ABSTRACT
Hypertension (HTN) impacts almost half of adults, predisposing them to cardiovascular disease and renal damage. Salt-sensitive HTN (SSHTN) and angiotensin II (A2)-induced HTN (A2HTN) both involve immune system activation and renal innate immune cell infiltration. Subpopulations of activated [Cluster of differentiation 38 (CD38)] innate immune cells, such as macrophages and dendritic cells (DCs), play distinct roles in modulating renal function and blood pressure. It is unknown how these cells become CD38+ or which subtypes are pro-hypertensive. When bone marrow-derived monocytes (BMDMs) were grown in granulocyte-macrophage colony stimulating factor (GM-CSF) and treated with salt or A2, CD38+ macrophages and CD38+ DCs increased. The adoptive transfer of GM-CSF-primed BMDMs into mice with either SSHTN or A2HTN increased renal CD38+ macrophages and CD38+ DCs. Flow cytometry revealed increased renal M1 macrophages and type-2 conventional DCs (cDC2s), along with their CD38+ counterparts, in mice with either SSHTN or A2HTN. These results were replicable in vitro. Either salt or A2 treatment of GM-CSF-primed BMDMs significantly increased bone marrow-derived (BMD)-M1 macrophages, CD38+ BMD-M1 macrophages, BMD-cDC2s, and CD38+ BMD-cDC2s. Overall, these data suggest that GM-CSF is necessary for the salt or A2 induction of CD38+ innate immune cells, and that CD38 distinguishes pro-hypertensive immune cells. Further investigation of CD38+ M1 macrophages and CD38+ cDC2s could provide new therapeutic targets for both SSHTN and A2HTN.
Subject(s)
Angiotensin II , Dendritic Cells , Granulocyte-Macrophage Colony-Stimulating Factor , Immunity, Innate , Macrophages , Animals , Angiotensin II/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Mice , Immunity, Innate/drug effects , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Hypertension/immunology , Mice, Inbred C57BL , ADP-ribosyl Cyclase 1/metabolism , Male , Monocytes/drug effects , Monocytes/metabolism , Monocytes/immunology , Kidney/immunology , Kidney/drug effectsABSTRACT
BACKGROUND: Mesenchymal stromal cells (MSCs) tropism for tumours allows their use as carriers of antitumoural factors and in vitro transcribed mRNA (IVT mRNA) is a promising tool for effective transient expression without insertional mutagenesis risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with antitumor properties by stimulating the specific immune response. The aim of this work was to generate modified MSCs by IVT mRNA transfection to overexpress GM-CSF and determine their therapeutic effect alone or in combination with doxorubicin (Dox) in a murine model of hepatocellular carcinoma (HCC). METHODS: DsRed or GM-CSF IVT mRNAs were generated from a cDNA template designed with specific primers followed by reverse transcription. Lipofectamine was used to transfect MSCs with DsRed (MSC/DsRed) or GM-CSF IVT mRNA (MSC/GM-CSF). Gene expression and cell surface markers were determined by flow cytometry. GM-CSF secretion was determined by ELISA. For in vitro experiments, the J774 macrophage line and bone marrow monocytes from mice were used to test GM-CSF function. An HCC model was developed by subcutaneous inoculation (s.c.) of Hepa129 cells into C3H/HeN mice. After s.c. injection of MSC/GM-CSF, Dox, or their combination, tumour size and mouse survival were evaluated. Tumour samples were collected for mRNA analysis and flow cytometry. RESULTS: DsRed expression by MSCs was observed from 2 h to 15 days after IVT mRNA transfection. Tumour growth remained unaltered after the administration of DsRed-expressing MSCs in a murine model of HCC and MSCs expressing GM-CSF maintained their phenotypic characteristic and migration capability. GM-CSF secreted by modified MSCs induced the differentiation of murine monocytes to dendritic cells and promoted a proinflammatory phenotype in the J774 macrophage cell line. In vivo, MSC/GM-CSF in combination with Dox strongly reduced HCC tumour growth in C3H/HeN mice and extended mouse survival in comparison with individual treatments. In addition, the tumours in the MSC/GM-CSF + Dox treated group exhibited elevated expression of proinflammatory genes and increased infiltration of CD8 + T cells and macrophages. CONCLUSIONS: Our results showed that IVT mRNA transfection is a suitable strategy for obtaining modified MSCs for therapeutic purposes. MSC/GM-CSF in combination with low doses of Dox led to a synergistic effect by increasing the proinflammatory tumour microenvironment, enhancing the antitumoural response in HCC.
