Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: Adjuvant conditioning (AC) shapes the adaptive immune response in vivo . A: Experimental design. 8-10-week-old male or female C57BL/6 mice were injected intraperitoneally (i.p.) with either Alum Imject™ (8mg in 200µL) or saline three times, every other day. 24 hours after the last injection, mice were adoptively transferred with 5×10 6 OT-II splenocytes intravenously, and 24 hours after the transfer, mice were immunized subcutaneously with 10 μg OVA adsorbed in 1.5mg of Alum Imject™, in a homologous prime (day 1) and boost (day 8) protocol. Seven days after the boost (day 15), mice were euthanized, and spleens were analyzed for OVA-specific Th1, Th2, Th17, and Treg total numbers. B: Flow cytometry plots and total number of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline or alum before the immunization. Flow cytometry plots and total number of OVA-specific Th1 (C) , Th2 (D) , Th17 (E) , and Tregs (F) , evidenced by the expression of CD4 + Vα + Tbet + , CD4 + Vα + GATA3 + , CD4 + Vα + RORγT + , and CD4 + Vα + Foxp3 + in splenocytes from mice treated with either saline or alum before the immunization. G: Flow cytometry plots and total number (H) of OVA-specific Th17 and Tregs, evidenced by the expression of CD4 + Vα + IL-17A and CD4 + Vα + LAP + in splenocytes treated with either saline or alum before the immunization and further cultured with OVAp (2μg/mL) in vitro for 48h in the presence of brefeldin-A. I: OVA-specific IgG titer in serum from mice treated with either saline or alum before the immunization. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in each graph. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, In Vivo, Injection, Saline, Flow Cytometry, Expressing, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: NLRP3 activation required for the effects of adjuvant conditioning in the adaptive immune response. A . Experimental design. 8-10-week-old male or female C57BL/6 or NLRP3 -/- mice were injected intraperitoneally (i.p.) with either Alum Imject™ (8mg in 200µL) or saline three times, every other day. 24 hours after the last injection, mice were adoptively transferred with 5×10 6 OT-II splenocytes intravenously, and 24 hours after the transfer, mice were immunized with 10 μg OVA adsorbed in Resiquimod (R848) (OVA-RSQ) (50µg), in a homologous prime (day 1) and boost (day 8) protocol. Seven days after the boost (day 15), mice were euthanized, and spleens were analyzed for OVA-specific Th1, Th2, Th17, and Treg total numbers. B: Total number of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline or alum before the immunization. C: OVA-specific IgG titer in serum from mice treated with either saline or alum before the immunization. D: Flow cytometry plots of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline or alum before the immunization. E: Total number of OVA-specific Th1, Th2, Th17 and Tregs, evidenced by the expression of CD4 + Vα + IFN-γ + , CD4 + Vα + IL-4 + , CD4 + Vα + IL-17A + and CD4 + Vα + LAP + respectively in splenocytes treated with either saline or alum before the immunization and further cultured with OVAp (2μg/mL) in vitro for 48h in the presence of brefeldin-A. Data shown represent three or more experiments, and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in the graphs. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Activation Assay, Adjuvant, Injection, Saline, Expressing, Flow Cytometry, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: NLRP3-dependent IL-1 signaling is involved in the effects of adjuvant conditioning in the adaptive immune response. A: Flow cytometry plots and total number (B) of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline, alum or alum + Anakinra (30mg/kg) (AC + AK) before the immunization. C: OVA-specific IgG titer in serum from mice treated with either saline, alum or alum + Anakinra (30mg/kg) (AC+AK) before the immunization. D: Total number of OVA-specific Th1, Th2, Th17 and Tregs, evidenced by the expression of CD4 + Vα + IFN -γ + , CD4 + Vα + IL-4 + , CD4 + Vα + IL-17A + and CD4 + Vα + LAP + in splenocytes treated with either saline, alum or alum + Anakinra (30mg/kg) (AC+AK) before the immunization and further cultured with OVAp (2μg/mL) in vitro for 48h in the presence of brefeldin-A. Data shown represent three or more experiments, and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in the graphs. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, Flow Cytometry, Expressing, Saline, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: Adjuvant conditioning induces the reprogramming of myeloid cells to a trained immunosuppression phenotype. A: Flow cytometry plots of CD11b + Ly6C + and CD11b + Ly6G + cells in the spleen 24h after saline or alum injections. B: TNF-α, IL-6 (C) , IL-10 (D) , and nitric oxide (E) production from CD11b + GR1 + cells isolated from saline or AC-treated mice cultured with either medium or LPS (200ng/mL) for 24h. F: Histograms of CD4 + Vα + cells in a suppression assay using isolated CD11b + GR1 + cells. Isolated MDSCs from spleens from AC-treated mice were cultured with OVAp (1µg/mL), C57BL/6 splenocytes (1×10 5 ) and isolated OT-II naïve CD4 T cells (2×10 5 ) in the ratios (MDSC : T cell) indicated in the graph. G: Percentage of CD4 + Vα + CTV low cells in a suppression assay using isolated CD11b + GR1 + cells and OT-II CD4 T cells. H: Percentage of CD4 + Vα + FVD + cells in a suppression assay using isolated CD11b + GR1 + cells and OT-II CD4 T cells. I: Flow cytometry plots of CD11b + Ly6C + and CD11b + Ly6G + cells in the spleen 1 day, 7 days, and 14 days after saline or alum injections. J: Total count of CD11b + Ly6C + and CD11b + Ly6G + cells in the spleen 1 day, 7 days, and 14 days after saline or alum injections. K: TNF-α, and nitric oxide (L) production from CD11b + GR1 + cells isolated 1 day, 7 days, and 14 days after saline or alum injections cultured with either medium or LPS (200ng/mL) for 24h. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in each graph. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, Flow Cytometry, Saline, Isolation, Cell Culture, Suppression Assay

