Sphingomyelin synthase 1 as a potential upstream amplifier of microglial CSF1R signaling in neuropathic pain

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised.

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1op/op mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r−/− and Csf1op/op mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r−/− colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r +/− male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r +/− female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.

Targeting CSF-1R represents an effective strategy in modulating inflammatory diseases

Regulation of Embryonic and Postnatal Development by the CSF-1 Receptor.

Microglial dynamics during brain development.

CSF1R signaling contributes to macrophage-associated CCL11 production and eosinophil recruitment in murine allergic conjunctivitis.

Molecular and cellular interactions among spinal dorsal horn neurons and microglia, the resident macrophages of the central nervous system, contribute to the induction and maintenance of neuropathic pain after peripheral nerve injury. Emerging evidence also demonstrates that reciprocal interactions between macrophages and nociceptive sensory neurons in the dorsal root ganglion contribute to the initiation and persistence of nerve injury-induced mechanical hypersensitivity (allodynia). We previously reported that sensory neuron-derived colony-stimulating factor 1 (CSF1), by engaging the CSF1 receptor (CSF1R) that is expressed by both microglia and macrophages, triggers the nerve injury-induced expansion of both resident microglia in the spinal cord and macrophages in the dorsal root ganglion and induces their respective contributions to the neuropathic pain phenotype. Here, we review recent research and discuss unanswered questions regarding CSF1/CSF1R-mediated microglial and macrophage signaling in the generation of neuropathic pain.

Purpose: Targeting tumor-associated macrophages with colony-stimulating factor 1 receptor (CSF-1R) inhibition reveals a strategy for cancer therapy. Here, we studied the impact of CSF1R germline genetic variant on CSF-1R signaling and the susceptibility to CSF-1R inhibitors.Experimental designs:CSF1R germline genetic variants were studied in 140 cancer patients. CSF-1R phosphorylation, endocytosis, and macrophage polarization were measured as the response to CSF-1 stimulation. Tumor-associated macrophages in surgical specimens and sensitivity to CSF-1R inhibitors were used to determine macrophage function.Results: A CSF1R c.1085A>G genetic variant causing the change of histidine to arginine in the domain of receptor dimerization was identified as a high allele frequency in Eastern Asian population. Cancer patients with this variant allele had less M2-like tumor-associated macrophages accompanied by low VEGF expression in tumor tissues. Importantly, CSF1R genetic variant was significantly associated with disease-free survival in colorectal, endometrial, and ovarian cancer. In terms of differentiation, macrophages with CSF1R c.1085A>G genetic variant displayed a refractory response to CSF-1 stimulation and macrophage survival was sensitive to CSF-1R inhibitors with IC50 of 0.1 to 1 nmol/L range. On contrast, CSF-1 induced a prominent phosphorylation and rapid endocytosis of CSF-1R, leading to an M2-like dominant polarization in macrophages with CSF1R c.1085 genotype A_A, in which CSF-1R inhibitors of PLX3397, BLZ945, and GW2580 inhibited macrophage survival with IC50 of 10 to 100 nmol/L range.Conclusions: The CSF1R c.1085A>G genetic variant regulates tumor immunity by altering the polarization and function of macrophages. This genetic variant confers the sensitivity to CSF-1R inhibitors, implying as a biomarker in targeting CSF-1R signaling for cancer treatment. Clin Cancer Res; 23(20); 6021-30. ©2017 AACR.

CSF-1 receptor signalling is governed by pre-requisite EHD1 mediated receptor display on the macrophage cell surface

Activation of the macrophage colony stimulating factor-1 receptor (CSF-1R) by CSF-1 stimulates pronounced macropinocytosis and drives proliferation of macrophages. Although the role of macropinocytosis in CSF-1R signaling remains unknown, we show here that, despite internalizing large quantities of plasma membrane, macropinosomes contribute little to the internalization of the CSF-1-CSF-1R complex. Rather, internalization of the CSF-1R in small endocytic vesicles that are sensitive to clathrin disruption, outcompetes macropinosomes for CSF-1R endocytosis. Following internalization, small vesicles carrying the CSF-1R underwent homotypic fusion and then trafficked to newly formed macropinosomes bearing Rab5. As these macropinosomes matured, acquiring Rab7, the CSF-1R was transported into their lumen and degraded. Inhibition of macropinocytosis delayed receptor degradation despite no disruption to CSF-1R endocytosis. These data indicate that CSF-1-stimulated macropinosomes are sites of multivesicular body formation and accelerate CSF-1R degradation. Furthermore, we demonstrate that macropinocytosis and cell growth have a matching dose dependence on CSF-1, suggesting that macropinosomes might be a central mechanism coupling CSF-1R signaling and macrophage growth.

