Expression Of CSF1R Research Articles

Myeloid Mir34a suppresses colitis-associated colon cancer: characterization of mediators by single-cell RNA sequencing.

We have previously shown that general deletion of the gene encoding the p53-inducible Mir34a microRNA enhances the number and invasion of colitis-associated colorectal cancers (CACs) in mice. Since the p53-pathway has been implicated in tumor-suppression mediated by cells in the tumor microenvironment (TME) we deleted Mir34a in myeloid cells and characterized CACs in these with scRNA-Seq (single cell RNA sequencing). This revealed an increase inspecificmacrophage subtypes, such as Cdk8+ macrophagesand Mrc1+,M2-like macrophages. The latterdisplayed elevated expression of 21 known Mir34a target mRNAs, including Csf1r, Axl, Foxp1, Ccr1, Nampt, and Tgfbr2, and 32 predicted Mir34a target mRNAs. Furthermore, Mir34a-deficient BMDMs showed enhanced migration, elevated expression of Csf1r and a shift towards M2-like polarization when compared to Mir34a-proficient BMDMs. Concomitant deletion of Csf1r or treatment with a Csf1r inhibitor reduced the CAC burden and invasion in these mice. Notably, loss of myeloid Mir34a function resulted in a prominent, inflammatory CAC cell subtype, which displayed epithelial and macrophage markers. These cells displayed high levels of the EMT transcription factor Zeb2 and may therefore enhance the invasiveness of CACs. Taken together, our results provide in vivo evidence for a tumor suppressive role of myeloid Mir34a in CACs which is, at least in part, mediated by maintaining macrophages in an M1-like state via repression of Mir34a targets, such as Csf1r. Collectively, these findings may serve to identify new therapeutic targets and approaches for treatment of CAC.

LncRNA018392 promotes the proliferation of Liaoning cashmere goat skin fibroblasts by upregulating CSF1R through binding to SPI1.

Liaoning cashmere goat is recognized as a valuable genetic resource breed, with restrictions on genetic outflow in China. Hair follicle development in the cashmere goat is influenced by melatonin and long non-coding RNAs (lncRNAs). However, the role of lncRNAs in facilitating melatonin-promoted cashmere growth remains poorly understood. Previous studies have identified a new lncRNA, lncRNA018392, which is involved in the melatonin-promoted proliferation of cashmere skin fibroblasts. Flow cytometry and CCK-8 assays confirmed that silencing lncRNA018392 negates the effects of melatonin on cell proliferation, and that proliferation was reduced when the gene CSF1R, located near lncRNA018392, was inhibited. Further investigation using a dual-luciferase reporter assay showed that lncRNA018392 could positively regulate the promoter of CSF1R. Results from RNA-binding protein immunoprecipitation (RIP) and chromatin immunoprecipitation sequencing (ChIP-Seq) revealed that lncRNA018392 interacts with the transcription factor SPI1, with CSF1R being a downstream target gene regulated by SPI1. This interaction was confirmed by ChIP-PCR, which demonstrated SPI1's binding to CSF1R. This study found that the melatonin-responsive lncRNA018392 accelerates the cell cycle and promotes cell proliferation by recruiting SPI1 to upregulate the expression of the neighboring gene CSF1R. These findings provide a theoretical foundation for elucidating the molecular mechanisms of cashmere growth and for the molecular breeding of cashmere goats.

JDF promotes the apoptosis of M2 macrophages and reduces epithelial-mesenchymal transition and migration of liver cancer cells by inhibiting CSF-1/PI3K/AKT signaling pathway

BackgroundThe interaction between cancer cells and the tumor microenvironment is of critical importance in liver cancer. Jiedu Granule formula (JDF) has been shown to minimize the risk of recurrence and metastasis following liver cancer resection. Investigating the mechanism underlying the therapeutic effects of JDF can extend its field of application and develop novel treatment approaches. MethodsWe established a rat liver orthotopic transplantation tumor model, and recorded the prognostic effects of JDF adjuvant therapy on the recurrence and metastasis of liver cancer. Liver and lung tissues were collected for immunofluorescence staining and H&E staining, respectively. In addition, THP-1 cells were incubated with PMA and IL-4 to induce them to differentiate into M2 macrophages. CSF-1 expression was knocked down using lentivirus to determine the function of CSF-1. Liver cancer cells were cultured with a conditioned medium (CM) or co-cultured with macrophages. Cell viability was determined using the MTT assay. The levels of CSF-1, CSF-1R, E-cadherin, N-cadherin, PI3K, AKT, and cleaved caspase-3 were detected using ELISA, Western blotting and qPCR. The ability of cells to migrate was assessed using cell scratch and transwell assays. Apoptosis was evaluated using flow cytometry. ResultsThe JDF treatment decreased the risk of liver cancer metastasis after surgery and the infiltration of CD206/CD68 cells in liver cancer tissue. In cell experiments, JDF showed effects in suppressing M2 macrophages activity and downregulating the expression of CSF-1 and CSF-1R. The concentration of CSF-1 in the supernatant was also lower in the JDF-treated group. Futhermore, M2-CM was found to promote cancer cell migration and epithelial-mesenchymal transition (EMT); however, these effects were weakened after administering JDF. Knocking down endogenous CSF-1 in M2 macrophages resulted in a comparable suppression of cancer cell migration and EMT. Additionally, JDF treatment inhibited activation of the PI3K/AKT pathway, thus promoting the apoptosis of M2 macrophages. ConclusionsTreatment with JDF reduced the EMT and migratory capacity of liver cancer cells, which might be attributed to the inhibition of M2 macrophage infiltration and interruption of the CSF-1/PI3K/AKT signaling pathway. This mechanism may hold significant implications for mitigating the risk of metastatic spread in the aftermath of hepatic surgery.

