Pulmonary DWCNT Exposure Causes Sustained Local and Low-Level Systemic Inflammatory Changes in Mice

Carbon nanotubes (CNTs) represent promising vectors to facilitate cellular drug delivery and to overcome biological barriers, but some types may also elicit persistent pulmonary inflammation based on their fibre characteristics. Here, we show the pulmonary response to aqueous suspensions of block copolymer dispersed, double-walled carbon nanotubes (DWCNT, length 1–10 lm) in mice by bronchoalveolar lavage (BAL) analysis, and BAL and blood cytokine and lung antioxidant profiling. The intratracheally instilled dose of 50 lg DWCNT caused significant pulmonary inflammation that was not resolved during a 7- day observation period. Light microscopy investigation of the uptake of DWCNT agglomerates revealed no particle ingestion for granulocytes, but only for macrophages. Accumulating macrophage, multinucleated macrophage and lymphocyte numbers in the alveolar region further indicated ineffective resolution with chronification of the inflammation. The local inflammatory impairment of the lung was accompanied by pulmonary antioxidant depletion and haematological signs of systemic inflammation. While the observed inflammation during its acute phase was dominated by neutrophils and neutrophil recruiting cytokines, the contribution of macrophages and lymphocytes with related cytokines became more significant after day 3 of exposure. This study confirms that acute pulmonary toxicity can occur on exposure of high doses of DWCNT agglomerates and offers further insight for improved nanotube design parameters to avoid potential long-term toxicity

Rolle der NFkB p-50 Untereinheit in Partikel-induzierter, aseptischer Lungenentzündung

We tested the hypothesis that pulmonary exposure to sterile carbon nanoparticles (CNP), a main constituent of urban air pollution, generates an acute inflammatory response by involving the p50 subunit of NFkB1, a pathway generally crucial for inflammatory gene expression in sepsis. Exposing wt and p50-deficinet mice to CNP caused an acute, aseptic neutrophilic alveolitis. While the storm of acute phase cytokines was much more pronounced in p50 mice, no genotype related difference in the number of to the airspace recruited inflammatory leukocytes could be detected. Nuclear profiling of CNP exposed lung however detected NFkB activation only for WT and not p50 mice. Even the activation potential of alveolar macrophages (AM), which are known to be the primary source of inflammatory cytokine production in infected lungs, revealed a rather antiinflammatory skewed phenotype for p50 AMs. Finally our results indicate that inflammatory pathways described for infectious, septic triggers might differ from those during aseptic, sterile inflammation and may thus require further investigations.Die Inhalation von sterilen, Kohlenstoff-Nanoteilchen einem wichtigen Bestandteil urbanen Luftverschmutzung, bewirkt eine lokale Entzündungsreaktion. Da der NFkB Transkriptionsfaktor für septische Entzündungsreaktion bestens beschrieben ist, wurde hier untersucht inwieweit die NFkB p50-Untereinheit für die Ausprägung der aseptischen, Partikelinduzierten Entzündungsreaktion von Bedeutung ist. Partikelexposition von wt und p50-defizienten Mäusen bewirkte in Lungen von Mutanten eine verstärkte Expression und Freisetzung von Akutphase-Cytokinen. Die zelluläre Entzündungsreaktion, d.h. die Akkumulation von Leukozyten im Alveolarbereich war dagegen unabhängig vom Genotyp und NFkB-Aktivierung wurde nur in wt aber nicht p50-Lungen beobachtet. Die Aktivierbarkeit von Alveolarmakrophagen, den Zellen der Cytokinfreisetzung, zeigen ebenfalls einen antiinflammatorischen Phänotyp für p50 Mäuse auf. Die Signalwege der septischen Entzündungsreaktion scheinen damit denen der aseptischen nicht unbedingt zu entsprechen und bedürfen genauerer Untersuchung

Combination of the immunization with the sequence close to the consensus sequence and two DNA prime plus one VLP boost generate H5 hemagglutinin specific broad neutralizing antibodies.

