Colony-stimulating Factor 1 Receptor Inhibitor Research Articles

Microglial and neuronal fates following inhibition of CSF-1R in synucleinopathy mouse model

Synucleinopathies are age-related neurological disorders characterized by the abnormal accumulation of α-synuclein (α-syn) in neuronal and non-neuronal cells. It has been proposed that microglial cells play an important role in synucleinopathy neuroinflammation, as well as homeostatically, such as in the clearance of α-syn aggregates in the brain. Here, we examined the effects of microglia on the pathogenesis of synucleinopathies by cell depletion in a mouse model of synucleinopathies. For this purpose, we treated non-transgenic (Non-tg) and α-synuclein transgenic (α-syn-tg) mice with pexidartinib (PLX3397), a tyrosine kinase inhibitor of colony-stimulating factor 1 receptor (CSF-1R). Neuropathological and immunoblot analysis confirmed that Iba-1 immunoreactive microglial cells were decreased by 95% following PLX3397 treatment in Non-tg and α-syn-tg mice. The level of total α-syn in the Triton X-insoluble fraction of brain homogenate was significantly decreased by microglial depletion in the α-syn-tg mice, while the level of Triton X-soluble human α-syn was not affected. Furthermore, the number of p-α-syn immunoreactive inclusions was reduced in α-syn-tg mice treated with PLX3397. Microglial depletion also ameliorated neuronal and synaptic degeneration in α-syn-tg mice, thereby resulted partially improving the motor behavioral deficit in α-syn-tg mice. Moreover, we demonstrated that microglia that survived post-PLX3397 treatment (PLX-resistant microglia) have lower expressions of CSF-1R, and microglial transcriptome analysis further elucidated that PLX-resistant microglia have unique morphology and transcriptomic signatures relative to vehicle-treated microglia of both genotypes; these include differences in definitive microglial functions such as their immune response, cell mobility, cell–cell communications, and regulation of neural homeostasis. Therefore, we suggest that microglia play a critical role in the pathogenesis of synucleinopathies, and that modulation of microglial status might be an effective therapeutic strategy for synucleinopathies.

Switch to phagocytic microglia by CSFR1 inhibition drives amyloid-beta clearance from glutamatergic terminals rescuing LTP in acute hippocampal slices

Microglia, traditionally regarded as innate immune cells in the brain, drive neuroinflammation and synaptic dysfunctions in the early phases of Alzheimer disease (AD), acting upstream to Aβ accumulation. Colony stimulating factor 1-receptor (CSF-1R) is predominantly expressed on microglia and its levels are significantly increased in neurodegenerative diseases, possibly contributing to the chronic inflammatory microglial response. On the other hand, CSF-1R inhibitors confer neuroprotection in preclinical models of neurodegenerative diseases. Here, we determined the effects of the CSF-1R inhibitor PLX3397 on the Aβ-mediated synaptic alterations in ex vivo hippocampal slices. Electrophysiological findings show that PLX3397 rescues LTP impairment and neurotransmission changes induced by Aβ. In addition, using confocal imaging experiments, we demonstrate that PLX3397 stimulates a microglial transition toward a phagocytic phenotype, which in turn promotes the clearance of Aβ from glutamatergic terminals. We believe that the selective pruning of Aβ-loaded synaptic terminals might contribute to the restoration of LTP and excitatory transmission alterations observed upon acute PLX3397 treatment. This result is in accordance with the mechanism proposed for CSF1R inhibitors, that is to eliminate responsive microglia and replace it with newly generated, homeostatic microglia, capable of promoting brain repair. Overall, our findings identify a connection between the rapid microglia adjustments and the early synaptic alterations observed in AD, possibly highlighting a novel disease-modifying target.

