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Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

Kamenyeva O, Boularan C, Kabat J, Cheung GY, Cicala C, Yeh AJ, Chan JL, Periasamy S, Otto M, Kehrl JH - PLoS Pathog. (2015)

Bottom Line: They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis.Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders.Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

No MeSH data available.


Related in: MedlinePlus

Local immunization with S. aureus bioparticles recruits neutrophils to the LN.S. aureus bioparticles were opsonized and injected subcutaneously near the iLN. Neutrophil recruitment to the blood and lymphoid organs was analyzed by flow cytometry in C57BL/6 mice, or imaged using epifluorescent stereomicroscope or TP-LSM in LysM-GFP mice with adoptively transferred lymphocytes. (A) Kinetics of neutrophil recruitment to the blood (left panel) and to the iLN (right panel) between 0 and 24 h after immunization. N = 3 mice/6 iLNs; 3 independent experiments. Means ± SEM. (B) Fluorescent (main image) and bright field (lower right corner) images of immunized and PBS control LysM-GFP LNs are shown at 12 h after injection. Neutrophils: LysM-GFP, green. Image labeling: SCS (white dashed line), TZ (T), IFZ (IF, white arrows), LN follicles (B, dotted lines). Data is representative of 3 mice (6 iLNs) per group. (C) TP-LSM images of neutrophils (LysM-GFP, green) exiting blood vessels in the LN stroma (left panel, blue arrowheads) and accumulating in the SCS (right panel, blue arrowheads) between 2 h (left) and 4 h (right) after S. aureus (red arrowheads) injection are shown (S3 Movie, Mobilization). ILN border, white dashed line; HEVs, EB, gray. Scale bars: 50 μm. (D) Neutrophils phagocytizing S. aureus in the SCS (left panel, blue arrowheads) and swarming in the IFZ (right panel, white squares) between 3 and 4 h after S. aureus inoculation are shown (S3 Movie, Swarming). Scale bars: 25 μm (right), 20 μm (left). Data is representative of 12 imaging sessions. (E, F) DsRed B cells or CD4+ T cells were adoptively transferred 24 h prior to imaging. Neutrophil (green) interactions with (E) B cells (red) or (F) CD4+ T cells (red) at the T-B border (dashed line) at 12 h after S. aureus injection are shown. Blood vessels, EB (gray); collagen, second harmonic (blue). Scale bars: (C) 50 μm; (D, left) 50 μm; (D, right) 20 μm, (E, left) 50 μm; (E, right) 20 μm; (F, left) 70 μm; (F, right) 20 μm. (G) The percentages of short and long-lasting interactions formed by neutrophils with B cells (upper chart) and by neutrophils with CD4+ T cells (lower chart). Data is representative of 3 independent computations. (H) Analysis of S. aureus uptake by Ly6G+ and CD169+ populations in the iLN of isotype control or 1A8-injected mice between 0 and 48 h after immunization. N = 4 iLNs; 3 repeats. Means ± SEM.
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ppat.1004827.g002: Local immunization with S. aureus bioparticles recruits neutrophils to the LN.S. aureus bioparticles were opsonized and injected subcutaneously near the iLN. Neutrophil recruitment to the blood and lymphoid organs was analyzed by flow cytometry in C57BL/6 mice, or imaged using epifluorescent stereomicroscope or TP-LSM in LysM-GFP mice with adoptively transferred lymphocytes. (A) Kinetics of neutrophil recruitment to the blood (left panel) and to the iLN (right panel) between 0 and 24 h after immunization. N = 3 mice/6 iLNs; 3 independent experiments. Means ± SEM. (B) Fluorescent (main image) and bright field (lower right corner) images of immunized and PBS control LysM-GFP LNs are shown at 12 h after injection. Neutrophils: LysM-GFP, green. Image labeling: SCS (white dashed line), TZ (T), IFZ (IF, white arrows), LN follicles (B, dotted lines). Data is representative of 3 mice (6 iLNs) per group. (C) TP-LSM images of neutrophils (LysM-GFP, green) exiting blood vessels in the LN stroma (left panel, blue arrowheads) and accumulating in the SCS (right panel, blue arrowheads) between 2 h (left) and 4 h (right) after S. aureus (red arrowheads) injection are shown (S3 Movie, Mobilization). ILN border, white dashed line; HEVs, EB, gray. Scale bars: 50 μm. (D) Neutrophils phagocytizing S. aureus in the SCS (left panel, blue arrowheads) and swarming in the IFZ (right panel, white squares) between 3 and 4 h after S. aureus inoculation are shown (S3 Movie, Swarming). Scale bars: 25 μm (right), 20 μm (left). Data is representative of 12 imaging sessions. (E, F) DsRed B cells or CD4+ T cells were adoptively transferred 24 h prior to imaging. Neutrophil (green) interactions with (E) B cells (red) or (F) CD4+ T cells (red) at the T-B border (dashed line) at 12 h after S. aureus injection are shown. Blood vessels, EB (gray); collagen, second harmonic (blue). Scale bars: (C) 50 μm; (D, left) 50 μm; (D, right) 20 μm, (E, left) 50 μm; (E, right) 20 μm; (F, left) 70 μm; (F, right) 20 μm. (G) The percentages of short and long-lasting interactions formed by neutrophils with B cells (upper chart) and by neutrophils with CD4+ T cells (lower chart). Data is representative of 3 independent computations. (H) Analysis of S. aureus uptake by Ly6G+ and CD169+ populations in the iLN of isotype control or 1A8-injected mice between 0 and 48 h after immunization. N = 4 iLNs; 3 repeats. Means ± SEM.

