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Stress-Derived Corticotropin Releasing Factor Breaches Epithelial Endotoxin Tolerance.

Yu Y, Liu ZQ, Liu XY, Yang L, Geng XR, Yang G, Liu ZG, Zheng PY, Yang PC - PLoS ONE (2013)

Bottom Line: The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP).The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells.Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, the Second Hospital, Zhengzhou University, Zhengzhou, China.

ABSTRACT

Background and aims: Loss of the endotoxin tolerance of intestinal epithelium contributes to a number of intestinal diseases. The etiology is not clear. Psychological stress is proposed to compromise the intestinal barrier function. The present study aims to elucidate the role of the stress-derived corticotropin releasing factor (CRF) in breaching the established intestinal epithelial endotoxin tolerance.

Methods: Epithelial cells of HT-29, T84 and MDCK were exposed to lipopolysaccharide to induce the endotoxin tolerance; the cells were then stimulated with CRF. The epithelial barrier function was determined using as indicators of the endotoxin tolerant status. A water-avoid stress mouse model was employed to test the role of CRF in breaching the established endotoxin tolerance in the intestine.

Results: The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP). The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells. Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP. After treating mice with the 10-day chronic stress, the intestinal epithelial barrier function was markedly compromised, which could be prevented by blocking either CRF, or TLR4, or Cldn2.

Conclusions: Psychological stress-derived CRF can breach the established endotoxin tolerance in the intestinal mucosa.

No MeSH data available.


Related in: MedlinePlus

Exposure to CRF breaches the established endotoxin tolerance.Epithelial cells were cultured into monolayers, treated with or without LPS (100 ng/ml) overnight; washed with pre-warmed fresh culture medium; then, CRF (A and B) or LPS (C and D) was added to the culture at the indicated doses and cultured for 48 h. The TER was recorded at the 46th h later; the HRP flux was carried out between the 46th h to 48th h. A and C, the bars indicate the values of TER recorded from the monolayers. B and D, the bars indicate the contents of HRP (detected in basal chambers of transwells that passed through the monolayers from apical chambers). The annotations on X axis indicate the treatment of each group. The numbers on the X axis indicate the amounts (ng/ml) of CRF (A, C) or LPS (C, D) added to the culture medium. Naïve: Naïve confluent monolayers. Tolerant: The epithelial monolayers had been treated with LPS overnight. R2anta: The monolayers were pretreated with CRF R2 antagonist, astressin2B at a dose of 300 nM. TLR4shRNA: TLR4-deficient monolayers (by shRNA of TLR4). csh: The monolayers were treated with control shRNA. Cldn2shRNA: Cldn2-deficient monolayers (treated by shRNA of Cldn2). The data represent 3 separate experiments.
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pone-0065760-g002: Exposure to CRF breaches the established endotoxin tolerance.Epithelial cells were cultured into monolayers, treated with or without LPS (100 ng/ml) overnight; washed with pre-warmed fresh culture medium; then, CRF (A and B) or LPS (C and D) was added to the culture at the indicated doses and cultured for 48 h. The TER was recorded at the 46th h later; the HRP flux was carried out between the 46th h to 48th h. A and C, the bars indicate the values of TER recorded from the monolayers. B and D, the bars indicate the contents of HRP (detected in basal chambers of transwells that passed through the monolayers from apical chambers). The annotations on X axis indicate the treatment of each group. The numbers on the X axis indicate the amounts (ng/ml) of CRF (A, C) or LPS (C, D) added to the culture medium. Naïve: Naïve confluent monolayers. Tolerant: The epithelial monolayers had been treated with LPS overnight. R2anta: The monolayers were pretreated with CRF R2 antagonist, astressin2B at a dose of 300 nM. TLR4shRNA: TLR4-deficient monolayers (by shRNA of TLR4). csh: The monolayers were treated with control shRNA. Cldn2shRNA: Cldn2-deficient monolayers (treated by shRNA of Cldn2). The data represent 3 separate experiments.

