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Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity.

Lee JY, Suh JS, Kim JM, Kim JH, Park HJ, Park YJ, Chung CP - Int J Nanomedicine (2015)

Bottom Line: Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner.Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model.Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to sites, such as the cytoplasm or nucleus, as hBD3-3 has the ability to be used as a carrier, and suggest a potential approach to effectively treat inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Chungcheongbuk-do, Republic of Korea.

ABSTRACT
Human beta-defensins (hBDs) are crucial factors of intrinsic immunity that function in the immunologic response to a variety of invading enveloped viruses, bacteria, and fungi. hBDs can cause membrane depolarization and cell lysis due to their highly cationic nature. These molecules participate in antimicrobial defenses and the control of adaptive and innate immunity in every mammalian species and are produced by various cell types. The C-terminal 15-mer peptide within hBD3, designated as hBD3-3, was selected for study due to its cell- and skin-penetrating activity, which can induce anti-inflammatory activity in lipopolysaccharide-treated RAW 264.7 macrophages. hBD3-3 penetrated both the outer membrane of the cells and mouse skin within a short treatment period. Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner. Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. Further investigation also revealed that hBD3-3 downregulated nuclear factor kappa B-dependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active nuclear factor kappa B p65. Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to sites, such as the cytoplasm or nucleus, as hBD3-3 has the ability to be used as a carrier, and suggest a potential approach to effectively treat inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus

Intracellular translocation of hBD3-3 in vitro.Notes: (A) Cellular localization of rhodamine-labeled peptide fragments in RAW 264.7 cells. Cells (1×104) were incubated for 10 minutes in medium containing the rhodamine-labeled peptides (50 μM) (original magnification 40×). (B) FACS analysis of cells treated with rhodamine-labeled peptides. Cells (1×106) were incubated for 10 minutes and 30 minutes in medium containing the rhodamine-labeled peptides (50 μM).Abbreviations: hBD3, human beta-defensin 3; FACS, fluorescence-activated cell sorting; DAPI, 4′,6-diamidino-2-phenylindole; min, minutes.
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f2-ijn-10-5423: Intracellular translocation of hBD3-3 in vitro.Notes: (A) Cellular localization of rhodamine-labeled peptide fragments in RAW 264.7 cells. Cells (1×104) were incubated for 10 minutes in medium containing the rhodamine-labeled peptides (50 μM) (original magnification 40×). (B) FACS analysis of cells treated with rhodamine-labeled peptides. Cells (1×106) were incubated for 10 minutes and 30 minutes in medium containing the rhodamine-labeled peptides (50 μM).Abbreviations: hBD3, human beta-defensin 3; FACS, fluorescence-activated cell sorting; DAPI, 4′,6-diamidino-2-phenylindole; min, minutes.

Mentions: hBD3 contains 45 amino acids. Three 15-amino-acids peptides (hBD3-1, hBD3-2, hBD3-3) were generated from the hBD3 protein starting at the N-terminus (Figure 1). The hBD3-3 peptide includes eight basic amino acids and features a higher net positive charge than the other peptides. The hBD3-3 sequence shows conserved identities to that of cell-penetrating peptides.16 Peptide charge, as determined by arginine, lysine, and histidine residues, helps to bind negatively charged cell surface molecules, and arginine, in particular, can trigger the cellular uptake through endocytosis. By far, the most commonly known CPP is transactivator of transcription, discovered from human immunodeficiency virus-type 1, which delivers various cargo molecules into cells and whose net charge is also determined by arginine.17,18 We investigated the ability of the three hBD3 peptide fragments to penetrate the outer membrane of cells, as well as mouse skin. The cellular internalization of the three peptides was visualized via confocal microscopy after 10 minutes and 30 minutes of treatment. Figure 2A indicates that cytosolic fluorescence could not be observed for hBD3-1, hBD3-2, or a mismatch peptide of hBD3-3. In contrast, hBD3-3 was significantly internalized by cells, consistent with its positive charge. We quantified peptide internalization via flow cytometry to more accurately evaluate hBD3-3-mediated uptake ability (Figure 2B). The internalization of hBD3-3 was measured based on the mean fluorescent intensity of each sample. The flow cytometry data correlated with confocal microscopy analyses, and the hBD3-3 was detected in the cells both after 10 minutes and 30 minutes of incubation. As shown in Figure 2A and B, the cell-uptake analyses clearly demonstrated that hBD3-3 effectively translocated into cells, including into the cytosol. The potential of hBD3-3 to penetrate the skin of nude mice was monitored via confocal microscopy (Figure 3). Skin loaded with PBS as a mock control exhibited slight autofluorescence. Treatment of the skin with rhodamine-labeled hBD3-1, hBD3-2, or the mismatch peptide resulted in fluorescent staining of the stratum cornea, with only very faint color being observed in the epidermis. However, in skin treated with rhodamine-labeled hBD3-3, we observed strong red fluorescence in the epidermis and weak fluorescence in the dermis (Figure 3). The thickness of the stratum cornea is 20 μm, and that of the epidermis is 100 μm.19,20 The tissue-penetration activity of hBD3-3 suggested the potential for transdermal application rather than injection to overcome the thicknesses of these tissues. These results were consistent with the in vitro cell penetration of hBD3-3.


Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity.

Lee JY, Suh JS, Kim JM, Kim JH, Park HJ, Park YJ, Chung CP - Int J Nanomedicine (2015)

Intracellular translocation of hBD3-3 in vitro.Notes: (A) Cellular localization of rhodamine-labeled peptide fragments in RAW 264.7 cells. Cells (1×104) were incubated for 10 minutes in medium containing the rhodamine-labeled peptides (50 μM) (original magnification 40×). (B) FACS analysis of cells treated with rhodamine-labeled peptides. Cells (1×106) were incubated for 10 minutes and 30 minutes in medium containing the rhodamine-labeled peptides (50 μM).Abbreviations: hBD3, human beta-defensin 3; FACS, fluorescence-activated cell sorting; DAPI, 4′,6-diamidino-2-phenylindole; min, minutes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554392&req=5

f2-ijn-10-5423: Intracellular translocation of hBD3-3 in vitro.Notes: (A) Cellular localization of rhodamine-labeled peptide fragments in RAW 264.7 cells. Cells (1×104) were incubated for 10 minutes in medium containing the rhodamine-labeled peptides (50 μM) (original magnification 40×). (B) FACS analysis of cells treated with rhodamine-labeled peptides. Cells (1×106) were incubated for 10 minutes and 30 minutes in medium containing the rhodamine-labeled peptides (50 μM).Abbreviations: hBD3, human beta-defensin 3; FACS, fluorescence-activated cell sorting; DAPI, 4′,6-diamidino-2-phenylindole; min, minutes.
Mentions: hBD3 contains 45 amino acids. Three 15-amino-acids peptides (hBD3-1, hBD3-2, hBD3-3) were generated from the hBD3 protein starting at the N-terminus (Figure 1). The hBD3-3 peptide includes eight basic amino acids and features a higher net positive charge than the other peptides. The hBD3-3 sequence shows conserved identities to that of cell-penetrating peptides.16 Peptide charge, as determined by arginine, lysine, and histidine residues, helps to bind negatively charged cell surface molecules, and arginine, in particular, can trigger the cellular uptake through endocytosis. By far, the most commonly known CPP is transactivator of transcription, discovered from human immunodeficiency virus-type 1, which delivers various cargo molecules into cells and whose net charge is also determined by arginine.17,18 We investigated the ability of the three hBD3 peptide fragments to penetrate the outer membrane of cells, as well as mouse skin. The cellular internalization of the three peptides was visualized via confocal microscopy after 10 minutes and 30 minutes of treatment. Figure 2A indicates that cytosolic fluorescence could not be observed for hBD3-1, hBD3-2, or a mismatch peptide of hBD3-3. In contrast, hBD3-3 was significantly internalized by cells, consistent with its positive charge. We quantified peptide internalization via flow cytometry to more accurately evaluate hBD3-3-mediated uptake ability (Figure 2B). The internalization of hBD3-3 was measured based on the mean fluorescent intensity of each sample. The flow cytometry data correlated with confocal microscopy analyses, and the hBD3-3 was detected in the cells both after 10 minutes and 30 minutes of incubation. As shown in Figure 2A and B, the cell-uptake analyses clearly demonstrated that hBD3-3 effectively translocated into cells, including into the cytosol. The potential of hBD3-3 to penetrate the skin of nude mice was monitored via confocal microscopy (Figure 3). Skin loaded with PBS as a mock control exhibited slight autofluorescence. Treatment of the skin with rhodamine-labeled hBD3-1, hBD3-2, or the mismatch peptide resulted in fluorescent staining of the stratum cornea, with only very faint color being observed in the epidermis. However, in skin treated with rhodamine-labeled hBD3-3, we observed strong red fluorescence in the epidermis and weak fluorescence in the dermis (Figure 3). The thickness of the stratum cornea is 20 μm, and that of the epidermis is 100 μm.19,20 The tissue-penetration activity of hBD3-3 suggested the potential for transdermal application rather than injection to overcome the thicknesses of these tissues. These results were consistent with the in vitro cell penetration of hBD3-3.

Bottom Line: Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner.Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model.Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to sites, such as the cytoplasm or nucleus, as hBD3-3 has the ability to be used as a carrier, and suggest a potential approach to effectively treat inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Chungcheongbuk-do, Republic of Korea.

ABSTRACT
Human beta-defensins (hBDs) are crucial factors of intrinsic immunity that function in the immunologic response to a variety of invading enveloped viruses, bacteria, and fungi. hBDs can cause membrane depolarization and cell lysis due to their highly cationic nature. These molecules participate in antimicrobial defenses and the control of adaptive and innate immunity in every mammalian species and are produced by various cell types. The C-terminal 15-mer peptide within hBD3, designated as hBD3-3, was selected for study due to its cell- and skin-penetrating activity, which can induce anti-inflammatory activity in lipopolysaccharide-treated RAW 264.7 macrophages. hBD3-3 penetrated both the outer membrane of the cells and mouse skin within a short treatment period. Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner. Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. Further investigation also revealed that hBD3-3 downregulated nuclear factor kappa B-dependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active nuclear factor kappa B p65. Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to sites, such as the cytoplasm or nucleus, as hBD3-3 has the ability to be used as a carrier, and suggest a potential approach to effectively treat inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus