dr Maciej Czerkies

Zakład Biosystemów i Miękkiej Materii (ZBiMM)
Pracownia Modelowania w Biologii i Medycynie (PMBM)
stanowisko: adiunkt
telefon: (+48) 22 826 12 81 wew.: 326
pokój: 221
e-mail: mczerkie
strona www: http://pmbm.ippt.pan.pl/web/Maciej_Czerkies

Doktorat
2012-09-10Udział receptora TLR4 i białka CD14 w internalizacji niskich i wysokich stężeń lipopolisacharydu   (IBD PAN)
promotor -- prof. dr hab. Katarzyna Kwiatkowska, IBD PAN
1323
 
Ostatnie publikacje
1.Urbanek O., Sajkiewicz P., Pierini F., Czerkies M., Kołbuk D., Structure and properties of polycaprolactone/chitosan nonwovens tailored by solvent systems, Biomedical Materials, ISSN: 1748-6041, DOI: 10.1088/1748-605X/aa5647, Vol.12, No.1, pp.015020-1-12, 2017
Urbanek O., Sajkiewicz P., Pierini F., Czerkies M., Kołbuk D., Structure and properties of polycaprolactone/chitosan nonwovens tailored by solvent systems, Biomedical Materials, ISSN: 1748-6041, DOI: 10.1088/1748-605X/aa5647, Vol.12, No.1, pp.015020-1-12, 2017

Abstract:
Electrospinning of chitosan blends is a reasonable idea to prepare fibre mats for biomedical applications. Synthetic and natural components provide, for example, appropriate mechanical strength and biocompatibility, respectively. However, solvent characteristics and the polyelectrolyte nature of chitosan influence the spinnability of these blends. In order to compare the effect of solvent on polycaprolactone/chitosan fibres, two types of the most commonly used solvent systems were chosen, namely 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and acetic acid (AA)/formic acid (FA). Results obtained by various experimental methods clearly indicated the effect of the solvent system on the structure and properties of electrospun polycaprolactone/chitosan fibres. Viscosity measurements confirmed different polymer–solvent interactions. Various molecular interactions resulting in different macromolecular conformations of chitosan influenced its spinnability and properties. HFIP enabled fibres to be obtained whose average diameter was less than 250 nm while maintaining the brittle and hydrophilic character of the nonwoven, typical for the chitosan component. Spectroscopy studies revealed the formation of chitosan salts in the case of the AA/FA solvent system. Chitosan salts visibly influenced the structure and properties of the prepared fibre mats. The use of AA/FA caused a reduction of Young's modulus and wettability of the proposed blends. It was confirmed that wettability, mechanical properties and the antibacterial effect of polycaprolactone/chitosan fibres may be tailored by selecting an appropriate solvent system. The MTT cell proliferation assay revealed an increase of cytotoxicity to mouse fibroblasts in the case of 25% w/w of chitosan in electrospun nonwovens.

Keywords:
chitosan, electrospinning, PCL/chitosan fibres, solvent system, chitosan salts

30p.
2.Korwek Z., Tudelska K., Nałęcz-Jawecki P., Czerkies M., Prus W., Markiewicz J., Kochańczyk M., Lipniacki T., Importins promote high-frequency NF-κB oscillations increasing information channel capacity, Biology Direct, ISSN: 1745-6150, DOI: 10.1186/s13062-016-0164-z, Vol.11, No.61, pp.1-21, 2016
Korwek Z., Tudelska K., Nałęcz-Jawecki P., Czerkies M., Prus W., Markiewicz J., Kochańczyk M., Lipniacki T., Importins promote high-frequency NF-κB oscillations increasing information channel capacity, Biology Direct, ISSN: 1745-6150, DOI: 10.1186/s13062-016-0164-z, Vol.11, No.61, pp.1-21, 2016

Abstract:
BACKGROUND:
Importins and exportins influence gene expression by enabling nucleocytoplasmic shuttling of transcription factors. A key transcription factor of innate immunity, NF-κB, is sequestered in the cytoplasm by its inhibitor, IκBα, which masks nuclear localization sequence of NF-κB. In response to TNFα or LPS, IκBα is degraded, which allows importins to bind NF-κB and shepherd it across nuclear pores. NF-κB nuclear activity is terminated when newly synthesized IκBα enters the nucleus, binds NF-κB and exportin which directs the complex to the cytoplasm. Although importins/exportins are known to regulate spatiotemporal kinetics of NF-κB and other transcription factors governing innate immunity, the mechanistic details of these interactions have not been elucidated and mathematically modelled.
RESULTS:
Based on our quantitative experimental data, we pursue NF-κB system modelling by explicitly including NF-κB-importin and IκBα-exportin binding to show that the competition between importins and IκBα enables NF-κB nuclear translocation despite high levels of IκBα. These interactions reduce the effective relaxation time and allow the NF-κB regulatory pathway to respond to recurrent TNFα pulses of 45-min period, which is about twice shorter than the characteristic period of NF-κB oscillations. By stochastic simulations of model dynamics we demonstrate that randomly appearing, short TNFα pulses can be converted to essentially digital pulses of NF-κB activity, provided that intervals between input pulses are not shorter than 1 h.
CONCLUSIONS:
By including interactions involving importin-α and exportin we bring the modelling of spatiotemporal kinetics of transcription factors to a more mechanistic level. Basing on the analysis of the pursued model we estimated the information transmission rate of the NF-κB pathway as 1 bit per hour.

