Commonly macrophages are categorized into M1 and M2 subsets. M1 macrophages are defined as inflammatory cells and may contribute to tissue injury, while, M2 macrophages play a central role in tissue remodeling and control of immune responses. It seems that the existence of these cells in graft location is effective for control of inflammation and improvement of transplantation consequences. Although the relative contribution of M2 cells in organ transplantation is not clear, the accumulation of these cells in acute and chronic injury models of transplantation was shown. In some cases, the depletion of M2 cells leads to the amelioration of disease; however in other causes, skewing the response in M2 cells leads to an augmentation of graft fibrosis and worsening of graft condition. In spite of these findings, the benefits of such strategies and their implications in human disease are not clearly understood. The purpose of this mini-review is to highlight the role of alternatively activated M2-type macrophages in the improvement or reduction of the kidney transplantation outcome.

Ovarian cancer is the common cause of death among gynecological cancers. Although ovarian cancer initially responds to chemotherapy, the frequent recurrence in patients remains a therapeutic challenge. Pyruvate kinase M2 (PKM2) plays a pivotal role in regulating cancer cell survival. However, its therapeutic roles remain unclear. Here, we investigated the anticancer effects of compound 3K, a specific PKM2 inhibitor, on autophagic and apoptotic pathway regulation in SK-OV-3 (PKM2-overexpressing human ovarian adenocarcinoma cell line). The anticancer effect of compound 3K was examined using the MTT and colony formation assay in SK-OV-3. The results of tissue microarray showed that PKM2 expression positively correlated with the severity of the tumor. Moreover, the expression of pro-apoptotic proteins increased in SK-OV-3 following compound 3K treatment. Compound 3K induced AMPK activation, which was accompanied by the inhibition of mTOR. Additionally, this compound inhibited glycolysis, resulting in reduced proliferation in SK-OV-3. Compound 3K treatment suppressed tumor progression in vivo xenograft model. Our findings suggest that the inhibition of PKM2 by compound 3K affected Warburg effects and induced autophagic cell death. Therefore, the use of specific PKM2 inhibitors to block the glycolytic pathway and target cancer cell metabolism represents a promising therapeutic approach for treating PKM2-overexpressing ovarian cancer.

Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis and is highly expressed in various cancer tissues. Although high PKM2 expression is observed in prostate cancer tissues, its functional role in cancer metabolism is unclear. Here, we investigated the role of PKM2 in regulating autophagy and its associated pathways in prostate cancer cells. PKM2 expression was silenced using various PKM2 small interfering RNAs (siRNAs) and then we measured PKM2-related cellular pathways associated with autophagy. PKM2 siRNA-transfected prostate cancer cells showed significantly reduced viability. Acridine orange staining and immunoblotting analysis showed that PKM2 downregulation markedly increased autophagic cell death. Results of western blotting analysis showed that PKM2 knockdown affected protein kinase B/mechanistic target of rapamycin 1 pathway, which consequently downregulated the expression of glycolytic enzymes lactate dehydrogenase A and glucose transporter 1. To the best of our knowledge, this is the first study to show that PKM2 inhibition alters cancer cell metabolism and induces autophagy. Thus, the present study provides a strategy for the development of PKM2-targeted novel anticancer drugs for the treatment of prostate cancer.

Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. IBs assembly is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We show that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1−P interaction, thereby affecting the relative mobility of proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.

Microglia are resident immune cells of the central nervous system such as brain-specific macrophages and also known to regulate the innate immune functions of astrocytes through secretory molecules. This conversation plays an important role in brain functions and homeostasis as well as in neuropathologic disease. In this study, we aimed to elucidate whether astrocytes and microglia can cross-talk to induce microglial polarization and proliferation, which can be further regulated under the brain stroke-mimic microenvironment. Microglia in mixed glial culture increased their survival and proliferation and altered to the M2 microglia, whose role was provided by CD11b-GFAP+ astrocytes by showing approximately tenfold increase in microglia cell proliferation after the astrocyte reconstitution. Furthermore, GM-CSF stimulated microglial proliferation approximately tenfold and induced to CCR7+ M1 microglia, whose phenotype could be suppressed by anti-inflammatory cytokines such as IL-4, IL-10, and Substance-P. Also, astrocyte in the microglia co-culture revealed A2 phenotype, which could be activated to A1 astrocyte by TNFα and IFNγ under the stroke-mimic condition. Altogether, astrocyte in the mixed glial culture stimulated the microglia proliferation and M2 polarization possibly through its acquisition of A2 phenotype, both of which could be converted to M1 microglia and A1 astrocytes under the inflammatory stroke-mimic environment. This study demonstrated that microglia and astrocyte can be polarized to M2 microglia and A2 astrocytes respectively through the cross-talk in vitro and provided a system to explore how microglia and astrocyte may behave in the inflammatory disease milieu after in vivo transplantation.

