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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 limit the humoral response following local immunization or infection.C57BL/6 mice were given PBS as a baseline control, immunized with S. aureus bioparticles, or infected with LAC-GFP. Isotype control or 1A8 antibody was injected intraperitoneally at 100 μg/mouse on day -1, 0 and 1 of immunization/infection. (A) Images of iLNs in isotype control (left) and neutrophil depleted (right) mice immunized with S. aureus bioparticles are shown at day 5 after immunization. Indicated are LN edges (black arrows), blood vessels (blue arrows). Scale bars: 5 mm. (B) Flow cytometry analysis of B and T cell populations in the iLN of isotype control or neutrophil-depleted mice 3 days after immunization. N = 4 iLNs; 3 repeats. Means ± SEM. (C) ELISA of total IgG and IgM produced by iLN B cells isolated from PBS control, immunized isotype control and immunized neutrophil-depleted (1A8) mice. B cells were isolated from the iLNs at day 6 after immunization and cultured for 3 days. N = 4 iLNs. Data are shown as fold change. 3 repeats. Means ± SEM. (D) ELISA of total IgG in the serum of immunized isotype control and immunized neutrophil-depleted mice measured at days 0, 7, 14, 21 and 28 after immunization. N = 4 mice; 2 repeats; means ± SEM. (E-G) Mice were depleted of neutrophils as above and infected with LAC-GFP. (E) ELISA of total IgG and IgM produced by iLN B cells. ILNs were harvested at day 5 after infection, B cells were isolated and cultured for 3 days. N = 5–7 iLNs. Data are shown as antibody concentration in B cell supernatants. Means ± SD. (F) Comparison of fold changes in IgG and IgM production in neutrophil-depleted and isotype iLNs, calculated for S. aureus immunized versus LAC-GFP infected mice. Data shown as fold increases (Means ± SD). (G) ELISA of LAC-specific (upper panel) and LAC spa-specific IgG and IgM. N = 5–7 iLNs. Means ± SD. (H) ELISA of IgM produced in vitro by LPS or S. aureus activated LN B cells in presence of neutrophils, activated correspondingly. No LPS in culture used as a baseline control. (I) ELISA of IgM produced by LPS activated iLN B cells in presence of LPS-activated neutrophils supernatants from LPS-activated (SN act) and non-activated neutrophils (SN NA), neutralizing anti-TGF-β1 antibody, and TGF-β1. (J) ELISA results measuring TGF-β1 produced by activated neutrophils, non-activated neutrophils or LPS-activated B cells in 24 h cultures. (H-J) Final concentration of LPS in all B cell cultures: 2 μg/mL. 3 to 5 repeats for each of in vitro experiments. Means ± SEM.
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ppat.1004827.g005: Neutrophils limit the humoral response following local immunization or infection.C57BL/6 mice were given PBS as a baseline control, immunized with S. aureus bioparticles, or infected with LAC-GFP. Isotype control or 1A8 antibody was injected intraperitoneally at 100 μg/mouse on day -1, 0 and 1 of immunization/infection. (A) Images of iLNs in isotype control (left) and neutrophil depleted (right) mice immunized with S. aureus bioparticles are shown at day 5 after immunization. Indicated are LN edges (black arrows), blood vessels (blue arrows). Scale bars: 5 mm. (B) Flow cytometry analysis of B and T cell populations in the iLN of isotype control or neutrophil-depleted mice 3 days after immunization. N = 4 iLNs; 3 repeats. Means ± SEM. (C) ELISA of total IgG and IgM produced by iLN B cells isolated from PBS control, immunized isotype control and immunized neutrophil-depleted (1A8) mice. B cells were isolated from the iLNs at day 6 after immunization and cultured for 3 days. N = 4 iLNs. Data are shown as fold change. 