Activity Of Macrophages Research Articles

Network pharmacology and multi-omics validation of the Jianpi-Yishen formula in the treatment of chronic kidney disease

ObjectiveChronic kidney disease (CKD) is a major global health problem. In clinical practice, the Chinese patent herbal medicine Jianpi-Yishen (JPYS) formula is commonly used to treat CKD. However, the molecular mechanisms by which JPYS targets and modulates the host immune response remain unclear.MethodsThis study utilized network pharmacology, RNA sequencing (RNA-seq), and metabolic analyses using in vivo and in vitro models to investigate the impact of the JPYS formula on inflammation and the immune system. Specifically, the study focused on macrophage polarization and metabolic changes that may slow down the progression of CKD.ResultsA total of 14,946 CKD-related targets were identified from the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases through network pharmacology analyses. 227 potential targets of the JPYS formula were predicted using the TCMSP database. Additionally, network diagram demonstrated that 11 targets were associated with macrophage activity. In vivo studies indicated that the JPYS formula could reduce blood urea nitrogen and serum creatinine in adenine-induced CKD rats. Furthermore, the formula inhibited inflammatory damage and abnormal macrophage infiltration in this CKD model. RNA-seq, proteomic and metabolic analyses identified the regulation of amino acid metabolism by betaine, specifically referring to glycine, serine, and threonine metabolism, as a key target of the JPYS formula in slowing the progression of CKD. In addition, in vitro studies suggested that JPYS may enhance tryptophan metabolism in M1 macrophage polarization and betaine metabolism in M2 macrophage polarization.ConclusionsThe JPYS formula has been shown to have beneficial impact on CKD; a key mechanism is the mitigation of inflammatory damage through the interaction between amino acid metabolism and macrophage polarization. Of specific importance in this context are the roles of tryptophan in M1 polarization and betaine in M2 polarization.

Deciphering key nano-bio interface descriptors to predict nanoparticle-induced lung fibrosis

BackgroundThe advancement of nanotechnology underscores the imperative need for establishing in silico predictive models to assess safety, particularly in the context of chronic respiratory afflictions such as lung fibrosis, a pathogenic transformation that is irreversible. While the compilation of predictive descriptors is pivotal for in silico model development, key features specifically tailored for predicting lung fibrosis remain elusive. This study aimed to uncover the essential predictive descriptors governing nanoparticle-induced pulmonary fibrosis.MethodsWe conducted a comprehensive analysis of the trajectory of metal oxide nanoparticles (MeONPs) within pulmonary systems. Two biological media (simulated lung fluid and phagolysosomal simulated fluid) and two cell lines (macrophages and epithelial cells) were meticulously chosen to scrutinize MeONP behaviors. Their interactions with MeONPs, also referred to as nano-bio interactions, can lead to alterations in the properties of the MeONPs as well as specific cellular responses. Physicochemical properties of MeONPs were assessed in biological media. The impact of MeONPs on cell membranes, lysosomes, mitochondria, and cytoplasmic components was evaluated using fluorescent probes, colorimetric enzyme substrates, and ELISA. The fibrogenic potential of MeONPs in mouse lungs was assessed by examining collagen deposition and growth factor release. Random forest classification was employed for analyzing in chemico, in vitro and in vivo data to identify predictive descriptors.ResultsThe nano-bio interactions induced diverse changes in the 4 characteristics of MeONPs and had variable effects on the 14 cellular functions, which were quantitatively evaluated in chemico and in vitro. Among these 18 quantitative features, seven features were found to play key roles in predicting the pro-fibrogenic potential of MeONPs. Notably, IL-1β was identified as the most important feature, contributing 27.8% to the model’s prediction. Mitochondrial activity (specifically NADH levels) in macrophages followed closely with a contribution of 17.6%. The remaining five key features include TGF-β1 release and NADH levels in epithelial cells, dissolution in lysosomal simulated fluids, zeta potential, and the hydrodynamic size of MeONPs.ConclusionsThe pro-fibrogenic potential of MeONPs can be predicted by combination of key features at nano-bio interfaces, simulating their behavior and interactions within the lung environment. Among the 18 quantitative features, a combination of seven in chemico and in vitro descriptors could be leveraged to predict lung fibrosis in animals. Our findings offer crucial insights for developing in silico predictive models for nano-induced pulmonary fibrosis.

