Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids.

BACKGROUND: Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. METHODS: Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. RESULTS: We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. CONCLUSION: Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Stromal B Lymphocytes Affecting Prognosis in Triple-Negative Breast Cancer by Opal/TSA Multiplexed Immunofluorescence.

Objective: Immune cells play a key role in tumor microenvironment. The purpose of this study was to investigate the infiltration and clinical indication of immune cells including their combined prognostic value in microenvironment of triple negative breast cancer. Methods: We investigated 100 patients with triple negative breast cancer by Opal/Tyramide Signal Amplification multispectral immunofluorescence between 2003 and 2017 at Zhejiang Provincial people's Hospital. Intratumoral and stromal immune cells of triple negative breast cancer were classified and quantitatively analyzed. Survival outcomes were compared using the Kaplan-Meier method and further analyzed with multivariate analysis. Results: Infiltration level of stromal B lymphocytes, stromal and intratumoral CD8+ T cells, stromal CD4+ T cells, stromal PD-L1 and intratumoral tumor associated macrophages 2 cells were shown as independent factors affecting disease-free survival and overall survival in univariate analysis. Stromal B lymphocytes, T stage, N stage and pathological type were independent predictive factors for both DFS and OS in multivariate analysis. We firstly found that patients with B lymphocytes-enriched subtypes have a better prognosis than those with T lymphocytes-enriched subtypes and tumor-associated macrophage-enriched subtypes. Conclusion: The present study identified a bunch of immune targets and subtypes, which could be exploited in future combined immunotherapy/chemotherapy strategies for triple negative breast cancer patients.

Mechanical gated ion channel Piezo1: Function, and role in macrophage inflammatory response.

Macrophages are present in many mechanically active tissues and are often subjected to varying degrees of mechanical stimulation. Macrophages play a crucial role in resisting pathogen invasion and maintaining tissue homeostasis. Piezo-type mechanosensitive channel component 1 (Piezo1) is the main cation channel involved in the rapid response to mechanical stimuli in mammals. This channel plays a crucial role in controlling blood pressure and motor performance and regulates urinary osmotic pressure and epithelial cell proliferation and division. In recent years, numerous studies have shown that in macrophages, Piezo1 not only plays a role in regulating the aforementioned physiological processes but also participates in multiple pathological processes such as inflammation and cancer. In this review, we summarize the research progress on Piezo1-mediated regulation of macrophage-mediated inflammatory responses through downstream signalling pathways and the aerobic glycolysis pathway.

Neutrophil and macrophage crosstalk might be a potential target for liver regeneration.

The regenerative capability of the liver is remarkable, but further research is required to understand the role that neutrophils play in this process. In the present study, we reanalyzed single-cell RNA sequencing data from a mouse partial hepatectomy (PH) model to track the transcriptional changes in hepatocytes and non-parenchymal cells. Notably, we unraveled the regenerative capacity of hepatocytes at diverse temporal points after PH, unveiling the contributions of three distinct zones in the liver regeneration process. In addition, we observed that the depletion of neutrophils reduced the survival and liver volume after PH, confirming the important role of neutrophils in liver regeneration. CellChat analysis revealed an intricate crosstalk between neutrophils and macrophages promoting liver regeneration and, using weighted gene correlation network analysis, we identified the most significant genetic module associated with liver regeneration. Our study found that hepatocytes in the periportal zone of the liver are more active than in other zones, suggesting that the crosstalk between neutrophils and macrophages might be a potential target for liver regeneration treatment.

Corrigendum: Sulforaphane diminishes moonlighting of pyruvate kinase M2 and interleukin 1ß expression in M1 (LPS) macrophages.

[This corrects the article DOI: 10.3389/fimmu.2022.935692.].

Ultrasound Neuromodulation of an Anti-Inflammatory Pathway at the Spleen Improves Experimental Pulmonary Hypertension.

