Expression Of Colony-stimulating Factor Research Articles

Hypoxia inducible factor 2α promotes tolerogenic macrophage development during cardiac transplantation through transcriptional regulation of colony stimulating factor 1 receptor

Solid organ transplantation mobilizes myeloid cells, including monocytes and macrophages, which are central protagonists of allograft rejection. However, myeloid cells can also be functionally reprogrammed by perioperative costimulatory blockade to promote a state of transplantation tolerance. Transplantation tolerance holds promise to reduce complications from chronic immunosuppression and promote long-term survival in transplant recipients. We sought to identify different mediators of transplantation tolerance by performing single-cell RNA sequencing of acute rejecting or tolerized cardiac allografts. This led to the unbiased identification of the transcription factor, hypoxia inducible factor (HIF)-2α, in a subset of tolerogenic monocytes. Using flow cytometric analyses and mice with conditional loss or gain of function, we uncovered that myeloid cell expression of HIF-2α was required for costimulatory blockade-induced transplantation tolerance. While HIF-2α was dispensable for mobilization of tolerogenic monocytes, which were sourced in part from the spleen, it promoted the expression of colony stimulating factor 1 receptor (CSF1R). CSF1R mediates monocyte differentiation into tolerogenic macrophages and was found to be a direct transcriptional target of HIF-2α in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2α in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2α activation in myeloid cells as a therapeutic strategy for transplantation tolerance.

Baricitinib ameliorates inflammatory and neuropathic pain in collagen antibody-induced arthritis mice by modulating the IL-6/JAK/STAT3 pathway and CSF-1 expression in dorsal root ganglion neurons

BackgroundJanus kinase (JAK) inhibitors, such as baricitinib, are widely used to treat rheumatoid arthritis (RA). Clinical studies show that baricitinib is more effective at reducing pain than other similar drugs. Here, we aimed to elucidate the molecular mechanisms underlying the pain relief conferred by baricitinib, using a mouse model of arthritis.MethodsWe treated collagen antibody-induced arthritis (CAIA) model mice with baricitinib, celecoxib, or vehicle, and evaluated the severity of arthritis, histological findings of the spinal cord, and pain-related behaviours. We also conducted RNA sequencing (RNA-seq) to identify alterations in gene expression in the dorsal root ganglion (DRG) following baricitinib treatment. Finally, we conducted in vitro experiments to investigate the direct effects of baricitinib on neuronal cells.ResultsBoth baricitinib and celecoxib significantly decreased CAIA and improved arthritis-dependent grip-strength deficit, while only baricitinib notably suppressed residual tactile allodynia as determined by the von Frey test. CAIA induction of inflammatory cytokines in ankle synovium, including interleukin (IL)-1β and IL-6, was suppressed by treatment with either baricitinib or celecoxib. In contrast, RNA-seq analysis of the DRG revealed that baricitinib, but not celecoxib, restored gene expression alterations induced by CAIA to the control condition. Among many pathways changed by CAIA and baricitinib treatment, the interferon-alpha/gamma, JAK-signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) pathways were considerably decreased in the baricitinib group compared with the celecoxib group. Notably, only baricitinib decreased the expression of colony-stimulating factor 1 (CSF-1), a potent cytokine that causes neuropathic pain through activation of the microglia–astrocyte axis in the spinal cord. Accordingly, baricitinib prevented increases in microglia and astrocytes caused by CAIA. Baricitinib also suppressed JAK/STAT3 pathway activity and Csf1 expression in cultured neuronal cells.ConclusionsOur findings demonstrate the effects baricitinib has on the DRG in relation to ameliorating both inflammatory and neuropathic pain.

Inhibition of non-small cell lung cancer metastasis by knocking down APE1 through regulating myeloid-derived suppressor cells-induced immune disorders.

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. 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. 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. 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.

Mesenchymal Stem Cells Induce an Immunosuppressive Microenvironment in Pituitary Tumors.

The tumor microenvironment (TME) includes diverse cellular components such as mesenchymal stem cells (MSCs) and immune cells, among others. MSC have been isolated from different tumors and they favor tumor cell growth; however, their role in pituitary tumors (PTs) remains unknown. Herein we report the presence of MSCs in 2 adrenocorticotropin (ACTH)-secreting PTs causing Cushing disease (MCU), 2 nonfunctioning adenomas of gonadotrope differentiation (MNF), and 2 nontumoral pituitary glands (MS). We have analyzed the transcriptomic profiles by RNA sequencing and compared MSCs in terms of their immunosuppressive effects against lymphoid T-cell and macrophage populations by means of cocultures and flow cytometry. Our transcriptomic analysis revealed molecular differences between MSCs derived from nontumoral pituitaries and MSCs derived from PTs. Two distinct subpopulations of MSC emerged: one displaying immunosuppressive properties and the other with increased proproliferative capabilities, regardless of their origin. MSCs derived from ACTH- and nonfunctioning PTs, but not those derived from nontumoral glands, significantly inhibited the proliferation of activated T cells, favored the generation of regulatory T cells, and promoted M2 macrophage polarization. Such immunosuppressive effects were correlated with an upregulation of programmed death ligand 1 and intracellular expression of macrophage colony-stimulating factor (M-CSF) and interleukin-10. Importantly, MSC derived from ACTH-PTs showed a higher immunosuppressive potential than MSC isolated from nonfunctioning tumors. This study demonstrates the presence of at least 2 MSC subpopulations in the pituitary gland and suggests that immunosuppressive effects of MSCs may have important implications in PT growth.

Abstract 5368: Targeting tumor-intrinsic S100A1 augments antitumour immunity and potentiates anti-PD-1 efficacy

Abstract Immune-checkpoint blockade (ICB) has revolutionized cancer treatment. However, only a minority of patients achieve the long-term, durable response to ICB, and the response rates in most advanced tumors is limited to 10%-25%. The unsatisfactory overall response to ICB and the great variation in response among individuals highlight the necessity for developing predictive biomarkers and overcoming ICB resistance. A potential mode of resistance is tumor-mediated immunosuppression leading to inhibition of T cell-mediated infiltration and immunity. Here, combining single-cell and bulk transcriptomic analysis with liquid biopsy of tumor patients, we showed that S100 calcium binding protein A1 (S100A1) was closely related to ICB response, and that detecting plasma S100A1 level could help predicting the response to ICB, implying that tumor-intrinsic S100A1 may contribute to immunosuppressive tumor microenvironment (TME) and resistance to ICB. Importantly, endogenous S100a1 knockdown (S100a1KD) promoted anti-tumor immunity, conferred tumor regression, and sensitized tumors to anti-PD-1 immunotherapy in multiple syngeneic murine models. By characterizing the immune landscape of S100a1KD syngeneic murine tumor models and non-small cell lung cancer (NSCLC) clinical samples, we demonstrated a negative relationship between tumor-intrinsic S100A1 expression and infiltration of M1-like macrophage and CD8+ T cells. Single cell and spatial transcriptomic analyses, together with functional studies, have revealed that ablation of tumor-intrinsic S100A1 exerts profound impacts on the M1-like polarization of macrophages and cytotoxicity of CD8+ T cells. Moreover, the conditioned medium from S100a1KD tumor cells promoted M1 polarization of both bone marrow-derived macrophages (BMDMs) and Raw264.7 cells, which in turn reinvigorated tumor-specific T cell immunity both in vitro and in vivo. Of note, as a secreted protein, introducing recombinant S100A1 into the medium from S100a1KD tumor cells could not reverse the effects on T cells and macrophages, suggesting that these effects were independent of extracelluar S100A1 secretion and that S100A1 may act as an intracellular signaling molecule. Mechanistically, S100A1 affects deubiquitination of NF-κB through interacting with the ubiquitin-like (Ubl) domains of ubiquitin specific protease 7 (USP7), leading to upregulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression. Collectively, these data identified the S100A1/USP7/p65/GM-CSF inhibitory axis as a critical immune suppressor on M1-like macrophage polarization and T cell immunity for the promotion of tumor progression and resistance to ICB. Our findings demonstrated the potential of plasma S100A1 expression in predicting the response to ICB, and provide a rationale for combining GM-CSF and anti-PD-1 immunotherapy in tumors with high S100A1 expression. Citation Format: Yufeng Guo, Hua Bai, Jie Wang. Targeting tumor-intrinsic S100A1 augments antitumour immunity and potentiates anti-PD-1 efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5368.

