The Human Antimicrobial Peptide LL-37 Transfers Extracellular DNA Plasmid to the Nuclear Compartment of Mammalian Cells via Lipid Rafts and Proteoglycan-dependent Endocytosis

Abstract

Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37·DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans. Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37·DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans. Several in vitro and in vivo studies have provided compelling evidence for the importance of antimicrobial peptides in the innate host defense of multicellular organisms against microbial intruders (1Boman H.G. Scand. J. Immunol. 1998; 48: 15-25Crossref Immunol. Antimicrobial peptides were from and in organisms in which H.G. Immunol. J. Immunol. antimicrobial peptides is the to where of and acids are in of the of bacterial cells to peptide to the of in the membrane as to that of and cells, which of J. and the are the of antimicrobial peptides in of a and a antimicrobial J. LL-37, a peptide with is the known human antimicrobial of LL-37, and are in cells but are in and epithelial cells of a of in host defense J. H.G. J. J. J. J. 1998; LL-37 in and the of is J. Immunol. expression and of LL-37 are or both in and in epithelial cells at J. J. J. the LL-37 in from with the of J. Immunol. of as as is sensitive to the cytotoxic activity of LL-37 at peptide concentrations activity is by in or which to the that LL-37 in J. J. 1998; LL-37 activity was in the of from a associated with extracellular and of LL-37 bacterial to in J. to bacterial cell LL-37 to bacterial and to effects on which by which LL-37 microbial J. J. J. J. J. J. J. a by J. H.G. it was shown that in bacterial infections with expression of LL-37 and is or which the associated with the that plasmid from by the of LL-37, was a of antimicrobial peptide of bacterial with of LL-37, whereas bacterial plasmid the the of the antimicrobial peptide LL-37 to with extracellular DNA and to uptake and nuclear transfer of in mammalian cells. of membrane J. and cell surface cholera toxin flow serum human cholera toxin flow serum human in LL-37-mediated DNA is was by peptide was by and reporter gene was provided by and the was from DNA was with to the provided by the was from and with and cholera toxin were from were the as J. was as by J. was from and were from were from cells and human were from the cells were in with and in a and cells were in with the as DNA plasmid was in with serum in the or in the of LL-37 at for the was by the of by and to the were by with and a from mammalian cell and the was as cells were in at and to for in by a of in was and cells were for at in with or LL-37 at the was from the of to the cells as in a at bacterial cells provided by in were in with or concentrations of LL-37 for at were and in and on of were DNA plasmid or were with LL-37 at concentrations in for at was to cell in and the were to for of at in at of the and with the cells were with by with to extracellular the cells were in and for fluorescence in a cell by were on at in of for LL-37·DNA plasmid DNA and LL-37 were by and for at was to cell the of the at the was and with of of for the cells were with and in of cell lysis and for at expression was in of the cell a was by at the of membrane in LL-37·DNA complex cells were with or for at cells were with DNA or and LL-37 in the of the at the which and were as no or in cell as a of were were at in and to and were with and LL-37 to the was with and LL-37 for cells were from extracellular by with in by with cells were in in for the were with and and cells were for or in for and with serum in cells were for with for with in and for with in were with and and cells were a and a microscopy were in the activity and is the from at the were the DNA to the of the of LL-37, DNA plasmid was by serum whereas the of LL-37 efficiently protected plasmid DNA against serum nuclease activity complex DNA and the peptide LL-37 in LL-37·DNA complex efficient of DNA to mammalian cells, as shown for peptides, and J. the of LL-37 on the uptake of plasmid DNA in cells was the LL-37 DNA plasmid uptake to results were with cells, human and cells of LL-37 was for as uptake of extracellular was by LL-37 to that LL-37 binds to DNA that in the of DNA from serum nuclease and transfer of DNA the the that DNA uptake in the of LL-37 was a of membrane and cell with of the cell in were of the cell was at LL-37 concentrations to whereas as were efficiently killed by the LL-37 in uptake and efficient expression of plasmid DNA in mammalian cells. DNA or at was with LL-37 and and with cells for at of the the cells were with by with to extracellular cells