BACKGROUND: Inflammation is pathogenically implicated in pulmonary arterial hypertension; however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension. METHODS: Pulmonary hypertension was induced in rats either by Sugen 5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (sugen/hypoxia model), or by monocrotaline (60 mg/kg IP) injection (monocrotaline model). Animals were randomized to receive either 12-minute-long sessions of sFUS daily or sham stimulation for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry, and lung single-cell RNA sequencing were performed after treatment to assess the effects of sFUS. RESULTS: Splenic denervation right before induction of pulmonary hypertension results in a more severe disease phenotype. In both sugen/hypoxia and monocrotaline models, sFUS treatment reduces right ventricular systolic pressure by 25% to 30% compared with sham treatment, without affecting systemic pressure, and improves right ventricular function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and right ventricular fibrosis and hypertrophy and lowers BNP (brain natriuretic peptide). Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cell counts in the spleen and downregulates several inflammatory genes and pathways in nonclassical and classical monocytes and macrophages in the lung. Differentially expressed genes in those cell types are significantly enriched for human pulmonary arterial hypertension-associated genes. CONCLUSIONS: sFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory, and pathological manifestations in 2 models of experimental pulmonary hypertension. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.

Interactions between Aeromonas caviae and Yersinia enterocolitica isolated from a case of diarrhea: evaluation of antimicrobial susceptibility and immune response of infected macrophages.

Aeromonas species cause a wide spectrum of human diseases, primarily gastroenteritis, septicemia, and wound infections. Several studies have shown that about 40% of these cases involve mixed or polymicrobial infections between Aeromonas spp. and bacteria from other genera. However, the immune response of macrophages in front of the bacteria present in the mixed infections, as well as their impact on antimicrobial therapy, have not been investigated. This study evaluated the cell damage and immune response of the mouse macrophage BALB/c cell line (J774A.1) after performing a single and a mixed infection with a strain of Aeromonas caviae and Yersinia enterocolitica, both recovered from the same fecal sample from a patient with diarrhea. Macrophage cell damage was measured by the release of lactate dehydrogenase (LDH) while the immune response was evaluated studying the expression by RT-qPCR of six relevant immune-related genes. Additionally, the antimicrobial susceptibility pattern of the single and mixed strains in front of seventeen antibiotics was evaluated to determine the potential impact on the infection treatment. Macrophages infected with the mixture of the two strains showed a higher cell damage in comparison with the single infections and the immune-related genes, i.e., cytokines and chemokines genes (TNF-α, CCL20), and apoptotic and pyroptotic genes (TP53 and IL-1ß) were overexpressed. After infection with the mixed cultures, an increase in the antimicrobial resistance was observed for ciprofloxacin, trimethoprim, chloramphenicol, gentamicin and ertapenem. This study increased the knowledge about the synergetic effect of the bacteria involved in mixed infection and on their potential impact on the treatment and evolution of the infection.

Activating innate immune responses repolarizes hPSC-derived CAR macrophages to improve anti-tumor activity.

Generation of chimeric antigen receptor macrophages (CAR-Ms) from human pluripotent stem cells (hPSCs) offers new prospects for cancer immunotherapy but is currently challenged by low differentiation efficiency and limited function. Here, we develop a highly efficient monolayer-based system that can produce around 6,000 macrophages from a single hPSC within 3 weeks. Based on CAR structure screening, we generate hPSC-CAR-Ms with stable CAR expression and potent tumoricidal activity in vitro. To overcome the loss of tumoricidal activity of hPSC-CAR-Ms in vivo, we use interferon-γ and monophosphoryl lipid A to activate an innate immune response that repolarizes the hPSC-CAR-Ms to tumoricidal macrophages. Moreover, through combined activation of T cells by hPSC-CAR-Ms, we demonstrate that activating a collaborative innate-adaptive immune response can further enhance the anti-tumor effect of hPSC-CAR-Ms in vivo. Collectively, our study provides feasible methodologies that significantly improve the production and function of hPSC-CAR-Ms to support their translation into clinical applications.

Novel IL-4/HB-EGF-dependent crosstalk between eosinophils and macrophages controls liver regeneration after ischaemia and reperfusion injury.