Knock down of APE1 suppressed gastric cancer metastasis via improving immune disorders caused by myeloid-derived suppressor cells

ABSTRACT Gastric cancer is a highly immunogenic malignancy. Immune tolerance facilitated by myeloid-derived suppressor cells (MDSCs) has been implicated in gastric cancer resistance mechanisms. The potential role of APE1 in regulating gastric cancer metastasis by targeting MDSCs remains uncertain. In this study, the plasmid Plxpsp-mGM-CSF was used to induce high expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in GES-1 cells. For tumor transplantation experiments, AGS, AGS+GM-CSF and AGS+GM-CSF-siAPE1 cell lines were established by transfection, followed by subcutaneous implantation of tumor cells. MDSCs, Treg cells, IgG, CD3 and CD8 levels were assessed. Transfection with siAPE1 significantly inhibited tumor growth compared to the AGS+GM-CSF group. APE1 gene knockdown modulated the immune system in gastric cancer mice, characterized by a decrease in MDSCs and an increase in Treg cells, IgG, CD3 and CD8. In addition, APE1 gene knockdown resulted in decreased levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12). Furthermore, APE1 gene knockdown inhibited proliferation, migration and invasion of AGS and MKN45 cells. AGS-GM-CSF cell transplantation increased MDSC levels and accelerated tumor growth, whereas APE1 knockdown reduced MDSC levels, inhibited tumor growth and attenuated inflammatory infiltration in gastric cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach to the prevention and treatment of gastric cancer, providing new insights into its management.

TPX2 influences the regulation of macrophage polarization via the NF-κB pathway in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Xklp2 targeting protein (TPX2), a crucial oncogene exhibits high expression levels in various cancers including LUAD, may serve as a potential target for clinical intervention. Additionally, the growth of lung cancer is significantly influenced by the tumor microenvironment (TME). However, there have been no reports on experiments investigating TPX2 in tumor-infiltrating immune cells (TIICs) in LUAD. Therefore, we verified the effect of TPX2 on macrophage polarization both in vitro and in vivo. MethodsWe silenced TPX2 the gene in A549 cells and collected supernatants for macrophage culture. We then used flow cytometry and Western blot analysis to assess macrophage polarization. Additionally, we verified the expression of macrophage colony-stimulating factor (M-CSF), and CD163 by immunohistochemistry (IHC) in tissue specimens from LUAD patients. Finally, pathways related to TPX2's regulatory function in macrophage polarization were analyzed through whole genome sequencing, Western blotting, and immunofluorescence (IF). ResultsSilencing TPX2 can affect the ratio of CD80+ M1/CD163+ M2 and reduce the polarization of M0 macrophages to CD163+ M2 macrophages mainly by inhibiting the expression of M-CSF. In human LUAD tissues, the expression levels of TPX2, M-CSF and CD163 increased with the degree of differentiation. Silencing TPX2 inhibits the NF-κB signaling pathway, thereby reducing the expression of M-CSF, and affecting macrophage polarization. ConclusionSilencing TPX2 can inhibit the expression of M-CSF by blocking the NF-κB signal, thereby reducing CD163+ M2 macrophage polarization. The TPX2/NF-κB/M-CSF signaling axis may be involved in regulating macrophage polarization.

The Notch1 signaling pathway directly modulates the human RANKL-induced osteoclastogenesis

Notch signaling is an evolutionary conserved pathway with a key role in tissue homeostasis, differentiation and proliferation. It was reported that Notch1 receptor negatively regulates mouse osteoclast development and formation by inhibiting the expression of macrophage colony-stimulating factor in mesenchymal cells. Nonetheless, the involvement of Notch1 pathway in the generation of human osteoclasts is still controversial. Here, we report that the constitutive activation of Notch1 signaling induced a differentiation block in human mononuclear CD14+ cells directly isolated from peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation to osteoclasts. Additionally, using a combined approach of single-cell RNA sequencing (scRNA-Seq) simultaneously with a panel of 31 oligo-conjugated antibodies against cell surface markers (AbSeq assay) as well as unsupervised learning methods, we detected four different cell stages of human RANKL-induced osteoclastogenesis after 5 days in which Notch1 signaling enforces the cell expansion of specific subsets. These cell populations were characterized by distinct gene expression and immunophenotypic profiles and active Notch1, JAK/STAT and WNT signaling pathways. Furthermore, cell–cell communication analyses revealed extrinsic modulators of osteoclast progenitors including the IL7/IL7R and WNT5a/RYK axes. Interestingly, we also report that Interleukin-7 receptor (IL7R) was a downstream effector of Notch1 pathway and that Notch1 and IL7R interplay promoted cell expansion of human RANKL-induced osteoclast progenitors. Taken together, these findings underline a novel cell pattern of human osteoclastogenesis, outlining the key role of Notch1 and IL-7R signaling pathways.

