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The role of peroxisome proliferator-activated receptor γ in immune responses to enteroaggregative Escherichia coli infection.

Philipson CW, Bassaganya-Riera J, Viladomiu M, Pedragosa M, Guerrant RL, Roche JK, Hontecillas R - PLoS ONE (2013)

Bottom Line: At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery.The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 in vivo.Our studies provide in vivo evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.

View Article: PubMed Central - PubMed

Affiliation: Nutritional Immunology and Molecular Medicine Laboratory, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT

Background: Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of persistent diarrhea and enteric disease worldwide. Mucosal immunity towards EAEC infections is incompletely understood due in part to the lack of appropriate animal models. This study presents a new mouse model and investigates the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the modulation of host responses to EAEC in nourished and malnourished mice.

Methods/principal findings: Wild-type and T cell-specific PPARγ C57BL/6 mice were fed protein-deficient diets at weaning and challenged with 5×10(9)cfu EAEC strain JM221 to measure colonic gene expression and immune responses to EAEC. Antigen-specific responses to E. coli antigens were measured in nourished and malnourished mice following infection and demonstrated the immunosuppressive effects of malnutrition at the cellular level. At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery. The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 in vivo.

Conclusions: Our studies provide in vivo evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.

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Related in: MedlinePlus

Immune responses during enteroaggregative Escherichia coli (EAEC) infection in peroxisome proliferator-activated receptor γ (PPARγ)-deficient mice associated with bacterial clearance.Antigen specific recall responses of spleenocytes from mice infected with EAEC were measured ex vivo using the lymphocyte blastogenesis test. EAEC JM221 whole cell and whole cell sonicate were used in parallel to two negative controls, E. coli HS and mutant EAEC Aff/I strains as well as one positive control, concanavalin A (ConA). Lymphocyte proliferation is expressed stimulation indexes which are calculated by dividing the counts per minute (CPM) of antigen-stimulated wells by the CPM of unstimulated wells (A). IL-17 expression was assessed in colonic lamina propria (B) and whole blood (C) CD4+ T cells by flow cytometry and in the colon by quantitative real time RT-PCR (D) 14 days PI. Mice per group: n = 10. Asterisks indicate values where differences are statistically significant (p<0.05) while bars connect groups where comparisons are made.
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pone-0057812-g002: Immune responses during enteroaggregative Escherichia coli (EAEC) infection in peroxisome proliferator-activated receptor γ (PPARγ)-deficient mice associated with bacterial clearance.Antigen specific recall responses of spleenocytes from mice infected with EAEC were measured ex vivo using the lymphocyte blastogenesis test. EAEC JM221 whole cell and whole cell sonicate were used in parallel to two negative controls, E. coli HS and mutant EAEC Aff/I strains as well as one positive control, concanavalin A (ConA). Lymphocyte proliferation is expressed stimulation indexes which are calculated by dividing the counts per minute (CPM) of antigen-stimulated wells by the CPM of unstimulated wells (A). IL-17 expression was assessed in colonic lamina propria (B) and whole blood (C) CD4+ T cells by flow cytometry and in the colon by quantitative real time RT-PCR (D) 14 days PI. Mice per group: n = 10. Asterisks indicate values where differences are statistically significant (p<0.05) while bars connect groups where comparisons are made.

Mentions: Overall lymphocyte proliferation was assessed in splenocytes using ex vivo antigen stimulation and the incorporation of titrated thymidine in a lymphocyte blastogenesis test. The loss of PPARγ enhanced the magnitude of antigen-specific recall responses to EAEC in nourished mice, whereas malnutrition abrogated responsiveness to antigens or to the mitogen ConA (Figure 2A) regardless of genotype. In addition, PPARγ deficiency led to an increase in colonic IL-17 expression and Th17 responses. IL-17 is one of the first cytokines released during innate responses and plays an essential role in mucosal defense against extracellular bacteria through neutrophil trafficking [23] which is critical for host defense against various pathogens [24]. Tissue from the whole colon was analyzed for IL-17 gene expression 14 days PI. Regardless of infection or diet several mice expressed low basal levels of IL-17 in the colon, however nourished MMTV-cre+ mice expressed significantly elevated levels of colonic IL-17 compared to all other groups (Figure 2D). Malnourished MMTV-cre+ mice also had a higher tendency to express IL-17. Flow cytometric analysis provided evidence that percentages of local CD4+ T cells expressing IL-17 (i.e., Th17 cells) at the colonic mucosa were increased in nourished and malnourished MMTV-cre+ and malnourished WT mice (Figure 2B). The systemic levels of Th17 cells were also significantly elevated in malnourished MMTV-cre+ mice (Figure 2C). These combined results suggest that EAEC infection may induce Th17 responses and the loss of PPARγ enhances the magnitude of Th17 responses. All mice except for the wild-type malnourished completely cleared colonization by day 14 post infection providing evidence that the enhanced effector responses facilitated bacterial clearance (data not shown).


The role of peroxisome proliferator-activated receptor γ in immune responses to enteroaggregative Escherichia coli infection.

Philipson CW, Bassaganya-Riera J, Viladomiu M, Pedragosa M, Guerrant RL, Roche JK, Hontecillas R - PLoS ONE (2013)

Immune responses during enteroaggregative Escherichia coli (EAEC) infection in peroxisome proliferator-activated receptor γ (PPARγ)-deficient mice associated with bacterial clearance.Antigen specific recall responses of spleenocytes from mice infected with EAEC were measured ex vivo using the lymphocyte blastogenesis test. EAEC JM221 whole cell and whole cell sonicate were used in parallel to two negative controls, E. coli HS and mutant EAEC Aff/I strains as well as one positive control, concanavalin A (ConA). Lymphocyte proliferation is expressed stimulation indexes which are calculated by dividing the counts per minute (CPM) of antigen-stimulated wells by the CPM of unstimulated wells (A). IL-17 expression was assessed in colonic lamina propria (B) and whole blood (C) CD4+ T cells by flow cytometry and in the colon by quantitative real time RT-PCR (D) 14 days PI. Mice per group: n = 10. Asterisks indicate values where differences are statistically significant (p<0.05) while bars connect groups where comparisons are made.
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pone-0057812-g002: Immune responses during enteroaggregative Escherichia coli (EAEC) infection in peroxisome proliferator-activated receptor γ (PPARγ)-deficient mice associated with bacterial clearance.Antigen specific recall responses of spleenocytes from mice infected with EAEC were measured ex vivo using the lymphocyte blastogenesis test. EAEC JM221 whole cell and whole cell sonicate were used in parallel to two negative controls, E. coli HS and mutant EAEC Aff/I strains as well as one positive control, concanavalin A (ConA). Lymphocyte proliferation is expressed stimulation indexes which are calculated by dividing the counts per minute (CPM) of antigen-stimulated wells by the CPM of unstimulated wells (A). IL-17 expression was assessed in colonic lamina propria (B) and whole blood (C) CD4+ T cells by flow cytometry and in the colon by quantitative real time RT-PCR (D) 14 days PI. Mice per group: n = 10. Asterisks indicate values where differences are statistically significant (p<0.05) while bars connect groups where comparisons are made.
Mentions: Overall lymphocyte proliferation was assessed in splenocytes using ex vivo antigen stimulation and the incorporation of titrated thymidine in a lymphocyte blastogenesis test. The loss of PPARγ enhanced the magnitude of antigen-specific recall responses to EAEC in nourished mice, whereas malnutrition abrogated responsiveness to antigens or to the mitogen ConA (Figure 2A) regardless of genotype. In addition, PPARγ deficiency led to an increase in colonic IL-17 expression and Th17 responses. IL-17 is one of the first cytokines released during innate responses and plays an essential role in mucosal defense against extracellular bacteria through neutrophil trafficking [23] which is critical for host defense against various pathogens [24]. Tissue from the whole colon was analyzed for IL-17 gene expression 14 days PI. Regardless of infection or diet several mice expressed low basal levels of IL-17 in the colon, however nourished MMTV-cre+ mice expressed significantly elevated levels of colonic IL-17 compared to all other groups (Figure 2D). Malnourished MMTV-cre+ mice also had a higher tendency to express IL-17. Flow cytometric analysis provided evidence that percentages of local CD4+ T cells expressing IL-17 (i.e., Th17 cells) at the colonic mucosa were increased in nourished and malnourished MMTV-cre+ and malnourished WT mice (Figure 2B). The systemic levels of Th17 cells were also significantly elevated in malnourished MMTV-cre+ mice (Figure 2C). These combined results suggest that EAEC infection may induce Th17 responses and the loss of PPARγ enhances the magnitude of Th17 responses. All mice except for the wild-type malnourished completely cleared colonization by day 14 post infection providing evidence that the enhanced effector responses facilitated bacterial clearance (data not shown).

Bottom Line: At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery.The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 in vivo.Our studies provide in vivo evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.

View Article: PubMed Central - PubMed

Affiliation: Nutritional Immunology and Molecular Medicine Laboratory, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT

Background: Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of persistent diarrhea and enteric disease worldwide. Mucosal immunity towards EAEC infections is incompletely understood due in part to the lack of appropriate animal models. This study presents a new mouse model and investigates the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the modulation of host responses to EAEC in nourished and malnourished mice.

Methods/principal findings: Wild-type and T cell-specific PPARγ C57BL/6 mice were fed protein-deficient diets at weaning and challenged with 5×10(9)cfu EAEC strain JM221 to measure colonic gene expression and immune responses to EAEC. Antigen-specific responses to E. coli antigens were measured in nourished and malnourished mice following infection and demonstrated the immunosuppressive effects of malnutrition at the cellular level. At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery. The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 in vivo.

Conclusions: Our studies provide in vivo evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.

Show MeSH
Related in: MedlinePlus