Shanmugam Nagarajan

Effects of intra-abdominal sepsis on atherosclerosis in mice.

IntroductionSepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis.MethodsWe performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE−/−) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE−/− mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation.ResultsApoE−/− mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (-6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05).ConclusionsUsing a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.

Anti‐Inflammatory Immune Skewing Is Atheroprotective: Apoe−/−FcγRIIb−/− Mice Develop Fibrous Carotid Plaques

Background Stroke, caused by carotid plaque rupture, is a major cause of death in the United States. Whereas vulnerable human plaques have higher Fc receptor (FcγR) expression than their stable counterparts, how FcγR expression impacts plaque histology is unknown. We investigated the role of FcγRIIb in carotid plaque development and stability in apolipoprotein (Apo)e−/− and Apoe−/−FcγRIIb−/− double knockout (DKO) animals. Methods and Results Plaques were induced by implantation of a shear stress‐modifying cast around the carotid artery. Plaque length and stenosis were followed longitudinally using ultrasound biomicroscopy. Immune status was determined by flow cytometry, cytokine release, immunoglobulin G concentration and analysis of macrophage polarization both in plaques and in vitro. Surprisingly, DKO animals had lower plaque burden in both carotid artery and descending aorta. Plaques from Apoe−/− mice were foam‐cell rich and resembled vulnerable human specimens, whereas those from DKO mice were fibrous and histologically stable. Plaques from DKO animals expressed higher arginase 1 (Arg‐1) and lower inducible nitric oxide synthase (iNOS), indicating the presence of M2 macrophages. Analysis of blood and cervical lymph nodes revealed higher interleukin (IL)‐10, immune complexes, and regulatory T cells (Tregs) and lower IL‐12, IL‐1β, and tumor necrosis factor alpha (TNF‐α) in DKO mice. Similarly, in vitro stimulation produced higher IL‐10 and Arg‐1 and lower iNOS, IL‐1β, and TNF‐α in DKO versus Apoe−/− macrophages. These results define a systemic anti‐inflammatory phenotype. Conclusions We hypothesized that removal of FcγRIIb would exacerbate atherosclerosis and generate unstable plaques. However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an anti‐inflammatory Treg/M2 polarization that is atheroprotective.

Wnt signaling regulates hepatobiliary repair following cholestatic liver injury in mice

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate‐tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up‐regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low‐density lipoprotein‐related protein 5/6, transgenic mice expressing stable β‐catenin, and in vitro studies, we show a role of Wnt signaling through β‐catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a β‐catenin‐independent manner. Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both β‐catenin‐dependent and ‐independent manners. (Hepatology 2016;64:1652‐1666)

IL-17 Induces Expression of Vascular Cell Adhesion Molecule Through Signalling Pathway of NF-κB, but not Akt1 and TAK1 in Vascular Smooth Muscle Cells

Interleukin‐17 (IL‐17) plays an important role in several autoimmune diseases. IL‐17 can induce the expression of vascular cell adhesion molecule (VCAM‐1) in aortic vascular smooth muscle cells (SMCs), which is important for the development of atherosclerosis. However, the signalling pathway of IL‐17‐induced VCAM‐1 expression remains unclear. In this study, we reported that IL‐17‐induced expression of VCAM‐1 in SMCs is dependent on NF‐κB, but independent of Akt1 and TAK1. This is because knocking down Akt1 or TAK1 by siRNA did not reduce IL‐17‐induced activation of NF‐κB and expression of VCAM‐1, whereas knocking down NF‐κB by siRNA markedly inhibited IL‐17‐mediated upregulation of VCAM‐1 expression. In addition, IL‐17‐induced expression of VCAM‐1 is partially dependent on activation of ERK1/2. Therefore, these signalling pathways of IL‐17‐mediated upregulation of VCAM‐1 expression might be therapeutic targets for treatment of IL‐17‐mediated inflammation.

Scavenger Receptor Function of Mouse Fcγ Receptor III Contributes to Progression of Atherosclerosis in Apolipoprotein E Hyperlipidemic Mice

Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis.

Reduced Atherosclerosis in apoE-inhibitory FcγRIIb–Deficient Mice Is Associated With Increased Anti-Inflammatory Responses by T Cells and Macrophages