Erythropoietin Treatment in Mice Enriches Bone Marrow Osteoclast Precursors, As Reflected By Increased Macrophage Colony-Stimulating Factor (MCSF) and Elevated Expression of Osteoclast Markers in Monocytes

The impact of erythropoietin (EPO) on erythropoiesis has long been established. However, recent studies have indicated that EPO may also exert regulatory effects on other hematopoietic lineages, including the monocyte lineage. In that respect, we have previously shown that EPO treatment in mice induces bone loss. Here, we studied the effect of EPO on bone marrow (BM) monocytes and its potential implications for osteoclastogenesis. To address the dynamic changes in monocyte populations following EPO administration, we injected EPO (180U) once, or 3 times on alternating days for one week. Mice were sacrificed 16 h after EPO injection. Our results revealed a significant 20 percent increase in the monocyte population (TER119 -, LY6G -, CD11b +) 16 hours after EPO injection, and a twofold increase in the levels of these cells after one week of 3 injections of EPO. Soluble macrophage colony-stimulating factor (MCSF) protein levels in the bone marrow were increased 1.5-fold 16 h after EPO injection, while MCSF mRNA expression levels remained unchanged. After 1-week of EPO administration (3 injections), although the global BM expression of MCSF was reduced, we observed a two-fold increase in MCSF expression levels in T cells (CD3 +). To further elucidate the functional consequences of EPO-induced monocyte expansion, we isolated monocytes from mice treated with EPO for one week. We characterized expression levels of key osteoclastic markers in these cells. Indeed, we found that in BM monocytes, EPO treatment led to the upregulation of Lymphocyte function-associated antigen 1 (LFA-1) and Receptor Activator of Nuclear Factor κ B (RANK). The former is implicated in osteoclastogenesis via its interaction with intercellular adhesion molecule-1 (ICAM−1), while the latter is the surface receptor for RANK ligand which regulates osteoclast differentiation and activation. These data suggest that EPO not only influences BM monocyte expansion, but also contributes to the regulation of osteoclast differentiation, thus linking erythropoiesis to bone homeostasis. Furthermore, we investigated the early effects of EPO treatment on the expression of transcription factors implicated in monocyte differentiation. Remarkably, we found that EPO administration for 16 hours resulted in the downregulation of KLF2 and KLF4, transcription factors involved in monocytic lineage commitment. The rapid EPO-induced expansion of the monocytic population, coupled with the EPO-regulated expression of MSCF, osteoclastic markers, and transcription factors, suggests a role for EPO in modulating monocyte-to-osteoclast differentiation. The current study opens avenues for further investigating the crosstalk between erythropoiesis, myelopoiesis, and bone homeostasis.

IGF2BP2-meidated m6A modification of CSF2 reprograms MSC to promote gastric cancer progression

The interaction between tumor cells and stromal cells within the tumor microenvironment plays a critical role in cancer progression. Mesenchymal stem cells (MSCs) are important tumor stromal cells that exhibit pro-oncogenic activities when reprogrammed by the tumor. However, the precise mechanisms underlying MSC reprogramming in gastric cancer remain not well understood. QRT-PCR, western blot, and immunohistochemistry were used to examine gene and protein expression levels. In vitro and in vivo experiments were conducted to assess the biological functions of gastric cancer cells. RNA-sequencing, RNA immunoprecipitation (RIP), and meRIP assays were performed to investigate underlying molecular mechanisms. We found a significant increase in the expression and N6-methyladenosine (m6A) modification levels of colony-stimulating factor 2 (CSF2) in gastric cancer MSCs. CSF2 gene overexpression induced the reprogramming of normal MSCs into cancer-promoting MSCs, thereby enhancing the proliferation, migration, and drug resistance of gastric cancer cells through the secretion of various pro-inflammatory factors. Additionally, we demonstrated that the m6A reader IGF2BP2 bound to and stabilized CSF2 mRNA in gastric cancer MSCs. Notably, overexpression of IGF2BP2 mimicked the effect of CSF2 on MSCs, promoting gastric cancer progression. Finally, we unveiled that CSF2 induced the ubiquitination of Notch1 to reprogram MSCs. Our study highlights a critical role of IGF2BP2-mediated m6A modification of CSF2 in reprogramming MSCs, which presents a promising therapeutic target for gastric cancer.

