Expression Of M2 Markers Research Articles

Macrophages with reduced expressions of classical M1 and M2 surface markers in human bronchoalveolar lavage fluid exhibit pro-inflammatory gene signatures

The classical M1/M2 polarity of macrophages may not be applicable to inflammatory lung diseases including chronic obstructive pulmonary disease (COPD) due to the complex microenvironment in lungs and the plasticity of macrophages. We examined macrophage sub-phenotypes in bronchoalveolar lavage (BAL) fluid in 25 participants with CD40 (a M1 marker) and CD163 (a M2 marker). Of these, we performed RNA-sequencing on each subtype in 10 patients using the Illumina NextSeq 500. Approximately 25% of the macrophages did not harbor classical M1 or M2 surface markers (double negative, DN), and these cells were significantly enriched in COPD patients compared with non-COPD patients (46.7% vs. 14.5%, p < 0.001). 1886 genes were differentially expressed in the DN subtype compared with all other subtypes at a 10% false discovery rate. The 602 up-regulated genes included 15 mitochondrial genes and were enriched in 86 gene ontology (GO) biological processes including inflammatory responses. Modules associated with cellular functions including oxidative phosphorylation were significantly down-regulated in the DN subtype. Macrophages in the human BAL fluid, which were negative for both M1/M2 surface markers, harbored a gene signature that was pro-inflammatory and suggested dysfunction in cellular homeostasis. These macrophages may contribute to the pathogenesis and manifestations of inflammatory lung diseases such as COPD.

ARG1 mRNA Level Is a Promising Prognostic Marker in Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV−) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV− tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers—cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)—was detected in HPV− patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

Exosomes from human umbilical cord mesenchymal stem cells attenuate the inflammation of severe steroid-resistant asthma by reshaping macrophage polarization

BackgroundSevere, steroid-resistant asthma (SSRA) is a serious clinical problem in asthma management. Affected patients have severe clinical symptoms, worsened quality of life, and do not respond to steroid, a mainstay steroid treatment of asthma. Thus, effective therapies are urgently needed. Exosomes derived from mesenchymal stem cell (MSC-Exo) has become attractive candidates for the lung inflammatory diseases through its immunomodulatory effects. In this study, we explored the therapeutic effects of MSC-Exo in SSRA and identified the therapeutic mechanism of MSC-Exo.MethodExosomes from human umbilical cord mesenchymal stem cell (hUCMSC) were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis and flow cytometry analysis. Effects of MSC-Exo on airway hyper responsiveness (AHR), inflammation, histopathology, and macrophage polarization in SSRA in mice were evaluated. Systematic depletion of macrophages determined the role of macrophages in the therapeutic effect of SSRA in mice. LPS-stimulated RAW 264.7 cell model was constructed to determine the underlying mechanism of MSC-Exo on macrophage polarization. qRT-PCR, Western blotting, immunofluorescence, and flow cytometry were performed to evaluate the expression of M1 or M2 markers. Tandem mass tags (TMT)-labeled quantitative proteomics were applied to explore the central protein during the regulation effect of MSC-Exo on macrophage polarization. Knockdown and overexpression of TRAF1 were used to further clarify the role of the central protein on macrophage polarization.ResultWe successfully isolated and characterized exosomes from hUCMSCs. We verified that the intratracheal administration of MSC-Exo reversed AHR, histopathology changes, and inflammation in SSRA mice. Systematic depletion of macrophages weakened the therapeutic effect of MSC-Exo. We found that MSC-Exo treatment inhibited M1 polarization and promoted M2 polarization in LPS-stimulated RAW 264.7 cells. Subsequently, tumor necrosis factor receptor-associated factor 1 (TRAF1) was determined as the central protein which may be closely related to the regulation of macrophage polarization from TMT-labeled quantitative proteomics analysis. Knockdown and overexpression of TRAF1 demonstrated that the effect of MSC-Exo treatment on macrophage polarization, NF-κB and PI3K/AKT signaling was dependent on TRAF1.ConclusionMSC-Exo can ameliorate SSRA by moderating inflammation, which is achieved by reshaping macrophage polarization via inhibition of TRAF1.