OBJECTIVE: Previous studies indicate that eosinophils are recruited into the allograft following orthotopic liver transplantation and protect from ischaemia reperfusion (IR) injury. In the current studies, we aim to explore whether their protective function could outlast during liver repair. DESIGN: Eosinophil-deficient mice and adoptive transfer of bone marrow-derived eosinophils (bmEos) were employed to investigate the effects of eosinophils on tissue repair and regeneration after hepatic IR injury. Aside from exogenous cytokine or neutralising antibody treatments, mechanistic studies made use of a panel of mouse models of eosinophil-specific IL-4/IL-13-deletion, cell-specific IL-4rα-deletion in liver macrophages and hepatocytes and macrophage-specific deletion of heparin-binding epidermal growth factor-like growth factor (hb-egf). RESULT: We observed that eosinophils persisted over a week following hepatic IR injury. Their peak accumulation coincided with that of hepatocyte proliferation. Functional studies showed that eosinophil deficiency was associated with a dramatic delay in liver repair, which was normalised by the adoptive transfer of bmEos. Mechanistic studies demonstrated that eosinophil-derived IL-4, but not IL-13, was critically involved in the reparative function of these cells. The data further revealed a selective role of macrophage-dependent IL-4 signalling in liver regeneration. Eosinophil-derived IL-4 stimulated macrophages to produce HB-EGF. Moreover, macrophage-specific hb-egf deletion impaired hepatocyte regeneration after IR injury. CONCLUSION: Together, these studies uncovered an indispensable role of eosinophils in liver repair after acute injury and identified a novel crosstalk between eosinophils and macrophages through the IL-4/HB-EGF axis.

Tissue-resident C1q + macrophages exert anti-aging potential through the Sirt1 pathway.

OBJECTIVE: Resident immune cells are at the forefront of sensory organ-specific signals, and changes in these cells are closely related to the aging process. The Sirt pathway can regulate NAD + metabolism during aging, thereby affecting the accumulation of ROS. However, the role of the Sirt pathway in resident immune cells in aged tissues is currently unclear. METHODS: We investigated Sirt1 signalling in resident immune cells during chronic inflammation in an aged mouse model. Integrated single-cell RNA sequencing data from young and aged mice were used to refine the characterization of immune cells in aged tissues RESULTS: We found that C1q + macrophages could affect chronic inflammation during aging. C1q + macrophages acted in an opposing manner to Il1b + macrophages and were responsible for anti-inflammatory effects during aging. Sirt1 agonists inhibited the decrease in C1qb in macrophages during aging, and anti-aging drugs could affect the expression of C1qb in macrophages via the Sirt1 pathway. CONCLUSIONS: In this study, we first identified the relevance of C1q + macrophages in chronic inflammation during aging. The potential anti-aging effect of C1q + macrophages was mediated by the Sirt1 pathway, suggesting new strategies for aging immunotherapy.

Macrophage ATG16L1 expression suppresses MASH progression by promoting lipophagy.

Background/Aims: metabolic dysfunction-associated steatohepatitis (MASH) is an unmet clinical challenge due to the rapid increased occurrence but lacking approved drugs. Autophagy-related protein 16-like 1 (ATG16L1) plays an important role in the process of autophagy, which is indispensable for proper biogenesis of the autophagosome, but its role in modulating macrophage-related inflammation and metabolism during MASH has not been documented. Here, we aimed to elucidate the role of ATG16L1 in the progression of MASH. Methods: Expression analysis was performed with liver samples from human and mice. MASH models were induced in myeloid-specific Atg16l1-deficient and myeloid-specific Atg16l1-overexpressed mice by high-fat and high-cholesterol diet or methionine- and choline-deficient diet to explore the function and mechanism of macrophage ATG16L1 in MASH. Results: Macrophage-specific Atg16l1 knockout exacerbated MASH and inhibited energy expenditure, whereas macrophage-specific Atg16l1 transgenic overexpression attenuated MASH and promotes energy expenditure. Mechanistically, Atg16l1 knockout inhibited macrophage lipophagy, thereby suppressing macrophage ß-oxidation and decreasing the production of 4-hydroxynonenal (4-HNE), which further inhibited stimulator of interferon genes (STING) carbonylation. STING palmitoylation was enhanced, STING trafficking from the ER to the Golgi was promoted, and downstream STING signaling was activated, promoting proinflammatory and profibrotic cytokines secretion, resulting in hepatic steatosis and HSCs activation. Moreover, Atg16l1-deficiency enhanced macrophage phagosome ability but inhibited lysosome formation, engulfing mtDNA released by pyroptotic hepatocytes. Increased mtDNA promoted cGAS/STING signaling activation. Moreover, pharmacological promotion of ATG16L1 substantially blocked MASH progression. Conclusions: ATG16L1 suppresses MASH progression by maintaining macrophage lipophagy, restraining liver inflammation, and may be a promising therapeutic target for MASH management.

