Co-culture Model Of Macrophages Research Articles

Effect of Perilla frutescens polyphenols against tight junction alterations and intestinal inflammation: a Caco-2/macrophage co-culture model-based study integrated with network pharmacology

The intestinal barrier plays important roles in the uptake of nutrients and prevention of harmful pathogens. However, intestinal inflammation causes barrier dysfunction, which may contribute to inflammatory bowel disease (IBD). Polyphenols from Perilla frutescens (L.) Britt. exhibit various bioactivities, though their protective effect on the intestinal barrier remains unclear. To address this, the study investigated the effect of Perilla frutescens (L.) Britt. polyphenols (PFP) against tight junction alterations and intestinal inflammation using an intestinal-like Caco-2/macrophage co-culture model. LC-MS/MS analysis identified 15 polyphenols in the PFP extract. Furthermore, PFP were transported across the apical Caco-2 monolayer, with apigenin, genistin, syringic acid and cinnamic acid showing relatively high transport rates (> 70%). The lipopolysaccharide (LPS)-induced tight junction alterations, evaluated by trans-epithelial electrical resistance (TEER) analysis, qPCR, and immunofluorescence assay, were significantly restored by medium and high doses of PFP (P < 0.05). In addition, network pharmacology analysis demonstrated that the core targets of PFP for intestinal inflammation were mostly located in the MAPK and NF-κB signaling pathways. The expression levels of the key proteins in these pathways were further validated by qPCR and western blot analyses. Overall, PFP exhibits great potential as a novel functional food component for treating intestinal inflammation.

Glutamine Attenuates Inflammation and Stimulates Amniotic Cell Proliferation in Premature Rupture of Membranes-related in vitro Models.

Premature rupture of membranes (PROM), with a prevalence of 15.3% in China, frequently results in adverse pregnancy outcomes. In this study, we aimed to identify amino acid metabolites that were differentially expressed in PROM versus healthy controls (HC) using targeted metabolomics and further explored their mechanisms of action with in vitro models.Inclusion and exclusion criteria were established to recruit 50 PROM and 50 HC cases for targeted metabolomics analysis. Twenty-three amino acid metabolites were quantified in the secretions of the posterior vaginal fornix of pregnant women between 31 and 36 weeks of gestation. Glutamine (0.0216 vs. 0.037μg/mg, P = 0.003, AUC = 72.1%) was identified as the most differentially expressed amino acid metabolite between PROM and HC groups, and had a negative correlation with the abundance of Gardnerella (r=-0.3868, P = 0.0055), Megasphaera (r=-0.3130, P = 0.0269), and Prevotella (r=-0.2944, P = 0.0380), respectively.In amniotic epithelial cell and macrophage co-culture model, Glutamine reduced inflammatory cytokines and chemokines expression and suppressed macrophage chemotaxis. In LPS stimulated RAW 264.7 inflammation model, Glutamine inhibited the expression of inflammatory proteins iNOS and COX-2, down-regulated mRNA transcription of TNF, IL-6, and IL-1β, and reduced the production of reactive oxygen species through inhibiting NF-κB signaling pathway, and therefore demonstrated its anti-inflammatory effect. Furthermore, Glutamine protected amniotic epithelial cell from autophagy and stimulated its proliferation, therefore may intensify fetal membrane and prevent PROM in vivo.Our results suggested that low Glutamine level in vaginal secretion can be used as an indicator for PROM, and local Glutamine supplementation is a potential intervention and prevention strategy for PROM.

Discovery of vitexin as a novel VDR agonist that mitigates the transition from chronic intestinal inflammation to colorectal cancer

