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Regulation of human cell viability by the host defence peptide LL-37

Svensson, Daniel LU (2017)
Abstract
Accumulating evidence suggests that the main human cathelicidin peptide, LL-37, may influence host cell viability and pro-inflammatory signaling when locally overexpressed. LL-37 has pro-inflammatory properties linked to the development of for example psoriasis, rosacea, SLE and atherosclerosis. However, the importance of LL-37-induced host cell permeabilization and cytotoxic effects are poorly understood. We hypothesize that these effects are implicated in the tissue destruction associated with inflammatory diseases such as chronic periodontitis (CP). CP, the number one reason for adult tooth loss globally, is associated with elevated LL-37 levels. Here, tooth detachment is caused by degradation of the supporting bone. We aimed to... (More)
Accumulating evidence suggests that the main human cathelicidin peptide, LL-37, may influence host cell viability and pro-inflammatory signaling when locally overexpressed. LL-37 has pro-inflammatory properties linked to the development of for example psoriasis, rosacea, SLE and atherosclerosis. However, the importance of LL-37-induced host cell permeabilization and cytotoxic effects are poorly understood. We hypothesize that these effects are implicated in the tissue destruction associated with inflammatory diseases such as chronic periodontitis (CP). CP, the number one reason for adult tooth loss globally, is associated with elevated LL-37 levels. Here, tooth detachment is caused by degradation of the supporting bone. We aimed to investigate effects of LL-37 on osteoblast cell viability in vitro and the underlying mechanism in order to explore the detrimental effects of LL-37 and the possible involvement in tissue destruction associated with CP. In agreement with this, the peptide was found to permeabilize and kill osteoblasts at concentrations relevant for the in vivo situation. Cell permeabilization by LL-37 was associated with LDH release, Ca2+-influx, attenuation of cell viability, accumulation of annexin V positive cells and caspase 3 activation, indicative of apoptosis in MG63 osteoblasts.
As LL-37 constitutes a possible drug target, we further investigated compounds and endogenous mechanisms that may serve to inactivate LL-37. We found that the protein p33 (gC1qR) may be added extracellularly to rescue osteoblasts from LL-37-evoked cytotoxicity. Additionally, endogenous p33 appears to be a mean by which host cells are protected from LL-37-induced cytotoxicity, as the extent of the toxicity correlates to p33 expression levels in various host cell types. Host cell sensitivity towards LL-37 may moreover be modulated through up- or down- regulation of p33 expression, mediated through transfection with a pcDNA3.1 expression vector and siRNA. p33 was found in the mitochondria, cytoplasm and in proximity to the cell membrane. It inactivates LL-37 intracellularly, as cell viability but not cellular LDH release is influenced by p33 expression, and additionally, LL-37 co-immunoprecipitates with cytoplasmic p33. This indicates that internalization of LL-37 is critical for host cell cytotoxicity. Furthermore, a comparison of LL-37's effects on various cell types indicates that LL-37-evoked cytotoxicity is not directly correlated to the degree of cell permeabilization.
The active form of vitamin D, 1,25D3, is a well-known mediator of LL-37 synthesis, although its possible role in dysregulated expression of LL-37 is yet to be elucidated. Stimulation of LL-37 expressing THP-1 monocytes with 1,25D3 was shown to attenuate cell viability both of co-cultured periodontal ligament (PDL) cells and the THP-1 cells themselves. This effect is reversed by addition of recombinant p33 to the cultures, implicating LL-37 as the mediator of the cytotoxic effect. In skin, RXRα protein levels were found to be critical for LL-37 expression: siRNA mediated silencing of RXRα attenuates 1,25D3-induced stimulation of LL-37 in HaCaT keratinocytes. Furthermore, gene expressions were analyzed in human skin and gingiva biopsies. Skin shows significantly higher LL-37 gene expression compared to gingiva and this difference correlates to RXRα mRNA levels.
In conclusion, our study contributes to the mechanistic understanding of LL-37 induced cytotoxicity in host cells, as well as the regulation of LL-37 expression by vitamin D. The in vitro results presented here support the hypothesis that LL-37 may be involved in the tissue destruction, observed locally in inflammatory diseases such as CP.
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author
supervisor
opponent
  • professor Agerberth, Birgitta, Department of Laboratory Medicine, Karolinska Institute, Stockholm
organization
publishing date
type
Thesis
publication status
published
subject
keywords
LL-37, cathelicidin, cytotoxicity, host defence peptides, autoimmune diseases, inflammation, vitamin D
pages
166 pages
publisher
Lund University, Faculty of Medicine
defense location
Segerfalksalen, BMC A10, Sölvegatan 17, Lund.
defense date
2017-02-17 09:00
ISBN
978-91-7619-393-8
language
English
LU publication?
yes
id
0a17b069-0d7c-44de-bea2-eb5bc056841d
date added to LUP
2017-01-25 14:11:19
date last changed
2018-01-03 00:01:01
@phdthesis{0a17b069-0d7c-44de-bea2-eb5bc056841d,
  abstract     = {Accumulating evidence suggests that the main human cathelicidin peptide, LL-37, may influence host cell viability and pro-inflammatory signaling when locally overexpressed. LL-37 has pro-inflammatory properties linked to the development of for example psoriasis, rosacea, SLE and atherosclerosis. However, the importance of LL-37-induced host cell permeabilization and cytotoxic effects are poorly understood. We hypothesize that these effects are implicated in the tissue destruction associated with inflammatory diseases such as chronic periodontitis (CP). CP, the number one reason for adult tooth loss globally, is associated with elevated LL-37 levels. Here, tooth detachment is caused by degradation of the supporting bone. We aimed to investigate effects of LL-37 on osteoblast cell viability in vitro and the underlying mechanism in order to explore the detrimental effects of LL-37 and the possible involvement in tissue destruction associated with CP. In agreement with this, the peptide was found to permeabilize and kill osteoblasts at concentrations relevant for the in vivo situation. Cell permeabilization by LL-37 was associated with LDH release, Ca2+-influx, attenuation of cell viability, accumulation of annexin V positive cells and caspase 3 activation, indicative of apoptosis in MG63 osteoblasts. <br/>As LL-37 constitutes a possible drug target, we further investigated compounds and endogenous mechanisms that may serve to inactivate LL-37. We found that the protein p33 (gC1qR) may be added extracellularly to rescue osteoblasts from LL-37-evoked cytotoxicity. Additionally, endogenous p33 appears to be a mean by which host cells are protected from LL-37-induced cytotoxicity, as the extent of the toxicity correlates to p33 expression levels in various host cell types. Host cell sensitivity towards LL-37 may moreover be modulated through up- or down- regulation of p33 expression, mediated through transfection with a pcDNA3.1 expression vector and siRNA. p33 was found in the mitochondria, cytoplasm and in proximity to the cell membrane. It inactivates LL-37 intracellularly, as cell viability but not cellular LDH release is influenced by p33 expression, and additionally, LL-37 co-immunoprecipitates with cytoplasmic p33. This indicates that internalization of LL-37 is critical for host cell cytotoxicity. Furthermore, a comparison of LL-37's effects on various cell types indicates that LL-37-evoked cytotoxicity is not directly correlated to the degree of cell permeabilization.<br/>The active form of vitamin D, 1,25D3, is a well-known mediator of LL-37 synthesis, although its possible role in dysregulated expression of LL-37 is yet to be elucidated. Stimulation of LL-37 expressing THP-1 monocytes with 1,25D3 was shown to attenuate cell viability both of co-cultured periodontal ligament (PDL) cells and the THP-1 cells themselves. This effect is reversed by addition of recombinant p33 to the cultures, implicating LL-37 as the mediator of the cytotoxic effect. In skin, RXRα protein levels were found to be critical for LL-37 expression: siRNA mediated silencing of RXRα attenuates 1,25D3-induced stimulation of LL-37 in HaCaT keratinocytes. Furthermore, gene expressions were analyzed in human skin and gingiva biopsies. Skin shows significantly higher LL-37 gene expression compared to gingiva and this difference correlates to RXRα mRNA levels.<br/>In conclusion, our study contributes to the mechanistic understanding of LL-37 induced cytotoxicity in host cells, as well as the regulation of LL-37 expression by vitamin D. The in vitro results presented here support the hypothesis that LL-37 may be involved in the tissue destruction, observed locally in inflammatory diseases such as CP.<br/>},
  author       = {Svensson, Daniel},
  isbn         = {978-91-7619-393-8},
  keyword      = {LL-37, cathelicidin, cytotoxicity, host defence peptides, autoimmune diseases, inflammation, vitamin D},
  language     = {eng},
  pages        = {166},
  publisher    = {Lund University, Faculty of Medicine},
  school       = {Lund University},
  title        = {Regulation of human cell viability by the host defence peptide LL-37},
  year         = {2017},
}