Subject(s)
Carcinoma, Hepatocellular , Doxorubicin , Granulocyte-Macrophage Colony-Stimulating Factor , Liver Neoplasms , Mesenchymal Stem Cells , RNA, Messenger , Animals , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Mesenchymal Stem Cells/metabolism , Mice , Liver Neoplasms/therapy , Liver Neoplasms/pathology , Liver Neoplasms/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Cell Line, Tumor , Mesenchymal Stem Cell Transplantation/methods , Humans , Mice, Inbred C3H , TransfectionABSTRACT
PROBLEM: Adenomyosis (AM) is associated with immune response and inflammation. However, the role of T cell subsets in AM development has not been thoroughly understood. METHOD OF STUDY: Patients with focal or diffuse AM were recruited. Serum cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). Different T cell subsets in the blood and ectopic endometrium were determined by flow cytometry. RESULTS: Serum interleukin-6 (IL-6) and macrophage-colony-stimulating factor (GM-CSF) were increased in patients with focal or diffuse AM before focused ultrasound ablation surgery (FUAS), but not after FUAS. Compared with the healthy control, the frequencies of CD8+ interferon-gamma (IFN-γ)-expressing cytotoxic T lymphocytes (CTLs), interleukin-17A (IL-17A)-expressing Tc17 cells, CD4+ T helper 1 (Th1) cells, and GM-CSF-expressing T helper (ThGM) cells were up-regulated in the blood of patients with AM, especially those with diffuse AM. However, these changes were eradicated after FUAS. Meanwhile, the frequencies of these T cell subsets were positively correlated with the CA-125 level. Furthermore, these T cell subsets were also increased in ectopic endometrium. CONCLUSIONS: Our study delineates for the first time the presence of CTLs, Tc17 cells, Th1, and ThGM cells in the blood and ectopic endometrium in AM. The results imply that T cell response might impact AM development.
Subject(s)
Adenomyosis , Endometrium , Granulocyte-Macrophage Colony-Stimulating Factor , Th1 Cells , Humans , Female , Endometrium/immunology , Endometrium/pathology , Adult , Adenomyosis/immunology , Adenomyosis/blood , Adenomyosis/pathology , Th1 Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/blood , T-Lymphocytes, Cytotoxic/immunology , Middle Aged , Interleukin-17/metabolism , Interleukin-17/blood , Interleukin-6/blood , Interleukin-6/metabolism , Th17 Cells/immunology , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
Coronary heart disease (CHD) is related to aberrant aggregation of immune cells in the plaques. This study focused on identification of abnormal T cell subtypes and inflammatory factors in CHD patients. To this end, the subtypes of T cells in peripheral blood of CHD patients (n=141) and healthy controls (n=46) were analyzed by flow cytometry. Plasma concentrations of cytokines were analyzed by multiplex assay. It was shown that the number of T helper cells producing granulocyte-macrophage CSF (GM-CSF) was higher in CHD patients in comparison with healthy controls. In addition, the fractions of Th1 and Th17 cells as well as the levels of IL-4, IL-5, IL-6, and IL-10 in CHD patients also surpassed the control values (p<0.05). However, the level of GM-CSF was insignificantly lower in CHD patients. Thus, we revealed a relationship between the number of T cells producing GM-CSF and the severity of CHD. Our results can be used to develop new potential biomarkers for CHD detection.
Subject(s)
Biomarkers , Coronary Disease , Granulocyte-Macrophage Colony-Stimulating Factor , Interleukin-6 , Humans , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Male , Female , Coronary Disease/immunology , Coronary Disease/blood , Middle Aged , Biomarkers/blood , Interleukin-6/blood , Case-Control Studies , Interleukin-10/blood , Th17 Cells/immunology , Th17 Cells/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Interleukin-4/blood , Aged , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Adult , Flow Cytometry , Interleukin-5ABSTRACT
Pulmonary alveolar proteinosis (PAP) is an ultra-rare disease caused by impaired pulmonary surfactant clearance due to the dysfunction of alveolar macrophages or their signaling pathways. PAP is categorized into autoimmune, congenital, and secondary PAP, with autoimmune PAP being the most prevalent. This article aims to present a comprehensive review of PAP classification, pathogenesis, clinical presentation, diagnostics, and treatment. The literature search was conducted using the PubMed database and a total of 67 articles were selected. The PAP diagnosis is usually based on clinical symptoms, radiological imaging, and bronchoalveolar lavage, with additional GM-CSF antibody tests. The gold standard for PAP treatment is whole-lung lavage. This review presents a summary of the most recent findings concerning pulmonary alveolar proteinosis, pointing out specific features that require further investigation.
