LOX-1 – dependent mitochondrial DNA damage and NLRP3 activation during systemic inflammation in mice

Background

Lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) is known to be involved in many pathophysiological events, such as inflammation.

Methods

To clarify the role of LOX-1 in mtDNA damage and NLRP3 inflammasome activation, we studied wild-type (WT) and LOX-1 knockout (KO) mice given thioglycollate, an inflammatory stimulus.

Results

We observed intense inflammatory response (CD45 and CD68 expression) and mtDNA damage in spleen and kidneys of WT mice given thioglycollate. The abrogation of LOX-1 (use of LOX-1 knockout mice) reduced the inflammatory response as well as mtDNA damage (P < 0.05 vs. WT mice). We also observed that mice with LOX-1 deletion had markedly reduced expression of caspase-1 (P10 and P20 subunits) as well as cleaved IL-1β and IL-18. These mice also had much less mtDNA damage and only limited NLRP3 inflammasome expression.

Conclusions

These in vivo observations indicate that LOX-1 plays a key role in mtDNA damage which then leads to NLRP3 inflammasome activation during inflammation.     

Leukocyte ABCA1 Remains Atheroprotective in Splenectomized LDL Receptor Knockout Mice

Aim

ATP-binding cassette transporter A1 (ABCA1) is an important mediator of macrophage cholesterol efflux. It mediates the efflux of cellular cholesterol to lipid-poor apolipoprotein A-I. LDL receptor (LDLr) knockout (KO) mice deficient for leukocyte ABCA1 (ABCA1 KO→LDLr KO) show increased atherosclerosis and splenic lipid accumulation despite largely attenuated serum cholesterol levels. In the present study, we aimed to explore the importance of the spleen for the atheroprotective effects of leukocyte ABCA1.

Methods

LDLr KO mice were transplanted with bone marrow from ABCA1 KO mice or wild-type (WT) controls. After 8 weeks recovery, mice were either splenectomized (SP-x) or underwent a sham operation, and were subsequently challenged with a Western-type diet (WTD).

Results

In agreement with previous studies, the atherosclerotic lesion area in ABCA1 KO→LDLr KO sham animals (655±82×10³ µm²) was 1.4-fold (p = 0.03) larger compared to sham WT→LDLr KO mice (459±33×10³ µm²) after 8 weeks WTD feeding, despite 1.7-fold (p<0.001) lower serum cholesterol levels. Interestingly, deletion of ABCA1 in leukocytes led to 1.6-fold higher neutrophil content in the spleen in absence of differences in circulating neutrophils. Levels of KC, an important chemoattractant for neutrophils, in serum, however, were increased 2.9-fold (p = 0.07) in ABCA1 KO→LDLr KO mice. SP-x induced blood neutrophilia as compared to WT→LDLr KO mice (1.9-fold; p<0.05), but did not evoke differences in serum cholesterol and anti-oxLDL antibody levels. Atherosclerotic lesion development, however, was 1.3-fold induced both in the presence and absence of leukocyte ABCA1 (WT: 614±106×10³ µm², ABCA1 KO: 786±44×10³ µm²). Two-way ANOVA revealed independent effects on atherosclerosis for both leukocyte ABCA1 deficiency and SP-x (p<0.05).

Conclusions

The observed splenic alterations induced by leukocyte ABCA1 deficiency do not play a significant role in the anti-atherogenic effects of leukocyte ABCA1 on lesion development.     

Macrophage activation state determines the response to rhinovirus infection in a mouse model of allergic asthma

Background

The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease.

Methods

Eight week-old wild type or IL-4 receptor knockout (IL-4R KO) mice were sensitized and challenged with OVA and inoculated with RV1B or sham HeLa cell lysate.

