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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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G-CSF treatment mimics the super-shedder phenotype by inhibiting IL-2 mediated TH1 expansion.A,B: Mice were identified as moderate (<106 cfu/gm) or super-shedder (>108 cfu/gm) between day 15 and day 30 post infection. After 30 days of infection, mice were injected with 15 µg IL-2 antibody complexed with 4.5 µg IL-2 cytokine in a 15 minute incubation period at room temperature. Data shown is from a single experiment with a total of 12 mice, 4 untreated and 8 treated. A: Splenocytes were fixed and permeabilized and pSTAT5 MFI quantified in all CD4 T cells. B: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells. Untreated mice (in square) showed no expansion and IL-2 antibody complex- treated mice included both super-shedders (open circles) and moderate-shedders (filled circles). IL-2 mediated TH1 expansion was significantly lower in super-shedder mice (p = 0.02), calculated using a one-tailed Mann Whitney U test. C, D, E: Moderate shedders were treated with either G-CSF or PBS for 3 days and then injected with IL-2 antibody complex on the 4th day. Mice were sacrificed on the 6th day, 36 days post-infection. Data shown is representative of 2 independent experiments with a total of 8 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 p<0.01. C: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 for 15 minutes and fixed and permeabilized and the frequency of pSTAT5+ CD4 T cells was quantified. D: Splenocytes were fixed and Ki-67+ CD4 T cells were quantified as a percentage of total CD4 T cells. E: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells in G-CSF treated mice (open squares) and control mice (closed squares). TH1 cells were significantly lower in the G-CSF pretreated mice (p = 0.02) calculated using a two-tailed Mann Whitney U test.
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ppat-1003408-g006: G-CSF treatment mimics the super-shedder phenotype by inhibiting IL-2 mediated TH1 expansion.A,B: Mice were identified as moderate (<106 cfu/gm) or super-shedder (>108 cfu/gm) between day 15 and day 30 post infection. After 30 days of infection, mice were injected with 15 µg IL-2 antibody complexed with 4.5 µg IL-2 cytokine in a 15 minute incubation period at room temperature. Data shown is from a single experiment with a total of 12 mice, 4 untreated and 8 treated. A: Splenocytes were fixed and permeabilized and pSTAT5 MFI quantified in all CD4 T cells. B: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells. Untreated mice (in square) showed no expansion and IL-2 antibody complex- treated mice included both super-shedders (open circles) and moderate-shedders (filled circles). IL-2 mediated TH1 expansion was significantly lower in super-shedder mice (p = 0.02), calculated using a one-tailed Mann Whitney U test. C, D, E: Moderate shedders were treated with either G-CSF or PBS for 3 days and then injected with IL-2 antibody complex on the 4th day. Mice were sacrificed on the 6th day, 36 days post-infection. Data shown is representative of 2 independent experiments with a total of 8 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 p<0.01. C: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 for 15 minutes and fixed and permeabilized and the frequency of pSTAT5+ CD4 T cells was quantified. D: Splenocytes were fixed and Ki-67+ CD4 T cells were quantified as a percentage of total CD4 T cells. E: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells in G-CSF treated mice (open squares) and control mice (closed squares). TH1 cells were significantly lower in the G-CSF pretreated mice (p = 0.02) calculated using a two-tailed Mann Whitney U test.

Mentions: Having observed that G-CSF mediated neutrophilia dampens IL-2 responsiveness across the CD4 T cell population; we next investigated whether TH1 and Treg CD4 T cell subsets differed in their IL-2 responsiveness with functional consequences. Both Tregs and TH1 cells activated by infection induce phosphorylation of STAT5 in response to IL-2 (Figure S7C). Previous studies have shown that IL-2 antibody complexed with IL-2 cytokine (hereafter referred to as IL-2 antibody complex) can induce expansion of Tregs in uninfected mice [28], [29]. When S. Typhimurium-infected mice were treated with IL-2 antibody complex, we observed expansion of both Tregs and TH1 cells (Figure 6B). This was accompanied by an increase in the number of pSTAT5+ total CD4 T cells both before (basal) and after ex vivo IL-2 stimulation (Figure 6A). Furthermore, IL-2 mediated TH1 expansion was significantly greater in moderate-shedders than super-shedders (p<0.05). These results indicate that high levels of gastrointestinal Salmonella burden and neutrophilia may be associated with an impairment in the ability of splenic TH1 cells to undergo IL-2 mediated proliferation.