IL-34 and its receptors as predictors of brain metastasis and prognosis in lung adenocarcinoma: Unveiling insights through bioinformatic and immunohistochemical investigations

BackgroundBrain metastasis (BM) is a prevalent form of metastasis in lung adenocarcinoma (LUAD), necessitating investigations into the underlying mechanisms. Interleukin 34 (IL-34) and its receptors, macrophage colony-stimulating factor-1 receptor (CSF-IR), Syndecan-1 (SDC-1), and protein-tyrosine phosphatase zeta receptor (PTPRZ1), are known to play pivotal roles in the metastasis of malignant tumors, thereby holding promise as potential biomarkers for studying BM in LUAD. MethodsWe performed immunohistochemistry to analyze the expression of IL-34, CSF-1R, SDC-1, and PTPRZ1 in 10 pairs of LUAD primary tissues and BMs, along with 96 unpaired primary tissues and 68 unpaired BMs. Subsequently, we evaluated the association between protein expression and the occurrence of BM. Furthermore, Kaplan-Meier survival curve analysis was conducted on both network and clinical data to explore the association between protein expression and patient prognosis and survival. ResultsAt the protein level, the expression of IL-34 and its receptors showed significant variation between paired primary tumors and BMs in 10 LUAD patients. The levels of IL-34, CSF-1R, and SDC-1 expression are typically elevated in brain metastatic lesions of LUAD compared to primary LUAD tumors. Furthermore, patients with high CSF-1R expression in primary LUAD are at a greater risk of developing brain metastases. High expression of IL-34 and CSF-1R in primary LUAD lesions indicated poor disease-free survival (DFS) and overall survival (OS), while high expression of SDC-1 indicated poor OS. Cox multivariate analysis further revealed that CSF-1R and IL-34+CSF-1R positivity independently affected LUAD OS. These findings were further substantiated in unpaired samples. ConclusionsOur results indicate significant alterations in the expression of IL-34 and its receptors, CSF-1R and SDC-1, between LUAD primary lesions and BMs, with increased expression observed in BMs. LUAD patients with positive CSF-1R expression in primary lesions exhibited a higher likelihood of developing BM, and high expression of IL-34, CSF-1R, and SDC-1 correlated with poor prognosis. These findings contribute novel insights towards identifying potential treatment or diagnostic targets for metastatic LUAD.

Alpha-synuclein inclusion responsive microglia are resistant to CSF1R inhibition

BackgroundParkinson’s disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously determined that microglia proximal to PFF-induced nigral α-syn inclusions increase in soma size, upregulate major-histocompatibility complex-II (MHC-II) expression, and increase expression of a suite of inflammation-associated transcripts. This microglial response is observed months prior to degeneration, suggesting that microglia reacting to α-syn inclusion may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether colony stimulating factor-1 receptor (CSF1R)-mediated microglial depletion impacts the magnitude of α-syn aggregation, nigrostriatal degeneration, or the response of microglial in the context of the α-syn PFF model.MethodsMale Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600 mg/kg), a CSF1R inhibitor, to deplete microglia for a period of either 2 or 6 months.ResultsCSF1R inhibition resulted in significant depletion (~ 43%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. However, CSF1R inhibition did not impact the increase in microglial number, soma size, number of MHC-II immunoreactive microglia or microglial expression of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, and Fcer1g associated with phosphorylated α-syn (pSyn) nigral inclusions. Further, accumulation of pSyn and degeneration of nigral neurons was not impacted by CSF1R inhibition. Paradoxically, long term CSF1R inhibition resulted in increased soma size of remaining Iba-1ir microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions.ConclusionsCollectively, our results suggest that CSF1R inhibition does not impact the microglial response to nigral pSyn inclusions and that CSF1R inhibition is not a viable disease-modifying strategy for PD.

Discrimination of distinct chicken M cell subsets based on CSF1R expression.

In mammals, a subset of follicle-associated epithelial (FAE) cells, known as M cells, conduct the transcytosis of antigens across the epithelium into the underlying lymphoid tissues. We previously revealed that M cells in the FAE of the chicken lung, bursa of Fabricius (bursa), and caecum based on the expression of CSF1R. Here, we applied RNA-seq analysis on highly enriched CSF1R-expressing bursal M cells to investigate their transcriptome and identify novel chicken M cell-associated genes. Our data show that, like mammalian M cells, those in the FAE of the chicken bursa also express SOX8, MARCKSL1, TNFAIP2 and PRNP. Immunohistochemical analysis also confirmed the expression of SOX8 in CSF1R-expressing cells in the lung, bursa, and caecum. However, we found that many other mammalian M cell-associated genes such as SPIB and GP2 were not expressed by chicken M cells or represented in the chicken genome. Instead, we show bursal M cells express high levels of related genes such as SPI1. Whereas our data show that bursal M cells expressed CSF1R-highly, the M cells in the small intestine lacked CSF1R and both expressed SOX8. This study offers insights into the transcriptome of chicken M cells, revealing the expression of CSF1R in M cells is tissue-specific.

Decreases in the number of microglia and neural circuit dysfunction elicited by developmental exposure to neonicotinoid pesticides in mice.

Neonicotinoids are insecticides widely used in the world. Although neonicotinoids are believed to be toxic only to insects, their developmental neurotoxicity in mammals is a concern. Therefore, we examined the effects of developmental exposure to neonicotinoids on immune system in the brain and post-developmental behaviors in this study. Imidacloprid or clothianidin was orally administered to dams at a dosage of 0.1 mg/kg/day from embryonic day 11 to postnatal day 21. Imidacloprid decreased sociability, and both imidacloprid and clothianidin decreased locomotor activity and induced anxiety, depression and abnormal repetitive behaviors after the developmental period. There was no change in the number of neurons in the hippocampus of mice exposed to imidacloprid. However, the number and activity of microglia during development were significantly decreased by imidacloprid exposure. Imidacloprid also induced neural circuit dysfunction in the CA1 and CA3 regions of the hippocampus during the early postnatal period. Exposure to imidacloprid suppressed the expression of csf1r during development. Collectively, these results suggest that developmental exposure to imidacloprid decreases the number and activity of microglia, which can cause neural circuit dysfunction and abnormal behaviors after the developmental period. Care must be taken to avoid exposure to neonicotinoids, especially during development.

CSF1R inhibition reprograms tumor-associated macrophages to potentiate anti-PD-1 therapy efficacy against colorectal cancer

PD-1 blockade therapy has made great breakthroughs in treatment of multiple solid tumors. However, patients with microsatellite-stable (MSS) colorectal cancer (CRC) respond poorly to anti-PD-1 immunotherapy. Although CRC patients with microstatellite instability (MSI) or microsatellite instability-high (MSI-H) can benefit from PD-1 blockade therapy, there are still some problems such as tumor recurrence. Tumor-associated macrophages (TAMs), most abundant immune components in tumor microenvironment (TME), largely limit the therapeutic efficacy of anti-PD-1 against CRC. The CSF1/CSF1R pathway plays a key role in regulating macrophage polarization, and blocking CSF1R signaling transduction may be a potential strategy to effectively reprogram macrophages and remodel TME. Here, we found that increasing expression of CSF1R in macrophages predicted poor prognosis in CRC cohort. Furthermore, we discovered a novel potent CSF1R inhibitor, PXB17, which significantly reprogramed M2 macrophages to M1 phenotype. Mechanically, PXB17 significantly blocked activation of PI3K/AKT/mTORC1 signaling, resulting in inhibition of cholesterol biosynthesis. Results from 3D co-culture system suggested that PXB17-repolarized macrophages could induce infiltration of CD8+ T lymphocytes in tumors and improve the immunosuppressive microenvironment. In vivo, PXB17 significantly halted CRC growth, with a stronger effect than PLX3397. In particular, PXB17 potently enhanced therapeutic activity of PD-1 mAb in CT-26 (MSS) model and prevented tumor recurrence in MC-38 (MSI-H) model by promoting formation of long-term memory immunity. Our study opens a new avenue for CSF1R in tumor innate and adaptive anti-tumor immunomodulatory activity and suggests that PXB17 is a promising immunotherapy molecule for enhancing the efficacy of PD-1 mAb or reducing tumor recurrence of CRC.

MRI-based Machine Learning Radiomics Can Predict CSF1R Expression Level and Prognosis in High-grade Gliomas.

The purpose of this study is to predict the mRNA expression of CSF1R in HGG non-invasively using MRI (magnetic resonance imaging) omics technology and to evaluate the correlation between the established radiomics model and prognosis. We investigated the predictive value of CSF1R in the Cancer Genome Atlas (TCGA) and The Cancer Imaging Archive (TCIA) database. The Support vector machine (SVM) and the Logistic regression (LR) algorithms were used to create a radiomics_score (Rad_score), respectively. The effectiveness and performance of the radiomics model was assessed in the training (n = 89) and tenfold cross-validation sets. We further analyzed the correlation between Rad_score and macrophage-related genes using Spearman correlation analysis. A radiomics nomogram combining the clinical factors and Rad_score was constructed to validate the radiomic signatures for individualized survival estimation and risk stratification. The results showed that CSF1R expression was markedly elevated in HGG tissues, which was related to worse prognosis. CSF1R expression was closely related to the abundance of infiltrating immune cells, such as macrophages. We identified nine features for establishing a radiomics model. The radiomics model predicting CSF1R achieved high AUC in training (0.768 in SVM and 0.792 in LR) and tenfold cross-validation sets (0.706 in SVM and 0.717 in LR). Rad_score was highly associated with tumor-related macrophage genes. A radiomics nomogram combining the Rad_score and clinical factors was constructed and revealed satisfactory performance. MRI-based Rad_score is a novel way to predict CSF1R expression and prognosis in high-grade glioma patients. The radiomics nomogram could optimize individualized survival estimation for HGG patients.

Inhibition of CSF1R and KIT With Pexidartinib Reduces Inflammatory Signaling and Cell Viability in Endometriosis.

Endometriosis is a common and debilitating disease, affecting ∼170 million women worldwide. Affected patients have limited therapeutic options such as hormonal suppression or surgical excision of the lesions, though therapies are often not completely curative. Targeting receptor tyrosine kinases (RTKs) could provide a nonhormonal treatment option for endometriosis. We determined that 2 RTKs, macrophage-colony stimulating factor 1 receptor (CSF1R) and mast/stem cell growth factor receptor KIT (KIT), are overexpressed in endometriotic lesions and could be novel nonhormonal therapeutic targets for endometriosis. The kinase activity of CSF1R and KIT is suppressed by pexidartinib, a small molecule inhibitor that was recently approved by the US Food and Drug Administration. Using immunohistochemistry, we detected CSF1R and KIT in endometriotic tissues obtained from peritoneal lesions, colorectal lesions, and endometriomas. Specifically, we show that KIT is localized to the epithelium of the lesions, while CSF1R is expressed in the stroma and macrophages of the endometriotic lesions. Given the high epithelial expression of CSF1R and KIT, 12Z endometriotic epithelial cells were used to evaluate the efficacy of dual CSF1R and KIT inhibition with pexidartinib. We found that pexidartinib suppressed activation in 12Z cells of JNK, STAT3, and AKT signaling pathways, which control key proinflammatory and survival networks within the cell. Using quantitative real-time polymerase chain reaction, we determined that pexidartinib suppressed interleukin 8 (IL8) and cyclin D1 (CCND1) expression. Lastly, we demonstrated that pexidartinib decreased cell growth and viability. Overall, these results indicate that pexidartinib-mediated CSF1R and KIT inhibition reduces proinflammatory signaling and cell viability in endometriosis.

CSF-1R in Cancer: More than a Myeloid Cell Receptor.

Colony-stimulating factor 1 receptor (CFS-1R) is a myeloid receptor with a crucial role in monocyte survival and differentiation. Its overexpression is associated with aggressive tumors characterized by an immunosuppressive microenvironment and poor prognosis. CSF-1R ligands, IL-34 and M-CSF, are produced by many cells in the tumor microenvironment (TME), suggesting a key role for the receptor in the crosstalk between tumor, immune and stromal cells in the TME. Recently, CSF-1R expression was reported in the cell membrane of the cancer cells of different solid tumors, capturing the interest of various research groups interested in investigating the role of this receptor in non-myeloid cells. This review summarizes the current data available on the expression and activity of CSF-1R in different tumor types. Notably, CSF-1R+ cancer cells have been shown to produce CSF-1R ligands, indicating that CSF-1R signaling is positively regulated in an autocrine manner in cancer cells. Recent research demonstrated that CSF-1R signaling enhances cell transformation by supporting tumor cell proliferation, invasion, stemness and drug resistance. In addition, this review covers recent therapeutic strategies, including monoclonal antibodies and small-molecule inhibitors, targeting the CSF-1R and designed to block the pro-oncogenic role of CSF-1R in cancer cells.

Effects of CSF1R/p-ERK1/2 signaling pathway on RF/6A cells under high glucose conditions.