Colitis-associated colorectal cancer (CAC) frequently develops in patients with inflammatory bowel disease (IBD) who have been exposed to a prolonged state of chronic inflammation. The investigation of pharmacological agents and their mechanisms to prevent precancerous lesions and inhibit their progression remains a significant focus and challenge in CAC research. Previous studies have demonstrated that vitexin effectively mitigates CAC, however, its precise mechanism of action warrants further exploration. This study reveals that the absence of the Vitamin D receptor (VDR) accelerates the progression from chronic colitis to colorectal cancer. Our findings indicate that vitexin can specifically target the VDR protein, facilitating its translocation into the cell nucleus to exert transcriptional activity. Additionally, through a co-culture model of macrophages and cancer cells, we observed that vitexin promotes the polarization of macrophages towards the M1 phenotype, a process that is dependent on VDR. Furthermore, ChIP-seq analysis revealed that vitexin regulates the transcriptional activation of phenazine biosynthesis-like domain protein (PBLD) via VDR. ChIP assays and dual luciferase reporter assays were employed to identify the functional PBLD regulatory region, confirming that the VDR/PBLD pathway is critical for vitexin-mediated regulation of macrophage polarization. Finally, in a mouse model with myeloid VDR gene knockout, we found that the protective effects of vitexin were abolished in mid-stage CAC. In summary, our study establishes that vitexin targets VDR and modulates macrophage polarization through the VDR/PBLD pathway, thereby alleviating the transition from chronic colitis to colorectal cancer.

Gallic acid mitigates intestinal inflammation and loss of tight junction protein expression using a 2D-Caco-2 and RAW 264.7 co-culture model

A 2D-intestinal epithelial Caco-2/RAW 264.7 macrophage co-culture model was developed to demonstrate the relative efficacy of different phenolic acids to mitigate changes in Caco-2 epithelial cell redox state initiated both directly by autoxidation products, H2O2, and indirectly through cell communication events originating from cytokine stimulated macrophage. An inducer cocktail (lipopolysaccharide + interferon gamma) was used to activate RAW 264.7 cells in the 2D- Caco-2/RAW co-culture and intracellular changes in Caco-2 cell redox signaling occurred in response to positive changes (p < 0.05) in inflammatory biomarkers derived in macrophage that included IL-6, TNF-α, nitric oxide and peroxynitrite, respectively. Phenolic acids varied in relative capacity to reduce NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in cocktail inflamed induced macrophage. This response in addition to the relative predisposition of gallic acid (GA) to undergo autoxidation to generate H2O2 activity (p < 0.05), culminated in downstream cell signaling in Caco-2 nuclear factor erythroid 2-related factor (Nrf2) activity (increase 26.9 %), altered monolayer integrity (increase 33.7 %), and release of interleukin 8 (IL-8) (decrease 80.5 %) (p < 0.05). It can be concluded that the co-culture model described herein was useful to assess the importance of communication between cytokine stimulated macrophage and intestinal cells. Moreover, the relative unique efficacy of GA, compared to other phenolic acids tested to protect against activated macrophage induced changes related to intestinal dysfunction were particularly relevant to epithelial redox signaling, intestinal permeability and regulation of tight junction proteins. This study concludes that phenolic acids are not equal in the capacity to protect against intestinal cell dysfunction despite some indication of biological activity.

An evaluation of a hepatotoxicity risk induced by the microplastic polymethyl methacrylate (PMMA) using HepG2/THP-1 co-culture model

Inappropriate disposal of plastic wastes and their durability in nature cause uncontrolled accumulation of plastic in land/marine ecosystems, also causing destructive effects by bioaccumulating along the food chain. Microplastics may cause chronic inflammation in relation to their permanent structures, especially through oxidative stress and cytotoxic cellular damage, which could increase the risk of cancer development. The accumulation of microplastics in the liver is a major concern, and therefore, the identification of the mechanisms of their hepatotoxic effects is of great importance. Polymethyl methacrylate (PMMA) is a widely used thermoplastic. It has been determined that PMMA disrupts lipid metabolism in the liver in various aquatic organisms and causes reproductive and developmental toxicity. PMMA-induced hepatotoxic effects in humans have not yet been clarified. In our study, the toxic effects of PMMA (in the range of 3–10 μm) on the human liver were investigated using the HepG2/THP-1 macrophage co-culture model, which is a sensitive immune-mediated liver injury model. Cellular uptake of micro-sized PMMA in the cells was done by transmission electron microscopy. Determination of its effects on cell viability and inflammatory response, oxidative stress, along with gene and protein expression levels that play a role in the mechanism pathways underlying the effects were investigated. The results concluded that inflammation, oxidative stress, and disruptions in lipid metabolism should be the focus of attention as important underlying causes of PMMA-induced hepatotoxicity. Our study, which points out the potential adverse effects of microplastics on human health, supports the literature information on the subject.

