Dissecting functional phenotypes of microglia and macrophages in the rat brain after transient cerebral ischemia.

Ischemic brain injury causes local inflammation, which involves activation of resident microglia, leukocyte, and monocyte infiltration. Involvement of peripheral immune cells in ischemia-induced damage and repair is debatable. Using flow cytometry, gene expression profiling, and immunocytochemistry, we show that microglia predominate in the ischemic brain and express inflammation mediators at Day 1 after transient middle cerebral artery occlusion (MCAo) in rats. At Day 3, both resident microglia and bone marrow (BM)-derived macrophages are detected in the ischemic hemispheres and display unique transcriptomic profiles. Functional groups enriched in BM-macrophages are indicative of the pro-regenerative, immunosuppressive phenotype. Transient depletion of peripheral macrophages with clodronate-filled liposomes reduced the number of Arg1+ Iba1+ expressing cells in the ischemic brain. The analysis of microglia and macrophage signature genes shows that each cell type maintains the expression of their identity genes, even if gene expression is modified in a response to environmental clues. At Day 7, infiltrating BM-macrophages exhibit the reduced expression of Arg1, the elevated expression of iNos and many inflammatory genes, as shown by RNA sequencing. This is consistent with their switch toward a pro-inflammatory phenotype. We propose that BM-macrophages recruited to the injured brain early after ischemia could contribute to functional recovery after stroke, but they switch toward a pro-inflammatory phenotype in the ischemic parenchyma. Our results point to the detrimental role of microglia in an ischemic brain and the primarily pro-regenerative role of infiltrating BM-macrophages.

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage.

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke.

Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas.

Glioblastoma (GBM) is the most aggressive primary brain tumor, with ineffective anti-tumor responses and a poor prognosis despite aggressive treatments. GBM immune microenvironment is heterogenous  and activation of specific immune populations in GBM is not fully characterized. Reliable animal models are critical for defining mechanisms of anti-tumor immunity. First we analyzed the immune subpopulations present in rat C6 gliomas. Using flow cytometry we determined kinetics of infiltration of myeloid cells and T lymphocytes into glioma-bearing brains. We found significant increases of the amoeboid, pro-tumorigenic microglia/macrophages, T helper (Th) and T regulatory (Treg) cells in tumor-bearing brains, and rare infiltrating T cytotoxic (Tc) cells. Transcriptomic analyses of glioma-bearing hemispheres revealed overexpression of invasion and immunosuppression-related genes, reflecting the immunosuppressive microenvironment. Microglia, sorted as CD11b+CD45low cells from gliomas, displayed the pro-invasive and immunosuppressive type of activation. Accumulation of Th and Treg cells combined with the reduced presence of Tc lymphocytes in rat gliomas may result in the lack of effective anti-tumor responses. Transcriptional profiles of CD11b+ cells and composition of immune infiltrates in C6 gliomas indicate that rat C6 gliomas employ similar immune system evasion strategies as human GBMs.

Immune microenvironment of gliomas.

High-grade gliomas are rapidly progressing tumors of the central nervous system (CNS) with a very poor prognosis despite extensive resection combined with radiation and/or chemotherapy. Histopathological and flow cytometry analyses of human and rodent experimental gliomas revealed heterogeneity of a tumor and its niche, composed of reactive astrocytes, endothelial cells, and numerous immune cells. Infiltrating immune cells consist of CNS resident (microglia) and peripheral macrophages, granulocytes, myeloid-derived suppressor cells (MDSCs), and T lymphocytes. Intratumoral density of glioma-associated microglia/macrophages (GAMs) and MDSCs is the highest in malignant gliomas and inversely correlates with patient survival. Although GAMs have a few innate immune functions intact, their ability to be stimulated via TLRs, secrete cytokines, and upregulate co-stimulatory molecules is not sufficient to initiate antitumor immune responses. Moreover, tumor-reprogrammed GAMs release immunosuppressive cytokines and chemokines shaping antitumor responses. Both GAMs and MDSCs have ability to attract T regulatory lymphocytes to the tumor, but MDSCs inhibit cytotoxic responses mediated by natural killer cells, and block the activation of tumor-reactive CD4+ T helper cells and cytotoxic CD8+ T cells. The presence of regulatory T cells may further contribute to the lack of effective immune activation against malignant gliomas. We review the immunological aspects of glioma microenvironment, in particular composition and various roles of the immune cells infiltrating malignant human gliomas and experimental rodent gliomas. We describe tumor-derived signals and mechanisms driving myeloid cell accumulation and reprogramming. Although, understanding the complexity of cell-cell interactions in glioma microenvironment is far from being achieved, recent studies demonstrated several glioma-derived factors that trigger migration, accumulation, and reprogramming of immune cells. Identification of these factors may facilitate development of immunotherapy for gliomas as immunomodulatory and immune evasion mechanisms employed by malignant gliomas pose an appalling challenge to brain tumor immunotherapy.

The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma.

Osteopontin (SPP1, a secreted phosphoprotein 1) is primarily involved in immune responses, tissue remodelling and biomineralization. However, it is also overexpressed in many cancers and regulates tumour progression by increasing migration, invasion and cancer stem cell self-renewal. Mechanisms of SPP1 overexpression in gliomas are poorly understood. We demonstrate overexpression of two out of five SPP1 isoforms in glioblastoma (GBM) and differential isoform expression in glioma cell lines. Up-regulated SPP1 expression is associated with binding of the GLI1 transcription factor to the promoter and OCT4 (octamer-binding transcription factor 4) to the first SPP1 intron of the SPP1 gene in human glioma cells but not in non-transformed astrocytes. GLI1 knockdown reduced SPP1 mRNA and protein levels in glioma cells. GLI1 and OCT4 are known regulators of stem cell pluripotency. GBMs contain rare cells that express stem cell markers and display ability to self-renew. We reveal that SPP1 is overexpressed in glioma initiating cells defined by high rhodamine 123 efflux, sphere forming capacity and stemness marker expression. Forced differentiation of human glioma spheres reduced SPP1 expression. Knockdown of SPP1, GLI1 or CD44 with siRNAs diminished sphere formation. C6 glioma cells stably depleted of Spp1 displayed reduced sphere forming capacity and downregulated stemness marker expression. Overexpression of the wild type Spp1, but not Spp1 lacking a Cd44 binding domain, rescued cell ability to form spheres. Our findings show re-activation of the embryonic-type transcriptional regulation of SPP1 in malignant gliomas and point to the importance of SPP1-CD44 interactions in self-renewal and pluripotency glioma initiating cells.

Myeloid-derived suppressor cells in gliomas.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of early myeloid progenitors and precursors at different stages of differentiation into granulocytes, macrophages, and dendritic cells. Blockade of their differentiation into mature myeloid cells in cancer results in an expansion of this population. High-grade gliomas are the most common malignant tumours of the central nervous system (CNS), with a poor prognosis despite intensive radiation and chemotherapy. Histopathological and flow cytometry analyses of human and rodent experimental gliomas revealed the extensive heterogeneity of immune cells infiltrating gliomas and their microenvironment. Immune cell infiltrates consist of: resident (microglia) and peripheral macrophages, granulocytes, myeloid-derived suppressor cells, and T lymphocytes. Intratumoural density of glioma-associated MDSCs correlates positively with the histological grade of gliomas and patient's survival. MDSCs have the ability to attract T regulatory lymphocytes to the tumour, but block the activation of tumour-reactive CD4+ T helper cells and cytotoxic CD8+ T cells. Immunomodulatory mechanisms employed by malignant gliomas pose an appalling challenge to brain tumour immunotherapy. In this mini-review we describe phenotypic and functional characteristics of MDSCs in humans and rodents, and their occurrence and potential roles in glioma progression. While understanding the complexity of immune cell interactions in the glioma microenvironment is far from being accomplished, there is significant progress that may lead to the development of immunotherapy for gliomas.

The CXCL12-3'A allele plays a favourable role in patients with multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterized by bone marrow infiltration and the presence of a monoclonal protein in serum and/or urine. CXCR4 and its ligand CXCL12 are essential for neoplastic cell homing to bone marrow in haematological malignancies. The JAK2/STAT3 pathway, which is activated after CXCL12 binding to CXCR4, takes part in many signalling cascades which are linked to cell proliferation and cell survival. Constitutive activation of this pathway plays an important role in tumourigenesis and malignant transformation. The present study aimed to determine the association between the polymorphic features located within the CXCR4 (rs2228014) and CXCL12 (rs1801157) encoding genes and disease susceptibility and progression. For this purpose 172 individuals including 54 patients with MM and 118 healthy controls were typed for the CXCL12 (A/G) and CXCR4 (C/T) alleles using the PCR-RFLP technique. The CXCL12 alleles and genotypes segregated similarly among patients and controls while the CXCR4 T variant was less frequently represented among patients (OR=0.074, p<0.001). All patients with the CXCR4 T allele and 16 out of 48 with wild type genotype presented with grade III of MM according to the International Staging System (ISS) (p=0.047). The CXCL12-3'A variant was more frequently detected in patients with less advanced MM (9/17 vs. 7/38, p=0.012 for patients in stage IA or IIA vs. IIB, IIIA and IIIB, respectively). Moreover, patients lacking the CXCL12-3'A variant more frequently presented with ISS II-III (32/38 vs. 5/13, p=0.003 for patients lacking CXCL12-3'A with ISS>I vs. ISS=I). This favourable effect of the CXCL12-3'A allele was also seen in the analysis of patient survival (p<0.05). The impact of the CXCL12-3'A allele was confirmed by multivariate analyses. In conclusion, these results imply that the CXCL12-3'A allele plays a favourable role in patients with multiple myeloma.

Genetic variant of the G-CSF receptor gene is associated with lower mobilization potential and slower recovery of granulocytes after transplantation of autologous peripheral blood progenitor cells.

Peripheral blood mobilized by cytokines (i.e. granulocyte colony stimulating factor, G-CSF) and chemotherapy has become a major source of hematopoietic stem and progenitor cells for transplantation (PBPCT). In this study the effect of the G-CSF receptor (CSF3R) gene polymorphism was investigated. The presence of the CSF3R variant (T allele, rs3917924) was related to CD34(+) mobilization yield and the pace of granulocyte recovery after autologous PBPCT. The mobilization yield was higher in patients lacking the CSF3R variant (OR=4.756, p=0.046) and those with multiple myeloma (OR=10.534, p=0.019). The pace of granulocyte recovery was found to be associated with the CSF3R polymorphism and was significantly slower in patients carrying the CSF3R-T variant than in CC homozygotes (median of 17 vs. 13 days, p<0.001). This association was confirmed (OR=4.445, p=0.014) by multiple regression analysis considering patient age and sex, the number of transplanted CD34(+) cells, diagnosis and CSF3R polymorphism. These results imply that CSF3R gene polymorphism plays a significant role in PBPCT.

[The role of the SDF-1-CXCR4 axis in hematopoiesis and the mobilization of hematopoietic stem cells to peripheral blood]. / Znaczenie interakcji miedzy SDF-1 i CXCR4 w hematopoezie i mobilizacji macierzystych komórek hematopoetycznych do krwi obwodowej.

The treatment of donors with a granulocyte colony-stimulating factor (G-CSF) induces the mobilization of hematopoietic stem cells (HSCs) from the bone marrow to the blood circulation and guarantees a sufficient number of peripheral blood progenitor cells (PBPCs). This mobilization and leukopheresis are very complicated and unclear processes because of the relationships between many factors from outside and inside of the body in the course of GCS-F's action. One of these factors is stromal cell derived factor-1 (SDF-1, CXCL12), a member of the CXC chemokine family, which influences normal development of the embryo and directs many processes in mature organisms. This chemokine plays an important role in hematopoiesis leading to the normal development of hematopoietic cells. Interaction between SDF-1 and its receptor CXCR4 influences HSCs, which constitute a part of the transplantation material. This review describes SDF-1 and its effects on hematopoiesis and mobilization. Interactions between HSCs and their microenvironment are very important in all clinical transplantation procedures, starting from the mobilization of hematopoietic stem cells in donors to the process of leukopheresis and transplantation, and ending with the last step when the organism regenerates all HSC populations. For this reason, part of this article is dedicated to the role of adhesion molecules (present on the surface of stem cells, hematopoietic stem cells, and bone marrow cells) and SDF-1. This article deals with a small component of clinical transplantation. We hope that a deeper understanding of the SDF-1-CXCR4 axis will allow considering this relation in clinical practice to improve transplantation protocols (i.e. through the use of SDF-1-antagonist as a mobilization factor).