Distinct Infiltration of Neutrophils in Lesion Shoulders in ApoE?/? Mice

Inflammation and activation of immune cells are key mechanisms in the development of atherosclerosis. Previous data indicate important roles for monocytes and T-lymphocytes in lesions. However, recent data suggest that neutrophils also may be of importance in atherogenesis. Here, we use apolipoprotein E (ApoE)-deficient mice with fluorescent neutrophils and monocytes (ApoE^−/−/Lys^EGFP/EGFP mice) to specifically study neutrophil presence and recruitment in atherosclerotic lesions. We show by flow cytometry and confocal microscopy that neutrophils make up for 1.8% of CD45⁺ leukocytes in the aortic wall of ApoE^−/−/Lys^EGFP/EGFP mice and that their contribution relative to monocyte/macrophages within lesions is approximately 1:3. However, neutrophils accumulate at sites of monocyte high density, preferentially in shoulder regions of lesions, and may even outnumber monocyte/macrophages in these areas. Furthermore, intravital microscopy established that a majority of leukocytes interacting with endothelium on lesion shoulders are neutrophils, suggesting a significant recruitment of these cells to plaque. These data demonstrate neutrophilic granulocytes as a major cellular component of atherosclerotic lesions in ApoE^−/− mice and call for further study on the roles of these cells in atherogenesis.Recruitment of immune cells to the arterial intima is central to atherogenesis. Current dogma emphasizes the role of macrophages and T-lymphocytes in promoting plaque development and destabilization.^1,2 However, the most abundant white blood cell in the circulation, the neutrophilic granulocyte, has until recently rarely been associated with the development of atherosclerosis. Nonetheless, proteins typically secreted by neutrophils are abundant in lesions,^3,4,5,6,7 and systemic neutrophil counts appear to correlate closely with severity of atherosclerosis in humans.⁸ Similar observations were also recently made in the murine system in which increased peripheral neutrophil count was associated with enhanced plaque size, whereas the opposite was true when neutrophils were depleted from the circulation.⁹ There are also data that indicate presence of neutrophils in lesions of low-density lipoprotein (LDL) receptor–deficient mice.⁵ Despite these findings, data on potential roles of neutrophils in atherogenesis are rare in the literature.We recently crossed apolipoprotein E–deficient ApoE^−/− mice with mice carrying a knock-in mutation for enhanced green fluorescence protein (EGFP) in the lysozyme locus (Lys^EGFP/EGFP mice)¹⁰ generating ApoE^−/−/Lys^EGFP/EGFP mice, which allow for sensitive detection of neutrophils in atherosclerotic plaques.¹¹ Here, we study the presence and spatial distribution of neutrophils in atherosclerotic arteries of these mice. We demonstrate that neutrophils are present in substantial numbers in aortic plaque. Moreover, their contribution is higher in shoulder regions of plaque, which are areas of high inflammatory activity. Intravital microscopy further revealed that neutrophils are the main cell population that interacts with atherosclerotic endothelium, suggesting an ongoing recruitment of neutrophils to lesions. These data demonstrate that neutrophils represent a major population of cells in atherosclerosis and underscore the possibility that these cells may play previously underestimated roles in atherogenesis.     

Induction of Bacterial Antigen-Specific Colitis by a Simplified Human Microbiota Consortium in Gnotobiotic Interleukin-10?/? Mice