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: AC-induced MDSCs inhibition of the adaptive immune response in vivo requires NLRP3 activation and IL-1 signaling. A: Experimental design. Briefly, 2×10 6 CD11b + GR1 + cells isolated from either AC, alum + Anakinra (30mg/kg) (AC+AK) or saline-injected C57BL/6 or NLRP3 -/- mice were adoptively transferred to C57BL/6 mice before the immunization protocol. B: Flow cytometry plots and total number of OVA-specific T cells (C) , evidenced by the expression of CD4 + Vα + in splenocytes from mice adoptively transferred with CD11b + GR1 + cells isolated from either AC, or saline-injected C57BL/6 before the immunization protocol. D: OVA-specific IgG titer in serum from mice adoptively transferred with CD11b + GR1 + cells isolated from either AC, or saline-injected C57BL/6 before the immunization protocol. E: Flow cytometry plots and total number of OVA-specific T cells (F) , evidenced by the expression of CD4 + Vα + in splenocytes from mice adoptively transferred with CD11b + GR1 + cells isolated from either alum + Anakinra (30mg/kg) (AC+AK) or saline-injected C57BL/6 or NLRP3 -/- mice before the immunization protocol. G: OVA-specific IgG titer in serum from mice adoptively transferred with CD11b + GR1 + cells isolated from either alum + Anakinra (30mg/kg) (AC + AK) or saline-injected C57BL/6 or NLRP3 -/- mice before the immunization protocol. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in the graphs. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Inhibition, In Vivo, Activation Assay, Isolation, Saline, Injection, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: Adjuvant conditioning promotes allogeneic tolerance dependent on NLRP3. A: Allogeneic pancreatic islet transplantation experimental design. Briefly, alum or saline-treated C57BL/6 or NLRP3 -/- mice were treated with streptozotocin (STZ) 4 days before the transplantation with Balb/c isolated pancreatic islets. Survival of the graft was assessed daily by checking blood glucose levels. B: Survival curve of alum or saline-treated C57BL/6 or NLRP3 -/- mice that underwent allogeneic pancreatic islet transplantation. C57BL/6 saline (n=5), AC (n=6), NLRP3 -/- saline (n=5), AC (n=6). Graft survival significance was assessed by Kaplan-Meier/Mantel-Cox log-rank test. C: Cutaneous hypersensitivity experimental design. Briefly, alum or saline-treated C57BL/6 or NLRP3 -/- mice were subcutaneously immunized with 20× 10 6 Balb/c splenocytes on the neck. Seven days later, mice were challenged with 20×10 6 Balb/c splenocytes injected subcutaneously into the base of the ear, as described by . D: Cutaneous allogeneic response was assessed by measuring ear pinna swelling (mm) at 2-, 4- and 24- hours post-challenge. P-values show the comparison between ear swelling at 24-hours post-challenge. n=10 for each group. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in each graph. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, Transplantation Assay, Saline, Isolation, Injection, Comparison