The role of microglia in neuronal and cognitive function during high altitude acclimatization

Due to their interactions with the neurovasculature, microglia are implicated in maladaptive responses to hypobaric hypoxia at high altitude (HA). To explore these interactions at HA, pharmacological depletion of microglia with the colony-stimulating factor-1 receptor inhibitor, PLX5622, was employed in male C57BL/6J mice maintained at HA or sea level (SL) for 3-weeks, followed by assessment of ex-vivo hippocampal long-term potentiation (LTP), fear memory recall and microglial dynamics/physiology. Our findings revealed that microglia depletion decreased LTP and reduced glucose levels by 25% at SL but did not affect fear memory recall. At HA, the absence of microglia did not significantly alter HA associated deficits in fear memory or HA mediated decreases in peripheral glucose levels. In regard to microglial dynamics in the cortex, HA enhanced microglial surveillance activity, ablation of microglia resulted in increased chemotactic responses and decreased microglia tip proliferation during ball formation. In contrast, vessel ablation increased cortical microglia tip path tortuosity. In the hippocampus, changes in microglial dynamics were only observed in response to vessel ablation following HA. As the hippocampus is critical for learning and memory, poor hippocampal microglial context-dependent adaptation may be responsible for some of the enduring neurological deficits associated with HA.

CSF1R inhibition by PLX5622 reduces pulmonary fungal infection by depleting MHCIIhi interstitial lung macrophages

PLX5622 is a small molecular inhibitor of the CSF1 receptor (CSF1R) and is widely used to deplete macrophages within the central nervous system (CNS). We investigated the impact of PLX5622 treatment in wild-type C57BL/6 mice and discovered that one-week treatment with PLX5622 was sufficient to deplete interstitial macrophages in the lung and brain-infiltrating Ly6Clow patrolling monocytes, in addition to CNS-resident macrophages. These cell types were previously indicated to act as infection reservoirs for the pathogenic fungus Cryptococcus neoformans. We found that PLX5622-treated mice had significantly reduced fungal lung infection and reduced extrapulmonary dissemination to the CNS but not to the spleen or liver. Fungal lung infection mapped to MHCIIhi interstitial lung macrophages, which underwent significant expansion during infection following monocyte replenishment and not local division. Although PLX5622 depleted CNS infiltrating patrolling monocytes, these cells did not accumulate in the fungal-infected CNS following pulmonary infection. In addition, Nr4a1-deficient mice, which lack patrolling monocytes, had similar control and dissemination of C. neoformans infection to wild-type controls. PLX5622 did not directly affect CD4 T-cell responses, or significantly affect production of antibody in the lung during infection. However, we found that mice lacking lymphocytes had reduced numbers of MHCIIhi interstitial macrophages in the lung, which correlated with reduced infection load. Accordingly, PLX5622 treatment did not alter fungal burdens in the lungs of lymphocyte-deficient mice. Our data demonstrate that PLX5622 may help reduce lung burden of pathogenic fungi that utilise CSF1R-dependent myeloid cells as infection reservoirs, an effect which is dependent on the presence of lymphocytes.

CSF1R inhibition depletes brain macrophages and reduces brain virus burden in SIV-infected macaques.

Perivascular macrophages (PVMs) and, to a lesser degree, microglia are targets and reservoirs of HIV and simian immunodeficiency virus (SIV) in the brain. Previously, we demonstrated that colony-stimulating factor 1 receptor (CSF1R) in PVMs was upregulated and activated in chronically SIV-infected rhesus macaques with encephalitis, correlating with SIV infection of PVMs. Herein, we investigated the role of CSF1R in the brain during acute SIV infection using BLZ945, a brain-penetrant CSF1R kinase inhibitor. Apart from three uninfected historic controls, nine Indian rhesus macaques were infected acutely with SIVmac251 and divided into three groups (n = 3 each): an untreated control and two groups treated for 20-30 days with low- (10 mg/kg/day) or high- (30 mg/kg/day) dose BLZ945. With the high-dose BLZ945 treatment, there was a significant reduction in cells expressing CD163 and CD206 across all four brain areas examined, compared with the low-dose treatment and control groups. In 9 of 11 tested regions, tissue viral DNA (vDNA) loads were reduced by 95%-99% following at least one of the two doses, and even to undetectable levels in some instances. Decreased numbers of CD163+ and CD206+ cells correlated significantly with lower levels of vDNA in all four corresponding brain areas. In contrast, BLZ945 treatment did not significantly affect the number of microglia. Our results indicate that doses as low as 10 mg/kg/day of BLZ945 are sufficient to reduce the tissue vDNA loads in the brain with no apparent adverse effect. This study provides evidence that infected PVMs are highly sensitive to CSF1R inhibition, opening new possibilities to achieve viral clearance.