We evaluated whether a simplified human microbiota consortium (SIHUMI) induces colitis in germfree (GF) 129S6/SvEv (129) and C57BL/6 (B6) interleukin-10-deficient (IL-10^−/−) mice, determined mouse strain effects on colitis and the microbiota, examined the effects of inflammation on relative bacterial composition, and identified immunodominant bacterial species in “humanized” IL-10^−/− mice. GF wild-type (WT) and IL-10^−/− 129 and B6 mice were colonized with 7 human-derived inflammatory bowel disease (IBD)-related intestinal bacteria and maintained under gnotobiotic conditions. Quantification of bacteria in feces, ileal and colonic contents, and tissues was performed using 16S rRNA gene selective quantitative PCR. Colonic segments were scored histologically, and gamma interferon (IFN-γ), IL-12p40, and IL-17 levels were measured in supernatants of unstimulated colonic tissue explants and of mesenteric lymph node (MLN) cells stimulated by lysates of individual or aggregate bacterial strains. Relative bacterial species abundances changed over time and differed between 129 and B6 mice, WT and IL-10^−/− mice, luminal and mucosal samples, and ileal and colonic or fecal samples. SIHUMI induced colitis in all IL-10^−/− mice, with more aggressive colitis and MLN cell activation in 129 mice. Escherichia coli LF82 and Ruminococcus gnavus lysates induced dominant effector ex vivo MLN TH1 and TH17 responses, although the bacterial mucosal concentrations were low. In summary, this study shows that a simplified human bacterial consortium induces colitis in ex-GF 129 and B6 IL-10^−/− mice. Relative concentrations of individual SIHUMI species are determined by host genotype, the presence of inflammation, and anatomical location. A subset of IBD-relevant human enteric bacterial species preferentially stimulates bacterial antigen-specific TH1 and TH17 immune responses in this model, independent of luminal and mucosal bacterial concentrations.     

Hepatocyte-Specific Expression of Human Lysosome Acid Lipase Corrects Liver Inflammation and Tumor Metastasis in lal?/? Mice

The liver is a major organ for lipid synthesis and metabolism. Deficiency of lysosomal acid lipase (LAL; official name Lipa, encoded by Lipa) in mice (lal^−/−) results in enlarged liver size due to neutral lipid storage in hepatocytes and Kupffer cells. To test the functional role of LAL in hepatocyte, hepatocyte-specific expression of human LAL (hLAL) in lal^−/− mice was established by cross-breeding of liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− knockout (KO) (LAP-Tg/KO) triple mice. Hepatocyte-specific expression of hLAL in LAP-Tg/KO triple mice reduced the liver size to the normal level by decreasing lipid storage in both hepatocytes and Kupffer cells. hLAL expression reduced tumor-promoting myeloid-derived suppressive cells in the liver of lal^−/− mice. As a result, B16 melanoma metastasis to the liver was almost completely blocked. Expression and secretion of multiple tumor-promoting cytokines or chemokines in the liver were also significantly reduced. Because hLAL is a secretory protein, lal^−/− phenotypes in other compartments (eg, blood, spleen, and lung) also ameliorated, including systemic reduction of myeloid-derived suppressive cells, an increase in CD4⁺ and CD8⁺ T and B lymphocytes, and reduced B16 melanoma metastasis in the lung. These results support a concept that LAL in hepatocytes is a critical metabolic enzyme in controlling neutral lipid metabolism, liver homeostasis, immune response, and tumor metastasis.Lysosomal acid lipase (LAL) (official name LIPA, encoded by LIPA) hydrolyzes cholesteryl esters (CEs) and triglycerides (TGs) in lysosomes. Mutation in LIPA results in Wolman disease (WD) as early infantile onset and CE storage disease (CESD) as late onset.^1–3 Infants with WD have massive accumulations of CEs and TGs in the lysosomes of hepatocytes and Kupffer cells, as well as in macrophages throughout the viscera, which lead to liver failure, severe hepatosplenomegaly, steatorrhea, pulmonary fibrosis, and adrenal calcification.² Patients with CESD have the major symptom of hepatomegaly with increased hepatic levels of CEs, which reveals microvesicular steatosis leading to fibrosis and cirrhosis in the liver and increases atherosclerosis and premature demise.^4–6An LAL knockout (KO) mouse model (lal^−/−) resembles human CESD, and its biochemical and histopathologic phenotypes mimic human WD.^7,8 The lal^−/− mice are normal appearing at birth but develop liver enlargement by 1.5 months and have a grossly enlarged abdomen with hepatosplenomegaly and lymph node enlargement.^7,8 One interesting character of lal^−/− mice is a systemic expansion (including the liver) of CD11b⁺Ly6G⁺ cells, which are similar to myeloid-derived suppressive cells (MDSCs) in tumorigenesis.^9–11 MDSCs influence the tissue microenvironment and contribute to local pathogenesis.^7–13 In humans, increased CD14⁺CD16⁺ and CD14⁺CD33⁺ cells (human subsets of MDSCs) have been linked to heterozygote carriers of LAL mutations.¹⁴ Hepatocellular carcinoma has been linked to chronic inflammation with elevated MDSC counts.^15–17To better understand the physiologic role of LAL in hepatocytes and the link to clinical application, hepatocyte-specific expression of human LAL (hLAL) in lal^−/− mice was achieved by crossbreeding a liver-activated promoter (LAP)–driven tTA transgene (LAP-tTA) and a (tetO)₇-CMV-hLAL transgene (Tet off system) into lal^−/− mice (LAP-Tg/KO mice). Histologic and tissue lipid analyses revealed a correction of lipid storage in the liver, spleen, and small intestine in doxycycline-untreated LAP-Tg/KO mice (hLAL induction turns on) compared with doxycycline-treated LAP-Tg/KO mice (hLAL induction turns off). Flow cytometry analyses revealed reduced tumor-promoting MDSCs in the liver of LAP-Tg/KO mice. Furthermore, hLAL overexpression in hepatocytes greatly reduced metastasis of B16 melanoma into the liver. These pathogenic phenotypes were associated with a decrease of inflammatory cytokine and chemokine levels. Together, these results indicate that hepatic LAL plays an important role in lipid metabolism, cytokine production, MDSCs influx into organs, and tumor metastasis in the liver.     

