InVivoMAb anti-mouse/human/rat CCL2 (MCP-1)

Catalog #BE0185
Product Citations:
36
Clone:
2H5
Reactivities:
Mouse, Human, Rat

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Product Details

The 2H5 antibody reacts with mouse, human, and rat CCL2 also known as monocyte chemoattractant protein 1 (MCP-1). CCL2 is a 13 kDa chemokine that is produced mainly by monocytes/macrophages but is also expressed by endothelial, fibroblasts, epithelial, smooth muscle, mesangial, astrocytic, and microglial cells. It can be expressed constitutively or after induction by oxidative stress, cytokines, or growth factors. CCL2 regulates the migration and infiltration of monocytes, memory T lymphocytes, and NK cells and is implicated in the pathogeneses of several diseases characterized by monocytic infiltrates, such as multiple sclerosis, psoriasis, rheumatoid arthritis, and atherosclerosis. The 2H5 antibody has been shown to neutralize the biological effects of CCL2 in vivo.

Specifications

Isotype Armenian Hamster IgG, κ
Recommended Isotype Control(s) InVivoMAb polyclonal Armenian hamster IgG
Recommended Dilution Buffer InVivoPure pH 7.0 Dilution Buffer
Conjugation This product is unconjugated. Conjugation is available via our Antibody Conjugation Services.
Immunogen CHO-expressed mouse MCP-1
Reported Applications in vivo CCL2 neutralization
Immunohistochemistry (frozen)
Formulation PBS, pH 7.0
Contains no stabilizers or preservatives
Endotoxin <2EU/mg (<0.002EU/μg)
Determined by LAL gel clotting assay
Purity >95%
Determined by SDS-PAGE
Sterility 0.2 µm filtration
Production Purified from cell culture supernatant in an animal-free facility
Purification Protein G
RRID AB_10950302
Molecular Weight 150 kDa
Storage The antibody solution should be stored at the stock concentration at 4°C. Do not freeze.
in vivo CCL2 neutralization
Berg, N. K., et al. (2021). "Hypoxia-inducible factor-dependent induction of myeloid-derived netrin-1 attenuates natural killer cell infiltration during endotoxin-induced lung injury" Faseb j 35(4): e21334. PubMed

Sepsis and sepsis-associated lung inflammation significantly contribute to the morbidity and mortality of critical illness. Here, we examined the hypothesis that neuronal guidance proteins could orchestrate inflammatory events during endotoxin-induced lung injury. Through a targeted array, we identified netrin-1 as the top upregulated neuronal guidance protein in macrophages treated with lipopolysaccharide (LPS). Furthermore, we found that netrin-1 is highly enriched in infiltrating myeloid cells, particularly in macrophages during LPS-induced lung injury. Transcriptional studies implicate hypoxia-inducible factor HIF-1α in the transcriptional induction of netrin-1 during LPS treatment. Subsequently, the deletion of netrin-1 in the myeloid compartment (Ntn1(loxp/loxp) LysM Cre) resulted in exaggerated mortality and lung inflammation. Surprisingly, further studies revealed enhanced natural killer cells (NK cells) infiltration in Ntn1(loxp/loxp) LysM Cre mice, and neutralization of NK cell chemoattractant chemokine (C-C motif) ligand 2 (CCL2) reversed the exaggerated lung inflammation. Together, these studies provide functional insight into myeloid cell-derived netrin-1 in controlling lung inflammation through the modulation of CCL2-dependent infiltration of NK cells.

in vivo CCL2 neutralization
Brunner, P. M., et al. (2015). "CCL7 contributes to the TNF-alpha-dependent inflammation of lesional psoriatic skin" Exp Dermatol 24(7): 522-528. PubMed

Chemokines are small chemotactic proteins that have a crucial role in leukocyte recruitment into tissue. Targeting these mediators has been suggested as a potential therapeutic option in inflammatory skin diseases such as psoriasis. Using quantitative RT-PCR, we found CCL7, a chemokine ligand known to interact with multiple C-C chemokine receptors, to be markedly increased in lesional psoriasis as opposed to atopic dermatitis, lichen planus, non-lesional psoriatic and normal control skin. Surprisingly, this increase in CCL7 mRNA expression exceeded that of all other chemokines investigated, and keratinocytes and dermal blood endothelial cells were identified as its likely cellular sources. In an imiquimod-induced psoriasis-like mouse model, CCL7 had a profound impact on myeloid cell inflammation as well as on the upregulation of key pro-psoriatic cytokines such as CCL20, IL-12p40 and IL-17C, while its blockade led to an increase in the antipsoriatic cytokine IL-4. In humans receiving the TNF-alpha-blocker infliximab, CCL7 was downregulated in lesional psoriatic skin already within 16 hours after a single intravenous infusion. These data suggest that CCL7 acts as a driver of TNF-alpha-dependent Th1/Th17-mediated inflammation in lesional psoriatic skin.