3 repeats. Means ± SEM. (D) ELISA of total IgG in the serum of immunized isotype control and immunized neutrophil-depleted mice measured at days 0, 7, 14, 21 and 28 after immunization. N = 4 mice; 2 repeats; means ± SEM. (E-G) Mice were depleted of neutrophils as above and infected with LAC-GFP. (E) ELISA of total IgG and IgM produced by iLN B cells. ILNs were harvested at day 5 after infection, B cells were isolated and cultured for 3 days. N = 5–7 iLNs. Data are shown as antibody concentration in B cell supernatants. Means ± SD. (F) Comparison of fold changes in IgG and IgM production in neutrophil-depleted and isotype iLNs, calculated for S. aureus immunized versus LAC-GFP infected mice. Data shown as fold increases (Means ± SD). (G) ELISA of LAC-specific (upper panel) and LAC spa-specific IgG and IgM. N = 5–7 iLNs. Means ± SD. (H) ELISA of IgM produced in vitro by LPS or S. aureus activated LN B cells in presence of neutrophils, activated correspondingly. No LPS in culture used as a baseline control. (I) ELISA of IgM produced by LPS activated iLN B cells in presence of LPS-activated neutrophils supernatants from LPS-activated (SN act) and non-activated neutrophils (SN NA), neutralizing anti-TGF-β1 antibody, and TGF-β1. (J) ELISA results measuring TGF-β1 produced by activated neutrophils, non-activated neutrophils or LPS-activated B cells in 24 h cultures. (H-J) Final concentration of LPS in all B cell cultures: 2 μg/mL. 3 to 5 repeats for each of in vitro experiments. Means ± SEM.

Mentions: The large influx of neutrophils and their observed interactions with B cells following local injection of S. aureus suggested that these interactions might influence the subsequent humoral response. To test this possibility we depleted neutrophils in vivo and measured antibody production by iLN B cells in mice immunized with S. aureus bioparticles or infected with LAC-GFP. The mice received an intraperitoneal injection of Ly6G-specific antibodies (1A8) or isotype control antibodies at day -1, 0 and 1 of immunization/infection with S. aureus. 24 h after first 1A8 injection, neutrophils were mobilized to the blood and LNs in S. aureus immunized isotype control-injected, but not 1A8-injected mice (S5A–S5C Fig). At day 5, the iLNs in neutrophil-depleted mice were larger, and more heavily vascularized than in isotype control mice (Fig 5A). Analysis of the kinetics of lymphocyte recruitment to S. aureus bioparticle-immunized iLN revealed an increase in B220+ cell population and decrease in CD4+ and CD8+ populations in neutrophil-depleted mice (Fig 5B). B220+ cell numbers increased in neutrophil-depleted mice correlating with the total iLN cell numbers (S5D Fig). We harvested the iLN B cells at days 5–6 post S. aureus injection, cultured them for 3 days and measured the levels of IgG and IgM in the supernatants. We compared amounts of antibodies produced by B cells derived from a single iLN (S5D Fig). In the LNs from mice injected with S. aureus bioparticles, neutrophil depletion caused a 12-fold increase in total IgG and 30-fold increase in total IgM production (Figs 5C and S5E). When quantified as amount of antibodies per B cell number, antibody production was also increased in B cell cultures derived from neutrophil-depleted mice (S5F Fig). Total IgG levels were elevated in the serum of neutrophil-depleted mice starting at day 14 after immunization with S. aureus bioparticles (Fig 5D). In the LNs harvested from LAC-GFP infected mice, neutrophil depletion resulted in over a 100-fold increases in both IgG and IgM production by LN B cells (Fig 5E). Thus, the fold increase in antibody production after neutrophil depletion was higher in LAC-GFP infected mice than in the S. aureus bioparticle immunized mice (Fig 5F). Using LAC or LAC spa lysates as antigens, we found that LAC-specific IgG and IgM responses were elevated in neutrophil-depleted mice (Fig 5G). At day 5 after infection, LAC was found in the LNs of neutrophil depleted mice but not of isotype control-injected mice (S3G 