Distinct Macrophage Populations Research Articles

Abstract IA008: Dissecting innate immune mechanisms of resistance to immune checkpoint blockade therapy in bladder cancer

Abstract We have explored the TME of bladder cancer (BC) leveraging single-cell RNA sequencing (scRNAseq) and clinical trial data to ascertain the complexity of tumor-infiltrated immune ecllls in BC. As a first step we identify distinct tumor-associated macrophage (TAM) populations associated with immune checkpoint blockade (ICB) response and resistance. Through plasma cytokine profiling, in vitro assays, and spatial analyses, we uncover novel cytokine-driven axes influencing these TAM populations and their impact on T cells, offering insights into enhancing anti-tumor T cell immunity in BC. Second, we investigate the potential of urine-derived cells (UDCs) as surrogates for monitoring the BC TME. Our scRNAseq atlas of BC patient UDCs reveals a diverse composition of immune cell populations, strikingly transcriptionally similar to tumor immune cells and containing immune subsets absent or rare in peripheral blood, suggesting their utility as biomarkers for response to therapeutic intervention. Collectively, these findings provide new insights into the immune landscape of BC and provide putative targets to enhance existing immunotherapy strategies in BC. Citation Format: Nina Bhardwaj. Dissecting innate immune mechanisms of resistance to immune checkpoint blockade therapy in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA008.

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

Tolerogenic probioticsLactobacillus delbrueckiiand Lactobacillus rhamnosus promote anti-inflammatory profile of macrophages-derived monocytes of newly diagnosed patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is known as an autoimmune disorder that is characterized by the breakdown of self-tolerance, resulting in disease onset and progression. Macrophages have been implicated as a factor in the development of SLE through faulty phagocytosis of dead cells or an imbalanced M1/M2 ratio. The study aimed to investigate the immunomodulatory effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on M1 and M2 macrophages in new case lupus patients. For this purpose, blood monocytes were collected from lupus patients and healthy people and were cultured for 5 days to produce macrophages. For 48 h, the macrophages were then cocultured with either probiotics or lipopolysaccharides (LPS). Flow cytometry and real-time polymerase chain reactionwere then used to analyze the expression of cluster of differentiation (CD) 14, CD80, and human leukocyte antigen - DR (HLADR)markers, as well as cytokine expression (interleukin [IL]1-β,IL-12,tumor necrosis factor α [TNF-α],IL-10, and transforming growth factor beta [TGF-β]).The results indicated three distinct macrophage populations, M0, M1, and M2. In both control and patient-derived macrophage-derived monocytes (MDMs), the probiotic groups showed a decrease in CD14, CD80, and HLADR expression compared to the LPS group. This decrease was particularly evident in M0 and M2 macrophages from lupus patients and M1 macrophages from healthy subjects. In addition, the probiotic groups showed increased levels of IL-10 and TGF-β and decreased levels of IL-12, IL1-β, and TNF-α in MDMs from both healthy and lupus subjects compared to the LPS groups. Although there was a higher expression of pro-inflammatory cytokines in lupus patients, there was a higher expression of anti-inflammatory cytokines in healthy subjects. In general, L. delbrueckii and L. rhamnosus could induce anti-inflammatory effects on MDMs from both healthy and lupus subjects.

GSK3α/β Restrain IFN-γ-Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation.

Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFN-γ is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFN-γ-activated cells using a new ex vivo model of AMs from mice, fetal liver-derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages. Our findings reveal that IFN-γ robustly activates both macrophage types; however, the profile of activated IFN-γ-stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFN-γ. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3α/β alters the IFN-γ response. GSK3α/β controlled distinct IFN-γ responses, and in AM-like cells, we found that GSK3α/β restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFN-γ is restricted in a GSK3α/β-dependent manner and that IFN-γ responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

Clotrimazole reverses macrophage M2 polarization by disrupting the PI3K/AKT/mTOR pathway