Subject(s)
Pulmonary Alveolar Proteinosis , Pulmonary Alveolar Proteinosis/therapy , Pulmonary Alveolar Proteinosis/diagnosis , Pulmonary Alveolar Proteinosis/pathology , Humans , Bronchoalveolar Lavage , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Pulmonary Surfactants/metabolism , Pulmonary Surfactants/therapeutic use , Macrophages, Alveolar/metabolismABSTRACT
Hepatocellular carcinoma is a refractory tumor with poor prognosis and high mortality. Many oncolytic viruses are currently being investigated for the treatment of hepatocellular carcinoma. Based on previous studies, we constructed a recombinant GM-CSF-carrying Sindbis virus, named SINV-GM-CSF, which contains a mutation (G to S) at amino acid 285 in the nsp1 protein of the viral vector. The potential of this mutated vector for liver cancer therapy was verified at the cellular level and in vivo, respectively, and the changes in the tumor microenvironment after treatment were also described. The results showed that the Sindbis virus could effectively infect hepatocellular carcinoma cell lines and induce cell death. Furthermore, the addition of GM-CSF enhanced the tumor-killing effect of the Sindbis virus and increased the number of immune cells in the intra-tumor microenvironment during the treatment. In particular, SINV-GM-CSF was able to efficiently kill tumors in a mouse tumor model of hepatocellular carcinoma by regulating the elevation of M1-type macrophages (which have a tumor-resistant ability) and the decrease in M2-type macrophages (which have a tumor-promoting capacity). Overall, SINV-GM-CSF is an attractive vector platform with clinical potential for use as a safe and effective oncolytic virus.
Subject(s)
Carcinoma, Hepatocellular , Granulocyte-Macrophage Colony-Stimulating Factor , Liver Neoplasms , Oncolytic Virotherapy , Oncolytic Viruses , Sindbis Virus , Tumor Microenvironment , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Carcinoma, Hepatocellular/therapy , Animals , Sindbis Virus/genetics , Sindbis Virus/physiology , Liver Neoplasms/therapy , Liver Neoplasms/virology , Liver Neoplasms/genetics , Mice , Oncolytic Virotherapy/methods , Humans , Oncolytic Viruses/genetics , Oncolytic Viruses/physiology , Cell Line, Tumor , Xenograft Model Antitumor Assays , Macrophages/metabolism , Macrophages/immunologyABSTRACT
Alpha-1-antitrypsin (AAT) plays a homeostatic role in attenuating excessive inflammation and augmenting host defense against microbes. We demonstrated previously that AAT binds to the glucocorticoid receptor (GR) resulting in significant anti-inflammatory and antimycobacterial consequences in macrophages. Our current investigation aims to uncover AAT-regulated genes that rely on GR in macrophages. We incubated control THP-1 cells (THP-1control) and THP-1 cells knocked down for GR (THP-1GR-KD) with AAT, performed bulk RNA sequencing, and analyzed the findings. In THP-1control cells, AAT significantly upregulated 408 genes and downregulated 376 genes. Comparing THP-1control and THP-1GR-KD cells, 125 (30.6%) of the AAT-upregulated genes and 154 (41.0%) of the AAT-downregulated genes were significantly dependent on GR. Among the AAT-upregulated, GR-dependent genes, CSF-2 that encodes for granulocyte-monocyte colony-stimulating factor (GM-CSF), known to be host-protective against nontuberculous mycobacteria, was strongly upregulated by AAT and dependent on GR. We further quantified the mRNA and protein of several AAT-upregulated, GR-dependent genes in macrophages and the mRNA of several AAT-downregulated, GR-dependent genes. We also discussed the function(s) of selected AAT-regulated, GR-dependent gene products largely in the context of mycobacterial infections. In conclusion, AAT regulated several genes that are dependent on GR and play roles in host immunity against mycobacteria.
Subject(s)
Macrophages , Receptors, Glucocorticoid , alpha 1-Antitrypsin , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolism , Receptors, Glucocorticoid/metabolism , Receptors, Glucocorticoid/genetics , Humans , Macrophages/metabolism , Macrophages/immunology , THP-1 Cells , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/geneticsABSTRACT
BACKGROUND AND OBJECTIVE: Acute exacerbation of fibrosing interstitial lung disease (AE-FILD) is a serious condition with a high mortality rate. We aimed to comprehensively analyze cytokines in bronchoalveolar lavage fluid and their association with the clinical course of AE-FILD. METHODS: We retrospectively enrolled 60 patients with AE-FILD who underwent bronchoalveolar lavage. We comprehensively measured 44 cytokines and chemokines in the obtained bronchoalveolar lavage fluid using a Luminex analyzer. Patients were grouped into those who died within 90 days (non-survival group) and survived beyond 90 days (survival group) to investigate the association of the levels of cytokines and chemokines with mortality. RESULTS: The levels of matrix metalloproteinase 1 (p = 0.003), granulocyte-macrophage colony-stimulating factor (p = 0.040), interleukin 6 (p = 0.047), interleukin 8 (p = 0.050), monocyte chemoattractant protein-1 (p = 0.043), and eotaxin (p = 0.044) were significantly higher in the non-survival group than in the survival group. In the receiver operating characteristic analysis, their areas under the curve were 0.80, 0.68, 0.71, 0.70, 0.70, and 0.72, respectively. Using machine learning with these six cytokines and chemokines, the predictive accuracy for the survival group was 0.94. CONCLUSIONS: Our study demonstrated that several cytokines and chemokines in bronchoalveolar lavage fluid could be prognostic predictors in patients with AE-FILD.
