Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production methods
Aim. Evidence-backed execution summary for Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production methods from Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production.
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Effect of BMSCs on plasma cytokine concentrations
reagent used in the protocol.
- Use
- We hypothesized that BMSCs might alter the immune response to infection; therefore, we studied TNF-α and IL-6, proinflammatory cytokines that have a central role in sepsis. Twenty-four hours after injection of BMSCs, there was a significant reduction in serum TNF-α and IL-6 concentrations in treated versu...
Involvement of immune cell subtypes in the effect of BMSCs
reagent used in the protocol.
- Use
- The observations summarized above suggested that BMSCs might quickly act to reprogram a specific population of cells involved in mediating the immune response. To test this hypothesis, we examined the effects of BMSCs in mice that genetically lack mature T and B cells ( Rag2 -/- ) or are depleted of natu...
Macrophage-derived IL-10 is key for the effect of BMSCs
reagent used in the protocol.
- Use
- Because IL-10 serum levels were increased in CLP mice that were treated with BMSCs as compared to sham controls, we asked whether IL-10 might be important for the actions of BMSCs. To test this hypothesis, we treated mice with an antibody to IL-10 or an antibody to the IL-10 receptor before CLP. In both of these gro...
Macrophage-derived IL-10 is key for the effect of BMSCs
reagent used in the protocol.
- Use
- Because IL-10 has been reported to inhibit the rolling, adhesion and transepithelial migration - of neutrophils, we examined the white cell counts in the circulation of treated and untreated CLP mice and found a significant increase in the number of circulating neutrophils in the treated mice ( ). It is possible tha...
Molecular basis of the BMSC-macrophage interaction
reagent used in the protocol.
- Use
- To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB activation 30 min after LPS stimulation ( ). In a number of cell types, NF-κB has been shown to induce prostaglandin production and re...
Molecular basis of the BMSC-macrophage interaction
reagent used in the protocol.
- Use
- To further examine the effect of BMSCs on macrophages, we cultured macrophages with BMSCs, added LPS and measured the IL-10 concentration in the culture medium. Because Toll-like receptor-4 (TLR4) is the receptor to which LPS binds and through which it acts, we were not surprised to see that BMSCs from Tlr4 -...
Molecular basis of the BMSC-macrophage interaction
reagent used in the protocol.
- Use
- Because it seemed that prostaglandin E 2 might mediate the effect of BMSCs on macrophage cytokine production, we asked whether specific EP receptors could be involved in transducing the prostaglandin signal. To answer this question, we used prostaglandin receptor antagonists and macrophages from EP receptor-kn...
Figures
reagent used in the protocol.
- Use
- Characterization of control and stimulated mononuclear cells in CLP mice in vivo and in vitro. ( a ) The distribution of monocytes (Mon), macrophages (Mac) and polymorphonuclear cells (PMN) among cells isolated from septic lungs. Error bars represent means ± s.e.m. ( b ) FACS plots of lung mononuclear cells. T...
Effect of BMSCs on plasma cytokine concentrations
To learn where injected BMSCs go and how long they remain detectable, we prelabeled the cells with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) fluorescent tracking dye and visualized them 1-24 h later. We found BMSCs in the blood up to 1 h after intravenous injections and saw many cells in the lu...
- Use
- To learn where injected BMSCs go and how long they remain detectable, we prelabeled the cells with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) fluorescent tracking dye and visualized them 1-24 h later. We found BMSCs in the blood up to 1 h after intravenous injections and saw many cells in the lu...
Macrophage-derived IL-10 is key for the effect of BMSCs
Because IL-10 serum levels were increased in CLP mice that were treated with BMSCs as compared to sham controls, we asked whether IL-10 might be important for the actions of BMSCs. To test this hypothesis, we treated mice with an antibody to IL-10 or an antibody to the IL-10 receptor before CLP. In both of these gro...
