The extracellular matrix of eggshell displays anti-inflammatory activities through NF-κB in LPS-triggered human immune cells

Abstract

Avian eggshell membrane (ESM) is a natural biomaterial that has been used as an alternative natural bandage on burned and cut skin injuries for >400 years in Asian countries, and is available in large quantities from egg industries. Our aim was to characterize ESM that was separated and processed from egg waste, and to study whether this material possesses anti-inflammatory properties, making it suitable as an ingredient in industrial production of low cost wound healing products. Our results show that the processed ESM particles retain a fibrous structure similar to that observed for the native membrane, and contain collagen, and carbohydrate components such as hyaluronic acid and sulfated glycosaminoglycans, as well as N-glycans, mostly with uncharged structures. Furthermore, both processed ESM powder and the ESM-derived carbohydrate fraction had immunomodulation properties in monocytes and macrophage-like cells. Under inflammatory conditions induced by lipopolysaccharide, the ESM powder and the isolated carbohydrate fraction reduced the activity of the transcription factor nuclear factor-κB. The expression of the immune regulating receptors toll-like receptor 4 and ICAM-1, as well as the cell surface glycoprotein CD44, all important during inflammation response, were down-regulated by these fractions. Interestingly, our experiments show that the two fractions regulated cytokine secretion differently: ESM depressed inflammation by increased secretion of the anti-inflammatory cytokine IL-10 while the carbohydrate fraction reduced secretions of the pro inflammatory cytokines IL-1β and IL-6. Also, the phosphorylation of p65 and p50 subunits of nuclear factor-κB, as well as nuclear localization, differed between processed ESM powder and carbohydrate fraction, suggesting different down-stream regulation during inflammation. In conclusion, processed ESM powder and its soluble carbohydrate components possess anti-inflammatory properties, demonstrating the potential of ESM as a novel biological wound dressing for treatment of chronic inflammatory wounds.

Keywords: NF-κB; THP-1 cells; anti-inflammation; carbohydrates; cytokine secretions; eggshell membrane; hyaluronic acid; signal transduction pathways; wound healing.

Figure 1

Structural characterization of the processed ESM powder. Notes: Scanning electron micrograph of ESM showing fibrillary fibers (A). Collagen contained in ESM powder is determined by Picrosirius red staining and examined with light microscope (Bi) and by using polarized light (Bii). Abbreviation: ESM, eggshell membrane.

Figure 2

The distribution of N-glycan structures for a particular glycan site (QLEELLNR) of the glycoprotein Clusterin as determined by mass spectrometry.

Figure 3

The effect of ESM powder on NF-κB activity and cell viability in LPS-induced inflammatory monocytes. Notes: U937-3xκB-LUC cells were incubated with either 1 μg/ml LPS alone (control) or in combination with 0.5 or 1 mg/mL of ESM in cell culture medium for 6 h. Luciferase activity and cell viability were measured (luminescence). The NF-κB coupled inflammatory response was evaluated by measuring the luciferase activity in these cells (A). Cell viability was measured using Cell-Titer Glo cell viability assay (B). Each bar represents the mean ± SEM of at least three experiments performed in triplicates. (*p<0.05 by paired t-test). Abbreviations: ESM, eggshell membrane; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa B; SEM, standard error mean.

Figure 4

The effect of ESM powder on immune receptors. Notes: PMA-differentiated THP-1 cells were incubated with 0.5 ng/ml LPS alone (control) or in combination with 1 mg/ml ESM for 20 h at 37°C. Cells were harvested and subjected to: (A) RT-PCR analysis for determining the mRNA expressions of TLR4, ICAM1 and CD44. The results are presented as mean ± SEM fold change relative to the control samples (n=3). (**p<0.01 and ****p<0.0001 by paired t-test), (B) Western blotting analysis for assessment of protein levels of TLR4, ICAM1 and CD44. A representative blot out of three independent experiment is presented. Tubulin was used as a loading control. Abbreviations: ESM, eggshell membrane; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; TLR4, toll-like receptor 4; ICAM1, intercellular adhesion protein 1; SEM, standard error mean.

Figure 5

ESM powder up-regulates the pro- and anti-inflammatory cytokines secretions. Notes: PMA-differentiated THP-1 cells were incubated with 0.5 ng/ml LPS alone (control) or in combination with 1 mg/ml ESM for 20 h at 37°C. Cell media were collected and the level of secreted pro inflammatory cytokines IL-1β (A) and IL-6 (B), and the anti-inflammatory cytokine IL-10 (C) were determined by ELISA. The results are presented as mean ± SEM protein level (n=5). (*p<0.05, **p<0.01 or ****p<0.0001 indicate a statistically significant change between control and ESM treated cells, determined by paired t-test). Abbreviations: ELISA, enzyme-linked immunosorbent assay; ESM, eggshell membrane; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; IL-1β, interleukin 1 beta; SEM, standard error mean.

Figure 6

ESM powder increases the expression of HO-1 involved in inducement of macrophage M2 phenotype. Notes: PMA-differentiated THP-1 cells were incubated with 0.5 ng/ml LPS alone (control) or in combination with 1 mg/ml ESM for 6 h and 20 h at 37°C. The cell media were collected and the cells were washed and lysed in RIPA buffer. (A) The protein level of HO-1 in cell lysates were determined by Western blotting and tubulin was used as loading control. A representative blot out of four independent experiments is shown. (B) The level of secreted anti-inflammatory cytokine IL-10 into the media after 6 h treatment were determined by ELISA. The results are presented as mean ± SEM protein level (n=3). Abbreviations: Ctrl, control; ESM, eggshell membrane; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; HO-1, heme oxygenase 1; RIPA, radioimmunoprecipitation assay buffer; SEM, standard error mean.

Figure 7

Effect of ESM derived carbohydrate fraction on NF-κB activity and cell viability in LPS-induced inflammatory monocytes. Notes: U937-3xκB-LUC cells were incubated with either 1 μg/mL LPS alone (control) or in combination with various concentrations of carbohydrate fraction in cell culture medium for 6 h. Luciferase activity and cell viability were measured (luminescence). (A) The NF-κB coupled inflammatory response was evaluated by measuring the luciferase activity in these cells. (B) Cell viability was measured using Cell-Titer Glo cell viability assay. Each bar represents the mean ± SEM of three experiments performed in triplicates. *p<0.05 and ****p<0.0001 indicate a statistically significant change determined by paired t-test. Abbreviations: ESM, eggshell membrane; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa B; SEM, standard error mean.

Figure 8

The effect of ESM derived carbohydrate fraction on immune receptors. Notes: PMA-differentiated THP-1 cells were incubated with 0.5 ng/mL LPS alone (control) or in combination with 0.125 mg/mL carbohydrate fraction for 20 h at 37°C. Cells were harvested and total RNA was isolated before subjected to RT-PCR for determining the mRNA expressions of TLR4, ICAM1 and CD44 (A). The results are presented as mean ± SEM fold change relative to the control samples (n=3). **p<0.01 and ****p<0.0001 indicate a statistically significant change determined by paired t-test. (B) Whole cell lysates were analyzed by Western blotting for assessment of TLR4, ICAM1 and CD44 protein levels. A representative blot out of four independent experiments is presented. Tubulin was used as a loading control. Abbreviations: Carb, carbohydrate; PMA, phorbol 12-myristate 13-acetate; ESM, eggshell membrane; LPS, lipopolysaccharide; TLR4, toll-like receptor 4; SEM, standard error mean.

Figure 9

ESM-derived carbohydrate fraction depressed the secretion of proinflammatory cytokines IL-1β and IL-6. Notes: PMA-differentiated THP-1 cells were incubated with 0.5 ng/mL LPS alone (control) or in combination with 0.125 mg/mL carbohydrate fraction for 20 h at 37°C. Cell media were collected and subjected to ELISA for measuring the level of secreted pro inflammatory cytokines IL-1β (A) and IL-6 (B), and the anti-inflammatory cytokine IL-10 (C). The results are presented as mean ± SEM protein level (n=5). (***p<0.001 and ****p<0.0001 indicate a statistically significant change determined by paired t-test). Abbreviations: ELISA, enzyme-linked immunosorbent assay; PMA, phorbol 12-myristate 13-acetate; ESM, eggshell membrane; LPS, lipopolysaccharide; IL-1β, interleukin 1 beta; SEM, standard error mean.

Figure 10

The activation of p65 and p50 subunits of NF-κB are differently regulated by ESM powder and carbohydrate fraction in macrophages. Notes: PMA-differentiated THP-1 cells were incubated with 0.5 ng/mL LPS alone (control) or in combination with ESM (1 mg/mL) or carbohydrate fraction (0.125 mg/mL) for 20 h at 37°C. (A) The cells were harvested and lysed in RIPA buffer. Equal volumes of whole cell lysates were subjected to Western blotting with antibodies against phospho-p65 and phospho-p50. A representative blot out of four experiments is shown. Tubulin was used as a loading control. (B) Cells were fixed in 4% PFA and then subjected to fluorescent immunostaining with primary antibodies against phospho-p65 or phospho-p50, and Alexa-546 conjugated goat-anti rabbit secondary antibodies (red). The nuclei were counterstained with Hoechst (blue). The boxed areas are presented at higher magnification and showed at the right panels. Scale bars as indicated. Abbreviations: Carb, carbohydrate; PMA, phorbol 12-myristate 13-acetate; ESM, eggshell membrane, LPS, lipopolysaccharide, NF-κB, nuclear factor kappa B; p-p50, phospho-p50; p-p65, phospho-p65; Tub, tubulin; PFA, paraformaldehyde; RIPA, radioimmunoprecipitation assay buffer.