Granulocyte-macrophage Colony-stimulating Factor Receptor Research Articles

Granulocyte Macrophage-Colony Stimulating Factor Reverses HIV Protein-Induced Mitochondrial Derangements in Alveolar Macrophages.

Despite the advent of antiretroviral therapy, people living with HIV suffer from a range of infectious and noninfectious pulmonary complications. HIV impairs antioxidant defenses and innate immune function of the alveolar macrophage by diminishing granulocyte macrophage-colony stimulating factor (GM-CSF) signaling. Since GM-CSF may be linked to mitochondria, we sought to determine the effects of HIV on GM-CSF receptor expression and alveolar macrophage mitochondrial function. At an academic medical center, studies were completed on alveolar macrophages isolated from both wild-type and HIV transgenic (HIV Tg) rats and human subjects with and without HIV. Primary macrophages were plated and evaluated for expression of GM-CSF receptor beta, phagocytic index, and mitochondrial function in the presence and absence of GM-CSF treatment. GM-CSF receptor expression and mitochondrial function were impaired in macrophages isolated from HIV Tg rats, and treatment with GM-CSF restored GM-CSF receptor expression and mitochondrial function. GM-CSF treatment of HIV Tg rats also increased alveolar macrophage levels of the mitochondrial proteins voltage-dependent anion-selective channel 1 (VDAC) and glucose-regulated protein 75 (Grp75). Similar to the HIV Tg rat model, impairments in mitochondrial bioenergetics were confirmed in alveolar macrophages isolated from human subjects with HIV. HIV-associated impairments in alveolar macrophage mitochondrial bioenergetics likely contribute to innate immune dysfunction in HIV infection, and GM-CSF treatment may offer a novel therapeutic strategy for mitigating these deleterious effects.

Ssu72 regulates alveolar macrophage development and allergic airway inflammation by fine-tuning of GM-CSF receptor signaling

Fine-tuning of immune receptor signaling is critical for the development and functioning of immune cells. Moreover, GM-CSF receptor (GM-CSFR) signaling plays an essential role in the development of certain myeloid lineage cells, including alveolar macrophages (AMs). However, the significance of fine-tuning of GM-CSFR signaling in AMs and its relevance in allergic inflammation have not been reported. Our aim was to explore whether phosphatase Ssu72, originally identified as a regulator of RNA polymerase II activity, regulates AM development and allergic airway inflammation by regulating GM-CSF signaling. To address these issues, we generated LysM-CreSsu72fl/fl and Cd11c-CreSsu72fl/fl mice and used ovalbumin- or house dust mite-induced allergic asthma models. Following GM-CSF stimulation, Ssu72 directly bound to the GM-CSFR β-chain in AMs, preventing phosphorylation. Consistently, mature Ssu72-deficient AMs showed higher phosphorylation of the GM-CSFR β-chain and downstream molecules, which resulted in greater dysregulation of cell cycle, cell death, cell turnover, mitochondria-related metabolism, and LPS responsiveness in AMs than in mature wild-type AMs. The dysregulation was restored by using a Janus kinase 2 inhibitor, which reduced GM-CSFR β-chain phosphorylation. LysM-CreSsu72fl/fl mice exhibited deficits in development and maturation of AMs, which were also seen postnatally in Cd11c-CreSsu72fl/fl mice. Furthermore, LysM-CreSsu72fl/fl mice were less responsive to ovalbumin- or house dust mite-induced allergic asthma models than the control mice were; however, their responsiveness was restored by adoptive transfer of JAK2 inhibitor-pretreated mature Ssu72-deficient AMs. Our results demonstrate that Ssu72 fine-tunes GM-CSFR signaling by both binding to and reducing phosphorylation of GM-CSFR β-chain, thereby regulating the development, maturation, and mitochondrial functions of AMs and allergic airway inflammation.

GM-CSF in the treatment of COVID-19: a new conductor in the pathogenesis of cytokine storm?

GM-CSF in the treatment of COVID-19: a new conductor in the pathogenesis of cytokine storm?