Keywords:
Karyopherins, Nucleocytoplasmic transport, Negative feedback, Channel information capacity, Mathematical modelling

35p.
3.Czerkies M., Borzęcka K., Zdioruk M.I., Płóciennikowska A., Sobota A., Kwiatkowska K., An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS, IMMUNOBIOLOGY, ISSN: 0171-2985, DOI: 10.1016/j.imbio.2013.04.005, Vol.218, pp.1217-1226, 2013
Czerkies M., Borzęcka K., Zdioruk M.I., Płóciennikowska A., Sobota A., Kwiatkowska K., An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS, IMMUNOBIOLOGY, ISSN: 0171-2985, DOI: 10.1016/j.imbio.2013.04.005, Vol.218, pp.1217-1226, 2013

Abstract:
Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000 ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES. The binding and internalization of high amounts of LPS was mediated by scavenger receptor A (SR-A) with participation of CD14 without an engagement of TLR4. Occupation of SR-A by dextran sulfate or anti-SR-A antibodies enhanced LPS-induced production of TNF-α and RANTES by about 70%, with CD14 as a limiting factor. Dextran sulfate also elevated the cell surface levels of TLR4 and CD14, which could have contributed to the upregulation of the pro-inflammatory responses. Silencing of SR-A expression inhibited the LPS-triggered TNF-α production whereas RANTES release was unchanged. These data indicate that SR-A is required for maximal production of TNF-α in cells stimulated with LPS, possibly by modulating the cell surface levels of TLR4 and CD14.

Keywords:
CD14, CTX-FITC, Endocytosis, HEPES-buffered saline, LBP, LPS conjugated with Alexa Fluor 488, LPS-AF488, LPS-binding protein, Lipopolysaccharide, PD buffer, SR-A, Scavenger receptor, TLR, poly(I:C), polyinosinic–polycytidylic acid, scavenger receptor A, subunit B of cholera toxin conjugated with FITC, transferrin conjugated with Alexa Fluor 647, transferrin-AF647

25p.
4.Czerkies M., Kwiatkowska K., Receptory toll-podobne (TLR) i ich udział we wrodzonej odpowiedzi odpornościowej na przykładzie aktywacji TLR4 przez lipopolisacharyd, POSTĘPY BIOLOGII KOMÓRKI, ISSN: 0324-833X, Vol.40, pp.39-64, 2013
Czerkies M., Kwiatkowska K., Receptory toll-podobne (TLR) i ich udział we wrodzonej odpowiedzi odpornościowej na przykładzie aktywacji TLR4 przez lipopolisacharyd, POSTĘPY BIOLOGII KOMÓRKI, ISSN: 0324-833X, Vol.40, pp.39-64, 2013

Abstract:
Mechanizmy wrodzonej odpowiedzi odpornościowej uruchamiane są w wyniku rozpoznania elementów budulcowych mikroorganizmów o strukturze zachowanej w toku ewolucji, nazywanych wzorcami molekularnymi. W ich wiązaniu uczestniczą wyspecjalizowane receptory inicjujące kaskady sygnałów, które prowadzą do produkcji białek o charakterze prozapalnym oraz do aktywacji i regulacji mechanizmów nabytej odpowiedzi odpornościowej. Do receptorów tego typu należą receptory Toll-podobne (TLR), rozpoznające elementy ścian komórkowych, aparatu ruchu, jedno- i dwuniciowe RNA oraz motywy DNA typowe dla mikroorganizmów. Wszystkie receptory TLR zbudowane są z domeny wiążącej ligandy, która zawiera liczne motywy bogate w leucynę, pojedynczej domeny transbłonowej oraz domeny sygnałowej TIR. Pomimo znacznego strukturalnego zróżnicowania ligandów ich związanie prowadzi do dimeryzacji receptorów TLR, co z kolei umożliwia interakcję domeny TIR z białkami adaptorowymi i zapoczątkowuje kaskady sygnałowe. Receptory TLR angażują cztery wspólne białka adaptorowe, około dziesięciu kinaz o charakterze sygnałowym oraz aktywują docelowo kilka czynników transkrypcyjnych, w tym NFκB, IRF i AP-1. Szczególną uwagę poświęca się receptorowi TLR4 aktywowanemu przez lipopolisacharyd (LPS), budulec błony zewnętrznej bakterii Gram-ujemnych, gdyż uruchomiona przez LPS reakcja zapalna może prowadzić do potencjalnie śmiertelnego w skutkach szoku septycznego. Badania ostatnich lat znacznie rozszerzyły naszą wiedzę na temat współdziałania szeregu białek, w tym CD14, kompleksu TLR4/MD-2 oraz receptorów zmiataczy w modulacji odpowiedzi komórek na LPS. Ugruntowały też przekonanie o dychotomii ścieżek sygnałowych receptora TLR4, które zależą od udziału białek adaptorowych MyD88 i TRIF, oraz prowadzą do ekspresji genów kodujących cytokiny prozapalne i interferony typu I. Kluczowym wydarzeniem regulującym generację sygnału na ścieżce zależnej od TRIF jest internalizacja receptora TLR4