Mentions: Next, we studied neutrophil influx to the iLN in response to a local injection of inactivated S. aureus Wood 46 strain. Analysis of recruitment kinetics was expanded to time points between 0 and 120 min, and at 2, 3, 6, 12 and 24 h. The neutrophils increased in the blood 1 h post-injection, continued to increase reaching a plateau at 6 h, and returned to baseline by 12 h (Fig 2A, left). Over the same interval we detected two waves of neutrophils infiltrating the iLN, 1st between 0 and 60 min with the peak at 30 min, and 2nd between 2 and 24 h with a plateau between 6 and 12 h. Their percentage had returned almost to baseline by 24 h (Fig 2A, right). Epifluorescent microscopy of intact LysM-GFP iLN showed abundant presence of GFPhi cells in the SCS (white dashed line) and IFZ (IF, white arrows) of immunized iLN at 12 h (Fig 2B). We also analyzed recruitment of neutrophils to distant LNs choosing the axillary and superficial cervical LNs and to the spleen at 4 and 12 h after immunization. Along with the massive influx of neutrophils to the iLN, we detected a significant recruitment to the spleen, but none to distant LNs (S2A and S2B Fig).


Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

Kamenyeva O, Boularan C, Kabat J, Cheung GY, Cicala C, Yeh AJ, Chan JL, Periasamy S, Otto M, Kehrl JH - PLoS Pathog. (2015)