Pulmonary alveolar proteinosis in children

Pulmonary alveolar proteinosis (PAP) is an umbrella term for a wide spectrum of conditions that have a very characteristic appearance on computed tomography. There is outlining of the secondary pulmonary lobules on the background of ground-glass shadowing and pathologically, filling of the alveolar spaces with normal or abnormal surfactant. PAP is rare and the common causes in children are very different from those seen in adults; autoimmune PAP is rare and macrophage blockade not described in children. There are many genetic causes of PAP, the best known of which are mutations in the genes encoding surfactant protein (SP)-B, SP-C, thyroid transcription factor 1, ATP-binding cassette protein 3, and the granulocyte–macrophage colony-stimulating factor (GM-CSF) receptor α- and β- chains. PAP may also be a manifestation of rheumatological and metabolic disease, congenital immunodeficiency, and haematological malignancy. Precise diagnosis of the underlying cause is essential in planning treatment, as well as for genetic counselling. The evidence base for treatment is poor. Some forms of PAP respond well to whole-lung lavage, and autoimmune PAP, which is much commoner in adults, responds to inhaled or subcutaneous GM-CSF. Emerging therapies based on studies in murine models of PAP include stem-cell transplantation for GM-CSF receptor mutations.Educational aimsTo understand when to suspect that a child has pulmonary alveolar proteinosis (PAP) and how to confirm that this is the cause of the presentation.To show that PAP is an umbrella term for conditions characterised by alveolar filling by normal or abnormal surfactant, and that this term is the start, not the end, of the diagnostic journey.To review the developmental differences in the spectrum of conditions that may cause PAP, and specifically to understand the differences between causes in adults and children.To discuss when to treat PAP with whole-lung lavage and/or granulocyte–macrophage colony-stimulating factor, and review potential promising new therapies.

Pulmonary alveolar proteinosis: from classification to therapy.

Pulmonary alveolar proteinosis (PAP) is a rare respiratory syndrome characterised by the accumulation of surfactant lipoproteins within the alveoli. According to various pathogenetic mechanisms and aetiologies, PAP is classified as primary, secondary or congenital. Primary PAP is led by a granulocyte–macrophage colony-stimulating factor (GM-CSF) signalling disruption; the autoimmune form is driven by the presence of anti GM-CSF autoantibodies and represents 90% of all the PAP cases; and the hereditary form is the result of mutations in genes encoding GM-CSF receptor. Secondary PAP is associated with various diseases causing a reduction in function and/or number of alveolar macrophages. Congenital PAP emerges as a consequence of corrupted surfactant production, due to mutations in surfactant proteins or lipid transporter, or mutations affecting lung development. The clinical manifestations are various, ranging from insidious onset to acute or progressive respiratory failure, including premature death within the first days of life in neonates with congenital surfactant production disorders. The diagnostic workup includes clinical and radiological assessment (respiratory function test, high-resolution chest computed tomography), laboratory tests (anti-GM-CSF autoantibodies dosage, GM-CSF serum level and GM-CSF signalling test), and genetic tests. Whole-lung lavage is the current gold standard of care of PAP; however, the therapeutic approach depends on the pathogenic form and disease severity, including GM-CSF augmentation strategies in autoimmune PAP and other promising new treatments.Educational aimsTo update knowledge about a rare respiratory syndrome, pulmonary alveolar proteinosis, in order to promote early diagnosis and correct management.To highlight recent treatment options based on pathogenesis and disease severity.

NK cell-derived GM-CSF potentiates inflammatory arthritis and is negatively regulated by CIS.

Despite increasing recognition of the importance of GM-CSF in autoimmune disease, it remains unclear how GM-CSF is regulated at sites of tissue inflammation. Using GM-CSF fate reporter mice, we show that synovial NK cells produce GM-CSF in autoantibody-mediated inflammatory arthritis. Synovial NK cells promote a neutrophilic inflammatory cell infiltrate, and persistent arthritis, via GM-CSF production, as deletion of NK cells, or specific ablation of GM-CSF production in NK cells, abrogated disease. Synovial NK cell production of GM-CSF is IL-18-dependent. Furthermore, we show that cytokine-inducible SH2-containing protein (CIS) is crucial in limiting GM-CSF signaling not only during inflammatory arthritis but also in experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. Thus, a cellular cascade of synovial macrophages, NK cells, and neutrophils mediates persistent joint inflammation via production of IL-18 and GM-CSF. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor. These findings shed new light on GM-CSF biology in sterile tissue inflammation and identify several potential therapeutic targets.

Granulocyte-Macrophage Colony Stimulating Factor As an Indirect Mediator of Nociceptor Activation and Pain.

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.

Improved Anti-Tumor Response of Chimeric Antigen Receptor T Cell (CART) Therapy after GM-CSF Inhibition Is Mechanistically Supported By a Novel Direct Interaction of GM-CSF with Activated Carts

<h3>Introduction</h3> Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, limitations include neurotoxicity (NT) and cytokine release syndrome (CRS). Emerging literature suggests that inhibitory myeloid cells and their cytokines induce CAR-T cell toxicities and inhibit effector functions. GM-CSF was specifically identified as a critical cytokine in the development of NT and CRS. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS, enhance CART anti-tumor activity, and reduce the presence of tumor associated macrophages. In addition, there appears to be a direct effect on CART19 cells. <h3>Objectives</h3> In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. <h3>Methods</h3> We used a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector to knock out GM-CSF during CART cell manufacturing. Edited and wildtype T cells and CART cells were sent for whole exome sequencing (WES) and RNA sequencing. We also used flow cytometry to assess the expression of GM-CSF receptors on CART cells, activated using CD3/CD28 beads or irradiated NALM6 cells. <h3>Results</h3> WES analysis revealed no significant difference in the single nucleotide variants or indel counts between GM-CSF^k/o and GM-CSF^wt CART19 (Fig 1A). WES was significant for only two alterations in the gene targeted by the gRNA (Fig 1B). This high efficiency and accuracy indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggests a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. Further investigation robustly indicates that while resting CART cells do not express any GM-CSF receptors, activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells were activated through their T cell receptor (Fig 1C) or CAR (Fig 1D). This was also seen in GM-CSF^k/o CART19 cells, indicating this is specifically induced by T cell stimulation. These data suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Finally, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSF^k/o CART19 revealed a distinct signature, including significant inhibition of the Fas death pathway (Fig 1E). This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion. <h3>Conclusion</h3> Our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSF^k/o CART19 revealed a distinct transcriptome signature compared to GM-CSF^wt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells.

TLR4-activated p38 and NF-κB and GM-CSF receptor-mediated JAK2/STAT5 pathways are important for microglial long-term survival

We have previously reported that the activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) induced rapid death of primary cultured rat microglia. However, a subpopulation of microglia survived much longer than two days, in which time all control cells had died. These surviving microglia may have neuroprotective functions because the neurons remained viable in co-cultures with these microglia. Moreover, the LPS-stimulated microglia may produce GM-CSF to promote survival. However, signaling mechanism of TLR4-mediated microglial long-term survival remains unknown. Therefore, in this study, we investigated TLR4 signaling pathways that control microglial survival, focusing on p38 MAP kinase and NF-κB, which are known to be important for innate immune response and control of apoptosis. Furthermore, we examined the involvement of GM-CSF receptor downstream signaling intermediates, JAK2 and STAT5, which are known to regulate the transcription of survival genes. LPS stimulation resulted in the phosphorylation of p38 MAP kinase, NF-κB and STAT5 in primary rat microglia. Moreover, a p38 MAP kinase inhibitor, SB202190, and a NF-κB inhibitor, BAY11-7082, suppressed LPS-stimulated microglial survival. Inhibition of JAK2 by NVP-BSK805 also inhibited the survival of these microglia. These results suggest that p38 and/or NF-κB pathways may play important roles in TLR4-mediated microglial survival. Furthermore, microglia-producing GM-CSF may activate cytoprotective JAK2/STAT5 signals to support their survival.

MiR-532-3p-CSF2RA Axis as a Key Regulator of Vulnerable Atherosclerotic Plaque Formation

BackgroundThe most dangerous atherosclerotic plaques, referred to as “vulnerable,” are most likely to trigger acute atherothrombotic events such as myocardial infarction (heart attack) and stroke. Our goal was to uncover the molecular drivers of vulnerable plaque formation. MethodsTo elucidate the functional gene modules that drive vulnerable plaque formation, we performed a weighted gene coexpression network analysis integrated with a protein-protein interaction network analysis in human atherosclerotic carotid samples, which identified the candidate gene granulocyte-macrophage colony-stimulating factor 2 (GM-CSF) receptor alpha subunit (CSF2RA). Follow-up in vitro experiments were performed to elucidate the regulatory relationship between CSF2RA and the microRNA miR-532-3p as well as modifiers of macrophagic miR-532-3p-CSF2RA axis expression. Microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) studies elucidated the effect of statins on carotid miR-532-3p-CSF2RA axis expression in patients with carotid atherosclerotic disease. Apoe−/−, Ldlr−/−, and Csf2ra mutant Apoe−/− mouse models of atherosclerosis were employed to assess the effects of agomiR-532-3p therapy in vivo. ResultsThe integrated weighted gene coexpression network analysis/protein-protein interaction network analysis revealed that the macrophagic GM-CSF receptor CSF2RA is significantly upregulated in macrophage-rich vulnerable plaques. Follow-up analysis identified the miR-532-3p-CSF2RA axis, as miR-532-3p downregulates CSF2RA via binding to CSF2RA’s 3′UTR. Macrophagic miR-532-3p-CSF2RA dysregulation was enhanced via modified low-density lipoprotein or tumor necrosis factor α exposure in vitro. Moreover, this miR-532-3p-CSF2RA dysregulation was observed in human vulnerable plaques and Apoe−/− mouse plaques, effects rescued by statin therapy. In vivo, agomiR-532-3p therapy suppressed murine plaque formation and promoted plaque stabilization in a Csf2ra-dependent manner. ConclusionMacrophagic miR-532-3p-CSF2RA axis dysregulation is a key driver in vulnerable plaque formation.

JAK2ex13InDel drives oncogenic transformation and is associated with chronic eosinophilic leukemia and polycythemia vera

AbstractThe V617F mutation in the JH2 domain of Janus kinase 2 (JAK2) is an oncogenic driver in several myeloproliferative neoplasms (MPNs), including essential thrombocythemia, myelofibrosis, and polycythemia vera (PV). Other mutations in JAK2 have been identified in MPNs, most notably exon 12 mutations in PV. Here, we describe a novel recurrent mutation characterized by a common 4-amino-acid deletion and variable 1-amino-acid insertion (Leu583-Ala586DelInsSer/Gln/Pro) within the JH2 domain of JAK2. All 4 affected patients had eosinophilia, and both patients with Leu583-Ala586DelInsSer fulfilled diagnostic criteria of both PV and chronic eosinophilic leukemia (CEL). Computational and functional studies revealed that Leu583-Ala586DelInsSer (herein referred to as JAK2ex13InDel) deregulates JAK2 through a mechanism similar to JAK2V617F, activates signal transducer and activator of transcription 5 and extracellular signal-regulated kinase, and transforms parental Ba/F3 cells to growth factor independence. In contrast to JAK2V617F, JAK2ex13InDel does not require an exogenous homodimeric type 1 cytokine receptor to transform Ba/F3 cells and is capable of activating β common chain family cytokine receptor (interleukin-3 receptor [IL-3R], IL-5R, and granulocyte-macrophage colony stimulating factor receptor) signaling in the absence of ligand, with the maximum effect observed for IL-5R, consistent with the clinical phenotype of eosinophilia. Recognizing this new PV/CEL-overlap MPN has significant clinical implications, as both PV and CEL patients are at high risk for thrombosis, and concomitant cytoreduction of red cells, neutrophils, and eosinophils may be required for prevention of thromboembolic events. Targeted next-generation sequencing for genes recurrently mutated in myeloid malignancies in patients with unexplained eosinophilia may reveal additional cases of Leu583-Ala586DelInsSer/Gln/Pro, allowing for complete characterization of this unique MPN.