The mechanism of lncRNA‐CRNDE in regulating tumour‐associated macrophage M2 polarization and promoting tumour angiogenesis

M2 macrophages can promote liver cancer metastasis by promoting tumour angiogenesis; however, the mechanism underlying macrophage polarization has not been completely revealed. In this study, we mainly explored the mechanism underlying long non‐coding RNA‐CRNDE (lncRNA‐CRNDE) in regulating M2 macrophage polarization and promoting liver cancer angiogenesis. The expression of CRNDE was up‐regulated or down‐regulated in THP‐1 cells (CRNDE‐/‐‐THP‐1 cells and pcDNA3.1‐CRNDE‐THP‐1). THP‐1 cells were co‐cultured with liver cancer cell line H22, and M2 polarization was induced in THP‐1 by IL‐4/13 to simulate tumour‐induced macrophage polarization. As a result, after CRNDE overexpression, THP‐1 cell viability was up‐regulated, the expression of M2 membrane marker CD163 was up‐regulated, and the proportion of F4/80 + CD163+ cells was also up‐regulated. ELISA assay showed that the expression of M2 markers (including TGF‐β1 and IL‐10) and chemokines (including CCl22 and CCL22) was up‐regulated, and the expression of key signals (including STAT6, JAK‐1, p‐AKT1, and Arg‐1) was also up‐regulated, which were significantly different compared with the control group (Con). In addition, the intervention effect of CRNDE on THP‐1 was consistent between co‐culture with H22 cells and IL‐4/13 induction assay. The induced M2 THP‐1 cells were co‐cultured with HUVEC. As a result, THP‐1 cells with CRNDE overexpression can promote the migration and angiogenesis of HUVEC cells in vitro and simultaneously up‐regulate the expression of Notch1, Dll4 and VEGFR2, indicating that THP‐1 M2 polarization induced by CRNDE could further promote angiogenesis. The H22 cell tumour‐bearing mouse model was constructed, followed by injection of CRNDE anti‐oligosense nucleotides and overexpression plasmids to interfere CRNDE expression in tumour‐bearing tissues. Consequently, down‐regulation of CRNDE could down‐regulate tumour volume, simultaneously down‐regulate the expression of CD163 and CD31 in tissues, decrease the expression of key proteins (including JAK‐1, STAT‐6, p‐STAT6 and p‐AKT1), and down‐regulate the expression of key angiogenesis‐related proteins (including VEGF, Notch1, Dll4 and VEGFR2). In this study, we found that CENDE could indirectly regulate tumour angiogenesis by promoting M2 polarization of macrophages, which is also one of the mechanisms of microenvironmental immune regulation in liver cancer.

Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection.

The outcome of Leishmania donovani infection depends upon the dynamic interchanges between M1 and M2 macrophages. Information of the involvement of microRNAs (miRNAs) and epigenetic modifiers in regulating macrophage plasticity during L. donovani infection is still elusive. Differential expression analysis of polarization-regulating miRNAs, revealed significant enrichment of miR146a-5p during Leishmania donovani infection. A sustained enrichment of miR146a-5p was observed in both infected bone marrow derived macrophages (BMDMs) and BALB/c mice organs. We found involvement of miR146a-5p in phagocytosis and survivability of parasites. Moreover, miR146a-5pgot enriched in interleukin 4- stimulated BMDMs, indicating its possible involvement in M2 polarization. Upon transfecting BMDMs with miRVANA anti-146a oligos, M2 markers (CCR7, YM-1, FIZZ-1, arginase-1, IL10 and IL4) and transcription factors (p-STAT6 and c/EBPβ) got depleted with concomitant augmentation of M1-polarizing transcription factors (p-STAT1, AP1 and IRF-1), miR146a target genes (TRAF6 and IRAK1), M1 cytokines (IL12 and TNFα), iNOS, nitric oxide, and nuclear translocation of phospho p-65 subunit. Neutralization of intracellular mature miR146a-5p pool in infected BALB/c mice lower organ parasite burden and expressions of M2 markers and IL10 with enrichment of M1 markers like iNOS and IL12. Additionally, we explored the novel role of super enhancer (SE), a cis-acting regulatory component, to enrich miR146a-5p expression during infection. Enhanced expression and nuclear retention of SE components like BET bromodomain 4 (BRD4) and p300 were found in infected BMDMs. Upon silencing BRD4, expressions of miR146a-5p and M2 markers were down regulated and TRAF6, IRAK1 and iNOS levels increased. STRING V.11 based predication and immune precipitation confirmed the strong interaction amongst BRD4, p300 and RNA pol II (RpbI). Chromatin immune precipitation studies suggested the recruitment of BRD4 at the enhancer loci of miR146a-5p gene during infection. Altogether, our findings revealed a novel role of BRD4/p300-depdendent super-enhancer in regulating miR146a expression during L. donovani infection which in turn mediates M2 polarization and immune-suppression.

Downregulation of triggering receptor expressed on myeloid cells 1 inhibits invasion and migration of liver cancer cells by mediating macrophage polarization.

Triggering receptor expressed on myeloid cells‑1 (TREM1) is a cell‑surface protein expressed on tumor‑associated macrophages (TAMs), the predominant inflammatory cells in the tumor microenvironment; however, the mechanisms for the influence of TREM1 on TAM polarization during liver cancer progression have not been investigated. In the present study, 20patients diagnosed with hepatocellular carcinoma(HCC) who underwent surgery were enrolled, and TREM1 expression on M1/M2macrophages and on M2macrophages was assessed by immunohistochemical staining. Human leukemia monocytic cells(THP‑1) were differentiated into M2macrophages using phorbol 12‑myristate 13‑acetate, IL‑4 and IL‑13. A specific short hairpin RNA was used to knockdown TREM1 expression. To investigate the effects of TREM1 downregulation in macrophages on the migration and invasion of liver cancer cells, HepG2 and MHCC97H cell lines were co‑cultured with specific conditioned media. Reverse transcription‑quantitative PCR and western blot analyses were used to detect M1 and M2macrophage marker expression. The expression levels of proteins of the PI3K/AKT/mTOR signaling pathway were analyzed by western blotting, revealing that TREM1 expression in HCC tissues was significantly elevated compared with that in adjacent normal tissues, and TREM1 was highly expressed on the cell membranes of M2macrophages in tumor tissues compared with in adjacent normal tissues. The present results demonstrated that TREM1 downregulation in macrophages shifted M2macrophages towards an M1 phenotype, as defined by higher expression levels of M1‑associated markers and decreased expression levels of M2‑associated markers. In addition, TREM1 downregulation in macrophages suppressed migration and invasion of HepG2 and MHCC97H cells. Furthermore, TREM1‑knockdown in macrophages inhibited PI3K/AKT/mTOR activation in the polarization of M2macrophages. In conclusion, downregulation of TREM1 expression in macrophages shifted M2macrophages towards a M1 phenotype via inhibiting PI3K/AKT signaling. In addition, migration and invasion of HepG2 and MHCC97H cells were inhibited when this signaling pathway was blocked. The present findings suggest TREM1 as a novel potential therapeutic target for liver cancer management.

Anti-inflammatory effects of Platycodin D on dextran sulfate sodium (DSS) induced colitis and E. coli Lipopolysaccharide (LPS) induced inflammation

Platycodin D (PLD) is a saponin found in Platycodon grandiflorum, which has been reported to have anti-inflammatory effects. However, the effects of PLD on ulcerative colitis (UC) remain unknown. In this study, PLD showed the potential to reduce inflammation, ameliorate intestinal damage, and maintain intestinal integrity in DSS-induced colitis. However, the beneficial effect of PLD was reduced when macrophages were depleted, indicating the key role of macrophages in the beneficial effect of PLD in DSS-induced colitis. Meanwhile, we found that PLD inhibited the expression of M1 markers and promoted the expression of M2 markers in colon. Similarly, we found PLD significantly attenuated the levels of pro-inflammatory cytokines, increased the level of anti-inflammatory cytokine and altered macrophage proportions in LPS-stimulated RAW 264.7 cells in vitro. Moreover, treating LPS-stimulated RAW 264.7 cells with PLD increased the activation of the PI3K/Akt signaling pathway and decreased activation of NF-κB pathway. Furthermore, we found that the anti-inflammatory and macrophage polarization regulatory effects of PLD was Adenosine 5′-monophosphate-activated protein kinase (AMPK)-dependent. These results indicate that PLD attenuates DSS-induced colitis and LPS-induced inflammation, and the mechanism behind the phenomenon may be regulating macrophage polarization via activation of AMPK. Our study provides a theoretical basis for PLD to be used as a potential treatment of colitis.