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 limit the humoral response following local immunization or infection.C57BL/6 mice were given PBS as a baseline control, immunized with S. aureus bioparticles, or infected with LAC-GFP. Isotype control or 1A8 antibody was injected intraperitoneally at 100 μg/mouse on day -1, 0 and 1 of immunization/infection. (A) Images of iLNs in isotype control (left) and neutrophil depleted (right) mice immunized with S. aureus bioparticles are shown at day 5 after immunization. Indicated are LN edges (black arrows), blood vessels (blue arrows). Scale bars: 5 mm. (B) Flow cytometry analysis of B and T cell populations in the iLN of isotype control or neutrophil-depleted mice 3 days after immunization. N = 4 iLNs; 3 repeats. Means ± SEM. (C) ELISA of total IgG and IgM produced by iLN B cells isolated from PBS control, immunized isotype control and immunized neutrophil-depleted (1A8) mice. B cells were isolated from the iLNs at day 6 after immunization and cultured for 3 days. N = 4 iLNs. Data are shown as fold change. 3 repeats. Means ± SEM. (D) ELISA of total IgG in the serum of immunized isotype control and immunized neutrophil-depleted mice measured at days 0, 7, 14, 21 and 28 after immunization. N = 4 mice; 2 repeats; means ± SEM. (E-G) Mice were depleted of neutrophils as above and infected with LAC-GFP. (E) ELISA of total IgG and IgM produced by iLN B cells. ILNs were harvested at day 5 after infection, B cells were isolated and cultured for 3 days. N = 5–7 iLNs. Data are shown as antibody concentration in B cell supernatants. Means ± SD. (F) Comparison of fold changes in IgG and IgM production in neutrophil-depleted and isotype iLNs, calculated for S. aureus immunized versus LAC-GFP infected mice. Data shown as fold increases (Means ± SD). (G) ELISA of LAC-specific (upper panel) and LAC spa-specific IgG and IgM. N = 5–7 iLNs. Means ± SD. (H) ELISA of IgM produced in vitro by LPS or S. aureus activated LN B cells in presence of neutrophils, activated correspondingly. No LPS in culture used as a baseline control. (I) ELISA of IgM produced by LPS activated iLN B cells in presence of LPS-activated neutrophils supernatants from LPS-activated (SN act) and non-activated neutrophils (SN NA), neutralizing anti-TGF-β1 antibody, and TGF-β1. (J) ELISA results measuring TGF-β1 produced by activated neutrophils, non-activated neutrophils or LPS-activated B cells in 24 h cultures. (H-J) Final concentration of LPS in all B cell cultures: 2 μg/mL. 3 to 5 repeats for each of in vitro experiments. Means ± SEM.
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ppat.1004827.g005: Neutrophils limit the humoral response following local immunization or infection.C57BL/6 mice were given PBS as a baseline control, immunized with S. aureus bioparticles, or infected with LAC-GFP. Isotype control or 1A8 antibody was injected intraperitoneally at 100 μg/mouse on day -1, 0 and 1 of immunization/infection. (A) Images of iLNs in isotype control (left) and neutrophil depleted (right) mice immunized with S. aureus bioparticles are shown at day 5 after immunization. Indicated are LN edges (black arrows), blood vessels (blue arrows). Scale bars: 5 mm. (B) Flow cytometry analysis of B and T cell populations in the iLN of isotype control or neutrophil-depleted mice 3 days after immunization. N = 4 iLNs; 3 repeats. Means ± SEM. (C) ELISA of total IgG and IgM produced by iLN B cells isolated from PBS control, immunized isotype control and immunized neutrophil-depleted (1A8) mice. B cells were isolated from the iLNs at day 6 after immunization and cultured for 3 days. N = 4 iLNs. Data are shown as fold change. 