Paracrine effects of mesenchymal stem cells-conditioned medium on microglial cytokines expression and nitric oxide production.

BACKGROUND/AIM: Microglia, the resident macrophages in the central nervous system, secrete various proinflammatory cytokines and undergo proliferation upon activation in various neurodegenerative diseases. Activation of microglia has been implicated in exacerbation of various neurodegenerative diseases. Recently, it has been proposed that mesenchymal stem cells (MSC) have immunosuppressive properties and the potential to moderate inflammation. This study aimed to elucidate the effects of MSC-conditioned medium (MSC-CM) in modulating microglial activation by analyzing microglial proinflammatory and anti-inflammatory factors [interleukin (IL)-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and IL-10], signaling pathway molecules [NFκB, c-Jun N-terminal kinase (JNK) and MKP-1) and NO production. METHODS: Immortalized murine microglia cell line, BV2 microglia and primary microglia isolated from C57BL/6 mouse pup brains were used in this study. Mouse MSC were isolated from the male C57BL/6 mouse tibia and fibula. The effects of MSC-CM on the expression of inflammatory cytokines and signaling molecules in microglia were elucidated using RT-PCR, immunofluorescence analysis and Western blot analysis. NO production in microglia was assessed using a Griess kit. RESULTS: MSC-CM significantly reduced the mRNA and protein expression levels of proinflammatory cytokines (IL-6 and TNF-α) in microglia activated by lipopolysaccharide (LPS). In addition, MSC-CM significantly reduced the protein expression of NFκB, JNK and c-Jun, but increased the expression levels of IL-10 and MKP-1 in activated BV2 microglia. NO production and iNOS expression by BV2 microglia in MSC-CM were increased. CONCLUSIONS: Overall, our findings suggest that MSC immunomodulate microglial activities through paracrine effects.

Mesenchymal stem cells exert anti-proliferative effect on lipopolysaccharide-stimulated BV2 microglia by reducing tumour necrosis factor-α levels.

BACKGROUND: Progression of neurodegenerative diseases occurs when microglia, upon persistent activation, perpetuate a cycle of damage in the central nervous system. Use of mesenchymal stem cells (MSC) has been suggested as an approach to manage microglia activation based on their immunomodulatory functions. In the present study, we describe the mechanism through which bone marrow-derived MSC modulate the proliferative responses of lipopolysaccharide-stimulated BV2 microglia. METHODS: BV2 microglia were cultured with MSC and stimulated with 1 µg/ml lipopolysaccharide. Using an inducible nitric oxide synthase inhibitor, tritiated thymidine (3H-TdR) incorporation assay was performed to determine the role of nitric oxide in the anti-proliferative effect of MSC. We also studied apoptosis and the cell cycle of both cell types using flow cytometry and explored their cytokine profile using protein and cytometric arrays. Moreover, the role of IL-6 and TNF-α in immunomodulation was deduced using specific blocking antibodies and recombinant proteins. RESULTS: MSC reduces microglia proliferation upon lipopolysaccharide stimulation by 21 to 28% and modulates the levels of nitric oxide, IL-6 and TNF-α. The role of nitric oxide in conferring the anti-proliferative effect of MSC was ruled out. Furthermore, we found that MSC exert their anti-proliferative effect by restoring the percentage of BV2 cells at S and G2/M phase to levels similar to unstimulated cells. MSC undergo a G0/G1 arrest while exerting this effect. We have also identified that MSC-mediated modulation of microglia is independent of IL-6, whilst reduction of TNF-α in co-culture is critical for inhibition of microglia proliferation. CONCLUSIONS: Our study demonstrates that MSC inhibit microglia proliferation independent of nitric oxide and IL-6, although reduction of TNF-α is critical for this effect. The inhibition of proliferation is through cell cycle modulation. These findings shed light on the mechanisms of microglial immunomodulation by MSC.

A review on the cultivation, bioactive compounds, health-promoting factors and clinical trials of medicinal mushrooms Taiwanofungus camphoratus, Inonotus obliquus and Tropicoporus linteus.