Subject(s)
Bronchoalveolar Lavage Fluid , Chemokines , Cytokines , Disease Progression , Lung Diseases, Interstitial , Humans , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Female , Male , Prognosis , Cytokines/metabolism , Aged , Retrospective Studies , Lung Diseases, Interstitial/mortality , Lung Diseases, Interstitial/metabolism , Lung Diseases, Interstitial/immunology , Lung Diseases, Interstitial/diagnosis , Chemokines/metabolism , Chemokines/analysis , Middle Aged , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Chemokine CCL2/metabolism , Chemokine CCL2/analysis , Interleukin-6/metabolism , Interleukin-6/analysis , Interleukin-8/metabolism , Interleukin-8/analysis , Chemokine CCL11/metabolism , Chemokine CCL11/analysis , Biomarkers/metabolism , Biomarkers/analysisABSTRACT
Chronic wounds are often caused by diabetes and present a challenging clinical problem due to vascular problems leading to ischemia. This inhibits proper wound healing by delaying inflammatory responses and angiogenesis. To address this problem, we have developed injectable particle-loaded hydrogels which sequentially release Granulocyte-macrophage- colony-stimulating-factor (GM-CSF) and Vascular endothelial growth factor (VEGF) encapsulated in polycaprolactone-lecithin-geleol mono-diglyceride hybrid particles. GM-CSF promotes inflammation, while VEGF facilitates angiogenesis. The hybrid particles (200-1000 nm) designed within the scope of the study can encapsulate the model proteins Bovine Serum Albumin 65 ± 5 % and Lysozyme 77 ± 10 % and can release stably for 21 days. In vivo tests and histological findings revealed that in the hydrogels containing GM-CSF/VEGF-loaded hybrid particles, wound depth decreased, inflammation phase increased, and fibrotic scar tissue decreased, while mature granulation tissue was formed on day 10. These findings confirm that the hybrid particles first initiate the inflammation phase by delivering GM-CSF, followed by VEGF, increasing the number of vascularization and thus increasing the healing rate of wounds. We emphasize the importance of multi-component and sequential release in wound healing and propose a unifying therapeutic strategy to sequentially deliver ligands targeting wound healing stages, which is very important in the treatment of the diabetic wounds.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Hydrogels , Vascular Endothelial Growth Factor A , Wound Healing , Vascular Endothelial Growth Factor A/metabolism , Animals , Wound Healing/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Hydrogels/chemistry , Drug Liberation , Mice , Male , Humans , PolyestersABSTRACT
BACKGROUND: Due to the size and location of the tumor, incomplete radiofrequency ablation (iRFA) of the target tumor inhibits tumor immunity. In this study, a murine herpes simplex virus (oHSV2-mGM) armed with granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed to explore its effect on innate and adaptive immunity during iRFA, and the inhibitory effect of programmed cell death-1 (PD1) on tumor. METHODS: We verified the polarization and activation of RAW264.7 cells mediated by oHSV2-mGM in vitro. Subsequently, we evaluated the efficacy of oHSV2-mGM alone and in combination with αPD1 in the treatment of residual tumors after iRFA in two mouse models. RNA-seq was used to characterize the changes of tumor microenvironment. RESULTS: oHSV2-mGM lysate effectively stimulated RAW264.7 cells to polarize into M1 cells and activated M1 phenotypic function. In the macrophage clearance experiment, oHSV2-mGM activated the immune response of tumor in mice. The results in vivo showed that oHSV2-mGM showed better anti-tumor effect in several mouse tumor models. Finally, oHSV2-mGM combined with PD1 antibody can further enhance the anti-tumor effect of oHSV2-mGM and improve the complete remission rate of tumor in mice. CONCLUSION: The application of oHSV2-mGM leads to the profound remodeling of the immune microenvironment of residual tumors. oHSV2-mGM also works in synergy with PD1 antibody to achieve complete remission of tumors that do not respond well to monotherapy at immune checkpoints. Our results support the feasibility of recombinant oncolytic virus in the treatment of residual tumors after iRFA, and propose a new strategy for oncolytic virus treatment of tumors.
Subject(s)
Neoplasm, Residual , Programmed Cell Death 1 Receptor , Tumor Microenvironment , Animals , Female , Humans , Mice , Cell Line, Tumor , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Macrophages/immunology , Macrophages/drug effects , Mice, Inbred BALB C , Mice, Inbred C57BL , Radiofrequency Ablation/methods , RAW 264.7 Cells , Tumor Microenvironment/drug effects , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/drug effects , Tumor-Associated Macrophages/metabolismABSTRACT
The second-leading cause of death, cancer, poses a significant threat to human life. Innovations in cancer therapies are crucial due to limitations in traditional approaches. Newcastle disease virus (NDV), a nonpathogenic oncolytic virus, exhibits multifunctional anticancer properties by selectively infecting, replicating, and eliminating tumor cells. To enhance NDV's antitumor activity, four oncolytic NDV viruses were developed, incorporating IL24 and/or GM-CSF genes at different gene loci using reverse genetics. In vitro experiments revealed that oncolytic NDV virus augmented the antitumor efficacy of the parental virus rClone30, inhibiting tumor cell proliferation, inducing tumor cell fusion, and promoting apoptosis. Moreover, NDV carrying the IL24 gene inhibited microvessel formation in CAM experiments. Evaluation in a mouse model of liver cancer confirmed the therapeutic efficacy of oncolytic NDV viral therapy. Tumors in mice treated with oncolytic NDV virus significantly decreased in size, accompanied by tumor cell detachment and apoptosis evident in pathological sections. Furthermore, oncolytic NDV virus enhanced T cell and dendritic cell production and substantially improved the survival rate of mice with hepatocellular carcinoma, with rClone30-IL24(P/M) demonstrating significant therapeutic effects. This study establishes a basis for utilizing oncolytic NDV virus as an antitumor agent in clinical practice.
