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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

mRNA expression of inflammatory markers in human omentum tissue and cultured omental cells.Altered expression of mRNA of genes involved in inflammation in omentum tissue and compared with human oral tissue (Figure 2 a). Alterations expression of marker genes in omentum derived cultured adipocytes exposed to LPS for 48 h (Figure 2 b). Real Time quantitative PCR (RTq-PCR) was performed in triplicate by 2× Power SYBR Green PCR master mix. β-actin was used as internal control to normalize the data and LPS induced alterations in mRNA expression are expressed in fold change. Normal oral submucosal tissue was used to compare the changes in cytokines. *P<0.05- significant, **P<0.01- highly significant.
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pone-0020446-g002: mRNA expression of inflammatory markers in human omentum tissue and cultured omental cells.Altered expression of mRNA of genes involved in inflammation in omentum tissue and compared with human oral tissue (Figure 2 a). Alterations expression of marker genes in omentum derived cultured adipocytes exposed to LPS for 48 h (Figure 2 b). Real Time quantitative PCR (RTq-PCR) was performed in triplicate by 2× Power SYBR Green PCR master mix. β-actin was used as internal control to normalize the data and LPS induced alterations in mRNA expression are expressed in fold change. Normal oral submucosal tissue was used to compare the changes in cytokines. *P<0.05- significant, **P<0.01- highly significant.

Mentions: To test our hypothesis that human omentum is capable of producing intrinsic anti-inflammatory response, we determined whether the major cytokines are induced in these tissues. Since our cytotoxicity data suggest that lower doses (5 and 10 ng/ml) of LPS do not significantly alter cell viability, we also treated adipocyte cells derived from human omentum tissues with these doses of LPS to determine whether cells are capable of inducing mRNA levels of specific cytokines even at these low doses. Our results are shown in Figure 2. We first compared the mRNA expression levels of inflammatory cytokines (IL-1β, IL-2, IL-4, IL-8, IL-10, TNF-α, and GM-CSF) of human omentum tissues with those of oral sub-mucosal tissues (Figure 2 a). Real Time PCR (qRT-PCR) was used to determine mRNA levels using specific primers (Table 1S) for each cytokine. It is evident from Figure 2 a that except for IL-2, IL-4 and IL-10, the levels of mRNA expression were significantly higher (∼2 fold or more; p≤0.01) for all other cytokines tested in omentum tissues when compared to oral sub-mucosal tissues. These results suggest that omentum tissues possess higher intrinsic level of inflammatory cytokines, which may be responsible for its wound healing properties. We further determined the levels of these cytokines in adipocyte cells derived from human omentum tissues and exposed to low non-toxic doses of LPS. Our results show that even at such low doses, LPS-induced alterations in the mRNA expression of genes associated with inflammatory cytokines were significantly higher. A dose- (5 and 10 ng/ml) dependent expression of these inflammatory cytokines in cells exposed to LPS for 48 h is shown in Figure 2 b. The expression levels were significantly higher for all the cytokines studied at 48 h in comparison to 24 h (data not shown for 24 h).


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)

mRNA expression of inflammatory markers in human omentum tissue and cultured omental cells.Altered expression of mRNA of genes involved in inflammation in omentum tissue and compared with human oral tissue (Figure 2 a). Alterations expression of marker genes in omentum derived cultured adipocytes exposed to LPS for 48 h (Figure 2 b). Real Time quantitative PCR (RTq-PCR) was performed in triplicate by 2× Power SYBR Green PCR master mix. β-actin was used as internal control to normalize the data and LPS induced alterations in mRNA expression are expressed in fold change. Normal oral submucosal tissue was used to compare the changes in cytokines. *P<0.05- significant, **P<0.01- highly significant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101256&req=5

pone-0020446-g002: mRNA expression of inflammatory markers in human omentum tissue and cultured omental cells.Altered expression of mRNA of genes involved in inflammation in omentum tissue and compared with human oral tissue (Figure 2 a). Alterations expression of marker genes in omentum derived cultured adipocytes exposed to LPS for 48 h (Figure 2 b). Real Time quantitative PCR (RTq-PCR) was performed in triplicate by 2× Power SYBR Green PCR master mix. β-actin was used as internal control to normalize the data and LPS induced alterations in mRNA expression are expressed in fold change. Normal oral submucosal tissue was used to compare the changes in cytokines. *P<0.05- significant, **P<0.01- highly significant.
Mentions: To test our hypothesis that human omentum is capable of producing intrinsic anti-inflammatory response, we determined whether the major cytokines are induced in these tissues. Since our cytotoxicity data suggest that lower doses (5 and 10 ng/ml) of LPS do not significantly alter cell viability, we also treated adipocyte cells derived from human omentum tissues with these doses of LPS to determine whether cells are capable of inducing mRNA levels of specific cytokines even at these low doses. Our results are shown in Figure 2. We first compared the mRNA expression levels of inflammatory cytokines (IL-1β, IL-2, IL-4, IL-8, IL-10, TNF-α, and GM-CSF) of human omentum tissues with those of oral sub-mucosal tissues (Figure 2 a). Real Time PCR (qRT-PCR) was used to determine mRNA levels using specific primers (Table 1S) for each cytokine. It is evident from Figure 2 a that except for IL-2, IL-4 and IL-10, the levels of mRNA expression were significantly higher (∼2 fold or more; p≤0.01) for all other cytokines tested in omentum tissues when compared to oral sub-mucosal tissues. These results suggest that omentum tissues possess higher intrinsic level of inflammatory cytokines, which may be responsible for its wound healing properties. We further determined the levels of these cytokines in adipocyte cells derived from human omentum tissues and exposed to low non-toxic doses of LPS. Our results show that even at such low doses, LPS-induced alterations in the mRNA expression of genes associated with inflammatory cytokines were significantly higher. A dose- (5 and 10 ng/ml) dependent expression of these inflammatory cytokines in cells exposed to LPS for 48 h is shown in Figure 2 b. The expression levels were significantly higher for all the cytokines studied at 48 h in comparison to 24 h (data not shown for 24 h).

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