Scutellarin suppresses the metastasis of triple-negative breast cancer via targeting TNFα/TNFR2-RUNX1-triggered G-CSF expression in endothelial cells

Triple-negative breast cancer (TNBC) is heterogeneous and aggressive, with high vascularity and frequent metastasis. We have already found natural flavonoid scutellarin (SC) suppressed spontaneous TNBC metastasis via normalizing tumor vasculature in vivo. In this study, supernatant from tumor necrosis factorα (TNFα)-treated human mammary microvascular endothelial cell (HMMEC) promoted cell migration and pseudopod formation in TNBC cells, but these phenomena were disappeared in SC-co-treated HMMEC. TNFα enhanced the expression of granulocyte colony-stimulating factor (G-CSF) and granulocyte–macrophage colony-stimulating factor (GM-CSF) in both HMMEC and human umbilical vein endothelial cell (HUVEC). G-CSF promoted TNBC migration and invasion in vitro, while G-CSF neutralization antibody and SC both inhibited TNBC metastasis in Balb/c mice. SC had no inhibition on the G-CSF-induced TNBC cell migration, but reduced G-CSF content in TNBC tumor tissues and TNFα-stimulated endothelial cells (ECs). SC restricted the nuclear translocation of runt-related transcription factor 1 (RUNX1) in TNBC tumor vessels and TNFα-treated ECs. RUNX1 was found to directly bind to the promoter of G-CSF in TNBC tumor vessels and regulated G-CSF expression. TNF receptor 2 (TNFR2) was crucial for regulating the TNFα-induced RUNX1 activation and G-CSF expression. Notably, SC hindered the interaction between TNFα and TNFR2 via binding to TNFR2. This work demonstrated that SC reduced TNBC metastasis by targeting TNFα/TNFR2-initiated RUNX1 activation and subsequent G-CSF production in TNBC-associated ECs.

The exploration of neuroinflammatory mechanism by which CRHR2 deficiency induced anxiety disorder

Inflammation stimulates the hypothalamic-pituitary adrenal (HPA) axis and triggers glial neuroinflammatory phenotypes, which reduces monoamine neurotransmitters by activating indoleamine 2,3-dioxygenase enzyme. These changes can induce psychiatric diseases, including anxiety. Corticotropin releasing hormone receptor 2 (CRHR2) in the HPA axis is involved in the etiology of anxiety. Omega(n)-3 polyunsaturated fatty acids (PUFAs) can attenuate anxiety through anti-inflammation and HPA axis modulation. However, the underlying molecular mechanism by CRHR2 modulates anxiety and its correlation with neuroinflammation remain unclear. Here, we first constructed a crhr2 zebrafish mutant line, and evaluated anxiety-like behaviors, gene expression associated with the HPA axis, neuroinflammatory response, neurotransmitters, and PUFAs profile in crhr2+/+ and crhr2−/− zebrafish. The crhr2 deficiency decreased cortisol levels and up-regulated crhr1 and down-regulated crhb, crhbp, ucn3l and proopiomelanocortin a (pomc a) in zebrafish. Interestingly, a significant increase in the neuroinflammatory markers, translocator protein (TSPO) and the activation of microglia M1 phenotype (CD11b) were found in crhr2−/− zebrafish. As a consequence, the expression of granulocyte-macrophage colony-stimulating factor, pro-inflammatory cytokines vascular endothelial growth factor, and astrocyte A1 phenotype c3 were up-regulated. While microglia anti-inflammatory phenotype (CD206), central anti-inflammatory cytokine interleukin-4, arginase-1, and transforming growth factor-β were downregulated. In parallel, crhr2-deficient zebrafish showed an upregulation of vdac1 protein, a TSPO ligand, and its downstream caspase-3. Furthermore, 5-HT/5-HIAA ratio was decreased and n-3 PUFAs deficiency was identified in crhr2−/− zebrafish. In conclusion, anxiety-like behavior displayed by crhr2-deficient zebrafish may be caused by the HPA axis dysfunction and enhanced neuroinflammation, which resulted in n-3 PUFAs and monoamine neurotransmitter reductions.

Soybean (Glycine max) INFOGEST Colonic Digests Attenuated Inflammatory Responses Based on Protein Profiles of Different Varieties.

