The crystal structure of titanium dioxide nanoparticles influences immune activity in vitro and in vivo

Abstract Background The use of engineered nanoparticles (NP) is widespread and still increasing. There is a great need to assess their safety. Newly engineered NP enter the market in a large variety; therefore safety evaluation should preferably be in a high-throughput fashion. In vitro screening is suitable for this purpose. TiO 2 NP exist in a large variety (crystal structure, coating and size), but information on their relative toxicities is scarce. TiO 2 NP may be inhaled by workers in e.g. paint production and application. In mice, inhalation of TiO 2 NP increases allergic reactions. Dendritic cells (DC) form an important part of the lung immune system, and are essential in adjuvant activity. The present study aimed to establish the effect of a variety of TiO 2 NP on DC maturation in vitro. Two NP of different crystal structure but similar in size, uncoated and from the same supplier, were evaluated for their adjuvant activity in vivo. Methods Immature DC were differentiated in vitro from human peripheral blood monocytes. Exposure effects of a series of fourteen TiO 2 NP on cell viability, CD83 and CD86 expression, and IL-12p40 and TNF-α production were measured. BALB/c mice were intranasally sensitized with ovalbumin (OVA) alone, OVA plus anatase TiO 2 NP, OVA plus rutile TiO 2 NP, and OVA plus Carbon Black (CB; positive control). The mice were intranasally challenged with OVA. OVA-specific IgE and IgG1 in serum, cellular inflammation in bronchoalveolar lavage fluid (BALF) and IL-4 and IL-5 production in draining bronchial lymph nodes were evaluated. Results All NP dispersions contained NP aggregates. The anatase NP and anatase/rutile mixture NP induced a higher CD83 and CD86 expression and a higher IL-12p40 production in vitro than the rutile NP (including coated rutile NP and a rutile NP of a 10-fold larger primary diameter). OVA-specific serum IgE and IgG1 were increased by anatase NP, rutile NP, and CB, in the order rutile<anatase<CB. The three particles similarly increased IL-4 and IL-5 production by bronchial LN cells and eosinophils and lymphocytes in the BALF. Neutrophils were induced by rutile NP and CB but not by anatase NP. Conclusions Our data show that measuring CD83 and CD86 expression and IL-12p40 and TNF-α production in DC in vitro may provide an efficient way to screen NP for potential adjuvant activity; future studies should establish whether this also holds for other NP. Based on antigen-specific IgE and IgG1, anatase NP have higher adjuvant activity than rutile NP, confirming our in vitro data. Other parameters of the allergic response showed a similar response for the two NP crystal structures. From the viewpoint of safe(r) by design products, rutile NP may be preferred over anatase NP, especially when inhalation exposure can be expected during production or application of the product

Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation

<div><p>The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable <i>in vitro</i> alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of <i>Bordetella pertussis</i> during cultivation. This system regulates the transcription of a range of virulence proteins, many of which are considered important for the induction of effective host immunity. The protein compositions and <i>in vivo</i> potencies of the vaccines were BvgASR dependent, with the vaccine containing the highest amount of virulence proteins having the highest <i>in vivo</i> potency. Here, the capacities of these vaccines to stimulate human Toll-like receptors (hTLR) 2 and 4 and the role these receptors play in wP vaccine-mediated activation of antigen-presenting cells <i>in vitro</i> were studied. Prolonged BvgASR suppression was associated with a decreased capacity of vaccines to activate hTLR4. In contrast, no significant differences in hTLR2 activation were observed. Similarly, vaccine-induced activation of MonoMac-6 and monocyte-derived dendritic cells was strongest with the highest potency vaccine. Blocking of TLR2 and TLR4 showed that differences in antigen-presenting cell activation could be largely attributed to vaccine-dependent variation in hTLR4 signalling. Interestingly, this BvgASR-dependent decrease in hTLR4 activation coincided with a reduction in GlcN-modified lipopolysaccharides in these vaccines. Accordingly, expression of the <i>lgmA-C</i> genes, required for this glucosamine modification, was significantly reduced in bacteria exposed to sulfate. Together, these findings demonstrate that the BvgASR status of bacteria during wP vaccine preparation is critical for their hTLR4 activation capacity and suggest that including such parameters to assess consistency of newly produced vaccines could bring <i>in vitro</i> testing of vaccine quality a step closer.</p></div

Secretion of IL-6 and IL-12p40 by MM6 cells stimulated with wP vaccines A_ref—E.

<p>MM6 cells were stimulated overnight with vaccines A_ref, B, C, D, E, LPS-EC (4 ng/mL) or PAM (40 ng/mL). Subsequently, IL-6 and IL-12p40 secretion was measured in culture supernatants. (A) Shown is the response of MM6 cells to 2-fold serial dilutions of vaccine A_ref, B, C, D and E (representative responses are shown from one out of three independent experiments). (B) Response of MM6 cells to vaccines A_ref, B, C, D and E at an OD_590nm of 0.00094. Each dot represents one value of three individually performed cell culture experiments. * = p < 0.05.</p

Expression of genes associated with LPS synthesis and modification in <i>B</i>. <i>pertussis</i> in response to sulfate exposure.

