Identification of the 90 KDa protein associated with NadA as hsp90 β by MS/MS.

<p>The picture shows a typical Coomassie G250 profile of the proteins co-isolated with NadA/anti-NadA immune complexes. The two arrows point to the 90 KDa protein and to 35 KDa NadA_Δ351–405, while H and L indicate antibody chains. The 90 KDa was excised from the gel for identification by MS/MS sequencing after trypsinization and identified as the human hsp90 β. The MS full scan (A) and data dependent MS/MS sequencing scans of the high mascot score peptide candidate 764.52 m/z (B) are shown, followed by a table of its Y and B ions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025089#pone-0025089-t001" target="_blank">Table 1</a>).</p

Effect of neutralizing anti-TLR4 and anti-TLR2 antibodies on NadA_Δ351–405 and NadA_Δ351–405/anti-hsp90 induced monocyte activation.

<p>Cells were incubated with NadA or with NadA and anti-hsp90 antibodies, after pre-treatment with neutralising antibodies to TLR4 or TLR2, as indicated. After 24 hours, the content of the three indicated cytokines was measured in the extracellular medium with a Bio-Plex suspension array. Data are expressed as percentage of the effect induced by adhesin or adhesin plus anti-hsp90 antibodies effects, in the absence of anti-TLR antibodies, and are the mean of three independent experiments run in triplicate +/− SE.</p

NadA_Δ351–405 binds to human hsp90 in an overlay-assay.

<p>A) Samples of total monocytes extracts obtained with 1% TX-100 were separated by SDS-PAGE and challenged or not as indicated with 1 µM NadA_Δ351–405 after blotting on nitrocellulose. After extensive washings, the polypeptide bands able to retain NadA were detected with antibodies to the whole adhesin or to NadA 52–70, as indicated. The picture is from a representative experiment out of four and arrows point to the NadA-interacting ∼90 KDa protein. B) Monocytes detergent extracts prepared as in A) were separated by SDS-PAGE, blotted on nitrocellulose and subjected to western blot with anti- grp94 and hsp90 antibodies or to an overlay assay with NadA/anti-NadA antibodies. The mobility of the NadA interacting protein and of hsp90 or grp94 was compared. C) Purified human hsp90 were subjected to overlay assay with (1 µM) or without NadA_Δ351–405 and HP-NAP as indicated. D) Whole TX-100 extracts from human monocytes were incubated with protein A sepharose-bound anti-NadA IgG in the presence or not of NadA_Δ351–405. After washings, beads were treated with LSB containing 4%SDS and 10% βme and analyzed by western blot after SDS-PAGE using anti-hsp90 antibodies and alkaline phosphatase-conjugated anti-IgG secondary antibodies. Arrow points to hsp90, H and L indicate antibody heavy and light chains respectively.</p

Model of the role of NadA_Δ351–405/hsp90 interaction in monocyte activation (discussed in the text).

<p>Model of the role of NadA_Δ351–405/hsp90 interaction in monocyte activation (discussed in the text).</p

Polymixin B inhibits the induction of cytokine/chemokine by NadA_Δ351–405 in monocytes.

<p>Human monocytes were stimulated for 24 hours with the indicated concentrations of NadA_Δ351–405 in the absence (open circles) or in the presence of 10 µg/ml polymixin B in DMEM plus 10% FCS. Cytokines/chemokines were subsequently quantified in the cells extracellular medium using a BioPlex assay. Data are from a representative experiment of three, run in duplicate and bars are ranges.</p

Anti-hsp90 antibodies increase NadA_Δ351–405 monocyte stimulation in a polymixin B insensitive-way.

<p>A) Cells were treated with NadA_Δ351–405 in the absence (black circles) or in the presence (open circles) of purified rabbit polyclonal antibodies directed to the COOH terminal domain of hsp90. Open squares refers to cells incubated with NadA_Δ351–405 in the presence of purified rabbit polyclonal antibodies from non immunized animals. The indicated cytokines/chemokines were analyzed in the extracellular medium by BioPlex suspension arrays. Data are the mean from a representative experiment out of four run in triplicate. Bars are +/− SE. Asterisk indicates signals significantly different (p<0.05) from control (with or without an unrelated antibody). B) Monocytes were stimulated or not with NadA_Δ351–405 (1.5 µM) as indicated, in the presence of polymixin B (black bars), antibodies to the COOH terminal of hsp90 (hatched bats) and in the presence of both polymixin B and anti-hsp90 antibodies (light gray bars). After 1 hour incubation, the indicated cytokines/chemokines were analyzed in the extracellular medium as indicated above. Data are the mean of two experiments run in triplicate. Bars represent +/− SE.</p

Peptides identified by the MS/MS tandem analysis.