Journal: mBio

Article Title: Autoantibody-Mediated Erythrophagocytosis Increases Tuberculosis Susceptibility in HIV Patients

doi: 10.1128/mBio.03246-19

Figure Lengend Snippet: RBCs from patients with HIV with a positive direct antiglobulin test (DAT) result increase erythrophagocytosis by macrophages. (A) RBCs labeled with a membrane-integrating dye (PKH-26) were assessed by confocal fluorescence microscopy (left; bars, 2 μm) and flow cytometry (right; labeling yield, 99.6%). (B and C) Ex vivo phagocytosis of PKH-26-labeled RBCs from healthy donors ( n = 33) and patients with HIV with negative ( n = 36) and positive ( n = 11) DAT results upon incubation with PMA-differentiated THP-1 macrophages for 6 h at RBC/macrophage (R/M) ratios of 10:1 and 100:1 revealed by forward scatter (FSC) data (B) and quantified by flow cytometry (C). (D) DAT results were classified as negative (−) or positive (with 1+ [+], 2+ [++], and 3+ [+++] degrees) corresponding to the RBC agglutination strength. (E) Correlation between erythrophagocytosis rate and DAT agglutination level at both R/M ratios (orange, R/M ratio = 10:1; purple, R/M ratio = 100:1), assessed using Spearman’s rank correlation. The data represent means ± standard deviations and are representative of results from at least three independent experiments. *** * , P < 0.0001.

Article Snippet: For the erythrophagocytosis assay, the PMA-differentiated THP-1 macrophages were pretreated with or without Fcγ receptor blocker (containing anti-CD16, anti-CD32, and anti-CD64; BioLegend) and anti-C1q receptor antibody (ImmunoWay Biotechnology Company) at dilution ratios of 1:1,000, 1:200, and 1:100 for 2 h and were then treated with PKH-26-labeled RBCs at the indicated RBC/macrophage (R/M) ratios of 10:1 and 100:1 for 0.5, 1, 2, 6, 12, and 24 h. The culture supernatants were then removed, and 1 ml RBC lysis buffer (Solarbio) was added to each well followed by incubation for 5 min at room temperature.

Techniques: Direct Antiglobulin Test, Labeling, Membrane, Fluorescence, Microscopy, Flow Cytometry, Ex Vivo, Incubation, Agglutination

Journal: mBio

Article Title: Autoantibody-Mediated Erythrophagocytosis Increases Tuberculosis Susceptibility in HIV Patients