Macrophages switch among different activation phenotypes according to distinct environmental stimuli, varying from pro-inflammatory (M1) to alternative (also named resolutive; M2) activation forms. M1-and M2-activated macrophages represent the two extremes of the activation spectrum involving multiple species, which vary in terms of function and the cytokines secreted. The consensus is that molecular characterization of the distinct macrophage population and the signals driving their activation will help in explaining disease etiology and formulating therapies. For instance, myeloid cells residing in the tumor microenvironment are key players in tumor progression and usually display an M2-like phenotype, which help tumor cells to evade local inflammatory processes. Therefore, these specific cells have been proposed as targets for tumor therapies by changing their activation profile. Furthermore, M2 polarized macrophages are phagocytic cells promoting tissue repair and wound healing and are therefore potential targets to treat different diseases. We have already shown that clotrimazole (CTZ) decreases tumor cell viability and thus tumor growth. The mechanism by which CTZ exerts its effects remains to be determined, but this drug is an inhibitor of the PI3K/AKT/mTOR pathway. In this study, we show that CTZ downregulated M2-activation markers in macrophages polarized to the M2 profile. This effect occurred without interfering with the expression of M1-polarized markers or pro-inflammatory cytokines and signaling. Moreover, CTZ suppressed NFkB pathway intermediates and disrupted PI3K/AKT/mTOR signaling. We concluded that CTZ reverses macrophage M2 polarization by disrupting the PI3K/AKT/mTOR pathway, which results in the suppression of NFkB induction of M2 polarization. In addition, we find that CTZ represents a promising therapeutic tool as an antitumor agent.

Clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in sickle cell anemia mice

AbstractAlthough it is caused by a single-nucleotide mutation in the β-globin gene, sickle cell anemia (SCA) is a systemic disease with complex, incompletely elucidated pathologies. The mononuclear phagocyte system plays critical roles in SCA pathophysiology. However, how heterogeneous populations of hepatic macrophages contribute to SCA remains unclear. Using a combination of single-cell RNA sequencing and spatial transcriptomics via multiplexed error-robust fluorescence in situ hybridization, we identified distinct macrophage populations with diversified origins and biological functions in SCA mouse liver. We previously found that administering the von Willebrand factor (VWF)–cleaving protease ADAMTS13 alleviated vaso-occlusive episode in mice with SCA. Here, we discovered that the ADAMTS13-cleaved VWF was cleared from the circulation by a Clec4f+Marcohigh macrophage subset in a desialylation-dependent manner in the liver. In addition, sickle erythrocytes were phagocytized predominantly by Clec4f+Marcohigh macrophages. Depletion of macrophages not only abolished the protective effect of ADAMTS13 but exacerbated vaso-occlusive episode in mice with SCA. Furthermore, promoting macrophage-mediated VWF clearance reduced vaso-occlusion in SCA mice. Our study demonstrates that hepatic macrophages are important in the pathogenesis of SCA, and efficient clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in SCA mice.

Human macrophages choreograph tissue development

Yolk sac-derived macrophages have been described to promote organogenesis and tissue function in animal models, but the relevance of these studies for humans has been debated. Wang et al. reveal that human macrophage development follows similar developmental trajectories with functionally distinct macrophage populations across tissues as observed in mice.

Spatial and Temporal Mapping of Breast Cancer Lung Metastases Identify TREM2 Macrophages as Regulators of the Metastatic Boundary.

Temporal and spatial single-cell analysis of metastasis stages revealed new players in modulating immune surveillance and suppression. Our study highlights distinct populations of TREM2 macrophages as modulators of the microenvironment in metastasis, and as the key immune determinant defining metastatic niches, pointing to myeloid checkpoints to improve therapeutic strategies. This article is featured in Selected Articles from This Issue, p. 2489.

Integrated transcriptomic and metabolomic analysis reveals the metabolic programming of GM-CSF- and M-CSF- differentiated mouse macrophages.

Macrophages play a critical role in the inflammatory response and tumor development. Macrophages are primarily divided into pro-inflammatory M1-like and anti-inflammatory M2-like macrophages based on their activation status and functions. In vitro macrophage models could be derived from mouse bone marrow cells stimulated with two types of differentiation factors: GM-CSF (GM-BMDMs) and M-CSF (M-BMDMs), to represent M1- and M2-like macrophages, respectively. Since macrophage differentiation requires coordinated metabolic reprogramming and transcriptional rewiring in order to fulfill their distinct roles, we combined both transcriptome and metabolome analysis, coupled with experimental validation, to gain insight into the metabolic status of GM- and M-BMDMs. The data revealed higher levels of the tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and urea and ornithine production from arginine in GM-BMDMs, and a preference for glycolysis, fatty acid storage, bile acid metabolism, and citrulline and nitric oxide (NO) production from arginine in M-BMDMs. Correlation analysis with the proteomic data showed high consistency in the mRNA and protein levels of metabolic genes. Similar results were also obtained when compared to RNA-seq data of human monocyte derived macrophages from the GEO database. Furthermore, canonical macrophage functions such as inflammatory response and phagocytosis were tightly associated with the representative metabolic pathways. In the current study, we identified the core metabolites, metabolic genes, and functional terms of the two distinct mouse macrophage populations. We also distinguished the metabolic influences of the differentiation factors GM-CSF and M-CSF, and wish to provide valuable information for in vitro macrophage studies.