- Use
- Because IL-10 serum levels were increased in CLP mice that were treated with BMSCs as compared to sham controls, we asked whether IL-10 might be important for the actions of BMSCs. To test this hypothesis, we treated mice with an antibody to IL-10 or an antibody to the IL-10 receptor before CLP. In both of these gro...
Molecular basis of the BMSC-macrophage interaction
Because it seemed that prostaglandin E 2 might mediate the effect of BMSCs on macrophage cytokine production, we asked whether specific EP receptors could be involved in transducing the prostaglandin signal. To answer this question, we used prostaglandin receptor antagonists and macrophages from EP receptor-kn...
- Use
- Because it seemed that prostaglandin E 2 might mediate the effect of BMSCs on macrophage cytokine production, we asked whether specific EP receptors could be involved in transducing the prostaglandin signal. To answer this question, we used prostaglandin receptor antagonists and macrophages from EP receptor-kn...
Figures
Characterization of control and stimulated mononuclear cells in CLP mice in vivo and in vitro. ( a ) The distribution of monocytes (Mon), macrophages (Mac) and polymorphonuclear cells (PMN) among cells isolated from septic lungs. Error bars represent means ± s.e.m. ( b ) FACS plots of lung mononuclear cells. T...
- Use
- Characterization of control and stimulated mononuclear cells in CLP mice in vivo and in vitro. ( a ) The distribution of monocytes (Mon), macrophages (Mac) and polymorphonuclear cells (PMN) among cells isolated from septic lungs. Error bars represent means ± s.e.m. ( b ) FACS plots of lung mononuclear cells. T...
Ex vivo studies on isolated lung monocytes and macrophages
We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pieces in RPMI 1640 medium with 1% penicillin-streptomycin and 1% glutamine for 30 min at 37 °C 5% CO 2 in the presence of collagenase typ...
- Use
- We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pieces in RPMI 1640 medium with 1% penicillin-streptomycin and 1% glutamine for 30 min at 37 °C 5% CO 2 in the presence of collagenase typ...
Macrophage-bone marrow stromal cells coculture experiments
First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh medium or fresh medium alone to the wells. As a control, we plated BMSCs without macrophages. After an overnight incubation, we added LPS to...
- Use
- First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh medium or fresh medium alone to the wells. As a control, we plated BMSCs without macrophages. After an overnight incubation, we added LPS to...
Blood chemistry, natural killer cell and macrophage depletion and IL-10 neutralization, microvascular permeability, b...
Detailed methodology is described in the.
- Use
- Detailed methodology is described in the.
Statistical analyses
We examined the differences between the groups for statistical significance by Student's t -test or analysis of variance with an appropriate correction. We compared survival curves with a log-rank test (Prism 4.0; Graphpad Software). A P value of <0.05 was accepted as statistically significant.
- Use
- We examined the differences between the groups for statistical significance by Student's t -test or analysis of variance with an appropriate correction. We compared survival curves with a log-rank test (Prism 4.0; Graphpad Software). A P value of <0.05 was accepted as statistically significant.
Statistical analyses
Software used for acquisition, scoring, statistics, or reporting.
- Use
- We examined the differences between the groups for statistical significance by Student's t -test or analysis of variance with an appropriate correction. We compared survival curves with a log-rank test (Prism 4.0; Graphpad Software). A P value of <0.05 was accepted as statistically significant.
Before you run
What should be confirmed before execution?
First confirmation
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Effect of BMSCs on plasma cytokine concentrations
Alterations in vascular permeability are central to the pathogenesis of sepsis-induced organ injury, and the benefits of BMSC treatment in reducing vascular permeability have already been reported -. We studied this in the peritoneum, lung, liver and kidney by measuring Evans blue dye leakage 24 h after CLP. CLP surgery significantly increased peritoneal, liver and renal vascular permeability ( P < 0.01, P < 0.01 and P < 0.001, respectively); all three were significantly ( P < 0.05, P < 0.05 and P < 0.05, respectively) decreased in mice treated with BMSCs ( online).
Molecular basis of the BMSC-macrophage interaction
To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB activation 30 min after LPS stimulation ( ). In a number of cell types, NF-κB has been shown to induce prostaglandin production and release through a pathway involving cyclooxygenase-2 (COX2; refs. - ). BMSCs have been shown to produce prostaglandin E 2 and possibly affect other immune cells via EP1-EP4 receptors,. We found a significant increase in the expression and activity of COX2 (which produces the substrate for prostaglandin synthase enzymes) in BMSCs 3 h and 5 h after LPS stimulation ( ), but when the BMSCs were treated with an antibody to TNF-α or collected from Tlr4 -/- mice, the COX2 expression did not change ( ). This suggested that prostaglandin E 2 might indeed be re...
METHODS
Mouse care was in full compliance with the US NIH criteria for the care and use of laboratory animals in research and all studies were approved by the Animal Care and Use Committee of the NIDDK and NIDCR, NIH. Aged (42-44 weeks old) male C57BL/6 mice (NIH) had free access to water and chow before and after surgery. In addition, 8-12-week-old males with a variety of genetic manipulations were used as listed in the online. All reagents used are summarized in online.
Polymicrobial cecal ligation and puncture sepsis
We performed CLP as previously described, with some modifications, in C57BL/6 mice,. We ligated the cecum by silk 4-0 and punctured it twice with a 21-gauge needle, gently squeezed it to express a small amount of fecal material and then returned it to the central abdominal cavity. In sham-operated mice, we located the cecum but neither ligated nor punctured it. We closed the abdominal incision in two layers with 6-0 nylon sutures. After surgery, we gave 1 ml per 30 g body weight of pre-warmed normal saline. All mice received antibiotic and fluid therapy subcutaneously (a combination of imipenem and cilastatin; 14 mg per kg in 1.5 ml of normal saline at 6 h and 7 mg per kg in 1.5 ml of normal saline at 18 h after surgery). The details of the tissue harvest are described in the.
Treatment of cecal ligation and puncture mice with bone marrow stromal cells
We injected one million BMSCs in 0.3 ml sterile PBS via the tail vein. We gave untreated control mice 0.3 ml sterile PBS with no cells. There was no difference in organ injury, cytokine abundance, Evans blue dye level or pathology in the sham-operated mice with or without BMSC injection. Details of cell isolation and culture are provided in the.
Survival studies
We assessed survival after surgery every 6 h within the first 48 h and then every 8 h for 4 d. We began antibiotic injection and fluid resuscitation 6 h after CLP by subcutaneous injection and repeated it every 12 h for 4 d. We killed all mice at the end of the fourth day.
Ex vivo studies on isolated lung monocytes and macrophages
We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pieces in RPMI 1640 medium with 1% penicillin-streptomycin and 1% glutamine for 30 min at 37 °C 5% CO 2 in the presence of collagenase type 1 (300 U ml -1 ) and DNase I (50 U ml -1 ) (Worthington Biochemicals). After the incubation, we filtered the cell suspension through a 70-µm cell strainer and then washed it with complete RPMI medium. We incubated the resulting cells for 15 min at 4 °C with CD11b magnetic beads and subsequently applied them to MS columns (Miltenyi) for the positive selection of CD11b cells. The cell purity of the CD11b + cells, as assessed by FACS analysis, was greater than 97%. After washing, we plated the isolated cells in 96-well plates at a concentration of 50,00...
Macrophage-bone marrow stromal cells coculture experiments
First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh medium or fresh medium alone to the wells. As a control, we plated BMSCs without macrophages. After an overnight incubation, we added LPS to the cocultures to reach a final concentration of 1 µg ml -1. We collected supernatants 1, 3, 5 and 7 h after the stimulation and performed ELISA (R&D Systems) on the samples to detect the released IL-10. For the transwell or conditioned medium experiments, we either added BMSCs on the insert membrane of the transwell system (HTS Transwell 0.4-µm pore size poly-carbonate membrane, from Corning Incorporated) or added conditioned medium diluted 1:1 with fresh medium to the macrophages. For generation of BMSC-conditioned medium, we cultured BMSCs in complete m...
