Blood-Brain Barrier Cellular Responses Toward Organophosphates: Natural Compensatory Processes and Exogenous Interventions to Rescue Barrier Properties

Organophosphorus compounds (OPs) are highly toxic chemicals widely used as pesticides (e.g., paraoxon (PX)- the active metabolite of the insecticide parathion) and as chemical warfare nerve agents. Blood-brain barrier (BBB) leakage has been shown in rodents exposed to PX, which is an organophosphate oxon. In this study, we investigated the cellular mechanisms involved in BBB reaction after acute exposure to PX in an established in vitro BBB system made of stem-cell derived, human brain-like endothelial cells (BLECs) together with brain pericytes that closely mimic the in vivo BBB. Our results show that PX directly affects the BBB in vitro both at toxic and non-toxic concentrations by attenuating tight junctional (TJ) protein expression and that only above a certain threshold the paracellular barrier integrity is compromised. Below this threshold, BLECs exhibit a morphological coping mechanism in which they enlarge their cell area thus preventing the formation of meaningful intercellular gaps and maintaining barrier integrity. Importantly, we demonstrate that reversal of the apoptotic cell death induced by PX, by a pan-caspase-inhibitor ZVAD-FMK (ZVAD) can reduce PX-induced cell death and elevate cell area but do not prevent the induced BBB permeability, implying that TJ complex functionality is hindered. This is corroborated by formation of ROS at all toxic concentrations of PX and which are even higher with ZVAD. We suggest that while lower levels of ROS can induce compensating mechanisms, higher PX-induced oxidative stress levels interfere with barrier integrity

The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC

Natural Killer Receptor 1 Dampens the Development of Allergic Eosinophilic Airway Inflammation

<div><p>The function of NCR1 was studied in a model of experimental asthma, classified as a type 1 hypersensitivity reaction, in mice. IgE levels were significantly increased in the serum of OVA immunized NCR1 deficient (<i>NCR1</i>^{<i>gfp/gfp</i>}) mice in comparison to OVA immunized wild type <i>(NCR1</i>^<i>+/+</i>) and adjuvant immunized mice. Histological analysis of OVA immunized <i>NCR1</i>^{<i>gfp/gfp</i>} mice revealed no preservation of the lung structure and overwhelming peribronchial and perivascular granulocytes together with mononuclear cells infiltration. OVA immunized <i>NCR</i>^<i>+/+</i> mice demonstrated preserved lung structure and peribronchial and perivascular immune cell infiltration to a lower extent than that in <i>NCR1</i>^{<i>gfp/gfp</i>} mice. Adjuvant immunized mice demonstrated lung structure preservation and no immune cell infiltration. OVA immunization caused an increase in PAS production independently of NCR1 presence. Bronchoalveolar lavage (BAL) revealed NCR1 dependent decreased percentages of eosinophils and increased percentages of lymphocytes and macrophages following OVA immunization. In the OVA immunized <i>NCR1</i>^{<i>gfp/</i>gfp} mice the protein levels of eosinophils’ (CCL24) and Th2 CD4⁺ T-cells’ chemoattractants (CCL17, and CCL24) in the BAL are increased in comparison with OVA immunized <i>NCR</i>^<i>+/+</i> mice. In the presence of NCR1, OVA immunization caused an increase in NK cells numbers and decreased NCR1 ligand expression on CD11c⁺GR1⁺ cells and decreased NCR1 mRNA expression in the BAL. OVA immunization resulted in significantly increased IL-13, IL-4 and CCL17 mRNA expression in <i>NCR1</i>^<i>+/+</i> and <i>NCR1</i>^{<i>gfp/</i>gfp} mice. IL-17 and TNFα expression increased only in OVA-immunized <i>NCR1</i>^<i>+/+</i>mice. IL-6 mRNA increased only in OVA immunized <i>NCR1</i>^{<i>gfp/gfp</i>} mice. Collectively, it is demonstrated that NCR1 dampens allergic eosinophilic airway inflammation.</p></div

NCR1 involvement in chemokine and Th2, type immune cytokines in response to allergic airway inflammation.

<p><i>NCR1</i>^<i>+/+</i> and <i>NCR1</i>^{<i>gfp/gfp</i>} mice were immunized with either OVA or adjuvant as described in Material and Methods, and 24 h following the last challenge the lungs were harvested and taken for RT-PCR. The bar graph represents levels of cytokine mRNA following either adjuvant or OVA immunization. Levels of cytokine mRNA were analyzed by RT-PCR and calibrated to mRNA level of HPRT (n = 3–7). (A) IL-4, (B) IL-13, (C) CCL17 (D) IL-17; (E). TNFα; (F) IL-6. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the adjuvant immunized <i>NCR1</i>^<i>+/+</i> group ± SD (two-tailed Student <i>t</i>-test).</p

A decrease in NCR1 mRNA expression and increase of NCR1 ligand expression in the BAL of OVA immunized mice.

