Supplementary File
Activation of neutrophils via IP3 pathway following exposure to
Demodex associated bacterial proteins.
Fred McMahon1, Nessa Banville2, David A. Bergin2, Christian Smedman3, Staffan
Paulie3, Emer Reeves2 and Kevin Kavanagh†*1
S1. Supplementary Materials and Methods.
S1.1 Analysis of Endotoxin Activity in Bacillus Protein Preparation by ELISpot
Fresh blood from healthy volunteers was collected in heparin-EDTA treated
tubes following ethical approval of the NUI Maynooth ethics committee (BSRESC2012-008) to isolate peripheral blood mononuclear cells (PBMC) according to the
method of Smedman et al. (2009). To confirm the absence of endotoxin
contamination in both the crude and pure B. oleronius protein preparations, a tumor
necrotic factor (TNF)-α ELISpot assay (Mabtech AB, Nacka Strand, Sweden) was
performed using isolated PBMC, at a density of 5 x 104 cells/well ± crude or pure B.
oleronius protein (2, 0.2, 0.002, and 0.0002 µg/ml), lipopolysaccharide (LPS) (10, 1,
0.1, and 0.001 ng/ml) and with the addition of polymyxin B (10 µg/ml) to specific
sample wells to inhibit LPS. Quantification of PBMC TNF-α secreted spots was
recorded using an ELISpot reader system (iSpot, AID, Strassberg, Germany)
S2. Supplementary Results.
S2.1 Assessment of Endotoxin Activity and TNF-α Spot Formation by Bacillus
Proteins Preparations
To ensure the effects on neutrophils of Bacillus protein preparations were not
caused by the presence of an endotoxin, the protein preparations were tested against
PBMC in a TNF-α ELISpot assay to detect minimal amounts of LPS and assess for
endotoxin activity. Addition of LPS led to an increase in the number of TNF-αsecreting cells which at 0.01 and 0.001 ng/mL LPS were effectively inhibited by the
action of polymyxin B (10 µg/ml), and the reduction of TNF-α-secreting cells was
calculated to be significant (p = 0.0032 and p = 0.0063, respectively) (Fig. S1). In
contrast, polymyxin B had little or no effect on the TNF-α-inducing capacity of
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PBMC exposed to both the pure (Fig. S2) and crude (Fig. S3) B. oleronius protein
preparations at 2, 0.2, 0.002, and 0.0002 µg/ml, demonstrating that the induction of
TNF-α
2
Figure S1. Assessment of endotoxin activity and TNF-α secretion in PBMC
exposed to LPS by ELISpot. (A) Investigating the endotoxin properties of pure and
crude B. oleronius protein preparations through TNF-α ELISpot, by performing
titrations of LPS (10 – 0.01 ng/ml) to examine spot formation induced by endotoxin
activity, and inhibition of endotixin activity by polymyxin B (10 μg/ml) at 0.1 ng/ml
LPS. The inhibition of spot formation at 0.1 ng/ml and 0.001 ng/ml LPS following the
addition of polymyxin B was significant. At 0.01 and 0.001 ng/ml LPS, TNF-α spot
formation induced by LPS was significantly decreased in the presence of polymyxin
B (p = 0.0032 and p = 0.0063, respectively). (B) Representative ELISpot images of
spot formation and inhibition were recorded. (Significance: ** = P <0.01).
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Figure S2. Assessment of endotoxin activity and TNF-α secretion in PBMC
exposed to pure B. oleronius protein by ELISpot. (A) Investigating the endotoxin
properties of pure B. oleronius protein preparations through TNF-α ELISpot. B.
oleronius protein and was observed not to inhibit TNF-α spot formation indicating a
non-endotoxin effect by the pure B. oleronius protein preparation (ns). (B)
Representative ELISpot images of spot formation and inhibition were recorded.
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Figure S3. Assessment of endotoxin activity and TNF-α secretion in PBMC
exposed to crude B. oleronius protein by ELISpot. (A) Investigating the endotoxin
properties of crude B. oleronius protein preparations through TNF-α ELISpot. B.
oleronius protein and was observed not to inhibit TNF-α spot formation indicating a
non-endotoxin effect by the pure B. oleronius protein preparation (ns). (B)
Representative ELISpot images of spot formation and inhibition were recorded.
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Demodex-associated bacterial proteins induce neutrophil activation. British Journal of
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Smedman, C., B. Gårdlund , K. Nihlmark, P. Gille-Johnson, J. Andersson, and S.
Paulie. 2009. ELISpot analysis of LPS-stimulated leukocytes: Human granulocytes
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