Measurement outputs
What raw and processed outputs should exist?
To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pie...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We examined the differences between the groups for statistical significance by Student's t -test or analysis of variance with an appropriate correction. We compared survival cur...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
We hypothesized that BMSCs might alter the immune response to infection; therefore, we studied TNF-α and IL-6, proinflammatory cytokines that have a central role in sepsis.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB...; We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pie...; First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh...; We examined the differences between the groups for statistical significance by Student's t -test or analysis of variance with an appropriate correction. We compared survival cur....
from paperStatistical comparison
We hypothesized that BMSCs might alter the immune response to infection; therefore, we studied TNF-α and IL-6, proinflammatory cytokines that have a central role in sepsis...; Because IL-10 serum levels were increased in CLP mice that were treated with BMSCs as compared to sham controls, we asked whether IL-10 might be important for the actions of BMS...; Because IL-10 has been reported to inhibit the rolling, adhesion and transepithelial migration - of neutrophils, we examined the white cell counts in the circulation of treated...; To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB...
from paperReporting output
Report representative outputs alongside summary comparisons for To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB..., We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pie..., First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh..., We examined the differences between the groups for statistical significance by Student's t -test or analysis of variance with an appropriate correction. We compared survival cur....
inferred from protocolStructured statistical methods
We hypothesized that BMSCs might alter the immune response to infection; therefore, we studied TNF-α and IL-6, proinflammatory cytokines that have a central role in sepsis...; Because IL-10 serum levels were increased in CLP mice that were treated with BMSCs as compared to sham controls, we asked whether IL-10 might be important for the actions of BMS...; Because IL-10 has been reported to inhibit the rolling, adhesion and transepithelial migration - of neutrophils, we examined the white cell counts in the circulation of treated...; To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Alterations in vascular permeability are central to the pathogenesis of sepsis-induced organ injury, and the benefits of BMSC treatment in reducing vascular permeability have already been reported -. We studied this in the peritoneum, lung, liver and kidney by measuring Evans blue dye leakage 24 h after CLP. CLP surgery significantly increased peritoneal, liver and renal vascular permeability ( P < 0.01, P < 0.01 and P < 0.001, respectively); all three were significantly ( P < 0.05, P < 0.05 and P < 0.05, respectively) decreased in mice treated with BMSCs ( online).
To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB activation 30 min after LPS stimulation ( ). In a number of cell types, NF-κB has been shown to induce prostaglandin production and release through a pathway involving cyclooxygenase-2 (COX2; refs. - ). BMSCs have been shown to produce prostaglandin E 2 and possibly affect other immune cells via EP1-EP4 receptors,. We found a significant increase in the expression and activity of COX2 (which produces the substrate for prostaglandin synthase enzymes) in BMSCs 3 h and 5 h after LPS stimulation ( ), but when the BMSCs were treated with an antibody to TNF-α or collected from Tlr4 -/- mice, the COX2 expression did not change ( ). This suggested that prostaglandin E 2 might indeed be responsible for reprogramming the macrophages. For this reason, we measured the amount of prostaglandin E 2 in the coculture medium and found a significant increase in its concentration after LPS stimulation ( ). This increase was eliminated if the BMSCs lacked TLR4 or if they were incubated with anti...
Mouse care was in full compliance with the US NIH criteria for the care and use of laboratory animals in research and all studies were approved by the Animal Care and Use Committee of the NIDDK and NIDCR, NIH. Aged (42-44 weeks old) male C57BL/6 mice (NIH) had free access to water and chow before and after surgery. In addition, 8-12-week-old males with a variety of genetic manipulations were used as listed in the online. All reagents used are summarized in online.