A mechanically stimulated co-culture in 3D composite scaffolds promotes osteogenic and anti-osteoclastogenic activity and M2 macrophage polarization

A mechanically stimulated co-culture in 3D composite scaffolds promotes osteogenic and anti-osteoclastogenic activity and M2 macrophage polarization

From pain to meningitis: bacteria hijack nociceptors to promote meningitis

Bacterial meningitis is a severe and life-threatening infection of the central nervous system (CNS), primarily caused by Streptococcus pneumoniae and Neisseria meningitidis. This condition carries a high risk of mortality and severe neurological sequelae, such as cognitive impairment and epilepsy. Pain, a central feature of meningitis, results from the activation of nociceptor sensory neurons by inflammatory mediators or bacterial toxins. These nociceptors, abundantly present in the meninges, trigger neuroimmune signaling pathways that influence the host immune response. However, the mechanisms by which bacteria hijack these nociceptors to promote CNS invasion and exacerbate the disease remain poorly understood. This review examines the interactions between bacteria and meningeal nociceptors, focusing on their direct and indirect activation via ion channels, such as transient receptor potential vanilloid-1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1), or through the release of neuropeptides like calcitonin gene-related peptide (CGRP). These interactions suppress immune defenses by inhibiting macrophage activity and neutrophil recruitment, thus facilitating bacterial survival and invasion of the CNS. Understanding this neuroimmune axis may open potential therapeutic targets for treating bacterial meningitis by enhancing host defenses and mitigating pain. Further research using advanced methodologies is essential to clarify the role of nociceptor-mediated immune modulation in this disease.

Cystatin A promotes the antitumor activity of T helper type 1 cells and dendritic cells in murine models of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis due to diagnostic and therapeutic limitations. We previously identified cystatin A (CSTA) as a PDAC biomarker and have conducted the present study to investigate the antitumor effects of CSTA. PDAC murine models were established with genetically modified PAN02 tumor cell lines to evaluate the antitumor immune response. PDAC mouse survival was significantly longer with CSTA, and its antitumor effect was mediated mainly by CD4+ cells and partly by CD8+ cells. We also observed an increased infiltration of CD4+ and CD8+ cells in tumors of mice overexpressing CSTA. Phenotypically, we confirmed higher T helper type 1 (Th1) cell activity and increased frequency and activity of M1 macrophages and dendritic cells (DCs) in CSTA-overexpressing mice. Gene expression analysis highlighted pathways related to interferon gamma (IFN-γ) induction and Th1 lymphocyte activation that were induced by CSTA. Macrophages and DCs shifted toward proinflammatory antitumor phenotypes. Furthermore, activated splenocytes of PDAC model mice expressing CSTA had increased proapoptotic activity. CSTA also promoted the selective migration of CD4+ and CD11c+ immune cells in an in vitro migration assay. In conclusion, CSTA exerts antitumor effects by enhancing Th1-mediated antitumor effects through promotion of DC and M1 macrophage activity, thereby increasing immune cell chemotaxis. CSTA could be a novel therapeutic candidate for PDAC.

Immuno-enhancement activity of meniran (Phyllanthus niruri L.) and temu mangga rhizome (Curcuma mangga Val.) combination extract in cyclophosphamide-induced immunodeficient mice.