Porphyromonas gingivalis lipopolysaccharide regulates macrophage polarization via triggering receptors expressed on myeloid cells-1

Objective: To investigate the relationship between pattern recognition receptor triggering receptors expressed on myeloid cells-1 (TREM-1) and M1/M2 polarization in macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS), so as to explore the mechanism of TREM-1 in periodontitis. Methods: Human monocytic cell line THP-1 were induced to differentiate into macrophages by phorbol-12-myristate-13-acetate and stimulated by 0 (blank control group) and 1 μg/ml Pg-LPS (LPS group), respectively. LP17, the TREM-1 inhibitor (LPS+LP17 group) and its control peptide (LPS+control peptide group) with final concentration of 0.1 μg/ml were added at the same time. After 24 hours stimulation, the expression of TREM-1, M1 markers and related cytokines [CD86, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β], M2 markers and related cytokines (CD206,IL-10) mRNA were detected by real-time quantitative-PCR (RT-qPCR), the level of TREM-1, CD86 and CD206 proteins were detected by Western blotting, and TNF-α, IL-1β and IL-10 in the macrophage culture supernatant were detected by enzyme linked immunosorbent assay. Results: After 24 h of cell culture, the relative expressions of TREM-1 mRNA (1.40±0.14) and protein (3.85±0.24) in macrophages in the LPS group increased compared with the blank control group (1.01±0.18 and 1.00±0.05, respectively) (P<0.05). Meanwhile, the expression levels of M1 markers CD86 mRNA and protein [LPS group vs blank control group were (1.42±0.01 vs 1.00±0.09) and (1.55±0.07 vs 1.00±0.10), respectively] were up-regulated (P<0.01), and the expressions of mRNA and protein of M1 related cytokines TNF-α and IL-1 increased (P<0.05). After the addition of TREM-1 blocker LP17, the levels of mRNA and protein of TREM-1 showed no significant changes (P>0.05), while the levels of CD86 mRNA (0.96±0.00) and protein (1.36±0.02) decreased (P<0.05), and the levels of TNF-α and IL-1 further decreased (P<0.05). For M2 marker CD206 and related cytokine IL-10, CD206 mRNA (0.56±0.05) and protein (0.25±0.04) were significantly down-regulated (P<0.01) compared with the blank control group (1.02±0.25 and 1.00±0.10, respectively), and IL-10 mRNA was up-regulated compared with the blank control group (P<0.05), with no significant change in protein (P>0.05). After the addition of LP17, the expressions of CD206 and IL-10 mRNA in the LPS+LP17 group were further down-regulated compared with the LPS group (P<0.05), and there was no statistically significant difference between the two groups in protein level (P>0.05). Conclusions: TREM-1 and its downstream signaling pathway might be involved in M1 polarization of Pg-LPS-mediated macrophages, thus playing a pro-inflammatory role in the development of periodontitis. There is no obvious evidence that TREM-1 is involved in regulating M2 polarization of Pg-LPS-mediated macrophages.

MiR-375 silencing attenuates pro-inflammatory macrophage response and foam cell formation by targeting KLF4

Macrophage mediated inflammation and foam cell formation play crucial roles in the development of atherosclerosis. MiR-375 is a small noncoding RNA that significantly implicated in multiple tumor regulation and has been emerged as a novel biomarker for type 2 diabetes. However, the exact role of miR-375 on macrophage activation remains unknown. In the present study, we observed that miR-375 expression showed an up-regulated expression in atherosclerotic aortas, as well as in bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs) isolated from ApoE deficiency mice and was gradually increased followed the Ox-LDL treated time. Functionally, miR-375 inhibition significantly decreased foam cell formation accompanied by up-regulated genes expression involved in cholesterol efflux but reduced genes expression implicated in cholesterol influx. Moreover, miR-375 silencing increased resolving M2 macrophage but reduced pro-inflammatory M1 macrophage markers expression. Such above effects can be reversed by miR-375 overexpression. Mechanistically, we noticed that miR-375 knockdown promoted KLF4 expression which was required for the ameliorated effect of miR-375 silencing on macrophage activation. Importantly, the consistent results in mRNA expression of M1 and M2 markers were observed in vivo, and miR-375−/−ApoE−/− mice significant decreased atherosclerotic lesions in the whole aorta and aortic sinus. Taken together, these evidences suggested that miR-375 knockdown attenuated macrophage activation partially through activation of KLF4-dependent mechanism.

Immunomodulatory Layered Double Hydroxide Nanoparticles Enable Neurogenesis by Targeting Transforming Growth Factor-β Receptor 2.

