During postnatal development, microglia, CNS resident innate immune cells, are essential for synaptic pruning, neuronal apoptosis and remodeling. During this period microglia undergo morphological and phenotypic transformations; however, little is known about how microglial number and density is regulated during postnatal CNS development. We found that after an initial increase during the first 14 postnatal days, microglial numbers in mouse brain began declining in the third postnatal week and were reduced by 50% by 6 weeks of age; these “adult” levels were maintained until at least 9 months of age. Microglial CD11b levels increased, whereas CD45 and ER-MP58 declined between P10 and adulthood, consistent with a maturing microglial phenotype. Our data indicate that both increased microglial apoptosis and a decreased proliferative capacity contribute to the developmental reduction in microglial numbers. We found no correlation between developmental reductions in microglial numbers and brain mRNA levels of Cd200, Cx3Cl1, M-Csf or Il-34. We tested the ability of M-Csf-overexpression, a key growth factor promoting microglial proliferation and survival, to prevent microglial loss in the third postnatal week. Mice overexpressing M-Csf in astrocytes had higher numbers of microglia at all ages tested. However, the developmental decline in microglial numbers still occurred, suggesting that chronically elevated M-CSF is unable to overcome the developmental decrease in microglial numbers. Whereas the identity of the factor(s) regulating microglial number and density during development remains to be determined, it is likely that microglia respond to a “maturation” signal since the reduction in microglial numbers coincides with CNS maturation.

Macrophage colony stimulating factor (CSF1) is a cytokine that is upregulated in several diseases of the central nervous system (CNS). To examine the effects of CSF1 over-expression on microglia, transgenic mice that over-express CSF1 in the glial fibrillary acidic protein (GFAP) compartment were generated. CSF1 over-expressing mice have increased microglial proliferation and increased microglial numbers compared to controls. Treatment with PLX3397, a small molecule inhibitor of the CSF1 receptor CSF1R and related kinases, decreases microglial numbers by promoting microglial apoptosis in both CSF1 over-expressing and control mice. Microglia in CSF1 over-expressing mice exhibit gene expression profiles indicating that they are not basally M1 or M2 polarized, but they do have defects in inducing expression of certain genes in response to the inflammatory stimulus lipopolysaccharide (LPS). These results indicate that the CSF1 over-expression observed in CNS pathologies likely has pleiotropic influences on microglia. Furthermore, small molecule inhibition of CSF1R has the potential to reverse CSF1-driven microglial accumulation that is frequently observed in CNS pathologies, but can also promote apoptosis of normal microglia.

Current therapies for high-grade gliomas extend survival only modestly. The glioma microenvironment, including glioma-associated microglia/macrophages (GAMs), is a potential therapeutic target. The microglia/macrophage cytokine CSF1 and its receptor CSF1R are overexpressed in human high-grade gliomas. To determine if the other known CSF1R ligand IL-34 is expressed in gliomas, we examined expression array data of human high-grade gliomas and performed RT-PCR on glioblastoma sphere-forming cell lines (GSCs). Expression microarray analyses indicated that CSF1, but not IL-34, is frequently overexpressed in human tumors. We found that while GSCs did express CSF1, most GSC lines did not express detectable levels of IL-34 mRNA. We therefore studied the impact of modulating CSF1 levels on gliomagenesis in the context of the GFAP-V12Ha-ras-IRESLacZ (Ras*) model. Csf1 deficiency deterred glioma formation in the Ras* model while CSF1 transgenic overexpression decreased the survival of Ras* mice and promoted the formation of high-grade gliomas. Conversely, CSF1 overexpression increased GAM density, but did not impact GAM polarization state. Regardless of CSF1 expression status, most GAMs were negative for the M2 polarization markers ARG1 and CD206; when present, ARG1+ and CD206+ cells were found in regions of peripheral immune cell invasion. Therefore, our findings indicate that CSF1 signaling is oncogenic during gliomagenesis through a mechanism distinct from modulating GAM polarization status.

To evaluate timing and duration differences in airway protection and esophageal opening after oral intubation and mechanical ventilation for acute respiratory distress syndrome (ARDS) survivors versus age-matched healthy volunteers. Orally intubated adult (≥ 18 years old) patients receiving mechanical ventilation for ARDS were evaluated for swallowing impairments via a videofluoroscopic swallow study (VFSS) during usual care. Exclusion criteria were tracheostomy, neurological impairment, and head and neck cancer. Previously recruited healthy volunteers (n = 56) served as age-matched controls. All subjects were evaluated using 5-ml thin liquid barium boluses. VFSS recordings were reviewed frame-by-frame for the onsets of 9 pharyngeal and laryngeal events during swallowing. Eleven patients met inclusion criteria, with a median (interquartile range [IQR]) intubation duration of 14 (9, 16) days, and VFSSs completed a median of 5 (4, 13) days post-extubation. After arrival of the bolus in the pharynx, ARDS patients achieved maximum laryngeal closure a median (IQR) of 184 (158, 351) ms later than age-matched, healthy volunteers (p < 0.001) and it took longer to achieve laryngeal closure with a median (IQR) difference of 151 (103, 217) ms (p < 0.001), although there was no significant difference in duration of laryngeal closure. Pharyngoesophageal segment opening was a median (IQR) of - 116 (- 183, 1) ms (p = 0.004) shorter than in age-matched, healthy controls. Evaluation of swallowing physiology after oral endotracheal intubation in ARDS patients demonstrates slowed pharyngeal and laryngeal swallowing timing, suggesting swallow-related muscle weakness. These findings may highlight specific areas for further evaluation and potential therapeutic intervention to reduce post-extubation aspiration.

Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.

Background Colony-stimulating factor 1 (CSF1) expression in the central nervous system (CNS) increases in response to a variety of stimuli, and CSF1 is overexpressed in many CNS diseases. In young adult mice, we previously showed that CSF1 overexpression in the CNS caused the proliferation of IBA1+ microglia without promoting the expression of M2 polarization markers. Methods Immunohistochemical and molecular analyses were performed to further examine the impact of CSF1 overexpression on glia in both young and aged mice. Results As CSF1 overexpressing mice age, IBA1+ cell numbers are constrained by a decline in proliferation rate. Compared to controls, there were no differences in expression of the M2 markers ARG1 and MRC1 (CD206) in CSF1 overexpressing mice of any age, indicating that even prolonged exposure to increased CSF1 does not impact M2 polarization status in vivo. Moreover, RNA-sequencing confirmed the lack of increased expression of markers of M2 polarization in microglia exposed to CSF1 overexpression but did reveal changes in expression of other immune-related genes. Although treatment with inhibitors of the CSF1 receptor, CSF1R, has been shown to impact other glia, no increased expression of oligodendrocyte lineage or astrocyte markers was observed in CSF1 overexpressing mice. Conclusions Our study indicates that microglia are the primary glial lineage impacted by CSF1 overexpression in the CNS and that microglia ultimately adapt to the presence of the CSF1 mitogenic signal.

Current therapies for high-grade gliomas only modestly extend survival. Given that intrinsic characteristics of glioma cancer stem cells (gCSCs) make them hard to effectively target with current therapies, cells in the tumor microenvironment such as microglia/macrophages are being investigated as therapy targets. We previously identified a microglial/macrophage growth factor, Macrophage colony stimulating factor (Csf1), as a candidate glioma oncogene in a mouse somatic mutagenesis screen. CSF1 overexpression, CSF1 receptor (CSF1R) overexpression, and phosphorylated CSF1R have also been detected in human gliomas. We are using genetic and pharmacological strategies to investigate the role of CSF1 signaling in gliomagenesis in immunocompetent autochthonous mouse models to examine the therapeutic utility of targeting the CSF1R signaling axis. To determine the relevant CSF1R ligand(s) in human gliomas, we performed semi-quantitative RT-PCR on patient-derived gCSCs to examine expression of splice variants encoding membrane bound or secreted CSF1 as well as the additional CSF1R ligand IL34. We found that gCSCs primarily express the mRNA splice variant encoding secreted CSF1, but a splice variant encoding a membrane bound form was also detected. Most gCSCs lines did not express detectable levels of IL34 mRNA. We therefore developed a tissue-specific secreted CSF1 overexpressing transgenic model to study the effect of modulating CSF1 levels in the central nervous system. Since CSF1 is a growth factor for microglia, we previously studied the effects of increased CSF1 levels on microglial phenotypes and found that CSF1 overexpression increased microglial numbers and promoted microglial proliferation in vivo, but did not inherently polarize microglia toward a M2-like phenotype. Moreover, CSF1 signaling is required for survival of adult microglia in vivo, since inhibition of CSF1 signaling by the small molecule inhibitor PLX3397 induced microglial apoptosis. We now show that CSF1 overexpression accelerates gliomagenesis in a genetic model of spontaneous gliomagenesis driven by activated Ras. Both high- and low-grade gliomas form in mice with activated Ras, but CSF1 overexpression causes a dramatic increase in the percentage of tumors that are high-grade. Additionally, gliomas do not form in mice expressing activated Ras that are also CSF1-deficient. Therefore our data in this in vivo model indicate that CSF1 signaling plays a fundamental role in glioma development. Additional studies are underway to determine the impact of CSF1 on microglial and glioma phenotypes, and to explore how to best target CSF1 signaling for glioma therapy. Citation Format: Ishani De, Megan D. Steffen, Emily Sokn, Clayton Patros, Suzanne Litscher, Paul A. Clark, John S. Kuo, Fausto J. Rodriguez, Lara S. Collier. CSF1 signaling is a potential therapeutic target for glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR06.

Increased microglial numbers and activated microglial phenotypes are associated with many central nervous system (CNS) disorders including gliomas, neurodegeneration and brain injury. Thus, microglia are being tested as potential drug targets. Macrophage Colony Stimulating Factor (CSF1), an important cytokine for microglia and macrophages, is upregulated in these CNS disorders, but the effects of CSF1 on microglial phenotypes have not been well‐characterized in vivo. To study the effects of CSF1 over‐expression on microglia in the unperturbed CNS in vivo, we generated a transgenic mouse model in which CSF1 is inducibly over‐expressed in the GFAP compartment. We found that CSF1 over‐expression results in increased microglial proliferation, increased microglial numbers and an altered microglial phenotype compared to control mice. Microglia in CSF1 over‐expressing mice do not exhibit gene expression profiles typical of either M1 or M2 polarization, however they are defective in their response to the inflammatory stimulus LPS. Treatment with a small molecule inhibitor of the CSF1 receptor (CSF1R) and related kinases, decreased microglial numbers through apoptosis in both CSF1 over‐expressing and control mice. These results indicate that CSF1 signaling has multiple effects on microglia in vivo, and pharmacological inhibition of this signaling pathway may have efficacy in reversing the pathological levels of microglial accumulation in various CNS diseases by promoting microglial apoptosis. Moreover, our CSF1 over‐expressing genetic model should have utility for studying the impact of CSF1 over‐expression in mouse models of human disease. Grant Funding Source: Goldhirsh Foundation, the University of Wisconsin Graduate School and the NIH