Macrophage polarisation in caesarean scar diverticulum

AimsTo determine immunohistochemical features and correlations between M1/M2 polarisation status with disease severity of post-caesarean scar diverticulum (CSD).MethodsHistological and immunohistological stainings were performed and inflammatory (CD16, CD163 and tumour necrosis factor-α (TNF-α)), fibrosis (α-smooth muscle actin (α-SMA)) and angiogenic (CD31) markers were examined in uterine tissues collected from patients with uterine scar diverticula (CSD) (n=37) and caesarean section (CS) (n=3).ResultsCSD tissues have higher expression of α-SMA, TNF-α, CD16 and CD31 and lower expression of CD163 than CS tissue (p<0.05). Compared with adjacent tissues, thick-walled blood vessels, glands and fibrotic sites have higher expression of α-SMA, TNF-α and CD16. Statistical correlation was observed between the expression of CD16 and TNF-α (R=0.693, p<0.001), α-SMA (R=0.404, p<0.05) and CD31 (R=0.253, p<0.05) in CSD tissues, especially with the ratio of CD16/CD163 (R=0.590, p<0.01). A more significant difference was observed between the expression of CD16/CD163 and α-SMA (R=0.556, p<0.001), TNF-α (R=0.633, p<0.0001) and CD31 (R=0.336, p<0.05).ConclusionsIn this study, TNF-α, α-SMA, CD16 and CD31 proteins were overexpressed in all CSD cases, and CD16/CD163 was positively correlated with tissue inflammation, fibrosis and neovascularisation. Abnormal mononuclear macrophage infiltration may be involved in the origin and progression of CSD.

Sarcodia suieae Acetyl-Xylogalactan Regulates Nile Tilapia (Oreochromis niloticus) Tissue Phagocytotic Activity and Serum Indices

Sarcodia suieae acetyl-xylogalactan was reported to induce macrophage polarisation, and could positively regulate macrophage activation. In this study, we evaluated the effect of Sarcodia suieae acetyl-xylogalactan on the Nile tilapia. First, we assessed the influence of acetyl-xylogalactan on the survival, glucose uptake, and phagocytic activity of tilapia head kidney (THK) melanomacrophage, and observed increased proliferation of these cells in the MTT assay after 12 and 24 h of treatment. Glucose uptake increased in THK melanomacrophage treated with 20 and 30 μg acetyl-xylogalactan for 24 h. Their phagocytic activity was positively enhanced following exposure to acetyl-xylogalactan. Nile tilapia were fed with acetyl-xylogalactan for 4 weeks. At the end of the experiment, Nile tilapia were sacrificed, and the lipopolysaccharide-induced liver and head-kidney apoptosis was examined under reducing conditions in comparison with controls. The phagocytic activities of liver and head-kidney cells were enhanced after 4 weeks of feeding. Blood biochemical analysis revealed a reduction in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels after 4 weeks of feeding. Combined with in vitro and in vivo experiments results, the extracted S. suieae acetyl-xylogalactan could directly induce THK melanomacrophage proliferation, glucose uptake, and phagocytic activity. Acetyl-xylogalactan was able to induce Nile tilapia liver and head-kidney resident macrophage activity, and reduced LPS-induced liver and head-kidney cell apoptosis. S. suieae acetyl-xylogalactan may modulate Nile tilapia macrophage activation by polarising them into M1 macrophages to improve the Nile tilapia nonspecific immune response.

Dual Role of p73 in Cancer Microenvironment and DNA Damage Response

Understanding the mechanisms that regulate cancer progression is pivotal for the development of new therapies. Although p53 is mutated in half of human cancers, its family member p73 is not. At the same time, isoforms of p73 are often overexpressed in cancers and p73 can overtake many p53 functions to kill abnormal cells. According to the latest studies, while p73 represses epithelial–mesenchymal transition and metastasis, it can also promote tumour growth by modulating crosstalk between cancer and immune cells in the tumor microenvironment, M2 macrophage polarisation, Th2 T-cell differentiation, and angiogenesis. Thus, p73 likely plays a dual role as a tumor suppressor by regulating apoptosis in response to genotoxic stress or as an oncoprotein by promoting the immunosuppressive environment and immune cell differentiation.

