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The systemic immune state of super-shedder mice is characterized by a unique neutrophil-dependent blunting of TH1 responses.

Gopinath S, Hotson A, Johns J, Nolan G, Monack D - PLoS Pathog. (2013)

Bottom Line: Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion.Finally, depletion of neutrophils led to an increase in the number of T-bet(+) T(H)1 cells and restored their ability to respond to IL-2.Typhimurium in the gastrointestinal tract.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism Salmonella enterica serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened T(H)1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (T(regs)), fewer T-bet(+) (T(H)1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened T(H)1 response with fewer T-bet(+) T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet(+) T(H)1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the TH1 immune response in the spleens of mice that are colonized by high levels of S. Typhimurium in the gastrointestinal tract.

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Neutrophil depletion results in increased CD4 pSTAT5 and Tbet expression.After 30 days of infection, mice were injected with 1 µg RB6 depletion antibody every day for 3 days and sacrificed on the 4th day. Data shown is representative of 2 independent experiments with a total of 8–10 mice in each condition. Significance was determined using a Two-tailed Mann-Whitney U test with one asterisk representing p<0.05 and two representing a p<0.01. A: Tbet+ CD4 T (TH1) cells from splenocytes were quantified as a percentage of CD4 T cells. B: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 and fixed and permeabilized and total CD4 T cell pSTAT5 MFI measured. C: FoxP3+ CD4 T cells (Tregs) were quantified as a percentage of CD4 T cells. D: Fecal pellets were collected and bacterial burden quantified.
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ppat-1003408-g004: Neutrophil depletion results in increased CD4 pSTAT5 and Tbet expression.After 30 days of infection, mice were injected with 1 µg RB6 depletion antibody every day for 3 days and sacrificed on the 4th day. Data shown is representative of 2 independent experiments with a total of 8–10 mice in each condition. Significance was determined using a Two-tailed Mann-Whitney U test with one asterisk representing p<0.05 and two representing a p<0.01. A: Tbet+ CD4 T (TH1) cells from splenocytes were quantified as a percentage of CD4 T cells. B: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 and fixed and permeabilized and total CD4 T cell pSTAT5 MFI measured. C: FoxP3+ CD4 T cells (Tregs) were quantified as a percentage of CD4 T cells. D: Fecal pellets were collected and bacterial burden quantified.

Mentions: We depleted neutrophils using the monoclonal antibody RB6, which targets cells expressing both Ly6C and Ly6G. Neutrophil depletion increased the levels of splenic TH1 cells from 10.2±5.8% in control mice to 24.1±8.8% in RB6-treated mice (Figure 4A). Similar results were obtained with a Ly6G-specific depletion antibody, IA8 (Figure S6A,B). An increase was observed in the frequency of pSTAT5+ CD4 T cells that responded to ex vivo IL-2 stimulation regardless of infection, indicating that neutrophils suppress CD4 T cell responsiveness to IL-2 (Figure 4B). Intriguingly, uninfected mice depleted of neutrophils also showed a similar increase in pSTAT5+ CD4 T cells, but without TH1 expansion. This indicates that the IL-2/pSTAT5 response may be an intermediate step to TH1 expansion and that TH1 biasing occurs only in the context of infection. The percentage of splenic Tregs in infected and uninfected mice did not statistically change upon neutrophil depletion, implying that the increase in IL-2 responsive CD4 cells was not due to Treg reduction (Figure 4C). In addition, RB6-treated mice had significantly higher bacterial burdens in the spleen compared to control mice (Figure S6C). This demonstrates that neutrophils are necessary to control splenic infection, and that the increased TH1 response is unable to compensate for neutrophil depletion. Surprisingly, there was no difference observed in fecal bacterial burden (Figure 4D) suggesting that there might be an organ specific function for neutrophils in persistent Salmonella infection. Taken together, the results indicate that high levels of neutrophils in the spleen are necessary for both dampened IL-2 responsiveness and a reduction in the levels of of TH1 cells.


