Optimization of cell density and LPS concentration for the evaluation of nitric oxide production on BV-2 cells in a Griess assay

Production of nitric oxide (NO) is one of the main responses elicited by a variety of immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation. Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the intensity of an immune response; and has been performed using different methods including the Griess assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype. Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2 microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS) for the Griess assay is demonstrated in this study. Methods:BV-2 microglia were cultured at different cell densities, and treated with LPS at three concentrations (1, 5, 10 μg/ml). NO production in culture supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT) was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose dependent, and NO levels are increased proportional to the LPS concentration up to 1.0μg/ml, whereas the higher LPS concentrations are associated with decreased cell viability may be caused by the high toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however, optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells

Comparative analysis of inflammatory markers produced by macrophages inoculated with invasive and colonizing strains of Streptococcus agalactiae (group B streptococcus) and evaluation of patients' clinical data

Introduction: Group B Streptococcus (GBS), infection and recurrence in newborns and pregnant women can lead to chronic medical illness resulting in significant morbidity, and mortality. Pathogenesis of GBS may be due to reasons such as activation of the immune system, followed by the production of inflammatory markers and toxic components by immune cells including macrophages. Methods: The studies on invasive and colonizing GBS strains inoculated either with peripheral or brain macrophages, the expression of nitric oxide (NO), cell viability, and CD40 were also measured by Griess assay, methyl tetrazolium assay (MTT), and flow cytometry, respectively. Furthermore, the clinical manifestations of the selected patients were also assessed for this study. Results: Outcome of inflammatory markers studies, after GBS inoculation indicated that, invasive GBS strains induced higher inflammatory markers in comparison to colonizing GBS strains. Furthermore, patients’ clinical data showed that patients with invasive GBS infections had severe condition unlike among patients with colonizing GBS strains. The fatality rate in patients with invasive GBS strain were 30.8% while there was no death among carriers. Conclusion: This study, aimed to understand the immune response to GBS, and strengthen the knowledge on GBS pathogenesis. It was concluded that invasive GBS strains not only showed higher expression of inflammatory markers on immune cells but also had higher pathogenesis effect in comparison to colonizing GBS strains

Mesenchymal stem cell-mediated immunomodulation of microglia

Neuroinflammation is a key pathogenic event of neurological diseases. The continuous inflammatory responses of microglia exacerbate disease by increasing neuronal damage, an effect that may warrant control. An approach for this is the utilisation of mesenchymal stem cells (MSC). This study explores the mechanisms through which MSC modulate inflammatory responses of microglia. For this, mouse bone marrowderived MSC and BV2 (a microglial cell line) were cocultured at different ratios. The anti-proliferative effect of MSC on microglia was deciphered by examining cell cycle, apoptosis and the role of nitric oxide (NO). Migration of both cell types was also examined along with differential expression of soluble mediators. Direct contact of BV2 with MSC at the 1:0.2 (BV2:MSC) seeding ratio inhibited proliferation of LPS-stimulated BV2 microglia to 71.2 ± 9.7% (p<0.05), an effect also conferred by MSC soluble factors. At the same 1:0.2 ratio, MSC also increased NO expression in cocultures, inducing a 25% surge from 56.94 ± 2.65μM to 76.59 ± 3.08μM at 48hrs (p<0.05). However, NO was not implicated in the anti-proliferative effect as inhibiting NO did not restore BV2 proliferation. Role of apoptosis in the reduction of BV2 proliferation was also ruled out as the number of Annexin-V-/PI- cells remained high. A slowdown of cell cycle was identified as the mechanism through which MSC exert their anti-proliferative effect on microglia. Coculture with MSC reduced the population of BV2 microglia in S-phase by 6.25 ± 1.5% (p<0.001) and restored the percentage of BV2 cells at the G2/Mphase to levels similar in unstimulated BV2 microglia. The immunomodulatory effects reported here were also accompanied by an MSC cell cycle arrest at G0/G1-phase (percentage of MSC in G0/G1-phase increased from 55.34 ± 2.6% to 86.32 ± 2.0% (p<0.001)). Using protein array, galectin-1 was identified as a possible immunomodulator as its levels increased significantly to 2.16-fold in coculture (p<0.05). Presence of MSC also increased IL-6 (by 30-fold, p<0.05) whereas TNF-α was reduced by 4-fold (p<0.05). The study then explored the role of IL-6 and TNF-α in MSC-mediated modulation of NO production and proliferation of microglia. By using neutralising antibodies, it was shown that IL-6 did not play a role in the NO production of cocultures or inhibition of microglial proliferation, whilst neutralisation of TNF-α abolished the NO surge although leaving proliferation unaffected in coculture. As blocking TNF-α reduced LPS-stimulated BV2 proliferation, proliferation of microglia was proposed to be mediated by TNF-α and that MSC inhibit microglial proliferation by downregulating TNF-α levels in coculture. This study then pursued the migratory properties of microglia and MSC. The results demonstrated that BV2 cells showed remarkable migration to MSC, paralleled by an increase in MMP-9 activity (12.35 ± 2.89-fold). It was also shown that MSC intrinsically migrated towards microglia (208.5 ± 10.6), and more so in inflammatory conditions (295.3 ± 43.8). MSC also migrated towards LPS-stimulated astrocytes. However, LPS alone did not influence MSC migration,indicating the importance of a cellular impetus that MSC requires for their migration and the reciprocity in migration of BV2 microglia and MSC. The findings from this study shed light on mechanisms of immunomodulatory functions of MSC on microglia which improve our understanding for MSC-mediated therapy of neuroinflammatory conditions

Reciprocal interactions of bone marrow-derived mesenchymal stem cells and BV2 microglia following lipopolysaccharide stimulation