Residual Microglia Following Short-term PLX5622 Treatment in 5xFAD Mice Exhibit Diminished NLRP3 Inflammasome and mTOR Signaling, and Enhanced Autophagy.

Chronic neuroinflammation represents a prominent hallmark of Alzheimer's disease (AD). While moderately activated microglia are pivotal in clearing amyloid beta (Aβ), hyperactivated microglia perpetuate neuroinflammation. Prior investigations have indicated that the elimination of ∼80% of microglia through a month-long inhibition of the colony-stimulating factor 1 receptor (CSF1R) during the advanced stage of neuroinflammation in 5xFamilial AD (5xFAD) mice mitigates synapse loss and neurodegeneration without impacting Aβ levels. Furthermore, prolonged CSF1R inhibition diminished the development of parenchymal plaques. Nonetheless, the immediate effects of short-term CSF1R inhibition during the early stages of neuroinflammation on residual microglial phenotype or metabolic fitness are unknown. Therefore, we investigated the effects of 10-day CSF1R inhibition in three-month-old female 5xFAD mice, a stage characterized by the onset of neuroinflammation and minimal Aβ plaques. We observed ∼65% microglia depletion in the hippocampus and cerebral cortex. The leftover microglia demonstrated a noninflammatory phenotype, with highly branched and ramified processes and reduced NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome complexes. Moreover, plaque-associated microglia were reduced in number with diminished Clec7a (dectin-1) expression. Additionally, both microglia and neurons displayed reduced mechanistic target of rapamycin (mTOR) signaling and autophagy. Biochemical assays validated the inhibition of NLRP3 inflammasome activation, decreased mTOR signaling, and enhanced autophagy. However, short-term CSF1R inhibition did not influence Aβ plaques, soluble Aβ-42 levels, or hippocampal neurogenesis. Thus, short-term CSF1R inhibition during the early stages of neuroinflammation in 5xFAD mice promotes the retention of homeostatic microglia with diminished inflammasome activation and mTOR signaling, alongside increased autophagy.

Evaluation of a Positron Emission Tomography Tracer Targeting Colony-Stimulating Factor 1 Receptor for Detecting Pulmonary Inflammation.

Colony-stimulating factor 1 receptor (CSF1R) is a type III receptor tyrosine kinase that is crucial for immune cell activation, survival, proliferation, and differentiation. Its expression significantly increases in macrophages during inflammation, playing a crucial role in regulating inflammation resolution and termination. Consequently, CSF1R has emerged as a critical target for both therapeutic intervention and imaging of inflammatory diseases. Herein, we have developed a radiotracer, 1-[4-((7-(dimethylamino)quinazolin-4-yl)oxy)phenyl]-3-(4-[18F]fluorophenyl)urea ([18F]17), for in vivo positron emission tomography (PET) imaging of CSF1R. Compound 17 exhibits a comparable inhibitory potency against CSF1R as the well-known CSF1R inhibitor PLX647. The radiosynthesis of [18F]17 was successfully performed by radiofluorination of aryltrimethyltin precursor with a yield of approximately 12% at the end of synthesis, maintaining a purity exceeding 98%. In vivo stability and biodistribution studies demonstrate that [18F]17 remains >90% intact at 30 min postinjection, with no defluorination observed even at 60 min postinjection. The PET/CT imaging study in lipopolysaccharide-induced pulmonary inflammation mice indicates that [18F]17 offers a more sensitive characterization of pulmonary inflammation compared to traditional [18F]FDG. Notably, [18F]17 shows a higher discrepancy in uptake ratio between mice with pulmonary inflammation and the sham group. Furthermore, the variations in [18F]17 uptake ratio observed on day 7 and day 14 correspond to lung density changes observed in CT imaging. Moreover, the expression levels of CSF1R on day 7 and day 14 follow a trend similar to the uptake pattern of [18F]17, indicating its potential for accurately characterizing CSF1R expression levels and effectively monitoring the pulmonary inflammation progression. These results strongly suggest that [18F]17 has promising prospects as a CSF1R PET tracer, providing diagnostic opportunities for pulmonary inflammatory diseases.