We performed CLP as previously described, with some modifications, in C57BL/6 mice,. We ligated the cecum by silk 4-0 and punctured it twice with a 21-gauge needle, gently squeezed it to express a small amount of fecal material and then returned it to the central abdominal cavity. In sham-operated mice, we located the cecum but neither ligated nor punctured it. We closed the abdominal incision in two layers with 6-0 nylon sutures. After surgery, we gave 1 ml per 30 g body weight of pre-warmed normal saline. All mice received antibiotic and fluid therapy subcutaneously (a combination of imipenem and cilastatin; 14 mg per kg in 1.5 ml of normal saline at 6 h and 7 mg per kg in 1.5 ml of normal saline at 18 h after surgery). The details of the tissue harvest are described in the.
We injected one million BMSCs in 0.3 ml sterile PBS via the tail vein. We gave untreated control mice 0.3 ml sterile PBS with no cells. There was no difference in organ injury, cytokine abundance, Evans blue dye level or pathology in the sham-operated mice with or without BMSC injection. Details of cell isolation and culture are provided in the.
We assessed survival after surgery every 6 h within the first 48 h and then every 8 h for 4 d. We began antibiotic injection and fluid resuscitation 6 h after CLP by subcutaneous injection and repeated it every 12 h for 4 d. We killed all mice at the end of the fourth day.
We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pieces in RPMI 1640 medium with 1% penicillin-streptomycin and 1% glutamine for 30 min at 37 °C 5% CO 2 in the presence of collagenase type 1 (300 U ml -1 ) and DNase I (50 U ml -1 ) (Worthington Biochemicals). After the incubation, we filtered the cell suspension through a 70-µm cell strainer and then washed it with complete RPMI medium. We incubated the resulting cells for 15 min at 4 °C with CD11b magnetic beads and subsequently applied them to MS columns (Miltenyi) for the positive selection of CD11b cells. The cell purity of the CD11b + cells, as assessed by FACS analysis, was greater than 97%. After washing, we plated the isolated cells in 96-well plates at a concentration of 50,000 cells per well per 200 µl RPMI medium (with 10% FBS, 1% glutamine and 1% penicillin-streptomycin) containing 10 µg ml -1 LPS (from Escherichia coli O111:B4, Sigma). We incubated cells for 1, 3, 5 and 7 h after stimulation and collected supernatants sequentially from wells dedicated...
First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh medium or fresh medium alone to the wells. As a control, we plated BMSCs without macrophages. After an overnight incubation, we added LPS to the cocultures to reach a final concentration of 1 µg ml -1. We collected supernatants 1, 3, 5 and 7 h after the stimulation and performed ELISA (R&D Systems) on the samples to detect the released IL-10. For the transwell or conditioned medium experiments, we either added BMSCs on the insert membrane of the transwell system (HTS Transwell 0.4-µm pore size poly-carbonate membrane, from Corning Incorporated) or added conditioned medium diluted 1:1 with fresh medium to the macrophages. For generation of BMSC-conditioned medium, we cultured BMSCs in complete medium. After 3 d, we collected supernatants, spun them down to remove possible cell contamination (500 g for 10 min) and stored the resulting supernatants at -20 °C until further use. Further details on ELISA and western blotting are in the.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production methods",
"description": "Evidence-backed execution summary for Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production methods from Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production.",
"totalTime": "PT105670M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Effect of BMSCs on plasma cytokine concentrations",
"text": "Alterations in vascular permeability are central to the pathogenesis of sepsis-induced organ injury, and the benefits of BMSC treatment in reducing vascular permeability have already been reported -. We studied this in the peritoneum, lung, liver and kidney by measuring Evans blue dye leakage 24 h after CLP. CLP surgery significantly increased peritoneal, liver and renal vascular permeability ( P < 0.01, P < 0.01 and P < 0.001, respectively); all three were significantly ( P < 0.05, P < 0.05 and P < 0.05, respectively) decreased in mice treated with BMSCs ( online)."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Molecular basis of the BMSC-macrophage interaction",
"text": "To understand the molecular basis of the interaction between BMSCs and macrophages, we performed a series of experiments in vitro and in vivo. Wild-type BMSCs showed NF-κB activation 30 min after LPS stimulation ( ). In a number of cell types, NF-κB has been shown to induce prostaglandin production and release through a pathway involving cyclooxygenase-2 (COX2; refs. - ). BMSCs have been shown to produce prostaglandin E 2 and possibly affect other immune cells via EP1-EP4 receptors,. We found a significant increase in the expression and activity of COX2 (which produces the substrate for prostaglandin synthase enzymes) in BMSCs 3 h and 5 h after LPS stimulation ( ), but when the BMSCs were treated with an antibody to TNF-α or collected from Tlr4 -/- mice, the COX2 expression did not change ( ). This suggested that prostaglandin E 2 might indeed be re..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "METHODS",
"text": "Mouse care was in full compliance with the US NIH criteria for the care and use of laboratory animals in research and all studies were approved by the Animal Care and Use Committee of the NIDDK and NIDCR, NIH. Aged (42-44 weeks old) male C57BL/6 mice (NIH) had free access to water and chow before and after surgery. In addition, 8-12-week-old males with a variety of genetic manipulations were used as listed in the online. All reagents used are summarized in online."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Polymicrobial cecal ligation and puncture sepsis",
"text": "We performed CLP as previously described, with some modifications, in C57BL/6 mice,. We ligated the cecum by silk 4-0 and punctured it twice with a 21-gauge needle, gently squeezed it to express a small amount of fecal material and then returned it to the central abdominal cavity. In sham-operated mice, we located the cecum but neither ligated nor punctured it. We closed the abdominal incision in two layers with 6-0 nylon sutures. After surgery, we gave 1 ml per 30 g body weight of pre-warmed normal saline. All mice received antibiotic and fluid therapy subcutaneously (a combination of imipenem and cilastatin; 14 mg per kg in 1.5 ml of normal saline at 6 h and 7 mg per kg in 1.5 ml of normal saline at 18 h after surgery). The details of the tissue harvest are described in the."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Treatment of cecal ligation and puncture mice with bone marrow stromal cells",
"text": "We injected one million BMSCs in 0.3 ml sterile PBS via the tail vein. We gave untreated control mice 0.3 ml sterile PBS with no cells. There was no difference in organ injury, cytokine abundance, Evans blue dye level or pathology in the sham-operated mice with or without BMSC injection. Details of cell isolation and culture are provided in the."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Survival studies",
"text": "We assessed survival after surgery every 6 h within the first 48 h and then every 8 h for 4 d. We began antibiotic injection and fluid resuscitation 6 h after CLP by subcutaneous injection and repeated it every 12 h for 4 d. We killed all mice at the end of the fourth day."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Ex vivo studies on isolated lung monocytes and macrophages",
"text": "We killed septic mice 6 h after CLP induction with or without intravenous injection of 1 million BMSCs. We removed the lungs, minced them into small pieces and incubated the pieces in RPMI 1640 medium with 1% penicillin-streptomycin and 1% glutamine for 30 min at 37 °C 5% CO 2 in the presence of collagenase type 1 (300 U ml -1 ) and DNase I (50 U ml -1 ) (Worthington Biochemicals). After the incubation, we filtered the cell suspension through a 70-µm cell strainer and then washed it with complete RPMI medium. We incubated the resulting cells for 15 min at 4 °C with CD11b magnetic beads and subsequently applied them to MS columns (Miltenyi) for the positive selection of CD11b cells. The cell purity of the CD11b + cells, as assessed by FACS analysis, was greater than 97%. After washing, we plated the isolated cells in 96-well plates at a concentration of 50,00..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Macrophage-bone marrow stromal cells coculture experiments",
"text": "First, we plated macrophages in 96-well plates at a concentration of 40,000 cells per 200 µl. After 1 h, we removed the supernatants and added either 40,000 BMSCs in fresh medium or fresh medium alone to the wells. As a control, we plated BMSCs without macrophages. After an overnight incubation, we added LPS to the cocultures to reach a final concentration of 1 µg ml -1. We collected supernatants 1, 3, 5 and 7 h after the stimulation and performed ELISA (R&D Systems) on the samples to detect the released IL-10. For the transwell or conditioned medium experiments, we either added BMSCs on the insert membrane of the transwell system (HTS Transwell 0.4-µm pore size poly-carbonate membrane, from Corning Incorporated) or added conditioned medium diluted 1:1 with fresh medium to the macrophages. For generation of BMSC-conditioned medium, we cultured BMSCs in complete m..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Effect of BMSCs on plasma cytokine concentrations"
},
{
"@type": "HowToTool",
"name": "Macrophage-derived IL-10 is key for the effect of BMSCs"
},
{
"@type": "HowToTool",
"name": "Molecular basis of the BMSC-macrophage interaction"
},
{
"@type": "HowToTool",
"name": "Figures"
},
{
"@type": "HowToTool",
"name": "Ex vivo studies on isolated lung monocytes and macrophages"
},
{
"@type": "HowToTool",
"name": "Macrophage-bone marrow stromal cells coculture experiments"
},
{
"@type": "HowToTool",
"name": "Blood chemistry, natural killer cell and macrophage depletion and IL-10 neutralization, microvascular permeability, b..."
},
{
"@type": "HowToTool",
"name": "Statistical analyses"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Effect of BMSCs on plasma cytokine concentrations"
},
{
"@type": "HowToSupply",
"name": "Involvement of immune cell subtypes in the effect of BMSCs"
},
{
"@type": "HowToSupply",
"name": "Macrophage-derived IL-10 is key for the effect of BMSCs"
},
{
"@type": "HowToSupply",
"name": "Macrophage-derived IL-10 is key for the effect of BMSCs"
},
{
"@type": "HowToSupply",
"name": "Molecular basis of the BMSC-macrophage interaction"
},
{
"@type": "HowToSupply",
"name": "Molecular basis of the BMSC-macrophage interaction"
},
{
"@type": "HowToSupply",
"name": "Molecular basis of the BMSC-macrophage interaction"
},
{
"@type": "HowToSupply",
"name": "Figures"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production",
"datePublished": "2008",
"author": [
{
"@type": "Person",
"name": "Krisztián Németh"
},
{
"@type": "Person",
"name": "Asada Leelahavanichkul"
},
{
"@type": "Person",
"name": "Peter S T Yuen"
},
{
"@type": "Person",
"name": "Balázs Mayer"
},
{
"@type": "Person",
"name": "Alissa Parmelee"
},
{
"@type": "Person",
"name": "Kent Doi"
},
{
"@type": "Person",
"name": "Pamela G Robey"
},
{
"@type": "Person",
"name": "Kantima Leelahavanichkul"
},
{
"@type": "Person",
"name": "Beverly H Koller"
},
{
"@type": "Person",
"name": "Jared M Brown"
},
{
"@type": "Person",
"name": "Xuzhen Hu"
},
{
"@type": "Person",
"name": "Ivett Jelinek"
},
{
"@type": "Person",
"name": "Robert A Star"
},
{
"@type": "Person",
"name": "Éva Mezey"
}
],
"identifier": "10.1038/nm.1905"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 -dependent reprogramming of host macrophages to increase their interleukin-10 production methods",
"item": "https://replicatescience.com/experiments/bone-marrow-stromal-cells-attenuate-sepsis-via-prostaglandin-e-2-8212-dependent-reprogramming-of-host-macrophages-to-increase-their-interleukin-10-production-methods-kriszti-225-n-/bone-marrow-stromal-cells-attenuate-sepsis-via-prostaglandin-e2-dependent-reprogramming-of-host-macr-mlpgveah"
}
]
}
]