INTRODUCTION: Mesenchymal stem cells (MSCs) are immunosuppressive, but we lack an understanding of how these adult stem cells are in turn affected by immune cells and the surrounding tissue environment. As MSCs have stromal functions and exhibit great plasticity, the influence of an inflamed microenvironment on their responses is important to determine. MSCs downregulate microglial inflammatory responses, and here we describe the mutual effects of coculturing mouse bone marrow MSCs with BV2 microglia in a lipopolysaccharide (LPS) inflammatory paradigm. METHODS: Mouse MSCs were cultured from femoral and tibial bone marrow aspirates and characterized. MSCs were cocultured with BV2 microglia at four seeding-density ratios (1:0.2, 1:0.1, 1:0.02, and 1:0.01 (BV2/MSC)), and stimulated with 1 μg/ml LPS. In certain assays, MSCs were separated from BV2 cells with a cell-culture insert to determine the influence of soluble factors on downstream responses. Inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) were measured in cocultures, and MSC and BV2 chemotactic ability determined by migration assays. RESULTS: We demonstrated MSCs to increase expression of NO and IL-6 and decrease TNF-α in LPS-treated cocultures. These effects are differentially mediated by soluble factors and cell-to-cell contact. In response to an LPS stimulus, MSCs display distinct behaviors, including expressing IL-6 and very high levels of the chemokine CCL2. Microglia increase their migration almost fourfold in the presence of LPS, and interestingly, MSCs provide an equal impetus for microglia locomotion. MSCs do not migrate toward LPS but migrate toward microglia, with their chemotaxis increasing when microglia are activated. Similarly, MSCs do not produce NO when exposed to LPS, but secrete large amounts when exposed to soluble factors from activated microglia. This demonstrates that certain phenotypic changes of MSCs are governed by inflammatory microglia, and not by the inflammatory stimulus. Nonetheless, LPS appears to "prime" the NO-secretory effects of MSCs, as prior treatment with LPS triggers a bigger NO response from MSCs after exposure to microglial soluble factors. CONCLUSIONS: These effects demonstrate the multifaceted and reciprocal interactions of MSCs and microglia within an inflammatory milieu

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-1low) to typical MSC phenotype (CD106+CD45-CD11b-Sca-1high) with increasing passages. CONCLUSION: Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation

Nitric oxide modulation in neuroinflammation and the role of mesenchymal stem cells

Nitric oxide is a versatile mediator formed by enzymes called nitric oxide synthases. It has numerous homeostatic functions and important roles in inflammation. Within the inflamed brain, microglia and astrocytes produce large amounts of nitric oxide during inflammation. Excessive nitric oxide causes neuronal toxicity and death and mesenchymal stem cells can be used as an approach to limit the neuronal damage caused by neuroinflammation. Mesenchymal stem cell therapy ameliorates inflammation and neuronal damage in disease models of Alzheimer’s disease, Parkinson’s disease, and other neuroinflammatory disorders. Interestingly, we have reported that in vitro, mesenchymal stem cells themselves contribute to a rise in nitric oxide levels through microglial cues. This may be an undesirable effect and highlights a possible need to explore acellular approaches for mesenchymal stem cell therapy in the central nervous system

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

Immunomodulatory properties of wharton’s jelly-derived mesenchymal stem cells from three anatomical segments of umbilical cord

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are reported to be immune-privileged and immuneevasive. MSCs are capable of differentiating into specific cell types for subsequent use in cell-based therapy. They express low levels of human leucocyte antigen (HLA)-ABC and no HLA-DR. Wharton’s jelly-derived MSCs (WJ-MSCs) were also found to express human leukocyte antigen G (HLA-G), which renders them immunosuppressive. This study aimed to determine whether cultured WJ-MSCs retain their immune-privileged and immune-evasive properties after cell differentiation, and whether these properties differ among MSCs derived from different anatomical segments of the umbilical cord. Umbilical cords of healthy pregnant mothers undergoing caesarean section were obtained and grouped by three anatomical segments: fetal, middle, and maternal segments. WJ-MSCs were isolated, culture-expanded, and differentiated into osteogenic cells. Expression of HLA-DR, HLA-ABC, and HLA-G were quantified using flow cytometry. Both undifferentiated and osteodifferentiated WJ-MSCs were subsequently co-cultured with allogeneic peripheral blood mononuclear cells with/without lipopolysaccharide (LPS) stimulation for five days. Lymphocyte proliferation assay was performed using carboxyfluorescein succinimidyl ester (CFSE) as a tracker. Our results showed no significant difference existed in the HLA profiles among WJ-MSCs from different segments and between WJ-MSCs with and without osteogenic differentiation. Mean levels for HLA-G, HLABC, and HLA-DR were 24.82±17.64, 52.50±18.41, and 1.00±1.68%, respectively. Stimulation with LPS and WJ-MSCs increased peripheral blooc mononuclear cells (PBMC) proliferation. However, PBMC proliferation was significantly lower when PBMCs were co-cultured with osteodifferentiated WJ-MSCs (p < .05; with LPS stimulation and p < .001 without LPS stimulation) than when they were co-cultured with undifferentiated WJ-MSCs. These findings suggest that cultured WJ-MSCs stimulate lymphocyte proliferation and are not immune-privileged. Osteodifferentiated WJ-MSCs reduced the immunogenicity of WJ-MSCs, and this reduction in PBMC proliferation was even more pronounced in the presence of LPS (p < .05). In conclusion, cultured WJ-MSCs are not immune-privileged. Osteodifferentiated WJ-MSCs are less immunogenic than undifferentiated WJ-MSCs, in which case hypoimmunogenicity is more profound under LPS-stimulated conditions