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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

Neutrophils arrive from HEVs to occupy IFZ, MR and SCS in immunized iLN.Mice were injected subcutaneously with CFA. C57BL/6 mice were subjected to flow cytometry analysis of whole blood and LN cell populations; LysM-GFP expression was imaged using confocal microscopy or TP-LSM. (A) Flow cytometry analysis of blood (upper panel) and iLN cells (lower panel) in immunized C57BL/6 mice between 0 and 24 h after injection. Percentages of Ly6Ghi/CD11bhi population in live cell gate are shown. N = 2 mice/4 iLNs, repeated 3 times. Means ± SEM (B, C) Mice were sacrificed 4 h after CFA or PBS injections. The iLNs were sectioned, immunostained and analyzed by confocal microscopy. Single Z stack images were collected and assembled to form a large tiled image of the whole iLN. TZ (T), IFZ (IF), medulla (MR), LN follicle (B), and SCS are labeled. Tiled confocal images of (B) immunized and (C) PBS injected control LysM-GFP iLNs with GFPhi neutrophils (green), B cells (B220, blue), lymphatics (LYVE-1, red) and blood vessels (VE-cadherin, gray) are shown. Scale bars: 300 μm; Z = 35 μm. (D) GFP (green) and B220 (blue) channels were split, profiles of intensities of fluorescence were plotted across the images of immunized and control iLNs and measured on a scale from 1 to 100. X axis: distance in mm. Representative for 10 random profiles plotted across each section. The images are representative of 10 mice analyzed. (E, F) For TP-LSM B cells (CMTPX, red) were adoptively transferred 24 h prior to imaging; blood vessels were visualized via intravenous injection of EB (gray); collagen fibers were seen as second harmonic generation (blue). TP-LSM images of (E) immunized (S1 Movie) and (F) PBS control iLN at 2 and 4 h after injections. Scale bars: 70 μm (left and middle panels). Single HEVs (white arrows) at 4 h after injections are shown. Scale bars: 50 μm (right panels). (G) An HEV volume was defined using Imaris, and neutrophils were distinguished as cells inside (green) or outside the blood vessel (orange) in immunized (left) and PBS control (right) iLN. (H) GFP intensity of cells inside HEVs was calculated for 5 random blood vessels in immunized versus PBS control iLN, and normalized for a blood vessel volume. 5 repeats; means ± SEM.
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ppat.1004827.g001: Neutrophils arrive from HEVs to occupy IFZ, MR and SCS in immunized iLN.Mice were injected subcutaneously with CFA. C57BL/6 mice were subjected to flow cytometry analysis of whole blood and LN cell populations; LysM-GFP expression was imaged using confocal microscopy or TP-LSM. (A) Flow cytometry analysis of blood (upper panel) and iLN cells (lower panel) in immunized C57BL/6 mice between 0 and 24 h after injection. Percentages of Ly6Ghi/CD11bhi population in live cell gate are shown. N = 2 mice/4 iLNs, repeated 3 times. Means ± SEM (B, C) Mice were sacrificed 4 h after CFA or PBS injections. The iLNs were sectioned, immunostained and analyzed by confocal microscopy. Single Z stack images were collected and assembled to form a large tiled image of the whole iLN. TZ (T), IFZ (IF), medulla (MR), LN follicle (B), and SCS are labeled. Tiled confocal images of (B) immunized and (C) PBS injected control LysM-GFP iLNs with GFPhi neutrophils (green), B cells (B220, blue), lymphatics (LYVE-1, red) and blood vessels (VE-cadherin, gray) are shown. Scale bars: 300 μm; Z = 35 μm. (D) GFP (green) and B220 (blue) channels were split, profiles of intensities of fluorescence were plotted across the images of immunized and control iLNs and measured on a scale from 1 to 100. X axis: distance in mm. Representative for 10 random profiles plotted across each section. The images are representative of 10 mice analyzed. (E, F) For TP-LSM B cells (CMTPX, red) were adoptively transferred 24 h prior to imaging; blood vessels were visualized via intravenous injection of EB (gray); collagen fibers were seen as second harmonic generation (blue). TP-LSM images of (E) immunized (S1 Movie) and (F) PBS control iLN at 2 and 4 h after injections. Scale bars: 70 μm (left and middle panels). Single HEVs (white arrows) at 4 h after injections are shown. Scale bars: 50 μm (right panels). (G) An HEV volume was defined using Imaris, and neutrophils were distinguished as cells inside (green) or outside the blood vessel (orange) in immunized (left) and PBS control (right) iLN. (H) GFP intensity of cells inside HEVs was calculated for 5 random blood vessels in immunized versus PBS control iLN, and normalized for a blood vessel volume. 5 repeats; means ± SEM.

Mentions: A previous study had shown neutrophil recruitment to the iLN following the local injection of Complete Freunds’s adjuvant (CFA) [36]. CFA is composed of inactivated and dead M. tuberculosis emulsified in mineral oil. It is commonly used to enhance humoral immunity and is part of some induction schemes for triggering autoimmunity in mice. To provide a basis for comparison to S. aureus injected mice, we assessed local neutrophil response following subcutaneous CFA injection near the iLN (S1A Fig). Analysis of cell mobilization kinetics indicated a peak of neutrophil recruitment approximately 4 h after CFA immunization both in the blood and in the iLN that subsided nearly to base line the following day (Fig 1A). Ly6G+/CD11b+ cell population increased 10 fold in the blood (S1B Fig) and 8 fold in the iLNs (S1C and S1D Fig). Both the percentage and overall number of B220+ cells also increased in the LN by 24 h after CFA injection, while CD4+ and CD8+ T cells numbers remained unchanged (S1E 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)