The systemic immune state of super-shedder mice is characterized by a unique neutrophil-dependent blunting of TH1 responses.

Gopinath S, Hotson A, Johns J, Nolan G, Monack D - PLoS Pathog. (2013)

Neutrophil depletion results in increased CD4 pSTAT5 and Tbet expression.After 30 days of infection, mice were injected with 1 µg RB6 depletion antibody every day for 3 days and sacrificed on the 4th day. Data shown is representative of 2 independent experiments with a total of 8–10 mice in each condition. Significance was determined using a Two-tailed Mann-Whitney U test with one asterisk representing p<0.05 and two representing a p<0.01. A: Tbet+ CD4 T (TH1) cells from splenocytes were quantified as a percentage of CD4 T cells. B: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 and fixed and permeabilized and total CD4 T cell pSTAT5 MFI measured. C: FoxP3+ CD4 T cells (Tregs) were quantified as a percentage of CD4 T cells. D: Fecal pellets were collected and bacterial burden quantified.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675027&req=5

ppat-1003408-g004: Neutrophil depletion results in increased CD4 pSTAT5 and Tbet expression.After 30 days of infection, mice were injected with 1 µg RB6 depletion antibody every day for 3 days and sacrificed on the 4th day. Data shown is representative of 2 independent experiments with a total of 8–10 mice in each condition. Significance was determined using a Two-tailed Mann-Whitney U test with one asterisk representing p<0.05 and two representing a p<0.01. A: Tbet+ CD4 T (TH1) cells from splenocytes were quantified as a percentage of CD4 T cells. B: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 and fixed and permeabilized and total CD4 T cell pSTAT5 MFI measured. C: FoxP3+ CD4 T cells (Tregs) were quantified as a percentage of CD4 T cells. D: Fecal pellets were collected and bacterial burden quantified.
Mentions: We depleted neutrophils using the monoclonal antibody RB6, which targets cells expressing both Ly6C and Ly6G. Neutrophil depletion increased the levels of splenic TH1 cells from 10.2±5.8% in control mice to 24.1±8.8% in RB6-treated mice (Figure 4A). Similar results were obtained with a Ly6G-specific depletion antibody, IA8 (Figure S6A,B). An increase was observed in the frequency of pSTAT5+ CD4 T cells that responded to ex vivo IL-2 stimulation regardless of infection, indicating that neutrophils suppress CD4 T cell responsiveness to IL-2 (Figure 4B). Intriguingly, uninfected mice depleted of neutrophils also showed a similar increase in pSTAT5+ CD4 T cells, but without TH1 expansion. This indicates that the IL-2/pSTAT5 response may be an intermediate step to TH1 expansion and that TH1 biasing occurs only in the context of infection. The percentage of splenic Tregs in infected and uninfected mice did not statistically change upon neutrophil depletion, implying that the increase in IL-2 responsive CD4 cells was not due to Treg reduction (Figure 4C). In addition, RB6-treated mice had significantly higher bacterial burdens in the spleen compared to control mice (Figure S6C). This demonstrates that neutrophils are necessary to control splenic infection, and that the increased TH1 response is unable to compensate for neutrophil depletion. Surprisingly, there was no difference observed in fecal bacterial burden (Figure 4D) suggesting that there might be an organ specific function for neutrophils in persistent Salmonella infection. Taken together, the results indicate that high levels of neutrophils in the spleen are necessary for both dampened IL-2 responsiveness and a reduction in the levels of of TH1 cells.

Bottom Line: Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion.Finally, depletion of neutrophils led to an increase in the number of T-bet(+) T(H)1 cells and restored their ability to respond to IL-2.Typhimurium in the gastrointestinal tract.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism Salmonella enterica serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened T(H)1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (T(regs)), fewer T-bet(+) (T(H)1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened T(H)1 response with fewer T-bet(+) T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet(+) T(H)1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the TH1 immune response in the spleens of mice that are colonized by high levels of S. Typhimurium in the gastrointestinal tract.

Show MeSH
Related in: MedlinePlus