Accumulation of Lipid Droplets Induced by Listeria monocytogenes in Macrophages: Implications for Survival and Evasion of Innate Immunity.

Listeriosis, caused by Listeria monocytogenes (L.m.), poses a significant public health concern as one of the most severe foodborne diseases. The pathogenesis of L.m. involves critical steps such as phagosome rupture and escape upon internalization. Throughout infection, L.m. influences various host processes, including lipid metabolism pathways, yet the role of lipid droplets (LDs) remains unclear. Here, we reported a rapid, time-dependent increase in LD formation in macrophages induced by L.m. LD biogenesis was found to be dependent on L.m. viability and virulence genes, particularly on the activity of the pore-forming protein listeriolysin O (LLO). The prevention of LD formation by inhibiting diacylglycerol O-acyltransferase 1 (DGAT1) and cytosolic phospholipase A2 (cPLA2) significantly reduced intracellular bacterial survival, impaired prostaglandin E2 (PGE2) synthesis, and decreased IL-10 production. Additionally, inhibiting LD formation led to increased levels of TNF-α and IFN-ß. Collectively, our data suggest a role for LDs in promoting L.m. cell survival and evasion within macrophages.

Mechanism of efferocytosis in atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory vascular disease that occurs in the intima of large and medium-sized arteries with the immune system's involvement. It is a common pathological basis for high morbidity and mortality of cardiovascular diseases. Abnormal proliferation of apoptotic cells and necrotic cells leads to AS plaque expansion, necrotic core formation, and rupture. In the early stage of AS, macrophages exert an efferocytosis effect to engulf and degrade apoptotic, dead, damaged, or senescent cells by efferocytosis, thus enabling the regulation of the organism. In the early stage of AS, macrophages rely on this effect to slow down the process of AS. However, in the advanced stage of AS, the efferocytosis of macrophages within the plaque is impaired, which leads to the inability of macrophages to promptly remove the apoptotic cells (ACs) from the organism promptly, causing exacerbation of AS. Moreover, upregulation of CD47 expression in AS plaques also protects ACs from phagocytosis by macrophages, resulting in a large amount of residual ACs in the plaque, further expanding the necrotic core. In this review, we discussed the molecular mechanisms involved in the process of efferocytosis and how efferocytosis is impaired and regulated during AS, hoping to provide new insights for treating AS.

Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

Strategies to reprogram anti-inflammatory macrophages towards pro-inflammatory macrophages to support cancer immunotherapies.

Tumor-associated myeloid cells, including macrophages and myeloid-derived suppressor cells, can be highly prevalent in solid tumors and play a significant role in the development of the tumor. Therefore, myeloid cells are being considered potential targets for cancer immunotherapies. In this review, we focused on strategies aimed at targeting tumor-associated macrophages (TAMs). Most strategies were studied preclinically but we also included a limited number of clinical studies based on these strategies. We describe possible underlying mechanisms and discuss future challenges and prospects.

Adipose tissue-selective ablation of ADAM10 results in divergent metabolic phenotypes following long-term dietary manipulation.

