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Persistently active microbial molecules prolong innate immune tolerance in vivo.

Lu M, Varley AW, Munford RS - PLoS Pathog. (2013)

Bottom Line: Mice that are unable to inactivate LPS, in contrast, remain tolerant for several months; during this time they respond sluggishly to Gram-negative bacterial challenge, with high mortality.We show here that prolonged macrophage reprogramming is maintained in vivo by the persistence of stimulatory LPS molecules within the cells' in vivo environment, where naïve cells can acquire LPS via cell-cell contact or from the extracellular fluid.Measures that disable microbial molecules might enhance resolution of tissue inflammation and help restore innate defenses in individuals recovering from many different infectious diseases.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America. Lum3@niaid.nih.gov

ABSTRACT
Measures that bolster the resolution phase of infectious diseases may offer new opportunities for improving outcome. Here we show that inactivation of microbial lipopolysaccharides (LPS) can be required for animals to recover from the innate immune tolerance that follows exposure to Gram-negative bacteria. When wildtype mice are exposed to small parenteral doses of LPS or Gram-negative bacteria, their macrophages become reprogrammed (tolerant) for a few days before they resume normal function. Mice that are unable to inactivate LPS, in contrast, remain tolerant for several months; during this time they respond sluggishly to Gram-negative bacterial challenge, with high mortality. We show here that prolonged macrophage reprogramming is maintained in vivo by the persistence of stimulatory LPS molecules within the cells' in vivo environment, where naïve cells can acquire LPS via cell-cell contact or from the extracellular fluid. The findings provide strong evidence that inactivation of a stimulatory microbial molecule can be required for animals to regain immune homeostasis following parenteral exposure to bacteria. Measures that disable microbial molecules might enhance resolution of tissue inflammation and help restore innate defenses in individuals recovering from many different infectious diseases.

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Related in: MedlinePlus

Aoah−/−Tlr4−/− macrophages can become tolerant in LPS-injected Aoah−/− mice.(A) CD45.2 Aoah−/−Tlr4−/− naïve peritoneal cells were transferred to the peritoneal cavities of CD45.1 Aoah+/+Tlr4+/+ and Aoah−/−Tlr4+/+ mice. After 18 hours, half of the recipient mice in each group were injected i.p. with 1 µg LPS. Fourteen days after injection, the peritoneal cells were harvested. Some of the cells were treated ex vivo with 40 µg/ml Micrococcus luteus plus 2.5 µg/ml poly I:C for 8 hours in the presence of Brefeldin A. IL-6 and TNF production by donor (D) and recipient (R) F4/80+ macrophages was measured using flow cytometry. (B and C) The remainder of the peritoneal cells was not re-stimulated ex vivo and was used to determine surface expression of F4/80 and CD86 on macrophages (F4/80+) by flow cytometry. **, P<0.01; ***, P<0.001. Data were combined from 2 independent experiments. n = 5–7. Aoah−/− Tlr4−/− macrophages, which are unable to respond to LPS, became tolerant to TLR2 and TLR3 ligands (A) and developed the tolerant surface phenotype (B and C) when they were transferred into LPS-primed Aoah−/− mice, supporting a role for non-LPS stimuli in promoting tolerance in vivo. Pink bars, Aoah−/−Tlr4−/− donor macrophages; Blue bars, Aoah+/+ recipient macrophages; red bars, Aoah−/− recipient macrophages; diagonal markings indicate in vivo LPS exposure.
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ppat-1003339-g008: Aoah−/−Tlr4−/− macrophages can become tolerant in LPS-injected Aoah−/− mice.(A) CD45.2 Aoah−/−Tlr4−/− naïve peritoneal cells were transferred to the peritoneal cavities of CD45.1 Aoah+/+Tlr4+/+ and Aoah−/−Tlr4+/+ mice. After 18 hours, half of the recipient mice in each group were injected i.p. with 1 µg LPS. Fourteen days after injection, the peritoneal cells were harvested. Some of the cells were treated ex vivo with 40 µg/ml Micrococcus luteus plus 2.5 µg/ml poly I:C for 8 hours in the presence of Brefeldin A. IL-6 and TNF production by donor (D) and recipient (R) F4/80+ macrophages was measured using flow cytometry. (B and C) The remainder of the peritoneal cells was not re-stimulated ex vivo and was used to determine surface expression of F4/80 and CD86 on macrophages (F4/80+) by flow cytometry. **, P<0.01; ***, P<0.001. Data were combined from 2 independent experiments. n = 5–7. Aoah−/− Tlr4−/− macrophages, which are unable to respond to LPS, became tolerant to TLR2 and TLR3 ligands (A) and developed the tolerant surface phenotype (B and C) when they were transferred into LPS-primed Aoah−/− mice, supporting a role for non-LPS stimuli in promoting tolerance in vivo. Pink bars, Aoah−/−Tlr4−/− donor macrophages; Blue bars, Aoah+/+ recipient macrophages; red bars, Aoah−/− recipient macrophages; diagonal markings indicate in vivo LPS exposure.