Keywords:
receptory Toll-podobne, przekazywanie sygnału, lipopolisacharyd, sepsa, wrodzona odpowiedź odpornościowa

15p.
5.Kleveta G., Borzęcka K., Zdioruk M., Czerkies M., Kuberczyk H., Sybirna N., Sobota A., Kwiatkowska K., LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility, JOURNAL OF CELLULAR BIOCHEMISTRY, ISSN: 0730-2312, DOI: 10.1002/jcb.23330, Vol.113, No.1, pp.80-92, 2012
Kleveta G., Borzęcka K., Zdioruk M., Czerkies M., Kuberczyk H., Sybirna N., Sobota A., Kwiatkowska K., LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility, JOURNAL OF CELLULAR BIOCHEMISTRY, ISSN: 0730-2312, DOI: 10.1002/jcb.23330, Vol.113, No.1, pp.80-92, 2012

Abstract:
Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5-bisphosphate. Fractionation of Triton X-100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin-regulatory proteins, paxillin on tyrosine 118 by 80% and N-WASP on serine 484/485 by 20%, and these events preceded activation of NF-κB. LPS-induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N-WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS-stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF-α production suggesting that LPS-induced cell motility is not required for TNF-α release.

Keywords:
ACTIN CYTOSKELETON, CELL MOTILITY, LPS, N-WASP, PAXILLIN, SRC KINASES

30p.
6.Józefowski S., Czerkies M., Sobota A., Kwiatkowska K., Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling, ANALYTICAL BIOCHEMISTRY, ISSN: 0003-2697, DOI: 10.1016/j.ab.2011.02.031, Vol.413, No.2, pp.185-191, 2011
Józefowski S., Czerkies M., Sobota A., Kwiatkowska K., Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling, ANALYTICAL BIOCHEMISTRY, ISSN: 0003-2697, DOI: 10.1016/j.ab.2011.02.031, Vol.413, No.2, pp.185-191, 2011

Abstract:
Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G–fluorescein isothiocyanate (IgG–FITC) for flow cytometry or with anti-rabbit IgG–peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with 125I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1–100 ng/ml LPS; however, it was reduced by approximately 30–40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.

Keywords:
Lipopolysaccharide, Toll-like receptor 4, ELISA, Radiolabeling, Flow cytometry

30p.
7.Józefowski S., Czerkies M., Łukasik A., Bielawska A., Bielawski J., Kwiatkowska K., Sobota A., Ceramide and Ceramide 1-Phosphate Are Negative Regulators of TNF-^5; Production Induced by Lipopolysaccharide, JOURNAL OF IMMUNOLOGY, ISSN: 0022-1767, DOI: 10.4049/jimmunol.0902926, Vol.185, No.11, pp.6960-6973, 2010
Józefowski S., Czerkies M., Łukasik A., Bielawska A., Bielawski J., Kwiatkowska K., Sobota A., Ceramide and Ceramide 1-Phosphate Are Negative Regulators of TNF-^5; Production Induced by Lipopolysaccharide, JOURNAL OF IMMUNOLOGY, ISSN: 0022-1767, DOI: 10.4049/jimmunol.0902926, Vol.185, No.11, pp.6960-6973, 2010

Abstract:
LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation

Keywords:
lipopolysaccharide, TNF alpha, MIP-2, ceramide. ceramide-1-phosphate, sphingomyelinase, J774, macrophages

32p.
8.Czerkies M., Raczkowska A., Brzostek K., Quo vadis Yersinia pestis? Ewolucja patogennych gatunków z rodzaju Yersinia [Quo vadis Yersinia pestis? The evolution of pathogenic species of the genus Yersinia], POSTĘPY MIKROBIOLOGII, ISSN: 0079-4252, Vol.48, No.3, pp.181-196, 2009
Czerkies M., Raczkowska A., Brzostek K., Quo vadis Yersinia pestis? Ewolucja patogennych gatunków z rodzaju Yersinia [Quo vadis Yersinia pestis? The evolution of pathogenic species of the genus Yersinia], POSTĘPY MIKROBIOLOGII, ISSN: 0079-4252, Vol.48, No.3, pp.181-196, 2009

Keywords:
dżuma, ewolucja, genomika, patogeneza, Yersinia