doi: 10.1128/mBio.03246-19

Figure Lengend Snippet: Enhanced erythrophagocytosis in patients with HIV can be blocked by neutralizing Fcγ receptors on macrophages. (A and B) RBCs from healthy donors were sensitized with different concentrations of anti-human RBC IgG (0, 25, 375, and 1,500 μg/ml) or isotype rabbit IgG (375 μg/ml) and were then labeled with PKH-26. In vitro phagocytosis of the indicated RBCs after incubation with PMA-differentiated THP-1 macrophages for 6 h at an RBC/macrophage (R/M) ratio of 100:1 was revealed by forward scatter data (A) and quantified by flow cytometry (B). (C) Erythrophagocytosis analysis was performed at various time points (0.5, 1, 2, 6, 12, and 24 h) after treatment of sensitized RBCs (sRBCs) (anti-human RBC IgG, 1,500 μg/ml) at an R/M ratio of 100:1, as assessed by flow cytometry. (D and E) PMA-differentiated THP-1 macrophages were treated with the indicated dilution ratios of Fcγ receptor blocker for 2 h followed by exposure to PKH-26-labeled RBCs from healthy donors ( n = 5) or from patients with HIV with a positive direct antiglobulin test (DAT) result ( n = 5) or to sRBCs for another 6 h. Healthy RBCs without sensitization were used as a control. The rate of erythrophagocytosis was quantified by flow cytometry. The data represent means ± standard deviations and are representative of results from at least three independent experiments. * * , P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: For the erythrophagocytosis assay, the PMA-differentiated THP-1 macrophages were pretreated with or without Fcγ receptor blocker (containing anti-CD16, anti-CD32, and anti-CD64; BioLegend) and anti-C1q receptor antibody (ImmunoWay Biotechnology Company) at dilution ratios of 1:1,000, 1:200, and 1:100 for 2 h and were then treated with PKH-26-labeled RBCs at the indicated RBC/macrophage (R/M) ratios of 10:1 and 100:1 for 0.5, 1, 2, 6, 12, and 24 h. The culture supernatants were then removed, and 1 ml RBC lysis buffer (Solarbio) was added to each well followed by incubation for 5 min at room temperature.

Techniques: Labeling, In Vitro, Incubation, Flow Cytometry, Direct Antiglobulin Test, Control

Journal: Blood

Article Title: Ligation of complement receptor 1 increases erythrocyte membrane deformability

doi: 10.1182/blood-2010-04-273904

Figure Lengend Snippet: RBCs express functional TRPC1. (A) RBC CR1 ligation promotes a SAC-dependent Ca++ influx. RBCs were loaded with Fluo-4 and incubated with buffer, 1 or 5μM GsMTX-4 for 30 minutes and then analyzed by flow cytometry for CR1-mediated Ca++ influx (arrow) as described above. (B) RBCs express TRPC1. RBCs were fixed, permeabilized, and incubated with either control (left panel) or rabbit monoclonal anti-TRPC1 (right panel) for 16 hours, washed, and incubated with goat anti–rabbit AlexaFluor488. Cells were imaged under the microscope using the fluorescein isothiocyanate/green fluorescent protein filter. (C) Immunoblotting detection of TRPC1 in RBCs. Testes tissue lysate (Santa Cruz Biotechnology; second lane) and RBC lysate (third lane) were separated by electrophoresis using Tris-HCl gels and probed with rabbit anti-TRPC1 Ab (Santa Cruz Biotechnology). TRPC1 is seen as a band at approximately 87 kDa (arrow) in both positive control and RBC lysate. Lane 1, Bio-Rad molecular weight markers. (D) Anti-TRPC1 inhibitory antibody T1E3 binds RBCs. RBC were incubated with either 1/500 dilution of normal rabbit serum (dotted histogram) or T1E3 Ab anti-serum (continuous histogram) for 10 minutes followed by AlexaFluor488 goat anti–rabbit. Cells were washed and examined by flow cytometry. (E) CR1 ligation triggers a Ca++ influx dependent on TRPC1. Fluo-4 loaded RBCs were preincubated with control serum or T1E3 for 20 minutes, washed twice, and then analyzed by flow cytometry for CR1-mediated Ca++ influx (arrow) as described above.

Article Snippet: Testes tissue lysate (Santa Cruz Biotechnology; second lane) and RBC lysate (third lane) were separated by electrophoresis using Tris-HCl gels and probed with rabbit anti-TRPC1 Ab (Santa Cruz Biotechnology).

Techniques: Functional Assay, Ligation, Incubation, Flow Cytometry, Control, Microscopy, Western Blot, Electrophoresis, Positive Control, Molecular Weight

Journal: Blood

Article Title: Ligation of complement receptor 1 increases erythrocyte membrane deformability

doi: 10.1182/blood-2010-04-273904

Figure Lengend Snippet: Ligation of RBC CR1 induces serine/threonine phosphorylation of β-spectrin and adducin. (A) Cross-linking of RBC CR1 induces time-dependent serine/threonine phosphorylation, as assessed by FACS. RBCs were incubated with either control Ab or anti-CR1 for various amounts of time, fixed, permeabilized, and serine/threonine phosphorylation levels assessed by flow cytometry. (B) Western blotting analysis of the serine/threonine phosphorylation of β-spectrin (arrow) was temporally concordant with the FACS analysis. Control represents the basal serine/threonine phosphorylation levels of β-spectrin in fresh RBCs at 4°C. The experiment was repeated 4 times with similar results. (C) Phosphorylation levels of adducin increases 10 minutes after ligation of CR1. Control represents basal levels of phospho-adducin (lane 1), and as positive control, RBCs were activated by 0.5μM PMA (lane 3). Extra bands present in the anti-CR1 lane represent heavy and light IgG chains or the cross-linking anti-CR1 Ab. Loading controls are represented by the total amount of adducin in RBC lysate probed with rabbit polyclonal antibody anti-adducin. These results are representative of 3 independent experiments. (D) CR1-mediated adducin phosphorylation depends on Ca++. RBCs were incubated with PMA or anti-CR1 in the presence or absence of Ca++ (Mg++ EGTA) and phosphorylation levels of adducin measured as above. Loading controls are represented by the levels of glyceraldehyde-3-phosphate dehydrogenase in RBC lysates.

Article Snippet: Testes tissue lysate (Santa Cruz Biotechnology; second lane) and RBC lysate (third lane) were separated by electrophoresis using Tris-HCl gels and probed with rabbit anti-TRPC1 Ab (Santa Cruz Biotechnology).

Techniques: Ligation, Phospho-proteomics, Incubation, Control, Flow Cytometry, Western Blot, Positive Control

Journal: Frontiers in Physiology

Article Title: The Relation Between Capillary Transit Times and Hemoglobin Saturation Heterogeneity. Part 2: Capillary Networks

doi: 10.3389/fphys.2018.01296

Figure Lengend Snippet: Distribution of the HS S v at the distal end of capillary paths in CN 1. Left: constant inflow value; right: random inflow value. (A,E) Computational model with moving RBCs; (B,F) differential equation model with functional tissue radii; (C,G) differential equation model with geometric tissue radii; (D,H) box plot with whiskers for the 5th and 95th percentile. The values from the differential equation model are weighted by the RBC flow in the distal capillaries. Error bars above the histograms: mean ± SD.

Article Snippet: These slight deviations most probably occur since the RBC flow through the network is permanently fluctuating (Schmid et al., ) and hence tissue P o 2 as well.

Techniques: Functional Assay

Journal: Frontiers in Physiology

Article Title: The Relation Between Capillary Transit Times and Hemoglobin Saturation Heterogeneity. Part 2: Capillary Networks

doi: 10.3389/fphys.2018.01296

Figure Lengend Snippet: Profiles of HS in four selected vessels from CN 1 downstream of a converging bifurcation. Thin green lines: individual RBCs; thin black lines: mean HS S ¯ ; thick black lines: S ¯ ± σ S . In panel (A) , the HS profiles slightly increase at the end of the capillary since it flows into another capillary with higher HS. In panel (C) , HS values are significantly lower since RBC flow in the parent vessels of the capillary is very low, hence the large upstream oxygen discharge.

Article Snippet: These slight deviations most probably occur since the RBC flow through the network is permanently fluctuating (Schmid et al., ) and hence tissue P o 2 as well.

Techniques: