Project description:RNAseq analysis of 12 human macrophage samples was performed using ribosomal-depleted total RNA to analyze long non-coding RNA and coding transcript expression profiles comparing macrophage in vitro subtypes (M2, M1 and TAM) obtained from peripheral blood normal monocytes. Overall design: Dataset included RNA-seq data (Four Fastq files per sample as paired end sequencing was performed and two replicates per sample were included to increase the total number of reads to 100M) from ribosomal-depleted total RNA of macrophage in vitro subtypes obtained from peripheral blood normal monocytes to analyze their long non-coding RNA and coding transcript expression profiles. Sample metadata is referred to the in vitro treatments performed on to the different monocyte samples to obtain the macrophage subtypes of interest: M2 using M-CSF (N=3), M1 using GM-CSF (N=6), TAM using conditioned media from the follicular lymphoma cell line WSU-FSCCL (N=3).
Project description:Therapeutics against coronavirus disease 2019 (COVID-19) are urgently needed. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a myelopoietic growth factor and pro-inflammatory cytokine, plays a critical role in alveolar macrophage homeostasis, lung inflammation and immunological disease. Both administration and inhibition of GM-CSF are currently being therapeutically tested in COVID-19 clinical trials. This Perspective discusses the pleiotropic biology of GM-CSF and the scientific merits behind these contrasting approaches.
Project description:Granulocyte-macrophage colony-stimulating factor (GM-CSF) has many more functions than its original in vitro identification as an inducer of granulocyte and macrophage development from progenitor cells. Key features of GM-CSF biology need to be defined better, such as the responding and producing cell types, its links with other mediators, its prosurvival versus activation/differentiation functions, and when it is relevant in pathology. Significant preclinical data have emerged from GM-CSF deletion/depletion approaches indicating that GM-CSF is a potential target in many inflammatory/autoimmune conditions. Clinical trials targeting GM-CSF or its receptor have shown encouraging efficacy and safety profiles, particularly in rheumatoid arthritis. This review provides an update on the above topics and current issues/questions surrounding GM-CSF biology.
Project description:Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders.
Project description:Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies.
Project description:Pre-clinical models and clinical trials demonstrate that targeting the action of the cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF), can be efficacious in inflammation/autoimmunity reinforcing the importance of understanding how GM-CSF functions; a significant GM-CSF-responding cell in this context is likely to be the monocyte. This article summarizes critically the literature on the downstream cellular pathways regulating GM-CSF interaction with monocytes (and macrophages), highlighting some contentious issues, and conclusions surrounding this biology. It also suggests future directions which could be undertaken so as to more fully understand this aspect of GM-CSF biology. Given the focus of this collection of articles on monocytes, the following discussion in general will be limited to this population or to its more mature progeny, the macrophage, even though GM-CSF biology is broader than this.
Project description:The main cytokine induced by the interaction of oral epithelial cells with C. glabrata is granulocyte monocyte colony-stimulating factor (GM-CSF); however, the mechanisms regulating this response are unknown. Based on previously published information on the interactions of C. albicans with oral epithelial cells, we hypothesized that interaction with viable C. glabrata triggers GM-CSF synthesis via NF-kappaB activation. We found that C. glabrata-induced GM-CSF synthesis was adhesion-dependent, enhanced by endocytosis, and required fungal viability. NF-kappaB activation was noted during interaction of epithelial cells with C. glabrata, and pre-treatment with an NF-kappaB inhibitor partly inhibited GM-CSF synthesis. Blocking TLR4 with anti-TLR4 antibody did not inhibit GM-CSF production. In contrast, an anti-CDw17 antibody triggered significant inhibition of NF-kappaB activation and GM-CSF synthesis. beta-glucans did not stimulate GM-CSF synthesis, suggesting that the CDw17/NF-kappaB/GM-CSF pathway may be beta-glucan-independent. This study provides new insights into the mechanism of GM-CSF induction by C. glabrata.
Project description:Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, we obtained a homologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells--i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. We therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the alpha and beta subunits of the GM-CSF receptor, respectively.
Project description:Comparison of the DNA methylation profiles of CD14+ monocytes from human peripheral blood with derived dendritic cells (DCs) and macrophages (MACs) obtained by exposure with GM-CSF/IL-4 and GM-CSF, respectively, and with mature DCs and MACs after lipopolysaccharide (LPS) exposure The methylation profiles of bisulfite-modified DNA of human CD14+ monocytes were compared with derived immature dendritic cells (iDCs) and macrophages (iMACs) following GM-CSF/IL-4 and GM-CSF incubation, and then activation/maturation with lypopolysaccharyde (LPS) using the Infinium HumanMethylation450 BeadChips (Illumina, Inc., San Diego, CA,). This platform allows the interrogation of >485,000 methylation sites per sample at single-nucleotide resolution, and comprises an average of 17 CpG sites per gene in the 99% of RefSeq genes. 96% of CpG islands are covered, with additional coverage in CpG island shores and the regions flanking them. The samples were hybridized in the array following the manufacturerâs instructions. Total DNA isolated by standard procedures from CD14+ cells (total monocytes, MOs) corresponding to three sets of samples of monocytes (MOs), derived immature DCs and MACs (iDCS and iMACS) and activated/mature DCs and MACs following incubation with LPS (mDCS and mMACs)
Project description:Analysis of genes induced in DC precursors and in BM cells and monocytes treated with GM-CSF For progenitor arrays, bone marrow progenitors (CMP, GMP, CDP, and pre-cDC) were harvested from WT C57Bl/6 mice. For culture arrays, BM was cultured in the presence of GM-CSF or M-CSF and adherent and non-adherent cells sorted. For monocyte cultures, sorted BM monocytes were treated with GM-CSF for 0, 24 or 48 hours.