A novel in vitro model for investigating oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions: Impact of microglial depletion and repopulation

Experimental models of multiple sclerosis (MS) have significantly contributed to our understanding of pathophysiology and the development of therapeutic interventions. Various in vivo animal models have successfully replicated key features of MS and associated pathophysiological processes, shedding light on the sequence of events leading to disease initiation, progression, and resolution. Nevertheless, these models often entail substantial costs and prolonged treatment periods. In contrast, in vitro models offer distinct advantages, including cost-effectiveness and precise control over experimental conditions, thereby facilitating more reproducible results. We have developed a novel in vitro model tailored to the study of oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions, which encompasses all the cell types present in the central nervous system (CNS). Of note, our model enables the evaluation of microglial cell commitment through a protocol involving their depletion and subsequent repopulation. Given that the development and survival of microglia are critically reliant on colony-stimulating factor-1 receptor (CSF-1R) signaling, we have employed CSF-1R inhibition to effectively deplete microglia. This versatile model holds promise for the assessment of potential therapies aimed at promoting oligodendroglial differentiation to safeguard and repair myelin, hence mitigate neurodegenerative processes.

Single-cell transcriptomics predicts colony stimulating factor-1 regulation of age-dependent intestinal epithelial restitution

Intestinal diseases associated with ischemic injury, which damages the principal barrier against noxious luminal contents, result in unacceptably poor outcomes in newborns. Ischemia-induced loss of the intestinal epithelial barrier predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. There is an age-dependency of intestinal recovery in that neonates are the most susceptible to succumb to disease of the intestinal barrier versus older patients. While this age-dependence in repair has not been demonstrated in traditional rodent models, we have developed a highly translational pig model of intestinal ischemic injury and repair that does reflect this difference. We have shown that, while juvenile (weaned) pigs recover rapidly after ischemic intestinal injury, barrier repair is markedly underdeveloped in neonatal (nursing) pigs due to complete failure of epithelial restitution. Importantly, we found that restitution in neonates can be rescued by the direct application of homogenized mucosa from ischemia-injured small intestine from juvenile pigs. The mechanisms that allow for this restitution and rescue remain to be defined. We hypothesized that by identifying a subpopulation of restituting enterocytes by their expression of cell migration transcriptional pathways, we can then predict novel upstream regulators of age-dependent restitution response programs. Superficial mucosal epithelial cells collected from recovering ischemic jejunum of juvenile pigs were processed for single cell RNA sequencing, unbiased clustering and upstream regulator analysis. A porcine intestinal epithelial cell line (IPEC-J2) and banked tissues from prior rescue experiments were qualitatively and functionally assessed for activity of predicted upstream regulators. Single cell transcriptomics in recovering juvenile epithelium revealed a subcluster of absorptive enterocytes that express several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation by many potential molecules, including colony stimulating factor-1 (CSF-1) which is known to induce cell migration in non-intestinal epithelial tissues. To begin validating this prediction, we demonstrated that CSF-1 was enriched in the ischemic juvenile mucosa which rescues neonatal restitution and documented expression of the CSF-1 receptor (CSF1R) in both neonatal and juvenile epithelium, indicating that these cells are equipped to respond to CSF-1. CSF-1 and CSF1R co-localized in ischemic juvenile, but not neonatal, wound-adjacent epithelial cells and in the restituted epithelium of juveniles and rescued (but not control) neonates. Further, the CSF1R inhibitor BLZ945 reduced restitution in scratch wounded IPEC-J2 cells. Single cell transcriptomics have the power to inform potential novel therapeutic targets, such as CSF-1, to improve mucosal recovery in neonates with intestinal failure in this unique and powerful pig model. NIH K01 OD028207, NIH R01 HD095876, NIH U01 TR002953, NIH T32 OD011130, NIH P30 DK034987, USDA NIFA 1007263 and 07985. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alpha-synuclein inclusion responsive microglia are resistant to CSF1R inhibition