Soybean compounds have been established to modulate inflammation, but less is known about how whole soybean compositions work together after digestion. The objective was to evaluate and compare the anti-inflammatory responses of different soybean varieties under simulated gastrointestinal digestion, with additional consideration of the glycinin:β-conglycinin ratio (GBR). Soybean colonic digests (SCD) inhibited cyclooxygenase (COX)-2 (25-82%), 5-lipoxidase (LOX) (18-35%), and inducible nitric oxide (iNOS) (8-61%). Varieties 88, GN3, and 93 were the most effective inhibitors. SCD (1 mg/mL) of varieties 81 and GN1 significantly (p < 0.05) reduced nitrite production by 44 and 47%, respectively, compared to lipopolysaccharide (LPS)-stimulated macrophages. SCD effectively reduced pro-inflammatory cytokine interleukin (IL)-6 (50 and 80% for 96 and GN1, respectively). Western blot results showed a decrease in the expression of iNOS, p65, and p50. The GBR was in the range of 0.05-1.57. Higher ratio correlated with higher production of IL-1β (r = 0.44) and tumor necrosis factor-alpha (TNF-α, r = 0.56). Inflammatory microarray results showed a significant decrease in expression of markers granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6 in cells treated with GN1 SCD compared to LPS. The results suggested that SCD exerted its anti-inflammatory potential through nuclear factor kappa B (NF-κΒ) pathway inhibition by decreasing the levels of NF-κB-dependent cytokines and subunits, and inhibition of pro-inflammatory enzyme activity.

Decidual lymphatic endothelial cell-derived granulocyte-macrophage colony-stimulating factor induces M1 macrophage polarization via the NF-κB pathway in severe pre-eclampsia.

Direct interactions between macrophages and lymphatic vessels have been shown previously.In pre-eclampsia (PE), macrophages are dominantly polarized into a proinflammatory M1 phenotype and lymphangiogenesis is defective in the decidua.Here, we investigated whether decidual lymphatic endothelial cells (dLECs) affect macrophage polarization in PE. THP-1 macrophages were cocultured with dLECs or cultured in the conditioned medium (CM) of dLECs. Macrophage polarization was measured using flow cytometry. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in dLECs was measured using qRT-PCR and ELISA. The activation of nuclear translocation of nuclear factor-κ (NF-κB), an upstream signaling molecule of GM-CSF, was assessed by immunocytochemical localization of p65. Through GM-CSF knockdown and NF-κB inhibition in dLEC, we evaluated whether the GM-CSF/NF-κB pathway of PE dLEC affects decidual macrophage polarization. The ratio of inflammatory M1 macrophages with HLA-DR+ /CD80+ markers significantly increased following coculturing with PE dLECs or culturing in PE dLEC CM, indicating that the PE dLEC-derived soluble factor acts in a paracrine manner. GM-CSF expression was significantly upregulated in PE dLECs. Recombinant human GM-CSF induced macrophage polarization toward an M1-like phenotype, whereas its knockdown in PE dLECs suppressed it, suggesting PE dLECs induce M1 macrophage polarization by secreting GM-CSF. The NF-κB p65 significantly increased in PE dLECs compared to the control, and pretreatment with an NF-κB inhibitor significantly suppressed GM-CSF production from PE dLECs. In PE, dLECs expressing high levels of GM-CSF via the NF-κB-dependent pathway play a role in inducing decidual M1 macrophage polarization.

Simultaneous Expression of Chicken Granulocyte Monocyte Colony-Stimulating Factor and the Hemagglutinin-Neuraminidase Epitope of the Virulent Newcastle Disease Virus Genotype VII C22 Strain in a Functional Synthetic Recombinant Adenovirus as a Genotype-Matched Vaccine with Potential Antiviral Activity.

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-β, IFN-γ, interleukin-1β (IL-1β), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.

Mesenchymal Stem/Stromal Cells Derived from Cervical Cancer Promote M2 Macrophage Polarization

Macrophages with the M2 phenotype promote tumor development through the immunosuppression of antitumor immunity. We previously demonstrated the presence of mesenchymal stem/stromal cells (MSCs) in cervical cancer (CeCa-MSCs), suggesting an immune protective capacity in tumors, but to date, their effect in modulating macrophage polarization remains unknown. In this study, we compared the capacities of MSCs from normal cervix (NCx) and CeCa to promote M2 macrophage polarization in a coculture system. Our results demonstrated that CeCa-MSCs, in contrast to NCx-MSCs, significantly decreased M1 macrophage cell surface marker expression (HLA-DR, CD80, CD86) and increased M2 macrophage expression (CD14, CD163, CD206, Arg1) in cytokine-induced CD14+ monocytes toward M1- or M2-polarized macrophages. Interestingly, compared with NCx-MSCs, in M2 macrophages generated from CeCa-MSC cocultures, we observed an increase in the percentage of phagocytic cells, in the intracellular production of IL-10 and IDO, the capacity to decrease T cell proliferation and for the generation of CD4+CD25+FoxP3+ Tregs. Importantly, this capacity to promote M2 macrophage polarization was correlated with the intracellular expression of macrophage colony-stimulating factor (M-CSF) and upregulation of IL-10 in CeCa-MSCs. Furthermore, the presence of M2 macrophages was correlated with the increased production of IL-10 and IL-1RA anti-inflammatory molecules. Our in vitro results indicate that CeCa-MSCs, in contrast to NCx-MSCs, display an increased M2-macrophage polarization potential and suggest a role of CeCa-MSCs in antitumor immunity.