Inhalation of panaxadiol alleviates lung inflammation via inhibiting TNFA/TNFAR and IL7/IL7R signaling between macrophages and epithelial cells

BackgroundLung inflammation occurs in many lung diseases, but has limited effective therapeutics. Ginseng and its derivatives have anti-inflammatory effects, but their unstable physicochemical and metabolic properties hinder their application in the treatment. Panaxadiol (PD) is a stable saponin among ginsenosides. Inhalation administration may solve these issues, and the specific mechanism of action needs to be studied. MethodsA mouse model of lung inflammation induced by lipopolysaccharide (LPS), an in vitro macrophage inflammation model, and a coculture model of epithelial cells and macrophages were used to study the effects and mechanisms of inhalation delivery of PD. Pathology and molecular assessments were used to evaluate efficacy. Transcriptome sequencing was used to screen the mechanism and target. Finally, the efficacy and mechanism were verified in a human BALF cell model. ResultsInhaled PD reduced LPS-induced lung inflammation in mice in a dose-dependent manner, including inflammatory cell infiltration, lung tissue pathology, and inflammatory factor expression. Meanwhile, the dose of inhalation was much lower than that of intragastric administration under the same therapeutic effect, which may be related to its higher bioavailability and superior pharmacokinetic parameters. Using transcriptome analysis and verification by a coculture model of macrophage and epithelial cells, we found that PD may act by inhibiting TNFA/TNFAR and IL7/IL7R signaling to reduce macrophage inflammatory factor-induced epithelial apoptosis and promote proliferation. ConclusionPD inhalation alleviates lung inflammation and pathology by inhibiting TNFA/TNFAR and IL7/IL7R signaling between macrophages and epithelial cells. PD may be a novel drug for the clinical treatment of lung inflammation.

The Overexpression of Fibronectin 1 Promotes Cancer Progression and Associated with M2 Macrophages Polarization in Head and Neck Squamous Cell Carcinoma Patients

PurposeThis study aimed to investigate the biological roles of fibronectin 1 (FN1) in head and neck squamous cell carcinoma (HNSCC) and its effects on macrophage M2 polarization.MethodsWe analyzed FN1 expression pattern and examined its clinical relevance in HNSCC progression by bioinformatic analysis. Small interfering RNA (siRNA) was utilized to silence FN1 in HNSCC cells. Cell counting kit-8 (CCK-8) assay, colony formation assay, Transwell assay and wound healing assay were performed to reveal the effect of FN1 on malignant behaviors of HNSCC cells. Moreover, a co-culture model of macrophages and HNSCC cells was established to investigate whether FN1 induce macrophage M2 polarization. Finally, we used bioinformatic methods to explore the possible FN1-related pathways in HNSCC.ResultsFN1 is significantly overexpressed in HNSCC patients and has been obviously correlated with higher pathological stage and poor prognosis. Downregulation of FN1 suppressed the proliferation, migration and invasion of HNSCC cells, and inhibited macrophage M2 polarization in vitro. In addition, “PI3K-Akt” and “MAPK” signaling pathways may be involved in the malignant process of FN1 in HNSCC.ConclusionThe overexpression of FN1 promotes HNSCC progression and induces macrophages M2 polarization. FN1 may serve as a promising prognostic biomarker and therapeutic target in HNSCC.

Mannose Attenuates Colitis-Associated Colorectal Tumorigenesis by Targeting Tumor-Associated Macrophages.

Mannose has recently drawn extensive attention for its substantial anti-cancer activities, but the underlying mechanism remains largely unclear. The aim of this study was to investigate the effects of mannose on experimental colitis-associated colorectal tumorigenesis and underlying mechanisms. Data clearly showed that at plasma concentrations achieved after oral administration, mannose slightly affected malignancy of tumor cells or tumor promoter-induced transformation of pre-neoplastic cells, but substantially suppressed manifestation of the M2-like phenotype of tumor-associated macrophages (TAMs) in a cancer cell and macrophage co-culture model. Mechanistically, mannose might greatly impair the production of tumor cell-derived lactate which has a critical role in the functional polarization of TAMs. Importantly, oral administration of mannose protected mice against colitis-associated colorectal tumorigenesis by normalizing TAM polarization. Collectively, these findings highlight the importance of TAMs in colorectal tumorigenesis, and provide a rationale for introducing mannose supplementation to patients suffering from inflammatory bowel diseases.