Results

In contrast to OVA-treated wild-type mice with both neutrophilic and eosinophilic airway inflammation, OVA-treated IL-4R KO mice showed increased neutrophilic inflammation with few eosinophils in the airways. Like wild-type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV. There was a shift in lung cytokines from a type 2-predominant response to a type 1 response, including production of IL-12p40 and TNF-α. IL-17A was also increased. RV infection of OVA-treated IL-4R KO mice further increased neutrophilic inflammation. Bronchoalveolar macrophages showed an M1 polarization pattern and ex vivo RV infection increased macrophage production of TNF-α, IFN-γ and IL-12p40. Finally, lung cells from OVA-treated IL-4R KO mice showed reduced CD206+ CD301+ M2 macrophages, decreased IL-13 and increased TNF-α and IL-17A production by F4/80+, CD11b+ macrophages.

Conclusions

OVA-treated IL-4R KO mice show neutrophilic airway inflammation constituting a model of allergic, type 1 cytokine-driven neutrophilic asthma. In the absence of IL-4/IL-13 signaling, RV infection of OVA-treated mice increased type 1 cytokine and IL-17A production from conventionally-activated macrophages, augmenting neutrophilic rather than eosinophilic inflammation. In mice with allergic airways inflammation, IL-4R signaling determines macrophage activation state and the response to subsequent RV infection.     

The Liver-Selective Thyromimetic T-0681 Influences Reverse Cholesterol Transport and Atherosclerosis Development in Mice

Background

Liver-selective thyromimetics have been reported to efficiently reduce plasma cholesterol through the hepatic induction of both, the low-density lipoprotein receptor (LDLr) and the high-density lipoprotein (HDL) receptor; the scavenger receptor class B type I (SR-BI). Here, we investigated the effect of the thyromimetic T-0681 on reverse cholesterol transport (RCT) and atherosclerosis, and studied the underlying mechanisms using different mouse models, including mice lacking LDLr, SR-BI, and apoE, as well as CETP transgenic mice.

Methodology/Principal Findings

T-0681 treatment promoted bile acid production and biliary sterol secretion consistently in the majority of the studied mouse models, which was associated with a marked reduction of plasma cholesterol. Using an assay of macrophage RCT in mice, we found T-0681 to significantly increase fecal excretion of macrophage-derived neutral and acidic sterols. No positive effect on RCT was found in CETP transgenic mice, most likely due to the observed decrease in plasma CETP mass. Studies in SR-BI KO and LDLr KO mice suggested hepatic LDLr to be necessary for the action of T-0681 on lipid metabolism, as the compound did not have any influence on plasma cholesterol levels in mice lacking this receptor. Finally, prolonged treatment with T-0681 reduced the development of atherosclerosis by 60% in apoE KOs on Western type diet. In contrast, at an earlier time-point T-0681 slightly increased small fatty streak lesions, in part due to an impaired macrophage cholesterol efflux capacity, when compared to controls.

Conclusions/Significance

The present results show that liver-selective thyromimetics can promote RCT and that such compounds may protect from atherosclerosis partly through induction of bile acid metabolism and biliary sterol secretion. On-going clinical trials will show whether selective thyromimetics do prevent atherosclerosis also in humans.     

Glutamate transporter type 3 regulates mouse hippocampal GluR1 trafficking

Background

Rapid trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) to the plasma membrane is considered a fundamental biological process for learning and memory. GluR1 is an AMPAR subunit. We have shown that mice with knockout of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, have impaired learning and memory. The mechanisms for this impairment are not known and may be via regulation of AMPAR trafficking.

Methods

Freshly prepared 300 μm coronal hippocampal slices from wild-type or EAAT3 knockout mice were incubated with or without 25 mM tetraethylammonium for 10 min. The trafficking of GluR1, an AMPAR subunit, to the plasma membrane and its phosphorylation were measured.

Results

Tetraethylammonium increased the trafficking of GluR1 and EAAT3 to the plasma membrane in the wild-type mouse hippocampal slices but did not cause GluR1 trafficking in the EAAT3 knockout mice. Tetraethylammonium also increased the phosphorylation of GluR1 at S845, a protein kinase A (PKA) site, in the wild-type mice but not in the EAAT3 knockout mice. The PKA antagonist KT5720 attenuated tetraethylammonium-induced GluR1 phosphorylation and trafficking in the wild-type mice. The PKA agonist 6-BNz-cAMP caused GluR1 trafficking to the plasma membrane in the EAAT3 knockout mice. In addition, EAAT3 was co-immunoprecipitated with PKA.