<p>Peptides identified by the MS/MS analysis and recognized by Mascot. In the columns are indicated, from left to right: the relative sequence position; the experimental m/z value (m/z Obs); the theoretical molecular weight, in Dalton, obtained from the Swiss-Prot and ExPASy databases (Mr_exp); the relative molecular mass calculated from the matched peptide (Mr_calc); the difference between the experimental and calculated masses (Delta); the Mascot score for the identified protein (Score) and the sequence of the peptide. The peptide with the highest score is highlighted in bold and used as example of MS/MS scan analysis in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025089#pone-0025089-g002" target="_blank">Fig. 2</a>. All shown peptides belong to human hsp90 β.</p

Polymixin B binds to NadA_Δ351–405, inhibits the formation of the NadA_Δ351–405/hsp90 complex but does not hamper NadA_Δ351–405 monocyte binding.

<p>A) 0.1 ml of NadA_Δ351–405 solution in PBS (10 µg/ml) was incubated with polymixin B agarose at room temperature for 2 hours. Beads were recovered by centrifugation, and corresponding supernatant and pellets, washed in PBS, were subjected to SDS-PAGE to reveal NadA presence by Coomassie staining (arrow). A reference lane containing 1 µg NadA_Δ351–405 was run in parallel. B) Monocyte detergent extracts were subjected to co-immune isolation with proteinA-sepharose bound anti-NadA antibodies and NadA_Δ351–405 plus or minus 10 µg/ml polymixin B, as indicated. Proteins were separated by SDS-PAGE and silver stained. The arrows points to hsp90 or to NadA; H and L indicate the chains of anti-NadA antibodies. C, D) Monocytes were incubated with the indicated concentrations of NadA_Δ351–405 at 0° C (C) or 37°C (D) in the presence (black circles) or in the absence (open circles) of 20 µg/ml polymixin B for three hours in culture medium. After washings with PBS, at 0°C, cells were further incubated with anti-hsp90 antibodies and with PE-conjugated secondary antibodies. Cell fluorescence (MFI), quantified by flow-cytofluorimetry, corresponds to the mean of three experiments run in triplicate and bars represent +/− S.E. Asterisk indicates signals significantly different (p<0.05) from control (no adhesin present in the system). E) Monocytes were incubated with 600 nM Alexa NadA_Δ351–405 at 0°C or 37°C in the presence of increasing concentrations of polymixin B. Cells were washed and treated at 4°C with anti-hsp90 and anti-IgG PE-conjugated antibodies keeping polymixin B in the solutions where necessary. Cell fluorescence (MFI), quantified by flow-cytofluorimetry, corresponds to the mean of three experiments run in triplicate and bars represent +/− S.E.</p

Analysis of the reciprocal interference of NadA_Δ351–405 and anti-hsp90 antibodies to the monocyte surface at 37° and 0°C.

<p>A) Human monocytes were incubated for 1 hours at 0°C in RPMI, 10% FCS, containing NadA_Δ351–405 at the indicated concentrations. After washing at 0°C cells were further incubated with anti-hsp90 antibody and anti-IgG PE-labeled secondary antibodies. MFI was then analyzed by flow-cytofluorimetry. Data are from an experiment representative of four, and bars represent +/− SE. Asterisk indicates signals significantly different (p<0.05) from control (0°C). B) Monocytes were pre-treated or not with anti-hsp90 antibodies (40 µg/ml) for 1 hour at 0°C, washed as above and further incubated at the indicated temperature with Alexa-labeled NadA_Δ351–405. Graphs are made with mean values from a representative experiment of four. Bars are +/− SE.</p

C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes

Poly­(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly­(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and <i>in vivo</i> performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity (<i>K</i>_d = 11 ± 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps