Sclerostin blockade inhibits bone resorption through PDGF receptor signaling in osteoblast lineage cells.

While sclerostin-neutralizing antibodies (Scl-Abs) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Abs control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anticatabolic effect of Scl-Abs could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effects on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin coactivates PDGFRs independently of Wnt/ß-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in preosteoblasts. In turn, Scl-Ab prevents sclerostin-mediated coactivation of PDGFR signaling and consequent M-CSF upregulation in preosteoblast cultures, thereby inhibiting osteoclast activity in preosteoblast/osteoclast coculture assays. These results provide a potential mechanism explaining the dissociation between anabolic and antiresorptive effects of long-term Scl-Ab.

Mechanical stretch preconditioned adipose-derived stem cells elicit polarization of anti-inflammatory M2-like macrophages and improve chronic wound healing.

Transplantation of adipose-derived stem cells (ASCs) is a promising option in the field of chronic wounds treatment. However, the effectiveness of ASCs therapies has been hampered by highly inflammatory environment in chronic wound areas. These problems could be partially circumvented using efficient approaches that boost the survival and anti-inflammatory capacity of transplanted ASCs. Here, by application of mechanical stretch (MS), we show that ASCs exhibits increased survival and immunoregulatory properties in vitro. MS triggers the secretion of macrophage colony stimulating factor (M-CSF) from ASCs, a chemokine that is linked to anti-inflammatory M2-like macrophages polarization. When the MS-ASCs were transplanted to chronic wounds, the wound area yields significantly faster closure rate and lower inflammatory mediators, largely due to macrophages polarization driven by transplanted MS-ASCs. Thus, our work shows that mechanical stretch can be harnessed to enhance ASCs transplantation efficiency in chronic wounds treatment.

A Robust In Vitro Culture Model and Generation of Memory Monocytes.

Innate monocytes can be trained or reprogrammed to adopt distinct memory states, such as low-grade inflammation and immune exhaustion, bearing fundamental relevance to the pathogenesis of both acute diseases such as sepsis as well as chronic diseases such as atherosclerosis. Therefore, it is critically important to develop a regimen for generating memory monocytes in vitro in order to better define key monocyte memory states with diverse potentials for proliferation, differentiation, and activation, as well as underlying mechanisms. Here, we describe an efficient in vitro system to propagate a large number of highly purified murine memory monocytes through sustaining bone marrow-derived monocytes with macrophage colony-stimulating factor (M-CSF, 10 ng/mL)-containing medium, together with other polarization agents such as lipopolysaccharide (LPS) for a 5-day period. This method can yield high-purity monocytes, capable of exhibiting dynamic memory behaviors upon training with various polarizing agents.

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

Optical controlled and nuclear targeted CECR2 competitor to downregulate CSF-1 for metastatic breast cancer immunotherapy.

The crosstalk between breast cancer cells and tumor associated macrophages (TAMs) greatly contributes to tumor progression and immunosuppression. In this work, cat eye syndrome chromosome region candidate 2 (CECR2) is identified to overexpress in breast cancer patients, which can recognize v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) and activate nuclear factor κB (NF-κB) to release colony stimulating factor-1 (CSF-1). Pharmacological inhibition of CECR2 by the bromodomain competitor (Bromosporine, Bro) can downregulate CSF-1 to inhibit M2 type TAMs. To amplify the immunotherapeutic effect, a chimeric peptide-based and optical controlled CECR2 competitor (designated as N-PB) is constructed to enhance the nuclear targeted delivery of Bro and initiate an immunogenic cell death (ICD). In vivo results indicate a favorable breast cancer targeting ability and primary tumor suppression effect of N-PB under optical irradiation. Importantly, N-PB downregulates CSF-1 by competitive inhibition of CECR2 and NF-κB(RelA) interactions, thus inhibiting immunosuppressive M2-like TAMs while improving the antitumorigenic M1-like phenotype. Ultimately, the systemic anti-tumor immunity is activated to suppress the metastatic breast cancer in an optical controlled manner. This study provides a promising therapeutic target and reliable strategy for metastatic breast cancer treatment by interrupting immunosuppressive crosstalk between tumor cells and macrophages.

Tumor microenvironment restricts IL-10 induced multipotent progenitors to myeloid-lymphatic phenotype.

Lymphangiogenesis is induced by local pro-lymphatic growth factors and bone marrow (BM)-derived myeloid-lymphatic endothelial cell progenitors (M-LECP). We previously showed that M-LECP play a significant role in lymphangiogenesis and lymph node metastasis in clinical breast cancer (BC) and experimental BC models. We also showed that differentiation of mouse and human M-LECP can be induced through sequential activation of colony stimulating factor-1 (CSF-1) and Toll-like receptor-4 (TLR4) pathways. This treatment activates the autocrine interleukin-10 (IL-10) pathway that, in turn, induces myeloid immunosuppressive M2 phenotype along with lymphatic-specific proteins. Because IL-10 is implicated in differentiation of numerous lineages, we sought to determine whether this pathway specifically promotes the lymphatic phenotype or multipotent progenitors that can give rise to M-LECP among other lineages. Analyses of BM cells activated either by CSF-1/TLR4 ligands in vitro or orthotopic breast tumors in vivo showed expansion of stem/progenitor population and coincident upregulation of markers for at least four lineages including M2-macrophage, lymphatic endothelial, erythroid, and T-cells. Induction of cell plasticity and multipotency was IL-10 dependent as indicated by significant reduction of stem cell markers and those for multiple lineages in differentiated cells treated with anti-IL-10 receptor (IL-10R) antibody or derived from IL-10R knockout mice. However, multipotent CD11b+/Lyve-1+/Ter-119+/CD3e+ progenitors detected in BM appeared to split into a predominant myeloid-lymphatic fraction and minor subsets expressing erythroid and T-cell markers upon establishing tumor residence. Each sub-population was detected at a distinct intratumoral site. This study provides direct evidence for differences in maturation status between the BM progenitors and those reaching tumor destination. The study results suggest preferential tumor bias towards expansion of myeloid-lymphatic cells while underscoring the role of IL-10 in early BM production of multipotent progenitors that give rise to both hematopoietic and endothelial lineages.

Dietary Salicornia ramosissima improves the European seabass (Dicentrarchus labrax) inflammatory response against Photobacterium damselae piscicida.

Introduction: Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources, competition for the limited cultivation space for 22 vegetable crops. In that sense, halophytes have the added value of being rich in valuable bioactive compounds and salt tolerant. This study assessed the inclusion of non-food fractions of S. ramosissima in European seabass diets. Methods: Different levels (2.5%, 5%, and 10%) were incorporated into seabass diets, replacing wheat meal (diets ST2.5, ST5, and ST10) or without inclusion (CTRL). Experimental diets were administered to seabass juveniles (8.62 ± 0.63 g) for 34 and 62 days and subsequent inflammatory responses to a heat-inactivated Photobacterium damselae subsp. piscicida (Phdp) were evaluated in a time-course manner (4, 24, 48, and 72 h after the challenge). At each sampling point, seabass haematological profile, plasma immune parameters, and head-kidney immune-related gene expression were evaluated. Results: After both feeding periods, most parameters remained unaltered by S. ramosissima inclusion; nonetheless, seabass fed ST10 showed an upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and cluster of differentiation 8 (cd8ß) compared with those fed CTRL after 62 days of feeding. Regarding the inflammatory response, seabass fed ST10 showed lower plasma lysozyme levels than their counterparts fed ST2.5 and ST5 at 24 h following injection, while 4 h after the inflammatory stimulus, seabass fed ST10 presented higher numbers of peritoneal leucocytes than fish fed CTRL. Moreover, at 4 h, fish fed ST2.5, ST5, and ST10 showed a higher expression of interleukin 1ß (il1ß), while fish fed ST5 showed higher levels of ornithine decarboxylase (odc) than those fed CTRL. An upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and glutathione peroxidase (gpx) was also observed at 72 h in fish fed ST10 or ST5 and ST10 compared with CTRL, respectively. Discussion: In conclusion, incorporating up to 10% of the non-food fraction S. ramosissima in feed did not compromise seabass growth or immune status after 62 days, aligning with circular economy principles. However, S. ramosissima inclusion improved the leucocyte response and upregulated key immune-related genes in seabass challenged with an inactivated pathogen.

SPI1 exacerbates iron accumulation and promotes osteoclast formation through inhibiting the expression of Hepcidin.

BACKGROUND: Osteoporosis (OP) can be caused by an overactive osteoclastic function. Anti-osteoporosis considerable therapeutic effects in tissue repair and regeneration because bone resorption is a unique osteoclast function. In this study, we mainly explored the underlying mechanisms of osteoclasts' effects on osteoporosis. METHODS: RAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl's iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: We found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice. CONCLUSION: Therefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.

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.

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.

The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

Single-Cell Analysis Reveals a Subset of High IL-12p40-Secreting Dendritic Cells within Mouse Bone Marrow-Derived Macrophages Differentiated with M-CSF.

Macrophages and dendritic cells (DCs), although ontogenetically distinct, have overlapping functions and exhibit substantial cell-to-cell heterogeneity that can complicate their identification and obscure innate immune function. In this study, we report that M-CSF-differentiated murine bone marrow-derived macrophages (BMDMs) exhibit extreme heterogeneity in the production of IL-12, a key proinflammatory cytokine linking innate and adaptive immunity. A microwell secretion assay revealed that a small fraction of BMDMs stimulated with LPS secrete most IL-12p40, and we confirmed that this is due to extremely high expression of Il12b, the gene encoding IL-12p40, in a subset of cells. Using an Il12b-YFP reporter mouse, we isolated cells with high LPS-induced Il12b expression and found that this subset was enriched for genes associated with the DC lineage. Single-cell RNA sequencing data confirmed a DC-like subset that differentiates within BMDM cultures that is transcriptionally distinct but could not be isolated by surface marker expression. Although not readily apparent in the resting state, upon LPS stimulation, this subset exhibited a typical DC-associated activation program that is distinct from LPS-induced stochastic BMDM cell-to-cell heterogeneity. Overall, our findings underscore the difficulty in distinguishing macrophages and DCs even in widely used in vitro murine BMDM cultures and could affect the interpretation of some studies that use BMDMs to explore acute inflammatory responses.

Differentiated mesenchymal stem cells-derived exosomes immobilized in decellularized sciatic nerve hydrogels for peripheral nerve repair.

Peripheral nerve injury (PNI) and the limitations of current treatments often result in incomplete sensory and motor function recovery, which significantly impact the patient's quality of life. While exosomes (Exo) derived from stem cells and Schwann cells have shown promise on promoting PNI repair following systemic administration or intraneural injection, achieving effective local and sustained Exo delivery holds promise to treat local PNI and remains challenging. In this study, we developed Exo-loaded decellularized porcine nerve hydrogels (DNH) for PNI repair. We successfully isolated Exo from differentiated human adipose-derived mesenchymal stem cells (hADMSC) with a Schwann cell-like phenotype (denoted as dExo). These dExo were further combined with polyethylenimine (PEI), and DNH to create polyplex hydrogels (dExo-loaded pDNH). At a PEI content of 0.1%, pDNH showed cytocompatibility for hADMSCs and supported neurite outgrowth of dorsal root ganglions. The sustained release of dExos from dExo-loaded pDNH persisted for at least 21 days both in vitro and in vivo. When applied around injured nerves in a mouse sciatic nerve crush injury model, the dExo-loaded pDNH group significantly improved sensory and motor function recovery and enhanced remyelination compared to dExo and pDNH only groups, highlighting the synergistic regenerative effects. Interestingly, we observed a negative correlation between the number of colony-stimulating factor-1 receptor (CSF-1R) positive cells and the extent of PNI regeneration at the 21-day post-surgery stage. Subsequent in vitro experiments demonstrated the potential involvement of the CSF-1/CSF-1R axis in Schwann cells and macrophage interaction, with dExo effectively downregulating CSF-1/CSF-1R signaling.

Granulocyte macrophage colony-stimulating factor-induced macrophages of individuals with autism spectrum disorder adversely affect neuronal dendrites through the secretion of pro-inflammatory cytokines.