BackgroundParkinson’s disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously determined that microglia proximal to PFF-induced nigral α-syn inclusions increase in soma size, upregulate major-histocompatibility complex-II (MHC-II) expression, and increase expression of a suite of inflammation-associated transcripts. This microglial response is observed months prior to degeneration, suggesting that microglia reacting to α-syn inclusion may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether colony stimulating factor-1 receptor (CSF1R)-mediated microglial depletion impacts the magnitude of α-syn aggregation, nigrostriatal degeneration, or the response of microglial in the context of the α-syn PFF model.MethodsMale Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600 mg/kg), a CSF1R inhibitor, to deplete microglia for a period of either 2 or 6 months.ResultsCSF1R inhibition resulted in significant depletion (~ 43%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. However, CSF1R inhibition did not impact the increase in microglial number, soma size, number of MHC-II immunoreactive microglia or microglial expression of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, and Fcer1g associated with phosphorylated α-syn (pSyn) nigral inclusions. Further, accumulation of pSyn and degeneration of nigral neurons was not impacted by CSF1R inhibition. Paradoxically, long term CSF1R inhibition resulted in increased soma size of remaining Iba-1ir microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions.ConclusionsCollectively, our results suggest that CSF1R inhibition does not impact the microglial response to nigral pSyn inclusions and that CSF1R inhibition is not a viable disease-modifying strategy for PD.

An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

BackgroundInduced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R.MethodsSerial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL.ResultsThe newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls.ConclusionsWe optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.Graphical

Targeting the CSF-1/CSF-1R Axis: Exploring the Potential of CSF1R Inhibitors in Neurodegenerative Diseases.

We report herein the potential of colony-stimulating factor-1 receptor (CSF1R) inhibitors as therapeutic agents in neuroinflammatory diseases, with a focus on Alzheimer's disease (AD). Employing a carefully modified scaffold, N-(4-heterocycloalkyl-2-cycloalkylphenyl)-5-methylisoxazole-3-carboxamide, we identify highly selective and potent CSF1R inhibitors─7dri and 7dsi. Molecular docking studies shed light on the binding modes of these key compounds within the CSF1R binding site. Remarkably, kinome-wide selectivity assessment underscores the impressive specificity of 7dri for CSF-1R. Notably, 7dri emerges as a potent CSF-1R inhibitor with favorable cellular activity and minimal cytotoxicity among the synthesized compounds. Demonstrating efficacy in inhibiting CSF1R phosphorylation in microglial cells and successfully mitigating neuroinflammation in an in vivo LPS-induced model, 7dri establishes itself as a promising antineuroinflammatory agent.

Abstract 6521: Macrophage-targeting immunotherapy enhances chemotherapy response in primary and metastatic triple-negative breast cancer models