Immune microenvironment amelioration and reconstruction by functional biomaterials has become a promising strategy for spinal cord injury (SCI) recovery. In this study, we evaluated the neural regeneration and immunoregulation functions of Mg/Al layered double hydroxide (Mg/Al-LDH) nanoparticles in completely transected and excised mice and revealed the immune-related mechanisms. LDH achieved significant performance in accelerating neural stem cells (NSCs) migration, neural differentiation, L-Ca2+ channel activation, and inducible action potential generation. In vivo, the behavioral and electrophysiological performance of SCI mice was significantly improved by LDH implantation, with BrdU+ endogenous NSCs and neurons clearly observed in the lesion sites. According to RNA-seq and ingenuity pathway analysis, transforming growth factor-β receptor 2 (TGFBR2) is the key gene through which LDH inhibits inflammatory responses and accelerates neural regeneration. Significant colocalization of TGFBR2 and LDH was found on the cell membranes of NSCs both in vitro and in vivo, and LDH increased the expression of TGF-β2 in NSCs and activated the proliferation of precursor neural cells. LDH decreased the expression of M1 markers and increased the expression of M2 markers in both microglia and bone marrow-derived macrophages, and these effects were reversed by a TGFBR2 inhibitor. In addition, as a carrier, LDH loaded with NT3 exhibited better recovery effects with regard to the basso mouse scale score, motor evoked potential performance, and regenerated neural cell numbers than LDH itself. Thus, we have developed Mg/Al-LDH that can be used to construct a suitable immune microenvironment for SCI recovery and have revealed the targeted receptor.

Behaviour at the peritoneal interface of next-generation prosthetic materials for hernia repair.

When using a prosthetic material in hernia repair, the behaviour of the mesh at the peritoneal interface is especially important for implant success. Biomaterials developed for their intraperitoneal placement are known as composites and are made up of two different-structure materials, one is responsible for good integration within host tissue and the other is responsible to make contact with the viscera. This study examines the behaviour at the peritoneal level of two composites, the fully degradable Phasix-ST® and the partially degradable Symbotex®. A polypropylene mesh (Optilene®) served as control. Sequential laparoscopy from 3 to 90days, in a preclinical model in the New Zealand white rabbit, allowed monitoring adhesion formation. Morphological studies were performed to analyse the neoperitoneum formed in the repair process. Total macrophages were identified by immunohistochemical labelling. To identify the different macrophage phenotypes, complementary DNAs were amplified by qRT-PCR using specific primers for M1 (TNF-α/CXCL9) and M2 (MRC1/IL-10) macrophages. The percentage of firm and integrated adhesions remained very high in the control group over time. Both composites showed a significant decrease in adhesions at all study times and in qualitative terms were mainly loose. Significant differences were also observed from 7days onwards between the two composites, increasing the values in Phasix over time. Neoperitoneum thickness for Phasix was significantly greater than those of the other meshes, showing mature and organized neoformed connective tissue. Immunohistochemically, a significantly higher percentage of macrophages was observed in Symbotex. mRNA expression levels for the M2 repair-type macrophages were highest for Phasix but significant differences only emerged for IL-10. Fewer adhesions formed to the Symbotex than Phasix implants. Ninety days after implant, total macrophage counts were significantly higher for Symbotex, yet Phasix showed the greater expression of M2 markers related to the tissue repair process.

MicroRNA-125a-5p modulates macrophage polarization by targeting E26 transformation-specific variant 6 gene during orthodontic tooth movement

ObjectiveThe aim of this study was to investigate the role of microRNA-125a-5p (miR-125a-5p) in macrophages during orthodontic tooth movement (OTM). DesignPeriodontal ligament tissues were collected from patients underwent OTM. Periodontal ligament cells were isolated from periodontal ligament tissues. Periodontal ligament stem cells were isolated from normal human impacted third molars. The miR-125-5p levels were measured by real-time quantitative polymerase chain reaction. The impact of miR-125-5p on macrophage polarization was tested by alizarin red staining assay. The effects of miR-125-5p and E26 transformation-specific variant 6 gene (ETV6) on M1/M2 macrophages phenotype markers were determined by real-time quantitative polymerase chain reaction, western blot, and flow cytometry analyses. The interaction between miR-125-5p and ETV6 was verified using luciferase reporter and RNA immunoprecipitation assays. ResultsPeriodontal miR-125a-5p was upregulated under the force. Macrophage polarization facilitated osteogenesis by cocultured system. Moreover, miR-125a-5p was upregulated in macrophages polarized with M2 conditions. MiR-125a-5p downregulation promoted the expression of M1 phenotype markers, while suppressed the expression of M2 markers. Mechanistically, ETV6 was confirmed to be a target of miR-125a-5p. ETV6 overexpression increased the expression of M1 polarized markers, while decreased the expression of M2 polarized markers. Furthermore, ETV6 knockdown reversed the effects of miR-125a-5p inhibitor on both M1 macrophages and M2 macrophages. ConclusionsOverall, miR-125a-5p facilitates bone healing by targeting ETV6 to promote macrophage M2 polarization.

