The characterization of human microglia has been hampered by poor availability of human cell sources. However, microglia is involved in the physiopathology of multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, HIV dementia, retinal degenerative diseases, cancer, and many other conditions. Therefore, there is an important need to have experimental paradigms of human microglia characterized and usable to study the role of microglia in the different pathologies in which it is involved. In the present work, we carried out an extensive characterization of Immortalized Human Microglia—SV40 cell line (IMhu), marketed by Applied Biological Material. The functional response of IMhu to a large variety of stimuli was studied. In particular, we investigated morphology, mortality, and changes in the production of different cytokines and chemokines, both under basal conditions and after stimulation. Moreover, western blotting analysis was conducted on phospho-mTOR (Ser 2448) and downstream parameters, p-P70S6K and 4EBP1, in order to understand if IMhu can be used for evaluations of mTOR pathway. In conclusion, IMhu cells proved to be a useful experimental model to investigate the physiopathology of inflammatory disease that involved microglia cells, including pathological conditions that involved the mTOR pathway.

The glioblastoma (GB) microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs) that are still poorly characterized. A potential role on the mechanisms regulating GAM activity might be played by the endoplasmic reticulum protein ERp57/PDIA3 (protein disulfide-isomerase A3), the modulation of which has been reported in a variety of cancers. Moreover, by using The Cancer Genome Atlas database, we found that overexpression of PDIA3 correlated with about 55% reduction of overall survival of glioma patients. Therefore, we analyzed the expression of ERp57/PDIA3 using specimens obtained after surgery from 18 GB patients. Immunohistochemical analysis of tumor samples revealed ERp57/PDIA3 expression in GB cells as well as in GAMs. The ERp57/PDIA3 levels were higher in GAMs than in the microglia present in the surrounding parenchyma. Therefore, we studied the role of PDIA3 modulation in microglia–glioma interaction, based on the ability of conditioned media collected from human GB cells to induce the activation of microglial cells. The results indicated that reduced PDIA3 expression/activity in GB cells significantly limited the microglia pro-tumor polarization towards the M2 phenotype and the production of pro-inflammatory factors. Our data support a role of PDIA3 expression in GB-mediated protumor activation of microglia.

No abstract

The anti-vascular endothelial growth factor-A (VEGF-A) monoclonal antibody (mAb) bevacizumab is an FDA-approved monotherapy for the treatment of recurrent glioblastoma (GB), a highly angiogenic and infiltrative tumour. However, bevacizumab does not increase overall survival and blockade of VEGF-A/VEGF receptor (VEGFR)-2 signal transduction is associated with severe adverse effects due to inhibition of physiological angiogenesis. Conversely, VEGFR-1 does not play a relevant role in physiological angiogenesis in the adult. VEGFR-1 is activated by both VEGF-A and placenta growth factor (PlGF), a protein involved in tumour growth and progression. In previous studies, it was demonstrated that inhibition of VEGFR-1 using a specific mAb developed in our laboratories reduced angiogenesis and GB cell chemotaxis and increased the survival of tumour-bearing mice. Failure of treatments directed toward the VEGF-A/VEGFR-2 axis could in part be due to inefficient targeting of the tumour microenvironment. In the present study, VEGFR-1 expression was investigated in GB-associated microglia/macrophages (GAMs) by analysing surgical specimens collected from 42 patients with GB. Data obtained from The Cancer Genome Atlas (TCGA) database revealed that upregulation of the VEGFR-1 ligands VEGF-A and PlGF was associated with a significant reduction in overall survival for patients with GB, highlighting the potential relevance of this receptor in the aggressiveness of GB. Immunohistochemical analysis indicated that VEGFR-1 is expressed not only in GB tissue but also in GAMs. Furthermore, the percentage of VEGFR-1-positive GAMs was significantly higher in the tumour region compared with that noted in the surrounding parenchyma. Thus, VEGFR-1 represents a potential therapeutic target for the treatment of GB, being present not only in GB and endothelial cells, but also in GAMs that are involved in tumour progression.

No abstract

No abstract

Previous studies in the rat suggest that microglial cells represent a potential druggable target for nerve growth factor (NGF) in the brain. The painless human Nerve Growth Factor (hNGFp) is a recombinant mutated form of human nerve growth factor (hNGF) that shows identical neurotrophic and neuroprotective properties of wild-type NGF but displays at least 10-fold lower algogenic activity. From the pharmacological point of view, hNGFp is a biased tropomyosin receptor kinase A (TrkA) agonist and displays a significantly lower affinity for the p75 neurotrophin receptor (p75NTR). This study aimed to evaluate the expression of TrkA and p75NTR NGF receptors in two different human microglia cell lines, and to investigate the effects of hNGFp and wild-type NGF (NGF) on L-arginine metabolism, taken as a marker of microglia polarization. Both NGF receptors are expressed in human microglia cell lines and are effective in transducing signals triggered by NGF and hNGFp. The latter and, to a lesser extent, NGF inhibit cytokine-stimulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these cells. Conversely NGF but not hNGFp stimulates arginase-mediated urea production.

Background: Glioblastoma (GB - grade IV glioma) is the most aggressive and common cancer of central nervous system with an overall survival of 14-16 months. The GB tumor microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs). It is known that between GAMs and GB cells there is a double interaction, but the role of GAMs is still poorly characterized. The endoplasmic reticulum (ER) protein ERp57, also known as PDIA3, is a thiol oxidoreductase with main function related on glycoprotein folding in endoplasmic reticulum. However, PDIA3 shows different functions. In fact, the various subcellular localizations and binding partners of PDIA3 affect numerous physiological processes and diseases: different regulation and modulation of PDIA3 has been reported in multiple pathologies including neurodegenerative diseases and cancer. Methods: In the present work, we evaluated in both GB cells and microglia-macrophage cells the expression of PDIA3 using specimens collected after surgical from 18 GB patients. In addition, we studied in vitro microglia-glioma interaction to determine the role of PDIA3 in viability and the activation of both GB and microglia cells. The study was carried using PDIA3-silenced T98G cells and/or using a pharmacological inhibitor of PDIA3 activity (Punicalagin-PUN).Results: We initially investigated the role of the PDIA3 in GB survival by inquiring The Cancer Genome Atlas dataset. The results indicated that 352 out of 690 patients reported over-expression of PDIA3, which significantly correlated with a ~55% reduction of overall survival. Subsequently, for the first time, we investigated the PDIA3 expression in the tumor and the nearby parenchyma of 18 GB patients and our data showed a significant upregulation (15% vs 10%) of ERp57/PDIA3 in GAMs of tumor specimens respect the microglia present in parenchyma. In addition, we show that conditioned medium (CMs) obtained from both wild type T98G and PDIA3 silenced T98G induced an activation of microglia cells, but the PDIA3 silenced-T98G CMs significant limited the microglia pro-tumor activation probably through a IL-6-STAT3-PDIA3 dependent mechanism. Conclusion: Our data support the relevant role of PDIA3 expression in GB pathology and link the different activation of microglia to a mechanism a IL-6-STAT3-PDIA3 dependent.