To investigate the effects of G-CSF or GM-CSF therapy in non-neutropenic patients with sepsis.

Heat-killed (HK) Mycobacterium obuense (NCTC13365) is currently being evaluated in the clinic as an immunotherapeutic agent for cancer treatment. Yet, the molecular underpinnings underlying immunomodulatory properties of HK M. obuense are still largely undefined. To fill this void, we sought to perform immunophenotyping, chemokine/cytokine release analysis and genome-wide characterization of monocyte-derived macrophages (MDM) in which monocytes were originally isolated from healthy donors and differentiated by HK M. obuense (Mob-MDM) relative to macrophage colony-stimulating factor (M-MDM) and granulocyte/macrophage colony-stimulating factor (GM-MDM). Immunophenotyping and cytokine release analysis revealed downregulated surface expression of CD36, decreased spontaneous release of CCL2 and increased spontaneous secretion of CCL5, CXCL8/IL-8, IL-6, and TNF-α in Mob-MDM relative to M-MDM and GM-MDM. Analysis of cytostatic activity showed that Mob-MDM exhibited similar growth inhibitory effects on immortalized and malignant epithelial cells compared with GM-MDM but at an elevated rate relative to M-MDM. To understand global cues in Mob-MDM, we performed comparative RNA-sequencing (RNA-Seq) analysis of Mob-MDM relative to GM-MDM and M-MDM (n = 4 donors). Clustering analysis underscored expression profiles (n = 256) that were significantly modulated in Mob-MDM versus both M-MDM and GM-MDM including, among others, chemokines/cytokines and their receptors, enzymes and transcriptions factors. Topological functional analysis of these profiles identified pathways and gene sets linked to Mob-MDM phenotype including nitric oxide production, acute phase response signaling and microbe recognition pathways as well as signaling cues mediated by the proinflammatory cytokine, interferon-gamma, and the intracellular pattern recognition receptor, nucleotide-binding oligomerization domain-containing protein 2. Taken together, our study highlights molecular immune phenotypes and global signaling cues in Mob-MDM that may underlie immunomodulatory properties of HK M. obuense. Such properties could be of valuable use in immunotherapy approaches such as adoptive cell therapy against cancer.

Macrophage colony-stimulating factor receptor (M-CSFR) is found in cells of the mononuclear phagocyte lineage and is aberrantly expressed in a range of tumours, in addition to tumour-associated macrophages. Consequently, a variety of cancer therapies directed against M-CSFR are under development. We set out to engineer chimeric antigen receptors (CARs) that employ the natural ligands of this receptor, namely M-CSF or interleukin (IL)-34, to achieve specificity for M-CSFR-expressing target cells. Both M-CSF and IL-34 bind to overlapping regions of M-CSFR, although affinity of IL-34 is significantly greater than that of M-CSF. Matched second- and third-generation CARs targeted using M-CSF or IL-34 were expressed in human T-cells using the SFG retroviral vector. We found that both M-CSF- and IL-34-containing CARs enable T-cells to mediate selective destruction of tumour cells that express enforced or endogenous M-CSFR, accompanied by production of both IL-2 and interferon (IFN)-γ. Although they contain an additional co-stimulatory module, third-generation CARs did not outperform second-generation CARs. M-CSF-containing CARs mediated enhanced cytokine production and cytolytic activity compared to IL-34-containing CARs. These data demonstrate the feasibility of targeting M-CSFR using ligand-based CARs and raise the possibility that the low picomolar affinity of IL-34 for M-CSFR is detrimental to CAR function.

We investigated the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on behavioral and pathological outcomes in Alzheimer's disease (AD) and non-transgenic mice. GM-CSF treatment in AD mice reduced brain amyloidosis, increased plasma Aβ, and rescued cognitive impairment with increased hippocampal expression of calbindin and synaptophysin and increased levels of doublecortin-positive cells in the dentate gyrus. These data extend GM-CSF pleiotropic neuroprotection mechanisms in AD and include regulatory T cell-mediated immunomodulation of microglial function, Aβ clearance, maintenance of synaptic integrity, and induction of neurogenesis. Together these data support further development of GM-CSF as a neuroprotective agent for AD.