Prolonged Infusion of Angiotensin II in apoE?/? Mice Promotes Macrophage Recruitment with Continued Expansion of Abdominal Aortic Aneurysm

Angiotensin II (AngII) infusion initiates abdominal aortic aneurysm (AAA) development due to medial disruption and results in luminal dilation and thrombus formation. The objective of this study was to determine whether AAA progressed during protracted AngII infusion. Male apoE^−/− mice were infused with AngII using miniosmotic pumps. On day 27, suprarenal aortic luminal diameters were ultrasonically measured to identify mice exhibiting AAAs. Mice were designated to three groups with similar mean luminal dilation. Group 1 mice were sacrificed on day 28. Group 2 and 3 mice were subsequently infused with saline or AngII, respectively, for an additional 56 days. In Group 2, saline infusion—after the initial 28 days of AngII infusion—led to an immediate decrease in systolic blood pressure. Over the subsequent 56 days of saline infusion, there were no aneurysm-related deaths or significant changes in luminal diameter. In contrast, continuous AngII infusion in Group 3 maintained persistently increased systolic blood pressure, with aneurysmal rupture–associated deaths, increased luminal diameters, and tissue remodeling. Aortic aneurysmal segments that expanded during continuous AngII infusion exhibited macrophage accumulation in regions of medial disruption, predominantly on the adventitial aspect. Macrophages immunostained for CD206 more than for iNOS, consistent with an M2 phenotype. In conclusion, prolonged AngII infusion promotes AAA expansion, and is associated with enhanced rupture rates and increased macrophage infiltration.Infusion of angiotensin II (AngII) into mice promotes rapid formation of abdominal aortic aneurysms (AAAs) within 14 days.^1–11 Initiation of AngII-induced AAA is associated with medial disruption leading to a localized thrombus.¹² There is a rapid luminal dilation between days 3 and 8 of AngII infusion with subsequent, gradual luminal expansion.¹² Although many laboratories have demonstrated that AngII consistently initiates AAA development in both hyper- and normocholesterolemic mice,^13–16 its role in aneurysmal progression, expansion, and rupture has not been defined in prospective human studies. However, there are conflicting data from retrospective analyses on AAA expansion and rupture in humans.^17–19Many factors have been associated with the development of AAAs in animal models.²⁰ Most of these studies have invoked mechanisms that prevent the initiation phase of AAAs. However, pharmacological treatment of AAAs in humans would be initiated in aortas with established disease. Currently, it is unknown whether pharmacological approaches that suppress initiation of disease would also inhibit progression of established aneurysms. Therefore, there is a need to determine mechanisms of AAA progression, expansion, and rupture since these may differ from disease initiation.The purpose of this current study was to determine whether chronic AngII infusion led to AAA expansion and rupture. This was accomplished by prolonged infusion of AngII for 84 days with sequential luminal diameter measurements and histological characterization of aortas at termination. This group was compared to one in which AAAs were initiated by AngII infusion, but were subsequently implanted with miniosmotic pumps infusing saline. The data demonstrate that continuous AngII infusion increased aortic dilation and aneurysmal rupture. Sequential sectioning of aneurysmal tissues demonstrated morphological heterogeneity, and increased macrophage recruitment localized to regions of medial disruption.     