We report that neurons in the central nervous system express colony stimulating factor-1 receptor (CSF-1R) mRNA and protein and that the expression has regional specificity. The presence of CSF-1R in neurons was demonstrated by the use of four different types of antibodies to CSF-1R and the presence of CSF-1R mRNA by in situ hybridization using oligonucleotide probe. In the steady state in most areas of the brain, CSF-1R is weakly expressed in only a few neurons. In the cerebellum, brainstem, and spinal cord, however, CSF-1R is expressed constitutively in greater numbers of neurons. After cerebral cortex ischemic injury, neurons in the area next to the ischemic lesion markedly upregulate CSF-1R. It is also upregulated in the contralateral cortex and in many other areas of the brain and spinal cord. We demonstrated that in cultures the ligand CSF-1 binds to its receptor (CSF-1R) in neurons and that reduction of the number of apoptotic neurons and potentiation of neuron survival is CSF-1 dose dependent. We propose that CSF-1/CSF-1R signaling is an important regulatory pathway between neurons, microglia, and astrocytes.

Colony Stimulating Factor-1 (CSF-1)/Colony Stimulating Factor-1 Receptor (CSF-1R) signaling axis plays an essential role in the development, maintenance, and proliferation of macrophage lineage cells. Within the central nervous system, CSF-1R signaling primarily maintains microglial homeostasis. Microglia, being the resident macrophage and first responder to any neurological insults, plays critical importance in overall health of the human brain. Aberrant and sustained activation of microglia along with continued proliferation and release of neurotoxic proinflammatory cytokines have been reported in various neurological and neurodegenerative diseases. Therefore, halting the neuroinflammatory pathway via targeting microglial proliferation, which depends on CSF-1R signaling, has emerged as a potential therapeutic target for neurological disorders. However, apart from regulating the microglial function, recently it has been discovered that CSF-1R has much broader role in central nervous system. These findings limit the therapeutic utility of CSF-1R inhibitors but also highlight the need for a complete understanding of CSF-1R function within the central nervous system. Moreover, it has been found that selective inhibitors of CSF-1R may be more efficient in avoiding non-specific targeting and associated side effects. Short-term depletion of microglial population in diseased conditions have also been found to be beneficial; however, the dose and therapeutic window for optimum effects may need to be standardized further.This review summarizes the present understanding of CSF-1R function within the central nervous system. We discuss the CSF-1R signaling in the context of microglia function, crosstalk between microglia and astroglia, and regulation of neuronal cell function. We also discuss a few of the neurological disorders with a focus on the utility of CSF-1R inhibitors as potential therapeutic strategy for halting the progression of neurological diseases.

Interaction between astrocytic colony stimulating factor and its receptor on microglia mediates central sensitization and behavioral hypersensitivity in chronic post ischemic pain model

KMT2A-rearranged infant leukaemia is one of the most severe malignancies in infants and children, and is characterised by a very aggressive phenotype and lineage plasticity. KMT2A::MLLT3 is among the most common translocations initiating leukaemia in infants, where it can manifest with a myeloid or lymphoid leukaemia phenotype. The cell-of-origin and the mechanisms driving lineage choice in KMT2A::MLLT3+ infant leukaemia are poorly understood. In this study, we show that a subset of foetal lymphoid-primed multipotent progenitors (LMPPs) expressing the Colony-Stimulating Factor 1 receptor (CSF1R) gives rise to acute myeloid leukaemia (AML) upon KMT2A::MLLT3 induction in a mouse model, with the myeloid phenotype, at least in part, being dependent on CSF1R signalling. In line with their leukaemia-propagating properties, KMT2A::MLLT3 + CSF1R+ LMPPs possess a stemcell-like and myeloid-biased expression signature and require autophagy to expand and form blast-like colonies in methylcellulose. Interrogation of public datasets confirms the existence of a human foetal-restricted CSF1R+ LMPP population at early stages of embryonic development. Finally, CSF1R inhibition on a KMT2A::MLLT3+ paediatric leukaemia cell line resulted in significant cell death, suggesting that CSF1R could be therapeutically targeted in these patients. Our findings suggest that KMT2A::MLLT3+ infant AML may originate from foetal liver CSF1R+ LMPPs, and that these patients may benefit from anti-CSF1R-CAR-T cell therapy.

Heterozygous missense CSF1R variants hamper in vitro CD34+-derived dendritic cell generation but not in vivo dendritic cell development