Conclusions

These results suggest that EAAT3 is upstream of PKA in a pathway to regulate GluR1 trafficking.

General significance

Our results provide initial evidence for the involvement of EAAT3 in the biochemical cascade of learning and memory.     

MicroRNA-21 Knockout Improve the Survival Rate in DSS Induced Fatal Colitis through Protecting against Inflammation and Tissue Injury

Background

MicroRNA-21 (miR-21) is overexpressed in most inflammatory diseases, but its physiological role in gut inflammation and tissue injury is poorly understood. The goal of this work is to understand the role of miR-21 in colitis and damage progression of intestine in a genetically modified murine model.

Methods

Experimental colitis was induced in miR-21 KO and wild-type (WT) mice by 3.5% dextran sulphate sodium (DSS) administration for 7 days. Disease activity index(DAI), blood parameters, intestinal permeability, histopathologic injury, cytokine and chemokine production, and epithelial cells apoptosis were examined in colons of miR-21 KO and WT mice.

Results

miR-21 was overexpressed in intestine of inflammatory bowel diseases (IBD) and acute intestinal obstruction (AIO) patients when compared with normal intestinal tissues. Likewise, miR-21 was up-regulated in colon of IL-10 KO mice when compared with control mice. WT mice rapidly lost weight and were moribund 5 days after treatment with 3.5% DSS, while miR-21 KO mice survived for at least 6 days. Elevated leukocytes and more severe histopathology were observed in WT mice when compared with miR-21 KO mice. Elevated levels of TNF-α and macrophage inflammatory protein-2(MIP-2) in colon culture supernatants from WT mice exhibited significant higher than miR-21 KO mice. Furthermore, CD3 and CD68 positive cells, intestinal permeability and apoptosis of epithelial cells were significantly increased in WT mice when compared with miR-21 KO mice. Finally, we found that miR-21 regulated the intestinal barrier function through modulating the expression of RhoB and CDC42.

Conclusion

Our results suggest that miR-21 is overexpressed in intestinal inflammation and tissue injury, while knockout of miR-21 in mice improve the survival rate in DSS-induced fatal colitis through protecting against inflammation and tissue injury. Therefore, attenuated expression of miR-21 in gut may prevent the onset or progression of inflammatory bowel disease in patients.     

Intracellular Ca2+ transients in delta-sarcoglycan knockout mouse skeletal muscle

Background

δ-Sarcoglycan (δ-SG) knockout (KO) mice develop skeletal muscle histopathological alterations similar to those in humans with limb muscular dystrophy. Membrane fragility and increased Ca²⁺ permeability have been linked to muscle degeneration. However, little is known about the mechanisms by which genetic defects lead to disease.

Methods

Isolated skeletal muscle fibers of wild-type and δ-SG KO mice were used to investigate whether the absence of δ-SG alters the increase in intracellular Ca²⁺ during single twitches and tetani or during repeated stimulation. Immunolabeling, electrical field stimulation and Ca²⁺ transient recording techniques with fluorescent indicators were used.

Results

Ca²⁺ transients during single twitches and tetani generated by muscle fibers of δ-SG KO mice are similar to those of wild-type mice, but their amplitude is greatly decreased during protracted stimulation in KO compared to wild-type fibers. This impairment is independent of extracellular Ca²⁺ and is mimicked in wild-type fibers by blocking store-operated calcium channels with 2-aminoethoxydiphenyl borate (2-APB). Also, immunolabeling indicates the localization of a δ-SG isoform in the sarcoplasmic reticulum of the isolated skeletal muscle fibers of wild-type animals, which may be related to the functional differences between wild-type and KO muscles.

Conclusions

δ-SG has a role in calcium homeostasis in skeletal muscle fibers.

General significance

These results support a possible role of δ-SG on calcium homeostasis. The alterations caused by the absence of δ-SG may be related to the pathogenesis of muscular dystrophy.     

Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice

Background

CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.

Methods

Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.

Results

Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.

Conclusions

We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.     

Macrophage elastase (MMP-12) in expanding murine adipose tissue

Background

Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue.

Methods

The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks.