Objective— Fc&ggr; receptors (Fc&ggr;Rs) are classified as activating (Fc&ggr;RI, III, and IV) and inhibitory (Fc&ggr;RII) receptors. We have reported that deletion of activating Fc&ggr;Rs in apolipoprotein E (apoE) single knockout mice attenuated atherosclerosis. In this report, we investigated the hypothesis that deficiency of inhibitory Fc&ggr;RIIb exacerbates atherosclerosis. Approach and Results— ApoE-Fc&ggr;RIIb double knockout mice, congenic to the C57BL/6 (apoE-Fc&ggr;RIIbB6 −/−), were generated and atherosclerotic lesions were assessed. In contrary to our hypothesis, when compared with apoE single knockout mice, arterial lesions were significantly decreased in apoE-Fc&ggr;RIIbB6 −/− male and female mice fed chow or high-fat diets. Chimeric mice generated by transplanting apoE-Fc&ggr;RIIbB6 −/− marrow into apoE single knockout mice also developed reduced lesions. CD4+ T cells from apoE-Fc&ggr;RIIbB6 −/− mice produced higher levels of interleukin-10 and transforming growth factor-&bgr; than their apoE single knockout counterparts. As our findings conflict with a previous report using apoE-Fc&ggr;RIIb129/B6 −/− mice on a mixed genetic background, we investigated whether strain differences contributed to the anti-inflammatory response. Macrophages from Fc&ggr;RIIb129/B6 −/− mice on a mixed genetic background produced more interleukin-1&bgr; and MCP-1 (monocyte chemoattractant protein-1) in response to immune complexes, whereas congenic Fc&ggr;RIIbB6 −/− mice generated more interleukin-10 and significantly less interleukin-1&bgr;. Interestingly, the expression of lupus-associated slam genes, located in proximity to fcgr2b in mouse chromosome 1, is upregulated only in mixed Fc&ggr;RIIb129/B6 −/− mice. Conclusions— Our findings demonstrate a detrimental role for Fc&ggr;RIIb signaling in atherosclerosis and the contribution of anti-inflammatory cytokine responses in the attenuated lesions observed in apoE-Fc&ggr;RIIbB6 −/− mice. As 129/sv genome–derived lupus-associated genes have been implicated in lupus phenotype in Fc&ggr;RIIb129/B6 −/− mice, our findings suggest possible epistatic mechanism contributing to the decreased lesions.

Mice lacking liver-specific β-catenin develop steatohepatitis and fibrosis after iron overload

BACKGROUND & AIMS Iron overload disorders such as hereditary hemochromatosis and iron loading anemias are a common cause of morbidity from liver diseases and increase risk of hepatic fibrosis and hepatocellular carcinoma (HCC). Treatment options for iron-induced damage are limited, partly because there is lack of animal models of human disease. Therefore, we investigated the effect of iron overload in liver-specific β-catenin knockout mice (KO), which are susceptible to injury, fibrosis and tumorigenesis following chemical carcinogen exposure. METHODS Iron overload diet was administered to KO and littermate control (CON) mice for various times. To ameliorate an oxidant-mediated component of tissue injury, N-Acetyl-L-(+)-cysteine (NAC) was added to drinking water of mice on iron overload diet. RESULTS KO on iron diet (KO +Fe) exhibited remarkable inflammation, followed by steatosis, oxidative stress, fibrosis, regenerating nodules and occurrence of occasional HCC. Increased injury in KO +Fe was associated with activated protein kinase B (AKT), ERK, and NF-κB, along with reappearance of β-catenin and target gene Cyp2e1, which promoted lipid peroxidation and hepatic damage. Addition of NAC to drinking water protected KO +Fe from hepatic steatosis, injury and fibrosis, and prevented activation of AKT, ERK, NF-κB and reappearance of β-catenin. CONCLUSIONS The absence of hepatic β-catenin predisposes mice to hepatic injury and fibrosis following iron overload, which was reminiscent of hemochromatosis and associated with enhanced steatohepatitis and fibrosis. Disease progression was notably alleviated by antioxidant therapy, which supports its chemopreventive role in the management of chronic iron overload disorders. LAY SUMMARY Lack of animal models for iron overload disorders makes it hard to study the disease process for improving therapies. Feeding high iron diet to mice that lack the β-catenin gene in liver cells led to increased inflammation followed by fat accumulation, cell death and wound healing that mimicked human disease. Administration of an antioxidant prevented hepatic injury in this model.

Role and regulation of p65/β-catenin association during liver injury and regeneration: a 'complex' relationship.

An important role for β-catenin in regulating p65 (a subunit of NF-κB) during acute liver injury has recently been elucidated through use of conditional β-catenin knockout mice, which show protection from apoptosis through increased activation of p65. Thus, we hypothesized that the p65/β-catenin complex may play a role in regulating processes such as cell proliferation during liver regeneration. We show through in vitro and in vivo studies that the p65/β-catenin complex is regulated through the TNF-α pathway and not through Wnt signaling. However, this complex is unchanged after partial hepatectomy (PH), despite increased p65 and β-catenin nuclear translocation as well as cyclin D1 activation. We demonstrate through both in vitro silencing experiments and chromatin immunoprecipitation after PH that β-catenin, and not p65, regulates cyclin D1 expression. Conversely, using reporter mice we show p65 is activated exclusively in the nonparenchymal (NPC) compartment during liver regeneration. Furthermore, stimulation of macrophages by TNF-α induces activation of NF-κB and subsequent secretion of Wnts essential for β-catenin activation in hepatocytes. Thus, we show that β-catenin and p65 are activated in separate cellular compartments during liver regeneration, with p65 activity in NPCs contributing to the activation of hepatocyte β-catenin, cyclin D1 expression, and subsequent proliferation.