BACKGROUND: A growing body of evidence suggests that immune dysfunction and inflammation in the peripheral tissues as well as the central nervous system are associated with the neurodevelopmental deficits observed in autism spectrum disorder (ASD). Elevated expression of pro-inflammatory cytokines in the plasma, serum, and peripheral blood mononuclear cells of ASD has been reported. These cytokine expression levels are associated with the severity of behavioral impairments and symptoms in ASD. In a prior study, our group reported that tumor necrosis factor-α (TNF-α) expression in granulocyte-macrophage colony-stimulating factor-induced macrophages (GM-CSF MΦ) and the TNF-α expression ratio in GM-CSF MΦ/M-CSF MΦ (macrophage colony-stimulating factor-induced macrophages) was markedly higher in individuals with ASD than in typically developed (TD) individuals. However, the mechanisms of how the macrophages and the highly expressed cytokines affect neurons remain to be addressed. METHODS: To elucidate the effect of macrophages on human neurons, we used a co-culture system of control human-induced pluripotent stem cell-derived neurons and differentiated macrophages obtained from the peripheral blood mononuclear cells of five TD individuals and five individuals with ASD. All participants were male and ethnically Japanese. RESULTS: Our results of co-culture experiments showed that GM-CSF MΦ affect the dendritic outgrowth of neurons through the secretion of pro-inflammatory cytokines, interleukin-1α and TNF-α. Macrophages derived from individuals with ASD exerted more severe effects than those derived from TD individuals. LIMITATIONS: The main limitations of our study were the small sample size with a gender bias toward males, the use of artificially polarized macrophages, and the inability to directly observe the interaction between neurons and macrophages from the same individuals. CONCLUSIONS: Our co-culture system revealed the non-cell autonomous adverse effects of GM-CSF MΦ in individuals with ASD on neurons, mediated by interleukin-1α and TNF-α. These results may support the immune dysfunction hypothesis of ASD, providing new insights into its pathology.

Parasite-induced IFN-γ regulates host defense via CD115 and mTOR-dependent mechanism of tissue-resident macrophage death.

Host resistance to a common protozoan parasite Toxoplasma gondii relies on a coordinated immune response involving multiple cell types, including macrophages. Embryonically seeded tissue-resident macrophages (TRMs) play a critical role in maintaining tissue homeostasis, but their role in parasite clearance is poorly understood. In this study, we uncovered a crucial aspect of host defense against T. gondii mediated by TRMs. Through the use of neutralizing antibodies and conditional IFN-γ receptor-deficient mice, we demonstrated that IFN-γ directly mediated the elimination of TRMs. Mechanistically, IFN-γ stimulation in vivo rendered macrophages unresponsive to macrophage colony-stimulating factor (M-CSF) and inactivated mTOR signaling by causing the shedding of CD115 (CSFR1), the receptor for M-CSF. Further experiments revealed the essential role of macrophage IFN-γ responsiveness in host resistance to T. gondii. The elimination of peritoneal TRMs emerged as an additional host defense mechanism aimed at limiting the parasite's reservoir. The identified mechanism, involving IFN-γ-induced suppression of CD115-dependent mTOR signaling in macrophages, provides insights into the adaptation of macrophage subsets during infection and highlights a crucial aspect of host defense against intracellular pathogens.

Keratinocyte integrin α3ß1 induces expression of the macrophage stimulating factor, CSF-1, through a YAP/TEAD-dependent mechanism.

The development of wound therapy targeting integrins is hampered by inadequate understanding of integrin function in cutaneous wound healing and the wound microenvironment. Following cutaneous injury, keratinocytes migrate to restore the skin barrier, and macrophages aid in debris clearance. Thus, both keratinocytes and macrophages are critical to the coordination of tissue repair. Keratinocyte integrins have been shown to participate in this coordinated effort by regulating secreted factors, some of which crosstalk to distinct cells in the wound microenvironment. Epidermal integrin α3ß1 is a receptor for laminin-332 in the cutaneous basement membrane. Here we show that wounds deficient in epidermal α3ß1 express less epidermal-derived macrophage colony-stimulating factor 1 (CSF-1), the primary macrophage-stimulating growth factor. α3ß1-deficient wounds also have fewer wound-proximal macrophages, suggesting that keratinocyte α3ß1 may stimulate wound macrophages through the regulation of CSF-1. Indeed, using a set of immortalized keratinocytes, we demonstrate that keratinocyte-derived CSF-1 supports macrophage growth, and that α3ß1 regulates Csf1 expression through Src-dependent stimulation of Yes-associated protein (YAP)-Transcriptional enhanced associate domain (TEAD)-mediated transcription. Consistently, α3ß1-deficient wounds in vivo display a substantially reduced number of keratinocytes with YAP-positive nuclei. Overall, our current findings identify a novel role for epidermal integrin α3ß1 in regulating the cutaneous wound microenvironment by mediating paracrine crosstalk from keratinocytes to wound macrophages, implicating α3ß1 as a potential target of wound therapy.