Mentions: Intestinal epithelial cells naturally developed tolerance to the commensal microbes in the intestinal tract. Such tolerance may be breached under pathological circumstances, such as in the intestine of patients with inflammatory bowel disease (IBD) [25]. It is observed that exposure to microbial products, such as LPS, is associated with intestinal chronic inflammation [26]; LPS is the ligand of TLR4; we postulated that the increase in TLR4 expression in the intestinal epithelial cells might exaggerate the responses of intestinal epithelial cells to the stimulus of LPS and thus breached the established endotoxin tolerance. To test the inference, following published procedures [27], we firstly treated the epithelial cell monolayers (including HT-29, T84 and MDCK) with LPS (100 ng/ml, overnight) to establish the endotoxin tolerance (the LPS dose “0” group of Fig. 2). The tolerant epithelial monolayers were re-exposed to LPS in the presence or absence of CRF in the culture for 48 h. The results showed that the endotoxin tolerance of the epithelial monolayer was breached by the presence of CRF as shown by the increase in the permeability to a protein tracer, HRP, and the decrease in TER. To confirm the role of CRF in the breaching the established endotoxin tolerance, some epithelial monolayers were pretreated with CRF-R2 antagonist, astressin2B; the results showed that the epithelial barrier function was not affected by the exposure to LPS. Similar results were observed in TLR4-deficient epithelial cell monolayers (Fig.S1A) after stimulated by CRF (Fig. 2). The results indicate that the established endotoxin tolerance in epithelial cells can be breached by the presence of CRF via increasing the expression of TLR4 in epithelial cells. In other words, the silencing TLR4 gene can “avoid” the decrease in TER induced by CRF. With the barrier function as indicators, we also checked the response to LPS-stimulation in the tolerant epithelial cells. The results showed that the barrier function was not significantly altered in the tolerant epithelial monolayers, but markedly compromised in naïve epithelial monolayers (Fig. 2C–D).


Stress-Derived Corticotropin Releasing Factor Breaches Epithelial Endotoxin Tolerance.

Yu Y, Liu ZQ, Liu XY, Yang L, Geng XR, Yang G, Liu ZG, Zheng PY, Yang PC - PLoS ONE (2013)