Exosomes from adipose-derived stem cells alleviate the inflammation and oxidative stress via regulating Nrf2/HO-1 axis in macrophages

ADSCs exosomes, an important means of intercellular communication, can regulate an array of biological processes, including promoting tissue repairs and regeneration, and attenuating inflammation. In this study, we found that ADSCs exosomes could polarize macrophage to an anti-inflammatory phenotype via regulating the expression of Nrf2 and HO-1, and improve inflammatory reaction and injury of multi-organ in sepsis. We revealed that ADSCs exosomes could alleviate LPS induced accumulation of ROS and the expression of inflammatory cytokines IL-1β, TNF-α, and IL-6 in macrophages. Western blot and Flow cytometry results indicated that expression of M1 markers (iNOS and CD86) in LPS stimulated macrophages were significantly declined, while M2 (Arg1 and CD206) were enhanced when pretreated with ADSCs exosomes. Besides, the stress-related molecule HO-1 was upregulated when pretreated with ADSCs exosomes. Further H0-1 interference experiment indicated that anti-inflammatory effect of ADSCs exosomes was dependent on HO-1. Moreover, ADSCs exosomes enhanced expression and nucleus translocation of Nrf2, while downregulated its negative mediator Keap1. In in vivo sepsis models, intravenous injection of ADSCs exosomes relieved inflammatory cytokines storm and organ injury, while promoted expression of HO-1. In conclusion, we proved that ADSCs exosomes alleviated LPS induced inflammation and exerted protective effect in sepsis via regulating Nrf2/HO-1 expression.

Progranulin induces immune escape in breast cancer via up-regulating PD-L1 expression on tumor-associated macrophages (TAMs) and promoting CD8+ T cell exclusion

BackgroundProgranulin (PGRN), as a multifunctional growth factor, is overexpressed in multiple tumors, but the role of PGRN on tumor immunity is still unclear. Here, we studied the effect of PGRN on breast cancer tumor immunity and its possible molecular mechanism.MethodsThe changes of macrophage phenotypes after PGRN treatment were detected by western blot, quantitative polymerase chain reaction (PCR) and flow cytometry. Western blot was used to study the signal molecular mechanism of PGRN regulating this process. The number and localization of immune cells in Wild-type (WT) and PGRN−/− breast cancer tissues were analyzed by immunohistochemical staining and immunofluorescence techniques. The activation and proliferation of CD8+ T cells were measured by flow cytometry.ResultsAfter being treated with PGRN, the expressions of M2 markers and programmed death ligand 1 (PD-L1) on macrophages increased significantly. Signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitor Stattic significantly inhibited the expression of PD-L1 and M2 related markers induced by PGRN. In WT group, CD8 were co-localized with macrophages and PD-L1, but not tumor cells. The number of immune cells in PGRN−/− breast cancer tissue increased, and their infiltration into tumor parenchyma was also enhanced. Moreover, in the co-culture system, WT peritoneal macrophages not only reduced the ratio of activated CD8+ T cells but also reduced the proportion of proliferating CD8+ T cells. The addition of programmed death receptor 1 (PD-1) and PD-L1 neutralizing antibodies effectively reversed this effect and restored the immune function of CD8+ T cells.ConclusionThese results demonstrate that PGRN promotes M2 polarization and PD-L1 expression by activating the STAT3 signaling pathway. Furthermore, through PD-1/PD-L1 interaction, PGRN can promote the breast tumor immune escape. Our research may provide new ideas and targets for clinical breast cancer immunotherapy.

MicroRNA-21 Inhibition Suppresses Alveolar M2 Macrophages in an Ovalbumin-Induced Allergic Asthma Mice Model