A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10), is involved in several metabolic and inflammatory pathways. We speculated that ADAM10 plays a modulatory role in adipose tissue inflammation and metabolism. To this end, we studied adipose tissue-specific ADAM10 knock-out mice (aKO). While young, regular chow diet-fed aKO mice showed increased insulin sensitivity, following prolonged (33 weeks) high-fat diet (HFD) exposure, aKO mice developed obesity and insulin resistance. Compared to controls, aKO mice showed less inflammatory adipokine profile despite the significant increase in adiposity. In brown adipose tissue, aKO mice on HFD had changes in CD8+ T cell populations indicating a lesser inflammatory pattern. Following HFD, both aKO and control littermates demonstrated decreased adipose tissue pro-inflammatory macrophages, and increased anti-inflammatory accumulation, without differences between the genotypes. Collectively, our observations indicate that selective deletion of ADAM10 in adipocytes results in a mitigated inflammatory response, leading to increased insulin sensitivity in young mice fed with regular diet. This state of insulin sensitivity, following prolonged HFD, facilitates energy storage resulting in increased fat accumulation which ultimately leads to the development of a phenotype of obesity and insulin resistance. In conclusion, the data indicate that ADAM10 has a modulatory effect of inflammation and whole-body energy metabolism.

Salvimulticanol from Salvia multicaulis suppresses LPS-induced inflammation in RAW264.7 macrophages: in vitro and in silico studies.

Sustained inflammatory responses can badly affect several vital organs and lead to chronic inflammation-related disorders, such as atherosclerosis, pneumonia, rheumatoid arthritis, obesity, diabetes, Alzheimer's disease, and cancers. Salvia multicaulis is one of the widely distributed plants that contains several biologically active phytochemicals and diterpenoids with anti-inflammatory effects. Therefore, finding alternative and safer natural plant-extracted compounds with good curative anti-inflammatory efficiencies is an urgent need for the clinical treatment of inflammation-related diseases. In the current study, S. multicaulis Vahl was used to extract and isolate two compounds identified as salvimulticanol and candesalvone B methyl ester to examine their effects against inflammation in murine macrophage RAW264.7 cells that were induced by lipopolysaccharide (LPS). Accordingly, after culturing RAW264.7 cells and induction of inflammation by LPS (100 ng/ml), cells were exposed to different concentrations (9, 18, 37.5, 75, and 150 µM) of each compound. Then, Griess assay for detection of nitric oxide (NO) levels and western blotting for the determination of inducible nitric oxide synthase (iNOS) expression were performed. Molecular docking and molecular dynamics (MD) simulation studies were employed to investigate the anti-inflammatory mechanism. Our obtained results validated that the level of NO was significantly decreased in the macrophage cell suspensions as a response to salvimulticanol treatment in a dose-dependent manner (IC50: 25.1 ± 1.2 µM) as compared to the methyl ester of candesalvone B which exerted a weaker inhibition (IC50: 69.2 ± 3.0 µM). This decline in NO percentage was comparable with a down-regulation of iNOS expression by western blotting. Salvimulticanol strongly interacted with both the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex and the inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKß) to disrupt their inflammatory activation due to the significant hydrogen bonds and effective interactions with amino acid residues present in the target proteins' active sites. S.multicaulis is a rich natural source of the aromatic abietane diterpenoid, salvimulticanol, which exerted a strong anti-inflammatory effect through targeting iNOS and diminishing NO production in LPS-induced RAW264.7 cells in a mechanism that is dependent on the inhibition of TLR4-MD-2 and IKKß as activators of the classical NF-κB-mediated inflammatory pathway.

Fluorinated hydroxyapatite conditions a favorable osteo-immune microenvironment via triggering metabolic shift from glycolysis to oxidative phosphorylation.