Results

MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass. Adipocyte and blood vessel size and density in SC and GON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GON adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GON adipose tissue.

Conclusions

Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS.

General Significance

MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes.     

Secreted Protein Acidic and Rich in Cysteine (SPARC) Exacerbates Colonic Inflammatory Symptoms in Dextran Sodium Sulphate-Induced Murine Colitis

Background

Secreted Protein Acidic and Rich in Cysteine (SPARC) is expressed during tissue repair and regulates cellular proliferation, migration and cytokine expression. The aim was to determine if SPARC modifies intestinal inflammation.

Methods

Wild-type (WT) and SPARC-null (KO) mice received 3% dextran sodium sulphate (DSS) for 7 days. Inflammation was assessed endoscopically, clinically and histologically. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12/IL23p40, TNF-α, IFN-γ, RANTES, MCP-1, MIP-1α, MIP-1β, MIG and TGF-β1 levels were measured by ELISA and cytometric bead array. Inflammatory cells were characterised by CD68, Ly6G, F4/80 and CD11b immunofluorescence staining and regulatory T cells from spleen and mesenteric lymph nodes were assessed by flow cytometry.

Results

KO mice had less weight loss and diarrhoea with less endoscopic and histological inflammation than WT animals. By day 35, all (n = 13) KO animals completely resolved the inflammation compared to 7 of 14 WT mice (p<0.01). Compared to WTs, KO animals at day 7 had less IL1β (p = 0.025) and MIG (p = 0.031) with higher TGFβ1 (p = 0.017) expression and a greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals (p<0.01). KO mice also had fewer CD68+ and F4/80+ macrophages, Ly6G+ neutrophils and CD11b+ cells infiltrating the inflamed colon.

Conclusions

Compared to WT, SPARC KO mice had less inflammation with fewer inflammatory cells and more regulatory T cells. Together, with increased TGF-β1 levels, this could aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa suggesting that the presence of SPARC increases intestinal inflammation.     

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

Background

The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics).

Methods

Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin–luciferase based real-time luminometry.

Results

Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%.Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux.Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers.

Conclusions

MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers.

General significance

Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.     

Effects of deletion of macrophage ABCA7 on lipid metabolism and the development of atherosclerosis in the presence and absence of ABCA1

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen.     

Role of CD14 in a Mouse Model of Acute Lung Inflammation Induced by Different Lipopolysaccharide Chemotypes

Background

Recognition of lipopolysaccharide (LPS) is required for effective defense against invading gram-negative bacteria. Recently, in vitro studies revealed that CD14 is required for activation of the myeloid differentiation factor (MyD)88-dependent Toll-like receptor (TLR)4 signaling pathway by smooth (S)-LPS, but not by rough (R)-LPS. The present study investigated the role of CD14 in induction of lung inflammation in mice by these different LPS chemotypes.

Methodology/Results

Neutrophil accumulation and tumor necrosis factor (TNF) release in bronchoalveolar lavage fluid were determined 6 hours after intranasal treatment of wild type (WT) and CD14 knock-out (KO) mice with different doses S-LPS or R-LPS. The contribution of CD14 to lung inflammation induced by S-LPS or R-LPS depended on the LPS dose. At low doses, S-LPS and R-LPS induced neutrophil influx in a CD14-dependent manner. Low dose S-LPS-induced cytokine release also depended on CD14. Strikingly, neutrophil influx and TNF release induced by high dose S-LPS or R-LPS was diminished in the presence of CD14. Intranasal administration of sCD14 to CD14 KO mice treated with S-LPS partially reversed the inflammatory response to the response observed in WT mice.

Conclusions

In conclusion, CD14 modulates effects of both S-LPS and R-LPS within the lung in a similar way. Except for R-LPS-induced TNF release, S-LPS and R-LPS at low dose induced acute lung inflammation in a CD14-dependent manner, while the inflammatory response triggered by high dose S-LPS or R-LPS was diminished by CD14.     

Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

Background and aim

CD200:CD200 receptor (CD200R) interactions lead to potent immunosuppression and inhibition of autoimmune inflammation. We investigated the effect of "knockout"of CD200 or CD200R, or over-expression of CD200, on susceptibility to dextran sodium sulfate (DSS)—induced colitis, a mouse model of inflammatory bowel disease (IBD).

