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Aging Leads to Increased Monocytes and Macrophages With Altered CSF-1 Receptor Expression and Earlier Tumor-Associated Macrophage Expansion in Murine Mesothelioma

ABSTRACT: Increased cancer incidence occurs with the emergence of immunosenescence, highlighting the indispensability of the immune system in preventing cancer and its dysregulation with aging. Tumor-associated macrophages (TAMs) are often present in high numbers and are associated with poor clinical outcomes in solid cancers, including mesothelioma. Monocytes and macrophages from the bone marrow and spleen can respond to tumor-derived factors, such as CSF-1, and initiation of the CSF-1R signaling cascade results in their proliferation, differentiation, and migration to the tumor. Age-related changes occur in monocytes and macrophages in terms of numbers and function, which in turn can impact tumor initiation and progression. Whether this is due to changes in CSF-1R expression with aging is currently unknown and was investigated in this study. We examined monocytes and macrophages in the bone marrow and spleen during healthy aging in young (3–4 months) and elderly (20–24 months) female C57BL/6J mice. Additionally, changes to these tissues and in TAMs were examined during AE17 mesothelioma tumor growth. Healthy aging resulted in an expansion of Ly6Chigh monocytes and macrophages in the bone marrow and spleen. CSF-1R expression levels were reduced in elderly splenic macrophages only, suggesting differences in CSF-1R signaling between both cell type and tissue site. In tumor-bearing mice, Ly6Chigh monocytes increased with tumor growth in the spleen in the elderly and increased intracellular CSF-1R expression occurred in bone marrow Ly6Chigh monocytes in elderly mice bearing large tumors. Age-related changes to bone marrow and splenic Ly6Chigh monocytes were reflected in the tumor, where we observed increased Ly6Chigh TAMs earlier and expansion of Ly6Clow TAMs later during AE17 tumor growth in the elderly compared to young mice. F4/80high TAMs increased with tumor growth in both young and elderly mice and were the largest subset of TAMs in the tumor. Together, this suggests there may be a faster transition of Ly6Chigh towards F4/80high TAMs with aging. Amongst TAM subsets, expression of CSF-1R was lowest in F4/80high TAMs, however Ly6Clow TAMs had higher intracellular CSF-1R expression. This suggests downstream CSF-1R signaling may vary between macrophage subsets, which can have implications towards CSF-1R blockade therapies targeting macrophages in cancer.

SUBMITTER: Duong L

PROVIDER: S-EPMC9261395 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of SLC7A7/y+LAT1 impair system y+L transport activity for cationic amino acids. A severe complication of LPI is a form of Pulmonary Alveolar Proteinosis (PAP), in which alveolar spaces are filled with lipoproteinaceous material because of the impaired surfactant clearance by resident macrophages. The pathogenesis of LPI-associated PAP remains still obscure. The present study investigates for the first time the expression and function of y+LAT1 in monocytes and macrophages isolated from a patient affected by LPI-associated PAP. A comparison with mesenchymal cells from the same subject has been also performed. METHODS: Monocytes from peripheral blood were isolated from a 21-year-old patient with LPI. Alveolar macrophages and fibroblastic-like mesenchymal cells were obtained from a whole lung lavage (WLL) performed on the same patient. System y+L activity was determined measuring the 1-min uptake of [3H]-arginine under discriminating conditions. Gene expression was evaluated through qRT-PCR. RESULTS: We have found that: 1) system y+L activity is markedly lowered in monocytes and alveolar macrophages from the LPI patient, because of the prevailing expression of SLC7A7/y+LAT1 in these cells; 2) on the contrary, fibroblasts isolated from the same patient do not display the transport defect due to compensation by the SLC7A6/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of SLC7A7, suggesting that the gene is a target of the cytokine; 4) GM-CSF-induced differentiation of LPI monocytes is comparable to that of normal cells, demonstrating that GM-CSF signalling is unaltered; 5) general and respiratory conditions of the patient, along with PAP-associated parameters, markedly improved after GM-CSF therapy through aerosolization. CONCLUSIONS: Monocytes and macrophages, but not fibroblasts, derived from a LPI patient clearly display the defect in system y+L-mediated arginine transport. The different transport phenotypes are referable to the relative levels of expression of SLC7A7 and SLC7A6. Moreover, the expression of SLC7A7 is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.

Project description:ObjectiveTo develop a simple and efficient method for producing homogeneous populations of monocytes and macrophages from human embryonic stem cells (hES).Materials and methodsHuman embryonic stem cell lines KCL001, KCL002, and HUES-2 were differentiated into monocytes by coculture-free differentiation with two growth factors using a three-step method. The method involved embryoid body (EB) formation in hES media, directed differentiation with macrophage colony-stimulating factor and interleukin (IL)-3, and harvest of nonadherent monocytes from the culture supernatants. hES monocytes (esMCs) were analyzed by microscopy, flow cytometry, transcriptome analysis, and tested for the ability to differentiate into macrophages. hES monocyte-derived macrophages (esMDM) were analyzed for phagocytosis and endocytosis by microscopy and flow cytometry, cytokine secretion by multiplex cytokine assay, and for interferon (IFN)-gamma and IL-4 activation by flow cytometry.ResultsHomogeneous esMCs (>90% CD14-positive) that did not require any additional purification steps were produced after 18.7 +/- 7.7 days (mean +/- SD, n = 19). Production continued for several months when growth factors were replaced, with a total yield of 3.4 x 10(5) +/- 2.0 esMCs (mean +/- SD, n = 9) per EB. Transcriptome analysis of the esMC and the esMDM revealed a distinct myeloid signature that correlated with primary adult blood-derived monocytes and spleen tissue samples but not with other tissue samples tested. We found that esMCs and esMDMs expressed well-defined markers of the mononuclear phagocyte system including PU-1, C/EBPalpha, EMR1, and EMR2, MPEG1, CD1c, CD4, CD18, CD32, CD33, CD68, cathepsins and serine carboxypeptidase. Finally, esMCs differentiated into functional macrophages that could endocytose acetylated low-density lipoprotein, phagocytose opsonized yeast particles, secrete specific cytokines in response to lipopolysaccharide, and be activated differentially with IFN-gamma and IL-4.ConclusionsWe have developed a simple and efficient method for producing homogeneous populations of monocytes and macrophages from hES cells. esMCs have a myeloid signature and can differentiate into functional macrophages. The method should prove useful in answering experimental questions regarding monocyte and macrophage development and biology.

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Aging Leads to Increased Monocytes and Macrophages With Altered CSF-1 Receptor Expression and Earlier Tumor-Associated Macrophage Expansion in Murine Mesothelioma

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