Immunomodulators play a crucial role in maintaining overall health and enhancing the body's defense against various diseases. In Indonesian traditional medicinal practices, meniran (Phyllanthus niruri L.) and temu mangga rhizome (Curcuma mangga Val) are well-regarded for their health benefits and therapeutic properties. Notably, meniran is utilized in jamu, an ancient Indonesian herbal tonic with widespread use in traditional healthcare. This study aimed to formulate a synergistic herbal blend by combining ethanolic extracts of meniran and temu mangga rhizome, assessing their efficacy against Cyclophosphamide (CYP)-induced immunosuppression in a murine model and elucidating potential mechanisms for immune restoration. We conducted quantitative and qualitative investigations of rutin and demethoxycurcumin composition in the combination extracts using high-performance thin-layer chromatography (HPTLC). An in vivo investigation was performed using a CYP-induced immunosuppressed murine model to assess the immunomodulatory effects of the combination extracts. The immunostimulant effect was investigated by analyzing hematology, macrophage activity (Nitric Oxide secretion, phagocytic capacity and index), lymphocyte proliferation (MTT assay), cytokine levels (IL-6 and TNF-α by ELISA), and analysis of CD4+ and CD8+ T cell populations using flow cytometry. Results demonstrated that the combined extracts effectively mitigated the immunosuppressive effects of CYP, significantly enhancing various immune indicators, including red blood cell count, hemoglobin levels, macrophage activity, lymphocyte proliferation, and cytokine serum levels. However, negligible effects were observed on erythrocyte indexes compared to the CYP-induced group. In conclusion, the combination of meniran (Phyllanthus niruri L.) and temu mangga rhizome (Curcuma mangga Val) substantially enhances various aspects of immune function, such as red blood cell count, hemoglobin levels, and macrophage activity, suggesting promising potential for immunomodulation in therapeutic contexts.

Identification of BMVC-8C3O as a novel Pks13 inhibitor with anti-tuberculosis activity.

Given the increasing prevalence of drug-resistant tuberculosis (TB), there is an urgent demand in developing novel anti-TB medications with highly effective, safe, and utilize innovative mechanisms of action. Blocking the mycolic acid synthesis pathway is well-established to be a significant strategy in developing anti-TB drugs, and Pks13 was identified as a crucial enzyme in this process. Importantly, the modes of action of recognized Pks13 inhibitors differ from traditional anti-TB medications, highlighting Pks13 as a potential and promising target in drug development within TB treatment. In this study, we discovered a compound named BMVC-8C3O that effectively inhibited the activity of Pks13 with a 6.94μM IC50 value. The binding between BMVC-8C3O and Pks13 was validated through surface plasmon resonance (SPR) assay as well as molecular docking analysis. Moreover, the SPR assay showed that the mutation of Asn1640 and Ser1533 resulted in decreased affinity of BMVC-8C3O to Pks13. Additionally, BMVC-8C3O not only exhibited activity against Mycobacterium tuberculosis (MTB), but also displayed potential intracellular anti-TB activity in macrophages. In summary, our findings indicate that BMVC-8C3O holds great potential as a lead compound against TB.

Shaoyao decoction alleviates DSS-induced colitis by inhibiting IL-17a-mediated polarization of M1 macrophages