Methods

Acute or chronic colitis was induced by administration of dextran sodium sulfate (DSS) in four groups of age-matched C57BL/6 female mice: (1) CD200-transgenic mice (CD200^tg); (2) wild-type (WT) mice; (3) CD200 receptor 1-deficient (CD200R1KO) mice; and (4) CD200-deficient (CD200KO) mice. The extent of colitis was determined using a histological scoring system. Colon tissues were collected for quantitative RT-PCR and Immunohistochemical staining. Supernatants from colonic explant cultures and mononuclear cells isolated from colonic tissue were used for ELISA.

Results

CD200KO and CD200R1KO mice showed greater sensitivity to acute colitis than WT mice, with accelerated loss of body weight, significantly higher histological scores, more severe infiltration of macrophages, neutrophils and CD3⁺ cells, and greater expression of macrophage-derived inflammatory cytokines, whose production was inhibited in vitro (in WT/CD200KO mouse cells) by CD200. In contrast, CD200^tg mice showed less sensitivity to DSS compared with WT mice, with attenuation of all of the features seen in other groups. In a chronic colitis model, greater infiltration of Foxp3⁺ regulatory T (Treg) cells was seen in the colon of CD200^tg mice compared to WT mice, and anti-CD25 mAb given to these mice attenuated protection.

Conclusions

The CD200:CD200R axis plays an immunoregulatory role in control of DSS induced colitis in mice.     

Orp8 Deficiency in Bone Marrow-Derived Cells Reduces Atherosclerotic Lesion Progression in LDL Receptor Knockout Mice

Introduction

Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity.

Conclusions

Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.     

The inhibitory effect of naringenin on atopic dermatitis induced by DNFB in NC/Nga mice

Aims

Atopic dermatitis (AD) is a chronic and relapsing inflammatory dermatitis characterized by pruritic and eczematous skin lesions. Here, we investigated the therapeutic effect of the fruit flavonoid naringenin on DNFB induced atopic dermatitis mice model.

Main methods

AD-like skin lesion was induced by repetitive skin contact with DNFB in NC/Nga mice and the effects of the fruit flavonoid naringenin were evaluated on the basis of histopathological findings of skin, ear swelling and cytokine production of CD4⁺T cells.

Key findings

Intraperitoneal injection of naringenin for one week after DNFB challenge significantly lowered ear swelling and improved back skin lesions. In addition, naringenin significantly suppressed production of interferon-gamma (IFN-γ) by activated CD4⁺ T cells and serum IgE level. Furthermore, naringenin reduced DNFB-induced infiltration of eosinophils, mast cells, CD4⁺ T cells, and CD8⁺ T cells in skin lesions.

Significance

Naringenin may suppress the development of AD-like skin lesions in DNFB-treated NC/Nga mice by reducing IFN-γ production of activated CD4⁺ T cells, serum IgE levels and infiltration of immune cells to skin lesion.     

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity

Background

Osteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.

Aims

To investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.

Methods

We compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.

Results

OPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.

Conclusions

OPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.     

Peritoneal mast cell degranulation differently affected thioglycollate-induced macrophage phenotype and activity in Dark Agouti and Albino Oxford rats

Aims

Macrophages are heterogeneous population of inflammatory cells and, in response to the microenvironment, become differentially activated. The objective of the study was to explore macrophage effector functions during different inflammatory conditions in two rat strains.

Main methods

We have investigated the effects of in vivo treatment with mast cell-degranulating compound 48/80 and/or thioglycollate on peritoneal macrophage phagocytosis and capacity to secrete hydrogen peroxide (H₂O₂), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in Dark Agouti (DA) and Albino Oxford (AO) rat strains. Besides, fresh peritoneal cells were examined for the expression of ED1, ED2 and CD86 molecules.