Could Increased Expression of Hsp27, an "Anti-Inflammatory" Chaperone, Contribute to the Monocyte-Derived Dendritic Cell Bias towards Tolerance Induction in Breast Cancer Patients?

Dendritic cells (DCs) are the most efficient antigen-presenting cells and link the innate immune sensing of the environment to the initiation of adaptive immune responses, which may be directed to either acceptance or elimination of the recognized antigen. In cancer patients, though DCs would be expected to present tumor antigens to T lymphocytes and induce tumor-eliminating responses, this is frequently not the case. The complex tumor microenvironment subverts the immune response, blocks some effector mechanisms, and drives others to support tumor growth. Chronic inflammation in a tumor microenvironment is believed to contribute to the induction of such regulatory/tolerogenic response. Among the various mediators of the modulatory switch in chronic inflammation is the “antidanger signal” chaperone, heat shock protein 27 (Hsp27), that has been described, interestingly, to be associated with cell migration and drug resistance of breast cancer cells. Thus, here, we investigated the expression of Hsp27 during the differentiation of monocyte-derived DCs (Mo-DCs) from healthy donors and breast cancer patients and evaluated their surface phenotype, cytokine secretion pattern, and lymphostimulatory activity. Surface phenotype and lymphocyte proliferation were evaluated by flow cytometry, interferon- (IFN-) γ, and interleukin- (IL-) 10 secretion, by ELISA and Hsp27 expression, by quantitative polymerase chain reaction (qPCR). Mo-DCs from cancer patients presented decreased expression of DC maturation markers, decreased ability to induce allogeneic lymphocyte proliferation, and increased IL-10 secretion. In coculture with breast cancer cell lines, healthy donors' Mo-DCs showed phenotype changes similar to those found in patients' cells. Interestingly, patients' monocytes expressed less GM-CSF and IL-4 receptors than healthy donors' monocytes and Hsp27 expression was significantly higher in patients' Mo-DCs (and in tumor samples). Both phenomena could contribute to the phenotypic bias of breast cancer patients' Mo-DCs and might prove potential targets for the development of new immunotherapeutic approaches for breast cancer.

Improved Anti-Tumor Response of Chimeric Antigen Receptor T Cell (CART) Therapy after GM-CSF Inhibition Is Mechanistically Supported By a Novel Direct Interaction of GM-CSF with Activated Carts

Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, the therapy is limited by the development of severe life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS). Additionally, durable efficacy following CART19 therapy is not optimal. Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CAR-T cell toxicities and also contribute to the inhibition of their effector functions. Specifically, GM-CSF was identified as a critical cytokine in the development of NT and CRS after CART19 therapy. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS and enhance CART anti-tumor activity through modulation of myeloid cell behavior, resulting in reduction of tumor associated macrophages. In addition to the predominant effect of GM-CSF on myeloid cells, there appears to be a direct effect on CART19 cells. In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Our initial finding of enhanced anti-tumor activity of CART19 cells after GM-CSF inhibition suggested a direct effect of GM-CSF on CART cells (Sterner et al. 2019, Blood). In these experiments, a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector was used to knock out GM-CSF during CART cell manufacturing. This resulted in a disruption efficiency of approximately 70% of the GM-CSF gene. Using a high tumor burden xenograft model for relapsed acute lymphoblastic leukemia established through the engraftment of the CD19+ luciferase+ NALM6 cell line (1x106 cells intravenously) in immunocompromised NOD-SCID-γ-/- mice, treatment with low doses of GM-CSFk/o CART19 resulted in improved anti-tumor activity and overall survival compared to GM-CSFwt CART19. The lack of myeloid cells in this model pointed to an intrinsic effect of GM-CSF on CAR-T cells. To ensure that this was not related to an off-target effect of the gRNA, whole exome sequencing (WES) of the modified cells was performed. There was no difference in the single nucleotide variants or indel counts between GM-CSFk/o CART19 and GM-CSFwt CART19 (Figure 1A). WES was significant for only two alterations in the exon 3 targeted by the gRNA (Figure 1B). The high efficiency and accuracy of targeting exon 3 of GM-CSF indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggested the possibility of a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. To investigate this interaction, we first assessed the expression of GM-CSF receptors on CART cells. While resting CART cells do not express any GM-CSF receptors, our analysis robustly indicates that activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells are activated through their T cell receptor with CD3/CD28 beads (Figure 1C) or through the CAR with irradiated NALM6 cells (Figure 1D). Additionally, activated GM-CSFk/o CART19 cells also upregulated GM-CSF receptors, indicating this upregulation is induced by T cell stimulation. These results suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Having demonstrated that 1) GM-CSF depletion enhances CART19 efficacy in xenograft models in the absence of monocytes and 2) T cell activation increases GM-CSF receptor expression, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature including a significant inhibition of the Fas death pathway, a known critical pathway in inducing CART cell apoptosis. This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion (Figure 1E). In summary, our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells. Disclosures Cox: Humanigen: Patents &amp; Royalties. Sterner:Humanigen: Patents &amp; Royalties. Sakemura:Humanigen: Patents &amp; Royalties. Ahmed:Humanigen: Employment. Chappell:Humanigen: Employment. Durrant:Humanigen: Employment. Parikh:Acerta Pharma: Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Kite/Gilead: Research Funding; Morphosys: Research Funding.

SCIDOT-26. THE ROLE OF TUMOR-DERIVED GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF/CSF2) IN REGULATION OF MICROGLIA-DEPENDENT INVASION IN GLIOMAS

Abstract Brain resident immune cells (microglia) and peripheral macrophages accumulate in malignant gliomas and constitute for 30–50% of the tumor mass. These immune cells are polarized by factors released by glioma and become the pro-invasive, immunosuppressive cells that support tumor progression. We have previously found that tumor-derived granulocyte macrophage colony stimulating factor (GM-CSF/Csf-2) is a crucial factor controlling accumulation of microglia and macrophages in murine gliomas. The analysis of TCGA dataset revealed overexpression of the CSF2 gene (encoding GM-CSF) in a set of mesenchymal glioblastomas (most aggressive WHO grade IV gliomas) and its association with high immune gene expression. To study the role of GM-CSF in microglia-stimulated glioma invasion, we used a co-culture system, which mimics microglia interactions with tumor cells. We silenced the expression of CSF2 in glioma cells and found reduced microglia-dependent invasion of glioma cells. To translate those results into clinically relevant setting, we designed and tested humanized short peptides interfering with binding of GM-CSF to its receptor. Selected peptide effectively inhibited binding of GM-CSF to its receptor as demonstrated with different methods. We selected the non-cytotoxic peptides that potently blocked microglia-dependent glioma invasion in cell co-cultures. Blocking GM-CSF-receptor signaling pathway with a neutralizing antibody against a GM-CSF receptor also inhibited microglia-dependent invasion of glioma cells. Altogether, our results demonstrate that glioma-derived GM-CSF supports pro-tumorigenic polarization of microglia turning them into cells that facilitate glioma growth and shape the immune microenvironment. The study was supported by grant 2014/15/B/NZ3/04704 from The National Science Centre, Poland.

