Multi-walled carbon nanotubes induce arachidonate 5-lipoxygenase expression and enhance the polarization and function of M1 macrophages <i>in vitro</i>

Fibrogenic carbon nanotubes (CNTs) induce the polarization of M1 and M2 macrophages in mouse lungs. Polarization of the macrophages regulates the production of proinflammatory and pro-resolving lipid mediators (LMs) to mediate acute inflammation and its resolution in a time-dependent manner. Here we examined the molecular mechanism by which multi-walled CNTs (MWCNTs, Mitsui-7) induce M1 polarization in vitro. Treatment of murine macrophages (J774A.1) with Mitsui-7 MWCNTs increased the expression of Alox5 mRNA and protein in a concentration- and time-dependent manner. The MWCNTs induced the expression of CD68 and that induction persisted for up to 3 days post-exposure. The expression and activity of inducible nitric oxide synthase, an intracellular marker of M1, were increased by MWCNTs. Consistent with M1 polarization, the MWCNTs induced the production and secretion of proinflammatory cytokines tumor necrosis factor-α and interleukin-1β, and proinflammatory LMs leukotriene B4 (LTB4) and prostaglandin E2 (PGE2). The cell-free media from MWCNT-polarized macrophages induced the migration of neutrophilic cells (differentiated from HL-60), which was blocked by Acebilustat, a specific leukotriene A4 hydrolase inhibitor, or LY239111, an LTB4 receptor antagonist, but not NS-398, a cyclooxygenase 2 inhibitor, revealing LTB4 as a major mediator of neutrophil chemotaxis from MWCNT-polarized macrophages. Knockdown of Alox5 using specific small hairpin-RNA suppressed MWCNT-induced M1 polarization, LTB4 secretion, and migration of neutrophils. Taken together, these findings demonstrate the polarization of M1 macrophages by Mitsui-7 MWCNTs in vitro and that induction of Alox5 is an important mechanism by which the MWCNTs promote proinflammatory responses by boosting M1 polarization and production of proinflammatory LMs.</p

Effect of diet on plasma amino acid levels (µM) in C57BL/6J and apoE^−/− mice.

<p>n = 4 for HF groups and n = 3 for all other groups. Values are means ± SE. *p<0.05 vs strain-specific standard diet.</p

Effect of HF and HC diets on plasma triglycerides and cholesterol.

<p>Values are means ± SE for n = 5 mice in each group. Key to symbols: apoE^−/− (open bars); C57BL/6J (black bars); *p<0.05 vs strain specific standard diet; ^$p<0.05 vs strain-specific HF diet; ^#p<0.05 for apoE^−/− vs C57BL/6J fed same diet.</p

Induction of tissue arginase activities in apoE^−/− mice on HC diet.

<p>Values are means ± SE for n = 6 in each group. *p<0.05 vs standard diet.</p

HF and HC diets reduce global arginine bioavailability.

<p>Values are means ± SE for n = 3–4 in each group. Abbreviations: ARG, arginine; ORN, ornithine; CIT, citrulline. Symbols are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015253#pone-0015253-g002" target="_blank">Figure 2</a> legend.</p

Effect of HF and HC diets on plasma levels of ALT.

<p>Values are means ± SE for n = 5 mice in each group. Symbols are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015253#pone-0015253-g002" target="_blank">Figure 2</a> legend.</p

Induction of arginase mRNA and protein in apoE^−/− mice on HC diet.

<p>(A) Effect of HC diet on arginase I and II mRNAs in heart, lung, spleen and kidney. mRNA levels are expressed relative to the levels in apoE^−/− mice on standard diet (arbitrarily set to 1.0 for each tissue and indicated by dotted line). Values are means ± SE for n = 5–7 in each group. *p<0.05 vs standard diet. (B) Effect of HC diet on arginase II protein abundance in lung, spleen and kidney. For each tissue, the upper panel represents arginase II and the lower panel GAPDH. Western blots of extracts from tissues of 5 representative animals on each diet are shown. Amounts of protein loaded in each lane were 10 µg (lung and kidney) or 25 µg (spleen). An extract of C57BL/6J whole kidney (20 µg for lung and kidney blots, 3 µg for spleen blot) was used in the first lane of each blot as positive control (+CT) for arginase II. Twenty µg protein from kidney of the arginase II knockout mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015253#pone.0015253-Shi1" target="_blank">[47]</a> was included in the second lane of the kidney blot in order to establish identity of the lowest band as arginase II. (C) Effect of HC diet on arginase I protein abundance in spleen. The upper panel represents arginase I and the lower panel GAPDH. The Western blot represents extracts from spleen (50 µg each lane) of 5 representative animals on each diet and 100 ng of C57BL/6J liver extract (+CT) as positive control. (D) Densitometry of Western blots, represented in arbitrary units (Arginase/GAPDH), was analyzed by Image Quant 5.2 Software. *p<0.01 vs standard diet. Molecular weights are indicated by the following symbols: solid triangles, 39 kDa; solid diamonds, 37 kDa; open triangles, 37 kDa.</p

Correlation between activities of ALT and arginase in plasma of apoE^−/− mice on different diets.

<p>Regression analysis of ALT and arginase activities for the different diets (n = 5 in each group) is indicated by the solid line. Key to symbols: solid circles, standard diet; solid diamonds, HF diet; open circles, HC diet.</p

Effect of HF and HC diets on plasma arginase activity (nmol/ml/min).

<p>Values are means ± SE for n = 10-15 for standard and HC diet and n = 6 for HF diet. Key to symbols: apoE^−/− (open bars); C57BL/6J (black bars); *p<0.05 vs standard diet; ^$p<0.05 vs strain-specific HF diet.</p

Additional file 1: of Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells

Figure S1. TEM of BEAS-2B cell exposed to 2.4 μg/mL MWCNT-7 for 24 h. The nuclear envelope is indicated by the black arrows. Some of the MWCNT that are enclosed in the cell cytoplasm are indicated by green arrows. Several of the indicated MWCNT at the top of the micrograph (larger green arrows) appear to be within membrane bound vesicles while other MWCNTs within the cell cytoplasm (small green arrows) at the bottom of the micrograph are not membrane bound. The single red arrow indicates a MWCNT within the nucleus. The MWCNT within the nucleus is not bound by a lipid membrane. Magnification bar is 2.5 μm. (TIF 948 kb