Hemagglutinin (HA) head has long been considered to be able to elicit only a narrow, strain-specific antibody response as it undergoes rapid antigenic drift. However, we previously showed that a heterologous prime-boost strategy, in which mice were primed twice with DNA encoding HA and boosted once with virus-like particles (VLP) from an H5N1 strain A/Thailand/1(KAN)-1/2004 (noted as TH DDV), induced anti-head broad cross-H5 neutralizing antibody response. To explain why TH DDV immunization could generate such breadth, we systemically compared the neutralization breadth and potency between TH DDV sera and immune sera elicited by TH DDD (three times of DNA immunizations), TH VVV (three times of VLP immunizations), TH DV (one DNA prime plus one VLP boost) and TK DDV (plasmid DNA and VLP derived from another H5N1 strain, A/Turkey/65596/2006). Then we determined the antigenic sites (AS) on TH HA head and the key residues of the main antigenic site. Through the comparison of different regiments, we found that the combination of the immunization with the sequence close to the consensus sequence and two DNA prime plus one VLP boost caused that TH DDV immunization generate broad neutralizing antibodies. Antigenic analysis showed that TH DDV, TH DV, TH DDD and TH VVV sera recognize the common antigenic site AS1. Antibodies directed to AS1 contribute to the largest proportion of the neutralizing activity of these immune sera. Residues 188 and 193 in AS1 are the key residues which are responsible for neutralization breadth of the immune sera. Interestingly, residues 188 and 193 locate in classical antigen sites but are relatively conserved among the 16 tested strains and 1,663 HA sequences from NCBI database. Thus, our results strongly indicate that it is feasible to develop broad cross-H5 influenza vaccines against HA head

Pulmonary DWCNT Exposure Causes Sustained Local and Low-Level Systemic Inflammatory Changes in Mice

Carbon nanotubes (CNTs) represent promising vectors to facilitate cellular drug delivery and to overcome biological barriers, but some types may also elicit persistent pulmonary inflammation based on their fibre characteristics. Here, we show the pulmonary response to aqueous suspensions of block copolymer dispersed, double-walled carbon nanotubes (DWCNT, length 1–10 lm) in mice by bronchoalveolar lavage (BAL) analysis, and BAL and blood cytokine and lung antioxidant profiling. The intratracheally instilled dose of 50 lg DWCNT caused significant pulmonary inflammation that was not resolved during a 7- day observation period. Light microscopy investigation of the uptake of DWCNT agglomerates revealed no particle ingestion for granulocytes, but only for macrophages. Accumulating macrophage, multinucleated macrophage and lymphocyte numbers in the alveolar region further indicated ineffective resolution with chronification of the inflammation. The local inflammatory impairment of the lung was accompanied by pulmonary antioxidant depletion and haematological signs of systemic inflammation. While the observed inflammation during its acute phase was dominated by neutrophils and neutrophil recruiting cytokines, the contribution of macrophages and lymphocytes with related cytokines became more significant after day 3 of exposure. This study confirms that acute pulmonary toxicity can occur on exposure of high doses of DWCNT agglomerates and offers further insight for improved nanotube design parameters to avoid potential long-term toxicity

Residue polymorphism in AS1 at positions 158,159,160,188 and 193 among 16 strains.

<p>Residue polymorphism in AS1 at positions 158,159,160,188 and 193 among 16 strains.</p

IC₅₀ values of TH DDV, TH DDD, TH VVV and TH DV sera against head/stem chimera and parental viruses.

<p>IC₅₀ values of TH DDV, TH DDD, TH VVV and TH DV sera against head/stem chimera and parental viruses.</p

The panel of HPAI H5N1 pseudotypes used in current study^a.

<p>The panel of HPAI H5N1 pseudotypes used in current study<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176854#t001fn001" target="_blank">^a</a>.</p

The number and percentage of different groups in 1,663 H5HA sequences.

<p>The number and percentage of different groups in 1,663 H5HA sequences.</p

Antigenic sites recognized by TH DDV, TH DDD, TH VVV and TH DV sera.

<p>(A) Location and classification of the amino acids in the antigenic sites on TH HA and HK5052 HA. The different residues in the antigenic sites between HK5052 HA and TH HA are shown in red. (B) Structural and spatial modeling of the four antigenic sites on an H5N1 influenza strain A/Vietnam/1203/2004 HA (PDB: 2FKO). (C) IC₅₀ values of TH DDV, TH DDD, TH VVV and TH DV sera against chimera and parental viruses. Data collected from three independent experiments are presented and best fit values for IC₅₀ along with the 95% Confidence Intervals.</p