Rescue of Impaired Fracture Healing in COX-2?/? Mice via Activation of Prostaglandin E2 Receptor Subtype 4

Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2^−/− mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2^−/− mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2^−/− callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2^−/− mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2^−/− fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2^−/− mice.Fracture healing is a complex process orchestrated by precise presentation of growth factors and cytokines that control activation, proliferation, and differentiation of the local mesenchymal stem/progenitor cells. Fracture healing begins with hematoma formation and an inflammatory response. The activated stem/progenitor cells proliferate and further differentiate into osteoblasts and chondrocytes. Endochondral bone formation takes place toward the most central avascular region of the callus. Chondrogenesis initiates directly adjacent to the surface of the cortical bone and is surrounded by less-differentiated mesenchymal progenitor cells. The subsequent expansion of the callus involves the conversion of the lingering mesenchymal progenitor cells into chondrocytes and further proliferation and differentiation of chondrocytes into a calcified cartilage template that permits vascular invasion and bone formation. Areas of intramembranous bone formation flank the area of endochondral ossification, particularly along the bone surface furthest from the central fracture site where the blood supply is typically better preserved. The coordinated endochondral and intramembranous bone formation pathways eventually result in a bridging mineralized callus that re-establishes the integrity of the skeletal element.^1,2,3COX-2 is the inducible isoform of cyclooxygenase, the key rate-limiting enzyme in the prostaglandin biosynthesis pathway.^4,5 Cyclooxygenase catalyzes the bis-oxygenation of arachidonic acid to form prostaglandin (PG)G2 and subsequent reduction of PGG2 to form PGH2. PGH2 is further metabolized by specific isomerases to produce various derivatives, eg, PGE2, PGD2, PGA2, thromboxanes, and prostacyclins, collectively called prostanoids. Prostanoids are labile molecules that act locally as important microenvironmental hormones mediating autocrine and/or paracrine functions. Among them, PGE2 has been shown to have both anabolic and catabolic effects on bone metabolism,^6,7 acting via four distinct EP receptors (EP1, EP2, EP3, and EP4) that belong to G-protein-coupled receptors.⁸ EP1 signaling is coupled to phospholipase C/inositol trisphosphate and leads to mobilization of intracellular calcium, whereas EP2 and EP4 receptor signaling generates increased intracellular cAMP via coupling to G_sα proteins. EP3 has generally been thought to couple to a G_i protein, leading to reduction in intracellular cAMP levels.⁹ EP2 and EP4 receptors are the major mediators for catabolic and anabolic effects of PGE2 in bone.^10,11 Both EP2 and EP4 mediate induction of RANKL through cAMP in osteoclastogenesis.^12,13 EP2 and EP4 also mediate PGE2-induced bone formation via osteoblastogenesis in animal models.^{11,14,15,16,17}We and others have previously shown that genetic ablation of COX-2 results in delayed and impaired fracture healing in mouse models.^18,19 COX-2^−/− fracture healing is characterized by marked reduction of bone formation, persistence of cartilaginous tissue, and high incidence of nonunions. Consistent with COX-2^−/− phenotypes, inhibitors of COX-2 are found to delay or impair healing in animal models.^{19,20,21,22,23} In one such study, COX-2 inhibitor was shown to impair fracture repair when used during the early inflammatory phase of healing.²⁵ A recent study using retrovirus overexpressing COX-2 shows that overexpression of COX-2 locally accelerates fracture healing and bone union.²⁵ Together, these studies provide overwhelming evidence to demonstrate that COX-2 is critical for bone fracture repair.The goal of this study was to further understand the molecular mechanisms and downstream targets of COX-2 during fracture repair. To this end, we used the COX-2^−/− mouse model and selective EP agonists that have been shown to enhance bone formation in animal models.^14,16,26 By performing detailed analyses using micro computed tomography (CT) and histology we show that COX-2 is induced early in the bone-healing milieu. Deficiency in COX-2 impairs initiation and completion of endochondral bone repair. Local injection of an EP2 agonist, CP-463755 marginally improved fracture healing in COX-2^−/− mice whereas delivery of an EP4 agonist, CP734432, reversed the defective bone fracture healing in COX-2^−/− mice. Further in vitro analyses demonstrated a differential role of EP receptor in chondrogenesis and induction of MMP-9, a matrix metalloproteinase (MMP) essential for angiogenesis during endochondral ossification. Together, the results pinpoint a key role of the COX-2/PGE2/EP4 pathway in fracture repair.     