Lipoxin A4 Regulates M1/M2 Macrophage Polarization via FPR2-IRF Pathway

Lipoxin A4 (LXA4) has been shown to have anti-inflammatory activity, but its underlying molecular mechanisms are not clear. Herein, our team investigated the potential role of LXA4 in the macrophage polarisation and elucidated its possible molecular mechanism. The RAW264.7 macrophage cell line was subjected to pre-treatment with LXA4 with or without lipopolysaccharides (LPS) and interleukin-4 (IL-4). In cultured macrophages, LXA4 inhibits LPS-induced inflammatory polarization, thereby decreasing the release of proinflammation cell factors (IL-1β, IL-6, TNF-α) and increasing the release of antiinflammation cytokines (IL-4 and IL-10). Notably, the inhibitory effect of LXA4 on inflammation macrophage polarisation was related to the downregulation of p-NF-κB p65 and IRF5 activity, thereby downregulating LPS-induced phenotypic and functional polarization of macrophage M1 via the FPR2/IRF5 signaling pathway. Moreover, LXA4 also promotes the IL-4-induced polarization of M2 macrophages by promoting the FPR2/IRF4 signaling pathway. Therefore, Lipoxin A4 regulates M1/M2 polarization of macrophages via FPR2-IRF pathway.

Characterisation of Macrophage Polarisation in Mice Infected with Ninoa Strain of Trypanosoma cruzi

Macrophages (MΦ) play a key role in the development of the protective immune response against Trypanosoma cruzi infection. To determine the role of MΦ subtypes M1 and M2 in the development of immunity against the Mexican strain of T. cruzi (Ninoa strain), we have analysed in a time course the infection and characterised the M1 and M2 subtypes in two mouse models, BALB/c and C57BL/6. After infection, BALB/c mice developed an increased blood parasite load and the parasites were cleared from the blood one week later than in C57BL/6 mice. However, similar cellular infiltrate and cardiac alterations were observed between BALB/c and C57BL/6 mice. At 36 days, the T. cruzi infection differentially modulated the expression of immune cells, and both the BALB/c and C57BL/6 mice significantly reduced TCD4+ cells. However, BALB/c mice produced significantly more TCD8+ than C57BL/6 mice in the spleen and lymph nodes. Furthermore, BALB/c mice produce significantly more MΦ in the spleen, while C57BL/6 produce similar levels to uninfected mice. The M1 MΦ ratio increased significantly at 3–5 days post-infection (dpi), but then decreased slightly. On the contrary, the M2 MΦ were low at the beginning of the infection, but the proportion of M1 and M2 MΦ at 36 dpi was similar. Importantly, the MΦ subtypes M2c and M2d significantly increased the induction of tissue repair by the end of the acute phase of the infection. These results indicate that the Ninoa strain has developed strategies to modulate the immune response, with fine differences depending on the genetic background of the host.

Selective CDK9 inhibition resolves neutrophilic inflammation and enhances cardiac regeneration in larval zebrafish

Sustained neutrophilic inflammation is detrimental for cardiac repair and associated with adverse outcomes following myocardial infarction (MI). An attractive therapeutic strategy to treat MI is to reduce or remove infiltrating neutrophils to promote downstream reparative mechanisms. CDK9 inhibitor compounds enhance the resolution of neutrophilic inflammation, however, their effects on cardiac repair/regeneration are unknown. Our laboratory has devised a cardiac injury model to investigate inflammatory and regenerative responses in larval zebrafish using heartbeat-synchronised light sheet fluorescence microscopy. We used this model to test two clinically approved CDK9 inhibitors, AT7519 and Flavopiridol, examining their effects on neutrophils, macrophages and cardiomyocyte regeneration. We found AT7519 and Flavopiridol resolve neutrophil infiltration by inducing reverse migration from the cardiac lesion. While continuous exposure to AT7519 or Flavopiridol caused adverse phenotypes, transient treatment accelerated neutrophil resolution while avoiding these effects. Transient treatment with AT7519, but not Flavopiridol, augmented wound-associated macrophage polarisation, which enhanced macrophage-dependent cardiomyocyte number expansion and the rate of myocardial wound closure. Using cdk9−/- knockout mutants we showed AT7519 is a selective CDK9 inhibitor, revealing the potential of such treatments to promote cardiac repair/regeneration.