in vivo CCL2 neutralization
Singh, M., et al. (2014). "Effective innate and adaptive antimelanoma immunity through localized TLR7/8 activation" J Immunol 193(9): 4722-4731. PubMed

Intratumoral immune activation can induce local and systemic antitumor immunity. Imiquimod is a cream-formulated, TLR7 agonist that is Food and Drug Administration approved for the treatment of nonmelanoma skin cancers, but it has limited activity against melanoma. We studied the antitumor activity and mechanism of action of a novel, injectable, tissue-retained TLR7/8 agonist, 3M-052, which avoids systemic distribution. Intratumoral administration of 3M-052 generated systemic antitumor immunity and suppressed both injected and distant, uninjected wild-type B16.F10 melanomas. Treated tumors showed that an increased level of CCL2 chemokines and infiltration of M1 phenotype-shifted macrophages, which could kill tumor cells directly through production of NO and CCL2, were essential for the antitumor activity of 3M-052. CD8(+) T cells, B cells, type I IFN, IFN-gamma, and plasmacytoid dendritic cells were contributed to efficient tumor suppression, whereas perforin, NK cells, and CD4 T cells were not required. Finally, 3M-052 therapy potentiated checkpoint blockade therapy with anti-CTLA-4 and anti-programmed death ligand 1 Abs, even when checkpoint blockade alone was ineffective. Our findings suggest that intratumoral treatment with 3M-052 is a promising approach for the treatment of cancer and establish a rational strategy and mechanistic understanding for combination therapy with intratumoral, tissue-retained TLR7/8 agonist and checkpoint blockade in metastatic cancer.

Immunohistochemistry (frozen)
Tominaga, T., et al. (2009). "Blocking mast cell-mediated type I hypersensitivity in experimental allergic conjunctivitis by monocyte chemoattractant protein-1/CCR2" Invest Ophthalmol Vis Sci 50(11): 5181-5188. PubMed

PURPOSE: To characterize the roles played by monocyte chemoattractant protein-1 and its preferential receptor CCR2 (MCP-1/CCL2) in acute allergic inflammation. METHODS: The direct effects of MCP-1 were evaluated histologically after a subconjunctival injection of recombinant MCP-1 into naive mice. The mice were sensitized to ragweed pollen, and allergic conjunctivitis was induced by an allergen challenge. The location of the induced MCP-1 was determined by immunohistochemistry. Anti-MCP-1 antibody and CCR2-specific antagonist, RS 504393, were used to determine whether an inhibition of MCP-1 or CCR2 signals would suppress the allergen-induced immediate hypersensitivity reaction. The effect of blocking CCR2 was tested in vitro with isolated mast cells from connective tissue, to evaluate the co-stimulatory signals mediated by CCR2 in mast cells directly. RESULTS: A subconjunctival injection of MCP-1 stimulated conjunctival mast cell degranulation and recruited monocytes/macrophages. In the allergic conjunctivitis model, the allergen-induced MCP-1 protein was located in the monocytes/macrophages in the substantia propria of the conjunctiva. Blocking MCP-1 significantly suppressed the allergen-induced clinical signs and mast cell degranulation without affecting the allergen-specific IgE, or the release of Th2 cytokine from the isolated draining lymph node cells. Inhibition of CCR2 similarly suppressed the acute inflammatory responses. Consistent with the outcome of the disease model, inhibition of CCR2 suppressed allergen-specific degranulation of IgE-primed, isolated conjunctival mast cells. CONCLUSIONS: Stimulation of the co-stimulatory axis of CCR2 by MCP-1 is essentially required for mast cell-mediated hypersensitivity reactions in mouse eyes.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Immunology and Microbiology
    Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

    In Immunity on 10 October 2023 by Pekayvaz, K., Gold, C., et al.

    PubMed

    Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche. Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

    • Immunology and Microbiology
    The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries.

    In Immunity on 11 April 2023 by Hanna, B. S., Wang, G., et al.

