NADPH Oxidase-Derived Peroxynitrite Drives Inflammation in Mice and Human Nonalcoholic Steatohepatitis via TLR4-Lipid Raft Recruitment

Abstract

The molecular events that link NADPH oxidase activation and the induction of Toll-like receptor (TLR)-4 recruitment into hepatic lipid rafts in nonalcoholic steatohepatitis (NASH) are unclear. We hypothesized that in liver, NADPH oxidase activation is key in TLR4 recruitment into lipid rafts, which in turn up-regulates NF-κB translocation to the nucleus and subsequent DNA binding, leading to NASH progression. Results from confocal microscopy showed that liver from murine and human NASH had NADPH oxidase activation, which led to the formation of highly reactive peroxynitrite, as shown by 3-nitrotyrosine formation in diseased liver. Expression and recruitment of TLR4 into the lipid rafts were significantly greater in rodent and human NASH. The described phenomenon was NADPH oxidase, p47phox, and peroxynitrite dependent, as liver from p47phox-deficient mice and from mice treated with a peroxynitrite decomposition catalyst [iron(III) tetrakis(p-sulfonatophenyl)porphyrin] or a peroxynitrite scavenger (phenylboronic acid) had markedly less Tlr4 recruitment into lipid rafts. Mechanistically, peroxynitrite-induced TLR4 recruitment was linked to increased IL-1β, sinusoidal injury, and Kupffer cell activation while blocking peroxynitrite-attenuated NASH symptoms. The results strongly suggest that NADPH oxidase-mediated peroxynitrite drove TLR4 recruitment into hepatic lipid rafts and inflammation, whereas the in vivo use of the peroxynitrite scavenger phenylboronic acid, a novel synthetic molecule having high reactivity with peroxynitrite, attenuates inflammatory pathogenesis in NASH.

Figure 1

NADPH oxidase activation and peroxynitrite-mediated tyrosine nitration. A and B: Representative images of 5-μm-thick immunostained liver sections, imaged using immunofluorescence microscopy. Nuclear stain (blue) is due to ProLong Gold Antifade Reagent (Life Technologies, Carlsbad, CA) with DAPI. A: Secondary antibodies used were Alexa Fluor 568 (Invitrogen; red) against α-gp91phox primary antibody and Alexa Fluor 488 (green) against α-p47phox primary antibody. Yellow spots/patches indicate alignment of cytoplasmic subunit of NADPH oxidase, p47phox (red), with membrane subunit glycoprotein (gp)91phox (green). B: Secondary antibody used was Alexa Fluor 633 (red) against α-3-nitrotyrosine primary antibody. C and D: Morphometry performed on three independent fields for each sample, including representative images in A for gp91phox/p47phox colocalization events per 300 cells (C) and in B for percentage of area showing positive immunoreactivity of 3-nitrotyrosine, calculated in arbitrary units (D). ^∗∗P < 0.01. Apo, diet-induced obesity wild-type (DIO) mouse exposed to bromodichloromethane (BDCM) and injected with apocynin; DIO + BDCM, DIO mouse exposed to BDCM; FBA, DIO mouse exposed to BDCM and phenylboronic acid; FeTPPS, DIO mouse exposed to BDCM and iron(III) tetrakis(p-sulfonatophenyl)porphyrin; p47phox KO, high-fat diet–fed mouse with p47phox gene knockout exposed to BDCM; Tlr4 KO, high-fat diet–fed mouse with Tlr4 knockout exposed to BDCM.

Figure 2

NADPH oxidase and peroxynitrite–driven Toll-like receptor (TLR)-4 recruitment into lipid rafts in hepatic cell membranes. A: Representative images of 5-μm-thick immunostained liver sections, imaged using immunofluorescence microscopy. Secondary antibodies used were Alexa Fluor 633 (Invitrogen; red) against α-Tlr4 primary antibody and Alexa Fluor 488 (green) against α-flotillin primary antibody. Nuclear stain (blue) is due to ProLong Gold Antifade Reagent (Life Technologies, Carlsbad, CA) with DAPI. Yellow spots/patches indicate alignment of Tlr4 (red), with lipid raft protein flotillin (green). B: Morphometry performed on three independent fields for each sample, including representative images in A for Tlr4–flotillin colocalization events per 300 cells. C: Confocal laser scan of Tlr4–flotillin colocalization in the membranes of liver cells with nonalcoholic steatohepatitis (NASH). D: Colocalization analysis using LSM image browser version 4.2.0.0121 (Carl Zeiss, Oberkochen, Germany). ^∗∗P < 0.01. Original magnification ×60 (A). Apo, diet-induced obesity wild-type (DIO) mouse exposed to bromodichloromethane (BDCM) and apocynin; DIO + BDCM, DIO mouse exposed to BDCM; FBA, DIO mouse exposed to BDCM and phenylboronic acid; FeTPPS, DIO mouse exposed to BDCM and iron(III) tetrakis(p-sulfonatophenyl)porphyrin; p47phox KO, high-fat diet–fed mouse with p47phox gene knockout exposed to BDCM.

Figure 3

NADPH oxidase and peroxynitrite–driven NF-κB translocation and DNA-binding via Toll-like receptor (Tlr)-4 signaling. A: Results of NF-κB activation assay in murine samples of toxin-induced nonalcoholic steatohepatitis (NASH) model. NF-κB activation index (assessed from nuclear translocation and DNA binding of p65 subunit) is normalized against diet-induced obesity wild-type mouse (DIO) sample. B: Results of NF-κB activation assay in murine samples of diet-induced NASH model and human samples. NF-κB activation index is normalized against appropriate and respective control samples. ^∗P < 0.05. BCDM, bromodichloromethane.

Figure 4

NADPH oxidase and peroxynitrite–driven hepatic sinusoidal injury via Toll-like receptor (Tlr)-4 signaling. A and B: Representative images of 5-μm-thick immunostained liver sections, imaged using immunofluorescence microscopy. Secondary antibody used was Alexa Fluor 568 (Invitrogen; red) against α-intercellular adhesion molecule (Icam)-1 primary antibody (A) and against α-E-selectin primary antibody (B) (both Icam-1 and E-selectin are sinusoidal injury markers). Nuclear stain (blue) is due to ProLong Gold Antifade Reagent (Life Technologies, Carlsbad, CA) with DAPI. C and D: Morphometry performed on three independent fields for each sample including representative images in for percentage of area showing positive immunoreactivity of Icam-1 in A (C) and of E-selectin in B (D), calculated in arbitrary units. ^∗∗P < 0.01. Original magnification, ×20 (A and B). Apo, diet-induced obesity wild-type (DIO) mouse exposed to bromodichloromethane (BDCM) and injected with apocynin; DIO + BDCM, DIO mouse exposed to BDCM; FBA, DIO mouse exposed to BDCM and phenylboronic acid; FeTPPS, DIO mouse exposed to BDCM and iron(III) tetrakis(p-sulfonatophenyl)porphyrin; p47phox KO, high-fat diet–fed mouse with p47phox gene knockout exposed to BDCM; Tlr4 KO, high-fat diet–fed mouse with Tlr4 knockout exposed to BDCM.

Figure 5

Toll-like receptor (Tlr)-4 signaling–mediated cytokine release and Kupffer cell activation. A–C: Representative images of 5-μm-thick immunostained liver sections, imaged using bright-field microscopy. Positive immunoreactivities (brown) are due to diaminobenzidine binding to respective biotinylated secondary antibodies via streptavidin–horseradish peroxidase. Nuclei (blue) counterstained with Mayer's hematoxylin solution. Immunoreactivities detected are for proinflammatory cytokines Il-1β (A), monocyte chemotactic protein (MCP)-1 (B), and Kupffer cell activation marker CD68 (C). D–F: Morphometry performed on three independent fields for each sample, including representative images for percentage of area showing positive immunoreactivity of Il-1β in A (D) and of MCP-1 in B (E) and CD68 in C (F), calculated in arbitrary units. ^∗∗P < 0.01. Original magnification, ×20 (A–C). DIO, diet-induced obesity wild-type mouse; DIO + BDCM, DIO mouse exposed to bromodichloromethane (BDCM); FeTPPS, DIO mouse exposed to BDCM and iron(III) tetrakis(p-sulfonatophenyl)porphyrin; p47phox KO, high-fat diet–fed mouse with p47phox gene knockout exposed to BDCM; Tlr4 KO, high-fat diet–fed mouse with Tlr4 knockout exposed to BDCM.

Figure 6

A–C: Representative images of 5-μm-thick immunostained liver sections, imaged using bright-field microscopy. Positive immunoreactivities (brown) are due to diaminobenzidine binding to respective biotinylated secondary antibodies via streptavidin–horseradish peroxidase. Nuclei (blue) counterstained with Mayer's hematoxylin solution. Immunoreactivities detected are for stellate cell proliferation marker α-smooth muscle actin (SMA). D–F: Morphometries performed on three independent fields for each sample including representative images for percentage of area showing positive immunoreactivity of α-SMA in A (D), B (E), and C (F), calculated in arbitrary units. ^∗P < 0.05, ^∗∗P < 0.01. Original magnification, ×20 (A–C). DIO, diet-induced obesity wild-type mouse; DIO + BDCM, DIO mouse exposed to bromodichloromethane (BDCM); FeTPPS, DIO mouse exposed to BDCM and iron(III) tetrakis(p-sulfonatophenyl)porphyrin; Hu Ctrl, healthy human control; Hu NASH, human nonalcoholic steatohepatitis; MCD, methionine- and choline-deficient diet–fed mouse; MCS, methionine- and choline-sufficient diet–fed mouse; p47phox KO, high-fat diet–fed mouse with p47phox gene knockout exposed to BDCM; Tlr4 KO, high-fat diet–fed mouse with Tlr4 knockout exposed to BDCM.