Inflammasome Activation in the Hip Synovium of Rapidly Destructive Coxopathy Patients and Its Relationship with the Development of Synovitis and Bone Loss

Rapidly destructive coxopathy (RDC), a rare disease of unknown etiology, is characterized by the rapid destruction of the hip joint. In the current study, the potential involvement of inflammasome signaling in the progression of RDC was investigated. Histopathologic changes and the gene expression of inflammasome activation markers in hip synovial tissues collected from patients with RDC were evaluated and compared with those of osteoarthritis and osteonecrosis of the femoral head patients. The synovial tissues of patients with RDC exhibited remarkable increases in the number of infiltrated macrophages and osteoclasts, and the expression of inflammasome activation markers was also increased compared with those of osteoarthritis and osteonecrosis of the femoral head patients. To further understand the histopathologic changes in the joint, a co-culture model of macrophages and synoviocytes that mimicked the joint environment was developed. Remarkably, the gene expression levels of NLRP3, GSDMD, IL1B, TNFA, ADMTS4, ADMTS5, MMP3, MMP9, and RANKL were significantly elevated in the synoviocytes that were co-cultured with activated THP-1 macrophages, suggesting the association between synovitis and inflammasome activation. Consistent with these findings, osteoclast precursor cells that were co-cultured with stimulated synoviocytes exhibited an increased number of tartrate-resistant acid phosphatase-positive cells, compared with cells that were co-cultured with non-stimulated synoviocytes. These findings suggest that the activation of inflammasome signaling in the synovium results in an increase in local inflammation and osteoclastogenesis, thus leading to the rapid bone destruction in RDC.

A comparative study on the model of PM2.5 direct or indirect interaction with bronchial epithelial cells.

The impact of PM2.5 on epithelial cells is a pivotal process leading to many lung pathological changes and pulmonary diseases. In addition to PM2.5 direct interaction with epithelia, macrophages that engulf PM2.5 may also influence the function of epithelial cells. However, among the toxic researches of PM2.5, there is a lack of evaluation of direct or indirect exposure model on human bronchial epithelial cell against PM2.5. In this present research, PM2.5-exposed human bronchial epithelial cell line (BEAS-2B) serves as the direct interaction model. By contrast, a PM2.5-stimulated co-culture model of macrophages and epithelial cells based on the transwell system was adopted as indirect stimulation model. By comparing these two models of interaction, we examined the viability of BEAS-2B and mRNA/protein expression profile of oxidative stress and inflammatory response-related transcription factors Nrf2, NF-kB, and according inflammatory indicators such as IL-1, IL-6, and IL-8, with a view to evaluating the effects of different interaction models of PM2.5 on epithelial cell damage in vitro. Our results indicated that under the same doses, the direct stimulation model of PM2.5 could inhibit the viability of BEAS-2B. Furthermore, the indirect stimulation model strengthen inflammation response of epithelia under the higher concentration of PM2.5 and induce epithelia to undergo EMT under the lower concentration of PM2.5. Overall, we have found that macrophage involvement may protect epithelia from PM2.5 cytotoxic effect, while it strengthens the inflammation response and induce epithelia to undergo EMT.

Senegalia macrostachya seed polysaccharides attenuate inflammation-induced intestinal epithelial barrier dysfunction in a Caco-2 and RAW264.7 macrophage co-culture model by inhibiting the NF-κB/MLCK pathway.

The intestinal barrier dysfunction associated with chronic inflammation is a major health concern. This work aimed to investigate the protective effect and molecular mechanism of Senegalia macrostachya seed polysaccharide fraction (SMSP2) on inflammation-induced barrier dysfunction using Caco-2 cells and RAW264.7 macrophage co-culture model. The results showed that LPS stimulation of the basolateral RAW264.7 compromised the integrity of the apical differentiated Caco-2 cells monolayer, resulting in decreased transepithelial electrical resistance (TER) and increased inflammatory markers. SMSP2 apical treatment maintained a higher TER value and reduced the epithelial permeability to lucifer yellow (LY) dye. In addition, the SMSP2 group showed a significant decrease in the mRNA level of inflammatory factors such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin (IL)-8, and tumor necrosis factor-α (TNF-α) in the Caco-2 monolayer compared to the LPS group. Moreover, SMSP2 increased the expression of the tight junctions (TJ) zonula occludens (ZO-1), occludin, and claudin-1 at the mRNA and protein levels. Furthermore, the immunofluorescence assay showed that SMSP2 ameliorated the overall distribution of the TJ proteins in the Caco-2 monolayer. SMSP2 application also resulted in the downregulation of the nuclear factor kappa-B (NF-κB) phosphorylation and the myosin light chain kinase (MLCK) expression, which implies that SMSP2 preserved the monolayer integrity from the inflammation-induced barrier disruption through the inhibition of the NF-kB-mediated MLCK signaling pathway activation. Senegalia macrostachya seeds could therefore be a promising functional food that could be used to improve intestinal barrier function.

