Journal: bioRxiv

Article Title: An Unexpected Role of Neutrophils in Clearing Apoptotic Hepatocytes In Vivo

doi: 10.1101/2023.02.08.527616

Figure Lengend Snippet: (A) Intravital microscopy images of liver tissues from WT and MRP8cre/DTR mice. Neutrophils are labeled with an i.v. injection of anti-Ly6G antibody (green), KCs are labeled with anti-F4/80 antibody (red), and apoptotic cells are labeled with Annexin V (blue). Neutrophils inside and KCs associated with apoptotic hepatocytes are detected and analyzed with IMARIS software as described in Methods. The distances from neutrophils to the apoptotic hepatocyte border are recorded in table S6. A total of 24 apoptotic cells were observed in the WT liver with an average of 2 burrowed neutrophils. Scale bar, 100 μm. (B) Intravital image sequences of neutrophils phagocytosing apoptotic hepatocytes in mouse livers at indicated time points. Neutrophils are labeled with an i.v. injection of anti-Ly6G antibody (green), and apoptotic cells are labeled with Annexin V (red). A total of 13 apoptotic cells with burrowed neutrophils were observed in 12 WT mouse livers. Scale bar, 20 μm. (C) Electron microscopy images of apoptotic mouse hepatocytes occupied by neutrophils. The apoptotic hepatic nucleus (AN) is evident by distorted nuclear membrane (pointed by black arrowheads). The neutrophils are indicated by white arrowheads with a characteristic multilobed nucleus. 29 apoptotic cells with burrowed neutrophils were observed. Scale bar, 5 μm. Data are representative of three independent experiments.

Article Snippet: For antibody-based depletion, wild-type mice were injected i.p. with anti-mouse Ly6G clone 1A8 (Bio X Cell) supernatant containing 0.5 mg protein every 48 h for 1 month.

Techniques: Intravital Microscopy, Labeling, Injection, Software, Electron Microscopy, Membrane

Journal: Acta Pharmacologica Sinica

Article Title: Hederacoside C ameliorates colitis via restoring impaired intestinal barrier through moderating S100A9/MAPK and neutrophil recruitment inactivation

doi: 10.1038/s41401-022-00933-3

Figure Lengend Snippet: Rats were enteric injected with TNBS (80 mg/kg) once, and then intraperitoneally injected with HSC (0.625, 1.25, 2.5 mg/kg) twice per day for 7 days. Mesalazine was intragastrically administered at the dose of 200 mg/kg once a day for 7 consecutive days. Rats were sacrificed on the 8th day after colitis induction. n = 8 per group. a Neutrophils in colon tissue were analyzed by immunohistochemistry with Ly6G and the results was photographed by microscope. Black arrows indicated Ly6G. Scale bar: 100 μm. Images were from three rats in one group ( n = 3 per group). b MPO in colon tissues was detected by ELISA kits. Data were shown as mean ± SD ( n = 3). # P < 0.05 vs control group; * P < 0.05 vs TNBS group. c Sections of colon tissue were immunostained with FITC-S100A9 (red) and FITC-CD11b (green) and the immunofluorescence was detected by confocal laser-scanning microscope. Images were from three rats in one group ( n = 3 per group).

Article Snippet: The sections were blocked with 2.5% normal horse serum and then were incubated overnight at 4 °C in blocking buffer with the following primary antibodies: Ly6G (1:200, #orb322983, Biorbyt, Cambridge, CB4 0WY, UK), ZO-1 (1:200, #29274, Signalway Antibody, Maryland, USA), Occludin (1:200, #27260-1-AP, Proteintech, Inc, Rosement, IL, USA) and Claudin-1 (1:200, #28674-1-AP, Proteintech) which was followed by incubation with a secondary antibody (#A0192, Beyotime).

