Tumor Necrosis Factor Alpha-Induced Interleukin-1 Alpha Synthesis and Cell Death Is Increased in Mouse Epithelial Cells Infected With Chlamydia muridarum.

Chlamydia trachomatis-genital infection in women can be modeled in mice using Chlamydia muridarum. Using this model, it has been shown that the cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1α lead to irreversible tissue damage in the oviducts. In this study, we investigated the contribution of TNFα on IL-1α synthesis in infected epithelial cells. We show that C muridarum infection enhanced TNFα-induced IL-1α expression and release in a mouse epithelial cell line. In addition to IL-1α, several TNFα-induced inflammatory genes were also highly induced, and infection enhanced TNF-induced cell death. In the mouse model of genital infection, oviducts from mice lacking the TNFα receptor displayed minimal staining for IL-1α compared with wild-type oviducts. Our results suggest TNFα and IL-1α enhance each other's downstream effects resulting in a hyperinflammatory response to chlamydial infection. We propose that biologics targeting TNF-induced IL-1α synthesis could be used to mitigate tissue damage during chlamydial infection.

Loss of Wnt Secretion by Macrophages Promotes Hepatobiliary Injury after Administration of 3,5-Diethoxycarbonyl-1, 4-Dihydrocollidine Diet.

Exposure of mice to a diet containing 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) induces porphyrin accumulation, cholestasis, immune response, and hepatobiliary damage mimicking hepatic porphyria and sclerosing cholangitis. Although ß-catenin signaling promotes hepatocyte proliferation, and macrophages are a source of Wnts, the role of macrophage-derived Wnts in modulating hepatobiliary injury/repair remains unresolved. We investigated the effect of macrophage-specific deletion of Wntless, a cargo protein critical for cellular Wnt secretion, by feeding macrophage-Wntless-knockout (Mac-KO) and wild-type littermates a DDC diet for 14 days. DDC exposure induced Wnt11 up-regulation in macrophages. Mac-KO mice on DDC showed increased serum alkaline phosphatase, aspartate aminotransferase, direct bilirubin, and histologic evidence of more cell death, inflammation, and ductular reaction. There was impaired hepatocyte proliferation evidenced by Ki-67 immunostaining, which was associated with decreased hepatocyte ß-catenin activation and cyclin-D1 in Mac-KO. Mac-KO also showed increased CD45, F4/80, and neutrophil infiltration after DDC diet, along with increased expression of several proinflammatory cytokines and chemokines. Gene expression analyses of bone marrow-derived macrophages from Mac-KO mice and F4/80+ macrophages isolated from DDC-fed Mac-KO livers showed proinflammatory M1 polarization. In conclusion, this study shows that a lack of macrophage Wnt secretion leads to more DDC-induced hepatic injury due to impaired hepatocyte proliferation and increased M1 macrophages, which promotes immune-mediated cell injury.

Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration.

Platelet-derived growth factor receptor α (PDGFRα), a tyrosine kinase receptor, is up-regulated in hepatic stellate cells (HSCs) during chronic liver injury. HSCs mediate hepatic fibrosis through their activation from a quiescent state partially in response to profibrotic growth factors. HSC activation entails enhanced expression of profibrotic genes, increase in proliferation, and increase in motility, which facilitates migration within the hepatic lobule. We show colocalization of PDGFRα in murine carbon tetrachloride, bile duct ligation, and 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine models of chronic liver injury, and investigate the role of PDGFRα on proliferation, profibrotic gene expression, and migration in primary human HSCs (HHSteCs) using the PDGFRα-specific inhibitory monoclonal antibody olaratumab. Although lacking any effects on HHSteC transdifferentiation assessed by gene expression of ACTA2, TGFB1, COL1A1, SYP1, and FN1, olaratumab specifically reduced HHSteC proliferation (AlamarBlue assay) and cell migration (transwell migration assays). Using phospho-specific antibodies, we show that olaratumab attenuates PDGFRα activation in response to PDGF-BB, and reduced phosphorylation of extracellular signal-regulated kinase 1 and 2, Elk-1, p38, Akt, focal adhesion kinase, mechanistic target of rapamycin, C10 regulator of kinase II, and C10 regulator of kinase-like, suggesting that PDGFRα contributes to mitogenesis and actin reorganization through diverse downstream effectors. Our findings support a distinct contribution of PDGFRα signaling to HSC proliferation and migration and provide evidence that inhibition of PDGFRα signaling could alter the pathogenesis of hepatic fibrosis.

Mice lacking liver-specific ß-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 ß-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 ß-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 ß-catenin. CONCLUSIONS: The absence of hepatic ß-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 ß-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.

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 ß-catenin, and in vitro studies, we show a role of Wnt signaling through ß-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a ß-catenin-independent manner. CONCLUSION: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both ß-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).

Role and Regulation of p65/ß-Catenin Association During Liver Injury and Regeneration: A "Complex" Relationship.