Notch Signaling Ligand Jagged1 Enhances Macrophage-Mediated Response to Helicobacter pylori.

Helicobacter pylori (H. pylori) is one of the gram-negative bacteria that mainly colonize the stomach mucosa and cause many gastrointestinal diseases, such as gastritis, peptic ulcer, and gastric cancer. Macrophages play a key role in eradicating H. pylori. Recent data have shown that Notch signaling could modulate the activation and bactericidal activities of macrophages. However, the role of Notch signaling in macrophages against H. pylori remains unclear. In the present study, in the co-culture model of macrophages with H. pylori, the inhibition of Notch signaling using γ-secretase decreased the expression of inducible nitric oxide synthase (iNOS) and its product, nitric oxide (NO), and downregulated the secretion of pro-inflammatory cytokine and attenuated phagocytosis and bactericidal activities of macrophages to H. pylori. Furthermore, we identified that Jagged1, one of Notch signaling ligands, was both upregulated in mRNA and protein level in activated macrophages induced by H. pylori. Clinical specimens showed that the number of Jagged1+ macrophages in the stomach mucosa from H. pylori-infected patients was significantly higher than that in healthy control. The overexpression of Jagged1 promoted bactericidal activities of macrophages against H. pylori and siRNA-Jagged1 presented the opposite effect. Besides, the addition of exogenous rJagged1 facilitated the pro-inflammatory mediators of macrophages against H. pylori, but the treatment of anti-Jagged1 neutralizing antibody attenuated it. Taken together, these results suggest that Jagged1 is a promoting molecule for macrophages against H. pylori, which will provide insight for exploring Jagged1 as a novel therapeutic target for the control of H. pylori infection.

Acai berry extract as a regulator of intestinal inflammation pathways in a Caco-2 and RAW 264.7 co-culture model.

The aim of this study was to assess the anti-inflammatory effects of acai berries in a Caco-2 and RAW 264.7 macrophage co-culture model. The acai berry extract (ABE) was prepared using 70% ethanol, and total anthocyanin, polyphenol, and flavonoid contents in ABE were analyzed. To the antioxidant activity of ABE, we measured radical scavenging activity as well as ferric reducing antioxidant power values. Prior to inducing inflammation, Caco-2 cells were co-cultured with RAW 264.7. Inflammation was induced using lipopolysaccharides (LPS) in RAW 264.7 cells. The transepithelial electrical resistance value was significantly recovered and the mRNA level of tight junction proteins, including ZO-1, JAM-1, and claudin-4, tended to increase compared with that in the LPS group. LPS-induced interleukin (IL)-6, IL-8, and prostaglandin E2 levels reduced significantly following treatment with the highest ABE concentration. In the highest ABE concentration, the phosphorylation of p65, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase was downregulated compared with the LPS group. The phosphorylation of extracellular signal-regulated kinase showed a decreased tendency. These results suggest that acai berry may improve gastrointestinal health. PRACTICAL APPLICATIONS: Acai berry is known to have abundant anthocyanin, which has many biological activities, including anti-inflammatory, antioxidant, antihypertensive, and anticytotoxic/cytoprotective activities. This study demonstrated the anti-inflammatory effects of acai berry extracts via TEER value, expression of tight junction protein, and production of inflammatory mediators and cytokines in the Caco-2 and RAW 264.7 co-culture model. Therefore, acai berry has the potential to prevent intestinal inflammatory diseases.

