Correction to: Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease.

[This corrects the article DOI: 10.1186/s12953-018-0131-y.].

Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE-/-) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001). Conclusion: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.

Effect of 2-amino-9H-pyrido[2,3-b]indole (AalphaC), a carcinogenic heterocyclic amine present in food, on atherosclerotic plaque development in apoE deficient mice.

There is experimental and epidemiological evidence demonstrating that polycyclic aromatic hydrocarbons (PAHs) are involved in the pathogenesis of cardiovascular diseases. However, heterocyclic amines (HAs), a class of carcinogenic compounds present in food, which share many biochemical features with PAHs, have not received much attention. Previous reports have shown that the heterocyclic amine 2-amino-9H-pyrido[2,3-b]indole (AalphaC) binds and metabolically affects endothelial cells in animal models suggesting a potential role in vascular remodeling. The present study investigates the effect of exposure to HAs on atherosclerotic plaque development in the apoE(-/-) mice. We observed that animals treated with AalphaC developed atherosclerotic lesions characterized by lower lipid content but richer in inflammatory cells and collagen content when compared with control animals. Moreover, atherosclerotic plaques from AalphaC-treated apoE(-/-) mice were also smaller with a marked reduction in the tunica media thickness. Furthermore, total cholesterol levels were significantly reduced in AalphaC-treated apoE(-/-) mice. In contrast to what has been previously reported for PAHs, we provide for the first time evidence that HAs may protect against cardiovascular disease by inducing stable atherosclerotic plaques and reducing circulating cholesterol levels. These results open new avenues to further investigate the role of these food-borne carcinogens in cardiovascular physiology and pathology.

Comparison of MMP-2 and MMP-9 secretion from T helper 0, 1 and 2 lymphocytes alone and in coculture with macrophages.

Metalloproteinases (MMPs) participate in extracellular matrix remodelling and regulatory signalling during chronic inflammatory states such as atherosclerosis formation. However, the sources and mediators of MMP upregulation need clarification. We investigated whether proinflammatory mouse T helper type 1 (Th1) lymphocytes are more active in MMP secretion than naïve Th0 or anti-inflammatory Th2 phenotypes, in the absence of specific antigenic stimulation, under baseline conditions and after contact with irradiated macrophages. We also compared the effect of Th0, Th1 or Th2 lymphocyte-conditioned medium and irradiated lymphocytes on MMP production from macrophages. Finally, we investigated whether CD40-CD40 ligand (CD40L) interactions were involved in T-cell-stimulated MMP secretion from macrophages. Under baseline conditions, MMP-2 messenger RNA (mRNA) and protein levels were greater in Th1 than Th0 or Th2 lymphocytes; MMP-9 mRNA, but not protein, was also upregulated. In the presence of irradiated macrophages MMP-2 and MMP-9 production from Th1 and Th2 was greater than from Th0 lymphocytes. Conditioned media from Th1 but not Th0 or Th2 cells increased MMP-9 secretion from macrophages. Irradiated Th1 lymphocytes stimulated both MMP-2 and MMP-9 secretion from macrophages more than irradiated Th2 or Th0 cells; this activation was independent of CD40-CD40L interaction. These findings demonstrate for the first time greater MMP secretion by Th1 than Th2 or Th0 lymphocytes and their greater ability to upregulate macrophage MMP secretion in the absence of specific antigenic stimulation. These mechanisms could promote matrix turnover in inflammatory states and, for example, promote atherosclerotic plaque rupture.

Antigen-induced immunomodulation in the pathogenesis of atherosclerosis.

Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of monocytes/macrophages, smooth muscle cells, and lymphocytes within the arterial wall in response to the release of proinflammatory molecules. Such accumulation results in the formation of the atherosclerotic plaque, which would eventually evolve to complications such as total artery occlusion, rupture, calcification, or aneurysm. Although the molecular mechanism responsible for the development of atherosclerosis is not completely understood, it is clear that the immune system plays a key role in the development of the atherosclerotic plaque and in its complications. There are multiple antigenic stimuli that have been associated with the pathogenesis of atherosclerosis. Most of these stimuli come from modified self-molecules such as oxidised low-density lipoproteins (oxLDLs), beta2glycoprotein1 (beta2GP1), lipoprotein a (LP(a)), heat shock proteins (HSPs), and protein components of the extracellular matrix such as collagen and fibrinogen in the form of advanced glycation-end (AGE) products. In addition, several foreign antigens including bacteria such as Porphyromonas gingivalis and Chlamydia pneumoniae and viruses such as enterovirus and cytomegalovirus have been associated with atherosclerosis as potentially causative or bystander participants, adding another level of complexity to the analysis of the pathophysiology of atherosclerosis. The present review summarises the most important scientific findings published within the last two decades on the importance of antigens, antigen stimulation, and adaptive immune responses in the development of atherosclerotic plaques.