    PubMed

    Specific microbial signals induce the differentiation of a distinct pool of RORγ+ regulatory T (Treg) cells crucial for intestinal homeostasis. We discovered highly analogous populations of microbiota-dependent Treg cells that promoted tissue regeneration at extra-gut sites, notably acutely injured skeletal muscle and fatty liver. Inflammatory meditators elicited by tissue damage combined with MHC-class-II-dependent T cell activation to drive the accumulation of gut-derived RORγ+ Treg cells in injured muscle, wherein they regulated the dynamics and tenor of early inflammation and helped balance the proliferation vs. differentiation of local stem cells. Reining in IL-17A-producing T cells was a major mechanism underlying the rheostatic functions of RORγ+ Treg cells in compromised tissues. Our findings highlight the importance of gut-trained Treg cell emissaries in controlling the response to sterile injury of non-mucosal tissues. Copyright © 2023 Elsevier Inc. All rights reserved.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Immunology and Microbiology
    Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation.

    In The Journal of Experimental Medicine on 3 April 2023 by Petkova, M., Kraft, M., et al.

    PubMed

    Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type-specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3caH1047R-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3caH1047R mice. Expression of pro-inflammatory genes, including monocyte/macrophage chemokine Ccl2, in Pik3caH1047R-iLECs was associated with recruitment of VEGF-C-producing macrophages. Macrophage depletion, CCL2 blockade, or anti-inflammatory COX-2 inhibition limited Pik3caH1047R-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk involving iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes.© 2023 Petkova et al.

    • Mus musculus (House mouse)
    Shp2 Deficiency in Kupffer Cells and Hepatocytes Aggravates Hepatocarcinogenesis by Recruiting Non-Kupffer Macrophages.

    In Cellular and Molecular Gastroenterology and Hepatology on 25 February 2023 by Du, L., Ji, Y., et al.

    PubMed

    Complex communications between hepatocytes and Kupffer cells (KCs) are known to drive or suppress hepatocarcinogenesis, with controversial data in the literature. In previous experiments that aimed to decipher hepatocyte/KC interactions, we unexpectedly unveiled a tumor-suppressing effect of polyinosinic-polycytidylic acid, a widely used inducer of MX dynamin like GTPase 1 (Mx1)-cre expression, which questioned a theory of interleukin 1a/6 cytokine circuit in hepatocyte/KC communication. The goal of this study was to clarify the controversy and decipher unique functions of KCs and non-KC macrophages in liver tumorigenesis. We used the C-type lectin domain family 4 member F (Clec4f)-cre system to delete Src-homology 2 domain-containing tyrosine phosphatase 2 (Shp2)/protein tyrosine phosphatase nonreceptor 11 (Ptpn11) in KCs, and a combination of Clec4f-cre and adeno-associated virus-cre to delete Shp2 in KCs and hepatocytes to investigate the effects on hepatocellular carcinoma development and immune cell compositions/activities. Ablating Shp2 in KCs generated a tumor-promoting niche, which was exacerbated further by concurrent removal of Shp2 in both KCs and hepatocytes. Shp2 deficiency induced KC apoptosis and decreased its numbers, which induced compensatory recruitment of bone marrow-derived monocytes into liver. These newly recruited monocytes differentiated into non-KC macrophages with tumor-associated macrophage function, leading to aggravated tumor progression through down-regulation of CD8 T cells. Tumor-associated macrophage blockade by anti-chemokine (C-C motif) ligand 2 (CCL2) antibody inhibited hepatocellular carcinoma progression, while depletion of all macrophages had a tumor-promoting effect by increasing myeloid-derived suppressor cells (M-MDSCs) and decreasing CD8 T cells. Shp2 loss in KCs or hepatocytes generated a protumorigenic microenvironment, which was exacerbated by its removal in both cell types. These results show the complexity of intercellular signaling events in liver tumorigenesis and raises caution on the use of specific Shp2 inhibitor in liver cancer therapy. Transcript profiling: RNA sequencing data are available at Gene Expression Omnibus (GSE222594). Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

    • Cell Biology
    • ,
    • Immunology and Microbiology
    Age-associated adipose tissue inflammation promotes monocyte chemotaxis and enhances atherosclerosis.

    In Aging Cell on 1 February 2023 by Song, J., Farris, D., et al.