Gold nanoclusters treat intracellular bacterial infections: Eliminating phagocytic pathogens and regulating cellular immune response

Intracellular bacterial infection is underlying many serious human disorders, leading to high morbidity and mortality. The development of safe and efficient therapeutic agents is the most effective solutions to combat intracellular bacterial infections. Recently, ultrasmall gold nanoclusters (AuNCs) have emerged as an innovative nanoantibiotics against multidrug-resistant bacterial infections due to their inherent antibacterial activity. However, the therapeutic effects of AuNCs on intracellular bacterial infections and their effects on host cells still remain unvisited. Here, we demonstrate the therapeutic potential of 4,6-diamino-2-mercaptopyrimidine-functionalized AuNCs (AuDAMP) for intracellular multidrug-resistant infections in a co-culture model of macrophages and methicillin-resistant Staphylococcus aureus (MRSA). The AuNCs were found to show a superior intracellular antibacterial capability, which can eliminate most of the MRSA phagocytosed by macrophages, and without exhibiting obvious cytotoxicity on host RAW 264.7 macrophages at tested concentrations. More importantly, treatment of AuDAMP exerts critical roles on enhancing the innate immune response to defend against pathogens invading inside the host cells and alleviating the bacterial infection-induced inflammatory response to avoid pyroptosis by up-regulating significantly xenophagy level in macrophages. Taken together, our results suggest that AuNCs hold great potential for the treatment of intracellular bacterial infections.

Abstract PO021: Lung cancer cells and cancer-associated fibroblasts drive macrophage polarization in a co-culture model

Abstract Introduction: Macrophages are key architects of the immune landscape in the tumor microenvironment (TME), and the plasticity of macrophage phenotype correlates with prognosis in non-small cell lung cancer (NSCLC). M2-like macrophages promote immunosuppression and facilitate tumor progression, while M1-like macrophages may drive an inflammatory antitumor immune response. We investigated if NSCLC oncogenotype impacts macrophage phenotype in a novel tumor cell, cancer-associated fibroblast (CAF) and macrophage co-culture model. We hypothesize that macrophage-mediated immunosuppression is driven in part through tumor-secreted signaling molecules that, if identified and blocked, may re-sensitize cancer cells to immune surveillance and enhance antitumor immunity. Methods: We developed an in vitro co-culture system (patient-derived NSCLC cells, human CAFs, and mouse macrophages) to interrogate impact of NSCLC cells and CAFs on macrophage phenotype. Through species specific qPCR we measured the expression salient macrophage genes (i.e. Arginase, NOS2, IL-1β, IL-6, CHIL-3, SOCS3) in the co-cultures. We also performed whole genome RNA sequencing (RNAseq) in select cases and evaluated protein expression using cytokine arrays measuring expression of 40 inflammatory cytokines. Positive controls included stimulation of macrophages with LPS and IL-4. Results: We characterized the effect of over 70 NSCLC cell lines in the co-culture assay. Three highly reproducible clusters of macrophage phenotypes were identified: high Arginase (immunosuppressive), high IL-1β (inflammatory) or high SOCS3 (inflammatory, involved in JAK-STAT3 pathway). Major NSCLC oncogenotypes (i.e. KRAS, TP53, STK11, EGFR, BRAF mutation) did not correlate with macrophage phenotype. Analyses of differences between the 3 clusters is ongoing. We selected 10 exemplar NSCLC lines representing each of these 3 clusters for RNA sequencing (mouse genes) and cytokine array protein (human) profiling. Across all clusters, we found suppression of macrophage endocytosis pathways and activation of scavenger receptor A (SRA) signaling (M2-like phenotype) and increased expression of human IL-6, IL-8, and MCP1, which are implicated in suppression of innate immune sensing of tumor cells. RNAseq of CAF lines demonstrated mixed inflammatory and fibroblastic phenotype, with increased expression of genes associated with macrophage recruitment and activation including: IL-6, CSF-1, CXCL6, CCL2, and CCL7. Conclusions: We demonstrate in a novel co-culture model that patient-derived NSCLC cells reproducibly induce three major macrophage phenotypes. Cytokine release from NSCLC cells and CAFs are implicated in this process. This co-culture model provides a robust, physiologically consistent experimental platform to identify tumor cell and CAF features that drive macrophage phenotype. Citation Format: Josiah Flaming, Raghav Chandra, Luc Girard, Debolina Ganguly, Jason Toombs, John D. Minna, Rolf A. Brekken. Lung cancer cells and cancer-associated fibroblasts drive macrophage polarization in a co-culture model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO021.