Sepsis-associated encephalopathy (SAE) is a common brain dysfunction following sepsis that often results in severe cognitive and neurological sequelae and increases the mortality rate in patients with sepsis. Microglia are resident macrophages in the brain that play essential roles in the pathological and physiological processes of SAE. Depending on the nature of the stimulus, microglia can adopt two polarization states (M1/M2), which correspond to altered microglial morphology, gene expression, and function. Therefore, we systematically described the pathogenesis, morphology, function, and phenotype of microglial activation in SAE. We demonstrated that microglia are closely related to occurrence and development of SAE and concomitant cognitive impairement. Finally, we outlined some potential therapeutic approaches that can prime microglia and neuroinflammation towards the beneficial restorative microglial phenotype in SAE.

AIM Non-alcoholic　fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Pemafibrate, a selective peroxisome proliferator-activated receptor α modulator (SPPARMα), has been reported to ameliorate liver function among patients with dyslipidemia. However, there are not many reports of the clinical effects of the pemafibrate. This study aims to summarize the experience of using pemafibrate and analyze the effects on liver function in patients with dyslipidemia. METHODS One hundred twelve cases of hyperlipidemia receiving pemafibrate 0.2 mg/day were retrospectively enrolled in this study. Age, gender, BMI, complications, concomitant medications, serum parameters (TG, HDL-C, LDL-C, AST, ALT, γGTP, ALP, platelets, M2BPGi, Cre, eGFR, HbA1c, blood glucose level at any time) were investigated and evaluated. RESULTS Pemafibrate administration significantly improved serum TG and HDL-C, but not in LDL-C. Serum AST, ALT, γGTP, and ALP were also significantly improved. The fib-4 index, a liver fibrosis score, did not change significantly, but M2-BPGi, an index of fibrosis, decreased significantly. No correlation was observed between each lipid parameter and ALT, and ALT decreased independently of the lipid parameters. Conclusions As we expected, pemafibrate demonstrated a lipid-improving effect without adversely affecting hepatic and renal functions. An unexpected finding was the decrease in ALT that was independent of lipid parameters.

Background: Although autophagy is a pro-survival process of tumor cells, in particular conditions and when differently regulated by specific signals it can stimulate cell death. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (M2 mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N-8-Iper. Methods: To assess the activation of autophagy, we analyzed the expression of LC3B by Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by Caspases 3 and 9 protein expression. Results: Our data indicate that activation of M2 mAChR by N-8-Iper promotes autophagy in both U251 and GB7 cells lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by the pAMPK expression. Downstream autophagy activation, the increase of apoptosis was also observed in both cell lines after treatment with the two M2 agonists. Conclusions: N-8-Iper treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N-8-Iper.

The characteristic lesion of primary tuberculosis is the granuloma as is widely studied in human tissues and animal models. Post-primary tuberculosis is different. It develops only in human lungs and begins as a prolonged subclinical obstructive lobular pneumonia that slowly accumulates mycobacterial antigens and host lipids in alveolar macrophages with nearby highly sensitized T cells. After several months, the lesions undergo necrosis to produce a mass of caseous pneumonia large enough to fragment and be coughed out to produce a cavity or be retained as the focus of a post-primary granuloma. Here we extend these findings with the demonstration of mycobacterial antigen, but not AFB, of M. tuberculosis in the cytoplasm of ciliated bronchiolar epithelium and alveolar pneumocytes in association with elements of the programmed death ligand 1 (PD-L1), cyclo-oxygenase (COX)-2, and fatty acid synthase (FAS) pathways in the early lesion. This suggests that M. tuberculosis uses its secreted antigens to coordinate prolonged subclinical development of the early le-sions in preparation for a necrotizing reaction sufficient to produce a cavity, post-primary granulomas and fibrocaseous disease.

