highly-efficient-transfection-human-thp-1-macrophages

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Friedrich Schiller University Jena

Cell-penetrating peptides (CPPs) are used to transport peptides, proteins, different types of ribonucleic acids (or mimics of these molecules), and DNA into live cells, both plant and mammalian. Leishmania belongs to the class of protozoa having, in comparison to mammalian cells, a different lipid composition of the membrane, proteoglycans on the surface, and signal pathways. We investigated the uptake of two different and easily detectable proteins into the non-pathogenic strain Leishmania tarentolae. From the large number of CPPs available, six and a histone were chosen specifically for their ability to form non-covalent complexes. For Leishmania we used the enzyme β-galactosidase and fluorescent labeled bovine serum albumin as cargoes. The results are compared to similar internalization studies using mammalian cells [Mussbach et al., ]. Leishmania cells can degrade CPPs by a secreted and membrane-bound chymotrypsin-like protease. Both cargo proteins were internalized with sufficient efficiency and achieved intramolecular concentrations similar to mammalian cells. The transport efficiencies of the CPPs differed from each other, and showed a different rank order for both cargoes. The intracellular distribution of fluorescent-labeled bovine serum albumin showed highest concentrations in the nucleus and kinetoplast. Leishmania are susceptible to high concentrations of some CPPs, although comparably dissimilar to mammalian cells. MPG-peptides are more cytotoxic in Leishmania than in mammalian cells, acting as antimicrobial peptides. Our results contribute to a better understanding of molecular interactions in Leishmania cells and possibly to new treatments of leishmaniasis.

Transduction of proteins into leishmania tarentolae by formation of non-covalent complexes with cell-penetrating peptides.

Macrophages are versatile cells of the immune system which react to various external stimuli through different polarization patterns which adjust the cells to the required function whether it is removal of pathogens or necrotic cells, tissue repair or propagation of inflammation. As much of macrophage behavior is determined by their polarization, appropriate models to study macrophage polarization are required. Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48h using 100ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48h of differentiation with 100ng/ml PMA. When we applied this protocol to study interleukin (IL) 10 dependent polarization, the cells were inert to the IL10 stimulus, as indicated by a failure to induce IL10 target genes such as SOCS3. Further investigation revealed that the cells were classically activated by the differentiation agent phorbol 12-myristate 13-acetate (PMA) as shown by induction of chemokine receptor CCR7. Subsequent reduction of PMA concentration during THP-1 macrophage differentiation significantly improved their response to IL10 as SOCS3 increased more than 40-fold. This increased responsiveness of the THP-1 macrophages was also confirmed for polarization with LPS and IFNγ. Up-regulation of classical activation markers CCL3, CCR7 and TNFα was enhanced from 18-, 21- and 70-fold, respectively, to 48-, 222- and 951-fold, respectively. Reduction of PMA concentration did not negatively affect macrophage differentiation or transfection efficiency. Expression of macrophage differentiation markers CD11b and CD68 as well as cell morphology remained unchanged. In addition transfection efficiency and rates of apoptosis and necrosis remained unaffected. Thus our revised protocol combines high transfection efficiency and cell vitality with a strong response to polarizing stimuli and therefore constitutes a suitable model for studying macrophage polarization in vitro.

Article	Year
Transduction of proteins into leishmania tarentolae by formation of non-covalent complexes with cell-penetrating peptides. Journal of cellular biochemistry\| PubMed ID: 24038170	2014
Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli. Journal of immunological methods\| PubMed ID: 24269601	2014

Berith Wittig

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