Leukotriene B4 Loaded in Microspheres Inhibits Osteoclast Differentiation and Activation

Abstract To investigate osteoclast formation in vivo and if leukotriene B4 (LTB4) loaded in microspheres (MS) could be used as a therapeutical strategy to promote a sustained delivery of the mediator and prevent osteoclast differentiation. Methods: In vivo, apical periodontitis was induced in mice to investigate osteoclast differentiation and signaling in absence of 5-lipoxygenase (5-LO). In vitro, LTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 macrophages were cultured in the presence of monocyte colony-stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity, in vitro MS-LTB4 uptake, osteoclast formation and gene expression were measured. Results: We found that 5-LO negatively regulates osteoclastic formation in vivo during apical periodontitis development. In vitro, LTB4-MS were up-taken by macrophages and were not cytotoxic to the cells. LTB4-MS inhibited osteoclast formation and the synthesis of osteoclastogenic genes Acp5, Mmp9, Calcr and Ctsk. LTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus.

Resumo O objetivo deste trabalho foi Investigar a formação de osteoclastos in vivo e se o leucotrieno B4 (LTB4) incorporado em microesferas (MS) poderia ser usado como estratégia terapêutica para promover uma entrega sustentada do mediador e prevenir a diferenciação dos osteoclastos. Métodos: In vivo, a periodontite apical foi induzida em camundongos para investigar a diferenciação e sinalização de osteoclastos na ausência de 5-lipoxigenase (5-LO). In vitro, LTB4-MS foi preparado usando um processo de evaporação e extração de solvente de emulsão de óleo em água. A caracterização e a eficiência do encapsulamento do LTB4 foram investigadas. Macrófagos J774A.1 foram cultivados na presença de fator estimulador de colônia de monócitos (M-CSF) e ligante para o receptor ativador do fator nuclear kappa B (RANKL) e, então, estimulados com LTB4-MS. Citotoxicidade, captação in vitro de MS-LTB4, formação de osteoclastos e expressão gênica foram avaliadas. Resultados: A via 5-LO regula negativamente a formação de osteoclastos in vivo durante o desenvolvimento da periodontite apical. In vitro, LTB4-MS foram fagocitadas pelos macrófagos e não foram citotóxicos para as células. LTB4-MS inibiu a formação de osteoclastos e a síntese dos genes pró-osteoclastogênicos Acp5, Mmp9, Calcr e Ctsk. Conclusões: LTB4-MS inibiu a diferenciação de macrófagos em um fenótipo osteoclástico e a ativação celular sob estímulo de M-CSF e RANKL.

La sucralosa promueve la polarización a macrófagos proinflamatorios M1

RESUMEN La sucralosa es un edulcorante no calórico de amplio consumo a nivel mundial, es considerado como un aditivo seguro, debido a que es eliminado en periodos cortos de tiempo. Recientemente se evidenció su bioacumulación en tejido adiposo, donde se encuentran inmersos macrófagos, células del sistema inmune involucradas en el desarrollo de la inflamación sistémica de bajo grado. A la fecha, no se cuenta con suficiente información para demostrar si los edulcorantes potencian los procesos inflamatorios alterando la función de células presentes en tejido y/o contribuyen en el desarrollo de patologías metabólicas. Por lo anterior, en nuestro trabajo se evaluó el efecto de la sucralosa en la viabilidad de los macrófagos diferenciados de la línea celular monocítica THP-1, por azul de tripán y ensayos de MTT, así como su efecto en la polarización M1/M2 por PCR según la expresión de IRF4, IRF5, STAT1, STAT6, perfil de expresión de IL-6, IL-12, TNF-α, TGF-β, IL-10 y SOCS3 por qPCR, y la cuantificación de la quimiocina IP-10 por ELISA. Los resultados indicaron que la sucralosa no tiene efectos citotóxicos, pero disminuye el número de células viables metabólicamente activas determinadas por MTT de manera dependiente de la concentración. La sucralosa incrementa la concentración de la quimiocina IP-10 y la expresión génica del factor de transcripción IRF5 y disminuye la expresión de IRF4 y STAT6, favoreciendo la polarización hacia poblaciones M1. La bioacumulación de sucralosa en tejido adiposo, y su interacción con macrófagos, podría inducir su polarización a M1.

ABSTRACT Sucralose is a non-nutritive sweetener widely consumed worldwide; it is considered a safe additive because it is eliminated quickly. Recently its bioaccumulation in adipose tissue was evidenced, where macrophages, cells of the immune system involved in developing low-grade systemic inflammation, are found. To date, there is a paucity of information regarding whether sweeteners potentiate inflammatory processes by altering the function of cells present in tissue and/or contribute to the development of metabolic pathologies. We evaluate the effect of sucralose on the viability of differentiated macrophages of the monocytic cell line THP-1, by trypan blue and MTT assays, respectively, as well as its effect on M1/ M2 by PCR according to the expression of IRF4, IRF5, STAT1, STAT6, expression profile of IL6, IL-12, TNF-α, TGF-β, IL-10 and SOCS3 by qPCR, and the quantification of the chemokine IP-10 by ELISE. The results indicated that sucralose has no cytotoxic effects but decreases the number of metabolically active viable cells determined by MTT of macrophages in a concentration-dependent manner. Sucralose increased the concentration of the chemokine IP-10 and the gene expression of the transcription factors IRF5 and decreased the expression of IRF4 and STAT 6 gene expression, favoring polarization towards M1 populations. The bioaccumulation of sucralose in adipose tissue, and its interaction with macrophages, could induce its polarization to M1.