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks targeted treatment options. While immune checkpoint blockade leads to significant benefits in the treatment of TNBC when in combination with chemotherapy, recurrence, metastasis, and chemoresistance still remain major challenges. The purpose of this study was to assess the ability of a colony-stimulating factor 1 receptor inhibitor (CSF-1Ri) to shift the phenotype of the tumor microenvironment (TME) in murine models of TNBC, and the potential benefit of such in enhancing standard of care chemotherapy. We focused on targeting tumor-associated macrophages (TAMs) as they are the most abundant immune infiltrate in the TNBC TME and are associated with worsening overall survival (OS) and metastasis free survival (MFS). Targeting TAMs, specifically M2-like TAMs, has been shown to inhibit tumor progression and improve OS in the clinic. We selected pexidartinib (PLX) as the CSF-1Ri of choice, given its translational potential (FDA approved) and gemcitabine (GEM) as chemotherapy, given its ability to target myeloid derived suppressor cells and M2-like TAMs. For primary TNBC we employed 4T1 mammary carcinoma cells transfected with luciferase reporter gene in BALB/c mice as an immunocompetent model. Mice were injected with 100,000 cells in the 4th mammary fat pad, and tumors established over the course of fifteen days. Mice were then exposed to daily administrations of PLX (8 mg/kg), and weekly administrations of GEM (60 mg/kg). Tumor volumes were recorded every other day. The TME was analyzed with flow cytometry (FC), immunohistochemistry (IHC), and multiplex immunofluorescence (mIF). For the metastatic model, mice were inoculated with 100,000 4T1-luc cells in the flank. On day six, mice were exposed to treatments as outlined above. Mice underwent survival surgery to remove the primary tumor on day thirteen and were then treated with adjuvant therapy. Metastasis and OS were tracked. FC analysis indicated only combination therapy (11.8% ± 2.1) significantly decreased total TAMs compared to vehicle (22% ± 2) in the primary tumor. Combination therapy also showed the greatest depletion of M2-like TAMs (2.2% ± 0.6), with a significant decrease compared to both vehicle (15.6% ± 0.4) and GEM (6.6% ± 0.9). This TME modulation was confirmed using IHC and mIF. After four cycles of treatment, combination therapy (232.9 mm3 ± 130.8) had a significant effect on tumor burden compared to GEM alone (369.7 mm3 ± 165.8), indicating a correlation between tumor phenotype and therapeutic benefit. Furthermore, combination therapy led to the greatest prolongation of MFS and OS in the resection model, indicating that combination therapy has efficacy in the neoadjuvant and adjuvant setting. In conclusion, targeting TAMs in TNBC primes the tumor for combination with chemotherapy in TNBC. Citation Format: Matthew Emilio Fernandez, Yasir Alshehry, Laura Graham, Jennifer E. Koblinski, Harry D. Bear, Doug H. Sweet, Sandro R. da Rocha. Macrophage-targeting immunotherapy enhances chemotherapy response in primary and metastatic triple-negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6521.

Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization.

Alveolar bone regeneration has been strongly linked to macrophage polarization. M1 macrophages aggravate alveolar bone loss, whereas M2 macrophages reverse this process. Berberine (BBR), a natural alkaloid isolated and refined from Chinese medicinal plants, has shown therapeutic effects in treating metabolic disorders. In this study, we first discovered that culture supernatant (CS) collected from BBR-treated human bone marrow mesenchymal stem cells (HBMSCs) ameliorated periodontal alveolar bone loss. CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro. To clarify the underlying mechanism, the bioactive materials were applied to different animal models. We discovered macrophage colony-stimulating factor (M-CSF), which regulates macrophage polarization and promotes bone formation, a key macromolecule in the CS. Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats. Colony-stimulating factor 1 receptor (CSF1R) inhibitor or anti-human M-CSF (M-CSF neutralizing antibody, Nab) abolished the therapeutic effects of the CS of BBR-treated HBMSCs. Moreover, AKT phosphorylation in macrophages was activated by the CS, and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab. These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis. Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets. Overall, our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

CSF1R-mediated myeloid cell depletion shifts the ratio of motor cortical excitatory to inhibitory neurons in a multiple system atrophy model

Motor cortical circuit functions depend on the coordinated fine-tuning of two functionally diverse neuronal populations: glutamatergic pyramidal neurons providing synaptic excitation and GABAergic interneurons adjusting the response of pyramidal neurons through synaptic inhibition. Microglia are brain resident macrophages which dynamically refine cortical circuits by monitoring perineuronal extracellular matrix and remodelling synapses. Previously, we showed that colony-stimulating factor 1 receptor (CSF1R)-mediated myeloid cell depletion extended the lifespan, but impaired motor functions of MBP29 mice, a mouse model for multiple system atrophy. In order to better understand the mechanisms underlying these motor deficits we characterized the microglial involvement in the cortical balance of GABAergic interneurons and glutamatergic pyramidal neurons in 4-months-old MBP29 mice following CSF1R inhibition for 12 weeks. Lack of myeloid cells resulted in a decreased number of COUP TF1 interacting protein 2-positive (CTIP2+) layer V pyramidal neurons, however in a proportional increase of calretinin-positive GABAergic interneurons in MBP29 mice. While myeloid cell depletion did not alter the expression of important presynaptic and postsynaptic proteins, the loss of cortical perineuronal net area was attenuated by CSF1R inhibition in MBP29 mice. These cortical changes may restrict synaptic plasticity and potentially modify parvalbumin-positive perisomatic input. Collectively, this study suggests, that the lack of myeloid cells shifts the neuronal balance toward an increased inhibitory connectivity in the motor cortex of MBP29 mice thereby potentially deteriorating motor functions.