Neutrophils arrive from HEVs to occupy IFZ, MR and SCS in immunized iLN.Mice were injected subcutaneously with CFA. C57BL/6 mice were subjected to flow cytometry analysis of whole blood and LN cell populations; LysM-GFP expression was imaged using confocal microscopy or TP-LSM. (A) Flow cytometry analysis of blood (upper panel) and iLN cells (lower panel) in immunized C57BL/6 mice between 0 and 24 h after injection. Percentages of Ly6Ghi/CD11bhi population in live cell gate are shown. N = 2 mice/4 iLNs, repeated 3 times. Means ± SEM (B, C) Mice were sacrificed 4 h after CFA or PBS injections. The iLNs were sectioned, immunostained and analyzed by confocal microscopy. Single Z stack images were collected and assembled to form a large tiled image of the whole iLN. TZ (T), IFZ (IF), medulla (MR), LN follicle (B), and SCS are labeled. Tiled confocal images of (B) immunized and (C) PBS injected control LysM-GFP iLNs with GFPhi neutrophils (green), B cells (B220, blue), lymphatics (LYVE-1, red) and blood vessels (VE-cadherin, gray) are shown. Scale bars: 300 μm; Z = 35 μm. (D) GFP (green) and B220 (blue) channels were split, profiles of intensities of fluorescence were plotted across the images of immunized and control iLNs and measured on a scale from 1 to 100. X axis: distance in mm. Representative for 10 random profiles plotted across each section. The images are representative of 10 mice analyzed. (E, F) For TP-LSM B cells (CMTPX, red) were adoptively transferred 24 h prior to imaging; blood vessels were visualized via intravenous injection of EB (gray); collagen fibers were seen as second harmonic generation (blue). TP-LSM images of (E) immunized (S1 Movie) and (F) PBS control iLN at 2 and 4 h after injections. Scale bars: 70 μm (left and middle panels). Single HEVs (white arrows) at 4 h after injections are shown. Scale bars: 50 μm (right panels). (G) An HEV volume was defined using Imaris, and neutrophils were distinguished as cells inside (green) or outside the blood vessel (orange) in immunized (left) and PBS control (right) iLN. (H) GFP intensity of cells inside HEVs was calculated for 5 random blood vessels in immunized versus PBS control iLN, and normalized for a blood vessel volume. 5 repeats; means ± SEM.
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ppat.1004827.g001: Neutrophils arrive from HEVs to occupy IFZ, MR and SCS in immunized iLN.Mice were injected subcutaneously with CFA. C57BL/6 mice were subjected to flow cytometry analysis of whole blood and LN cell populations; LysM-GFP expression was imaged using confocal microscopy or TP-LSM. (A) Flow cytometry analysis of blood (upper panel) and iLN cells (lower panel) in immunized C57BL/6 mice between 0 and 24 h after injection. Percentages of Ly6Ghi/CD11bhi population in live cell gate are shown. N = 2 mice/4 iLNs, repeated 3 times. Means ± SEM (B, C) Mice were sacrificed 4 h after CFA or PBS injections. The iLNs were sectioned, immunostained and analyzed by confocal microscopy. Single Z stack images were collected and assembled to form a large tiled image of the whole iLN. TZ (T), IFZ (IF), medulla (MR), LN follicle (B), and SCS are labeled. Tiled confocal images of (B) immunized and (C) PBS injected control LysM-GFP iLNs with GFPhi neutrophils (green), B cells (B220, blue), lymphatics (LYVE-1, red) and blood vessels (VE-cadherin, gray) are shown. Scale bars: 300 μm; Z = 35 μm. (D) GFP (green) and B220 (blue) channels were split, profiles of intensities of fluorescence were plotted across the images of immunized and control iLNs and measured on a scale from 1 to 100. X axis: distance in mm. Representative for 10 random profiles plotted across each section. The images are representative of 10 mice analyzed. (E, F) For TP-LSM B cells (CMTPX, red) were adoptively transferred 24 h prior to imaging; blood vessels were visualized via intravenous injection of EB (gray); collagen fibers were seen as second harmonic generation (blue). TP-LSM images of (E) immunized (S1 Movie) and (F) PBS control iLN at 2 and 4 h after injections. Scale bars: 70 μm (left and middle panels). Single HEVs (white arrows) at 4 h after injections are shown. Scale bars: 50 μm (right panels). (G) An HEV volume was defined using Imaris, and neutrophils were distinguished as cells inside (green) or outside the blood vessel (orange) in immunized (left) and PBS control (right) iLN. (H) GFP intensity of cells inside HEVs was calculated for 5 random blood vessels in immunized versus PBS control iLN, and normalized for a blood vessel volume. 5 repeats; means ± SEM.
Mentions: A previous study had shown neutrophil recruitment to the iLN following the local injection of Complete Freunds’s adjuvant (CFA) [36]. CFA is composed of inactivated and dead M. tuberculosis emulsified in mineral oil. It is commonly used to enhance humoral immunity and is part of some induction schemes for triggering autoimmunity in mice. To provide a basis for comparison to S. aureus injected mice, we assessed local neutrophil response following subcutaneous CFA injection near the iLN (S1A Fig). Analysis of cell mobilization kinetics indicated a peak of neutrophil recruitment approximately 4 h after CFA immunization both in the blood and in the iLN that subsided nearly to base line the following day (Fig 1A). Ly6G+/CD11b+ cell population increased 10 fold in the blood (S1B Fig) and 8 fold in the iLNs (S1C and S1D Fig). Both the percentage and overall number of B220+ cells also increased in the LN by 24 h after CFA injection, while CD4+ and CD8+ T cells numbers remained unchanged (S1E 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