    PubMed

    Although aging enhances atherosclerosis, we do not know if this occurs via alterations in circulating immune cells, lipid metabolism, vasculature, or adipose tissue. Here, we examined whether aging exerts a direct pro-atherogenic effect on adipose tissue in mice. After demonstrating that aging augmented the inflammatory profile of visceral but not subcutaneous adipose tissue, we transplanted visceral fat from young or aged mice onto the right carotid artery of Ldlr-/- recipients. Aged fat transplants not only increased atherosclerotic plaque size with increased macrophage numbers in the adjacent carotid artery, but also in distal vascular territories, indicating that aging of the adipose tissue enhances atherosclerosis via secreted factors. By depleting macrophages from the visceral fat, we identified that adipose tissue macrophages are major contributors of the secreted factors. To identify these inflammatory factors, we found that aged fat transplants secreted increased levels of the inflammatory mediators TNFα, CXCL2, and CCL2, which synergized to promote monocyte chemotaxis. Importantly, the combined blockade of these inflammatory mediators impeded the ability of aged fat transplants to enhance atherosclerosis. In conclusion, our study reveals that aging enhances atherosclerosis via increased inflammation of visceral fat. Our study suggests that future therapies targeting the visceral fat may reduce atherosclerosis disease burden in the expanding older population. © 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    Long-term exposure to house dust mites accelerates lung cancer development in mice.

    In Journal of Experimental & Clinical Cancer Research : CR on 21 January 2023 by Wang, D., Li, W., et al.

    PubMed

    Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).  METHODS: The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1β (IL-1β), and C-C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.  RESULTS: Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1β signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1β, and CCL2 neutralization, or ICS treatment. Collectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC). © 2023. The Author(s).

    • Cancer Research
    • ,
    • Neuroscience
    Astrocyte immunometabolic regulation of the tumour microenvironment drives glioblastoma pathogenicity.

    In Brain on 14 September 2022 by Perelroizen, R., Philosof, B., et al.

    PubMed

    Malignant brain tumours are the cause of a disproportionate level of morbidity and mortality among cancer patients, an unfortunate statistic that has remained constant for decades. Despite considerable advances in the molecular characterization of these tumours, targeting the cancer cells has yet to produce significant advances in treatment. An alternative strategy is to target cells in the glioblastoma microenvironment, such as tumour-associated astrocytes. Astrocytes control multiple processes in health and disease, ranging from maintaining the brain's metabolic homeostasis, to modulating neuroinflammation. However, their role in glioblastoma pathogenicity is not well understood. Here we report that depletion of reactive astrocytes regresses glioblastoma and prolongs mouse survival. Analysis of the tumour-associated astrocyte translatome revealed astrocytes initiate transcriptional programmes that shape the immune and metabolic compartments in the glioma microenvironment. Specifically, their expression of CCL2 and CSF1 governs the recruitment of tumour-associated macrophages and promotes a pro-tumourigenic macrophage phenotype. Concomitantly, we demonstrate that astrocyte-derived cholesterol is key to glioma cell survival, and that targeting astrocytic cholesterol efflux, via ABCA1, halts tumour progression. In summary, astrocytes control glioblastoma pathogenicity by reprogramming the immunological properties of the tumour microenvironment and supporting the non-oncogenic metabolic dependency of glioblastoma on cholesterol. These findings suggest that targeting astrocyte immunometabolic signalling may be useful in treating this uniformly lethal brain tumour. © The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

    • Immunology and Microbiology
    • ,
    • ELISA
    • ,
    • Homo sapiens (Human)
    cGAS and DDX41-STING mediated intrinsic immunity spreads intercellularly to promote neuroinflammation in SOD1 ALS model.

    In IScience on 17 June 2022 by Tan, H. Y., Yong, Y. K., et al.

    PubMed

    Neuroinflammation exacerbates the progression of SOD1-driven amyotrophic lateral sclerosis (ALS), although the underlying mechanisms remain largely unknown. Herein, we demonstrate that misfolded SOD1 (SOD1Mut)-causing ALS results in mitochondrial damage, thus triggering the release of mtDNA and an RNA:DNA hybrid into the cytosol in an mPTP-independent manner to activate IRF3- and IFNAR-dependent type I interferon (IFN-I) and interferon-stimulating genes. The neuronal hyper-IFN-I and pro-inflammatory responses triggered in ALS-SOD1Mut were sufficiently robust to cause a strong physiological outcome in vitro and in vivo. cGAS/DDX41-STING-signaling is amplified in bystander cells through inter-neuronal gap junctions. Our results highlight the importance of a common DNA-sensing pathway between SOD1 and TDP-43 in influencing the progression of ALS. © 2022.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    Macrophage-targeted anti-CCL2 immunotherapy enhances tumor sensitivity to 5-fluorouracil in a Balb/c-CT26 murine colon carcinoma model measured using diffuse reflectance spectroscopy.