TMIC-64. THE ROLE OF TUMOR-DERIVED GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF/CSF2) IN REGULATION OF MICROGLIA-DEPENDENT INVASION IN GLIOMAS

Abstract Brain resident immune cells (microglia) and peripheral macrophages accumulate in malignant gliomas and constitute for 30–50% of the tumor mass. These immune cells are polarized by factors released by glioma and become the pro-invasive, immunosuppressive cells that support tumor progression. We have previously found that tumor-derived granulocyte macrophage colony stimulating factor (GM-CSF/CSF2) is a crucial factor controlling accumulation of microglia and macrophages in murine gliomas. The analysis of TCGA dataset revealed overexpression of the CSF2 gene (encoding GM-CSF) in a set of mesenchymal glioblastomas and its association with high immune gene expression. To study the role of GM-CSF in microglia-stimulated glioma invasion, we used a co-culture system, which mimics microglia interactions with tumor cells. We silenced the expression of CSF2 in glioma cells and found reduced microglia-dependent invasion of glioma cells. To translate those results into clinically relevant setting, we designed and tested humanized short peptides interfering with binding of GM-CSF to its receptor. Selected peptide effectively inhibited binding of GM-CSF to its receptor as demonstrated with different methods. We selected the non-cytotoxic peptides that potently blocked microglia-dependent glioma invasion in cell co-cultures. Blocking GM-CSF-receptor signaling pathway with a neutralizing antibody against a GM-CSF receptor also inhibited microglia-dependent invasion of glioma cells. Altogether, our results demonstrate that glioma-derived GM-CSF supports pro-tumorigenic polarization of microglia turning them into cells that facilitate glioma growth and shape the immune microenvironment. This work was supported by statutory budget of Nencki Institute of Experimental Biology and grant PBS3/B7/19/2015 from The National Centre for Research and Development, Poland.

Siglec-F is induced by granulocyte-macrophage colony-stimulating factor and enhances interleukin-4-induced expression of arginase-1 in mouse macrophages.

Siglecs are cell surface lectins that recognize sialic acids and are primarily expressed in hematopoietic cells. Previous studies showed that some Siglecs regulate macrophage function. In the present study, we examined the induction and putative roles of mouse Siglec-F in bone-marrow-derived macrophages in mice. A quantitative RT-PCR analysis showed that the basal expression of Siglec-F was weak in bone-marrow-derived macrophages differentiated by macrophage colony-stimulating factor. However, a 24-hr stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced Siglec-F expression. GM-CSF also enhanced Siglec-F expression in thioglycollate-induced peritoneal macrophages. The inhibition of signal transducer and activator of transcription 5 (STAT5), but not that of phosphoinositide 3-kinase or mitogen-activated protein kinase kinase, significantly reduced the induction of Siglec-F. Interleukin-3, which uses a common β-chain shared with the GM-CSF receptor to stimulate the STAT5 pathway, also enhanced Siglec-F expression. The knockdown of Siglec-F by a specific small interfering RNA enhanced GM-CSF-induced STAT5 phosphorylation, suggesting that Siglec-F down-regulates its own expression upon prolonged GM-CSF stimulation. Furthermore, the knockdown of Siglec-F reduced the STAT6 phosphorylation and expression of arginase-1 in interleukin-4-stimulated macrophages. These results suggest that Siglec-F fine-tunes the immune responses of macrophages.

Signal Integration and Transcriptional Regulation of the Inflammatory Response Mediated by the GM-/M-CSF Signaling Axis in Human Monocytes

In recent years, the macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF) cytokines have been identified as opposing regulators of the inflammatory program. However, the two cytokines are simultaneously present in the inflammatory milieu, and it is not clear how cells integrate these signals. In order to understand the regulatory networks associated with the GM/M-CSF signaling axis, we analyzed DNA methylation in human monocytes. Our results indicate that GM-CSF induces activation of the inflammatory program and extensive DNA methylation changes, while M-CSF-polarized cells are in a less differentiated state. This inflammatory program is mediated via JAK2 associated with the GM-CSF receptor and the downstream extracellular signal-regulated (ERK) signaling. However, PI3K signaling is associated with a negative regulatory loop of the inflammatory program and M-CSF autocrine signaling in GM-CSF-polarized monocytes. Our findings describe the regulatory networks associated with the GM/M-CSF signaling axis and how they contribute to the establishment of the inflammatory program associated with monocyte activation.

Effective hematopoietic stem cell-based gene therapy in a murine model of hereditary pulmonary alveolar proteinosis

Hereditary pulmonary alveolar proteinosis due to GM-CSF receptor deficiency (herPAP) constitutes a life-threatening lung disease characterized by alveolar deposition of surfactant protein secondary to defective alveolar macrophage function. As current therapeutic options are primarily symptomatic, we have explored the potential of hematopoietic stem cell-based gene therapy. Using Csf2rb−/− mice, a model closely reflecting the human herPAP disease phenotype, we here demonstrate robust pulmonary engraftment of an alveolar macrophage population following intravenous transplantation of lentivirally corrected hematopoietic stem and progenitor cells. Engraftment was associated with marked improvement of critical herPAP disease parameters, including bronchoalveolar fluid protein, cholesterol and cytokine levels, pulmonary density on computed tomography scans, pulmonary deposition of Periodic Acid-Schiff+ material as well as respiratory mechanics. These effects were stable for at least nine months. With respect to engraftment and alveolar macrophage differentiation kinetics, we demonstrate the rapid development of CD11c+/SiglecF+ cells in the lungs from a CD11c–/SiglecF+ progenitor population within four weeks after transplantation. Based on these data, we suggest hematopoietic stem cell-based gene therapy as an effective and cause-directed treatment approach for herPAP.

Long-Term Safety and Efficacy of Gene-Pulmonary Macrophage Transplantation Therapy of PAP in Csf2ra-/- Mice.

Hereditary pulmonary alveolar proteinosis (PAP) is a genetic lung disease characterized by surfactant accumulation and respiratory failure arising from disruption of GM-CSF signaling. While mutations in either CSF2RA or CSF2RB (encoding GM-CSF receptor α or β chains, respectively) can cause PAP, α chain mutations are responsible in most patients. Pulmonary macrophage transplantation (PMT) is a promising new cell therapy in development; however, no studies have evaluated this approach for hereditary PAP (hPAP) caused by Csf2ra mutations. Here, we report on the preclinical safety, tolerability, and efficacy of lentiviral-vector (LV)-mediated Csf2ra expression in macrophages and PMT of gene-corrected macrophages (gene-PMT therapy) in Csf2ra gene-ablated (Csf2ra-/-) mice. Gene-PMT therapy resulted in a stable transgene integrationand correction of GM-CSF signaling and functions in Csf2ra-/- macrophages invitro and invivo and resulted in engraftment and long-term persistence of gene-corrected macrophages in alveoli; restoration of pulmonary surfactant homeostasis; correction of PAP-specific cytologic, histologic, and biomarker abnormalities; and reduced inflammation associated with disease progression in untreated mice. No adverse consequences of gene-PMT therapy in Csf2ra-/- mice were observed. Results demonstrate that gene-PMT therapy of hPAP in Csf2ra-/- mice was highly efficacious, durable, safe, and well tolerated.

TRAF Regulation of IL-17 Cytokine Signaling.

Tumor necrosis factor receptor (TNFR)-associated factors or (TRAFs) are important mediators of Interleukin-17 (IL-17) cytokine signaling and contribute to driving tissue responses that are crucial for protective immunity but are often implicated in immunopathology. By amplifying tissue immune activity, IL-17 cytokine pathways contribute to maintaining barrier function as well as activation of innate and adaptive immunity necessary for host defense. IL-17 receptors signaling is orchestrated in part, by the engagement of TRAFs and the subsequent unlocking of downstream cellular machinery that can promote pathogen clearance or contribute to immune dysregulation, chronic inflammation, and disease. Originally identified as signaling adaptors for TNFR superfamily, TRAF proteins can mediate the signaling of a variety of intercellular and extracellular stimuli and have been shown to regulate the downstream activity of many cytokine receptors including receptors for IL-1β, IL-2, IL-6, IL-17, IL-18, IL-33, type I IFNs, type III IFNs, GM-CSF, M-CSF, and TGF-β Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I- like receptors, and C-type lectin receptors. This review will focus on discussing studies that reveal our current understanding of how TRAFs mediate and regulate biochemical activities downstream of the IL-17 cytokines signaling.