Mentions: Bioactive LPS is thus present in the peritoneum of LPS-injected Aoah−/− mice, where it is sufficient to prevent macrophages from regaining responsiveness. Do LPS-induced mediators also play a role in maintaining tolerance? We transferred CD45.2 Aoah−/−Tlr4−/− peritoneal cells to CD45.1 Aoah+/+ or Aoah−/− (both Tlr4+/+) recipient mice, injected LPS i.p., and asked whether the Aoah−/−Tlr4−/− donor macrophages became tolerant in the peritoneum of tolerant Aoah−/− recipient mice. Fourteen days after LPS injection, we harvested recipient peritoneal cells and treated them ex vivo with Micrococcus luteus plus poly I:C (LPS-exposed Aoah−/− macrophages express hetero-tolerance [36] to M. luteus [TLR2 agonist] and poly I:C [TLR3 agonist] [25]). Aoah−/−Tlr4−/− donor macrophages (F4/80+) harvested from LPS-injected Aoah−/− recipients had significantly lower IL-6 and TNF responses to ex vivo re-challenge (Fig. 8A), and they also had less surface F4/80 and CD86 expression (Fig. 8B, C). Since Aoah−/−Tlr4−/− donor macrophages cannot sense LPS, these results suggested that LPS-induced mediators in the peritoneum are also important for prolonging tolerance in Aoah−/− mice. Aoah−/−Tlr4−/− donor cells were less tolerant than were the recipient's Aoah−/−Tlr4+/+ macrophages, again reinforcing the prominent direct role played by fully acylated LPS.


Persistently active microbial molecules prolong innate immune tolerance in vivo.

Lu M, Varley AW, Munford RS - PLoS Pathog. (2013)