    In BMC Immunology on 23 April 2022 by Bess, S. N., Greening, G. J., et al.

    PubMed

    Immunotherapy in colorectal cancer (CRC) regulates specific immune checkpoints and, when used in combination with chemotherapy, can improve patient prognosis. One specific immune checkpoint is the recruitment of circulating monocytes that differentiate into tumor-associated macrophages (TAMs) and promote tumor angiogenesis. Changes in vascularization can be non-invasively assessed via diffuse reflectance spectroscopy using hemoglobin concentrations and oxygenation in a localized tumor volume. In this study, we examine whether blockade of monocyte recruitment via CCL2 (macrophage chemoattractant protein-1) leads to enhanced sensitivity of 5-fluorouracil (5-FU) in a CT26-Balb/c mouse model of CRC. It was hypothesized that the blockade of TAMs will alter tumor perfusion, increasing chemotherapy response. A subcutaneous tumor model using Balb/c mice injected with CT26 colon carcinoma cells received either a saline or isotype control, anti-CCL2, 5-FU, or a combination of anti-CCL2 and 5-FU. Findings show that 12 days post-treatment, monocyte recruitment was significantly reduced by approximately 61% in the combination group. This shows that the addition of anti-CCL2 to 5-FU slowed the fold-change (change from the original measurement to the final measurement) in tumor volume from Day 0 to Day 12 (~ 5 fold). Modest improvements in oxygen saturation (~ 30%) were observed in the combination group. The findings in this work suggest that the blockade of CCL2 is sufficient in the reduction of TAMs that are recruited into the tumor microenvironment and has the ability to modestly alter tumor perfusion during early-tumor response to treatment even though the overall benefit is relatively modest. © 2022. The Author(s).

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    • ,
    • Stem Cells and Developmental Biology
    Immune deconvolution and temporal mapping identifies stromal targets and developmental intervals for abrogating murine low-grade optic glioma formation.

    In Neuro-oncology Advances on 22 February 2022 by de Andrade Costa, A., Chatterjee, J., et al.

    PubMed

    Brain tumor formation and progression are dictated by cooperative interactions between neoplastic and non-neoplastic cells. This stromal dependence is nicely illustrated by tumors arising in the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome, where children develop low-grade optic pathway gliomas (OPGs). Using several authenticated Nf1-OPG murine models, we previously demonstrated that murine Nf1-OPG growth is regulated by T cell function and microglia Ccl5 production, such that their inhibition reduces tumor proliferation in vivo. While these interactions are critical for established Nf1-OPG tumor growth, their importance in tumor formation has not been explored. A combination of bulk and single-cell RNA mouse optic nerve sequencing, immunohistochemistry, T cell assays, and pharmacologic and antibody-mediated inhibition methods were used in these experiments. We show that T cells and microglia are the main non-neoplastic immune cell populations in both murine and human LGGs. Moreover, we demonstrate that CD8+ T cells, the predominant LGG-infiltrating lymphocyte population, are selectively recruited through increased Ccl2 receptor (Ccr4) expression in CD8+, but not CD4+, T cells, in a NF1/RAS-dependent manner. Finally, we identify the times during gliomagenesis when microglia Ccl5 production (3-6 weeks of age) and Ccl2-mediated T cell infiltration (7-10 weeks of age) occur, such that temporally-restricted Ccl2 or Ccl5 inhibition abrogates tumor formation >3.5 months following the cessation of treatment. Collectively, these findings provide proof-of-concept demonstrations that targeting stromal support during early gliomagenesis durably blocks murine LGG formation. © The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.

    • Cancer Research
    • ,
    • Immunology and Microbiology
    Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity

    Preprint on BioRxiv : the Preprint Server for Biology on 28 January 2022 by Barreira da Silva, R., Leitao, R., et al.

    PubMed

    h4>ABSTRACT/h4> Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an enzyme that mediates N-terminal modification of several subtrates, including the monocyte-chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody blockade of the receptor CSF1R, which more broadly eliminates tissue macrophages, reversed tumor growth inhibition in Qpctl −/− mice, and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8 + T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    A GATA4-regulated secretory program suppresses tumors through recruitment of cytotoxic CD8 T cells.

    In Nature Communications on 11 January 2022 by Patel, R. S., Romero, R., et al.

    PubMed

    The GATA4 transcription factor acts as a master regulator of development of multiple tissues. GATA4 also acts in a distinct capacity to control a stress-inducible pro-inflammatory secretory program that is associated with senescence, a potent tumor suppression mechanism, but also operates in non-senescent contexts such as tumorigenesis. This secretory pathway is composed of chemokines, cytokines, growth factors, and proteases. Since GATA4 is deleted or epigenetically silenced in cancer, here we examine the role of GATA4 in tumorigenesis in mouse models through both loss-of-function and overexpression experiments. We find that GATA4 promotes non-cell autonomous tumor suppression in multiple model systems. Mechanistically, we show that Gata4-dependent tumor suppression requires cytotoxic CD8 T cells and partially requires the secreted chemokine CCL2. Analysis of transcriptome data in human tumors reveals reduced lymphocyte infiltration in GATA4-deficient tumors, consistent with our murine data. Notably, activation of the GATA4-dependent secretory program combined with an anti-PD-1 antibody robustly abrogates tumor growth in vivo. © 2022. The Author(s).

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Immunology and Microbiology
    GM-CSF production by non-classical monocytes controls antagonistic LPS-driven functions in allergic inflammation.

    In Cell Reports on 28 December 2021 by Kaur, K., Bachus, H., et al.

    PubMed

    Lipopolysaccharide (LPS) can either promote or prevent T helper 2 (Th2) cell allergic responses. However, the underlying mechanism remains unknown. We show here that LPS activity switches from pro-pathogenic to protective depending on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by non-classical monocytes. In the absence of GM-CSF, LPS can favor pathogenic Th2 cell responses by supporting the trafficking of lung-migratory dendritic cells (mDC2s) into the lung-draining lymph node. However, when non-classical monocytes produce GM-CSF, LPS and GM-CSF synergize to differentiate monocyte-derived DCs from classical Ly6Chi monocytes that instruct mDC2s for Th2 cell suppression. Importantly, only allergens with cysteine protease activity trigger GM-CSF production by non-classical monocytes. Hence, the therapeutic effect of LPS is restricted to allergens with this enzymatic activity. Treatment with GM-CSF, however, restores the protective effects of LPS. Thus, GM-CSF produced by non-classical monocytes acts as a rheostat that fine-tunes the pathogenic and therapeutic functions of LPS.Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

    • In Vivo
    • ,
    • Mus musculus (House mouse)
    • ,
    • Cancer Research
    A novel CXCR4 antagonist counteracts paradoxical generation of cisplatin-induced pro-metastatic niches in lung cancer.

    In Molecular Therapy on 6 October 2021 by Bertolini, G., Cancila, V., et al.

    PubMed

    Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases. In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

    • Block
    • ,
    • Mus musculus (House mouse)
    • ,
    • Immunology and Microbiology
    • ,
    • Neuroscience
    High-parameter cytometry unmasks microglial cell spatio-temporal response kinetics in severe neuroinflammatory disease.

    In Journal of Neuroinflammation on 26 July 2021 by Spiteri, A. G., Terry, R. L., et al.

    PubMed

    Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a 'microglia-like' phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is important to understand their temporal contribution to disease and inform effective intervention in its pathogenesis. During West Nile virus (WNV) encephalitis, widespread neuronal infection drives substantial CNS infiltration of inflammatory monocytes, causing severe immunopathology and/or death, but the role of microglia in this remains unclear. Using high-parameter cytometry and dimensionality-reduction, we devised a simple, novel gating strategy to identify microglia and infiltrating myeloid cells during WNV-infection. Validating our strategy, we (1) blocked the entry of infiltrating myeloid populations from peripheral blood using monoclonal blocking antibodies, (2) adoptively transferred BM-derived monocytes and tracked their phenotypic changes after infiltration and (3) labelled peripheral leukocytes that infiltrate into the brain with an intravenous dye. We demonstrated that myeloid immigrants populated only the identified macrophage gates, while PLX5622 depletion reduced all 4 subsets defined by the microglial gates. Using this gating approach, we identified four consistent microglia subsets in the homeostatic and WNV-infected brain. These were P2RY12hi CD86-, P2RY12hi CD86+ and P2RY12lo CD86- P2RY12lo CD86+. During infection, 2 further populations were identified as 'inflammatory' and 'microglia-like' macrophages, recruited from the bone marrow. Detailed kinetic analysis showed significant increases in the proportions of both P2RY12lo microglia subsets in all anatomical areas, largely at the expense of the P2RY12hi CD86- subset, with the latter undergoing compensatory proliferation, suggesting replenishment of, and differentiation from this subset in response to infection. Microglia altered their morphology early in infection, with all cells adopting temporal and regional disease-specific phenotypes. Late in disease, microglia produced IL-12, downregulated CX3CR1, F4/80 and TMEM119 and underwent apoptosis. Infiltrating macrophages expressed both TMEM119 and P2RY12 de novo, with the microglia-like subset notably exhibiting the highest proportional myeloid population death. Our approach enables detailed kinetic analysis of resident vs infiltrating myeloid cells in a wide range of neuroinflammatory models without non-physiological manipulation. This will more clearly inform potential therapeutic approaches that specifically modulate these cells. © 2021. The Author(s).