CD4+ T lymphocyte subsets express connexin 43 and establish gap junction channel communication with macrophages in vitro.

Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary lymphoid organ-derived lymphocytes. These channels in the plasma membrane play key roles in a range of lymphocyte functions exemplified by the synthesis and secretion of Igs and cytokines and during transmigration across the endothelium. Most recently, their involvement in antigen cross-presentation has also been established. We report here for the first time the expression of mRNA and protein encoding Cx43 in mouse-derived CD4+ Th0, Th1, and Th2 lymphocyte subpopulations and demonstrate the establishment gap junction channel formation with primary macrophages in vitro. We show that this mode of direct communication is particularly favored in Th1-macrophage interactions and that LPS inhibits lymphocyte-macrophage cross-talk independently of the subset of lymphocyte involved. Our work suggests that gap junction-mediated communication can be modulated in the absence of specific antigenic stimulation. Therefore, a further mechanism featuring gap junction-mediated communication may be implicated in immune regulation.

Interactions of 12-lipoxygenase with phospholipase A2 isoforms following platelet activation through the glycoprotein VI collagen receptor.

Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethylphosphocholine , but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A2 (PLA2) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA2 (sPLA2). Finally, venoms containing sPLA2 acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA2 and that 12-LOX functionally associates with both PLA2 isoforms.

Immune responses elicited by apoB-100-derived peptides in mice.

Peptides derived from apolipoprotein B (apoB)-100 have been previously used in vaccine preparations to treat atherosclerosis. Such vaccines have been shown to reduce atherosclerotic plaque development by 50 % in experimental animals, and this effect is associated with induction of T helper (Th)2 immune responses. In this study we immunised apolipoprotein E-deficient (apoE(-/-)) mice with apoB-100-derived peptides P2, P45 and P210. Animals received BSA-conjugated peptides or peptide-loaded bone marrow-derived dendritic cells (DCs). We used enzyme-linked immunosorbent assays to assess the synthesis of anti-peptide-specific IgG1 and IgG2a as well as the levels of interleukin (IL-)10 and interferon gamma (IFN-γ) in plasma of immunised animals. We also measured the effect of immunisation on the number of spleen-derived CD4(+) and CD8(+) regulatory T cells (Tregs) in these animals. Peptide and peptide-loaded DC immunisation significantly increased the levels of peptide-specific immunoglobulins and the number of Tregs in apoE(-/-) mice. This was accompanied by a significant increase in the secretion of IL-10 with no effect on IFN-γ levels. The results also show that the peptides can modulate the homing properties of DCs. Altogether, this study provides novel evidence for the immune mechanisms excerpted by apoB-100-derived peptides and their effect on Tregs and DCs relevant to their use in vaccine preparations.

Influenza vaccination promotes stable atherosclerotic plaques in apoE knockout mice.

OBJECTIVE: Current evidence suggests a relationship between seasonal Influenza viral infection and cardiovascular disease (CVD). Experimental animals inoculated with Influenza A virus have shown to develop thrombotic complications similar to those seen in humans. Conversely, several epidemiological studies and clinical trials have suggested that Influenza vaccination may have a protective effect on CVD. However, the potential mechanisms behind this protective effect remain unstudied. We aimed to study the effect of Influenza vaccination on atherosclerotic plaque development in apoE(-/-) mice. METHODS AND RESULTS: The effect of immunization with increasing doses of Influenza vaccine (0.38, 1.8, 9 and 45 µg/0.5 mL Vaxigrip®, Sanofi-Aventis) on atherogenesis was compared with that of animals immunized with Pneumo23® (pneumococcus vaccine, Sanofi-Aventis) and control group inoculated with phosphate buffered saline (PBS). Animals vaccinated with 45 µg/0.5 mL Vaxigrip®, (the same dose used to immunize humans adults against Influenza) developed smaller atherosclerotic lesions with lower lipid content but richer in smooth muscle cells and collagen when compared with control animals. Concomitantly, they showed lower levels of interferon gamma (IFNγ), interleukin (IL)-2 and tumor necrosis factor alpha (TNFα) but higher levels of IL-4. Furthermore, we found increased levels of anti-Influenza immunoglobulin (Ig) G1 or anti-Pneumo23® IgM specific antibodies in a time and dose dependent fashion in animals immunized with these vaccines. CONCLUSIONS: These results indicate that vaccination against Influenza may protect against the development of CVD by promoting smaller and stable atherosclerotic plaques and by inducing atheroprotective immune responses.

The effect of Chlamydophila pneumoniae Major Outer Membrane Protein (MOMP) on macrophage and T cell-mediated immune responses.

The Major Outer Membrane Protein (MOMP) belongs to the membrane complex of cysteine-rich proteins of Chlamydophila pneumoniae. Although MOMP can elicit strong immune responses it fails to confer long-term protection against infection in animal models. This effect has been attributed, at least in part, to an inadequate induction of protective Th1-mediated immune responses. In an effort to understand the cellular mechanisms associated to the immunomodulatory properties of MOMP we studied the effect of this protein on mouse macrophages and naïve T-lymphocytes. We found that incubation of mouse macrophages with recombinant MOMP (rMOMP) results in an increased secretion of MMP-9 and a down-regulation of MHC class II, CD86 and CD40. This was accompanied by an increase in IL-10 and IFNγ but not in IL-12 secretion. rMOMP induced a down-regulation of the expression of CD69 and CD154 markers by activated CD4(+) T cells, and enhanced the secretion of IL-2 and IL-10 by these cells. Conversely, rMOMP-treated macrophages up-regulated the expression of CD69 but not CD154, inhibited the synthesis of IL-10 and up-regulated the production of IFNγ by activated CD8(+) T cells. Immunization of mice with MOMP induced the synthesis only of MOMP-specific IgG1 but no differences in cytokine profile were observed compared to controls. Our results provide new evidence on the role of MOMP in modulating T cell-mediated immune responses.

Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase.

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

Gene expression alterations of human peripheral blood monocytes induced by medium-term treatment with the TH2-cytokines interleukin-4 and -13.

The TH2-cytokines interleukins-4 and -13 severely alter gene expression of monocytic cells. We quantified the impact of interleukins-4 and -13 on the gene expression pattern of human peripheral blood monocytes applying a strategy that involved microarray hybridization, RT-PCR, immunohistochemistry and activity assays. After 3 days of continuous cytokine exposure the six most strongly upregulated gene products (15-lipoxygenase-1, fibronectin, monoamine oxidase-A, CD1c, CD23A, coagulation factor XIII) included four proteins with potential anti-inflammatory properties: (i) 15-lipoxygenase-1 (290-fold upregulation), (ii) fibronectin (180-fold upregulation), (iii) monoamine oxidase-A (56-fold upregulation) and (iv) coagulation factor XIII (35-fold upregulation). In addition, a number of other gene products, the expression of which is consistent with inflammatory resolution (annexin 1, collagen 1alpha2, laminin alpha5, TIMP3, heme oxygenase-1, CCL22, heat shock protein A8), were upregulated to a lower extent. In contrast, expression of classical pro-inflammatory gene products, such as tumor necrosis factor alpha, monocyte chemotactic protein-1, interleukins-1, -6, -8, -18, cyclooxygenase-2, as well as enzymes and receptors of the leukotriene cascade (5-lipoxygenase, 5-lipoxygenase activating protein, leukotriene B(4) receptor, cysteinyl leukotriene receptor 2) were significantly downregulated. These data suggest that medium-term treatment of human peripheral blood monocytes with interleukins-4/13 alters the gene expression pattern so that the cells might adopt a resolving phenotype.

Elevated endothelial nitric oxide bioactivity and resistance to angiotensin-dependent hypertension in 12/15-lipoxygenase knockout mice.

12/15-Lipoxygenase (12/15-LOX) plays a pathogenic role in atherosclerosis. To characterize whether 12/15-LOX also contributes to endothelial dysfunction and hypertension, regulation of vessel tone and angiotensin II (ang II) responses were characterized in mice deficient in 12/15-LOX. There was a twofold increase in the magnitude of l-nitroarginine-methyl ester-inhibitable, acetylcholine-dependent relaxation or phenylephrine-dependent constriction in aortic rings isolated from 12/15-LOX(-/-) mice. Plasma NO metabolites and aortic endothelial NO synthase (eNOS) expression were also elevated twofold. Angiotensin II failed to vasoconstrict 12/15-LOX(-/-) aortic rings in the absence of L-nitroarginine-methyl ester, and ang II impaired acetylcholine-induced relaxation in wild-type, but not 12/15-LOX(-/-) rings. In vivo, 12/15-LOX(-/-) mice had similar basal systolic blood pressure measurements to wild type, however, blood pressure elevations in response to ang II infusion (1.1 mg/kg/day) were significantly attenuated (maximal pressure, 143.4 +/- 4 mmHg versus 122.1 +/- 5.3 mmHg for wild type and 12/15-LOX(-/-), respectively). In contrast, vascular hypertrophic responses to ang II, and ang II type 1 receptor (AT1-R) expression were similar in both strains. This study shows that 12/15-LOX(-/-) mice have increased NO biosynthesis and impaired ang II-dependent vascular responses in vitro and in vivo, suggesting that 12/15-LOX signaling contributes to impaired NO bioactivity in vascular disease in vivo.