Loss of Proliferation and Antigen Presentation Activity following Internalization of Polydispersed Carbon Nanotubes by Primary Lung Epithelial Cells

Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells

Uptake of fluorescence tagged AF-SWCNTs by PMs and PLE cells.

<p>Peritoneal macrophages and PLE cells were cultured in 48 well plates in presence of fluorescent tagged AF-SWCNTs (5 µg/ml). At different time periods, cells were washed with PBS and harvested by trypsinization. Percentage of cells positive for AF-SWCNTs was determined by using a flow cytometer. Each point represents Mean ±SEM of values obtained from 3 replicate assays.</p

Effect of AF-SWCNTs on antigen presentation activity of PMs and PLE cells.

<p>Peritoneal macrophages (panel A) and PLE cells (panel B) (2×10⁵) were cultured with BCG sonicate (sBCG, equivalent to a MOI of 100∶1) with or without AF-SWCNTs (50 µg/ml) for 24 h. Excess antigen and particles were removed by washing and fixation was performed with glutaraldehyde and quenched with L-lysine. CD4⁺ T cells (3×10⁵ cells, purity >98%) isolated from spleens of BCG infected mice were added to the wells containing BCG pulsed PMs or PLE cells. The culture supernatants were collected after 24 h and the amount of IL-2 determined by ELISA. Each value represents Mean ± SEM of IL2 levels in 3 replicate assay wells. *(p<0.05).</p

In vivo Uptake of fluorescence tagged AF-SWCNTs by lung epithelial cells and bronchoalveolar lavage (BAL) cells.

<p>Fluorescenated AF-SWCNTs (20 µg) were intratracheally administered in C57Bl6 mice. After 2, 24 and 48 h of the instillation of AF-SWCNTs, BAL cells were harvested and lung tissue processed for isolation of PLE cells. Uptake of AF-SWCNTs as percentage of cells positive for AF-SWCNT fluorescence was assessed flow cytometrically in PLE cells (left panel) and BAL cells (right panel). Each value represents Mean ± SEM of observations from 4 mice.</p

Effect of cytochalasin D on the uptake of AF-SWCNTs by PMs and PLE cells.

<p>PMs and PLE cells were cultured in 48 well culture plates in the presence or absence of 2.5 µg/ml cytochalasin D for 1 h. Fluorescence probe tagged AF-SWCNTs 5 µg/ml) were added and incubation continued for an additional 24 h. At the end of the incubation period, cells were washed with PBS, harvested by trypsinization and analyzed on FACS. Representative flow histograms for AF-SWCNTs uptake in control and cytolchalasin D treated PMs and PLE cells are shown in panel A where cells in M2 window represent AF-SWCNT⁺cells. Bar histograms in the lower panel represents Mean ± SEM of results obtained from 3 independent experiments.</p

Effect of AF-SWCNTs on the recovery of PMs and PLE cells in culture.

<p>PMs and PLE cells were cultured in 48 well plate with or without AF-SWCNTs (10 or 50 µg/ml). After 24, 48 and 72 h, cells were washed, detached by trypsinization and suspended in 0.2% trypan blue solution in PBS. Recoveries of viable cell numbers of PMs (panel A) and PLE cells (panel B) were assessed by using a hemocytometer. Each point represents Mean ±SEM values obtained from 3 replicate assay wells. *p<0.05 for difference between control and AF-SWCNTs treated cell cultures.</p

Examination by confocal microscopy of uptake of fluorescence tagged AF-SWCNTs by PMs and PLE cells.

<p>PMs and PLE cells were cultured on glass cover slip. After one day cells were washed with complete media and continued in culture for 2 more days. Fluorescence labeled AF-SWCNTs (5 µg/ml) were added to cell cultures and after 24 h cells were washed with PBS, fixed with paraformaldehyde, washed twice with quencher (Ammonium chloride) and examined by Confocal Laser Scanning Microscope. Two z-sections each of PLE cells (top two panels) and PMs (bottom two panels) show the presence of fluorescenated AF-SWCNTs in cytoplasm. (Magnification 60×).</p

Effect of AF-SWCNTs on PLE cell cycle.