    • Endocrinology and Physiology
    • ,
    • Immunology and Microbiology
    Viral infection of the ovaries compromises pregnancy and reveals innate immune mechanisms protecting fertility.

    In Immunity on 13 July 2021 by Tomac, J., Mazor, M., et al.

    PubMed

    Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility. Copyright © 2021 Elsevier Inc. All rights reserved.

    • Neuroscience
    High-parameter Cytometry Unmasks Microglial Cell Spatio-temporal Response Kinetics in Severe Neuroinflammatory Disease.

    Preprint on Research Square on 7 April 2021 by Spiteri, A. G., Terry, R. L., et al.

    PubMed

    h4>Background: /h4> Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a ‘microglia-like’ phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is important to understand their temporal contribution to disease and inform effective intervention in its pathogenesis. During West Nile virus (WNV) encephalitis, widespread neuronal infection drives substantial CNS infiltration of inflammatory monocytes, causing severe immunopathology and/or death, but the role of microglia in this remains unclear. h4>Methods: /h4>: Using high-parameter cytometry and dimensionality-reduction, we devised a simple, novel gating strategy to identify microglia and infiltrating myeloid cells under extreme inflammatory conditions. Validating our strategy we 1) blocked the entry of infiltrating myeloid populations from peripheral blood using monoclonal blocking antibodies, 2) adoptively transferred BM-derived monocytes and tracked their phenotypic changes after infiltration and 3) labelled peripheral leukocytes that infiltrate into the brain with an intravenous dye. We demonstrated that myeloid immigrants populated only the identified macrophage gates, while PLX5622 depletion reduced all 4 subsets defined by the microglial gates. h4>Results: /h4>: Using this novel gating approach, we identified four consistent microglia subsets in the homeostatic and WNV-infected brain. These were P2RY12 hi CD86 - , P2RY12 hi CD86 + , and P2RY12 lo CD86 - P2RY12 lo CD86 + . During infection, 2 further populations were identified as inflammatory and microglia-like macrophages, recruited from the bone marrow. Detailed kinetic analysis showed significant increases in the proportions of both P2RY12 lo microglia subsets in all anatomical areas, largely at the expense of the P2RY12 hi CD86 - subset, with the latter undergoing compensatory proliferation, suggesting replenishment of, and differentiation from this subset in response to infection. Microglia altered their morphology early in infection, with all cells adopting temporal and regional disease-specific phenotypes. Late in disease, microglia produced IL-12, downregulated CX3CR1, F4/80 and TMEM119 and underwent apoptosis. Infiltrating macrophages expressed both TMEM119 and P2RY12 de novo, with the microglia-like subset notably exhibiting the highest proportional myeloid population death. h4>Conclusions: /h4>: Our approach enables detailed kinetic analysis of resident vs infiltrating myeloid cells in a wide range of neuroinflammatory models without non-physiological manipulation. This will more clearly inform potential therapeutic approaches that specifically modulate these cells.

    • In Vivo
    • ,
    • Neutralization
    • ,
    • Mus musculus (House mouse)
    Hypoxia-inducible factor-dependent induction of myeloid-derived netrin-1 attenuates natural killer cell infiltration during endotoxin-induced lung injury.

    In The FASEB Journal on 1 April 2021 by Berg, N. K., Li, J., et al.