Aoah−/−Tlr4−/− macrophages can become tolerant in LPS-injected Aoah−/− mice.(A) CD45.2 Aoah−/−Tlr4−/− naïve peritoneal cells were transferred to the peritoneal cavities of CD45.1 Aoah+/+Tlr4+/+ and Aoah−/−Tlr4+/+ mice. After 18 hours, half of the recipient mice in each group were injected i.p. with 1 µg LPS. Fourteen days after injection, the peritoneal cells were harvested. Some of the cells were treated ex vivo with 40 µg/ml Micrococcus luteus plus 2.5 µg/ml poly I:C for 8 hours in the presence of Brefeldin A. IL-6 and TNF production by donor (D) and recipient (R) F4/80+ macrophages was measured using flow cytometry. (B and C) The remainder of the peritoneal cells was not re-stimulated ex vivo and was used to determine surface expression of F4/80 and CD86 on macrophages (F4/80+) by flow cytometry. **, P<0.01; ***, P<0.001. Data were combined from 2 independent experiments. n = 5–7. Aoah−/− Tlr4−/− macrophages, which are unable to respond to LPS, became tolerant to TLR2 and TLR3 ligands (A) and developed the tolerant surface phenotype (B and C) when they were transferred into LPS-primed Aoah−/− mice, supporting a role for non-LPS stimuli in promoting tolerance in vivo. Pink bars, Aoah−/−Tlr4−/− donor macrophages; Blue bars, Aoah+/+ recipient macrophages; red bars, Aoah−/− recipient macrophages; diagonal markings indicate in vivo LPS exposure.
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ppat-1003339-g008: Aoah−/−Tlr4−/− macrophages can become tolerant in LPS-injected Aoah−/− mice.(A) CD45.2 Aoah−/−Tlr4−/− naïve peritoneal cells were transferred to the peritoneal cavities of CD45.1 Aoah+/+Tlr4+/+ and Aoah−/−Tlr4+/+ mice. After 18 hours, half of the recipient mice in each group were injected i.p. with 1 µg LPS. Fourteen days after injection, the peritoneal cells were harvested. Some of the cells were treated ex vivo with 40 µg/ml Micrococcus luteus plus 2.5 µg/ml poly I:C for 8 hours in the presence of Brefeldin A. IL-6 and TNF production by donor (D) and recipient (R) F4/80+ macrophages was measured using flow cytometry. (B and C) The remainder of the peritoneal cells was not re-stimulated ex vivo and was used to determine surface expression of F4/80 and CD86 on macrophages (F4/80+) by flow cytometry. **, P<0.01; ***, P<0.001. Data were combined from 2 independent experiments. n = 5–7. Aoah−/− Tlr4−/− macrophages, which are unable to respond to LPS, became tolerant to TLR2 and TLR3 ligands (A) and developed the tolerant surface phenotype (B and C) when they were transferred into LPS-primed Aoah−/− mice, supporting a role for non-LPS stimuli in promoting tolerance in vivo. Pink bars, Aoah−/−Tlr4−/− donor macrophages; Blue bars, Aoah+/+ recipient macrophages; red bars, Aoah−/− recipient macrophages; diagonal markings indicate in vivo LPS exposure.
Mentions: Bioactive LPS is thus present in the peritoneum of LPS-injected Aoah−/− mice, where it is sufficient to prevent macrophages from regaining responsiveness. Do LPS-induced mediators also play a role in maintaining tolerance? We transferred CD45.2 Aoah−/−Tlr4−/− peritoneal cells to CD45.1 Aoah+/+ or Aoah−/− (both Tlr4+/+) recipient mice, injected LPS i.p., and asked whether the Aoah−/−Tlr4−/− donor macrophages became tolerant in the peritoneum of tolerant Aoah−/− recipient mice. Fourteen days after LPS injection, we harvested recipient peritoneal cells and treated them ex vivo with Micrococcus luteus plus poly I:C (LPS-exposed Aoah−/− macrophages express hetero-tolerance [36] to M. luteus [TLR2 agonist] and poly I:C [TLR3 agonist] [25]). Aoah−/−Tlr4−/− donor macrophages (F4/80+) harvested from LPS-injected Aoah−/− recipients had significantly lower IL-6 and TNF responses to ex vivo re-challenge (Fig. 8A), and they also had less surface F4/80 and CD86 expression (Fig. 8B, C). Since Aoah−/−Tlr4−/− donor macrophages cannot sense LPS, these results suggested that LPS-induced mediators in the peritoneum are also important for prolonging tolerance in Aoah−/− mice. Aoah−/−Tlr4−/− donor cells were less tolerant than were the recipient's Aoah−/−Tlr4+/+ macrophages, again reinforcing the prominent direct role played by fully acylated LPS.

Bottom Line: Mice that are unable to inactivate LPS, in contrast, remain tolerant for several months; during this time they respond sluggishly to Gram-negative bacterial challenge, with high mortality.We show here that prolonged macrophage reprogramming is maintained in vivo by the persistence of stimulatory LPS molecules within the cells' in vivo environment, where naïve cells can acquire LPS via cell-cell contact or from the extracellular fluid.Measures that disable microbial molecules might enhance resolution of tissue inflammation and help restore innate defenses in individuals recovering from many different infectious diseases.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America. Lum3@niaid.nih.gov

ABSTRACT
Measures that bolster the resolution phase of infectious diseases may offer new opportunities for improving outcome. Here we show that inactivation of microbial lipopolysaccharides (LPS) can be required for animals to recover from the innate immune tolerance that follows exposure to Gram-negative bacteria. When wildtype mice are exposed to small parenteral doses of LPS or Gram-negative bacteria, their macrophages become reprogrammed (tolerant) for a few days before they resume normal function. Mice that are unable to inactivate LPS, in contrast, remain tolerant for several months; during this time they respond sluggishly to Gram-negative bacterial challenge, with high mortality. We show here that prolonged macrophage reprogramming is maintained in vivo by the persistence of stimulatory LPS molecules within the cells' in vivo environment, where naïve cells can acquire LPS via cell-cell contact or from the extracellular fluid. The findings provide strong evidence that inactivation of a stimulatory microbial molecule can be required for animals to regain immune homeostasis following parenteral exposure to bacteria. Measures that disable microbial molecules might enhance resolution of tissue inflammation and help restore innate defenses in individuals recovering from many different infectious diseases.

Show MeSH
Related in: MedlinePlus