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)

G-CSF treatment mimics the super-shedder phenotype by inhibiting IL-2 mediated TH1 expansion.A,B: Mice were identified as moderate (<106 cfu/gm) or super-shedder (>108 cfu/gm) between day 15 and day 30 post infection. After 30 days of infection, mice were injected with 15 µg IL-2 antibody complexed with 4.5 µg IL-2 cytokine in a 15 minute incubation period at room temperature. Data shown is from a single experiment with a total of 12 mice, 4 untreated and 8 treated. A: Splenocytes were fixed and permeabilized and pSTAT5 MFI quantified in all CD4 T cells. B: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells. Untreated mice (in square) showed no expansion and IL-2 antibody complex- treated mice included both super-shedders (open circles) and moderate-shedders (filled circles). IL-2 mediated TH1 expansion was significantly lower in super-shedder mice (p = 0.02), calculated using a one-tailed Mann Whitney U test. C, D, E: Moderate shedders were treated with either G-CSF or PBS for 3 days and then injected with IL-2 antibody complex on the 4th day. Mice were sacrificed on the 6th day, 36 days post-infection. Data shown is representative of 2 independent experiments with a total of 8 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 p<0.01. C: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 for 15 minutes and fixed and permeabilized and the frequency of pSTAT5+ CD4 T cells was quantified. D: Splenocytes were fixed and Ki-67+ CD4 T cells were quantified as a percentage of total CD4 T cells. E: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells in G-CSF treated mice (open squares) and control mice (closed squares). TH1 cells were significantly lower in the G-CSF pretreated mice (p = 0.02) calculated using a two-tailed Mann Whitney U test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675027&req=5

ppat-1003408-g006: G-CSF treatment mimics the super-shedder phenotype by inhibiting IL-2 mediated TH1 expansion.A,B: Mice were identified as moderate (<106 cfu/gm) or super-shedder (>108 cfu/gm) between day 15 and day 30 post infection. After 30 days of infection, mice were injected with 15 µg IL-2 antibody complexed with 4.5 µg IL-2 cytokine in a 15 minute incubation period at room temperature. Data shown is from a single experiment with a total of 12 mice, 4 untreated and 8 treated. A: Splenocytes were fixed and permeabilized and pSTAT5 MFI quantified in all CD4 T cells. B: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells. Untreated mice (in square) showed no expansion and IL-2 antibody complex- treated mice included both super-shedders (open circles) and moderate-shedders (filled circles). IL-2 mediated TH1 expansion was significantly lower in super-shedder mice (p = 0.02), calculated using a one-tailed Mann Whitney U test. C, D, E: Moderate shedders were treated with either G-CSF or PBS for 3 days and then injected with IL-2 antibody complex on the 4th day. Mice were sacrificed on the 6th day, 36 days post-infection. Data shown is representative of 2 independent experiments with a total of 8 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 p<0.01. C: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 for 15 minutes and fixed and permeabilized and the frequency of pSTAT5+ CD4 T cells was quantified. D: Splenocytes were fixed and Ki-67+ CD4 T cells were quantified as a percentage of total CD4 T cells. E: TH1 and Treg levels were expressed as a percentage of total splenic CD4 T cells in G-CSF treated mice (open squares) and control mice (closed squares). TH1 cells were significantly lower in the G-CSF pretreated mice (p = 0.02) calculated using a two-tailed Mann Whitney U test.
Mentions: Having observed that G-CSF mediated neutrophilia dampens IL-2 responsiveness across the CD4 T cell population; we next investigated whether TH1 and Treg CD4 T cell subsets differed in their IL-2 responsiveness with functional consequences. Both Tregs and TH1 cells activated by infection induce phosphorylation of STAT5 in response to IL-2 (Figure S7C). Previous studies have shown that IL-2 antibody complexed with IL-2 cytokine (hereafter referred to as IL-2 antibody complex) can induce expansion of Tregs in uninfected mice [28], [29]. When S. Typhimurium-infected mice were treated with IL-2 antibody complex, we observed expansion of both Tregs and TH1 cells (Figure 6B). This was accompanied by an increase in the number of pSTAT5+ total CD4 T cells both before (basal) and after ex vivo IL-2 stimulation (Figure 6A). Furthermore, IL-2 mediated TH1 expansion was significantly greater in moderate-shedders than super-shedders (p<0.05). These results indicate that high levels of gastrointestinal Salmonella burden and neutrophilia may be associated with an impairment in the ability of splenic TH1 cells to undergo IL-2 mediated proliferation.

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