BACKGROUND: Biological-derived hydroxyapatite is widely used as a bone substitute for addressing bone defects, but its limited osteoconductive properties necessitate further improvement. The osteo-immunomodulatory properties hold crucial promise in maintaining bone homeostasis, and precise modulation of macrophage polarization is essential in this process. Metabolism serves as a guiding force for immunity, and fluoride modification represents a promising strategy for modulating the osteoimmunological environment by regulating immunometabolism. In this context, we synthesized fluorinated porcine hydroxyapatite (FPHA), and has demonstrated its enhanced biological properties and osteogenic capacity. However, it remains unknown whether and how FPHA affects the immune microenvironment of the bone defects. METHODS: FPHA was synthesized and its composition and structural properties were confirmed. Macrophages were cultured with FPHA extract to investigate the effects of FPHA on their polarization and the related osteo-immune microenvironment. Furthermore, total RNA of these macrophages was extracted, and RNA-seq analysis was performed to explore the underlying mechanisms associated with the observed changes in macrophages. The metabolic states were evaluated with a Seahorse analyzer. Additionally, immunohistochemical staining was performed to evaluate the macrophages response after implantation of the novel bone substitutes in critical size calvarial defects in SD rats. RESULTS: The incorporation of fluoride ions in FPHA was validated. FPHA promoted macrophage proliferation and enhanced the expression of M2 markers while suppressing the expression of M1 markers. Additionally, FPHA inhibited the expression of inflammatory factors and upregulated the expression of osteogenic factors, thereby enhancing the osteogenic differentiation capacity of the rBMSCs. RNA-seq analysis suggested that the polarization-regulating function of FPHA may be related to changes in cellular metabolism. Further experiments confirmed that FPHA enhanced mitochondrial function and promoted the metabolic shift of macrophages from glycolysis to oxidative phosphorylation. Moreover, in vivo experiments validated the above results in the calvarial defect model in SD rats. CONCLUSION: In summary, our study reveals that FPHA induces a metabolic shift in macrophages from glycolysis to oxidative phosphorylation. This shift leads to an increased tendency toward M2 polarization in macrophages, consequently creating a favorable osteo-immune microenvironment. These findings provide valuable insights into the impact of incorporating an appropriate concentration of fluoride on immunometabolism and macrophage mitochondrial function, which have important implications for the development of fluoride-modified immunometabolism-based bone regenerative biomaterials and the clinical application of FPHA or other fluoride-containing materials.

Tectorigenin inhibits inflammatory responses in murine inflammatory bowel disease and LPS-stimulated macrophages via inactivating MAPK signaling pathway.

BACKGROUND: Considering the antihepatitis effects of Tectorigenin (TEC), and the same adenosine mitogen-activated protein kinase (MAPK) pathway in both hepatitis and inflammatory bowel disease (IBD) models, exploring the role of TEC in IBD is contributive to develop a new treatment strategy against IBD. METHODS: The IBD mouse model was constructed by feeding with dextran sodium sulfate (DSS) and injection of TEC. Afterward, the mouse body weight, colon length, and disease activity index (DAI) were tested to assess the enteritis level. Mouse intestine lesions were detected by hematoxylin and eosin staining. Murine macrophages underwent lipopolysaccharide (LPS) induction to establish an inflammation model. Cell viability was determined by cell counting kit-8 assay. Enzyme-linked immunosorbent assay was performed to measure interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) levels. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions were quantified via quantitative reverse transcription polymerase chain reaction. Levels of MAPK pathway-related proteins (p-P38, P38, p-Jun N-terminal kinase (JNK), JNK, signal-regulated kinase (ERK), p-ERK), COX-2 and iNOS were quantitated by Western blot. RESULTS: TEC improved the inflammatory response through ameliorating weight loss, shortening colon, and increasing DAI score in IBD mouse. Expressions of intestinal inflammatory factors (IL-6, TNF-α, iNOS and COX-2) and MAPK pathway-related proteins (p-P38, p-JNK, and p-ERK) were increased both in DSS-induced mouse intestinal tissue, but TEC inhibited expressions of inflammatory factors. The same increased trend was identified in LPS-induced macrophages, but TEC improved macrophage inflammation, as evidenced by downregulation of inflammatory factors. CONCLUSION: TEC mitigates IBD and LPS-induced macrophage inflammation in mice via inhibiting MAPK signaling pathway.

Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites ß-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.