Effects of DNA sequence, endosomal pH, and lipid rafts/caveolae on uptake of human cathelicidin LL‐37/dsDNA complexes by monocytic cells

Abstract

LL‐37, the only human homolog of the class of cathelicidin cationic peptides, has many beneficial roles in the immune system as an antimicrobial and pathogen‐sensing molecule. However, altered levels of LL‐37 have been linked to a growing number of diseases, including autoimmune diseases, cancer, cystic fibrosis, and asthma. LL‐37 forms complexes with bacterial and self‐DNA that are internalized by many immune cell types, and formation of these complexes both protects DNA from degradation by nucleases and increases internalization of LL‐37. LL‐37 and self‐DNA uptake appears be related to autoimmune disease because it is a pathway by which the immune system may recognize and respond to self‐DNA. LL‐37 can bind to DNA and RNA and bring them into endosomes, activating innate immune receptors known as toll‐like receptors 3 and 9 (TLR3 and TLR9). In order to examine this mechanism, we have used fluorescence microscopy to observe the uptake of 5‐FAM‐LC‐LL‐37 (10 μM) into THP‐1 monocytic cells in the absence and presence of 24‐mer dsDNA oligonucleotides (0.6 μM). We investigate sequence effects and observe that complexes formed with CpG and poly A enhance LL‐37 uptake, but that GpC and poly G are less effective. We have also examined the effect of endosome acidification on uptake and intracellular association of LL‐37 and dsDNA by performing uptake in the presence or absence of inhibitors of endosome acidification. To investigate the possible role of cholesterol‐rich lipid rafts and caveolae in uptake, we pre‐treated the cells with the cholesterol‐depleting agents methyl‐β‐cyclodextrin (MCD) and nystatin known to disrupt raft/caveolar structures. The mechanism for LL‐37/DNA uptake is an interesting area of study that may reveal ways to facilitate LL‐37‐mediated immune responses and help to create more specialized medicines.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.