Macrophage and monocyte subsets in response to ischemic stroke.

Ischemic stroke is a leading cause of disability and mortality. Despite extensive efforts in stroke research, the only pharmacological treatment currently available is arterial recanalization, which has limited efficacy only in the acute phase of stroke. The neuroinflammatory response to stroke is believed to provide a wider time window than recanalization and has therefore been proposed as an attractive therapeutic target. In this review, we provide an overview of recent advances in the understanding of cellular and molecular responses of distinct macrophage populations following stroke, which may offer potential targets for therapeutic interventions. Specifically, we discuss the role of local responders in neuroinflammation, including the well-studied microglia as well as the emerging players, border-associated macrophages, and macrophages originating from the skull bone marrow. Additionally, we focus on the behavior of monocytes stemming from distant tissues, such as the bone marrow and spleen. Finally, we highlight aging as a crucial factor modulating the immune response, which is often neglected in animal studies. This article is protected by copyright. All rights reserved.

Studying Macrophages in the Murine Steatotic Liver Using Flow Cytometry and Confocal Microscopy.

The study of macrophage functions in the context of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction associated steatohepatitis (MASH) has been hampered by the fact that until recently all macrophages in the liver were thought to be Kupffer cells, the resident macrophages of the liver. With the advent of single-cell technologies, it is now clear that the steatotic liver harbors many distinct populations of macrophages, likely each with their own unique functions as well as subsets of monocytes and dendritic cells which can be difficult to discriminate from one another. Here, we detail the protocols we utilize to (i) induce MASLD/MASH in mice, (ii) isolate cells from the steatotic liver, and (iii) describe reliable gating strategies, which can be used to identify the different subsets of myeloid cells. Finally, we also discuss the issue of increased autofluorescence in the steatotic liver and the techniques we use to minimize this both for flow cytometry and confocal microscopy analyses.

Isolation and Characterization of Testis Macrophages Using Flow Cytometry.

Testis-resident macrophages are first responders of the innate immune system against pathogens. They also exert day-to-day functions that are poorly understood. To study testis macrophages, several techniques are used, among which we can find flow cytometry.Flow cytometry is a powerful tool that enables analysis of macrophages at a cellular as well as population level. To analyze testis macrophages using flow cytometry, a specific tissue processing is necessary to extract them. In this protocol, we explain how to extract and analyze the distinct macrophage populations.

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.

Leukolectin is expressed in lectophages, a distinct population of zebrafish embryonic macrophages

Leukolectins (LL) belong to the tectonin-family of proteins, with functions in innate immunity. Fish larvae compensating for loss of maternal chorionic protection post-hatching, provide a model-system for studying how lectins contribute to immunity. Atlantic salmon (Ssal) LL-proteins function after secretion in mucus from dermal lectocytes, as this mucus envelops embryos and larvae. The Ssalll-gene possesses multiple putative binding sites for diverse transcription-factors, suggestive of LL-functions in non-epithelial cells. Since zebrafish (zF) perivitelline fluid (PVF) contains LL-proteins, this study aims to characterize zF-leukolectins, their cellular origin, expression and gene structure. Extracts of (10 hpf) zF-embryos contained LL-proteins, and whole mount immuno-histochemistry revealed dispersed LL-positive cells including zF-lectocytes, accounting for exocrine LL-secretion by embryos. Lectocytes are lcp1-negative, but other zF-cells co-expressed LL-proteins and lcp1-transcripts, which (at this stage) identified such non-lectocytes as early macrophages (termed lectophages). In sections, LL-expression characterized large macrophage-progenitors and smaller colonizing macrophages. RT- and RACE-PCR yielded zF-LLcDNA including parts of untranslated regions. ORF encoded 255 AAs including (19 AA) signal peptide. Processing of a primary LL-transcript to (∼1.300 nt) LL-mRNA was suggested by Northern blots. Most zebrafish-egg lectins (zFELs) possess four TECPR-domains, while five TECPR-domains were predicted for zF-LL. Minor sequence variations suggested nearly identical zF-LL isoforms. Alignment of zFEL-proteins predicted a zFEL-tree with a separate leukolectin-branch. LL-amplification using zF-DNA, revealed five exons and four introns. Predicted structures of zF- and Ssal-leukolectins showed strong structural conservation (92% sequence-identity) with shorter zF-introns 2&4, but identical introns 1&3. Non-lectocytic LL-functions were investigated further by dual in situ hybridization, revealing that only some embryonic lcp1-expressing cells in early zF-embryos co-expressed LL-transcripts. Macrophages from erythro-myeloid progenitor (EMP) are known to colonize zebrafish tissues as resident macrophages (TRM), e.g. nervous system (CNS) and epiderm. Unlike Ssal-larvae relying on yolk for months, zF-larvae switch within days to nutrition from the digestive-tract, necessitating additional immuno-protection possibly from TRMs. EMP also gives rise to microglia, the TRM of CNS. The neural tube of zF-embryos exhibited numerous small, LL-positive cells, presumably stemming from lectophage-progenitors. Functions of these LL-positive embryonic microglia (lectoglia) appear more relevant for tissue remodelling than for pathogenic threats. Lectoglia sustaining CNS-neurons suggests physiological LL-roles relevant for adult health and disease. The data focus the need for resolving whether lectophages represent an unrecognized myelogenic lineage, or whether instead, LL-expression occurs in a subpopulation of the early macrophage-lineage.

#3186 INVESTIGATING TREM2 MACROPHAGES IN CHRONIC KIDNEY DISEASE

Abstract Background and Aims Kidney macrophages have been implicated in interstitial fibrosis and inflammation in various forms of chronic kidney disease (CKD), but subsets with different markers and putative functions have been identified through the application of single cell technologies. A recent study from the Greka Lab revealed distinct macrophage populations in the human kidney bearing lymphatic endothelium hyaluronan receptor-1 (LYVE1) and triggering receptor expressed on myeloid cells-2 (TREM2). Notably, TREM2+ macrophages, which have been implicated in limiting metabolic toxicity in chronic diseases, were enriched in diabetic mouse kidneys and human kidneys from obese individuals (Subramanian et al. BioRvix 2021). To support these findings, we aimed at characterizing LYVE1+ and TREM2+ macrophages in kidney samples taken from subjects with CKD-associated co-morbidities, including hypertension, diabetes mellitus and obesity. Method We performed immunofluorescence microscopy of samples collected from the cortex of kidneys donated for transplantation but deemed unsuitable for implantation. Each sample was stained with anti-CD163, -LYVE1, and -TREM2 antibodies, plus Hoechst to stain nuclei. Two identically sized fields of view were imaged in each sample. Macrophages were identified based on CD163 expression and the expression of the LYVE1 and TREM2 in macrophages delineated. Macrophage subsets were compared according to age, BMI, eGFR and the presence of hypertension, obesity and diabetes. Results of 49 donors assessed, 19 (39%) were “healthy”, six (12%) “hypertensive” without obesity or diabetes, 16 (33%) “obese” with no evidence of diabetes, and 8 (16%) “diabetic” (all had type 2 diabetes and 6/8 were also obese) The median eGFR at donation was 95 mL/min/1.73 m2 and none of the donors had a history of CKD. Gender, median age and cause of death were not significantly different as well (Table 1). “Healthy”, “hypertensive” and “obese” donors had similar numbers of total macrophages, LYVE1+ and TREM2+ macrophages per region of interest. In contrast, “diabetic” subjects had a tendency towards a higher macrophage count (P = .10) and significantly higher numbers of LYVE1+ (P = .02) and TREM2+ macrophages (P = .002), compared to “healthy” ones (see Figure 1). The median LYVE1+/CD163+ ratio was 0.69 and did not differ substantially between the groups (P = .18), while the TREM2+/CD163+ ratio was higher among “diabetic” compared to “healthy” donors (0.43 vs 0.34, P = .06). Conclusion Diabetes is associated with an increase in kidney macrophages, particularly in those expressing TREM2. Further investigation is required to determine the role these cells may play in CKD progression.

Tetramerization of STAT5 regulates monocyte differentiation and the dextran sulfate sodium-induced colitis in mice.

In response to external stimuli during immune responses, monocytes can have multifaceted roles such as pathogen clearance and tissue repair. However, aberrant control of monocyte activation can result in chronic inflammation and subsequent tissue damage. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces monocyte differentiation into a heterogenous population of monocyte-derived dendritic cells (moDCs) and macrophages. However, the downstream molecular signals that dictate the differentiation of monocytes under pathological conditions is incompletely understood. We report here that the GM-CSF-induced STAT5 tetramerization is a critical determinate of monocyte fate and function. Monocytes require STAT5 tetramers to differentiate into moDCs. Conversely, the absence of STAT5 tetramers results in a switch to a functionally distinct monocyte-derived macrophage population. In the dextran sulfate sodium (DSS) model of colitis, STAT5 tetramer-deficient monocytes exacerbate disease severity. Mechanistically, GM-CSF signaling in STAT5 tetramer-deficient monocytes results in the overexpression of arginase I and a reduction in nitric oxide synthesis following stimulation with lipopolysaccharide. Correspondingly, the inhibition of arginase I activity and sustained supplementation of nitric oxide ameliorates the worsened colitis in STAT5 tetramer-deficient mice. This study suggests that STAT5 tetramers protect against severe intestinal inflammation through the regulation of arginine metabolism.

Testicular macrophages are recruited during a narrow fetal time window and promote organ-specific developmental functions

A growing body of evidence demonstrates that fetal-derived tissue-resident macrophages have developmental functions. It has been proposed that macrophages promote testicular functions, but which macrophage populations are involved is unclear. Previous studies showed that macrophages play critical roles in fetal testis morphogenesis and described two adult testicular macrophage populations, interstitial and peritubular. There has been debate regarding the hematopoietic origins of testicular macrophages and whether distinct macrophage populations promote specific testicular functions. Here our hematopoietic lineage-tracing studies in mice show that yolk-sac-derived macrophages comprise the earliest testicular macrophages, while fetal hematopoietic stem cells (HSCs) generate monocytes that colonize the gonad during a narrow time window in a Sertoli-cell-dependent manner and differentiate into adult testicular macrophages. Finally, we show that yolk-sac-derived versus HSC-derived macrophages have distinct functions during testis morphogenesis, while interstitial macrophages specifically promote adult Leydig cell steroidogenesis. Our findings provide insight into testicular macrophage origins and their tissue-specific roles.

Single-cell RNA sequencing reveals immunosuppressive myeloid cell diversity during malignant progression in a murine model of glioma

Recent studies have shown the importance of the dynamic tumor microenvironment (TME) in high-grade gliomas (HGGs). In particular, myeloid cells are known to mediate immunosuppression in glioma; however, it is still unclear if myeloid cells play a role in low-grade glioma (LGG) malignant progression. Here, we investigate the cellular heterogeneity of the TME using single-cell RNA sequencing in a murine glioma model that recapitulates the malignant progression of LGG to HGG. LGGs show increased infiltrating CD4+ and CD8+ Tcells and natural killer (NK) cells in the TME, whereas HGGs abrogate this infiltration. Our study identifies distinct macrophage clusters in the TME that show an immune-activated phenotype in LGG but then evolve to an immunosuppressive state in HGG. We identify CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Targeting these intra-tumoral macrophages in the LGG stage may attenuate their immunosuppressive properties and impair malignant progression.