Liver X receptor activation attenuates plaque formation and improves vasomotor function of the aortic artery in atherosclerotic ApoE?/? mice

Aim

The severity of atherosclerosis is primarily determined by overall lipid metabolism and the degree of inflammation present within the vessel wall. We evaluated the effects of T-0901317, a liver X receptor agonist, on the atherosclerosis process, and especially on the endothelial function in ApoE^?/? mice.

Methods and results

ApoE^?/? mice were treated with LXR agonist T-0901317 (1?μmol/L) for 6?weeks. ApoE^?/? mice receiving T-0901317 were found to have markedly improved overall serum lipid profiles, albeit increased serum triglycerides. MRI imaging demonstrated that T-0901317 attenuated the atherosclerotic plaque burden in the aorta of ApoE^?/? mice. Transmission electron microscopy and immunohistochemistry revealed attenuated ultrastructural changes as well as enhanced expression of the ATP-binding cassette transporter ABCA1. In addition, treatment with the LXR agonist improved the vasomotor function of atherosclerotic arteries, as assessed by KCl/norepinephrine-induced vasoconstrictive and acetylcholine-induced vasorelaxation functional assays. In vitro studies showed increased ABCG1, phospho-Akt and phospho-eNOS expression in ApoE^?/? mice aorta endothelial cells (ECs) after T0901317 treatment.

Conclusion

The present study suggest that LXR agonists protect the endothelium against atherosclerotic insults by increasing ABCA1 and ABCG1 expression, and improve the endothelial-dependent vasomotor function probably by promoting Akt and eNOS phosphorylation.     

Invasion of Oral and Aortic Tissues by Oral Spirochete Treponema denticola in ApoE?/? Mice Causally Links Periodontal Disease and Atherosclerosis

Treponema denticola is a predominantly subgingival oral spirochete closely associated with periodontal disease and has been detected in atherosclerosis. This study was designed to evaluate causative links between periodontal disease induced by chronic oral T. denticola infection and atherosclerosis in hyperlipidemic ApoE^−/− mice. ApoE^−/− mice (n = 24) were orally infected with T. denticola ATCC 35404 and were euthanized after 12 and 24 weeks. T. denticola genomic DNA was detected in oral plaque samples, indicating colonization of the oral cavity. Infection elicited significantly (P = 0.0172) higher IgG antibody levels and enhanced intrabony defects than sham infection. T. denticola-infected mice had higher levels of horizontal alveolar bone resorption than sham-infected mice and an associated significant increase in aortic plaque area (P ≤ 0.05). Increased atherosclerotic plaque correlated with reduced serum nitric oxide (NO) levels and increased serum-oxidized low-density lipoprotein (LDL) levels compared to those of sham-infected mice. T. denticola infection altered the expression of genes known to be involved in atherosclerotic development, including the leukocyte/endothelial cell adhesion gene (Thbs4), the connective tissue growth factor gene (Ctgf), and the selectin-E gene (Sele). Fluorescent in situ hybridization (FISH) revealed T. denticola clusters in both gingival and aortic tissue of infected mice. This is the first study examining the potential causative role of chronic T. denticola periodontal infection and vascular atherosclerosis in vivo in hyperlipidemic ApoE^−/− mice. T. denticola is closely associated with periodontal disease and the rapid progression of atheroma in ApoE^−/− mice. These studies confirm a causal link for active oral T. denticola infection with both atheroma and periodontal disease.     