<p>Freshly isolated PLE cells were cultured in 6-well plate with or without of AF-SWCNTs (50 µg/ml). After 24 h, cells were washed and isolated by trypsinization. Cell were fixed, treated with RNase and stained with propidium iodide for flow cytometric analysis.. Data was analyzed by using Modfit software that enumerated proportion of cells in G1/Go phase (left peaks in all histograms), S phase (cross hatched peaks) and G2/M phase (right dark peaks in all histograms).</p

Intranasal curcumin and its evaluation in murine model of asthma

Curcumin, a phytochemical present in turmeric, rhizome of Curcuma longa, has been shown to have a wide variety of pharmacological activities including anti-inflammatory, anti-allergic and anti-asthmatic properties. Curcumin is known for its low systemic bioavailability and rapid metabolization through oral route and has limited its applications. Over the recent decades, the interest in intranasal delivery as a non-invasive route for drugs has increased as target tissue for drug delivery since nasal mucosa offers numerous benefits. In this study, we evaluated intranasal curcumin following its absorption through nasal mucosa by a sensitive and validated high-performance liquid chromatography (HPLC) method for the determination of intranasal curcumin in mouse blood plasma and lung tissue. Intranasal curcumin has been detected in plasma after 15 min to 3 h at pharmacological dose (5 mg/kg, i.n.), which has shown anti-asthmatic potential by inhibiting bronchoconstriction and inflammatory cell recruitment to the lungs. At considerably lower doses has proved better than standard drug disodium cromoglycate (DSCG 50 mg/kg, i.p.) by affecting inflammatory cell infiltration and histamine release in mouse model of asthma. HPLC detection revealed that curcumin absorption in lungs has started after 30 min following intranasal administration and retained till 3 h then declines. Present investigations suggest that intranasal curcumin (5.0 mg/kg, i.n.) has effectively being absorbed and detected in plasma and lungs both and suppressed airway inflammations at lower doses than the earlier doses used for detection (100–200 mg/kg, i.p.) for pharmacological studies (10–20 mg/kg, i.p.) in mouse model of asthma. Present study may prove the possibility of curcumin as complementary medication in the development of nasal drops to prevent airway inflammations and bronchoconstrictions in asthma without any side effect

<i>M. tuberculosis</i>induces miR-155 in macrophages

Mycobacterium tuberculosis (M.tb.) replicates in host macrophages to cause tuberculosis. We have investigated the role of miRNAs in M.tb.-infected murine RAW264.7 cells and bone marrow-derived macrophages (BMDMs), focusing on miR-155, the most highly upregulated miRNA. We observed that miR-155 upregulation is directly linked to the attenuation of expression of BTB and CNC homology 1 (Bach1) and SH2-containing inositol 5'-phosphatase (SHIP1). Bach1 is a transcriptional repressor of haem oxygenase-1 (HO-1), whereas SHIP1 inhibits the activation of the serine/threonine kinase AKT. We hypothesize that M.tb.-induced miR-155 induction leads to repression of Bach1, which augments the expression of HO-1, a documented activator of the M.tb. dormancy regulon. SHIP1 repression facilitates AKT activation, which is required for M.tb. survival. In addition, M.tb.-induced miR-155 inhibits expression of cyclooxygenase-2 (Cox-2) and interleukin-6 (Il-6), two modulators of the innate immune response. Importantly, we observed that the virulence-associated secreted protein ESAT-6 plays a key role in miR-155 induction and its subsequent effects on Bach1 and SHIP1 repression. Inhibition of miR-155 hindered survival of M.tb. in RAW264.7 and in murine BMDMs. Thus, our results offer new insights into the role of miRNAs in modulation of the host innate immune response by M.tb. for its own benefit