Resident and recruited macrophages control the development and proliferation of the liver. We have previously shown in multiple species that treatment with a macrophage colony stimulating factor (CSF1)-Fc fusion protein initiated hepatocyte proliferation and promoted repair in models of acute hepatic injury in mice. Here, we investigated the impact of CSF1-Fc on resolution of advanced fibrosis and liver regeneration, using a non-resolving toxin-induced model of chronic liver injury and fibrosis in C57BL/6J mice. Co-administration of CSF1-Fc with exposure to thioacetamide (TAA) exacerbated inflammation consistent with monocyte contributions to initiation of pathology. After removal of TAA, either acute or chronic CSF1-Fc treatment promoted liver growth, prevented progression and promoted resolution of fibrosis. Acute CSF1-Fc treatment was also anti-fibrotic and pro-regenerative in a model of partial hepatectomy in mice with established fibrosis. The beneficial impacts of CSF1-Fc treatment were associated with monocyte-macrophage recruitment and increased expression of remodelling enzymes and growth factors. These studies indicate that CSF1-dependent macrophages contribute to both initiation and resolution of fibrotic injury and that CSF1-Fc has therapeutic potential in human liver disease.

We have produced an Fc conjugate of colony-stimulating factor (CSF) 1 with an improved circulating half-life. CSF1-Fc retained its macrophage growth-promoting activity, and did not induce proinflammatory cytokines in vitro. Treatment with CSF1-Fc did not produce adverse effects in mice or pigs. The impact of CSF1-Fc was examined using the Csf1r-enhanced green fluorescent protein (EGFP) reporter gene in MacGreen mice. Administration of CSF1-Fc to mice drove extensive infiltration of all tissues by Csf1r-EGFP positive macrophages. The main consequence was hepatosplenomegaly, associated with proliferation of hepatocytes. Expression profiles of the liver indicated that infiltrating macrophages produced candidate mediators of hepatocyte proliferation including urokinase, tumor necrosis factor, and interleukin 6. CSF1-Fc also promoted osteoclastogenesis and produced pleiotropic effects on other organ systems, notably the testis, where CSF1-dependent macrophages have been implicated in homeostasis. However, it did not affect other putative CSF1 targets, notably intestine, where Paneth cell numbers and villus architecture were unchanged. CSF1 has therapeutic potential in regenerative medicine in multiple organs. We suggest that the CSF1-Fc conjugate retains this potential, and may permit daily delivery by injection rather than continuous infusion required for the core molecule.

To determine the presence and spatial distribution of different macrophage phenotypes, governed by granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) skewing signals, in giant cell arteritis (GCA) lesions.

Oncolytic virotherapy was approved as a localized treatment for advanced melanoma by the US Food and Drug Administration (FDA) in 2015. Granulocyte macrophage colony stimulating factor (GM-CSF) encoded by clinical virus-infected tumor cells, acting as a pro-inflammatory cytokine or growth factor, increases tumor antigen presentation, leading to the activation of macrophages and T cells. Notably, tumor-secreted lactate can promote the suppressive functions of M2-polarized tumor-associated macrophages and subsequently promote tumor growth. Furthermore, the consumption of tumor-secreted lactate has been implicated in the beneficial polarization of macrophages. Here, we report that GM-CSF-encoded recombinant adeno-associated virus (AAV2-GM-CSF) infection in B16-F10 mouse melanoma cells combined with lactate oxidase (LOX) leads to the recruitment of M1 macrophages for the inhibition of cancer cell growth. This study suggests that GM-CSF combined with LOX has potential as cancer virotherapy.

Treatment with growth factors could be beneficial in both inflammatory bowel disease and experimental colitis. The aim of this study was to investigate the effect of Colony Stimulating Factor (CSF), and Recombinant Human (rHu) Granulocyte Stimulating Factor (GSF) in experimental colitis in rats.

Multiple sclerosis is an inflammatory neurodegenerative disease of the central nervous system (CNS) and the most frequent cause of non-traumatic disability in adults in the Western world. Currently, several drugs have been approved for the treatment of multiple sclerosis. While the newer drugs are more effective, they have less favourable safety profiles. Thus, there is a need to identify new targets for effective and safe therapies, particularly in patients with progressive disease for whom no treatments are available. One such target is granulocyte-macrophage colony-stimulating factor (GM-CSF) or its receptor. In this article we review data on the potential role of GM-CSF and GM-CSF inhibition in MS. We discuss the expression and function of GM-CSF and its receptor in the CNS, as well as data from animal studies and clinical trials in MS.