This study analyzed how high glucose affects CSF1R and p-ERK1/2 expression in RF/6A cells. The cells were cultured as high glucose (HG) and normal control (C) groups, and CSF1R shRNA was introduced. Real time PCR was used to detect the expression of CSF1R and p-ERK1/2 mRNA. Western blot was used to detect the expression of CSF1R and p-ERK1/2 proteins. Cell Counting Kit 8 (CCK-8) method was used to detect cell proliferation, while flow cytometry was used to detect apoptosis in HREC. Real-time PCR showed significantly raised CSF1R mRNA expression in HG. CSF1R inhibition lowered HG + LV shCSF1R CSF1R mRNA levels. Western blotting revealed higher CSF1R and p-ERK1/2 protein expression in HG than in C. Their expression level dropped after CSF1R inhibition. The number of tube-forming cells was higher in HG than in C, which reduced after CSF1R suppression. Inhibiting CSF1R also decreased cell proliferation and raised apoptosis. Overall, under high glucose, CSF1R and p-ERK1/2 were highly expressed, leading to reduced cellular activity, and CSF1R inhibition helped alleviate this effect.

Xpo-1 Antagonism Impairs CSF-1R Expression and Depletes Lymphoma-Associated Macrophages in T-Cell Lymphomas

Background: Lymphoma-associated macrophages (LAM) provide ligands for antigen, costimulatory, and cytokine receptors expressed by malignant T cells and directly promote T-cell lymphomagenesis. LAM also play an important role in suppressing host anti-tumor immunity and indirectly promote T-cell lymphomagenesis. Therefore, LAM are a therapeutic vulnerability in T-cell lymphomas (TCL), and their depletion from the tumor microenvironment (TME) is a rational therapeutic goal. Methods: XPO1 and CD163 expression were examined by immunohistochemical staining. In order to determine the extent to which XPO1 antagonism depletes LAM, a pharmacologic strategy was adopted using selinexor. Viability of human monocyte-derived and TCL conditioned macrophages upon selinexor treatment was examined in vitro using standard methods. RNA binding to eIF4e was determined by RNA immunoprecipitation (RIP) using eIF4e and IgG antibodies and quantification performed by qPCR. Nuclear export of eIF4e client mRNA upon XPO1 antagonism with selinexor was examined by qRT-PCR in nuclear and cytoplasmic pools of RNA. LAM depletion upon selinexor treatment was examined in vivo using a previously described GEM ( Smarcb1-deficient) model and CD68-GFP reporter mice. Results: As we had previously demonstrated that CD163 identifies LAM that are alternatively polarized and proliferative, co-staining was performed with CD163 and XPO1, and CD163+XPO-1+ LAM identified in 69% of PTCL cases (n=26) examined. We previously demonstrated that LAM within a PTCL context are resistant to inhibition by selective CSF-1R antagonists. Therefore, we performed an unbiased, high-throughput screen using monocytes cultured in the presence of cell-free conditioned media (CFCM) obtained from T-cell lymphoma cell lines and primary specimens. This screen identified selinexor as a potential therapeutic vulnerability. Therefore, we cultured normal-donor monocytes in RPMI media alone, or in media supplemented with cell free conditioned media (CFCM) obtained from PTCL/CTCL cell lines (n=2) or primary specimens (n=5) and determined cell viability. Consistent with our prior findings, CFCM from PTCL/CTCL cells promoted the growth and survival of monocyte-derived macrophages (MDM), but this effect was abrogated upon selinexor treatment. These findings converged with the observation that CSF-1R transcripts are eIF4e clients in ALCL cells. Therefore, the extent to which transcripts required for macrophage survival (i.e. CSF-1R), proliferation (i.e. PU.1), and polarization (i.e. STAT3) are eIF4e clients was examined by eIF4e-RIP in MDM. These experiments demonstrated that CSF-1R, PU.1 and STAT3 are eIF4e clients, and selinexor treatment significantly decreased their nuclear export in MDM. Given the obvious clinical relevance of CSF-1R as a therapeutic target, CSF-1R expression in MDM following selinexor treatment was examined, and compatible with these results, a significant decrease in CSF-1R expression observed upon selinexor treatment. We previously employed CD68-GFP reporter mice in a Smarcb1-deficient GEM model, demonstrating that GFP+ macrophages in this TCL model are transcriptionally reprogrammed and proliferative. Selective CSF-1R antagonists minimally impair LAM survival in this model. In contrast, selinexor significantly depleted LAM ex vivo in this model and impaired the viability of bone marrow derived macrophages. In order to extend these findings, lymphoma-bearing GEM mice were treated with selinexor and the abundance of CD11b+Ly6G- LAM determined by flow cytometry, whereby a significant reduction in LAM abundance and TCL progression were observed, compatible with our in vitro/ex vivo findings. Conclusions: Therefore, we concluded that XPO1 inhibition indirectly impairs T-cell lymphoma growth and survival by depleting LAM, which are a dependency in these lymphomas.

Elevated Functional Hematopoietic Stem and Progenitor Cells in the Peripheral Blood of G-CSF-Mobilized Dnmt3a-Mutant Mice

Clonal hematopoiesis (CH) arising from somatic mutations in hematopoietic stem cells (HSCs) is associated with increased risk of developing several non-hematologic and hematologic diseases. In the context of stem cell transplantation, donor HSCs carrying CH-associated somatic mutations can be passed from donor to recipient during stem cell transplantation. Most literature supports that there is no negative impact on recipient survival or outcomes after allogeneic stem cell transplantation (allo-HSCT) from donors with CH. However, it is less clear how allo-HSCT donors with CH respond to common mobilization methods such as Neupogen (G-CSF). As hematopoietic stem and progenitor cells (HSPCs) with CH mutations have been found to respond to external stimuli such as inflammation in a manner distinct from wild-type HSPCs, it is plausible that CH-mutant HSPCs also distinctly respond to G-CSF mobilization. It is important to understand whether the process of mobilization differentially enriches for CH-mutant HSPCs in the mobilized peripheral blood product. To address this gap in knowledge, we used a murine model of Dnmt3a-mutant CH. After five consecutive days of Neupogen (G-CSF) administration, modeling the procedure for human HSC mobilization, we observed that Dnmt3a-mutant mice had significantly increased white blood cell, neutrophil and monocyte counts compared to control mice. We observed a significant increase in cell surface marker-defined HSPCs in the peripheral blood and spleen of G-CSF-treated Dnmt3a-mutant mice compared to G-CSF-treated control mice. Using in vitro colony-forming unit assays, the peripheral blood of G-CSF-treated Dnmt3a-mutant mice was found to contain a greater number of functional hematopoietic progenitors compared to G-CSF-treated control mice. To determine if the peripheral blood of G-CSF-treated Dnmt3a-mutant mice had a greater abundance of functional HSCs, we transplanted equal volumes of mobilized blood from Dnmt3a-mutant and control mice into primary and secondary lethally irradiated wild-type recipient mice. We observed increased long-term engraftment of mobilized blood from Dnmt3a-mutant mice compared to control mice in both primary and secondary recipient animals. At a mechanistic level, HSC mobilization is facilitated by myeloid cells that respond directly to G-CSF via the G-CSF receptor ( Csf1r). In Dnmt3a-mutant mice, we observed increased Csf1r expression in myeloid progenitor and mature myeloid cells compared to control mice, providing a putative mechanism by which increased mobilization is enabled. Together, our data support differential mobilization potential of Dnmt3a-mutant compared to control HSCs in response to G-CSF, which may have implications in the context of allo-HSCT.

Identification and validation of hub genes involved in foam cell formation and atherosclerosis development via bioinformatics.

Foam cells play crucial roles in all phases of atherosclerosis. However, until now, the specific mechanisms by which these foam cells contribute to atherosclerosis remain unclear. We aimed to identify novel foam cell biomarkers and interventional targets for atherosclerosis, characterizing their potential mechanisms in the progression of atherosclerosis. Microarray data of atherosclerosis and foam cells were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expression genes (DEGs) were screened using the "LIMMA" package in R software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) annotation were both carried out. Hub genes were found in Cytoscape after a protein-protein interaction (PPI) enrichment analysis was carried out. Validation of important genes in the GSE41571 dataset, cellular assays, and tissue samples. A total of 407 DEGs in atherosclerosis and 219 DEGs in foam cells were identified, and the DEGs in atherosclerosis were mainly involved in cell proliferation and differentiation. CSF1R and PLAUR were identified as common hub genes and validated in GSE41571. In addition, we also found that the expression of CSF1R and PLAUR gradually increased with the accumulation of lipids and disease progression in cell and tissue experiments. CSF1R and PLAUR are key hub genes of foam cells and may play an important role in the biological process of atherosclerosis. These results advance our understanding of the mechanism behind atherosclerosis and potential therapeutic targets for future development.

SERPINB5 is a novel serum diagnostic biomarker for gastric high-grade intraepithelial neoplasia and plays a role in regulation of macrophage phenotypes

BackgroundGastric cancer (GC) develops from gastric precancerous lesions (GPL), and early diagnosis and treatment at the premalignant stage may achieve a higher benefit‒cost ratio with a reduced necessity for surgery. However, reliable noninvasive screening biomarkers of GPL are currently lacking. MethodsThe marker genes of GPL encoding extracellular proteins were identified by bioinformatics analysis and further verified by immunofluorescence and immunohistochemistry assays. Serum samples were collected to measure the levels of SERPINB5, the diagnostic efficacy of which was assessed by the area under the receiver operating characteristic (ROC) curve (AUC). Finally, the effect of SERPINB5 on the phenotypic conversion of macrophages was verified by public data and in vitro experiments. ResultsSERPINB5 was identified as an extracellular biomarker of GPL that had good diagnostic efficacy. High expression of SERPINB5 was observed in the epithelial cells and adjacent extracellular matrix on sections of gastric high-grade intraepithelial neoplasia (HGIN). Importantly, SERPINB5 determined in serum was significantly increased in the HGIN group, and the AUC for discriminating between HGIN and chronic gastritis or low-grade intraepithelial neoplasia was 0.9936 and 0.9750, respectively. Moreover, SERPINB5 expression was positively correlated with macrophage infiltration, and M1 marker NOS2 expression, but negatively correlated with M2 marker CSF1R expression. In THP-1-derived macrophages, SERPINB5 upregulated expression of M1-related cytokines TNF-α and IL-12, and M1 marker CD86, but suppressed production of M2-related cytokines TGF-β and IL-10. ConclusionsOur study provides evidence that SERPINB5 may serve as a promising noninvasive serum biomarker for gastric HGIN screening and regulate macrophage phenotype conversion.

#5466 CSF-1R SYSTEM ACTIVATES PARIETAL EPITHELIAL CELLS LEADING TO FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS)

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is the most common glomerular cause leading to end-stage kidney disease. Actual treatment of primary FSGS by immunosuppressive agents presents inconsistent results. Thus, new innovative strategies different from those used to date by taking into consideration podocyte renewal and maintenance or even strategies complementary to immunosuppression are needed. In FSGS, parietal epithelial cells (PECs) switch to an activated phenotype (aPECs) in response to podocyte damage promoting glomerulosclerosis. Colony-stimulating factor (CSF-1) is a hematopoietic growth factor that acts via its specific receptor, the tyrosine kinase receptor CSF-1R. CSF-1 has been detected in sera and renal biopsies from patients with different renal complications, including FSGS, attributing their role and presence to macrophage but not to glomerular cells. The potential cellular and molecular mechanisms involved are also unknown. In this work, we evaluated the implication of CSF-1/CSF-1R axis in the pathogenesis of FSGS and its potential as new pharmacological target point by using specific CSF-1R inhibitors. Specifically, we focused on the modulation of PECS activation by de novo production of CD44 and the preservation of podocyte loss. Method We evaluated the role of the CSF-1/CSF-1R axis as a driver of glomerular damage in FSGS in adriamycin-induced nephropathy (ADR) in mice, the main experimental model to study human FSGS. To this end, we treated or not ADR-animals with CSF-1R specific inhibitors, GW2580 or Ki20227 (n = 5-7 group). We determined the expression and localization of CSF-1R in the glomerulus in tissue by triple immunofluorescence (WT-1, SSeCks and CSF-1R) and their relevance in glomerulosclerosis (PAS and pro-fibrotic genes), the determination of de novo CD44 formation and its correlation with ERK1/2 pathway by immunohistochemistry (IH). We detected podocyte in the glomerulus by WT-1 IH and the localization of aPECS in the glomerular tuff by Claudin-1 and SSeCkS markers. Finally, we used isolated human kidney progenitor cells with and without CSF-1 treatment (n = 6/group) to identify potential key interactors of CSF-1 by RNAseq. We validated genes of interest in the FSGS experimental model. Results We observed a constitutively expression of CSF-1R in the glomerulus of control mice that significantly increased in ADR-treated mice, specifically in podocytes (WT1) and PECs (SSeCks), confirmed with mRNA expression. ADR-treated mice showed important events of sclerotic glomerulus and pro-fibrotic genes as collagen that was significantly reversed by the treatment with CSF-1R inhibitors (p<0.001). Results reflected a CSF-1R inhibitor-dependent renal function recovery with the use of the inhibitors with a reduction of proteinuria and an increase of glomerular filtration rate (p<0.001). We found a positive CD44 staining both in Bowman's capsule and inner the tuff of ADR-treated mice, accompanied by an increase of ERK1/2 activation. CSF-1R inhibitors significantly reduced the percentage of glomeruli with de novo CD44 production. Remarkably, podocyte depletion was also preserved with very similar levels to non-treated animals (p<0.001). For RNAseq results, 227 differentially expressed genes (considering a criterion of a probability of differential expression >0.9 and a |M| > 1) were subjected to enrichment analysis. The top significant gene ontology terms were mainly involved with interferon-induced genes. Genes involved in this pathway were validated in the FSGS model, showing an increase in mice treated with ADR compared to controls and alleviated with CSF-1R inhibitors (p<0.001). Conclusion In this study we propose a novel therapeutic strategy to FSGS-associated pathology based on the inhibition of CSF1-R activity having an impact on reducing aPECs, glomerulosclerosis, proteinuria, improving renal function and preserving the podocyte-progenitor phenotype, thus, in podocyte preservation against damage.

Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis.

Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease.