SARS-CoV-2 Spike S1 glycoprotein is a TLR4 agonist, upregulates ACE2 expression and induces pro-inflammatory M1 macrophage polarisation.

Background and aims: TLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesis in vitro and demonstrate that the SARS-CoV-2 spike S1 domain is a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages. Methods: Adult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFN-γ, S1/IFN-γ or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2. Results: TLR4 activation by spike S1 or LPS resulted in the upregulation of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore, suggesting ACE2 expression is mediated by the alternative endosomal/β-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-γ treatment of THP-1-derived macrophages induced pro-inflammatory M1 polarisation as shown by an increase in IL-1-β and IL-6 mRNA. Conclusions: These results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In addition, pro-inflammatory M1 macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.

Endoplasmic Reticulum Stress Promotes The Release of Exosomal PD-L1 From Head and Neck Cancer Cells and Facilitates M2 Macrophage Differentiation

Background Endoplasmic reticulum stress (ER stress) fosters cancer cell escape from immune surveillance and upregulate PD-L1 expression, but the mechanisms remain unclear. Methods We analyzed protein levels by immunofluorescence and Western blotting, RNA levels by qRT-PCR. Exosomes were injected intravenously through the tail vein into 6-week-old nude mice once every other day for a total of 10 injections Results Expression of some ER stress markers, including GRP78 (glucose-regulated protein 78), ATF6 (activating transcription factor 6) and PERK (PKR-like endoplasmic reticulum kinase), was upregulated in OSCC tissues and correlated with poor overall survival. The level of ER stress-related proteins positively correlated with a cluster of PD-L1 expression and macrophage infiltration in OSCC tissues. PD-L1 expression in OSCC tissues was negatively correlated with cumulative survival. Incubation with Exo-ER upregulated PD-L1 levels in macrophages in vitro and vivo, and upregulation of PD-L1 promoted macrophage polarisation towards the M2 subtype. Conclusions ER stress induced exosome secretion by OSCC cells and PD-L1 expression in macrophages to promote M2 macrophage differentiation. A novel exosome-modulated mechanism was delineated for OSCCs-macropahge crosstalk that drove tumor growth and should be explored for its therapeutic utility.

Radiosensitivity index emerges as a potential biomarker for combined radiotherapy and immunotherapy

In the era of immunotherapy, there lacks of a reliable genomic predictor to identify optimal patient populations in combined radiotherapy and immunotherapy (CRI). The purpose of this study is to investigate whether genomic scores defining radiosensitivity are associated with immune response. Genomic data from Merged Microarray-Acquired dataset (MMD) were established and the Cancer Genome Atlas (TCGA) were obtained. Based on rank-based regression model including 10 genes, radiosensitivity index (RSI) was calculated. A total of 12832 primary tumours across 11 major cancer types were analysed for the association with DNA repair, cellular stemness, macrophage polarisation, and immune subtypes. Additional 585 metastatic tissues were extracted from MET500. RSI was stratified into RSI-Low and RSI-High by a cutpoint of 0.46. Proteomic differential analysis was used to identify significant proteins according to RSI categories. Gene Set Variance Analysis (GSVA) was applied to measure the genomic pathway activity (18 genes for T-cell inflamed activity). Kaplan-Meier analysis was performed for survival analysis. RSI was significantly associated with homologous DNA repair, cancer stemness and immune-related molecular features. Lower RSI was associated with higher fraction of M1 macrophage. Differential proteomic analysis identified significantly higher TAP2 expression in RSI-Low colorectal tumours. In the TCGA cohort, dominant interferon-γ (IFN-γ) response was characterised by low RSI and predicted better response to programmed cell death 1 (PD-1) blockade. In conclusion, in addition to radiation response, our study identified RSI to be associated with various immune-related features and predicted response to PD-1 blockade, thus, highlighting its potential as a candidate biomarker for CRI.