ETHNOPHARMACOLOGICAL RELEVANCEThe efficacy of Shaoyao decoction (SYD), a traditional Chinese medicine prescription, in alleviating colonic mucosal inflammation in ulcerative colitis (UC) has been established. However, the specific mechanism underlying the therapeutic effects of SYD on UC is not clear. AIM OF STUDYIn this study, we demonstrated the therapeutic effect of SYD on the polarization of M1 macrophages and elucidated the underlying mechanism. MATERIALS AND METHODSUC mice were induced with 3% DSS for one week and subsequently treated with SYD for another week. The composition of SYD was determined by HPLC. To assess the therapeutic efficacy of SYD, key parameters, including body weight changes, DAI scores, colon length, and histological alterations in colonic tissues, were monitored. ELISA was performed to quantify inflammatory cytokines, while Western blotting and immunofluorescence analyses were performed to quantify tight junction protein expression and M1 macrophage infiltration, respectively. Functional assessments focused on lysosomal activity and glucose oxidation in primary macrophages and RAW 264.7 cells exposed to LPS, IL-17a, and SYD using dedicated kits. To elucidate the mechanisms underlying the action of SYD, the data derived from TMT-based proteomics were analyzed to screen and predict its potential targets and regulated pathways. RESULTSAfter treatment for seven days, SYD significantly mitigated colitis symptoms in mice, as determined by a decrease in body weight loss, an increase in colon length, and a decrease in disease activity index (DAI) score. The results of histopathological analysis showed substantial improvements in the integrity of colonic tissue. Additionally, SYD treatment significantly decreased the levels of proinflammatory cytokines, including IL-17a, IL-6, IL-1β, and TNF-α. This effect was accompanied by a reduction in the infiltration of CD86+ macrophages and restoration of occludin and ZO-1 levels, thus improving colonic mucosal permeability. SYD treatment also reversed the upregulation of cathepsin (CTS) E, CTSS, lysosomal-associated membrane protein (LAMP)–1, and LAMP-2 expression observed in CD86+ macrophages and RAW 264.7 cells treated with IL-17a. These changes were accompanied by an increase in lysosomal acidification and the enzymatic activities of CTSE and CTSS, suggesting that SYD can restore lysosomal function. SYD also corrected the metabolic alterations in M1 macrophages, characterized by an increase in the extracellular acidification rate (ECAR) and a decrease in oxygen consumption rate (OCR). This was confirmed by a decrease in the ADP/ATP ratio, downregulation of pyruvate dehydrogenase kinase 4 (PDK4) and lactate dehydrogenase (LDH) expression, and a decrease in the concentrations of intracellular pyruvate and lactate. These findings showed that SYD promotes glucose oxidation in macrophages. The data derived from TMT-based proteomics showed that the PPAR pathway was a key target in regulating the polarization of M1 macrophages. SYD was also found to regulate the expression of the PPAR-α, PPAR-γ, and PPAR-δ proteins. CONCLUSION: SYD inhibited the polarization of M1 macrophages induced by IL-17a. This effect occurred due to the restoration of lysosomal activity and glucose oxidation via activation of the PPAR/NF-κB pathway. Our findings provided novel insights into the mechanisms underlying the therapeutic effects of SYD in UC.

Proteolytic activity of secreted proteases from pathogenic leptospires and effects on phagocytosis by murine macrophages.

Leptospirosis is a zoonosis caused by spirochete Leptospira. Pathogenic leptospires evade the Complement System, enabling their survival upon contact with normal human serum in vitro. In a previous study, we demonstrated that proteases secreted by pathogenic leptospires cleave several Complement proteins, including C3 and the opsonins C3b and iC3b. We hypothesize that these Leptospira proteases, such as thermolysin and leptolysin, may decrease the phagocytic activity of murine peritoneal macrophages. We observed decreased amounts of CR3 and CR4 using flow cytometry when these cells were treated with supernatant from the culture of pathogenic leptospires (SPL) for 24h. Through confocal microscopy, we observed a reduction in TLR2, CD11b, and CD206 (mannose receptor) levels when these cells were treated with SPL or recombinant thermolysin for 24h. Furthermore, opsonins such as C3b/iC3b deposited on the surface of pathogenic leptospires were clearly degraded in the presence of recombinant thermolysin or recombinant leptolysin. Consequently, when opsonized bacteria and macrophages were previously incubated with these proteases, phagocytic activity was diminished. These observations lead us to suggest that proteases secreted by pathogenic leptospires could degrade opsonins present in normal serum or deposited on the bacterial membrane, as well as cleave or inhibit macrophage surface molecules. Therefore, these proteases could interfere with the recognition and internalization by murine macrophages, favoring the spread of leptospires in the host.

X-ray irradiation reduces ATP-dependent activation of NLRP3 inflammasome by inhibiting TWIK2 activity in macrophages.