3 repeats. Means ± SEM. (D) ELISA of total IgG in the serum of immunized isotype control and immunized neutrophil-depleted mice measured at days 0, 7, 14, 21 and 28 after immunization. N = 4 mice; 2 repeats; means ± SEM. (E-G) Mice were depleted of neutrophils as above and infected with LAC-GFP. (E) ELISA of total IgG and IgM produced by iLN B cells. ILNs were harvested at day 5 after infection, B cells were isolated and cultured for 3 days. N = 5–7 iLNs. Data are shown as antibody concentration in B cell supernatants. Means ± SD. (F) Comparison of fold changes in IgG and IgM production in neutrophil-depleted and isotype iLNs, calculated for S. aureus immunized versus LAC-GFP infected mice. Data shown as fold increases (Means ± SD). (G) ELISA of LAC-specific (upper panel) and LAC spa-specific IgG and IgM. N = 5–7 iLNs. Means ± SD. (H) ELISA of IgM produced in vitro by LPS or S. aureus activated LN B cells in presence of neutrophils, activated correspondingly. No LPS in culture used as a baseline control. (I) ELISA of IgM produced by LPS activated iLN B cells in presence of LPS-activated neutrophils supernatants from LPS-activated (SN act) and non-activated neutrophils (SN NA), neutralizing anti-TGF-β1 antibody, and TGF-β1. (J) ELISA results measuring TGF-β1 produced by activated neutrophils, non-activated neutrophils or LPS-activated B cells in 24 h cultures. (H-J) Final concentration of LPS in all B cell cultures: 2 μg/mL. 3 to 5 repeats for each of in vitro experiments. Means ± SEM.
Mentions: The large influx of neutrophils and their observed interactions with B cells following local injection of S. aureus suggested that these interactions might influence the subsequent humoral response. To test this possibility we depleted neutrophils in vivo and measured antibody production by iLN B cells in mice immunized with S. aureus bioparticles or infected with LAC-GFP. The mice received an intraperitoneal injection of Ly6G-specific antibodies (1A8) or isotype control antibodies at day -1, 0 and 1 of immunization/infection with S. aureus. 24 h after first 1A8 injection, neutrophils were mobilized to the blood and LNs in S. aureus immunized isotype control-injected, but not 1A8-injected mice (S5A–S5C Fig). At day 5, the iLNs in neutrophil-depleted mice were larger, and more heavily vascularized than in isotype control mice (Fig 5A). Analysis of the kinetics of lymphocyte recruitment to S. aureus bioparticle-immunized iLN revealed an increase in B220+ cell population and decrease in CD4+ and CD8+ populations in neutrophil-depleted mice (Fig 5B). B220+ cell numbers increased in neutrophil-depleted mice correlating with the total iLN cell numbers (S5D Fig). We harvested the iLN B cells at days 5–6 post S. aureus injection, cultured them for 3 days and measured the levels of IgG and IgM in the supernatants. We compared amounts of antibodies produced by B cells derived from a single iLN (S5D Fig). In the LNs from mice injected with S. aureus bioparticles, neutrophil depletion caused a 12-fold increase in total IgG and 30-fold increase in total IgM production (Figs 5C and S5E). When quantified as amount of antibodies per B cell number, antibody production was also increased in B cell cultures derived from neutrophil-depleted mice (S5F Fig). Total IgG levels were elevated in the serum of neutrophil-depleted mice starting at day 14 after immunization with S. aureus bioparticles (Fig 5D). In the LNs harvested from LAC-GFP infected mice, neutrophil depletion resulted in over a 100-fold increases in both IgG and IgM production by LN B cells (Fig 5E). Thus, the fold increase in antibody production after neutrophil depletion was higher in LAC-GFP infected mice than in the S. aureus bioparticle immunized mice (Fig 5F). Using LAC or LAC spa lysates as antigens, we found that LAC-specific IgG and IgM responses were elevated in neutrophil-depleted mice (Fig 5G). At day 5 after infection, LAC was found in the LNs of neutrophil depleted mice but not of isotype control-injected mice (S3G 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