Industrial PM2.5 cause pulmonary adverse effect through RhoA/ROCK pathway

According to the Chinese Ministry of Health, industrial pollution-induced health impacts have been the leading cause of death in China. While industrial fine particulate matter (PM2.5) is associated with adverse health effects, the major action mechanisms of different compositions of PM2.5 are currently unclear. In this study, we treated normal human lung epithelial BEAS-2B cells with industrial organic and water-soluble PM2.5 extracts under daily alveolar deposition dose to elucidate the molecular mechanisms underlying adverse pulmonary effects induced by PM2.5, including oxidative damage, inflammatory response, lung epithelial barrier dysfunction, and the recruitment of macrophages. We found that water-soluble PM2.5 extracts caused more severe cytotoxic effects on BEAS-2B cells compared with that of organic extracts. Both organic and water-soluble PM2.5 extracts induced activation of the RhoA/ROCK pathway. Inflammatory response, epithelial barrier dysfunction, and the activation of NF-кB caused by both PM2.5 extracts were attenuated by ROCK inhibitor Y-27632. This indicated that both PM2.5 extracts could cause damage to epithelial cells through RhoA/ROCK-dependent NF-кB activation. Furthermore, the upregulation of macrophage adhesion induced by both PM2.5 extracts was also attenuated by Y-27632 in a co-culture model of macrophages and the epithelial cells. Therefore, our results support that industrial PM2.5 extracts-induced activation of the RhoA/ROCK-dependent NF-кB pathway induces pulmonary adverse effect. Thus, pharmacological inhibition of ROCK activation might have therapeutic potential in preventing lung disease associated with PM2.5.

Sodium tanshinone IIA sulfonate suppresses pulmonary fibroblast proliferation and activation induced by silica: role of the Nrf2/Trx pathway.

Alveolar macrophages are believed to induce oxidative stress via reactive oxygen species (ROS) when silica particles are inhaled. This process can contribute to the pathogenesis of silicosis, but the mechanism is unclear. A traditional Chinese herbal derivative, sodium tanshinone IIA sulfonate (STS), displays significant antioxidant effects. Here, we determine whether STS can attenuate the oxidative stress induced by silica. Traditionally, studies on the toxic effects of silica have focused on monocultures of macrophages or fibroblasts. A coculture model of macrophages (Raw 264.7) and pulmonary fibroblasts (MRC-5) was used in this study to mimic a more in vivo-like environment. We investigated the protective effects of STS on the abnormal proliferation of MRC-5 fibroblasts in an in vitro model. The results showed that fibroblast viability increased with the accumulation of intracellular ROS induced by cocultured Raw 264.7 cells after silica exposure. Treatment with STS markedly ameliorated the silica-induced cell proliferation and oxidative stress. Western blotting and immunofluorescence analysis of the Nrf2 and thioredoxin (Trx) system were conducted, and the results confirmed that treatment with STS enhanced nuclear Nrf2 accumulation and mediated antioxidant Trx system expression. These findings suggest that silica exposure might induce some level of oxidative stress in fibroblasts and that STS might augment antioxidant activities via up-regulation of the Nrf2 and Trx system pathways in MRC-5 cells in vitro.

Exposure of tumor-associated macrophages to apoptotic MCF-7 cells promotes breast cancer growth and metastasis.

Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer. To clarify the mechanisms underlying the crosstalk between TAMs and cancer stem cells (CSCs) in breast cancer recurrence and metastasis, we used a co-culture model of macrophages and apoptotic human breast cancer cell line MCF-7 cells to investigate the effects of TAMs on MCF-7 in vitro and in vivo. Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay. The macrophages exposed to apoptotic cells also induce an increase in the proportion of CD44+/CD24− cancer stem-like cells, as well as their proliferative ability accompanied with an increase in mucin1 (MUC1) expression. During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-β1 and HIF-1α. Our results indicate that when cancer cells endure chemotherapy induced apoptosis, macrophages in their microenvironment can then activate cancer stem cells to promote cancer growth and metastasis by secreting IL-6, which activates STAT3 phosphorylation to regulate the transcription of its downstream target genes.