The spleen, as the body's largest peripheral immune organ and a crucial source of circulating monocytes, plays a significant role in the acute inflammatory response of spleen-derived macrophages to diseases. Therefore, studying the impact and mechanism of X-ray irradiation on spleen-derived macrophages' inflammatory responses is of great importance. Extracted and identified mice splenic macrophages were divided into four groups: control group, LPS and ATP co-stimulated non-irradiated group, LPS and ATP co-stimulated group irradiated after 6h, and LPS and ATP co-stimulated group irradiated after 12h. In the LPS and ATP co-stimulated groups, LPS (1μg/ml) and ATP (5mmol/L) were added to establish an inflammatory model in mice splenic macrophages. The irradiated groups were exposed to a medical linear accelerator (Elekta Synergy), while the non-irradiated groups were placed under the light source for the same duration without irradiation. Protein extraction was performed in each group at 6h and 12h post-treatment for subsequent analysis using Western blot, ELISA, RT-qPCR and other relevant methods. (1) Compared with the non-irradiated group, the cell activity in the groups irradiated for 6h and 12h at 8Gy showed a significant increase (P<0.01). (2) In the LPS and ATP co-stimulated groups irradiated after 6h and 12h, the expression of NLRP3 mRNA and protein, IL-18 and IL-1β showed a notable decrease compared to the LPS and ATP co-stimulated non-irradiated group (P<0.05). Additionally, caspase-1 expression of caspase-1 mRNA and protein in the 12h post-irradiation group also decreased considerably when compared with the LPS and ATP co-stimulated non-irradiated group (P<0.05). In the groups irradiated after 6h and 12h, (3) there was a remarkable decrease in the expression of TWIK mRNA and TWIK2, (4) as well as Gq mRNA and protein, when compared to the LPS and ATP co-stimulated non-irradiated group (P<0.05). Particularly, the 12h post-irradiation group exhibited a notable reduction in PKC expression (P<0.05). X-ray irradiation is capable of inhibiting the activation of ATP-dependent NLRP3 inflammasomes in splenic macrophages.

Immunosenescence and age-related immune cells: causes of age-related diseases.

Immunosenescence is a weakening of the immune system due to aging, characterized by changes in immune cells and dysregulated immune function. Age-related immune cells are increasing with aging. They are associated with chronic prolonged inflammation, causing tissue dysfunction and age-related diseases. Here, we discuss increased pro-inflammatory activity of aged macrophages, accumulation of lymphocytes with an age-associated phenotype, and specific alterations in both functions and characteristics of these immune cells. These cellular changes are associated with development of age-related diseases. Additionally, we reviewed various therapeutic strategies targeting age-related immunosenescence, providing pathways to mitigate effects of age-related diseases.

Chronic Low-Level IFN-γ Expression Disrupts Mitochondrial Complex I Activity in Renal Macrophages: An Early Mechanistic Driver of Lupus Nephritis Pathogenesis.

Mitochondrial dysfunction and macrophage dysregulation are well recognized as significant contributors to the pathogenesis of autoimmune diseases. However, the detailed mechanisms connecting these two factors remain poorly understood. This study hypothesizes that low but chronic interferon-gamma (IFN-γ) plays a critical role in these processes. To explore this, we utilized ARE-Del mice, a model characterized by sustained low-level IFN-γ expression and lupus nephritis (LN)-like symptoms. Age- and tissue-dependent gene expression analyses in ARE-Del mice revealed significant suppression of mitochondrial complex I components and activities, particularly in the kidneys. The genotype-dependent suppression of mitochondrial complex I indicates early disruption, which leads to macrophage dysfunction. Notably, remission restored gene expression of mitochondrial complex I and macrophage dysfunction in isolated renal macrophages from NZB/W lupus-prone mice. These findings suggest that chronic low-level IFN-γ disrupts mitochondrial complex I activity in macrophages, highlighting its role in the early pathogenesis of autoimmune diseases like lupus nephritis. This provides new insights into the molecular interactions underlying autoimmune pathogenesis and suggests potential targets for therapeutic intervention.

Copper homeostasis; A rapier between mycobacteria and macrophages.