<p>Relative gene expression of a panel of 35 genes associated with LPS synthesis or modification in <i>B</i>. <i>pertussis</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161428#pone.0161428.ref025" target="_blank">25</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161428#pone.0161428.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161428#pone.0161428.ref041" target="_blank">41</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161428#pone.0161428.ref044" target="_blank">44</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161428#pone.0161428.ref050" target="_blank">50</a>]. Each row represents the relative transcript abundance of a single gene in <i>B</i>. <i>pertussis</i> bacteria harvested before and at different time points after sulfate addition to the growth medium. Column names (t = -24 –t = 24) correspond to the time points after the addition of sulfate at which bacteria were harvested (0 = vaccine A_ref, 2h = vaccine B, 6h = vaccine C, 12h = vaccine D, 24h = E). The colour scale indicates gene regulation ranging from strong downregulation (dark green), to no regulation (yellow), and strong upregulation (dark red). Genes for which the expression changed significantly throughout the production process are marked with an asterisk (p < 0.05).</p

Activation of MM6 cells and moDC by wP vaccines is primarily mediated by hTLR4 signalling.

<p>MM6 cells and moDC were pre-treated for 3 hours with the TLR4 antagonist LPS-RS (1 μg/mL), a blocking antibody against human TLR2 (0.5 μg/mL) or a combination of both. Subsequently, MM6 cells were stimulated with wP vaccine A_ref, C, E (OD_590nm of 0.00094), PAM (100 ng/mL), LPS-EC (4 ng/mL) or medium overnight. Similarly, moDC were stimulated for 2 days with vaccine A_ref, C and E (OD_590nm of 0.00047), LPS-EC (100 ng/mL), PAM (1 μg/mL) or only medium. (A) Secretion of IL-6 and IL-12p40 by MM6 cells measured in culture supernatants (one experiment out of two experiments with similar results is shown). (B) Secretion of IL-12p40 by moDC measured in culture supernatants (one experiment out of two experiments with similar results is shown). * = p < 0.05.</p

Negative-ion ESI mass spectra of LPS isolated from wP vaccine preparations A_ref—E.

<p>The triply charged (M-3H)3- molecular ion regions of the mass spectra of LPS from vaccine preparations A_ref, B, C, D and E are shown. The box on top of the mass spectra contains a simplified representation of the chemical structure of the LPS from <i>B</i>. <i>pertussis</i> reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161428#pone.0161428.ref035" target="_blank">35</a>]. This structure has been assigned to the ion of m/z 1351. The ions highlighted by an asterisk correspond to LPS with a glucosamine substitution of the lipid A phosphate. Abbreviations: Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Hep, L-glycero-D-manno-heptose; Glc, glucose; GlcN, glucosamine; GlcNAc, N-acetyl glucosamine; Fuc2NAc4NMe, 2-acetamido-4-N-methyl-2,4,6-deoxy-galactose; GalNA, galactosaminuronic acid; GlcA, glucuronic acid; Man2NAc3NAcA, 2-acetamido-3-acetamido-2,3-dideoxy-mannuronic acid; PPEA, pyrophosphoethanolamine; P, phosphate; C14OH, 3-hydroxy-tetradecanoic acid; C14, tetradecanoic acid; C10OH, 3-hydroxy-decanoic acid.</p

Activation of hTLR4- and hTLR2-mediated signalling by wP vaccines A_ref—E.

<p>HB-hTLR2, HB-hTLR4 and HB-Null-1 cells were stimulated overnight with wP vaccines A_ref, B, C, D, E, LPS-EC (0.8 ng/mL) or PAM (40 ng/mL). Shown is the SEAP activity in supernatants of HB-hTLR2, HB-hTLR4 (A) and HB-Null-1 (B) cells in response to 2-fold serial dilutions of the vaccines (representative responses are shown from one out of three independent experiments). (C) Shown is the SEAP activity of HB-hTLR2 and HB-hTLR4 cells in response to vaccines A_ref, B, C, D and E at an OD_590nm of 0.00094. Each dot represents one value of three individually performed cell culture experiments. * = p < 0.05.</p

Bvg status of <i>B</i>. <i>pertussis</i> bacteria at time of harvest affects protein composition of the resulting wP vaccines.

<p>Amounts of proteins (A) and LPS (B) present in wP vaccines A_ref, B, C, D, E (harvested 0, 2, 6, 12, 24 hours after the addition of sulfate, respectively) derived from three individual <i>B</i>. <i>pertussis</i> culture runs were measured by ELISA using specific monoclonal antibodies directed against individual proteins or LPS. (C) Fatty acid composition of vaccines A_ref, B, C, D, and E analysed using a modified gas chromatography method (in duplicate). The black lines indicate the background levels measured in PBS only by ELISA. * = p < 0.05.</p