Local immunization with S. aureus bioparticles recruits neutrophils to the LN.S. aureus bioparticles were opsonized and injected subcutaneously near the iLN. Neutrophil recruitment to the blood and lymphoid organs was analyzed by flow cytometry in C57BL/6 mice, or imaged using epifluorescent stereomicroscope or TP-LSM in LysM-GFP mice with adoptively transferred lymphocytes. (A) Kinetics of neutrophil recruitment to the blood (left panel) and to the iLN (right panel) between 0 and 24 h after immunization. N = 3 mice/6 iLNs; 3 independent experiments. Means ± SEM. (B) Fluorescent (main image) and bright field (lower right corner) images of immunized and PBS control LysM-GFP LNs are shown at 12 h after injection. Neutrophils: LysM-GFP, green. Image labeling: SCS (white dashed line), TZ (T), IFZ (IF, white arrows), LN follicles (B, dotted lines). Data is representative of 3 mice (6 iLNs) per group. (C) TP-LSM images of neutrophils (LysM-GFP, green) exiting blood vessels in the LN stroma (left panel, blue arrowheads) and accumulating in the SCS (right panel, blue arrowheads) between 2 h (left) and 4 h (right) after S. aureus (red arrowheads) injection are shown (S3 Movie, Mobilization). ILN border, white dashed line; HEVs, EB, gray. Scale bars: 50 μm. (D) Neutrophils phagocytizing S. aureus in the SCS (left panel, blue arrowheads) and swarming in the IFZ (right panel, white squares) between 3 and 4 h after S. aureus inoculation are shown (S3 Movie, Swarming). Scale bars: 25 μm (right), 20 μm (left). Data is representative of 12 imaging sessions. (E, F) DsRed B cells or CD4+ T cells were adoptively transferred 24 h prior to imaging. Neutrophil (green) interactions with (E) B cells (red) or (F) CD4+ T cells (red) at the T-B border (dashed line) at 12 h after S. aureus injection are shown. Blood vessels, EB (gray); collagen, second harmonic (blue). Scale bars: (C) 50 μm; (D, left) 50 μm; (D, right) 20 μm, (E, left) 50 μm; (E, right) 20 μm; (F, left) 70 μm; (F, right) 20 μm. (G) The percentages of short and long-lasting interactions formed by neutrophils with B cells (upper chart) and by neutrophils with CD4+ T cells (lower chart). Data is representative of 3 independent computations. (H) Analysis of S. aureus uptake by Ly6G+ and CD169+ populations in the iLN of isotype control or 1A8-injected mice between 0 and 48 h after immunization. N = 4 iLNs; 3 repeats. Means ± SEM.
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ppat.1004827.g002: Local immunization with S. aureus bioparticles recruits neutrophils to the LN.S. aureus bioparticles were opsonized and injected subcutaneously near the iLN. Neutrophil recruitment to the blood and lymphoid organs was analyzed by flow cytometry in C57BL/6 mice, or imaged using epifluorescent stereomicroscope or TP-LSM in LysM-GFP mice with adoptively transferred lymphocytes. (A) Kinetics of neutrophil recruitment to the blood (left panel) and to the iLN (right panel) between 0 and 24 h after immunization. N = 3 mice/6 iLNs; 3 independent experiments. Means ± SEM. (B) Fluorescent (main image) and bright field (lower right corner) images of immunized and PBS control LysM-GFP LNs are shown at 12 h after injection. Neutrophils: LysM-GFP, green. Image labeling: SCS (white dashed line), TZ (T), IFZ (IF, white arrows), LN follicles (B, dotted lines). Data is representative of 3 mice (6 iLNs) per group. (C) TP-LSM images of neutrophils (LysM-GFP, green) exiting blood vessels in the LN stroma (left panel, blue arrowheads) and accumulating in the SCS (right panel, blue arrowheads) between 2 h (left) and 4 h (right) after S. aureus (red arrowheads) injection are shown (S3 Movie, Mobilization). ILN border, white dashed line; HEVs, EB, gray. Scale bars: 50 μm. (D) Neutrophils phagocytizing S. aureus in the SCS (left panel, blue arrowheads) and swarming in the IFZ (right panel, white squares) between 3 and 4 h after S. aureus inoculation are shown (S3 Movie, Swarming). Scale bars: 25 μm (right), 20 μm (left). Data is representative of 12 imaging sessions. (E, F) DsRed B cells or CD4+ T cells were adoptively transferred 24 h prior to imaging. Neutrophil (green) interactions with (E) B cells (red) or (F) CD4+ T cells (red) at the T-B border (dashed line) at 12 h after S. aureus injection are shown. Blood vessels, EB (gray); collagen, second harmonic (blue). Scale bars: (C) 50 μm; (D, left) 50 μm; (D, right) 20 μm, (E, left) 50 μm; (E, right) 20 μm; (F, left) 70 μm; (F, right) 20 μm. (G) The percentages of short and long-lasting interactions formed by neutrophils with B cells (upper chart) and by neutrophils with CD4+ T cells (lower chart). Data is representative of 3 independent computations. (H) Analysis of S. aureus uptake by Ly6G+ and CD169+ populations in the iLN of isotype control or 1A8-injected mice between 0 and 48 h after immunization. N = 4 iLNs; 3 repeats. Means ± SEM.
Mentions: Next, we studied neutrophil influx to the iLN in response to a local injection of inactivated S. aureus Wood 46 strain. Analysis of recruitment kinetics was expanded to time points between 0 and 120 min, and at 2, 3, 6, 12 and 24 h. The neutrophils increased in the blood 1 h post-injection, continued to increase reaching a plateau at 6 h, and returned to baseline by 12 h (Fig 2A, left). Over the same interval we detected two waves of neutrophils infiltrating the iLN, 1st between 0 and 60 min with the peak at 30 min, and 2nd between 2 and 24 h with a plateau between 6 and 12 h. Their percentage had returned almost to baseline by 24 h (Fig 2A, right). Epifluorescent microscopy of intact LysM-GFP iLN showed abundant presence of GFPhi cells in the SCS (white dashed line) and IFZ (IF, white arrows) of immunized iLN at 12 h (Fig 2B). We also analyzed recruitment of neutrophils to distant LNs choosing the axillary and superficial cervical LNs and to the spleen at 4 and 12 h after immunization. Along with the massive influx of neutrophils to the iLN, we detected a significant recruitment to the spleen, but none to distant LNs (S2A and S2B Fig).

Bottom Line: They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis.Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders.Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

No MeSH data available.


Related in: MedlinePlus