Anlisis de artculo:

Neutrfilos humanos son

activados por un fragmento de
pptido de la TcdB de C. difficile
probablemente via rcp formil
pptido (FPR-1)

Cellular Microbiology 2015

Abstract
La cintica y perfiles del aumento intracellular de Ca2+ libre y produccin de ROS inducido por
TcdB son similares a los inducidos por fMLF (que activa FPR que reconoce secuencias de pptidos
bacterianos formilados).
Transfection assays with the FPR-1 isoform hFPR26 in HEK293 cells, heterologous desensitization
experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR-1.
Domain analyses revealed that the N-terminal glucosyltransferase domain of TcdB is a potent
activator of FPR pointing towards an additional mechanism that might contribute to
pathogenesis. This pro-inflammatory ligand effect can be triggered even by cleaved and, thus,
non-cytotoxic toxin.
In summary, we report
(i) a ligand effect on neutrophils as completely new molecular mode of action,
(ii) pathogenic potential of truncated or proteolytically cleaved non- cytotoxic fragments
(iii) an interaction of the N-terminal glucosyltransferase domain instead of the C-terminal
receptor binding domain of TcdB with target cells
INTRO
The present study describes a new effect of TcdB that activates
neutrophils by targeting the formyl peptide receptor (FPR). The
isolated glucosyltransferase domain or limited proteolytical digested
toxin were able to activate neutrophils emphasizing that non-
cytotoxic fragments of TcdB might also contribute to the
inflammatory process associated with CDIs
RESULTADOS
Revisar resultados de ros y degranulacin
Revisar bien bien metodologa
TcdB induce la entrada de Ca2+ extracelular
en neutrfilos humanos
Fura-2: es una tincin fluorescente ratiomtrica (proporcional) que se une al Ca2+ intracelular libre. Se excita a
340 nm and 380 nm de luz, y la proporcin de las emisiones a esas longitudes de onda se relaciona directamente a la
concentracin de Calcio intracelular.
Contaminants are not
responsible for the
observed effects
Conformation of TcdB is critical for neutrophil
activation
Glycosil transferase domain
Metodologa
Neutrfilos (cita Reher et al, 2012)
81 mL de sangre fresca en 9 tubos EDTA (9 mL c/u), diluidos con PBS (formula Dulbecco, sin Ca2+/Mg2+) vol. final de 280 mL
Se toman alicuotas de 35 mL de sangre diluida y se coloca en capa sobre 15 mL de Percoll (densidad: 1.077 g/ml) a temp ambiente
Se centrifuga a 430 x g sin freno por 30 min, luego se descartan PBMC, plasma y Percoll
Erythrocytes were removed by hypotonic shock lysis for 70 s in distilled water.
Lysis was stopped by adding 1/10 volume of 10-concentrated PBS followed by centrifugation at 430 g for 5 min.
The remaining cell pellet was treated with distilled water for additional 30 s followed by stop and centrifugation as described
above.
The resulting cell pellet was washed once with PBS and cells were counted using a hemocytometer. Then, per 5 107 cells 50 l
anti-CD16 micro beads and 50 l MACS running buffer were added. The cell suspension was incubated for 30 min at 4 C and
thereafter washed with 10 ml PBS.
Finally, the cells were resuspended in a total volume of 500 l MACS running buffer per 1 108 cells and separated into
neutrophils and eosinophils using the AutoMacs cell separator (Miltenyi Biotech).
Purity was determined by anti-CD16b staining and flow cytometry as follows. Cells (5 105 cells) were suspended in 100 l MACS
running buffer and 1 l of PE-conjugated CD16b antibody was added. After incubation for 30 min at 4 C, the cell suspension was
washed with 500 l MACS running buffer and resuspended in 200 l MACS running buffer. The labeled cells were analyzed using
the MACS Quant flow cytometer (Miltenyi Biotech). Eosinophils were defined as the CD16b-negative population, whereas
neutrophils were identified as CD16b-positive cells [52].
Metodologa: Generacin de ROS
ROS generation was determined as described elsewhere (Reher et al.,
2012). Priming of 105 isolated neutrophils in 100 l PBS was performed by
addition of 50 l PBS containing 1.2 g cytochalasin B or alternatively 400
ng ml1 IL-8, 4 mM CaCl2, 400 M cytochrome c per well in a 96-well plate.
Samples were preheated to 37C for 3 min and afterwards 50 l stimulus
per well was added. Absorption was measured at 550 nm for 30 min
(Reher et al., 2012)Eosinophils were resuspended in PBS (1 x 106/ml). 100
l/well of the cell suspension were added to a 96-well microplate
containing per well 10 l of 20-fold concentrated stimulus and 90 ml
reaction buffer (2.2 mM CaCl2, 0.23 mM cytochrome c and 1 g/ml
cytochalasin B). Superoxide anion (O2-) production was determined by
kinetic analysis (30 min) of superoxide dismutase- inhibitable reduction of
cytochrome c at 550 nm in a platereader (Biotek synergy 4, Winooski, VT,
USA)
Resultado: rTcdBBM induce produccin de ROS
y degranulacin de neutrfilos
Citometra de flujo
Surface CD35 as marker for degranulation
events was determined as described
earlier (Ward et al., 2000). Briefly, 5 106
neutrophils ml1 were stimulated as
indicated (Toxin solutions were added to
cell suspension in a volume ratio of 1:1
directly before starting data acquisition)
for 5 min at 37C, washed with ice-cold
buffer and incubated on ice for 30 min
with FITC mouse anti-human CD35 (BD
Pharmingen 555452). Surplus antibody
was removed by washing with ice-cold
PBS twice and cells were fixed with 4%
(v/v) formaldehyde and 4% (w/v) sucrose
in PBS for 20 min at room temperature.
Fluorescence intensity was determined by
flow cytometry and given as arbitrary
units (AU) in graphs.
For CD11b expression, 1 107 neutrophils
ml1 were stimulated as indicated for 10
min at 37C. Afterwards cells were stored
on ice for 10 min, unspecific binding was
blocked with 15% (v/v) anti-FcR (2.4G2)
hybridoma supernatant for 15 min prior
to addition of FITC-conjugated anti-CD11b
for 30 min. After staining with primary
antibodies surplus antibodies were
removed by washing with ice-cold PBS
twice, cells were resuspended in ice-cold
PBS and fluorescence was determined by
flow cytometry.
Burde et al, 1989. Histamine inhibits activation of
human neutrophils and HL-60 leukemic cells via
H2-receptors.
O2- formation was monitored by
continuous measurement of
ferricytochrome C reduction inhibitable
by superoxide dismutase, using an Uvikon
810 dual-beam spectrophotometer
(Kontron, Eching, FRG) (Markert et al.
1984; Seifert et al. 1989b). Reaction
mixtures (0.5 ml) contained 0.5-1.0 x 10 6
neutrophils or 2.5 x 10 6 HL-60 cells, 100
gM ferricytochrome C and a buffer
consisting of (mM) 138 NaCl, 6 KCl, 1
MgCl2, 1 CaCl2, 1 Na2HPO4, 5 NaHCO3,
5.5 glucose and 20 Hepes/NaOH, pH 7.4.
Reaction mixtures were preincubated for
5 min at 37 C in the presence of the
agents indicated. The absolute amounts
of O2- generated were calculated.
Aggregation of neutrophils:
Aggregation was measured by
turbidometry (Korchak et al. 1984; Seifert
et al. 1989c). Neutrophils (5 x 106 cells)
were suspended in 900 l of the buffer
described above. Cells were incubated for
3 min in the presence or absence of
various agents prior to the addition of
fMet-Leu-Phe. Aggregation experiments
were carried out under constant stirring
of cells at 103 rpm, using an Uvikon 810
dual-beam spectrophotometer. The
maximum extent of aggregation was
calculated.
 The activation profile of TcdB that comprises Ca2+ influx (subsequent
to relase from intracellular stores as described for fMLF; Demaurex
et al., 1994), ROS production and the degree of degranulation is
highly similar to that of fMLF.
In addition, cyclosporine H completely abolished TcdB-induced rise in
Ca2+ when applied at 10 M concentration and reduced TcdB-
induced ROS production when applied at 1 M, indi- cating
involvement of FPR1.