Subject(s)
Interleukins , Newcastle disease virus , Oncolytic Virotherapy , Oncolytic Viruses , Animals , Newcastle disease virus/genetics , Newcastle disease virus/physiology , Oncolytic Virotherapy/methods , Oncolytic Viruses/genetics , Oncolytic Viruses/physiology , Humans , Mice , Cell Line, Tumor , Interleukins/genetics , Interleukins/metabolism , Liver Neoplasms/therapy , Mice, Inbred BALB C , Carcinoma, Hepatocellular/therapy , Apoptosis , Neovascularization, Pathologic/therapy , Cell Proliferation , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Dendritic Cells/immunology , T-Lymphocytes/immunologyABSTRACT
BACKGROUND: Non-small cell lung cancer (NSCLC) represents a highly immunogenic malignancy. Immunologic tolerance facilitated by myeloid-derived suppressor cells (MDSCs) is implicated in primary or secondary resistance mechanisms in NSCLC. The potential role of APE1 in regulating NSCLC metastasis by targeting MDSCs remains uncertain. METHODS: This study utilized a plasmid, Plxpsp-mGM-CSF, to induce elevated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in A549 cells. Tumor transplantation experiments involved A549, A549+GM-CSF, and A549+GM-CSF-siAPE1 cell lines. Evaluation encompassed MDSCs, Treg cells, IgG, CD3, and CD8 levels. RESULTS: Notably, lung cancer tissues and cells displayed markedly reduced APE1 expression. siAPE1 transfection significantly curtailed tumor growth compared to the A549+GM-CSF group. APE1 knockdown orchestrated immune system modulation in lung tumor mice, characterized by diminished MDSCs but augmented Treg cells, IgG, CD3, and CD8. Additionally, APE1 knockdown led to reduced levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12) and a concurrent upregulation of the anti-MDSC cytokine IL-1ra. Furthermore, APE1 knockdown impeded cell viability in both A549 and H1650 cells. CONCLUSIONS: Transplantation of A549-GM-CSF amplified MDSC levels, fostering accelerated tumor growth, while mitigating MDSC levels through APE1 knockdown hindered tumor progression and alleviated inflammatory infiltration in lung cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach for lung cancer prevention and treatment, presenting novel insights for NSCLC management.
Subject(s)
Carcinoma, Non-Small-Cell Lung , DNA-(Apurinic or Apyrimidinic Site) Lyase , Granulocyte-Macrophage Colony-Stimulating Factor , Lung Neoplasms , Myeloid-Derived Suppressor Cells , Myeloid-Derived Suppressor Cells/immunology , Myeloid-Derived Suppressor Cells/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/metabolism , Animals , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Lung Neoplasms/metabolism , Humans , Mice , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , A549 Cells , Gene Knockdown Techniques , Neoplasm Metastasis , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Male , FemaleABSTRACT
BACKGROUND: The presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor in various testicular cells and spermatozoa suggests a potential role in enhancing spermatogonial and postmeiotic cell development. Moreover, GM-CSF activates the pivotal pathways implicated in sperm motility regulation and glucose metabolism. However, the impact of GM-CSF on testicular biopsies from patients with obstructive azoospermia (OA) remains unexplored. Therefore, this study aimed to investigate the in vitro effects of GM-CSF on the expression of genes related to glucose transporters and signaling pathways, sperm motility, and viability in testicular biopsies. METHODS AND RESULTS: Following testicular sperm extraction from 20 patients diagnosed with OA, each sample was divided into two parts: the experimental samples were incubated with medium containing 2 ng/ml GM-CSF at 37 °C for 60 min, and the control samples were incubated with medium without GM-CSF. Subsequently, the oocytes retrieved from the partner were injected with sperm from the treatment and control groups. The sperm parameters (motility and viability), the expression levels of sperm motility-related genes (PIK3R1, PIK3CA, and AKT1), and the expression levels of sperm energy metabolism-related genes (GLUT1, GLUT3, and GLUT14) were assessed. Furthermore, the fertilization and day 3 embryo development rate and embryo quality were evaluated. Compared with those in the nontreated group, the motility parameters and the mRNA expression levels of PIK3R1, AKT1, and GLUT3 in testicular sperm supplemented with GM-CSF were significantly greater (p < 0.05). However, no significant differences in the mRNA expression of PIK3CA, GLUT1, or GLUT14 were detected. According to the ICSI results, compared with the control group, the GM-CSF treatment group exhibited significantly greater fertilization rates (p = 0.027), Day 3 embryo development rate (p = 0.001), and proportions of good-quality embryos (p = 0.002). CONCLUSIONS: GM-CSF increased the expression of genes related to motility and the energy metabolism pathway and effectively promoted the motility of testis-extracted spermatozoa, consequently yielding positive clinical outcomes.