Copper is a vital trace element crucial for mediating interactions between Mycobacterium and macrophages. Within these immune cells, copper modulates oxidative stress responses and signaling pathways, enhancing macrophage immune functions and facilitating Mycobacterium clearance. Conversely, copper may promote Mycobacterium escape from macrophages through various mechanisms: inhibiting macrophage activity, diminishing phagocytic and bactericidal capacities, and supporting Mycobacterium survival and proliferation. This paradox has intensified research focus on the regulatory role of copper in immune cell-pathogen interactions. Interactions among metal ions can affect Mycobacterium concentration, distribution, and activity within an organism. In this review, we have elucidated the role of copper in these interactions, focusing on the mechanisms by which this metal influences both the immune defense mechanisms of macrophages and the survival strategies of Mycobacterium. The findings suggest that manipulating copper levels could enhance macrophage bactericidal functions and potentially limit Mycobacterium resistance. Therefore, elucidating the regulatory role of copper is pivotal for advancing our understanding of metal homeostasis in immune cell-pathogen dynamics and TB pathogenesis. Furthermore, we recommend further investigation into the role of copper in TB pathogenesis to advance tuberculosis diagnosis and treatment and gain comprehensive insights into metal homeostasis in infectious disease contexts.

Transient Receptor Potential Vanilloid 4 Calcium-Permeable Channel Contributes to Valve Stiffening in Aortic Stenosis.

Aortic valve stenosis (AVS) is a progressive disease characterized by fibrosis, inflammation, calcification, and stiffening of the aortic valve leaflets, leading to disrupted blood flow. If untreated, AVS can progress to heart failure and death within 2 to 5 years. Uncovering the molecular mechanisms behind AVS is key for developing effective noninvasive therapies. Emerging evidence highlights that matrix stiffness affect gene expression, inflammation, and cell differentiation. Activation of valvular interstitial cells into myofibroblasts, along with excessive extracellular matrix accumulation and remodeling, are major contributors to AVS progression. Inflammation further exacerbates the disease, as macrophages infiltrate valve leaflets, enhancing inflammation, activating valvular interstitial cells, and driving extracellular matrix remodeling. Our lab and others have shown that the activities of macrophages and fibroblasts are sensitive to matrix stiffness. Previously, we identified mechanosensitive transient receptor potential vanilloid 4 (TRPV4) channels as key regulators of fibrosis and macrophage activation, implicating TRPV4 in AVS as a potential stiffness sensor. Herein, we found elevated levels of TRPV4, α-smooth muscle actin, and cluster of differentiation 68 proteins in human AVS tissues compared with controls. Furthermore, the stiffening of human aortic valve tissue is associated with the levels of myofibroblasts, macrophages, and TRPV4 protein expression. In a mouse model, TRPV4 promoted valve stiffening during hypercholesterolemia-induced AVS. Additionally, TRPV4 mediated intracellular stiffness in valvular interstitial cells in response to transforming growth factor β1, which was blocked by the TRPV4 antagonist GSK2193874. These findings reveal a novel mechanism linking TRPV4 to valve stiffening, providing insights into how extracellular matrix mechanical properties drive inflammation and fibrosis in AVS.

Sphingosine 1-phosphate receptor 1signaling in macrophages reduces atherosclerosis in LDL receptor-deficient mice.

Sphingosine 1-phosphate (S1P) is a lysosphingolipid with antiatherogenic properties, but mechanisms underlying its effects remain unclear. We here investigated atherosclerosis development in cholesterol-rich diet-fed LDL receptor-deficient mice with high or low overexpression levels of S1P receptor 1 (S1P1) in macrophages. S1P1-overexpressing macrophages showed increased activity of transcription factors PU.1, interferon regulatory factor 8 (IRF8), and liver X receptor (LXR) and were skewed toward an M2-distinct phenotype characterized by enhanced production of IL-10, IL-1RA, and IL-5; increased ATP-binding cassette transporter A1- and G1-dependent cholesterol efflux; increased expression of MerTK and efferocytosis; and reduced apoptosis due to elevated B cell lymphoma 6 and Maf bZIP B. A similar macrophage phenotype was observed in mice administered S1P1-selective agonist KRP203. Mechanistically, the enhanced PU.1, IRF8, and LXR activity in S1P1-overexpressing macrophages led to downregulation of the cAMP-dependent PKA and activation of the signaling cascade encompassing protein kinases AKT and mTOR complex 1 as well as the late endosomal/lysosomal adaptor MAPK and mTOR activator 1. Atherosclerotic lesions in aortic roots and brachiocephalic arteries were profoundly or moderately reduced in mice with high and low S1P1 overexpression in macrophages, respectively. We conclude that S1P1 signaling polarizes macrophages toward an antiatherogenic functional phenotype and countervails the development of atherosclerosis in mice.

The microbial metabolite imidazole propionate dysregulates bone homeostasis by inhibiting AMP-activated protein kinase (AMPK) signaling

Microbial metabolites provide numerous benefits to the human body but can also contribute to diseases such as obesity, diabetes, cancer, and bone disorders. However, the role of imidazole propionate (ImP), a histidine-derived metabolite produced by the intestinal microbiome, in bone metabolism and the development of osteoporosis is still poorly understood. In this study, we investigated the role of ImP and its underlying mechanisms in regulating bone homeostasis. When ImP was administered to 8-week-old mice for 4 weeks, bone loss was observed, along with a decrease in alkaline phosphatase-positive osteoblast cells. Additionally, bone marrow stromal cells (BMSCs) isolated from ImP-treated mice exhibited reduced osteogenic potential. In BMSCs from control mice, ImP treatment inhibited BMP2-induced osteoblast differentiation while promoting adipocyte differentiation. However, ImP had no effect on RANKL-induced osteoclast differentiation or activity in bone marrow macrophages. Mechanistically, ImP treatment increased p38γ phosphorylation and decreased AMPK (T172) phosphorylation in BMSCs. Suppression of p38γ expression using si-p38γ reversed the inhibitory effects of ImP on osteoblast differentiation, with a concurrent increase in AMPK (T172) phosphorylation. Conversely, ImP stimulated adipocyte differentiation by decreasing AMPK (T172) phosphorylation. Treatment with the AMPK agonist metformin significantly reversed the inhibitory effects of ImP on osteoblast differentiation and the promotion of adipocyte differentiation, along with enhanced AMPK (T172) phosphorylation. These findings suggest that the microbial metabolite ImP may disrupt bone homeostasis by stimulating p38γ phosphorylation and inhibiting the AMPK pathway, presenting a potential therapeutic target for managing metabolic bone diseases.

Macrophages promote pre-metastatic niche formation of breast cancer through aryl hydrocarbon receptor activity

Macrophages that acquire an immunosuppressive phenotype play a crucial role in establishing the pre-metastatic niche (PMN), which is essential for facilitating breast cancer metastasis to distant organs. Our study showed that increased activity of the aryl hydrocarbon receptor (AHR) in lung macrophages plays a crucial role in establishing the immunosuppressive PMN in breast cancer. Specifically, AHR activation led to high expression of PD-L1 on macrophages by directly binding to the promoter of Pdl1. This upregulation of PD-L1 promoted the differentiation of regulatory T cells (Tregs) within the PMN, further enhancing immunosuppressive conditions. Mice with Ahr conditional deletion in macrophages had reduced lung metastasis of breast cancer. The elevated AHR levels in PMN macrophages were induced by GM-CSF, which was secreted by breast cancer cells. Mechanistically, the activated STAT5 signaling pathway induced by GM-CSF prevented AHR from being ubiquitinated, thereby sustaining its activity in macrophages. In breast cancer patients, the expression of AHR and PD-L1 was correlated with increased Treg cell infiltration, and higher levels of AHR were associated with a poor prognosis. These findings reveal that the crosstalk of breast cancer cells, lung macrophages, and Treg cells via the GM-CSF-STAT5-AHR-PD-L1 cascade modulates the lung pre-metastatic niche during breast cancer progression.

Molecular Mechanisms of Rett Syndrome: Emphasizing the Roles of Monoamine, Immunity, and Mitochondrial Dysfunction

Rett syndrome (RTT), which predominantly affects females, arises in most cases from mutations in the Methyl-CpG-binding Protein-2 (MECP2) gene. When MeCP2 is impaired, it disrupts the regulation of numerous genes, causing the production of dysfunctional proteins associated with various multi-systemic issues in RTT. In this review, we explore the current insights into molecular signaling related to monoamines, immune response, and mitochondrial function, and their implications for the pathophysiology of RTT. Research has shown that monoamines—such as dopamine, norepinephrine, epinephrine, serotonin, and histamine—exhibit alterations in RTT, contributing to a range of neurological symptoms. Furthermore, the immune system in RTT individuals demonstrates dysfunction through the abnormal activity of microglia, macrophages, lymphocytes, and non-immune cells, leading to the atypical release of inflammatory mediators and disruptions in the NF-κB signaling pathway. Moreover, mitochondria, essential for energy production and calcium storage, also show dysfunction in this condition. The delicate balance of producing and scavenging reactive oxygen species—termed redox balance—is disrupted in RTT. Targeting these molecular pathways presents a promising avenue for developing effective therapies.

Inflammatory Response of THP1 and U937 Cells: The RNAseq Approach.

THP1 and U937 are monocytic cell lines that are common bioassays to reflect monocyte and macrophage activities in inflammation research. However, THP-1 is a human monocytic leukemia cell line, and U937 originates from pleural effusion of histiocytic lymphoma; thus, even though they serve as bioassay in inflammation research, their response to agonists is not identical. Consequently, there has yet to be a consensus about the panel of strongly regulated genes in THP1 and U937 cells representing the inflammatory response to LPS and IFNG. Therefore, we have performed an RNAseq of THP1 and U937 exposed to LPS and IFNG to identify the most sensitive genes and the unique properties of each individual cell line. When applying a highly stringent threshold, we could identify 43, 8 up and 94, 103 down-regulated genes in THP1 and U937 cells, respectively. In THP1 cells, among the most strongly up-regulated genes are CCL1, CXCL2, CXCL3, IL1A, IL1B, IL6, and PTGES. In U937 cells, the strongest up-regulated genes include CSF2, CSF3, CXCL2, CXCL5, CXCL6, IL1A, IL19, IL36G, IL6, ITGA1, ITGA2, and PTGS2. Even though THP1 is considerably less responsive than U937, there are genes commonly upregulated by LPS and IFNG, including the CCL1, CCL3, CCL20, CXCL2, CXCL3, CXCL8, as well as IL1A, IL1B, IL23A, IL6, and genes of prostaglandin synthesis PTGES and PTGS2. Downregulated genes are limited to NRGN and CD36. This head-to-head comparison revealed that THP1 is less responsive than U937 cells to LPS and IFNG and identified a panel of highly regulated genes that can be applied in bioassays in inflammation research. Our data further propose bulk RNAseq as a standard method in bioassay research.

Polycytotoxic T cells mediate antimicrobial activity against intracellular Mycobacterium tuberculosis.

Protection against infections with intracellular bacteria requires the interaction of macrophages and T-lymphocytes, including CD8+ T cells. Recently, the expression of natural killer cell receptors NKG2A and NKG2C was introduced as markers of CD8+ T-cell subsets. The goal of this study was to functionally characterize human NKG2A and NKG2C-expressing T cells using the major pathogen Mycobacterium tuberculosis (Mtb) as a model organism. Sorted NKG2 populations were analyzed for their capacity to proliferate and degranulate and their intracellular expression of cytotoxic molecules. Cytokine release and the effect on bacterial growth were assessed after coculture of NKG2 populations with Mtb-infected macrophages. NKG2A+ T cells released higher levels of IFN-γ and IL-10, whereas NKG2C+ T cells released higher levels of IL-2, contained the greatest reservoir of intracellular granzyme B and showed a remarkable constitutive level of degranulation. Both subsets inhibited the intracellular growth of Mtb more efficiently than NKG2-negative CD8+ T cells. Antimicrobial activity of NKG2+ T cells was not associated with the release of cytokines or cytotoxic molecules. However, the frequency of polycytotoxic T cells (P-CTL), defined as CD8+ T cells co-expressing granzyme B, perforin, and granulysin, positively correlated with the ability of NKG2-expressing T cells to control Mtb-growth in macrophages. Our results highlight the potential of NKG2-expressing P-CTL to trigger the antibacterial activity of human macrophages. Targeting this population by preventive or therapeutic immune interventions could provide a novel strategy to combat severe infectious diseases such as tuberculosis.