An important role for ß-catenin in regulating p65 (a subunit of NF-κB) during acute liver injury has recently been elucidated through use of conditional ß-catenin knockout mice, which show protection from apoptosis through increased activation of p65. Thus, we hypothesized that the p65/ß-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/ß-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 ß-catenin nuclear translocation as well as cyclin D1 activation. We demonstrate through both in vitro silencing experiments and chromatin immunoprecipitation after PH that ß-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 ß-catenin activation in hepatocytes. Thus, we show that ß-catenin and p65 are activated in separate cellular compartments during liver regeneration, with p65 activity in NPCs contributing to the activation of hepatocyte ß-catenin, cyclin D1 expression, and subsequent proliferation.

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

OBJECTIVE: Fcγ receptors (FcγRs) are classified as activating (FcγRI, III, and IV) and inhibitory (FcγRII) receptors. We have reported that deletion of activating FcγRs in apolipoprotein E (apoE) single knockout mice attenuated atherosclerosis. In this report, we investigated the hypothesis that deficiency of inhibitory FcγRIIb exacerbates atherosclerosis. APPROACH AND RESULTS: ApoE-FcγRIIb double knockout mice, congenic to the C57BL/6 (apoE-FcγRIIbB6 (-/-)), 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-FcγRIIbB6 (-/-) male and female mice fed chow or high-fat diets. Chimeric mice generated by transplanting apoE-FcγRIIbB6 (-/-) marrow into apoE single knockout mice also developed reduced lesions. CD4(+) T cells from apoE-FcγRIIbB6 (-/-) mice produced higher levels of interleukin-10 and transforming growth factor-ß than their apoE single knockout counterparts. As our findings conflict with a previous report using apoE-FcγRIIb129/B6 (-/-) mice on a mixed genetic background, we investigated whether strain differences contributed to the anti-inflammatory response. Macrophages from FcγRIIb129/B6 (-/-) mice on a mixed genetic background produced more interleukin-1ß and MCP-1 (monocyte chemoattractant protein-1) in response to immune complexes, whereas congenic FcγRIIbB6 (-/-) mice generated more interleukin-10 and significantly less interleukin-1ß. Interestingly, the expression of lupus-associated slam genes, located in proximity to fcgr2b in mouse chromosome 1, is upregulated only in mixed FcγRIIb129/B6 (-/-) mice. CONCLUSIONS: Our findings demonstrate a detrimental role for FcγRIIb signaling in atherosclerosis and the contribution of anti-inflammatory cytokine responses in the attenuated lesions observed in apoE-FcγRIIbB6 (-/-) mice. As 129/sv genome-derived lupus-associated genes have been implicated in lupus phenotype in FcγRIIb129/B6 (-/-) mice, our findings suggest possible epistatic mechanism contributing to the decreased lesions.

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.

Effects of intra-abdominal sepsis on atherosclerosis in mice.

INTRODUCTION: Sepsis 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. METHODS: We 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. RESULTS: ApoE-/- 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). CONCLUSIONS: Using 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.

Soy protein inhibits inflammation-induced VCAM-1 and inflammatory cytokine induction by inhibiting the NF-κB and AKT signaling pathway in apolipoprotein E-deficient mice.

PURPOSE: Inflammation is a hallmark of many diseases, such as atherosclerosis, autoimmune diseases, obesity, and cancer. Isoflavone-free soy protein diet (SPI(-)) has been shown to reduce atherosclerotic lesions in a hyperlipidemic mouse model compared to casein (CAS)-fed mice, despite unchanged serum lipid levels. However, possible mechanisms contributing to the athero-protective effect of soy protein remain unknown. Therefore, we investigated whether and how SPI(-) diet inhibits inflammatory responses associated with atherosclerosis. METHODS: Apolipoprotein E knockout (apoE-/-) mice (5-week) were fed CAS or SPI(-) diet for 1 or 5 week to determine LPS- and hyperlipidemia-induced acute and chronic inflammatory responses, respectively. Expression of NF-κB-dependent inflammation mediators such as VCAM-1, TNF-α, and MCP-1 were determined in aorta and liver. NF-κB, MAP kinase, and AKT activation was determined to address mechanisms contributing to the anti-inflammatory properties of soy protein/peptides. RESULTS: Isoflavone-free soy protein diet significantly reduced LPS-induced VCAM-1 mRNA and protein expression in aorta compared to CAS-fed mice. Reduced VCAM-1 expression in SPI(-)-fed mice also paralleled attenuated monocyte adhesion to vascular endothelium, a critical and primary processes during inflammation. Notably, VCAM-1 mRNA and protein expression in lesion-prone aortic arch was significantly reduced in apoE-/- mice fed SPI(-) for 5 weeks compared with CAS-fed mice. Moreover, dietary SPI(-) potently inhibited LPS-induced NF-κB activation and the subsequent upregulation of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, and MCP-1. Interestingly, SPI(-) inhibited NF-κB-dependent inflammatory responses by targeting I-κB phosphorylation and AKT activation with no effect on MAP kinase pathway. Of the five putative soy peptides, four of the soy peptides inhibited LPS-induced VCAM-1, IL-6, IL-8, and MCP-1 protein expression in human vascular endothelial cells in vitro. CONCLUSIONS: Collectively, our findings suggest that anti-inflammatory properties of component(s) of soy protein/peptides may be a possible mechanism for the prevention of chronic inflammatory diseases such as atherosclerosis.