Identificación de macrófagos y virus Epstein-Barr en biopsias de pacientes con Síndrome de Sjögren

RESUMEN El síndrome de Sjögren (SS) es una enfermedad crónica mediada inmunológicamente. La presencia de macrófagos y el virus Epstein-Barr (VEB) se ha relacionado con su desarrollo y severidad. Los macrófagos contribuyen al proceso autoinmune local y la infección viral promueve el quiebre de la auto-tolerancia. Objetivos. Identificar la presencia de Macrófagos en el infiltrado inflamatorio y VEB en células inflamatorias, correlacionándolos con las características histológicas de glándulas salivales labiales. Metodología. En biopsias de glándulas salivales labiales (8 pacientes y 7 individuos controles) se realizó inmunohistoquímica antiCD-68 para identificar macrófagos. El conteo de macrófagos y células inflamatorias se efectuó en relación a su distribución en las glándulas salivales. La presencia del virus fue evaluada mediante hibridación in situ e inmunohistoquímica para LMP1. Se utilizó el test t no pareado y de Mann-Whitney para comparar los grupos, y coeficiente de correlación de Pearson para correlacionar con parámetros histológicos. Resultados. Se observó un mayor número de macrófagos en el infiltrado inflamatorio de pacientes (p=0,001**). Los macrófagos se distribuyeron difusamente en las glándulas de controles y en los focos inflamatorios de pacientes. En ambos grupos no se detectó la presencia del virus Epstein-Barr. Conclusión. Los pacientes con síndrome de Sjögren presentaron mayor presencia de macrófagos y su incremento es a expensas del foco inflamatorio.

ABSTRACT: Sjögren's syndrome (SS) is an immunologically mediated chronic disease of complex etiopathogenesis. Macrophages and Epstein-Barr virus are among the factors related to its development and severity. Macrophages contribute to the local autoimmune process and viral infection promotes the breakdown of self-tolerance. Objectives. Identify the presence of macrophages in the inflammatory infiltrate and Epstein-Barr virus in inflammatory cells, correlating them with the histological features of labial salivary glands. Methodology. In labial salivary glands biopsies of 8 patients and 7 control individuals, anti-CD-68 immunohistochemistry was performed to identify macrophages. The macrophages and inflammatory cells were counted in relation to their distribution in the salivary glands. The presence of the virus was evaluated by in situ hybridization for viral RNA and immunohistochemistry for latent membrane protein type 1. The comparison between both groups was made using the unpaired t-test and Mann-Whitney test. The correlations with histological parameters were established with the Pearson´s correlation coefficient. Results. A greater number of macrophages was observed in the inflammatory infiltrate of SS patients (p=0,001**). Macrophages in control individuals were diffusely distributed in the gland, while, SS in patients, they were mainly located in inflammatory foci. In both groups, no inflammatory or epithelial cells infected by the Epstein-Barr virus were identified. Conclusion. Patients with Sjögren's syndrome had a greater presence of macrophages and their increase is at the expense of the inflammatory focus.

Interleukin-17A is closely correlated with the progression of renal epithelial-mesenchymal transition in spontaneously hypertensive rats / 南方医科大学学报

OBJECTIVE@#To explore the role of interleukin-17A (IL-17A) in renal epithelial- mesenchymal transition (EMT) in essential hypertensive nephropathy.@*METHODS@#Four-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (control group) were both randomized into 4 groups (n=5) for observation at 4, 6, 10 and 30 weeks of age. Blood pressure of the rats was monitored using a noninvasive tail artery blood pressure measurement instrument. The percentage of Th17 cells in the splenocytes was analyzed using flow cytometry. The mRNA and protein expression levels of IL-17A, iNOS, Arg-1, E-cadherin, and α-SMA in the kidneys of the rats were detected using RT-PCR and immunohistochemical staining, respectively, and plasma levels of IL-17A were regularly detected using ELISA.@*RESULTS@#At the age of 6 weeks, the SHRs began to show significantly higher blood pressure with greater Th17 cell percentage in the splenocytes and high renal expression and plasma level of IL-17A than WKY rats (P < 0.05 or P < 0.01). At 30 weeks, renal expression of E-cadherin mRNA and protein was significantly lower and the expression of Arg-1 mRNA and protein was significantly higher in SHR than in WKY rats (P < 0.01). Compared with the WKY rats, the SHRs showed significantly higher mRNA and protein expressions of iNOS at 6 and 10 weeks (P < 0.05 or 0.01) and higher α-SMA mRNA and protein expressions since 10 weeks of age (P < 0.05 or 0.01). In SHRs older than 10 weeks, renal IL-17A mRNA and protein expression levels were negatively correlated with those of E-cadherin (r=-0.731, P < 0.05; r=-0.827, P < 0.01) and positively correlated with those of α-SMA (r=0.658, P < 0.05; r=0.968, P < 0.01).@*CONCLUSION@#IL-17A is closely correlated with the progression of renal EMT in SHR and plays its role possibly by mediating M1/M2 polarization of renal infiltrating macrophages.

Interference of P2X4 receptor expression in tumor-associated macrophages suppresses migration and invasion of glioma cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells.@*METHODS@#C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment.@*RESULTS@#The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA(P < 0.01)and protein (P < 0.01, P < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P < 0.05).@*CONCLUSION@#Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.

Estradiol inhibits differentiation of mouse macrophage into a pro-inflammatory phenotype by upregulating the IRE1α-XBP1 signaling axis / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway.@*METHODS@#Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR.@*RESULTS@#Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist.@*CONCLUSION@#Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.

Effect of hepatocellular carcinoma cell-derived exosomes on M2 polarization of tumor-associated macrophages / 临床肝胆病杂志

Objective To investigate the effect of exosomes derived from hepatocellular carcinoma cells on the polarization of tumor-associated macrophages (TAMs), and to reveal the novel mechanism of hepatocellular carcinoma formation. Methods Hepatocellular carcinoma cell-derived exosomes were isolated by ultracentrifugation, and the characteristics of exosomes were identified by transmission electron microscope (TEM), Dynamic Light Scattering (DLS), and Western blotting. The model of macrophage polarization was induced and verified by quantitative real-time PCR and Western blotting. The t -test was used for comparison of normally distributed continuous data between two groups. A one-way analysis of variance was used for comparison between multiple groups, and the LSD- t -test was used for further comparison between two groups. Results TEM showed that hepatocellular carcinoma cell-derived exosomes were round or oval vesicles, LDS showed that the exosomes had a particle size of 172.65±2.34 nm, and Western blotting showed highly positive expression of the biomarkers TSG101 and CD63 in exosomes. There was a significant increase in the expression of CD68 after the addition of 15 ng phorbol ester to induce human-derived mononuclear macrophages for 24 hours to achieve adherent growth (1.00±0.25 vs 6.67±0.98, t =11.20, P < 0.001). Western blotting showed that compared with the control group (L02 cell-derived exosomes), the hepatocellular carcinoma cell-derived exosomes (at low, middle, and high doses) induced M2 polarization of macrophages and increased the expression of the markers Arg-1 and CD163 (all P < 0.05). Conclusion Hepatocellular carcinoma cell-derived exosomes promote M2 polarization of TAMs.

Infiltration of tumor-associated macrophages and its association with prognosis of patients with diffuse large B-cell lymphoma based on gene expression profile data / 白血病·淋巴瘤

Objective:To analyze the infiltration of tumor-associated macrophages and their subtypes, and to investigate their association with prognosis of patients with diffuse large B-cell lymphoma (DLBCL) based on the gene chip expression database.Methods:The data were retrieved from microarray (Affymetrix U133 plus 2.0) database (No:GSE10846) of DLBCL patients in PubMed gene expression omnibus (GEO). The database included 414 DLBCL patients, among which 306 cases had complete clinical, cell of origin phenotype (COO subtyping), treatment and follow-up information. The data analysis was performed on the online computer program which could identify the cell-type (CIBERSORT) by estimating relative percentage of RNA transcripts. From the returned result file, the percentage of immune cells including macrophages subtypes of all cases in all identifiable immune cells in the microenvironment was identified in GSE10846 database. Taking the median percentage of macrophages subsets in all types of immune cells as cut-off value; ≥ cut-off value was high infiltration and < cut-off value was low infiltration. The median value of gene RNA expression level of myc, bcl-2, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2) of 414 DLBCL patients in the GSE10846 database was treated as the cut-off value; ≥ cut-off value was the high expression and < cut-off value was the low expression. The correlation of the expression levels of all subsets and total macrophages with clinical factors, gene expression, survival was analyzed; Cox proportional hazard model was used to make multivariate analysis of the prognosis for DLBCL patients. surv_cutpoint function of surv_miner package in R 4.0.4 software was used for the optimal cut-off value of the percentage of macrophages subsets in all immune cells in the microenvironment; the result less than the optimal cut-off value was statistically low infiltration and the result greater than or equal to the optimal cut-off value was statistically high infiltration.Results:CIBERSORT analysis showed that M0 macrophages [15.00% (0-44.41%)], M1 macrophages [7.46% (0-23.00%)] and M2 macrophages [6.28% (0-43.35%)] in the tumor microenvironment were identified in all 414 DLBCL cases. Among 306 patients with complete clinical and follow-up data, there were 155 cases (50.7%), 152 cases (49.7%), 156 cases (51.0%), 152 cases (49.7%), respectively in high infiltration patients with M0, M1, M2 and total macrophages; the high infiltration of M0 macrophages was correlated with COO subtyping germinal center B-cell (GCB) type and the high expression of PD-L1 gene, the absence of myc and bcl-2 double high expression at RNA level (R-DEL) (all P < 0.05); the high infiltration of M1 macrophages was correlated with female, the high expression of PD-L1 gene and PD-L2 gene (all P < 0.05); the high infiltration of M2 macrophages was correlated with COO subtyping GCB type, the high expression of PD-L2 gene (all P < 0.05); the high infiltration of total macrophages was correlated with female, COO subtyping GCB type, the high expression of PD-L1 gene and PD-L2 gene, the absence of R-DEL (all P < 0.05).The high expression of PD-L1 gene was associated with high infiltration of M0, M1 and total macrophages (all P < 0.01), and high PD-L2 gene expression was correlated with high infiltration of M1, M2 and total macrophages (all P < 0.01). The overall survival (OS) of M0 macrophage high infiltration group was better than that of the lower infiltration group ( P = 0.002); the OS of M2 macrophage low infiltration group was better than that of the high infiltration group ( P = 0.019). The OS of R-DEL group was worse than that of R-DEL absent group ( P = 0.001). The patients with low international prognostic index (IPI) score (0-2), COO subtyping GCB type, and treatment with rituximab had better OS (all P < 0.01). Multivariate Cox regression analysis showed that 60 years or above, COO subtyping non-GCB type, treatment without rituximab, M0 macrophage low infiltration, M2 macrophage high infiltration were all independent adverse prognostic factors for OS of DLBCL patients (all P < 0.05). The optimal cut-off value for M0 macrophages was 4.3%, and the optimal cut-off value for M2 macrophages was 4.8%, and the OS in the group with statistically low infiltration of M0 macrophage was worse ( P < 0.001), and so was the OS in the group with statistically high infiltration of M2 macrophage ( P = 0.001). Conclusions:Tumor-associated macrophage is confirmed as the most abundant immune cells in the tumor microenvironment of DLBCL. Patients with high infiltration of M2 macrophage have poor prognosis, while high infiltration of M0 macrophage indicates a better prognosis.