The CSF1-CSF1R pathway in the trigeminal ganglion mediates trigeminal neuralgia via inflammatory responses in mice.

Trigeminal neuralgia (TN) is the most severe type of neuropathic pain. The trigeminal ganglion (TG) is a crucial target for the pathogenesis and treatment of TN. The colony-stimulating factor 1 (CSF1) - colony-stimulating factor 1 receptor (CSF1R) pathway regulates lower limb pain development. However, the effect and mechanism of the CSF1-CSF1R pathway in TG on TN are unclear. Partial transection of the infraorbital nerve (pT-ION) model was used to generate a mouse TN model. Mechanical and cold allodynia were used to measure pain behaviors. Pro-inflammatory factors (IL-6, TNF-a) were used to measure inflammatory responses in TG. PLX3397, an inhibitor of CSF1R, was applied to inhibit the CSF1-CSF1R pathway in TG. This pathway was activated in naïve mice by stereotactic injection of CSF1 into the TG. The TN model activated the CSF1-CSF1R pathway in the TG, leading to exacerbated mechanical and cold allodynia. TN activated inflammatory responses in the TG manifested as a significant increase in IL-6 and TNF-a levels. After using PLX3397 to inhibit CSF1R, CSF1R expression in the TG declined significantly. Inhibiting the CSF1-CSF1R pathway in the TG downregulated the expression of IL-6 and TNF-α to reduce allodynia-related behaviors. Finally, mechanical allodynia behaviors were exacerbated in naïve mice after activating the CSF1-CSF1R pathway in the TG. The CSF1-CSF1R pathway in the TG modulates TN by regulating neuroimmune responses. Our findings provide a theoretical basis for the development of treatments for TN in the TG.

Pyrrolopyrimidine based CSF1R inhibitors: Attempted departure from Flatland

The colony-stimulating factor 1 receptor (CSF1R) is an attractive target for inflammation disorders and cancers. Based on a series of pyrrolo[2,3-d]pyrimidine containing two carbo-aromatic rings, we have searched for new CSF1R inhibitors having a higher fraction of sp3-atoms. The phenyl unit in the 4-amino group could efficiently be replaced by tetrahydropyran (THP) retaining inhibitor potency. Exchanging the 6-aryl group with cyclohex-2-ene units also resulted in highly potent compounds, while fully saturated ring systems at C-6 led to a loss of activity. The structure-activity relationship study evaluating THP containing pyrrolo[2,3-d]pyrimidine derivates identified several highly active inhibitors by enzymatic studies. A comparison of 11 pairs of THP and aromatic compounds showed that inhibitors containing THP had clear benefits in terms of enzymatic potency, solubility, and cell toxicity. Guided by cellular experiments in Ba/F3 cells, five CSF1R inhibitors were further profiled in ADME assays, indicating the para-aniline derivative 16t as the most attractive compound for further development.

CSF-1R inhibitor PLX3397 attenuates peripheral and brain chronic GVHD and improves functional outcomes in mice

Graft-versus-host disease (GVHD) is a serious complication of otherwise curative allogeneic haematopoietic stem cell transplants. Chronic GVHD induces pathological changes in peripheral organs as well as the brain and is a frequent cause of late morbidity and death after bone-marrow transplantation. In the periphery, bone-marrow-derived macrophages are key drivers of pathology, but recent evidence suggests that these cells also infiltrate into cGVHD-affected brains. Microglia are also persistently activated in the cGVHD-affected brain. To understand the involvement of these myeloid cell populations in the development and/or progression of cGVHD pathology, we here utilized the blood–brain-barrier permeable colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX3397 (pexidartinib) at varying doses to pharmacologically deplete both cell types. We demonstrate that PLX3397 treatment during the development of cGVHD (i.e., 30 days post-transplant) improves disease symptoms, reducing both the clinical scores and histopathology of multiple cGVHD target organs, including the sequestration of T cells in cGVHD-affected skin tissue. Cognitive impairments associated with cGVHD and neuroinflammation were also attenuated by PLX3397 treatment. PLX3397 treatment prior to the onset of cGVHD (i.e., immediately post-transplant) did not change in clinical scores or histopathology. Overall, our data demonstrate significant benefits of using PLX3397 for the treatment of cGVHD and associated organ pathologies in both the periphery and brain, highlighting the therapeutic potential of pexidartinib for this condition.

CSF1R inhibition promotes neuroinflammation and behavioral deficits during graft-versus-host disease in mice

AbstractChronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognized, in which brain-infiltrating donor major histocompatibility complex (MHC) class II+ bone marrow–derived macrophages (BMDM) drive pathology. BMDM are also mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of antibody blockade of colony-stimulating factor 1 receptor (CSF1R) to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. Increased blood-brain barrier permeability during acute GVHD (aGVHD) facilitated CNS antibody access and microglia depletion by anti-CSF1R treatment. However, CSF1R blockade early after transplant unexpectedly exacerbated aGVHD neuroinflammation. In established cGVHD, vascular changes and anti-CSF1R efficacy were more limited. Anti-CSF1R–treated mice retained donor BMDM, activated microglia, CD8+ and CD4+ T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients, in which CSF1R was conditionally depleted in donor CX3CR1+ BMDM. Notably, inhibition of CSF1R signaling after transplant failed to reverse GVHD-induced behavioral changes. Moreover, we observed aberrant behavior in non-GVHD control recipients administered anti-CSF1R blocking antibody and naïve mice lacking CSF1R in CX3CR1+ cells, revealing a novel role for homeostatic microglia and indicating that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast, transfer of Ifngr–/– grafts could reduce MHC class II+ BMDM infiltration, resulting in improved neurocognitive function. Our findings highlight unexpected neurological immune toxicity during CSF1R blockade and provide alternative targets for the treatment of cGVHD within the CNS.

Transient CSF1R Inhibition in the Early Stage of AD Retains Homeostatic Microglia with Reduced Inflammasomes and Increased autophagy in the 5XFAD Model

AbstractBackgroundChronic neuroinflammation is one of the most conspicuous features of Alzheimer’s disease (AD). While microglia play a vital role in phagocytosing amyloid beta (Aβ) and restraining plaque extension, disease‐associated microglia are known to perpetuate neuroinflammation. Previous studies have shown that ∼80% microglia elimination through a month‐long colony‐stimulating factor 1 receptor (CSF1R) inhibition in the advanced stage of AD in 5X Familial AD (5XFAD) mice does not reduce Aβ levels or plaques but restrains synapse loss and neurodegeneration. On the other hand, prolonged CSF1R inhibition has been shown to prevent parenchymal plaque development. However, the immediate effects of transient CSF1R inhibition in the early stage of AD on residual microglial morphology and their metabolic fitness are unknown.MethodWe investigated the impact of transient CSF1R inhibition in three‐month‐old 5XFAD mice, a stage at which neuroinflammation has commenced, but Aβ plaques were minimal. Microglia from the hippocampus and the somatosensory cortex (SSC) of 5XFAD mice receiving a diet containing either the CSF1R inhibitor PLX5622 or a standard diet for ten days were examined for morphology, markers of NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3) inflammasome complex, autophagy, and mechanistic target of rapamycin (mTOR) signaling.ResultTen days of CSF1R inhibition resulted in 67‐70% depletion of microglia in the hippocampus and SSC. The residual microglia displayed a noninflammatory phenotype, typified by highly branched and ramified processes, reduced incidence of NLRP3 inflammasome complexes, and increased autophagy. Interestingly, Aβ plaque load was reduced, and mTOR signaling was unaltered in microglia but reduced in neurons. However, transient CSF1R inhibition did not impact astrocyte hypertrophy or neurogenesis in the hippocampus.ConclusionThe results showed that transient CSF1R inhibition in the early stage of AD promotes the retention of homeostatic microglia with reduced inflammasomes and better metabolic fitness but with no changes in mTOR signaling, which can restrain the perpetuation of neuroinflammation and improve phagocytosis of Aβ by microglia.