Nicotinamide N -methyltransferase promotes M2 macrophage polarization by IL6 and MDSC conversion by GM-CSF in gallbladder carcinoma.

Nicotinamide N -methyltransferase (NNMT), an enzyme responsible for the methylation of nicotinamide, is involved in many metabolic pathways in adipose tissue and the liver. However, the role of NNMT in editing the tumor immune microenvironment is not well understood. Here, we identified that NNMT can promote IL6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by decreasing the tri-methyl-histone H3 levels on the promoters of IL6 and CSF2 (encoding GM-CSF) and CCAAT/Enhancer Binding Protein, an essential transcription factor for IL6 expression, thus promoting differentiation of macrophages into M2 type tumor-associated macrophages and generation of myeloid-derived suppressor cells from peripheral blood mononuclear cells. Treatment of xenografted tumor models overexpressing NNMT gallbladder carcinoma (GBC) cells with the NNMT inhibitor JBSNF-000088 resulted in compromised tumor development and decreased expression levels of IL6, GM-CSF, tumor-associated macrophage marker CD206, and myeloid-derived suppressor cell marker CD33 but increased expression levels of CD8. In addition, elevated expression of NNMT in tumors of patients with GBC was correlated with increased expression levels of CD206 and CD33 but with decreased levels of CD8 and survival of patients. These data highlight the critical role of NNMT in GBC progression. Inhibition of NNMT by JBSNF-000088 is a potential molecular target for GBC immunotherapy.

Epigenetic therapy reprograms M2-type tumor-associated macrophages into an M1-like phenotype by upregulating miR-7083-5p.

Reprogramming M2-type, pro-tumoral tumor-associated macrophages (TAMs) into M1-type, anti-tumoral macrophages is a key strategy in cancer therapy. In this study, we exploited epigenetic therapy using the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylation inhibitor trichostatin A (TSA), to reprogram M2-type macrophages into an M1-like phenotype. Treatment of M2-type macrophages with the combination of 5-aza-dC and TSA decreased the levels of M2 macrophage cytokines while increasing those of M1 macrophage cytokines, as compared to the use of either therapy alone. Conditioned medium of M2 macrophages treated with the combination of 5-aza-dC and TSA sensitized the tumor cells to paclitaxel. Moreover, treatment with the combination inhibited tumor growth and improved anti-tumor immunity in the tumor microenvironment. Depletion of macrophages reduced the anti-tumor growth activity of the combination therapy. Profiling of miRNAs revealed that the expression of miR-7083-5p was remarkably upregulated in M2 macrophages, following treatment with 5-aza-dC and TSA. Transfection of miR-7083-5p reprogrammed the M2-type macrophages towards an M1-like phenotype, and adoptive transfer of M2 macrophages pre-treated with miR-7083-5p into mice inhibited tumor growth. miR-7083-5p inhibited the expression of colony-stimulating factor 2 receptor alpha and CD43 as candidate targets. These results show that epigenetic therapy upon treatment with the combination of 5-aza-dC and TSA skews M2-type TAMs towards the M1-like phenotype by upregulating miR-7083-5p, which contributes to the inhibition of tumor growth.

Increased Tumor-Associated CD66b+ Myeloid-Derived Suppressor Cells in Waldenstrom Macroglobulinemia Inhibit T-Cell Immune Function

Waldenstrom macroglobulinemia (WM) is a discrete clinicopathologic entity resulting from the accumulation of lymphoplasmacytic lymphoma (LPL) cells in the bone marrow (BM) that secrete a monoclonal immunoglobulin (Ig)M protein. MYD88 (>90%) and CXCR4 (30-35%) mutations are the most commonly identified mutations in this disease. While much is known about the genomics of WM, far less is known about the role of the BM microenvironment in this disease. Emerging evidence has attested to the role of myeloid-derived suppressor cells (MDSCs) and their potent immunosuppressive activity, however, there is limited information about these cells in WM and monoclonal gammopathy of undetermined significance (MGUS). This study was therefore designed to explore the phenotype of MDSCs and their interaction with other cells in the BM in WM. A multiparametric approach was combined with single-cell genomic and proteomic tools to better understand MDSC immunology and the antitumor immune response in WM. In the present study, using flow cytometry, we first analyzed the number of MDSCs (by gating on Lin- HLA-DR- CD11b+ CD33+) in normal BM (NBM; n=11) and WM symptomatic /MGUS (n=18) and found an increased number of MDSCs in WM specimens compared to controls (p=0.005). We also found that most MDSCs in WM specimens had a granulocytic phenotype (Lin- HLA-DR- CD11b+ CD33+ CD15+). A detailed phenotypic study using CyTOF (mass spectrometry) was performed on MDSCs from NBM (n=4) and WM (n=8). The CyTOF analysis identified 3 subtypes of MDSCs including CD66b+ MDSCs with a granulocytic phenotype (G-MDSCs) and confirmed the expansion of CD66b+ MDSCs in WM symptomatic patients when compared to smoldering WM and normal controls. Our previous studies have shown that granulocyte colony-stimulating factor (G-CSF) expression is increased in the bone marrow of WM (Jalali et al., 2018). Therefore, to determine whether G-CSF accounted for the expansion of CD66b+ G-MDSCs, we treated WM-BM MDSCs with G-CSF and found that the CD66b+ G-MDSC population substantially increased (p=0.0017). Further, to establish a better understanding of the transcriptome profile and a unique phenotype of CD66b+ G-MDSCs, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on WM (n=3) and NBM (n=2) specimens. A significantly high expression of NLRP12, IGFBP7, CXCR4, IL2, CD55, SOX4, and FoxP1 genes was observed in CD66b+ MDSCs as compared to other MDSC populations. This gene expression signature suggested upregulation of inflammatory pathways potentially affecting immune function. Therefore, to evaluate the effect of CD66b+ G-MDSCs on the immune function of other cells in the BM, CD66b+ and CD66b- MDSCs were co-cultured with activated autologous T-cells. We found that while all populations of MDSCs suppressed T-cell activation, CD66b+ G-MDSCs had the greatest suppressive effect. In conclusion, this study has shown that CD66b+ G-MDSCs are expanded in WM patients, exhibit a unique inflammatory transcriptome, and substantially inhibit T-cell activation and proliferation. Furthermore, CD66b+ G-MDSCs are expanded due to increased G-CSF in the BM microenvironment in WM. Clinical strategies to inhibit the expansion of CD66b+ G-MDSCs may therefore enhance the efficacy of immunological therapies in patients with WM.

RNA-binding protein DHX9 promotes glioma growth and tumor-associated macrophages infiltration via TCF12.

Glioma is the most common malignant tumor of the central nervous system, with high heterogeneity, strong invasiveness, high therapeutic resistance, and poor prognosis, comprehending a serious challenge in neuro-oncology. Until now, the mechanisms underlying glioma progression have not been fully elucidated. The expression of DExH-box helicase 9 (DHX9) in tissues and cells was detected by qRT-PCR and western blot. EdU and transwell assays were conducted to assess the effect of DHX9 on proliferation, migration and invasion of glioma cells. Cocultured model was used to evaluate the role of DHX9 on macrophages recruitment and polarization. Animal study was performed to explore the role of DHX9 on macrophages recruitment and polarization in vivo. Bioinformatics analysis, dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay was used to explore the relation between DHX9 and TCF12/CSF1. DHX9 was elevated in gliomas, especially in glioblastoma multiforme (GBM). Besides promoting the proliferation, migration, and invasion of glioma cells, DHX9 facilitated the infiltration of macrophages into glioma tissues and polarization to M2-like macrophages, known as tumor-associated macrophages (TAMs). DHX9 silencing decreased the expression of colony-stimulating factor 1 (CSF1), which partially restored the inhibitory effect on malignant progress of glioma and infiltration of TAMs caused by DHX9 knockdown by targeting the transcription factor 12 (TCF12). Moreover, TCF12 could directly bind to the promoter region of CSF1. DHX9/TCF12/CSF1 axis regulated the increases in the infiltration of TAMs to promote glioma progression and might be a novel potential target for future immune therapies against gliomas.