The CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate neural progenitor cell maintenance and maturation.
ABSTRACT: The CSF-1 receptor (CSF-1R) regulates CNS microglial development. However, the localization and developmental roles of this receptor and its ligands, IL-34 and CSF-1, in the brain are poorly understood. Here we show that compared to wild type mice, CSF-1R-deficient (Csf1r-/-) mice have smaller brains of greater mass. They further exhibit an expansion of lateral ventricle size, an atrophy of the olfactory bulb and a failure of midline crossing of callosal axons. In brain, IL-34 exhibited a broader regional expression than CSF-1, mostly without overlap. Expression of IL-34, CSF-1 and the CSF-1R were maximal during early postnatal development. However, in contrast to the expression of its ligands, CSF-1R expression was very low in adult brain. Postnatal neocortical expression showed that CSF-1 was expressed in layer VI, whereas IL-34 was expressed in the meninges and layers II-V. The broader expression of IL-34 is consistent with its previously implicated role in microglial development. The differential expression of CSF-1R ligands, with respect to CSF-1R expression, could reflect their CSF-1R-independent signaling. Csf1r-/- mice displayed increased proliferation and apoptosis of neocortical progenitors and reduced differentiation of specific excitatory neuronal subtypes. Indeed, addition of CSF-1 or IL-34 to microglia-free, CSF-1R-expressing dorsal forebrain clonal cultures, suppressed progenitor self-renewal and enhanced neuronal differentiation. Consistent with a neural developmental role for the CSF-1R, ablation of the Csf1r gene in Nestin-positive neural progenitors led to a smaller brain size, an expanded neural progenitor pool and elevated cellular apoptosis in cortical forebrain. Thus our results also indicate novel roles for the CSF-1R in the regulation of corticogenesis.
Project description:Despite recent advances in diagnosis and treatment of lung cancers, the 5-year survival rate remains unsatisfactory, which necessitates the identification of novel factors that associates with disease progression and malignant degree for improving diagnostic and therapeutic strategies. Recent progress in cancer immunology research has unveiled critical roles for colony stimulating factor 1 receptor (CSF1R) in multiple aspects of the tumor microenvironment. CSF1R is expressed on tumor-associated macrophages (TAMs), and mediates important pro-tumorigenic functions. CSF1R also provides critical autocrine signals that promote cancer cell survival and proliferation. Activation of CSF1R can be achieved by two independent ligands; macrophage colony-stimulating factor (M-CSF) and interleukin 34 (IL-34). Accordingly, the expression of these ligands in cancer is expected to result in poor prognosis. In this study, we show that IL-34 and M-CSF expression correlates with poor survival in a cohort of lung cancer patients. Importantly, high co-expression of IL-34 and M-CSF associates with the poorest survival compared to cancers that show weak or absent expression of the two ligands. Furthermore, high expression of IL-34 and M-CSF associates with advanced stages of lung cancers. Together, these results indicate a correlation between IL-34/M-CSF expression with poor survival and disease progression in lung cancer patients.
Project description:Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.
Project description:Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.
Project description:CSF-1 is broadly expressed and regulates macrophage and osteoclast development. The action and expression of IL-34, a novel CSF-1R ligand, were investigated in the mouse. As expected, huIL-34 stimulated macrophage proliferation via the huCSF-1R, equivalently to huCSF-1, but was much less active at stimulating mouse macrophage proliferation than huCSF-1. Like muCSF-1, muIL-34 and a muIL-34 isoform lacking Q81 stimulated mouse macrophage proliferation, CSF-1R tyrosine phosphorylation, and signaling and synergized with other cytokines to generate macrophages and osteoclasts from cultured progenitors. However, they respectively possessed twofold and fivefold lower affinities for the CSF-1R and correspondingly, lower activities than muCSF-1. Furthermore, muIL-34, when transgenically expressed in a CSF-1-dependent manner in vivo, rescued the bone, osteoclast, tissue macrophage, and fertility defects of Csf1(op)/(op) mice, suggesting similar regulation of CSF-1R-expressing cells by IL-34 and CSF-1. Whole-mount IL34 in situ hybridization and CSF-1 reporter expression revealed that IL34 mRNA was strongly expressed in the embryonic brain at E11.5, prior to the expression of Csf1 mRNA. QRT-PCR revealed that compared with Csf1 mRNA, IL34 mRNA levels were lower in pregnant uterus and in cultured osteoblasts, higher in most regions of the brain and heart, and not compensatorily increased in Csf1(op/op) mouse tissues. Thus, the different spatiotemporal expression of IL-34 and CSF-1 allows for complementary activation of the CSF-1R in developing and adult tissues.
Project description:Cerebrovascular pathologies occur in up to 80% of cases of Alzheimer's disease; however, the underlying mechanisms that lead to perivascular pathology and accompanying blood-brain barrier (BBB) disruption are still not fully understood. We have identified previously unreported mutations in colony stimulating factor-1 receptor (CSF-1R) in an ultra-rare autosomal dominant condition termed adult-onset leucoencephalopathy with axonal spheroids and pigmented glia (ALSP). Cerebrovascular pathologies such as cerebral amyloid angiopathy (CAA) and perivascular p-Tau were some of the primary neuropathological features of this condition. We have identified two families with different dominant acting alleles with variants located in the kinase region of the CSF-1R gene, which confer a lack of kinase activity and signalling. The protein product of this gene acts as the receptor for 2 cognate ligands, namely colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34). Here, we show that depletion in CSF-1R signalling induces BBB disruption and decreases the phagocytic capacity of peripheral macrophages but not microglia. CSF-1R signalling appears to be critical for macrophage and microglial activation, and macrophage localisation to amyloid appears reduced following the induction of Csf-1r heterozygosity in macrophages. Finally, we show that endothelial/microglial crosstalk and concomitant attenuation of CSF-1R signalling causes re-modelling of BBB-associated tight junctions and suggest that regulating BBB integrity and systemic macrophage recruitment to the brain may be therapeutically relevant in ALSP and other Alzheimer's-like dementias.