Expression of Oct4 maintains cancer stem cell (CSC)-like properties in lung cancer cells and is correlated with poor prognosis of lung adenocarcinoma. M2-type tumor-associated macrophages (TAMs) promote cancer cell migration and metastasis. Tumor microenvironments promote monocyte differentiation into M2 TAMs via a complex cytokine-based connection. We explored the role of Oct4 in cytokine secretion in lung cancer and its impact on M2 TAM polarization.

Treatment of inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. IL-10 is a cytokine with therapeutic potential, but until now has not been as successful as previously anticipated. A reason for this may be that IL-10 responsiveness depends on the environment of the inflamed tissue. In this study we investigated whether GM-CSF is able to influence IL-10-mediated responses.

Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders.

Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) play key roles in the differentiation of macrophages and dendritic cells (DCs). We examined the effect of treatment with M-CSF-containing macrophage medium or GM-CSF-containing DC medium upon the phenotype of murine bone marrow-derived macrophages and DCs. Culture of macrophages for 5 days in DC medium reduced F4/80 expression and increased CD11c expression with cells effectively stimulating T cell proliferation in a mixed lymphocyte reaction. DC medium treatment of macrophages significantly reduced phagocytosis of both apoptotic cells and latex beads and strongly induced the expression of the chemokine receptor CCR7 known to be involved in DC trafficking to lymph nodes. Lysates of obstructed murine kidneys expressed both M-CSF and GM-CSF though M-CSF expression was dominant (M-CSF:GM-CSF ratio ∼30:1). However, combination treatment with both M-CSF and GM-CSF (ratio 30:1) indicated that small amounts of GM-CSF skewed macrophages towards a DC-like phenotype. To determine whether macrophage phenotype might be modulated in vivo we tracked CD45.1+ bone marrow-derived macrophages intravenously administered to CD45.2+ mice with unilateral ureteric obstruction. Flow cytometry of enzyme dissociated kidneys harvested 3 days later indicated CD11c and MHC Class II upregulation by adoptively transferred CD45.1+ cells with CD45.1+ cells evident in draining renal lymph nodes. Our data suggests that GM-CSF modulates mononuclear phagocyte plasticity, which likely promotes resolution of injury and healing in the injured kidney.

Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like zinc (Zn). The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF-activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription-factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H⁺ channel function and triggered reactive oxygen species generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.

We recently reported expression of hematopoietic growth factor GM-CSF and its receptor (GM-CSFR) in CNS neurons. Here we evaluated this system in learning and memory formation using GM-CSF deficient mice. In complementation, GM-CSF signalling was manipulated specifically in adult murine hippocampus by adeno-associated virus (AAV)-mediated GM-CSFR alpha overexpression or knock-down. GM-CSF ablation caused various hippocampus and amygdala-dependent deficits in spatial and fear memory while rendering intact basic parameters like motor function, inherent anxiety, and pain threshold levels. Corroborating these data, spatial memory of AAV-injected mice was positively correlated with GM-CSFRα expression levels. Hippocampal neurons of knock-out mice showed markedly pruned dendritic trees, reduced spine densities, and lower percentages of mature spines. Despite such morphological alterations, long-term potentiation (LTP) was unimpaired in the knock-out hippocampus. Collectively, these results suggest that GM-CSF signalling plays a major role in structural plasticity relevant to learning and memory.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease that disproportionately affects young adults, leading to disability and high costs to society. Infiltration of T cells and monocytes into the central nervous system (CNS) is critical for disease initiation and progression. However, despite a great deal of effort the molecular mechanisms by which immune cells initiate and perpetuate CNS damage in MS have not yet been elucidated. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by pathogenic Th1 and Th17 cells is critical for the recruitment of monocytes into the CNS during the initial stage of disease. We and others have recently shown that, compared with healthy individuals, MS patients have greater numbers of CD4+ and CD8+ T cells that produce GM-CSF. Here, we describe the expression of GM-CSF and its receptor, GM-CSFR, in normal brain and MS lesions. Our data show that in acute and chronic MS lesions, microglia and astrocytes have upregulated expression of GM-CSFR; in addition, we show that GM-CSF-associated molecules are also upregulated in MS lesions. These findings further strengthen the argument that GM-CSF signaling contributes to MS pathogenesis.