<p><i>NCR</i>^<i>+/+</i> and <i>NCR</i>^{<i>gfp/gfp</i>} mice were immunized with either adjuvant alone or with OVA/alum as described in the Material and Methods section. The mice were euthanized and the lung tissue was dissociated into single cells. (A) Total number of live immune cell. (B) NK cell number as determined by anti NK1.1 antibody. (C) qRT PCR performed with <i>NCR1</i> appropriate primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.s006" target="_blank">S1 Table</a>). (D) Five populations of cells carrying or lacking CD11c or GR1 were determined and stained with LY94-Ig fusion protein. *p > 0.05.</p

Increased allergic airway inflammation in the lungs of <i>NCR</i>^{<i>gfp/gfp</i>} mice.

<p><i>Ncr1</i>^<i>+/+</i> and <i>Ncr1</i>^{<i>gfp/gfp</i>} <i>C57Bl/6</i> mice were i.p. immunized with either OVA or adjuvant on days 0 and 14. Ten days after the second immunization, mice were challenged twice intranasally with OVA at days 24 and 27. 24 h following the second challenge, BALF was taken from each mouse and used in ELISA to detect levels of (A) CCL24 (n = 8 to 11), (B) CCL17 (n = 9 to 10) and CCL22 (n = 9–10). Results are the summary of 3 independent experiments and were normalized according to the OVA <i>NCR1</i>^<i>+/+</i> ± SEM group average that was considered as 1 in each experiment. **<i>p</i><0.01, ***<i>p<</i>0.001 (two-tailed Student <i>t-</i>test).</p

Increased inflammation in the lungs of <i>NCR1</i>^{<i>gfp/gfp</i>} mice (H&E+PAS x270).

<p>Mice were immunized and samples were process as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.g002" target="_blank">Fig 2</a>. (A) OVA immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x270). (B) Alum immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x270).</p

Increased inflammation in the lungs of <i>NCR1</i>^{<i>gfp/gfp</i>} mice H&E+PAS x180).

<p><i>NCR1</i>^{<i>gfp/gfp</i>} C57Bl/6 mice were intraperitoneally (i.p) immunized with either OVA/alum or adjuvant only on days 0 and 14. Ten days after the second immunization mice were challenged twice intranasally with OVA at days 24 and 27. Twenty-four hours following the second immunization mice were euthanized and the left lungs were harvested for histology and RT-PCR. Representative Hematoxylin& Eosin (H&E) and PAS stained sections from each group are shown. Inflammation severity was assessed blindly by the pathologist. (A) OVA/alum immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x180). (B) Alum immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x180).</p

NCR1 is involved in eosinophil and macrophage infiltration to the lung.

<p><i>NCR1</i>^<i>+/+</i> (Adjuvant n = 8, OVA n = 10) and <i>NCR1</i>^{<i>gfp/gfp</i>} (adjuvant n = 10, OVA n = 11) mice were immunized with either OVA or adjuvant as described in the Materials and Methods section. (A) The BAL was lavaged from each mouse, stained with PI and analyzed by flow cytometry for live cell count. The bar graph represents normalized number to the adjuvant immunized <i>NCR1</i>^<i>+/+</i> group whose average was considered as 1 of live cells in the BAL. (B-E) The bar graph represents an average percentage of eosinophils (B), macrophages (C), lymphocytes (D), and neutrophils (E) in the lung BAL (percentage of cells ± SEM; two-tailed Student <i>t</i>-test). These are the combined results of 2 experiments performed at different time points. * p < 0.05, **p < 0.01 *** p < 0.001.</p

Increased inflammation in the lungs of <i>NCR1</i>^{<i>gfp/gfp</i>} mice compared to <i>NCR1</i>^<i>+/+</i> mice (H&E+PAS x340).

<p>Mice were immunized and samples were process as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.g002" target="_blank">Fig 2</a>. (A) OVA immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x340). (B) OVA/Alum immunized <i>NCR</i>^<i>+/+</i> (H&E+PAS x340). A thick arrow with a short tail identifies mononuclear cells and thin arrow with a long tail identifies polymorphonuclear cells.</p