Engineered Microchannel Scaffolds with Instructive Niches Reinforce Endogenous Bone Regeneration by Regulating CSF-1/CSF-1R Pathway.

Structural and physiological cues provide guidance for the directional migration and spatial organization of endogenous cells. Here, a microchannel scaffold with instructive niches is developed using a circumferential freeze-casting technique with an alkaline salting-out strategy. Thereinto, polydopamine-coated nano-hydroxyapatite is employed as a functional inorganic linker to participate in the entanglement and crystallization of chitosan molecules. This scaffold orchestrates the advantage of an oriented porous structure for rapid cell infiltration and satisfactory immunomodulatory capacity to promote stem cell recruitment, retention, and subsequent osteogenic differentiation. Transcriptomic analysis as well as its in vitro and in vivo verification demonstrates that essential colony-stimulating factor-1 (CSF-1) factor is induced by this scaffold, and effectively bound to the target colony-stimulating factor-1 receptor (CSF-1R) on the macrophage surface to activate the M2 phenotype, achieving substantial endogenous bone regeneration. This strategy provides a simple and efficient approach for engineering inducible bone regenerative biomaterials.

Radical-Generating Activity, Phagocytosis, and Mechanical Properties of Four Phenotypes of Human Macrophages.

Macrophages are the major players and orchestrators of inflammatory response. Expressed proteins and secreted cytokines have been well studied for two polar macrophage phenotypes-pro-inflammatory M1 and anti-inflammatory regenerative M2, but little is known about how the polarization modulates macrophage functions. In this study, we used biochemical and biophysical methods to compare the functional activity and mechanical properties of activated human macrophages differentiated from monocyte with GM-CSF (M0_GM) and M-CSF (M0_M) and polarized into M1 and M2 phenotypes, respectively. Unlike GM-CSF, which generates dormant cells with low activity, M-CSF confers functional activity on macrophages. M0_M and M2 macrophages had very similar functional characteristics-high reactive oxygen species (ROS) production level, and higher phagocytosis and survival compared to M1, while M1 macrophages showed the highest radical-generating activity but the lowest phagocytosis and survival among all phenotypes. All phenotypes decreased their height upon activation, but only M1 and M2 cells increased in stiffness, which can indicate a decrease in the migration ability of these cells and changes in their interactions with other cells. Our results demonstrated that while mechanical properties differ between M0 and polarized cells, all four phenotypes of monocyte-derived macrophages differ in their functional activities, namely in cytokine secretion, ROS production, and phagocytosis. Within the broad continuum of human macrophages obtained in experimental models and existing in vivo, there is a diversity of phenotypes with varying combinations of both markers and functional activities.

Colony stimulating factor-1 (CSF-1) signalling is predictive of response to immune checkpoint inhibitors in advanced non-small cell lung cancer.

The identification of biomarkers related to treatment in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs) represents a significant challenge. The aim of this study was to determine the predictive value of macrophage-related markers assessed in plasma and tissue samples of patients with NSCLC undergoing ICI treatment. This bicentric study included a prospective cohort of 88 patients with advanced NSCLC who received first-line therapy with ICI (either as monotherapy or in combination with chemotherapy) or chemotherapy alone (CT). Samples were collected from the patients at baseline and during follow-up. Plasma levels of CSF-1 and IL-34 were measured using ELISA, while expression levels of the macrophage receptors CD163 and CSF-1-R were evaluated using immunohistochemistry on lung biopsies. At baseline, the median plasma CSF-1 expression was higher in patients who did not respond to immunotherapy compared to those who responded (8898 pg/mL vs. 14031 pg/mL, p = 0.0005). Importantly, high CSF-1 levels at the initial assessment were associated with disease progression regardless of the treatment received. Furthermore, high CSF-1 levels were associated with shorter progression-free survival (PFS) and overall survival (OS) in patients receiving ICI therapy, but not in those treated with chemotherapy. There was no correlation between IL-34, CSF-1R, CD163 and therapeutic response. We observed in vitro that the activation of lymphocytes mediated by pembrolizumab was hindered by the treatment of PBMC with recombinant CSF-1, suggesting that CSF-1 creates a systemic immunosuppressive state that interferes with ICI treatment. In conclusion, baseline CSF-1 levels represent a potential predictive marker to ICI treatment in NSCLC.

Efficacy and Safety of CSF-1 (0.4% Pilocarpine Hydrochloride) in Presbyopia: Pooled Results of the NEAR Phase 3 Randomized, Clinical Trials.

PURPOSE: This study was undertaken to evaluate the safety and efficacy of CSF-1 (0.4% pilocarpine hydrochloride ophthalmic solution) for use in individuals with presbyopia. METHODS: Two Phase 3 multicenter, randomized, double-masked, vehicle-controlled, parallel-group clinical trials were conducted in 35 private ophthalmology clinics in the United States from October 2020 to February 2022. Key inclusion criteria were the following: (1) age 45-64 years, (2) distance-corrected near visual acuity (DCNVA) at 40 cm ≥0.40 and ≤0.90 logarithm of the minimum angle of resolution (logMAR, approximately 20/50-20/160 Snellen) in at least 1 eye, (3) manifest refraction (MR) between -4.50 and +2.00 diopter (D) sphere in each eye with ≤2.00D difference between eyes, (4) <2.00D of cylinder MR in each eye, (5) ≤0.04 logMAR (20/20-2 or better) corrected distance visual acuity (CDVA) at 4 m in each eye. Key exclusion criteria were the following: (1) >0.14 logMAR (7 letters) improvement in post-vehicle treatment in monocular DCNVA in either eye at visit 1, (2) introcular pressure (IOP) <9 or >22 mm Hg, (3) average dark-adapted pupillometry <3.5 mm in either eye, (4) prior refractive surgery or intraocular lens (IOL) implantation. Participants applied CSF-1 or vehicle twice per day for 2 weeks. Efficacy and safety assessments were performed at several times on days 1, 8, and 15. Response was defined as ≥3-line gain in DCNVA without loss of ≥1-line in CDVA in the study eye under mesopic room lighting conditions. The primary efficacy endpoint was measured 1 hour post-dose 1 on day 8. Key secondary endpoints were 2 hours post-dose 1, and 1 and 2 hours post-dose 2, also on day 8. Safety endpoints were ocular and non-ocular treatment-related adverse events (TRAE), conjunctival redness, drop comfort, slit-lamp biomicroscopy, intraocular pressure, indirect fundoscopy, and CDVA at 4 m. FINDINGS: Six hundred thirteen participants were randomized to CSF-1 (n = 309) or vehicle (n = 304). Participants were predominantly White (80.8%) and female (62.0%), with mean age (standard deviation) of 54.7 (4.8). CSF-1 met the primary and key secondary endpoints. At the primary endpoint, 40.1% of the CSF-1 group achieved response versus 19.1% of the vehicle group (P < 0.0001). The percentage of responders was significantly greater in CSF-1 compared with vehicle at all tested times. Changes from baseline in all safety endpoints were comparable between groups. Most adverse events (AEs) were mild and transient. Neither serious nor severe AEs were reported with CSF-1. IMPLICATIONS: CSF-1, a low-dose pilocarpine ophthalmic solution, demonstrated superiority to vehicle in improving near vision in individuals with presbyopia without compromising distance vision. CSF-1 demonstrated a favorable safety profile. CLINICALTRIALS: gov identifier: NCT04599933 (NEAR-1), NCT04599972 (NEAR-2).