Emerging role of natural products in cancer immunotherapy

Cancer immunotherapy has become a new generation of anti-tumor treatment, but its indications still focus on several types of tumors that are sensitive to the immune system. Therefore, effective strategies that can expand its indications and enhance its efficiency become the key element for the further development of cancer immunotherapy. Natural products are reported to have this effect on cancer immunotherapy, including cancer vaccines, immune-check points inhibitors, and adoptive immune-cells therapy. And the mechanism of that is mainly attributed to the remodeling of the tumor-immunosuppressive microenvironment, which is the key factor that assists tumor to avoid the recognition and attack from immune system and cancer immunotherapy. Therefore, this review summarizes and concludes the natural products that reportedly improve cancer immunotherapy and investigates the mechanism. And we found that saponins, polysaccharides, and flavonoids are mainly three categories of natural products, which reflected significant effects combined with cancer immunotherapy through reversing the tumor-immunosuppressive microenvironment. Besides, this review also collected the studies about nano-technology used to improve the disadvantages of natural products. All of these studies showed the great potential of natural products in cancer immunotherapy.

Engineering a folic acid-decorated ultrasmall gemcitabine nanocarrier for breast cancer therapy: Dual targeting of tumor cells and tumor-associated macrophages

Combination of passive targeting with active targeting is a promising approach to improve the therapeutic efficacy of nanotherapy. However, most reported polymeric systems have sizes above 100 nm, which limits effective extravasation into tumors that are poorly vascularized and have dense stroma. This will, in turn, limit the overall effectiveness of the subsequent uptake by tumor cells via active targeting. In this study, we combined the passive targeting via ultra-small-sized gemcitabine (GEM)-based nanoparticles (NPs) with the active targeting provided by folic acid (FA) conjugation for enhanced dual targeted delivery to tumor cells and tumor-associated macrophages (TAMs). We developed an FA-modified prodrug carrier based on GEM (PGEM) to load doxorubicin (DOX), for co-delivery of GEM and DOX to tumors. The co-delivery system showed small particle size of ∼10 nm in diameter. The ligand-free and FA-targeted micelles showed comparable drug loading efficiency and a sustained DOX release profile. The FA-conjugated micelles effectively increased DOX uptake in cultured KB cancer cells that express a high level of folate receptor (FR), but no obvious increase was observed in 4T1.2 breast cancer cells that have a low-level expression of FR. Interestingly, in vivo, systemic delivery of FA-PGEM/DOX led to enhanced accumulation of the NPs in tumor and drastic reduction of tumor growth in a murine 4T1.2 breast cancer model. Mechanistic study showed that 4T1.2 tumor grown in mice expressed a significantly higher level of FOLR2, which was selectively expressed on TAMs. Thus, targeting of TAM may also contribute to the improved in vivo targeted delivery and therapeutic efficacy.

TGR5 deficiency activates antitumor immunity in non-small cell lung cancer via restraining M2 macrophage polarization

The bile acid-responsive G-protein-coupled receptor TGR5 is expressed in monocytes and macrophages, and plays a critical role in regulating inflammatory response. Our previous work has shown its role in promoting the progression of non-small cell lung cancer (NSCLC), yet the mechanism remains unclear. Here, using Tgr5-knockout mice, we show that TGR5 is required for M2 polarization of tumor-associated macrophages (TAMs) and suppresses antitumor immunity in NSCLC via involving TAMs-mediated CD8+ T cell suppression. Mechanistically, we demonstrate that TGR5 promotes TAMs into protumorigenic M2-like phenotypes via activating cAMP-STAT3/STAT6 signaling. Induction of cAMP production restores M2-like phenotypes in TGR5-deficient macrophages. In NSCLC tissues from human patients, the expression of TGR5 is associated with the infiltration of TAMs. The co-expression of TGR5 and high TAMs infiltration are associated with the prognosis and overall survival of NSCLC patients. Together, this study provides molecular mechanisms for the protumor function of TGR5 in NSCLC, highlighting its potential as a target for TAMs-centric immunotherapy in NSCLC.