Project description:Differential intestinal expression of the macrophage growth factors colony stimulating factor-1 (CSF-1), interleukin (IL)-34, and their shared CSF-1 receptor (CSF-1R) in inflammatory bowel disease (IBD) has been shown. Diverse expression between CSF-1 and IL-34, suggest that IL-34 may signal via an alternate receptor. Receptor-type protein-tyrosine phosphatase ? (PTPRZ1, RPTP-?), an additional IL-34 receptor, was recently identified. Here, we aimed to assess PTPRZ1 expression in IBD and non-IBD intestinal biopsies. Further, we aimed to investigate cellular PTPRZ1 and CSF-1R expression, and cytokine- and chemokine responses by IL-34 and CSF-1. The expression of PTPRZ1 was higher in non-IBD colon compared to ileum. PTPRZ1 expression was not altered with inflammation in IBD, however, correlated to IL34, CSF1, and CSF1R. The expression patterns of PTPRZ1 and CSF-1R differed in peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and intestinal epithelial cell line. PBMCs and monocytes of the same donors responded differently to IL-34 and CSF-1 with altered expression of tumor-necrosis factor ? (TNF-?), IL-1?, interferon ? (IFN-?), IL-13, IL-8, and monocyte chemotactic protein-1 (MCP-1) levels. This study shows that PTPRZ1 was expressed in bowel tissue. Furthermore, CSF-1R protein was detected in an intestinal epithelial cell line and donor dependently in primary PBMCs, monocytes, and macrophages, and first hints also suggest an expression in these cells for PTPRZ1, which may mediate IL-34 and CSF-1 actions.
Project description:Microglia are dependent on signaling through the colony stimulating factor-1 receptor (CSF-1R/CD115) for growth and survival. Activation of CSF-1R can lead to cell division, while blocking CSF-1R can lead to rapid microglia cell death. CSF-1R has two ligands, the growth factors colony stimulating factor-1 (CSF-1) and the more recently identified interleukin-34 (IL-34). Studies of IL-34 activation of rodent microglia and human macrophages have suggested it has different properties to CSF-1, resulting in an anti-inflammatory reparative phenotype. The goal of this study was to identify if the responses of human postmortem brain microglia to IL-34 differed from their responses to CSF-1 with the aim of identifying different phenotypes of microglia as a result of their responses. To approach this question, we also sought to identify differences between IL-34, CSF-1, and CSF-1R expression in human brain samples to establish whether there was an imbalance in Alzheimer's disease (AD). Using human brain samples [inferior temporal gyrus (ITG) and middle temporal gyrus (MTG)] from distinct cohorts of AD, control and high pathology, or mild cognitive impairment cases, we showed that there was increased expression of CSF-1R and CSF-1 mRNAs in both series of AD cases, and reduced expression of IL-34 mRNA in AD ITG samples. There was no change in expression of these genes in RNA from cerebellum of AD, Parkinson's disease (PD), or control cases. The results suggested an imbalance in CSF-1R signaling in AD. Using RNA sequencing to compare gene expression responses of CSF-1 and IL-34 stimulated human microglia, a profile of responses to CSF-1 and IL-34 was identified. Contrary to earlier work with rodent microglia, IL-34 induced primarily a classical activation response similar to that of CSF-1. It was not possible to identify any genes expressed significantly different by IL-34-stimulated microglia compared to CSF-1-stimulated microglia, but both cytokines did induce certain alternative activation-associated genes. These profiles also showed that a number of genes associated with lysosomal function and A? removal were downregulated by IL-34 and CSF-1 stimulation. Compared to earlier results our data indicate that CSF-1R stimulation by IL-34 or CSF-1 produced similar types of responses by elderly postmortem brain-derived microglia.
Project description:CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r+/- mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here, we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in humans. Our data provide insights into the mechanisms underlying ALSP. Because increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions.
Project description:CSF-1 and IL-34 share the CSF-1 receptor and no differences have been reported in the signaling pathways triggered by both ligands in human monocytes. IL-34 promotes the differentiation and survival of monocytes, macrophages and osteoclasts, as CSF-1 does. However, IL-34 binds other receptors, suggesting that differences exist in the effect of both cytokines. In the present study, we compared the differentiation and polarization abilities of human primary monocytes in response to CSF-1 or IL-34. CSF-1R engagement by one or the other ligands leads to AKT and caspase activation and autophagy induction through expression and activation of AMPK and ULK1. As no differences were detected on monocyte differentiation, we investigated the effect of CSF-1 and IL-34 on macrophage polarization into the M1 or M2 phenotype. We highlighted a striking increase in IL-10 and CCL17 secretion in M1 and M2 macrophages derived from IL-34 stimulated monocytes, respectively, compared to CSF-1 stimulated monocytes. Variations in the secretome induced by CSF-1 or IL-34 may account for their different ability to polarize naïve T cells into Th1 cells. In conclusion, our findings indicate that CSF-1 and IL-34 exhibit the same ability to induce human monocyte differentiation but may have a different ability to polarize macrophages.
Project description: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 Csf1(op/op) mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r(-/-) and Csf1(op/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.