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The anti-inflammatory and antibacterial basis of human omental defense: selective expression of cytokines and antimicrobial peptides.

Chandra A, Srivastava RK, Kashyap MP, Kumar R, Srivastava RN, Pant AB - PLoS ONE (2011)

Bottom Line: However, the underlying mechanisms of these effects are not well understood.The study shows significant higher levels of expression (mRNA and protein) of several specific cytokines, and antibacterial peptides in the omentum tissues when compared to oral sub-mucosal tissues.The altered expressions were more pronounced in cultured adipocytes cells when exposed to LPS as compared to the omentum tissue.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Gastroenterology, Erstwhile KG Medical College, CSM Medical University, Lucknow, India.

ABSTRACT

Background: The wound healing properties of the human omentum are well known and have extensively been exploited clinically. However, the underlying mechanisms of these effects are not well understood. We hypothesize that the omentum tissue promotes wound healing via modulation of anti-inflammatory pathways, and because the omentum is rich in adipocytes, the adipocytes may modulate the anti-inflammatory response. Factors released by human omentum may affect healing, inflammation and immune defense.

Methodology: Six human omentum tissues (non obese, free from malignancy, and any other systemic disorder) were obtained during diagnostic laparoscopies having a negative outcome. Healthy oral mucosa (obtained from routine oral biopsies) was used as control. Cultured adipocytes derived from human omentum were exposed to lipopolysaccharide (LPS) (1-50 ng/mL) for 12-72 hours to identify the non-cytotoxic doses. Levels of expression (mRNA and protein) were carried out for genes associated with pro- and anti-inflammatory cytokine responses and antibacterial/antimicrobial activity using qRT-PCR, western blotting, and cell-based ELISA assays.

Results: The study shows significant higher levels of expression (mRNA and protein) of several specific cytokines, and antibacterial peptides in the omentum tissues when compared to oral sub-mucosal tissues. In the validation studies, primary cultures of adipocytes, derived from human omentum were exposed to LPS (5 and 10 ng/mL) for 24 and 48 h. The altered expressions were more pronounced in cultured adipocytes cells when exposed to LPS as compared to the omentum tissue.

Conclusions/significance: Perhaps, this is the first report that provides evidence of expressional changes in pro- and anti-inflammatory cytokines and antibacterial peptides in the normal human omentum tissue as well as adipocytes cultured from this tissue. The study provides new insights on the molecular and cellular mechanisms of healing and defense by the omentum, and suggests the potential applicability of cultured adipocytes derived from the omentum for future therapeutic applications.

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Related in: MedlinePlus

Protein expression of markers associated with inflammation in human omentum tissue.(a) Alterations in the expression of proteins involved in the induction of inflammatory cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro and anti-inflammatory cytokines (Figure 4 a). Lane (1): Normal oral submucosal tissue; Lane (2): Human omentum tissue. Molecular weight of protein studied: IL-1β (17 kDa), IL-2 (15 kDa) IL-4 (17 kDa), IL-8 (11 kDa), IL-10 (20 kDa), TNF-α (26 kDa) GM-CSF (16 kDa) and β-Actin (42 kDa) for normalization. (b) Relative quantification of alterations in the protein expression of cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro-inflammatory cytokines. β-Actin was used as internal control to normalize the data. Quantification (densitometry) was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase™ FC StandAlone V.4.0 software. *P<0.05- significant, **P<0.01- highly significant.
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pone-0020446-g004: Protein expression of markers associated with inflammation in human omentum tissue.(a) Alterations in the expression of proteins involved in the induction of inflammatory cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro and anti-inflammatory cytokines (Figure 4 a). Lane (1): Normal oral submucosal tissue; Lane (2): Human omentum tissue. Molecular weight of protein studied: IL-1β (17 kDa), IL-2 (15 kDa) IL-4 (17 kDa), IL-8 (11 kDa), IL-10 (20 kDa), TNF-α (26 kDa) GM-CSF (16 kDa) and β-Actin (42 kDa) for normalization. (b) Relative quantification of alterations in the protein expression of cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro-inflammatory cytokines. β-Actin was used as internal control to normalize the data. Quantification (densitometry) was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase™ FC StandAlone V.4.0 software. *P<0.05- significant, **P<0.01- highly significant.

Mentions: We further determined whether these alterations in the level of mRNA expressions of these cytokines are taking place at the level of protein expression. To determine the level of protein expressions of each of these cytokines (IL-1β, IL-2, IL-4, IL-8, IL-10, TNF-α, and GM-CSF), we carried out immunoreactions using specific antibodies. Our results (Figure 4 a & 4 b) showed that except for IL-4 and IL-10, protein expression of all other cytokines tested in omentum tissues when compared to oral sub-mucosal tissues were significantly higher (∼2 fold or more; p≤0.01). These results are having similar trend to those observed for mRNA levels (Figure 2 a); however, the magnitude of induction in the expression is higher. Results of western blot analyses in omentum-derived cultured cells exposed to LPS (5 and 10 ng/ml for 48 h) showed significant alteration in protein expressions for all cytokines tested including IL-2, IL-4 and IL-10 when compared to untreated cells (Figure 5 a–g). However, the increased levels of expressions of these cytokines were significantly higher than those of elevated levels observed in omentum tissues. Together, our results demonstrate that higher intrinsic levels of cytokines observed at the mRNA levels were translated into protein expression.


The anti-inflammatory and antibacterial basis of human omental defense: selective expression of cytokines and antimicrobial peptides.

Chandra A, Srivastava RK, Kashyap MP, Kumar R, Srivastava RN, Pant AB - PLoS ONE (2011)

Protein expression of markers associated with inflammation in human omentum tissue.(a) Alterations in the expression of proteins involved in the induction of inflammatory cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro and anti-inflammatory cytokines (Figure 4 a). Lane (1): Normal oral submucosal tissue; Lane (2): Human omentum tissue. Molecular weight of protein studied: IL-1β (17 kDa), IL-2 (15 kDa) IL-4 (17 kDa), IL-8 (11 kDa), IL-10 (20 kDa), TNF-α (26 kDa) GM-CSF (16 kDa) and β-Actin (42 kDa) for normalization. (b) Relative quantification of alterations in the protein expression of cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro-inflammatory cytokines. β-Actin was used as internal control to normalize the data. Quantification (densitometry) was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase™ FC StandAlone V.4.0 software. *P<0.05- significant, **P<0.01- highly significant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101256&req=5

pone-0020446-g004: Protein expression of markers associated with inflammation in human omentum tissue.(a) Alterations in the expression of proteins involved in the induction of inflammatory cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro and anti-inflammatory cytokines (Figure 4 a). Lane (1): Normal oral submucosal tissue; Lane (2): Human omentum tissue. Molecular weight of protein studied: IL-1β (17 kDa), IL-2 (15 kDa) IL-4 (17 kDa), IL-8 (11 kDa), IL-10 (20 kDa), TNF-α (26 kDa) GM-CSF (16 kDa) and β-Actin (42 kDa) for normalization. (b) Relative quantification of alterations in the protein expression of cytokines in human omentum tissue. Normal oral submucosal tissue was used to compare the changes in protein of pro-inflammatory cytokines. β-Actin was used as internal control to normalize the data. Quantification (densitometry) was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase™ FC StandAlone V.4.0 software. *P<0.05- significant, **P<0.01- highly significant.
Mentions: We further determined whether these alterations in the level of mRNA expressions of these cytokines are taking place at the level of protein expression. To determine the level of protein expressions of each of these cytokines (IL-1β, IL-2, IL-4, IL-8, IL-10, TNF-α, and GM-CSF), we carried out immunoreactions using specific antibodies. Our results (Figure 4 a & 4 b) showed that except for IL-4 and IL-10, protein expression of all other cytokines tested in omentum tissues when compared to oral sub-mucosal tissues were significantly higher (∼2 fold or more; p≤0.01). These results are having similar trend to those observed for mRNA levels (Figure 2 a); however, the magnitude of induction in the expression is higher. Results of western blot analyses in omentum-derived cultured cells exposed to LPS (5 and 10 ng/ml for 48 h) showed significant alteration in protein expressions for all cytokines tested including IL-2, IL-4 and IL-10 when compared to untreated cells (Figure 5 a–g). However, the increased levels of expressions of these cytokines were significantly higher than those of elevated levels observed in omentum tissues. Together, our results demonstrate that higher intrinsic levels of cytokines observed at the mRNA levels were translated into protein expression.

Bottom Line: However, the underlying mechanisms of these effects are not well understood.The study shows significant higher levels of expression (mRNA and protein) of several specific cytokines, and antibacterial peptides in the omentum tissues when compared to oral sub-mucosal tissues.The altered expressions were more pronounced in cultured adipocytes cells when exposed to LPS as compared to the omentum tissue.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Gastroenterology, Erstwhile KG Medical College, CSM Medical University, Lucknow, India.

ABSTRACT

Background: The wound healing properties of the human omentum are well known and have extensively been exploited clinically. However, the underlying mechanisms of these effects are not well understood. We hypothesize that the omentum tissue promotes wound healing via modulation of anti-inflammatory pathways, and because the omentum is rich in adipocytes, the adipocytes may modulate the anti-inflammatory response. Factors released by human omentum may affect healing, inflammation and immune defense.

Methodology: Six human omentum tissues (non obese, free from malignancy, and any other systemic disorder) were obtained during diagnostic laparoscopies having a negative outcome. Healthy oral mucosa (obtained from routine oral biopsies) was used as control. Cultured adipocytes derived from human omentum were exposed to lipopolysaccharide (LPS) (1-50 ng/mL) for 12-72 hours to identify the non-cytotoxic doses. Levels of expression (mRNA and protein) were carried out for genes associated with pro- and anti-inflammatory cytokine responses and antibacterial/antimicrobial activity using qRT-PCR, western blotting, and cell-based ELISA assays.

Results: The study shows significant higher levels of expression (mRNA and protein) of several specific cytokines, and antibacterial peptides in the omentum tissues when compared to oral sub-mucosal tissues. In the validation studies, primary cultures of adipocytes, derived from human omentum were exposed to LPS (5 and 10 ng/mL) for 24 and 48 h. The altered expressions were more pronounced in cultured adipocytes cells when exposed to LPS as compared to the omentum tissue.

Conclusions/significance: Perhaps, this is the first report that provides evidence of expressional changes in pro- and anti-inflammatory cytokines and antibacterial peptides in the normal human omentum tissue as well as adipocytes cultured from this tissue. The study provides new insights on the molecular and cellular mechanisms of healing and defense by the omentum, and suggests the potential applicability of cultured adipocytes derived from the omentum for future therapeutic applications.

Show MeSH
Related in: MedlinePlus