Signaling via the colony-stimulating factor 1 receptor (CSF1R) controls the survival, differentiation, and proliferation of macrophages. Mutations in CSF1 or CSF1R in mice and rats have pleiotropic effects on postnatal somatic growth. We tested the possible application of pig CSF1-Fc fusion protein as a therapy for low birth weight (LBW) at term, using a model based on maternal dexamethasone treatment in rats. Neonatal CSF1-Fc treatment did not alter somatic growth and did not increase the blood monocyte count. Instead, there was a substantial increase in the size of liver in both control and LBW rats, and the treatment greatly exacerbated lipid droplet accumulation seen in the dexamethasone LBW model. These effects were reversed upon cessation of treatment. Transcriptional profiling of the livers supported histochemical evidence of a large increase in macrophages with a resident Kupffer cell phenotype and revealed increased expression of many genes implicated in lipid droplet formation. There was no further increase in hepatocyte proliferation over the already high rates in neonatal liver. In conclusion, treatment of neonatal rats with CSF1-Fc caused an increase in liver size and hepatic lipid accumulation, due to Kupffer cell expansion and/or activation rather than hepatocyte proliferation. Increased liver macrophage numbers and expression of endocytic receptors could mitigate defective clearance functions in neonates. NEW & NOTEWORTHY This study is based on extensive studies in mice and pigs of the role of CSF1/CSF1R in macrophage development and postnatal growth. We extended the study to neonatal rats as a possible therapy for low birth weight. Unlike our previous studies in mice and pigs, there was no increase in hepatocyte proliferation and no increase in monocyte numbers. Instead, neonatal rats treated with CSF1 displayed reversible hepatic steatosis and Kupffer cell expansion.

Neurodegeneration after traumatic brain injury is facilitated by innate and adaptive immunity and can be harnessed to affect brain repair. In mice subjected to controlled cortical impact (CCI), we show that treatment with granulocyte macrophage colony stimulating factor (GM-CSF) affects regulatory T cell numbers in the cervical lymph nodes coincident with decreased lesion volumes and increased cortical tissue sparing. This paralleled increases in neurofilament and diminished reactive microglial staining. Transcriptomic analysis showed that GM-CSF induces robust immune neuroprotective responses seven days following CCI. Together, these results support the therapeutic potential of GM-CSF for TBI.

Levels of circulating cytokines are elevated in inflammatory diseases. Previously, it was shown that interleukin (IL-)17A, in synergism with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and tumor necrosis factor α (TNFα), induces the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) by murine osteoblasts in vitro. In this study, we further analyzed the effects of GM-CSF on osteoclast development in vitro. The effects of IL-17A, TNFα, and 1,25(OH)2D3 on the regulation of osteoclast development were investigated in cocultures of bone marrow-derived osteoclast progenitor cells (OPC) and mouse calvarial osteoblasts. Additionally, OPC were grown for 3days in media containing macrophage colony-stimulating factor (M-CSF), GM-CSF, or M-CSF/GM-CSF. Subsequently, the osteoclastogenic potential and the capacity to dissolve amorphous calcium phosphate were assessed in each of the three populations of OPC. IL-17A, in synergism with TNFα and 1,25(OH)2D3, inhibited the development of osteoclasts in cocultures by stimulating the osteoblast lineage cells to release GM-CSF. GM-CSF-treated OPC expressed traits characteristic of dendritic cells. Upon removal of GM-CSF and supplementation of the culture media with M-CSF/RANKL, the cells lost their dendritic cell characteristics and differentiated into osteoclasts. OPC pretreated with GM-CSF and M-CSF/GM-CSF exhibited delayed development to osteoclasts and an extended proliferation phase. Elevated levels of GM-CSF in systemic inflammatory diseases may cause an expansion of the OPC pools in the bone, bone marrow, and blood. Upon homing to the bone, this may lead to an increase in the number of osteoclasts and in bone resorption.