Defining and targeting tumor-associated macrophages in malignant mesothelioma

Defining the ontogeny of tumor-associated macrophages (TAM) is important to develop therapeutic targets for mesothelioma. We identified two distinct macrophage populations in mouse peritoneal and pleural cavities, the monocyte-derived, small peritoneal/pleural macrophages (SPM), and the tissue-resident large peritoneal/pleural macrophages (LPM). SPM rapidly increased in tumor microenvironment after tumor challenge and contributed to the vast majority of M2-like TAM. The selective depletion of M2-like TAM by conditional deletion of the Dicer1 gene in myeloid cells (D-/-) promoted tumor rejection. Sorted SPM M2-like TAM initiated tumorigenesis invivo and invitro, confirming their capacity to support tumor development. The transcriptomic and single-cell RNA sequencinganalysis demonstrated that both SPM and LPM contributed to the tumor microenvironment by promoting the IL-2-STAT5 signaling pathway, inflammation, and epithelial-mesenchymal transition. However, while SPM preferentially activated the KRAS and TNF-α/NFkB signaling pathways, LPM activated the IFN-γ response. The importance of LPM in the immune response was confirmed by depleting LPM with intrapleural clodronate liposomes, which abrogated the antitumoral memory immunity. SPM gene signature could be identified in pleural effusion and tumor from patients with untreated mesothelioma. Five genes, TREM2, STAB1, LAIR1, GPNMB, and MARCO, could potentially be specific therapeutic targets. Accordingly, Trem2 gene deletion led to reduced SPM M2-like TAM with compensatory increase in LPM and slower tumor growth. Overall, these experiments demonstrate that SPM M2-like TAM play a key role in mesothelioma development, while LPM more specifically contribute to the immune response. Therefore, selective targeting of monocyte-derived TAM may enhance antitumor immunity through compensatory expansion of tissue-resident TAM.

PRO-INFLAMMATORY SIRPα-EXPRESSING MONOCYTES AND MACROPHAGES MEDIATE ANTI-TNF REFRACTORY CROHN'S DISEASE IN CHILDREN

Abstract BACKGROUND Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract with detrimental consequences on the pediatric population. Anti-tumor necrosis factor (aTNF) therapies are the only FDA-approved biologics for pediatric CD but one-third fail anti-TNF induction, and of those who initially respond, approximately 15% relapse every year. Consequently, there is a pressing need to elucidate the underlying immunogenetic mechanisms of anti-TNF refractory CD. METHODS A prospective cohort study was employed of treatment-naïve (TN-CD; n= 12), aTNF refractory (REF-CD; n=11), and endoscopic remission CD (REM-CD; n=16) along with non-IBD control (n=13) children. High dimensional flow cytometric analyses (HD FACS) of the blood, ileal and rectal biopsies were performed and analyzed by t-distributed stochastic neighbor embedding (t-SNE) clustering. Single-cell RNA sequencing (scRNA-seq) was conducted using the 10X Chromium platform with a 3’ reagent kit and sequenced on Illumina’s Novaseq600. The Seurat v4.1 workflow was used for the UMAP cell clustering algorithm. Effects of conditioned media (CM) from SIRPα cells on ileal organoids from non-IBD controls was assessed by enriching blood SIRPα+ cells from REF-CD via magnetic activated cell-sorting and stimulating with LPS (10 ng/mL) and flagellin (FLG; 10 ng/mL) for 96 hrs in culture. CM was collected and ileal organoids were treated for 72 hrs and re-stimulated with LPS+FLG followed by viability and intracellular cytokine staining. Descriptive statistics, one-way ANOVA with multiple comparisons testing, and univariate linear regression were utilized. RESULTS HD FACS revealed significant enrichment of SIRPα+CD11c+HLA-DR+CD64+ (SIRPα) cells in the ileum, rectum, and blood of REF-CD (P< 0.05). These cells expressed pro-inflammatory cytokines, IL-1β, IL-6, and TNFα. Deeper characterization with scRNA-seq revealed SIRPα cells to be macrophages based on expression of: CD14, FCGR3A, CD68, CD163, FCGR1A, and MRC1. In addition to pro-inflammatory cytokines detected by HD FACS, SIRPα macrophages expressed TGFB1 and CXCL8. There were significant positive correlations of intestinal SIRPα macrophages with fecal calprotectin (P<0.05) and CRP (P<0.05). CM from SIRPα+ cells of REF-CD induced significant cell death and intracellular cytokine expression of TNFα and IL-1β from non-IBD ileal organoids. CONCLUSION SIRPα cells are a distinct pro-inflammatory macrophage population enriched in the blood and intestinal lamina propria of REF-CD that may mediate resistance to anti-TNF therapy via secretion of pro-inflammatory cytokines and disruption of the intestinal epithelial barrier. These findings provide mechanistic insight into pathogenesis of anti-TNF refractory CD in children and highlight these cells to be a potential therapeutic target.