Targeting pathogenic macrophages by the application of SHP-1 agonists reduces inflammation and alleviates pulmonary fibrosis

Idiopathic pulmonary fibrosis is a progressive fibrotic disorder with no cure that is characterized by deterioration of lung function. Current FDA-approved drugs for IPF delay the decline in lung function, but neither reverse fibrosis nor significantly improve overall survival. SHP-1 deficiency results in hyperactive alveolar macrophages accumulating in the lung, which contribute to the induction of pulmonary fibrosis. Herein, we investigated whether employing a SHP-1 agonist ameliorates pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis murine model. Histological examination and micro-computed tomography images showed that SHP-1 agonist treatment alleviates bleomycin-induced pulmonary fibrosis. Reduced alveolar hemorrhage, lung inflammation, and collagen deposition, as well as enhanced alveolar space, lung capacity, and improved overall survival were observed in mice administered the SHP-1 agonist. The percentage of macrophages collected from bronchoalveolar lavage fluid and circulating monocytes in bleomycin-instilled mice were also significantly reduced by SHP-1 agonist treatment, suggesting that the SHP-1 agonist may alleviate pulmonary fibrosis by targeting macrophages and reshaping the immunofibrotic niche. In human monocyte-derived macrophages, SHP-1 agonist treatment downregulated CSF1R expression and inactivated STAT3/NFκB signaling, culminating in inhibited macrophage survival and perturbed macrophage polarization. The expression of pro-fibrotic markers (e.g., MRC1, CD200R1, and FN1) by IL4/IL13-induced M2 macrophages that rely on CSF1R signaling for their fate-determination was restricted by SHP-1 agonist treatment. While M2-derived medium promoted the expression of fibroblast-to-myofibroblast transition markers (e.g., ACTA2 and COL3A1), the application of SHP-1 agonist reversed the transition in a dose-dependent manner. Our report indicates that pharmacological activation of SHP-1 ameliorates pulmonary fibrosis via suppression of CSF1R signaling in macrophages, reduction of pathogenic macrophages, and the inhibition of fibroblast-to-myofibroblast transition. Our study thus identifies SHP-1 as a druggable target for the treatment of IPF, and suggests that the SHP-1 agonist may be developed as an anti-pulmonary fibrosis medication that both suppresses inflammation and restrains fibroblast-to-myofibroblast transition.

Artificial intelligence (AI) –powered spatial analysis of macrophages in tumor microenvironment and its association with interferon-gamma (IFNG) signature and immune phenotype (IP) in pan-cancer dataset.

2621 Background: Macrophages are known to play a crucial role in cancer progression, but few studies have reported macrophage spatial distribution and its association with genomic signatures and tumor-infiltrating lymphocytes (TIL). Here, we applied an AI-powered Whole Slide Image (WSI) analyzer in The Cancer Genome Atlas (TCGA) pan-cancer dataset to show correlation between specific localization of macrophage and anti-tumor activity, which is reflected by IFNG signature and IP based on spatial TIL density. Methods: For spatial macrophage analysis in TCGA pan-cancer dataset, we used Lunit SCOPE IO, an AI-powered H&E WSI analyzer, which identifies and quantifies various classes of cells such as tumor cell, macrophage, and TIL within cancer or stroma area. IPs were defined as follows: inflamed IP (IIP) as high intratumoral TIL (iTIL) and stromal TIL (sTIL); immune-excluded IP (IEP) as low iTIL and high sTIL; immune-desert IP (IDP) as low TIL overall. The immune cell profiling and quantification of the IFNG signature score were analyzed by using CIBERSORT and RSEM. Results: In TCGA dataset (N = 7,402), median macrophage density in total TME area (tMP) was 19.9 /mm2 (interquartile range [IQR] 9.0-44.9), and it was highly enriched in lung adenocarcinoma (median 65.1, IQR 34.3-110.0), lung squamous cell carcinoma (61, 32.4-101.3), and head and neck squamous cell carcinoma (39.4, 19.6-77.3). Based on IP classification, tMP was highly enriched in IIP (37.8, 18.7-73.8) compared to IEP (25.5, 13.8-48.6) and IDP (8.9, 4.4-17.1). Interestingly, spatial analysis showed that intratumoral macrophage density (iMP) in IIP was highly enriched compared to the others (mean fold change 2.57) and the difference of stromal macrophage density (sMP) among IPs was relatively smaller (mean fold change x1.85). Classified into four groups based on median values of iMP and sMP, high-iMP/high-sMP group had the highest proportion of IIP (47.3% vs 18.7%, p < 0.001), whereas low-iMP/high-sMP group had the highest one of IEP (54.9% vs 32.3%, p < 0.001) compared to the others, respectively. Moreover, the high-iMP/high-sMP group had the highest level of IFNG signature score among 4 groups (x2.13 times higher, p < 0.001). Notably, both iMP and sMP were significantly correlated with CD68 expression (rho 0.292 and 0.287) but CSF1R expression was more strongly correlated with iMP (rho 0.259) compared to sMP (rho 0.078). Conclusions: IIP, IFNG signature, and CSF1R expression are more strongly correlated with intratumoral macrophage density versus stromal macrophage density, suggesting a relationship between macrophage location and function in the TME warranting further investigation.