Bioresponsive immune-booster-based prodrug nanogel for cancer immunotherapy

The combination of chemotherapy and immunotherapy motivates a potent immune system by triggering immunogenic cell death (ICD), showing great potential in inhibiting tumor growth and improving the immunosuppressive tumor microenvironment (ITM). However, the therapeutic effectiveness has been restricted by inferior drug bioavailability. Herein, we reported a universal bioresponsive doxorubicin (DOX)-based nanogel to achieve tumor-specific co-delivery of drugs. DOX-based mannose nanogels (DM NGs) was designed and choosed as an example to elucidate the mechanism of combined chemo-immunotherapy. As expected, the DM NGs exhibited prominent micellar stability, selective drug release and prolonged survival time, benefited from the enhanced tumor permeability and prolonged blood circulation. We discovered that the DOX delivered by DM NGs could induce powerful anti-tumor immune response facilitated by promoting ICD. Meanwhile, the released mannose from DM NGs was proved as a powerful and synergetic treatment for breast cancer in vitro and in vivo, via damaging the glucose metabolism in glycolysis and the tricarboxylic acid cycle. Overall, the regulation of tumor microenvironment with DOX-based nanogel is expected to be an effectual candidate strategy to overcome the current limitations of ICD-based immunotherapy, offering a paradigm for the exploitation of immunomodulatory nanomedicines.

The disbalance of LRP1 and SIRPα by psychological stress dampens the clearance of tumor cells by macrophages

The relationship between chronic psychological stress and tumorigenesis has been well defined in epidemiological studies; however, the underlying mechanism remains underexplored. In this study, we discovered that impaired macrophage phagocytosis contributed to the psychological stress-evoked tumor susceptibility, and the stress hormone glucocorticoid (GC) was identified as a principal detrimental factor. Mechanistically, GC disturbed the balance of the "eat me" signal receptor (low-density lipoprotein receptor-related protein-1, LRP1) and the "don't eat me" signal receptor (signal regulatory protein alpha, SIRPα). Further analysis revealed that GC led to a direct, glucocorticoid receptor (GR)-dependent trans-repression of LRP1 expression, and the repressed LRP1, in turn, resulted in the elevated gene level of SIRPα by down-regulating miRNA-4695-3p. These data collectively demonstrate that stress induces the imbalance of the LRP1/SIRPα axis and entails the disturbance of tumor cell clearance by macrophages. Our findings provide the mechanistic insight into psychological stress-evoked tumor susceptibility and indicate that the balance of LRP1/SIRPα axis may serve as a potential therapeutic strategy for tumor treatment.

Tanshinone IIA prevents acute lung injury by regulating macrophage polarization / 中西医结合学报

OBJECTIVE@#Acute lung injury (ALI) is a serious respiratory dysfunction caused by pathogen or physical invasion. The strong induced inflammation often causes death. Tanshinone IIA (Tan-IIA) is the major constituent of Salvia miltiorrhiza Bunge and has been shown to display anti-inflammatory effects. The aim of the current study was to investigate the effects of Tan-IIA on ALI.@*METHODS@#A murine model of lipopolysaccharide (LPS)-induced ALI was used. The lungs and serum samples of mice were extracted at 3 days after treatment. ALI-induced inflammatory damages were confirmed from cytokine detections and histomorphology observations. Effects of Tan-IIA were investigated using in vivo and in vitro ALI models. Tan-IIA mechanisms were investigated by performing Western blot and flow cytometry experiments. A wound-healing assay was performed to confirm the Tan-IIA function.@*RESULTS@#The cytokine storm induced by LPS treatment was detected at 3 days after LPS treatment, and alveolar epithelial damage and lymphocyte aggregation were observed. Tan-IIA treatment attenuated the LPS-induced inflammation and reduced the levels of inflammatory cytokines released not only by inhibiting neutrophils, but also by macrophage. Moreover, we found that macrophage activation and polarization after LPS treatment were abrogated after applying the Tan-IIA treatment. An in vitro assay also confirmed that including the Tan-IIA supplement increased the relative amount of the M2 subtype and decreased that of M1. Rebalanced macrophages and Tan-IIA inhibited activations of the nuclear factor-κB and hypoxia-inducible factor pathways. Including Tan-IIA and macrophages also improved alveolar epithelial repair by regulating macrophage polarization.@*CONCLUSION@#This study found that while an LPS-induced cytokine storm exacerbated ALI, including Tan-IIA could prevent ALI-induced inflammation and improve the alveolar epithelial repair, and do so by regulating macrophage polarization.

Dental pulp stem cell-derived apoptotic bodies regulate macrophage polarization and inflammatory response / 口腔疾病防治

Objective@#To investigate the effects of apoptotic bodies (ABs) derived from dental pulp stem cells (DPSCs) on macrophage polarization and inflammation response in vivo. @*Methods @#Human DPSCs were extracted, cultured and identified. Staurosporine was used to apoptosis induction and differential methods were performed for ABs identification. The in vitro cultured macrophages were divided into 3 groups: solvent control, lipopolysaccharide (LPS), and the LPS+ABs. The macrophages were stimulated with LPS to induce inflammation followed by ABs treatment. In the untreated group, macrophages were added with an equal amount of solvent. The specific uptake of ABs by macrophages, the expression level of CD206 and the levels of inflammatory cytokines were analyzed. The mouse models of cutaneous wounds and dextran sulfate sodium (DSS)-induced colitis were established, and the mice were randomly divided into 3 groups: the PBS-treated group, the DPSCs-treated group, and the ABs-treated group. The mice were injected with the same volume of PBS, DPSCs and ABs, respectively. The body weight, histological pathology, the expression levels of CD206 and cytokines, and the extent of tissue regeneration were measured.@* Results @#DPSCs and ABs derived from DPSCs were successfully isolated and characterized. ABs could be taken up by macrophage. While lipopolysaccharide(LPS) induced production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), ABs significantly reduced the levels of these pro-inflammatory cytokines and increased the expression of transforming growth factor-β (TGF-β) and CD206 (P < 0.01). In the cutaneous inflammatory wound model, the wound closure rate in mice intravenously injected with ABs was significantly accelerated (P < 0.05). The administration of ABs markedly reduced the pro-inflammatory factors levels and increased the CD206+ cell number. In the colitis model, treatment with ABs markedly reduced the loss in bodyweight (P < 0.05), recovered the colon length (P < 0.01), and significantly increased the CD206+ cell number.@* Conclusion@# DPSCs-derived ABs could enhance macrophage M2 polarization and attenuate inflammation. Therefore, ABs could be used as a promising cell replacement for inflammatory regulation and tissue regeneration.

Effect of Huatan Qushi Huoxue prescription on lipopolysaccharide-induced pyroptosis of macrophages / 临床肝胆病杂志

Objective To investigate the effect of Huatan Qushi Huoxue prescription on lipopolysaccharide (LPS)-induced pyroptosis of RAW264.7 cells and its mechanism. Methods An in vitro cell model of LPS-induced activated RAW264.7 was established and divided into blank group, model group, high-, middle-, and low-dose Huatan Qushi Huoxue prescription groups, and control group. The corresponding drug-containing serum intervention was performed for 24 hours. A scanning electron microscope was used to observe cell morphology, and immunofluorescence assay was used to perform quantitative localization of GSDMD-N. Results The cells in the blank group were round and regular in shape with smooth surface, and those in the control group were swollen, with folds on the surface and gaps in the capsule, which were consistent with the morphology of cell pyroptosis. The cells in the control group had bubbles on the surface with obvious pseudopodia and pores in cell membrane, and those in the high-dose group were not swollen and had a rough surface with pseudopodia, with no obvious pores in cell membrane. The cells in the low- and middle-dose groups were swollen and had a rough surface of cell membrane with pores and pseudopodia. Immunofluorescence assay showed that compared with the blank group, the model group had a significant increase in the positive staining intensity of GSDMD-N, and compared with the model group, the control group and the Traditional Chinese medicine group had a reduction in the positive staining intensity of GSDMD-N. Conclusion Huatan Qushi Huoxue prescription can improve the pyroptosis of macrophages and reduce the expression of GSDMD-N.

Gastric cancer-derived mesenchymal stem cells regulate the M2 polarization of macrophages within gastric cancer microenvironment via JAK2/STAT3 signaling pathway / 中华肿瘤杂志

Objective: To investigate the role and mechanism of tumor-derived mesenchymal stem cells in regulating the M2 polarization of macrophages within gastric cancer microenvironment. Methods: Gastric cancer tissues and the adjacent non-cancerous tissues were collected from patients underwent gastric cancer resection in the First People's Hospital of Lianyungang during 2018. In our study, THP-1-differentiated macrophages were co-cultured with gastric cancer-derived mesenchymal stem cells (GC-MSCs). Then, the M2 subtype-related gene, the markers expressed on cell surface and the cytokine profile were analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry and Luminex liquid chip, respectively. The key cytokines mediating the inducing effect of GC-MSCs on macrophage polarization into the M2 subtype were detected and screened by Luminex liquid chip, which were further confirmed by the neutralizing antibody test. The expressions of macrophage proteins involved in M2 polarization-related signaling pathways under the different co-culture conditions of GC-MSCs were detected by western blot. Results: In Mac+ GC-MSC-culture medium (CM) group, the expression levels of Ym-1 and Fizz-1 (1.53±0.32 and 13.22±1.05, respectively), which are markers for M2 subtype, were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.60±0.41) was significantly lower than that of Mac group (1.06±0.38, P=0.023). In Mac+ GC-MSC-Transwell (TW) group, the expression levels of Ym-1 and Fizz-1 (1.47±0.09 and 13.16±2.77, respectively) were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.56±0.03) was significantly lower than that of Mac group (1.06±0.38, P=0.026). The ratios of CD163(+) /CD204(+) cells in Mac+ GC-MSC-CM and Mac+ GC-MSC-TW groups (3.80% and 4.40%, respectively) were both remarkably higher than that of Mac group (0.60%, P<0.05). The expression levels of IL-10, IL-6, MCP-1 and VEGF in Mac+ GC-MSC-CM group were (592.60±87.52), (1 346.80±64.70), (11 256.00±29.03) and (1 463.90±66.67) pg/ml, respectively, which were significantly higher than those of Mac group [(41.03±2.59), (17.35±1.79), (5 213.30±523.71) and (267.12±12.06) pg/ml, respectively, P<0.05]. The levels of TNF-α, IP-10, RANTES and MIP-1α were (95.57±9.34), (410.48±40.68), (6 967.30±1.29) and (1 538.70±283.04) pg/ml, which were significantly lower than those of Mac group [(138.01±24.31, (1 298.60±310.50), (14 631.00±4.21) and (6 633.20±1.47) pg/ml, respectively, P<0.05]. The levels of IL-6 and IL-8 in GC-MSCs [(11 185.02±2.82) and (12 718.03±370.17) pg/ml, respectively] were both strikingly higher than those of MSCs from adjacent non-cancerous gastric cancer tissues [(270.71±59.38) and (106.04±32.84) pg/ml, repectively, P<0.05]. The ratios of CD86(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (28.80% and 31.40%, respectively) were both higher than that of Mac+ GC-MSC-CM group (24.70%). Compared to Mac+ GC-MSC-CM group (13.70%), the ratios of CD204(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (9.90% and 8.70%, separately) were reduced. The expression levels of p-JAK2 and p-STAT3, which are proteins of macrophage M2 polarization-related signaling pathway, in Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, respectively) were significantly higher than those of Mac group (0.50±0.01 and 0.82±0.01, respectively, P<0.05). The expression levels of p-JAK2 in Mac+ IL-6-blocked-GC-MSC-CM group (0.47±0.02) were significantly lower those that of Mac+ GC-MSC-CM group (0.86±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-8-blocked-GC-MSC-CM group (0.50±0.01 and 0.85±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-6/IL-8-blocked-GC-MSC-CM group (0.37±0.01 and 0.65±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). Conclusion: GC-MSCs promote the activation of JAK2/STAT3 signaling pathway in macrophages via high secretions of IL-6 and IL-8, which subsequently induce the macrophage polarization into a pro-tumor M2 subtype within gastric cancer microenvironment.

The role of TSG-6 in inhibiting the secretion of IL-1β by bone-marrow-derived macrophages / 中国职业医学

@#Objective To investigate the role of tumor necrosis factor alpha stimulated gene/inducible protein 6（TSG-6）on free silica（SiO2 ）-induced secretion of pro-inflammatory cytokine interleukin（IL）-1β by bone-marrow-derived macrophages （BMDMs）. Methods i）BMDMs isolated from bone marrow were divided into eight groups：the control group was untreated； lipopolysaccharide （LPS） group was stimulated by LPS with a concentration of 50 µg/L；The TSG-6 control group was stimulated by 100 µg/L of recombinant mouse TSG-6. SiO2 model group was pre-stimulated with LPS for four hours，and then stimulated with SiO2 suspension at a final concentration of 250 mg/L；Different dose of TSG-6 groups were firstly treated with above concentrations of LPS and SiO2 suspension，then 10，20，50 and 100 µg/L of recombinant mouse TSG-6 were added respectively；After 16 hours of culture，the cells were collected and the level of IL-1β in BMDMs supernatant was detected by enzyme-linked immunosorbent assay（ELISA）to screen optimal concentration of TSG-6. ii）The cells of the control group，LPS group，SiO2 model group，TSG-6 optimal concentration group and TSG-6 control group were collected. The expression of IL-1β and components of its related pathways in BMDMs was detected by Western blot，including IL-1β，pro-IL-1β，caspase-1，pro dcaspase-1，asc type amino acid transport and NOD-like receptor protein 3（NLRP3）. Results i）Compared with the control group，the expression of IL-1β in SiO2 model group was increased significantly（P<0.01）. Compared with SiO2 model group，the expression of IL-1β in 20，50，100 µg/L dose of TSG-6 groups were decreased significantly（all P<0.01），and the optimal concentration of TSG-6 was found to be 100 µg/L. ii）Compared with the control group and LPS group，the relative expression levels of IL-1β，caspase-1 and NLRP3 in SiO2 model group were increased significantly （all P<0.05）. Compared with SiO2 model group，the expression levels of IL-1β、caspase-1 and NLRP3 were decreased in 100 µg/L TSG-6 group（all P<0.05）. Conclusion TSG-6 could inhibit BMDMs to secret pro-inflammatory cytokine IL-1β by down-regulating the expression of NLRP3 and caspase-1.

Bushen Jiedu Prescription Inhibits Metastasis of Colorectal Cancer by Regulating Polarization of M2-TAMs in Vivo / 中国实验方剂学杂志

ObjectiveTo explore the inhibitory effect of Bushen Jiedu prescription （BSJDP） on the metastasis of colorectal cancer （CRC） via activation of M2 tumor-associated macrophages （M2-TAMs） in vivo. MethodThe model of xenograft tumor was established with C57BL/6 mice， and then the model mice were randomly assigned into blank control group， Bushen Jiedu recipe-low dose （11.2 g·kg-1） group （BSJDP-L）， and Bushen Jiedu Recipe-high dose （22.4 g·kg-1） group （BSJDP-H）. Tumors were abolished when the volume reached 1.5-2.0 cm3. The mice were sacrificed 28 days post tumor abolishing and then the lung metastasis was observed. Histopathological changes in lung metastasis were examined by hematoxylin-eosin （HE） staining of metastasis tissues， and immunofluorescence （IF） staining was employed to observe the effect of BSJDP on tumor apoptosis and hypoxia. Flow cytometry was performed to analyze the macrophages M1/M2 ratio of tumor tissue. The expression levels of the genes （Arg1， CD206， and CD163） associated with the polarization of M2-TAMs were determined by Real-time fluorescent quantitative polymerase chain reaction （Real-time PCR）. ResultThe metastasis rate was 70%， 40%， and 10% in the blank control group， BSJDP-L group， and BSJDP-H group， respectively. The lower metastasis rates of BSJDP-L and BSJDP-H groups proved that BSJDP significantly inhibited lung metastasis of CRC. Compared with the blank control group， BSJDP-L and BSJDP-H reduced the tumor cell infiltration in tumor tissue （P<0.01）， increased the apoptosis of tumor cells， alleviated the hypoxic environment， and down-regulated the expression of Arg1， CD206， and CD163 in the tumor tissue （P<0.01）. In addition， the ratio of M2 macrophages ranked in a descending order of blank control group （34.867%） > BSJDP-L group （22.033%） > BSJDP-H group （11.633%） （P<0.01）. ConclusionBSJDP inhibits the lung metastasis of CRC by inhibiting the activation of M2-TAMs in the tumor microenvironment.

Pathological Mechanism of HSOS in Rats Induced by Different Doses of Monocrotaline / 中国实验方剂学杂志

ObjectiveTo observe the pathological changes of hepatic sinusoidal obstruction syndrome （HSOS） induced by different doses of monocrotaline （MCT） in rats， investigate the dose and duration of modeling， and elucidate the mechanism. MethodA total of 72 male SD rats were randomized into normal group （n=12）， and low-， medium-， and high-dose MCT groups （n=20 per group， 80，120，160 mg·kg-1， respecctively）. In the model groups， different doses of MCT were intragastrically administered to induce the HSOS in rats. After 48 h and 120 h separately， rats in each group were sacrificed and sampling was performed. The survival rate of rats in each group was calculated， and the body weight， liver weight， and and serum liver function indexes of the rats were examined. The histopathological changes of the liver were observed based on scanning electron microscopy， hematoxylin and eosin （HE） staining， and Sirius red （SR） staining. Glutathione S-transferase （GST） activity， total superoxide dismutase （T-SOD） activity， and malondialdehyde （MDA） content of liver tissue homogenate were measured with microplate method. The expression of liver tissue-related indexes was detected by real-time polymerase chain reaction （PCR）， Western blot， and immunohistochemistry. ResultThe activity of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in MCT groups rose with the increase in MCT dose （P<0.05， P<0.01） compared with that in the normal group. With the extension of modeling time， the activity of serum ALT and AST in the low-dose group decreased （P<0.01）， while the activity of them in the medium-dose and high-dose groups increased （P<0.01）. HE staining showed that hepatocyte necrosis， inflammatory cell infiltration， and erythrocyte accumulation in MCT groups. Electron microscopy demonstrated that fenestrae of liver sinusoidal endothelial cells widened and the sieve plates disappeared. Morever， the injury was worsened with the increase in MCT dose. In addition， the expression of CD44 in MCT groups was significantly reduced compared with that in the normal group （P<0.05， P<0.01）. SR staining showed that no positive staining was found in model groups after 48 h， while collagen deposition in portal areas and liver sinusoids could be seen in model groups after 120 h. MCT groups showed increase in MDA content and GST activity and decrease in T-SOD activity compared with the normal group， particularly the medium-dose and high-dose groups （P<0.01）， and the changes were dose-dependent after 120 h （P<0.01）. The protein expression of CD68 （pro-inflammatory macrophage marker） was raised with the increase in dosage， which was consistent with the results of immunohistochemistry （P<0.01）， while CD163 （anti-inflammatory macrophage marker） protein and mRNA expression was significantly decreased with the increase in dosage （P<0.01）. Western blot results showed that the expression of phosphorylated nuclear factor-κB/nuclear factor-κB （p-NF-κB/NF-κB） and phosphorylated protein kinase B/protein kinase B （p-Akt/t-Akt） was significantly increased in medium-dose and high-dose MCT groups （P<0.05，P<0.01）. The protein expression of α-smooth muscle actin （α-SMA） in liver tissues in MCT groups was significantly increased over time and with the increase in dose， and the mRNA expression of α-SMA， collagen type I α1 （Col1a1）， and collagen type Ⅳ α1 （Col4a1） showed the same trend （P<0.05， P<0.01）. The results of TUNEL staining showed that apoptotic cells were increased with the rise of MCT dose， while B-cell lymphoma-2（Bcl-2） /Bcl-2 associated X protein （Bax） was remarkably decreased （P<0.01）. ConclusionHSOS in rats induced by intragastric administration of different doses of MCT was aggravated with the increase of dosage. In the low-dose （80 mg·kg-1） MCT group， the liver healed spontaneously over time. However， liver damage caused by MCT of 120 mg·kg-1 and 160 mg·kg-1 aggravated over time， and even fibrosis and death occurred. The pathological mechanism of MCT-induced HSOS in rats may be that MCT triggered intense oxidative stress in liver tissue， thus activated pro-inflammatory macrophages to secrete large amounts of inflammatory factors， and further activated the NF-κB/Akt signalling pathway， leading to severe cell damage and death.