were in and DNA and were by are as B, plasmid and the concentrations of LL-37 were for at was to cell for at of for in activity was as are as of LL-37 on the of and mammalian of in a the of a reporter gene expression was to the of internalized LL-37·DNA shown in LL-37 peptide the expression of reporter plasmid in cells to that LL-37 nuclear transfer of DNA plasmid in mammalian cells with no cytotoxic was by fluorescence microscopy studies that nuclear of internalized DNA in the of LL-37 of fluorescence was to at the cell membrane and in the internalized DNA a in the with release from endocytotic and observed to to the nuclear compartment the of a DNA was observed in and LL-37-mediated DNA uptake was and temperature-dependent DNA uptake at results which is with endocytotic uptake targets plasmid DNA to the nuclear cells to were with and LL-37 for and cells were for DNA by as in are as B, cells were on and to for a of by with and LL-37 for and cells were for fluorescence microscopy as and to the of LL-37·DNA complex cells were with or both of which represent cholesterol-depleting agents for the of in J. J. and membrane as associated with caveolae, which are in of endocytosis J. shown in in of LL-37-mediated of both DNA and the of on the expression of internalized reporter gene plasmid was and reporter gene activity in the of LL-37 and with and both reporter gene expression of DNA plasmid and with and the that of by LL-37·DNA complex uptake expression of internalized DNA results were with known of that the expression of DNA plasmid it that effects were a of We the by J. to the of on LL-37-mediated DNA the concentrations of in no was observed gene transfer membrane cells were or with or for at by with LL-37 and or for agents were the of the cells were for DNA or by B, cells were with or for as by the B, by of with plasmid and LL-37 as in the of the cholesterol-depleting were with and the cells were in the of cholesterol-depleting for and for are as cells were with and LL-37 for cells were stained for and for fluorescence microscopy as cells were with and LL-37 for in the of and for fluorescence microscopy as fluorescence studies were to co-localization of internalized DNA with a marker of J. shown in was no co-localization and or the was of the of cell or or the in the of to microscopy co-localization of internalized DNA with in J. is a marker for lipid rafts it with which is from membrane a of found in the extracellular at the cell surface and in and are known to in the uptake of and the of we the of cell surface in LL-37·DNA complex cell in and we were to demonstrate a of expression in LL-37-mediated DNA was for as complex uptake reporter gene expression was in cells with LL-37·DNA as with cells gene transfer cell surface proteoglycans. cells or cells were with or in the of LL-37 for and by B, and cells were with plasmid in the or of LL-37 for by of and for are as we that concentrations of a human antimicrobial in and epithelial cells, plasmid DNA against serum nuclease and efficiently targets DNA to the nuclear compartment of mammalian cells. Furthermore, that LL-37·DNA mammalian cells endocytosis that lipid raft as as cell surface is the to evidence for of both rafts and in gene Moreover, important to the on the of of and peptides or they temperature-dependent endocytotic in of endocytosis peptides are to DNA or on cell surface and endocytosis for efficient which a for by and J. as shown that and DNA a J. and peptides have as as to LL-37, which efficiently bacterial the of LL-37 was shown to the expression of a cell surface in and studies in the effects of LL-37 on Furthermore, it is of to a membrane is in gene was that antimicrobial peptides with gene transfer activity have potential in the of as they bacterial and the gene of J. the that LL-37 and antimicrobial peptides the release of plasmid DNA from by of bacterial DNA from in the extracellular and the transfer of bacterial DNA to the nuclei of mammalian host cells for a the and in antimicrobial peptides and as the in the the transfer of bacterial to cells it that are with of LL-37 peptide and that of is in the epithelial of the J. on the of and that were in the human but were in the was that of to bacterial to gene the of in which gene in the the against gene transfer J. J. J. J. a for bacterial to gene targets bacterial DNA to the nuclear compartment of mammalian cells membrane raft with antimicrobial activity are from mammalian host cells and LL-37 peptides release bacterial DNA lysis of the by to and of bacterial DNA from by in the extracellular and the transfer of bacterial DNA to the nuclei of mammalian host cells in a process that rafts and cell surface and Several in vitro and in vivo studies have provided compelling evidence for the importance of antimicrobial peptides in the innate host defense of multicellular organisms against microbial intruders (1Boman H.G. Scand. J. Immunol. 1998; 48: 15-25Crossref Immunol. Antimicrobial peptides were from and in organisms in which H.G. Immunol. J. Immunol. antimicrobial peptides is the to where of and acids are in of the of bacterial cells to peptide to the of in the membrane as to that of and cells, which of J. and the are the of antimicrobial peptides in of a and a antimicrobial J. LL-37, a peptide with is the known human antimicrobial of LL-37, and are in cells but are in and epithelial cells of a of in host defense J. H.G. J. J. J. J. 1998; LL-37 in and the of is J. Immunol. expression and of LL-37 are or both in and in epithelial cells at J. J. J. the LL-37 in from with the of J. Immunol. of as as is sensitive to the cytotoxic activity of LL-37 at peptide concentrations activity is by in or which to the that LL-37 in J. J. 1998; LL-37 activity was in the of from a associated with extracellular and of LL-37 bacterial to in J. to bacterial cell LL-37 to bacterial and to effects on which by which LL-37 microbial J. J. J. J. J. J. J. a by J. H.G. it was shown that in bacterial infections with expression of LL-37 and is or which the associated with the that plasmid from by the of LL-37, was a of antimicrobial peptide of bacterial with of LL-37, whereas bacterial plasmid the the of the antimicrobial peptide LL-37 to with extracellular DNA and to uptake and nuclear transfer of in mammalian cells. of membrane J. and cell surface cholera toxin flow serum human cholera toxin flow serum human in LL-37-mediated DNA is was by peptide was by and reporter gene was provided by and the was from DNA was with to the provided by the was from and with and cholera toxin were from were the as J. was as by J. was from and were from were from cells and human were from the cells were in with and in a and cells were in with the as DNA plasmid was in with serum in the or in the of LL-37 at for the was by the of by and to the were by with and a from mammalian cell and the was as cells were in at and to for in by a of in was and cells were for at in with or LL-37 at the was from the of to the cells as in a at bacterial cells provided by in were in with or concentrations of LL-37 for at were and in and on of were DNA plasmid or were with LL-37 at concentrations in for at was to cell in and the were to for of at in at of the and with the cells were with by with to extracellular the cells were in and for fluorescence in a cell by were on at in of for LL-37·DNA plasmid DNA and LL-37 were by and for at was to cell the of the at the was and with of of for the cells were with and in of cell lysis and for at expression was in of the cell a was by at the of membrane in LL-37·DNA complex cells were with or for at cells were with DNA or and LL-37 in the of the at the which and were as no or in cell as a of were were at in and to and were with and LL-37 to the was with and LL-37 for cells were from extracellular by with in by with cells were in in for the were with and and cells were for or in for and with serum in cells were for with for with in and for with in were with and and cells were a and a microscopy were in the activity and is the from at the were the was by peptide was by and reporter gene was provided by and the was from DNA was with to the provided by the was from and with and cholera toxin were from were the as J. was as by J. was from and were from were from cells and human were from the cells were in with and in a and cells were in with the as DNA plasmid was in with serum in the or in the of LL-37 at for the was by the of by and to the were by with and a from mammalian cell and the was as cells were in at and to for in by a of in was and cells were for at in with or LL-37 at the was from the of to the cells as in a at bacterial cells provided by in were in with or concentrations of LL-37 for at were and in and on of were DNA plasmid or were with LL-37 at concentrations in for at was to cell in and the were to for of at in at of the and with the cells were with by with to extracellular the cells were in and for fluorescence in a cell by were on at in of for LL-37·DNA plasmid DNA and LL-37 were by and for at was to cell the of the at the was and with of of for the cells were with and in of cell lysis and for at expression was in of the cell a was by at the of membrane in LL-37·DNA complex cells were with or for at cells were with DNA or and LL-37 in the of the at the which and were as no or in cell as a of were were at in and to and were with and LL-37 to the was with and LL-37 for cells were from extracellular by with in by with cells were in in for the were with and and cells were for or in for and with serum in cells were for with for with in and for with in were with and and cells were a and a microscopy were in the activity and is the from at the were the DNA to the of the of LL-37, DNA plasmid was by serum whereas the of LL-37 efficiently protected plasmid DNA against serum nuclease activity complex DNA and the peptide LL-37 in LL-37·DNA complex efficient of DNA to mammalian cells, as shown for peptides, and J. the of LL-37 on the uptake of plasmid DNA in cells was the LL-37 DNA plasmid uptake to results were with cells, human and cells of LL-37 was for as uptake of extracellular was by LL-37 to that LL-37 binds to DNA that in the of DNA from serum nuclease and transfer of DNA the the that DNA uptake in the of LL-37 was a of membrane and cell with of the cell in were of the cell was at LL-37 concentrations to whereas as were efficiently killed by the LL-37 in of LL-37 on the of and mammalian of in a the of a reporter gene expression was to the of internalized LL-37·DNA shown in LL-37 peptide the expression of reporter plasmid in cells to that LL-37 nuclear transfer of DNA plasmid in mammalian cells with no cytotoxic was by fluorescence microscopy studies that nuclear of internalized DNA in the of LL-37 of fluorescence was to at the cell membrane and in the internalized DNA a in the with release from endocytotic and observed to to the nuclear compartment the of a DNA was observed in and LL-37-mediated DNA uptake was and temperature-dependent DNA uptake at results which is with endocytotic uptake targets plasmid DNA to the nuclear cells to were with and LL-37 for and cells were for DNA by as in are as B, cells were on and to for a of by with and LL-37 for and cells were for fluorescence microscopy as and to the of LL-37·DNA complex cells were with or both of which represent cholesterol-depleting agents for the of in J. J. and membrane as associated with caveolae, which are in of endocytosis J. shown in in of LL-37-mediated of both DNA and the of on the expression of internalized reporter gene plasmid was and reporter gene activity in the of LL-37 and with and both reporter gene expression of DNA plasmid and with and the that of by LL-37·DNA complex uptake expression of internalized DNA results were with known of that the expression of DNA plasmid it that effects were a of We the by J. to the of on LL-37-mediated DNA the concentrations of in no was observed gene transfer membrane cells were or with or for at by with LL-37 and or for agents were the of the cells were for DNA or by B, cells were with or for as by the B, by of with plasmid and LL-37 as in the of the cholesterol-depleting were with and the cells were in the of cholesterol-depleting for and for are as cells were with and LL-37 for cells were stained for and for fluorescence microscopy as cells were with and LL-37 for in the of and for fluorescence microscopy as fluorescence studies were to co-localization of internalized DNA with a marker of J. shown in was no co-localization and or the was of the of cell or or the in the of to microscopy co-localization of internalized DNA with in J. is a marker for lipid rafts it with which is from membrane a of found in the extracellular at the cell surface and in and are known to in the uptake of and the of we the of cell surface in LL-37·DNA complex cell in and we were to demonstrate a of expression in LL-37-mediated DNA was for as complex uptake reporter gene expression was in cells with LL-37·DNA as with cells gene transfer cell surface proteoglycans. cells or cells were with or in the of LL-37 for and by B, and cells were with plasmid in the or of LL-37 for by of and for are as LL-37 DNA to the of the of LL-37, DNA plasmid was by serum whereas the of LL-37 efficiently protected plasmid DNA against serum nuclease activity complex DNA and the peptide LL-37 in LL-37·DNA complex efficient of DNA to mammalian cells, as shown for peptides, and J. the of LL-37 on the uptake of plasmid DNA in cells was the LL-37 DNA plasmid uptake to results were with cells, human and cells of LL-37 was for as uptake of extracellular was by LL-37 to that LL-37 binds to DNA that in the of DNA from serum nuclease and transfer of DNA the the that DNA uptake in the of LL-37 was a of membrane and cell with of the cell in were of the cell was at LL-37 concentrations to whereas as were efficiently killed by the LL-37 in the of a reporter gene expression was to the of internalized LL-37·DNA shown in LL-37 peptide the expression of reporter plasmid in cells to that LL-37 nuclear transfer of DNA plasmid in mammalian cells with no cytotoxic was by fluorescence microscopy studies that nuclear of internalized DNA in the of LL-37 of fluorescence was to at the cell membrane and in the internalized DNA a in the with release from endocytotic and observed to to the nuclear compartment the of a DNA was observed in and LL-37-mediated DNA uptake was and temperature-dependent DNA uptake at results which is with endocytotic uptake LL-37-mediated and to the of LL-37·DNA complex cells were with or both of which represent cholesterol-depleting agents for the of in J. J. and membrane as associated with caveolae, which are in of endocytosis J. shown in in of LL-37-mediated of both DNA and the of on the expression of internalized reporter gene plasmid was and reporter gene activity in the of LL-37 and with and both reporter gene expression of DNA plasmid and with and the that of by LL-37·DNA complex uptake expression of internalized DNA results were with known of that the expression of DNA plasmid it that effects were a of We the by J. to the of on LL-37-mediated DNA the concentrations of in no was observed Confocal fluorescence studies were to co-localization of internalized DNA with a marker of J. shown in was no co-localization and or the was of the of cell or or the in the of to microscopy co-localization of internalized DNA with in J. is a marker for lipid rafts it with which is from membrane a of found in the extracellular at the cell surface and in and are known to in the uptake of and the of we the of cell surface in LL-37·DNA complex cell in and we were to demonstrate a of expression in LL-37-mediated DNA was for as complex uptake reporter gene expression was in cells with LL-37·DNA as with cells we that concentrations of a human antimicrobial in and epithelial cells, plasmid DNA against serum nuclease and efficiently targets DNA to the nuclear compartment of mammalian cells. Furthermore, that LL-37·DNA mammalian cells endocytosis that lipid raft as as cell surface is the to evidence for of both rafts and in gene Moreover, important to the on the of of and peptides or they temperature-dependent endocytotic in of endocytosis peptides are to DNA or on cell surface and endocytosis for efficient which a for by and J. as shown that and DNA a J. and peptides have as as to LL-37, which efficiently bacterial the of LL-37 was shown to the expression of a cell surface in and studies in the effects of LL-37 on Furthermore, it is of to a membrane is in gene was that antimicrobial peptides with gene transfer activity have potential in the of as they bacterial and the gene of J. the that LL-37 and antimicrobial peptides the release of plasmid DNA from by of bacterial DNA from in the extracellular and the transfer of bacterial DNA to the nuclei of mammalian host cells for a the and in antimicrobial peptides and as the in the the transfer of bacterial to cells it that are with of LL-37 peptide and that of is in the epithelial of the J. on the of and that were in the human but were in the was that of to bacterial to gene the of in which gene in the the against gene transfer J. J. J. J. a for bacterial to gene Here, we that concentrations of a human antimicrobial in and epithelial cells, plasmid DNA against serum nuclease and efficiently targets DNA to the nuclear compartment of mammalian cells. Furthermore, that LL-37·DNA mammalian cells endocytosis that lipid raft as as cell surface is the to evidence for of both rafts and in gene Moreover, important to the on the of of and peptides or they temperature-dependent endocytotic in of endocytosis peptides are to DNA or on cell surface and endocytosis for efficient which a for by and J. as shown that and DNA a J. and peptides have as as to LL-37, which efficiently bacterial the of LL-37 was shown to the expression of a cell surface in and studies in the effects of LL-37 on Furthermore, it is of to a membrane is in gene was that antimicrobial peptides with gene transfer activity have potential in the of as they bacterial and the gene of J. the that LL-37 and antimicrobial peptides the release of plasmid DNA from by of bacterial DNA from in the extracellular and the transfer of bacterial DNA to the nuclei of mammalian host cells for a the and in antimicrobial peptides and as the in the the transfer of bacterial to cells it that are with of LL-37 peptide and that of is in the epithelial of the J. on the of and that were in the human but were in the was that of to bacterial to gene the of in which gene in the the against gene transfer J. J. J. J. a for bacterial to gene We and at for of and flow and for