Key findings

In thioglycollate-elicited macrophages, increased proportion of ED1 + cells was accompanied with elevated phagocytosis of zymosan (DA strain), whereas increased expression level of CD86 molecule on ED2 + macrophages matched elevated secretory capacity for H₂O₂, TNF-α and NO (AO rats). Although mast cell degranulation induced by compound 48/80 increased the percentages of ED2 + macrophages in both rat strains, the proportion of ED2 + cells expressing CD86 molecule was decreased and increased in DA and AO rats, respectively. Furthermore, in DA strain compound 48/80 diminished macrophage secretion of NO, but stimulated all macrophage functions tested in AO strain. If applied concomitantly, the compound 48/80 additively increased macrophage activity induced by thioglycollate in AO rats.

Significance

Macrophages from DA and AO rat strains show different susceptibility to mediators released from mast cells, suggesting that strain-dependant predisposition(s) toward particular activation pattern is decisive for the macrophage efficacy in response to inflammatory agents.     

A role for B cells in organic dust induced lung inflammation

Background

Agriculture organic dust exposures induce lung disease with lymphoid aggregates comprised of both T and B cells. The precise role of B cells in mediating lung inflammation is unknown, yet might be relevant given the emerging role of B cells in obstructive pulmonary disease and associated autoimmunity.

Methods

Using an established animal model, C57BL/6 wild-type (WT) and B-cell receptor (BCR) knock-out (KO) mice were repetitively treated with intranasal inhalation of swine confinement organic dust extract (ODE) daily for 3 weeks and lavage fluid, lung tissues, and serum were collected.

Results

ODE-induced neutrophil influx in lavage fluid was not reduced in BCR KO animals, but there was reduction in TNF-α, IL-6, CXCL1, and CXCL2 release. ODE-induced lymphoid aggregates failed to develop in BCR KO mice. There was a decrease in ODE-induced lung tissue CD11c⁺CD11b⁺ exudative macrophages and compensatory increase in CD8⁺ T cells in lavage fluid of BCR KO animals. Compared to saline, there was an expansion of conventional B2-, innate B1 (CD19⁺CD11b⁺CD5^+/?)-, and memory (CD19⁺CD273^+/-CD73^+/?) B cells following ODE exposure in WT mice. Autoreactive responses including serum IgG anti-citrullinated protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) autoantibodies were increased in ODE treated WT mice as compared to saline control. B cells and serum immunoglobulins were not detected in BCR KO animals.

Conclusions

Lung tissue staining for citrullinated and MAA modified proteins were increased in ODE-treated WT animals, but not BCR KO mice. These studies show that agriculture organic dust induced lung inflammation is dependent upon B cells, and dust exposure induces an autoreactive response.

     

Heteroglucan-dendrimer glycoconjugate: a modulated construct with augmented immune responses and signaling phenomena

Background

Newer strategies for augmenting immune responses of pharmacologically active glucans may serve to improve the medicinal potential of these biomolecules. With this aim, the present work was focused on generating targeted high molecular size glucan particles with magnified immune response activity.

Methods

Heteroglucans were conjugated with PAMAM dendrimers using a Schiff base reductive amination reaction to generate a polytethered molecule with multiple glucan motifs. The modulated construct was characterized by FTIR, TEM, ¹H NMR and dynamic light scattering (DLS) methods. Effects of conjugated glucans were examined in RAW 264.7 macrophage cells as well as in S-180 murine tumor models.

Results

Dendrimer-conjugated glucans were found to exhibit a two-fold increase in immune stimulation in comparison to unconjugated glucans. This may be corroborated by the predominant enhancement in immunological functions such as nitric oxide production, ROS generation and immune directed tumor inhibition in murine models. Immune cell surface markers (CD4, CD8, CD19, MHC-II) and cytokine levels were also found to be highly up-regulated in the splenocytes of mice subjected to particulate glucan administration. Our study also demonstrated that conjugated glucan treatment to RAW 264.7 cells strongly enhanced the phosphorylation of two downstream signalling molecules of the mitogen activated protein kinase (MAPKs) family: p38 and MEK1/2 relative to single glucans thereby relating molecular mechanisms with enhanced immune stimulation.

Conclusions and general significance

The results obtained thus support that particulate format of soluble heteroglucan will thereby improve its functionality and identify leads in therapeutic competence.