The characteristic lesion of primary tuberculosis is the granuloma as is widely studied in human tissues and animal models. Post-primary tuberculosis is different. It develops only in human lungs and begins as a prolonged subclinical obstructive lobular pneumonia that slowly accumulates mycobacterial antigens and host lipids in alveolar macrophages with nearby highly sensitized T cells. After several months, the lesions under necrosis to produce a mass of caseous pneumonia large enough to fragment and be coughed out to produce a cavity or be retained as the focus of a post-primary granuloma. Here we extend these findings with the demonstration of mycobacterial antigen, but not AFB, of M. tuberculosis in the cytoplasm of ciliated bronchiolar epithelium and alveolar pneumocytes in association with elements of the programmed death ligand 1 (PD-L1), cyclo-oxygenase (COX)-2, and fatty acid synthase (FAS) pathways in the early lesion. This suggests that M. tuberculosis use its secreted antigens to coordinate prolonged subclinical development of the early lesions in preparation for a necrotizing reaction sufficient to produce a cavity, post-primary granulomas and fibrocaseous disease

Background: Recently we demonstrated that Astragalus polysaccharide (PG2), the active ingredient in dried roots of astragalus membranaceus, ameliorates cancer symptom clusters and improve quality of life (QoL) in patients with metastatic disease by modulating inflammatory cascade, against the background that inflammatory cells including macrophages, dendritic cells (DCs), and cytotoxic T lymphocytes (CTLs) in tumor initiation, metastasis, and progression. Nevertheless, the role of PG2 in the modulation of anticancer immunogenicity and therapeutic response remains relatively underexplored and unclear. Purpose: The present study investigates how and to what extent PG2 modulates cellular and biochemical components of the inflammatory cascade, enhance anticancer immunity, and the therapeutic implication of same in patients with lung cancer. Methods and Results: Herein, we demonstrated that PG2 significantly increased the M1/M2 macrophage polarization ratio in non-small cell carcinoma (NSCLC) H441 and H1299 cells. This PG2-induced preferential pharmacologic up-regulation of tumoral M1 population in vitro, positively correlated with the downregulation of tumor-promoting IL-6 and IL-10 expression in NSCLC cell-conditioned medium, with concomitant marked inhibition of cell proliferation, clonogenicity and tumorsphere formation. Our ex vivo results, using clinical sample from our NSCLC cohort, demonstrated that PG2 also promoted the functional maturation of DCs with consequent enhancement of T cell-mediated anticancer immune responses. Consistent with the in vitro and ex vivo results, our in vivo studies showed that mice treated with PG2 exhibited significant time-dependent depletion of the tumor-associated M2 population, synergistically enhanced the anti-M2-based anticancer effect of cisplatin, and inhibition of xenograft tumor growth in the NSCLC mice models. Moreover, in the presence of PG2, cisplatin-associated dyscresia and weight-loss was markedly suppressed. Conclusion: These results do indicate a therapeutically-relevant role for PG2 in modulating the M1/M2 macrophage pool, facilitating DC maturation, and synergistically enhancing the anticancer effect of conventional chemotherapeutic agent, cisplatin; thus laying the foundation for further exploration of the curative relevance of PG2 as surrogate immunotherapy and/or clinical feasibility of its use for maintenance therapy in patients with lung cancer.

Biphasic bone substitutes (BBS) are nowadays established biomaterials. Through their constant development even natural components like hyaluronic acid (HY) are added to improve both their handling and also their regenerative properties. However, low knowledge exists regarding the consequences of the HY addition for their biocompatibility and the inflammatory tissue reactions. Thus, the present study was conducted aiming to analyze the influence of two different amounts of high molecular weight HY (HMWHY) combined with a BBS on the in vitro biocompatibility and the in vivo tissue reaction. Established in vitro procedures using L929 cells were used for cytocompatibility analyses under the test conditions of DIN EN:ISO 10993-5. For the in vivo study part, calvarial defects were created in 20 Wistar rats and subsequently filled with the BBS and the BBS combined with two different HMWHY amounts, i.e., BBS+HY(L) and BBS+HY(H). As controls empty defects were used. Established histological, immunohistochemical and histomorphometrical methods were applied to analyze the tissue reactions to the three different materials, including the induction of pro- and anti-inflammatory macrophages and multinucleated giant cells (BMGCs). The in vitro results showed that none of the materials or compositions caused biological damages to the L929 cells and can considered to be non-toxic. The in vivo results showed that only the addition of high doses of HY to a biphasic bone substitute significantly decreases the occurrence of proinflammatory macrophages (* p < 0.05) comparable to the numbers found in the control group, while no significant differences within the three study groups for M2-macrophages nor BMGCs were detected. In conclusion, the addition of different amounts of HMWHY does not seem to affect the inflammation response to the BBS while improving the material handling properties.

Over the past several years there has been much debate with regards to the prognostic and clinical significance of pancreatic ductal adenocarcinoma (PDAC) with lymph nodes metastasis. The PDAC gene-expression knowledge and the biologic alterations underlying the lymph node involvement convey a clinical implication in dealing with the theranostic window.To this end, we provide an original bioinformatic dissection of the gene-expression differences of PDAC according to the nodal involvement from a large public available dataset. Comprehensive transcriptomic analysis from 143 RNA-seq patient’s derived samples indicated that WNT increased activation and a peculiar immune-microenvironment identify subjects with nodal involvement.In frame of this thinking, we validated the WNT pathway role in increasing the likelihood of lymphatic dissemination in vitro. Moreover, we demonstrated for the first time in a PDAC model the potential therapeutic window that XAV-939, a specific WNT pathway inhibitor, has in re-educating a tumour permissive immune system. Finally, we outline the potential implication on bystander molecular drivers exerted by WNT molecular inhibition, providing a picture of the proteomic oncogenic landscape changes elicited by XAV-939 on PDAC cells and their clinical implication. Our findings hold the promise to identify novel immune-based therapeutic strategies targeting WNT to enhance PDAC cytotoxicity and restore anti-PDAC immunity in nodes-positive disease.

Macrophages and dendritic cells (DCs) acquire functionally distinct properties in response to various environmental stimuli; the interaction of these cells with myeloid-derived suppressor cells (MDSCs) in tumor microenvironments regulates cancer progression. Immunodeficient mice lacking T cells are less likely to reject human cancer cells because of major histocompatibility complex (MHC) mismatches. The xenograft tumor microenvironment, comprising human cancer and mouse host cells, exhibits more complex bidirectional signaling and function than a syngeneic tumor microenvironment. Here human and mouse colorectal cancer cells were transplanted into nude mice to elucidate differences in macrophage, DC, and MDSC functions in human xenograft and mouse allograft tumor models. Plasma interferon-γ and interleukin-18 concentrations in the former model after intraperitoneal lipopolysaccharide (LPS) administration were significantly higher than those in the latter model and non-transplanted control group. Splenic MHC class I, II, and CD80 expression increased in CD11b+ and MDSC populations after LPS administration in only the xenograft tumor model. The number of CD80- and MRC1-expressing cells decreased upon LPS administration in only the xenograft tumor. These results suggxest that macrophages and DCs function normally in xenograft tumor models, whereas their functions in response to LPS administration vary in allograft tumor models.

Although most people are infected with Epstein-Barr Virus (EBV) during their lifetime, only a minority of them develop an EBV-associated malignancy. EBV acts in both direct and indirect ways to transform infected cells into tumor cells. There are multiple ways in which the EBV, host, and tumor environment interact to promote malignant transformation. This paper focuses on some of the mechanisms that EBV uses to transform the tumor microenvironment (TME) of EBV-associated gastric cancer (EBVaGC) for its benefit, including overexpression of IDO1, synergism between H pylori and EBV coinfection, and M1 to M2 switch. In this review, we expand on different modalities and combinatorial approaches to therapeutically target this mechanism.