Exposure to CRF breaches the established endotoxin tolerance.Epithelial cells were cultured into monolayers, treated with or without LPS (100 ng/ml) overnight; washed with pre-warmed fresh culture medium; then, CRF (A and B) or LPS (C and D) was added to the culture at the indicated doses and cultured for 48 h. The TER was recorded at the 46th h later; the HRP flux was carried out between the 46th h to 48th h. A and C, the bars indicate the values of TER recorded from the monolayers. B and D, the bars indicate the contents of HRP (detected in basal chambers of transwells that passed through the monolayers from apical chambers). The annotations on X axis indicate the treatment of each group. The numbers on the X axis indicate the amounts (ng/ml) of CRF (A, C) or LPS (C, D) added to the culture medium. Naïve: Naïve confluent monolayers. Tolerant: The epithelial monolayers had been treated with LPS overnight. R2anta: The monolayers were pretreated with CRF R2 antagonist, astressin2B at a dose of 300 nM. TLR4shRNA: TLR4-deficient monolayers (by shRNA of TLR4). csh: The monolayers were treated with control shRNA. Cldn2shRNA: Cldn2-deficient monolayers (treated by shRNA of Cldn2). The data represent 3 separate experiments.
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pone-0065760-g002: Exposure to CRF breaches the established endotoxin tolerance.Epithelial cells were cultured into monolayers, treated with or without LPS (100 ng/ml) overnight; washed with pre-warmed fresh culture medium; then, CRF (A and B) or LPS (C and D) was added to the culture at the indicated doses and cultured for 48 h. The TER was recorded at the 46th h later; the HRP flux was carried out between the 46th h to 48th h. A and C, the bars indicate the values of TER recorded from the monolayers. B and D, the bars indicate the contents of HRP (detected in basal chambers of transwells that passed through the monolayers from apical chambers). The annotations on X axis indicate the treatment of each group. The numbers on the X axis indicate the amounts (ng/ml) of CRF (A, C) or LPS (C, D) added to the culture medium. Naïve: Naïve confluent monolayers. Tolerant: The epithelial monolayers had been treated with LPS overnight. R2anta: The monolayers were pretreated with CRF R2 antagonist, astressin2B at a dose of 300 nM. TLR4shRNA: TLR4-deficient monolayers (by shRNA of TLR4). csh: The monolayers were treated with control shRNA. Cldn2shRNA: Cldn2-deficient monolayers (treated by shRNA of Cldn2). The data represent 3 separate experiments.
Mentions: Intestinal epithelial cells naturally developed tolerance to the commensal microbes in the intestinal tract. Such tolerance may be breached under pathological circumstances, such as in the intestine of patients with inflammatory bowel disease (IBD) [25]. It is observed that exposure to microbial products, such as LPS, is associated with intestinal chronic inflammation [26]; LPS is the ligand of TLR4; we postulated that the increase in TLR4 expression in the intestinal epithelial cells might exaggerate the responses of intestinal epithelial cells to the stimulus of LPS and thus breached the established endotoxin tolerance. To test the inference, following published procedures [27], we firstly treated the epithelial cell monolayers (including HT-29, T84 and MDCK) with LPS (100 ng/ml, overnight) to establish the endotoxin tolerance (the LPS dose “0” group of Fig. 2). The tolerant epithelial monolayers were re-exposed to LPS in the presence or absence of CRF in the culture for 48 h. The results showed that the endotoxin tolerance of the epithelial monolayer was breached by the presence of CRF as shown by the increase in the permeability to a protein tracer, HRP, and the decrease in TER. To confirm the role of CRF in the breaching the established endotoxin tolerance, some epithelial monolayers were pretreated with CRF-R2 antagonist, astressin2B; the results showed that the epithelial barrier function was not affected by the exposure to LPS. Similar results were observed in TLR4-deficient epithelial cell monolayers (Fig.S1A) after stimulated by CRF (Fig. 2). The results indicate that the established endotoxin tolerance in epithelial cells can be breached by the presence of CRF via increasing the expression of TLR4 in epithelial cells. In other words, the silencing TLR4 gene can “avoid” the decrease in TER induced by CRF. With the barrier function as indicators, we also checked the response to LPS-stimulation in the tolerant epithelial cells. The results showed that the barrier function was not significantly altered in the tolerant epithelial monolayers, but markedly compromised in naïve epithelial monolayers (Fig. 2C–D).

Bottom Line: The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP).The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells.Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, the Second Hospital, Zhengzhou University, Zhengzhou, China.

ABSTRACT

Background and aims: Loss of the endotoxin tolerance of intestinal epithelium contributes to a number of intestinal diseases. The etiology is not clear. Psychological stress is proposed to compromise the intestinal barrier function. The present study aims to elucidate the role of the stress-derived corticotropin releasing factor (CRF) in breaching the established intestinal epithelial endotoxin tolerance.

Methods: Epithelial cells of HT-29, T84 and MDCK were exposed to lipopolysaccharide to induce the endotoxin tolerance; the cells were then stimulated with CRF. The epithelial barrier function was determined using as indicators of the endotoxin tolerant status. A water-avoid stress mouse model was employed to test the role of CRF in breaching the established endotoxin tolerance in the intestine.

Results: The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP). The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells. Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP. After treating mice with the 10-day chronic stress, the intestinal epithelial barrier function was markedly compromised, which could be prevented by blocking either CRF, or TLR4, or Cldn2.

Conclusions: Psychological stress-derived CRF can breach the established endotoxin tolerance in the intestinal mucosa.

No MeSH data available.


Related in: MedlinePlus