Subject(s)
Azoospermia , Energy Metabolism , Granulocyte-Macrophage Colony-Stimulating Factor , Sperm Injections, Intracytoplasmic , Sperm Motility , Spermatozoa , Humans , Male , Sperm Motility/drug effects , Sperm Motility/genetics , Azoospermia/genetics , Azoospermia/drug therapy , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Sperm Injections, Intracytoplasmic/methods , Energy Metabolism/drug effects , Energy Metabolism/genetics , Spermatozoa/metabolism , Spermatozoa/drug effects , Adult , Testis/metabolism , Testis/drug effects , Glucose Transporter Type 3/genetics , Glucose Transporter Type 3/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Female , Gene Expression Regulation/drug effectsABSTRACT
Colony-stimulating factor 2 (CSF2) plays a regulatory role in numerous cancers. However, there is needed to investigate the role of CSF2 in oral squamous cell carcinoma (OSCC) malignant phenotype and the specific mechanisms of CSF2 N-6-methyladenosine (m6A) modification. Therefore, we investigated the regulatory mechanism of m6A-modified CSF2 by WT1-associated protein (WTAP) in OSCC via qRT-PCR, western blot, WTAP and CSF2 overexpression in OSCC. In a panel of OSCCs, Kaplan-Meier plot analysis indicated that high expression of CSF2 was associated with poorer prognosis. Cell functional experiments revealed that enrichment of CSF2 promoted the proliferation and migration of OSCC cells by activating the JAK/STAT3 pathway, whereas the reduced expression of CSF2 resulted in the malignant decline of OSCC cells by blocking the JAK/STAT3 pathway. This study also confirmed that WTAP enhanced the m6A level of CSF2 and facilitated the expression of CSF2 and that CSF2 silencing blocked the invasive phenotype of OSCC cells and reversed the malignancy induced by WTAP overexpression. Overall, this study demonstrated that WTAP mediates the m6A modification of CSF2 and the JAK/STAT3 pathway, which plays an oncogenic role in the development of OSCC and can be a target for the treatment of patients with OSCC.
Subject(s)
Adenosine , Carcinoma, Squamous Cell , Cell Proliferation , Disease Progression , Janus Kinases , Mouth Neoplasms , STAT3 Transcription Factor , Signal Transduction , Humans , STAT3 Transcription Factor/metabolism , Mouth Neoplasms/metabolism , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Adenosine/analogs & derivatives , Adenosine/metabolism , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Janus Kinases/metabolism , Cell Line, Tumor , Cell Movement , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Blotting, Western , PrognosisABSTRACT
The production of superoxide anions and other reactive oxygen species (ROS) by neutrophils is necessary for host defense against microbes. However, excessive ROS production can induce cell damage that participates in the inflammatory response. Superoxide anions are produced by the phagocyte NADPH oxidase, a multicomponent enzyme system consisting of two transmembrane proteins (gp91phox/NOX2 and p22phox) and four soluble cytosolic proteins (p40phox, p47phox, p67phox and the small G proteins Rac1/2). Stimulation of neutrophils by various agonists, such as the bacterial peptide formyl-Met-Leu-Phe (fMLF), induces NADPH oxidase activation and superoxide production, a process that is enhanced by the pro-inflammatory cytokines such as GM-CSF. The pathways involved in this GM-CSF-induced up-regulation or priming are not fully understood. Here we show that GM-CSF induces the activation of the prolyl cis/trans isomerase Pin1 in human neutrophils. Juglone and PiB, two selective Pin1 inhibitors, were able to block GM-CSF-induced priming of ROS production by human neutrophils. Interestingly, GM-CSF induced Pin1 binding to phosphorylated p47phox at Ser345. Neutrophils isolated from synovial fluid of patients with rheumatoid arthritis are known to be primed. Here we show that Pin1 activity was also increased in these neutrophils and that Pin1 inhibitors effectively inhibited ROS hyperproduction by the same cells. These results suggest that the prolyl cis/trans isomerase Pin1 may control GM-CSF-induced priming of ROS production by neutrophils and priming of neutrophils in synovial fluid of rheumatoid arthritis patients. Pharmacological targeting of Pin1 may be a valuable approach to the treatment of inflammation.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , NADPH Oxidases , NIMA-Interacting Peptidylprolyl Isomerase , Neutrophils , Humans , Neutrophils/immunology , Neutrophils/drug effects , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , NADPH Oxidases/metabolism , Reactive Oxygen Species/metabolism , Naphthoquinones/pharmacology , Inflammation/immunology , Cells, Cultured , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/drug therapyABSTRACT
Through Smad3-dependent signalings, transforming growth factor-ß (TGF-ß) suppresses the development, maturation, cytokine productions and cytolytic functions of NK cells in cancer. Silencing Smad3 remarkably restores the cytotoxicity of NK-92 against cancer in TGF-ß-rich microenvironment, but its effects on the immunoregulatory functions of NK cells remain obscure. In this study, we identified Smad3 functioned as a transcriptional repressor for CSF2 (GM-CSF) in NK cells. Therefore, disrupting Smad3 largely mitigated TGF-ß-mediated suppression on GM-CSF production by NK cells. Furthermore, silencing GM-CSF in Smad3 knockout NK cells substantially impaired their anti-lung carcinoma effects. In-depth study demonstrated that NK-derived GM-CSF strengthened T cell immune responses by stimulating dendritic cell differentiation and M1 macrophage polarization. Meanwhile, NK-derived GM-CSF promoted the survival of neutrophils, which in turn facilitated the terminal maturation of NK cells, and subsequently boosted NK-cell mediated cytotoxicity against lung carcinoma. Thus, Smad3-silenced NK-92 (NK-92-S3KD) may serve as a promising immunoadjuvant therapy with clinical translational value given its robust cytotoxicity against malignant cells and immunostimulatory functions to reinforce the therapeutic effects of other immunotherapies.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Killer Cells, Natural , Lung Neoplasms , Smad3 Protein , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Smad3 Protein/metabolism , Smad3 Protein/genetics , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/genetics , Animals , Humans , Mice , Mice, Inbred C57BL , Cell Line, Tumor , Dendritic Cells/immunology , Dendritic Cells/metabolism , Transforming Growth Factor beta/metabolism , Cell Differentiation , Macrophages/metabolism , Macrophages/immunology , Signal TransductionABSTRACT
Acute systemic inflammation critically alters the function of the immune system, often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus, systemic inflammation results in acute thymic atrophy and, consequently, impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here, we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy, while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs), while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs), where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture, indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis, TL1A, and IL-18 synergistically increased GM-CSF levels in the NTOC, which was mainly produced by thymic ILC1s. In support, TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade, revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together, our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Interleukin-18 , Thymus Gland , Tumor Necrosis Factor Ligand Superfamily Member 15 , Animals , Interleukin-18/metabolism , Thymus Gland/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Mice , Tumor Necrosis Factor Ligand Superfamily Member 15/metabolism , Mice, Inbred C57BL , Granulocytes/metabolism , Myelopoiesis , Neutrophils/immunology , Neutrophils/metabolism , Receptors, Notch/metabolism , Lymphopoiesis , AtrophyABSTRACT
Dendritic cell (DC)-based vaccines have emerged as a promising strategy in cancer immunotherapy due to low toxicity. However, the therapeutic efficacy of DC as a monotherapy is insufficient due to highly immunosuppressive tumor environment. To address these limitations of DC as immunotherapeutic agent, we have developed a polymeric nanocomplex incorporating (1) oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and (2) arginine-grafted bioreducible polymer with PEGylated paclitaxel (APP) to restore antitumor immune surveillance function in tumor milieu and potentiate immunostimulatory attributes of DC vaccine. Nanohybrid complex (oAd/APP) in combination with DC (oAd/APP+DC) induced superior expression level of antitumor cytokines (IL-12, GM-CSF, and interferon gamma) than either oAd/APP or DC monotherapy in tumor tissues, thus resulting in superior intratumoral infiltration of both endogenous and exogenous DCs. Furthermore, oAd/APP+DC treatment led superior migration of DC to secondary lymphoid organs, such as draining lymph nodes and spleen, in comparison with either monotherapy. Superior migration profile of DCs in oAd/APP+DC treatment group resulted in more prolific activation of tumor-specific T cells in these lymphoid organs and greater intratumoral infiltration of T cells. Additionally, oAd/APP+DC treatment led to lower subset of tumor infiltrating lymphocytes and splenocytes being immunosuppressive regulatory T cells than any other treatment groups. Collectively, oAd/APP+DC led to superior induction of antitumor immune response and amelioration of immunosuppressive tumor microenvironment to elicit potent tumor growth inhibition than either monotherapy.