Genes Required for the Fitness of Salmonella enterica Serovar Typhimurium during Infection of Immunodeficient gp91?/? phox Mice

Salmonella enterica causes systemic diseases (typhoid and paratyphoid fever), nontyphoidal septicemia (NTS), and gastroenteritis in humans and other animals worldwide. An important but underrecognized emerging infectious disease problem in sub-Saharan Africa is NTS in children and immunocompromised adults. A current goal is to identify Salmonella mutants that are not pathogenic in the absence of key components of the immune system such as might be found in immunocompromised hosts. Such attenuated strains have the potential to be used as live vaccines. We have used transposon-directed insertion site sequencing (TraDIS) to screen mutants of Salmonella enterica serovar Typhimurium for their ability to infect and grow in the tissues of wild-type and immunodeficient mice. This was to identify bacterial genes that might be deleted for the development of live attenuated vaccines that would be safer to use in situations and/or geographical areas where immunodeficiencies are prevalent. The relative fitness of each of 9,356 transposon mutants, representing mutations in 3,139 different genes, was determined in gp91^−/− phox mice. Mutations in certain genes led to reduced fitness in both wild-type and mutant mice. To validate these results, these genes were mutated by allelic replacement, and resultant mutants were retested for fitness in the mice. A defined deletion mutant of cysE was attenuated in C57BL/6 wild-type mice and immunodeficient gp91^−/− phox mice and was effective as a live vaccine in wild-type mice.     

Systemic Macrophage Depletion Inhibits Helicobacter bilis-Induced Proinflammatory Cytokine-Mediated Typhlocolitis and Impairs Bacterial Colonization Dynamics in a BALB/c Rag2?/? Mouse Model of Inflammatory Bowel Disease

Helicobacter bilis, an enterohepatic helicobacter, is associated with chronic hepatitis in aged immunocompetent inbred mice and inflammatory bowel disease (IBD) in immunodeficient mice. To evaluate the role of macrophages in H. bilis-induced IBD, Rag2^−/− BALB/c or wild-type (WT) BALB/c mice were either sham dosed or infected with H. bilis Missouri strain under specific-pathogen-free conditions, followed by an intravenous injection of a 0.2-ml suspension of liposomes coated with either phosphate-buffered saline (control) or clodronate (a macrophage depleting drug) at 15 weeks postinfection (wpi). At 16 wpi, the ceca of H. bilis-infected Rag2^−/− mice treated with control liposomes had significantly higher histopathological lesional scores (for cumulative typhlitis index, inflammation, edema, epithelial defects, and hyperplasia) and higher counts of F4/80⁺ macrophages and MPO⁺ neutrophils compared to H. bilis-infected Rag2^−/− mice treated with clodronate liposomes. In addition, cecal quantitative PCR analyses revealed a significant suppression in the expression of macrophage-related cytokine genes, namely, Tnfa, Il-1β, Il-10, Cxcl1, and iNos, in the clodronate-treated H. bilis-infected Rag2^−/− mice compared to the H. bilis-infected Rag2^−/− control mice. Finally, cecal quantitative PCR analyses also revealed a significant reduction in bacterial colonization in the clodronate-treated Rag2^−/− mice. Taken together, our results suggest that macrophages are critical inflammatory cellular mediators for promoting H. bilis-induced typhlocolitis in mice.