PurposeMicroRNA-21 (miR-21) influences the Th2 immune pathway by suppressing the expressions of interleukin (IL)-12 and interferon (IFN)-γ. The effects of miR-21 suppression on alveolar macrophage polarization and airway inflammation are not known.MethodsBALB/c and miR-21 knockout (KO) mice were sensitized and challenged with ovalbumin (OVA). The anti-miR-21 antagomir was administered to BALB/c mice by intranasal inhalation from the day of OVA sensitization. Changes in cell counts, cytokine levels in bronchoalveolar lavage fluid (BALF), and airway hyperresponsiveness (AHR) were examined. Total, M1, and M2 macrophages were examined in the lung tissues by immunohistochemistry (IHC). M2 macrophages from the OVA mice lung were inhaled into the anti-miR-21 antagomir-treated asthmatic mice. Moreover, the polarization of M0 to M2 macrophages upon IL-4 stimulation was analyzed after anti-miR-21 antagomir transfection.ResultsThe miR-21 KO mice showed decreases in AHR, total cell and eosinophil counts in BALF, and in the levels of IL-4, IL-5, IL-10, and IL-13. Expression of IL-12 and IFN-γ were increased in the miR-21 KO mice. Peribronchial inflammation and goblet cell dysplasia were significantly decreased in the lung tissues of miR-21 KO OVA mice compared to the wild type OVA mice. IHC for M1, M2, and total macrophage in the lung tissues showed that miR-21 inhalation suppressed alveolar M2 macrophages in KO mice. M2 macrophage inhalation restored AHR and eosinophilic airway inflammation in the miR-21 antagomir-treated mice. Moreover, anti-miR-21 antagomir transfection decreased the expression of M2 markers and increased the expression of M1 markers in M0 macrophages after IL-4 stimulation.ConclusionsThe results suggest that miR-21 antagonism could suppress alveolar M2 macrophage polarization, decreasing not only the Th2 eosinophilic airway inflammation but also AHR and airway remodeling process.

Renal fibrosis due to multiple cisplatin treatment is exacerbated by kinin B1 receptor antagonism.

Cisplatin is a widely used chemotherapeutic drug, but its side effects are a major limiting factor. Nephrotoxicity occurs in one third of patients undergoing cisplatin treatment. The acute tubular injury caused by cisplatin often leads to a defective repair process, which translates into chronic renal disorders. In this way, cisplatin affects tubular cells, and maladaptive tubules regeneration will ultimately result in tubulointerstitial fibrosis. Kinins are well known for being important peptides in the regulation of inflammatory stimuli, and kinin B1 receptor deficiency and antagonism have been shown to be beneficial against acute cisplatin nephrotoxicity. This study aimed to analyze the effects of kinin B1 receptor deletion and antagonism against repeated cisplatin-induced chronic renal dysfunction and fibrosis. Both the deletion and the antagonism of B1 receptor exacerbated cisplatin-induced chronic renal dysfunction. Moreover, the inhibition of B1 receptor increased tubular injury and tubulointerstitial fibrosis after repeated treatment with cisplatin. The balance between M1/M2 macrophage polarization plays an important role in renal fibrosis. Kinin B1 receptor antagonism had no impact on M1 markers when compared to cisplatin. However, YM1, an M2 marker and an important molecule for the wound healing process, was decreased in mice treated with kinin B1 receptor antagonist, compared to cisplatin alone. Endothelin-1 levels were also increased in mice with B1 receptor inhibition. This study showed that kinin B1 receptor inhibition exacerbated cisplatin-induced chronic renal dysfunction and fibrosis, associated with reduced YM1 M2 marker expression, thus possibly affecting the wound healing process.

Exosomes derived from macrophages upon Zn ion stimulation promote osteoblast and endothelial cell functions.

Osteogenesis and angiogenesis are both important for implant osseointegration, which can be tailored by immunomodulation of macrophages. Zn, a novel biodegradable material, can modulate macrophage functions in its ionic form. However, whether macrophage-derived exosomes, novel carriers of intracellular communication, participate in the process is still unclear. The present work shows that Zn ions in the concentration range of 0-100 μM have no significant influence on macrophage viability, proliferation, morphology, and secretion amount of exosomes, but generally downregulate the gene expression of both M1 and M2 markers. The exosomes can be ingested continuously by osteoblasts and endothelial cells. The osteoblasts show the highest alkaline phosphatase activity after ingesting the exosomes derived from macrophages upon 4 μM Zn ion stimulation. In contrast, the endothelial cells migrate the furthest distance after ingesting the exosomes upon 20 μM Zn ion stimulation. These results indicate that Zn ions may vary the composition of macrophage-derived exosomes, which in turn affects the osteogenesis and angiogenesis. These findings are meaningful for the surface design of immunomodulatory biomaterials from the perspective of macrophage-derived exosomes.