Subject(s)
Adenoviridae , Dendritic Cells , Oncolytic Virotherapy , Oncolytic Viruses , Paclitaxel , Dendritic Cells/immunology , Animals , Paclitaxel/pharmacology , Adenoviridae/genetics , Mice , Oncolytic Viruses/immunology , Oncolytic Viruses/genetics , Oncolytic Virotherapy/methods , Combined Modality Therapy , Cell Line, Tumor , Humans , Mice, Inbred C57BL , Cancer Vaccines/immunology , Immunotherapy/methods , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Female , Tumor Microenvironment/immunology , Tumor Microenvironment/drug effectsABSTRACT
BACKGROUND: Myeloid-derived suppressor cells (MDSCs) negatively modulate immune activity. Prior investigations have shown much promise in using MDSCs-assisted immunotherapy for organ transplantation patients. Additionally, owing to its immunosuppressive activity, MDSCs can also be used to manage immune-associated disorders. METHODS: Granulocyte-macrophage colony-stimulating factor (GM-CSF) was employed to stimulate myeloid progenitor cell differentiation. Triptolide (PG490) was introduced toward the later phases of in vitro MDSCs induction. Lastly, real-time PCR (RT-PCR) and flow cytometry were used to assess transcript expression and cell phenotype, and a mouse skin transplantation model was established to evaluate the MDSCs-mediated immune suppression in vivo. RESULTS: Co-stimulation with PG490 and GM-CSF potently induced myeloid-derived monocytes to form MDSCs, with remarkable immune-suppressive activity. The underlying mechanism involved downregulation of T cell proliferation, activation, enhancement of inflammatory cytokine release, as well as T cell conversion to Treg cells. PG490 strongly enhanced iNOS expression in MDSCs, and iNOS inhibition successfully reversed the immune-suppression. The PG490- and GM-CSF-induced MDSCs substantially extended survival duration of murine skin grafts, thereby validating their strong immune-suppressive activity in vivo. CONCLUSIONS: Herein, we presented a new approach involving MDSCs-based immunosuppression in vitro. PG490 and GM-CSF co-treatment strongly induced immuno-suppressive activity in MDSCs both in vitro and in vivo. Our findings highlight the promise of applying MDSCs-based therapy in clinical organ transplantation treatment.
Subject(s)
Cell Differentiation , Diterpenes , Epoxy Compounds , Granulocyte-Macrophage Colony-Stimulating Factor , Monocytes , Myeloid-Derived Suppressor Cells , Phenanthrenes , Diterpenes/pharmacology , Phenanthrenes/pharmacology , Epoxy Compounds/pharmacology , Myeloid-Derived Suppressor Cells/immunology , Myeloid-Derived Suppressor Cells/drug effects , Myeloid-Derived Suppressor Cells/metabolism , Animals , Humans , Monocytes/immunology , Monocytes/drug effects , Cell Differentiation/drug effects , Mice , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Skin Transplantation/methods , Nitric Oxide Synthase Type II/metabolism , Cell Proliferation/drug effects , Mice, Inbred BALB C , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Immunosuppressive Agents/pharmacology , Mice, Inbred C57BL , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Immunosuppression Therapy/methods , Cells, CulturedABSTRACT
Objective To investigate the effect of lysine 27 residue of histone H3 (H3K27) acetylation modification on the transcriptional promotion of long noncoding RNA OPA interacting protein 5-antisense RNA 1 (lncRNA OIP5-AS1) and apoptosis of nasal epithelial cells (NECs) in allergic rhinitis (AR) via regulating Toll-like receptor 4 (TLR4). Methods Interleukin-13 (IL-13) was used to treat NECs to establish an AR cell model. Real-time quantitative PCR was utilized to detect the expressions of OIP5-AS1 and TLR4 in nasal mucosal tissues of AR patients and in the in vitro cell model. The concentrations of macrophage colony-stimulating factor (GM-CSF), eotaxin-1, and mucin 5AC (MUC5AC) were detected by ELISA. The apoptosis of NECs was determined by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). A dual-luciferase report experiment was carried out to verify the relationship between OIP5-AS1 and TLR4. Chromatin immunoprecipitation (ChIP) assay was performed to verify H3K27 acetylation of histones in the OIP5-AS1 promoter region. Results Compared with healthy controls and untreated NECs, OIP5-AS1 and TLR4 were both up-regulated in nasal mucosal tissues from AR patients and IL-13-stimulated NECs. Knockdown of OIP5-AS1 decreased the level of TLR4 in IL-13-treated NECs, while overexpression of OIP5-AS1 increased the level of TLR4. Inhibition of OIP5-AS1 reduced the apoptosis rate, and inhibited the secretion of GM-CSF, eotaxin-1, and MUC5AC from IL-13-treated NECs, while overexpression of TLR4 partially reversed the effects of OIP5-AS1 knockdown on NEC apoptosis and the secretion of GM-CSF, eotaxin-1, and MUC5AC. In addition, H3K27 acetylation was markedly enriched in the promoter region of OIP5-AS1, and H3K27 acetylation promoted the expression of OIP5-AS1 in IL-13-treated NECs. Conclusion H3K27 acetylation promotes OIP5-AS1 transcription and induces NEC apoptosis in AR via upregulation of TLR4.