Techniques: Injection, Immunohistochemistry, Microscopy, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Laser-Scanning Microscopy

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Rapid recruitment of a Ly6G + population to the local draining lymph node following dendritic cell (DC) vaccination. Ex vivo -generated DC vaccines were injected into the hind footpads of female C57BL/6 mice (n = 2–6 per treatment), and the local draining lymph node was examined for Ly6G + cells. Ly6G+ cells were assessed by flow cytometry six, 12, 24, 48, and 120 h post-DC immunization. The number and percentage of Ly6G + cells in the lymph node of DC-immunized mice were compared with phosphate-buffered saline (PBS)-injected control mice. Significance was determined statistically using a two-way analysis of variance.; p -value < 0.05 (*), p -value < 0.0001 (****). Graphs show the mean and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Ex Vivo, Generated, Vaccines, Injection, Flow Cytometry, Saline, Control, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Ly6G + cell recruitment was limited to the vaccine-draining lymph node. Dendritic cell (DC) vaccines were injected into one hind footpad of each female C57BL/6 mouse and phosphate-buffered saline (PBS) into the other hind footpad. Both popliteal lymph nodes were examined for Ly6G + populations 12 h post-immunization, one representing the PBS-draining lymph node ( left panel ) and the other as a local vaccine-draining lymph node ( middle panel ). The inguinal lymph node on the vaccine-administered side of the mouse was also examined as a non-vaccine-draining lymph node ( right panel ). Representative flow cytometry dot plots of live cells in the lymph nodes are shown.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Injection, Saline, Flow Cytometry

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Neutrophils were recruited to the dendritic cell (DC) vaccine-draining lymph node. DC vaccines were generated ex vivo and administered into the hind footpads of female C57BL/6 mice (n = 2–4 per treatment). The vaccine-draining popliteal lymph nodes (labeled “DC”) were examined for neutrophils (Ly6G+ CD11b+) six-, 24-, and 48 h following DC vaccination and were compared with non-draining inguinal lymph nodes from the vaccinated side of mice (“DC non-draining”), and phosphate-buffered saline- draining lymph nodes (“PBS”). Statistical analysis was performed using a two-way analysis of variance; p -value < 0.05 (*), p -value < 0.001 (***), p -value < 0.0001 (****). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Generated, Ex Vivo, Labeling, Saline, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Neutrophil accumulation in the draining lymph nodes required maturation of dendritic cells (DCs). DC vaccines were prepared using DCs cultured ex vivo . The DCs were matured using lipopolysaccharide (LPS) as a source of pathogen-associated molecular pattern signaling that induces activation and maturation of DCs, and then they were loaded with antigens. DC cultures were prepared for immunization using different iterations of the manufacturing protocol. These included DCs stimulated with LPS only (DC LPS), DCs pulsed with antigens only (DC Ag), DCs pulsed with antigens and stimulated with LPS (DC Ag LPS), and DCs without LPS treatment or antigen-pulsing, which were termed “DC empty”. The number and percentage of neutrophils (Ly6G + CD11b + ) in the draining lymph nodes of the female C57BL/6 mice were examined one day following inoculations using flow cytometry. The data were analyzed for significance using a one-way analysis of variance; p -value < 0.005 (**), p -value < 0.001 (***), p -value < 0.0001 (****). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Soluble factor(s) produced by mature dendritic cells (DCs) were capable of recruiting neutrophils to the local draining lymph node. DCs were cultured ex vivo and half of the culture was matured using lipopolysaccharide, and the other half remained in an immature state. The DCs were plated in media and left overnight in an incubator at 37 °C. The supernatants of the immature and mature DCs were collected and injected into the hind footpads of female C57BL/6 mice (n = 4–7 per treatment), and media only was injected into negative control mice. Four hours post-injection, the draining lymph nodes were examined for neutrophils (Ly6G + CD11b + ). Significant differences were examined using a one-way analysis of variance; p -value > 0.0001 (****). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Produced, Cell Culture, Ex Vivo, Injection, Negative Control, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Neutrophils influenced dendritic cell (DC) vaccine recovery in the footpad and draining lymph node one day post-immunization. DC vaccines were prepared ex vivo and labelled with the fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). Female C57BL/6 mice (n = 8–16 per treatment) were treated with an anti-Ly6G antibody one day prior to and on the day of DC immunization to deplete neutrophils from the mice. The footpads and draining lymph nodes were examined for DC vaccine recovery at the injection site and at the draining lymph node one day following inoculation to evaluate the influence of neutrophils on the migratory capacities of the vaccine. The numbers of DCs in these tissues from mice that received anti-Ly6G and the DC vaccine were compared to mice that received the DC vaccine only, and negative control mice that received phosphate-buffered saline (PBS). Significant differences were determined using a one-way analysis of variance; p -value < 0.05 (*), p -value < 0.005 (**), p -value < 0.001 (***). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Ex Vivo, Injection, Negative Control, Saline, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Depletion of neutrophils influenced the accumulation of natural killer (NK) cells in the local draining lymph node one day following dendritic cell (DC) immunization. Anti-Ly6G was administered to female C57BL/6 mice (n = 4–12 per treatment) on the day before and the day of DC immunizations to deplete neutrophils. DC vaccines were prepared ex vivo and administered into the footpads of mice. One day post-DC immunization, the local draining lymph nodes were examined by flow cytometry for NK cell populations. Statistical analysis was performed using a one-way analysis of variance; p -value < 0.05 (*), p -value < 0.001 (***), p -value < 0.0001 (****). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Ex Vivo, Flow Cytometry, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Neutrophils influenced the functionality of natural killer (NK) cells in local draining lymph nodes two days following dendritic cell (DC) immunization. Half of the female C57BL/6 mice received anti-Ly6G to deplete neutrophils the day before and the day of immunizations, and then DC vaccines or phosphate-buffered saline (PBS) were administered (n = 4 per treatment) into hind food pads. ( a ) Two days later, the number and proportion of NK cells in the draining popliteal lymph nodes were compared between neutrophil-depleted and non-depleted mice. ( b ) NK cells were stimulated using IL-2, and the production of IFN γ (left panels) and CD107a (right panels) were measured using flow cytometry. A two-way analysis of variance was used to assess statistical significance; p -value < 0.05 (*), p -value < 0.005 (**), p -value < 0.001 (***), p -value < 0.0001 (****). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Saline, Flow Cytometry, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Influence of neutrophils on natural killer (NK) cell responses in the spleen one-week post-dendritic cell (DC) vaccination. Anti-Ly6G was given to half of the female C57BL/6 mice to deplete neutrophils (n = 8 per treatment). DC vaccines generated ex vivo were administered. Controls received phosphate-buffered saline (PBS). One week later, NK cell responses in the spleens were assessed. Splenocytes were stimulated with interleukin (IL)-2, and NK cell degranulation based on expression of CD107a and production of IFN γ were measured using flow cytometry. A two-way analysis of variance was used to evaluate statistical significance; p -value < 0.05 (*), p -value < 0.005 (**), p -value < 0.0001 (****). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Generated, Ex Vivo, Saline, Expressing, Flow Cytometry, Standard Deviation