Medicinal mushrooms, such as Taiwanofungus camphoratus, Inonotus obliquus, and Tropicoporus linteus, have been used in traditional medicine for therapeutic purposes and promotion of overall health in China and many East Asian countries for centuries. Modern pharmacological studies have demonstrated the large amounts of bioactive constituents (such as polysaccharides, triterpenoids, and phenolic compounds) available in these medicinal mushrooms and their potential therapeutic properties. Due to the rising demand for the health-promoting medicinal mushrooms, various cultivation methods have been explored to combat over-harvesting of the fungi. Evidence of the robust pharmacological properties, including their anticancer, hypoglycemic, hypolipidemic, antioxidant, and antiviral activities, have been provided in various studies, where the health-benefiting properties of the medicinal fungi have been further proven through numerous clinical trials. In this review, the cultivation methods, available bioactive constituents, therapeutic properties, and potential uses of T. camphoratus, I. obliquus and T. linteus are explored.

Response Profiles of BV2 Microglia to IFN-γ and LPS Co-Stimulation and Priming.

(1) Background: The latest research illustrates that microglia phenotype is not the binary 'resting' and 'activated' profiles. Instead, there is wide diversity in microglia states. Similarly, when testing different stimulation protocols for BV2 microglia, we discovered differences in the response of the cells in terms of the production of intracellular ROS (iROS), nitric oxide (NO), CD40 expression, and migratory capacity. (2) Methods: BV2 microglia were treated with single interferon gamma (IFN-γ) stimulation, LPS/IFN-γ co-stimulation, and priming with IFN-γ followed by stimulation with LPS for 24 h. The responses of BV2 microglia were then assessed using the H2DCFDA test for iROS, the Griess assay for NO, immunophenotyping for CD40/CD11b/MHC II, and migration using a transwell apparatus. (3) Results: Single stimulation with IFN-γ induced NO but not ROS in BV2 microglia. Co-stimulation with LPS200IFN-γ2.5 induced a higher iROS production (a 9.2-fold increase) and CD40 expression (28031 ± 8810.2 MFI), compared to priming with primedIFN-γ50LPS100 (a 4.0-fold increase in ROS and 16764 ± 1210.8 MFI of CD40). Co-stimulation also induced cell migration. On the other hand, priming BV2 microglia (primedIFN-γ50LPS100) resulted in a higher NO production (64 ± 1.4 µM) compared to LPS200IFN-γ2.5 co-stimulation (44 ± 1.7 µM). Unexpectedly, priming inhibited BV2 migration. (4) Conclusions: Taken together, the findings from this project reveal the ability of co-stimulation and priming in stimulating microglia into an inflammatory phenotype, and the heterogeneity of microglia responses towards different stimulating approaches.

Increased SPRY1 expression activates NF-κB signaling and promotes pancreatic cancer progression by recruiting neutrophils and macrophages through CXCL12-CXCR4 axis.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high mortality rate, in which about 90% of patients harbor somatic oncogenic point mutations in KRAS. SPRY family genes have been recognized as crucial negative regulators of Ras/Raf/ERK signaling. Here, we investigate the expression and role of SPRY proteins in PDAC. METHODS: Expression of SPRY genes in human and mice PDAC was analyzed using The Cancer Genome Atlas and Gene Expression Omnibus datasets, and by immunohistochemistry analysis. Gain-of-function, loss-of-function of Spry1 and orthotopic xenograft model were adopted to investigate the function of Spry1 in mice PDAC. Bioinformatics analysis, transwell and flowcytometry analysis were used to identify the effects of SPRY1 on immune cells. Co-immunoprecipitation and K-ras4B G12V overexpression were used to identify molecular mechanism. RESULTS: SPRY1 expression was remarkably increased in PDAC tissues and positively associated with poor prognosis of PDAC patients. SPRY1 knockdown suppressed tumor growth in mice. SPRY1 was found to promote CXCL12 expression and facilitate neutrophil and macrophage infiltration via CXCL12-CXCR4 axis. Pharmacological inhibition of CXCL12-CXCR4 largely abrogated the oncogenic functions of SPRY1 by suppressing neutrophil and macrophage infiltration. Mechanistically, SPRY1 interacted with ubiquitin carboxy-terminal hydrolase L1 to induce activation of nuclear factor κB signaling and ultimately increase CXCL12 expression. Moreover, SPRY1 transcription was dependent on KRAS mutation and was mediated by MAPK-ERK signaling. CONCLUSION: High expression of SPRY1 can function as an oncogene in PDAC by promoting cancer-associated inflammation. Targeting SPRY1 might be an important approach for designing new strategy of tumor therapy.

Immunotherapies for Parkinson's Disease: Progression of Clinical Development.

Parkinson's disease is a common neurodegenerative disease affecting the movement and well-being of most elderly. The manifestations of Parkinson's disease often include resting tremor, stiffness, bradykinesia, and muscular rigidity. The typical hallmark of Parkinson's disease is the destruction of neurons in the substantia nigra and the presence of Lewy bodies in different compartments of the central nervous system. Due to various limitations to the currently available treatments, immunotherapies have emerged to be the new approach to Parkinson's disease treatment. This approach shows some positive outcomes on the efficacy by removing the aggregated species of alpha-synuclein, which is believed to be one of the causes of Parkinson's disease. In this review, an overview of how alpha-synuclein contributes to Parkinson's disease and the effects of a few new immunotherapeutic treatments, including BIIB054 (cinpanemab), MEDI1341, AFFITOPE, and PRX002 (prasinezumab) that are currently under clinical development, will be discussed.

Effects of macrophage colony-stimulating factor on microglial responses to lipopolysaccharide and beta amyloid.

A challenge for studies involving microglia cultures is obtaining sufficient cells for downstream experiments. Macrophage colony-stimulating factor (M-CSF) has been used to improve yield of microglia in culture. However, the effects of M-CSF on activation profiles of microglia cultures are still unclear. Microglia activation is characterised by upregulation of co-stimulatory molecules and an inflammatory phenotype. The aim of this study is to demonstrate whether M-CSF supplementation alters microglial responses in resting and activated conditions. Microglia derived from mixed glia cultures and the BV-2 microglia cell line were cultivated with/without M-CSF and activated with lipopolysaccharide (LPS) and beta amyloid (Abeta). We show M-CSF expands primary microglia without affecting microglial responses to LPS and Abeta, as shown by the comparable expression of MHC class II and CD40 to microglia grown without this growth factor. M-CSF supplementation in BV-2 cells had no effect on nitric oxide (NO) production. Therefore, M-CSF can be considered for improving microglia yield in culture without introducing activation artefacts.

Immunophenotype and differentiation capacity of bone marrow-derived mesenchymal stem cells from CBA/Ca, ICR and Balb/c mice.

AIM: To assess the capacity to isolate and expand mesenchymal stem cells (MSC) from bone marrow of CBA/Ca, ICR and Balb/c mice. METHODS: Bone marrow of tibia and femur were flushed, cultured and maintained in supplemented Dulbecco's modified Eagle's medium. MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106, Sca-1 and CD44 and negativity to CD45, CD11b and MHC class II. Differentiation capacity of MSC towards osteogenic and adipogenic lineages were also assessed. RESULTS: MSC were successfully cultured from bone marrow of all 3 strains, albeit differences in the temporal expression of certain surface antigens. Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype (CD106(-)CD45(+)CD11b(+)Sca-1(low)) to typical MSC phenotype (CD106(+)CD45(-)CD11b(-)Sca-1(high)) with increasing passages. CONCLUSION: Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.

Bone marrow-derived mesenchymal stem cells modulate BV2 microglia responses to lipopolysaccharide.

The immunoregulatory properties of mesenchymal stem cells (MSC) have been demonstrated on a wide range of cells. Here, we describe the modulatory effects of mouse bone marrow-derived MSC on BV2 microglia proliferation rate, nitric oxide (NO) production and CD40 expression. Mouse bone marrow MSC were co-cultured with BV2 cells at various seeding density ratios and activated with lipopolysaccharide (LPS). We show that MSC exert an anti-proliferative effect on microglia and are potent producers of NO when stimulated by soluble factors released by LPS-activated BV2. MSC suppressed proliferation of both untreated and LPS-treated microglia in a dose-dependent manner, significantly reducing BV2 proliferation at seeding density ratios of 1:0.2 and 1:0.1 (p<.05). Co-culturing MSC with BV2 cells at different ratios revealed interesting dynamics in NO production. A high number of MSC significantly increases NO in co-cultures whilst a lower number reduces NO. The increased NO levels in co-cultures may be MSC-derived, as we also show that activated BV2 cells stimulate MSC to produce NO. Cell-cell interaction is not a requirement for this effect as soluble factors released by activated BV2 cells alone do stimulate MSC to produce high levels of NO. Although NO is implicated as a mediator for T cell proliferation, it does not appear to play a major role in the suppression of microglia proliferation. Additionally, MSC reduced the expression of the microglial co-stimulator molecule, CD40. Collectively, these regulatory effects of MSC on microglia offer insight into the potential moderating properties of MSC on inflammatory responses within the CNS.