    PubMed

    Sepsis and sepsis-associated lung inflammation significantly contribute to the morbidity and mortality of critical illness. Here, we examined the hypothesis that neuronal guidance proteins could orchestrate inflammatory events during endotoxin-induced lung injury. Through a targeted array, we identified netrin-1 as the top upregulated neuronal guidance protein in macrophages treated with lipopolysaccharide (LPS). Furthermore, we found that netrin-1 is highly enriched in infiltrating myeloid cells, particularly in macrophages during LPS-induced lung injury. Transcriptional studies implicate hypoxia-inducible factor HIF-1α in the transcriptional induction of netrin-1 during LPS treatment. Subsequently, the deletion of netrin-1 in the myeloid compartment (Ntn1loxp/loxp LysM Cre) resulted in exaggerated mortality and lung inflammation. Surprisingly, further studies revealed enhanced natural killer cells (NK cells) infiltration in Ntn1loxp/loxp LysM Cre mice, and neutralization of NK cell chemoattractant chemokine (C-C motif) ligand 2 (CCL2) reversed the exaggerated lung inflammation. Together, these studies provide functional insight into myeloid cell-derived netrin-1 in controlling lung inflammation through the modulation of CCL2-dependent infiltration of NK cells. © 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.

    • Cancer Research
    Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer.

    In Cancer Discovery on 1 February 2021 by Zhang, S., Iyer, S., et al.

    PubMed

    The paucity of genetically informed, immunocompetent tumor models impedes evaluation of conventional, targeted, and immune therapies. By engineering mouse fallopian tube epithelial organoids using lentiviral gene transduction and/or CRISPR/Cas9 mutagenesis, we generated multiple high-grade serous tubo-ovarian cancer (HGSC) models exhibiting mutational combinations seen in patients with HGSC. Detailed analysis of homologous recombination (HR)-proficient (Trp53-/-;Ccne1OE;Akt2OE;KrasOE ), HR-deficient (Trp53-/-;Brca1-/-;MycOE ), and unclassified (Trp53-/-;Pten-/-;Nf1-/- ) organoids revealed differences in in vitro properties (proliferation, differentiation, and "secretome"), copy-number aberrations, and tumorigenicity. Tumorigenic organoids had variable sensitivity to HGSC chemotherapeutics, and evoked distinct immune microenvironments that could be modulated by neutralizing organoid-produced chemokines/cytokines. These findings enabled development of a chemotherapy/immunotherapy regimen that yielded durable, T cell-dependent responses in Trp53-/-;Ccne1OE;Akt2OE;Kras HGSC; in contrast, Trp53-/-;Pten-/-;Nf1-/- tumors failed to respond. Mouse and human HGSC models showed genotype-dependent similarities in chemosensitivity, secretome, and immune microenvironment. Genotype-informed, syngeneic organoid models could provide a platform for the rapid evaluation of tumor biology and therapeutics. SIGNIFICANCE: The lack of genetically informed, diverse, immunocompetent models poses a major barrier to therapeutic development for many malignancies. Using engineered fallopian tube organoids to study the cell-autonomous and cell-nonautonomous effects of specific combinations of mutations found in HGSC, we suggest an effective combination treatment for the currently intractable CCNE1-amplified subgroup.This article is highlighted in the In This Issue feature, p. 211. ©2020 American Association for Cancer Research.

    • IHC-IF
    • ,
    • Mus musculus (House mouse)
    • ,
    • Immunology and Microbiology
    Keratinocyte-Macrophage Crosstalk by the Nrf2/Ccl2/EGF Signaling Axis Orchestrates Tissue Repair.

    In Cell Reports on 24 November 2020 by Villarreal-Ponce, A., Tiruneh, M. W., et al.

    PubMed

    Unveiling the molecular mechanisms underlying tissue regeneration provides new opportunities to develop treatments for diabetic ulcers and other chronic skin lesions. Here, we show that Ccl2 secretion by epidermal keratinocytes is directly orchestrated by Nrf2, a prominent transcriptional regulator of tissue regeneration that is activated early after cutaneous injury. Through a unique feedback mechanism, we find that Ccl2 from epidermal keratinocytes not only drives chemotaxis of macrophages into the wound but also triggers macrophage expression of EGF, which in turn activates basal epidermal keratinocyte proliferation. Notably, we find dysfunctional activation of Nrf2 in epidermal keratinocytes of diabetic mice after wounding, which partly explains regenerative impairments associated with diabetes. These findings provide mechanistic insight into the critical relationship between keratinocyte and macrophage signaling during tissue repair, providing the basis for continued investigation of the therapeutic value of Nrf2.Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

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