Journal: Cells

Article Title: Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

doi: 10.3390/cells12010121

Figure Lengend Snippet: Neutrophils did not influence dendritic (DC) vaccine-mediated education of antigen-specific CD8+ T cell degranulation or production of interferon (IFN γ ) and tumor necrosis factor (TNF) α . Female C57BL/6 mice (n = 12–16 per treatment) were given anti-Ly6G followed by DC vaccines. One week later, the spleens were examined for CD8+ T cell responses to stimulation with the vaccine-loaded epitopes, ovalbumin (OVA) 257-264 (SIIN), which is the immunodominant CD8+ T cell epitope, and OVA 323-339 (ISQ), which is the dominant CD4+ helper T cell epitope. The CD8+ T cell responses were measured by expression of CD107a as an indicator of degranulation, and production of IFN γ and TNF α using flow cytometry. The CD8+ T cell responses in the spleens of mice that received anti-Ly6G and the DC vaccine, the DC vaccine only, PBS only, and anti-Ly6G and PBS were compared using a two-way analysis of variance; p -value < 0.05 (*), p -value < 0.005 (**), p -value < 0.001 (***), p -value < 0.0001 (****). Graphs show the means and the standard deviation with each dot representing an individual mouse.

Article Snippet: Mice in the Ly6G depletion treatment group were given doses of 250 ug of InVivoPlus anti- mouse Ly6G (Bio X Cell, Burlington, Canada) in 200 uL PBS via intraperitoneal injection the day before DC immunization, the day of immunization, and every 2–3 days throughout the induction phase of the immune response.

Techniques: Vaccines, Expressing, Flow Cytometry, Standard Deviation