The administration of low-dose Curcuma longa extract induces M2 polarization in peritoneal macrophage culture

Curcuma longa (turmeric) has been widely used to accelerate wound healing, but the underlying mechanism remains unclear. Wound healing consists of four phases which are correlated and overlapping, i.e., coagulation, inflammation, proliferation and remodeling. Macrophages play an important role in most phases. Macrophage polarization to M2 initiates the proliferation phase, which is characterized by the production of several crucial growth factors. Since turmeric has been known to be an herb that accelerates wound closure, we investigated whether Curcuma longa extract administration in macrophage culture induces M2 macrophage switching. An in vitro study was performed using peritoneal macrophages from Swiss Webster strain mice. Peritoneal cells were collected and cultured in a 24-well plate. After 2 hours of incubation, macrophages (adherent cells) were treated with 0.5 ppm, 1.0 ppm and 5.0 ppm of ethanolic extract of Curcuma longa and incubated for 2 days. Quantitative real time PCR was performed to quantify M2 and M1 marker gene expression. The results revealed the upregulation of M2 marker (Arginase-1) expression upon administration of 0.5 ppm of Curcuma longa extract, but not of higher doses (1.0 and 5.0 ppm). In parallel, the ratio of Arg-1/Inos was high upon administration of 0. 5ppm of extract. In conclusion, Curcuma longa extract induces in vitro M2 polarization in low-dose administration.

The role of irreversible electroporation in promoting M1 macrophage polarization via regulating the HMGB1-RAGE-MAPK axis in pancreatic cancer

ABSTRACT Irreversible electroporation (IRE) is an effective method for treating pancreatic ductal adenocarcinoma (PDAC). It remains unclear whether IRE can induce a specific immune response by stimulating macrophages. Here, the associated markers of macrophages were analyzed after exposure to tumor culture supernatant (TSN) of tumor cells treated with electroporation. Subcutaneous and orthotopic PDAC models were also used to evaluate the effect of macrophage polarization induced by IRE. Aside from its direct killing effect, IRE could induce the immunogenic cell death of tumor cells by increasing the synthesis and secretion of damage associated molecular patterns. Moreover, IRE could increase the release of HMGB1, which activates the MAPK-p38 pathway and leads to the increased expression of M1 markers in macrophages, through binding to the receptor of the advanced glycation end-product (RAGE) receptor. M1 polarization was inhibited by the inhibitors of HMGB1 release, the RAGE receptor, and the MAPK-p38 signaling pathway, but it was activated by rHMGB1 or the stimulator of MAPK-p38. In addition, the promotion of M1 macrophage polarization was enhanced by the positive-feedback release or expression of HMGB1 and RAGE through the MAPK-ERK pathway in macrophages. The promotion of M1 macrophage polarization induced by IRE provided a specific rationale for the combination of IRE and immune therapy in treating PDAC.

Restoration of electrical microenvironment enhances bone regeneration under diabetic conditions by modulating macrophage polarization

Macrophage-mediated inflammation compromises bone repair in diabetic patients. Electrical signaling cues are known to regulate macrophage functions. However, the biological effects of electrical microenvironment from charged biomaterials on the immune response for regulating osteogenesis under diabetic conditions remain to be elucidated. Herein the endogeneous electrical microenvironment of native bone tissue was recapitulated by fabricating a ferroelectric BaTiO3/poly (vinylidene fluoridetrifluoroethylene) (BTO/P(VDF-TrFE)) nanocomposite membrane. In vitro, the polarized BaTiO3/poly (vinylidene fluoridetrifluoroethylene) (BTO/P(VDF-TrFE)) nanocomposite membranes inhibited high glucose-induced M1-type inflammation, by effecting changes in cell morphology, M1 marker expression and pro-inflammatory cytokine secretion in macrophages. This led to enhanced osteogenic differentiation of human bone marrow mesenchymal stem cells (BM-MSCs). In vivo, the biomimetic electrical microenvironment recapitulated by the polarized nanocomposite membranes switched macrophage phenotype from the pro-inflammatory (M1) into the pro-healing (M2) phenotype, which in turn enhanced bone regeneration in rats with type 2 diabetes mellitus. Mechanistic studies revealed that the biomimetic electrical microenvironment attenuated pro-inflammatory M1 macrophage polarization under hyperglycemic conditions by suppressing expression of AKT2 and IRF5 within the PI3K-AKT signaling pathway, thereby inducing favorable osteo-immunomodulatory effects. Our study thus provides fundamental insights into the biological effects of restoring the electrical microenvironment conducive for osteogenesis under DM conditions, and offers an effective strategy to design functionalized biomaterials for bone regeneration therapy in diabetic patients.