac-Derived Macrophages.

To better understand the distinct functions of yolk-sac-derived tissue-resident macrophages (TRMs) and bone-marrow-derived macrophages in homeostasis and disease, it is important to trace the ontogeny of these cells. The majority of TRMs originate from erythro-myeloid progenitors (EMPs). EMPs develop into pre-macrophages (pMacs), which can be detected starting at embryonic developmental day (E)9.0, and which give rise to all TRM during early development. pMacs start expressing the gene Cx3cr1, allowing us to genetically target the early yolk-sac wave of pMacs and their progeny. Here, we describe the protocol for the identification of yolk sac-derived TRMs utilizing in utero labelling of the inducible fate mapping Cx3cr1CreERT; Rosa26LSL-eYFP mouse model.

Fate-Mapping of Hematopoietic Stem Cell-Derived Macrophages.

Macrophages are cells of the innate immune system, which contribute to the maintenance of tissue homeostasis and form the first line of defense against pathogens. Tissue-resident macrophages that originate from erythro-myeloid-progenitors in the yolk sac colonize the organs early during development and self-maintain in most organs throughout adulthood. Under homeostatic and pathological conditions, circulating monocytes infiltrate the tissue, where they differentiate into macrophages. However, particularly upon inflammation, phenotyping of these distinct macrophage populations using surface markers or antibody stainings is insufficient as their phenotypes converge, at least transiently. A well-established method for the developmental origin of different cell types is the use of in vivo fate-mapping models, where a fluorescent reporter will be expressed under the control of a cell type-specific promoter. Here, we describe the Cxcr4CreERT2; Rosa26LSL-tdTomato mouse fate-mapping model, which labels hematopoietic stem cells and, thus, also monocytes and monocyte-derived macrophages while most tissue-resident macrophages are not targeted.