Cleavage of Osteopontin by Matrix Metalloproteinase-12 Modulates Experimental Autoimmune Encephalomyelitis Disease in C57BL/6 Mice

A role for osteopontin (OPN) in promoting disease activity of multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) has recently been suggested. As the biological activity of OPN is heavily influenced by posttranslational processing, we investigated the capacity of matrix metalloproteinase (MMP)-12 to cleave OPN and determined whether this influenced disease activity. We found that OPN mRNA and protein expression in the spinal cord increased with EAE disease in C57BL/6 mice concurrently with MMP-12 expression. A Western blot of EAE and control spinal cords revealed different OPN-immunoreactive bands, with a pattern that was similar to MMP-12 cleavage of recombinant OPN in vitro. In addition, OPN fragments in the spinal cord of EAE-afflicted mice were reduced in MMP-12^−/− mice compared with wild-type controls. However, examination of OPN^−/− mice in short- and long-term experiments revealed no difference in EAE outcomes from wild-type animals. OPN/MMP-12 double null mice were generated, and it was revealed that MMP-12^−/− mice had a worsening of disease compared with wild-type mice, which returned to wild-type levels in the OPN/MMP-12 double null mice. These results suggest that EAE disease activity may be modulated by the cleavage of OPN by MMP-12.Multiple sclerosis (MS) is a disease in which peripheral T cells infiltrate the central nervous system (CNS), where they become re-activated by presentation of CNS antigens by local antigen-presenting cells including microglia and dendritic cells to result in demyelination.^1,2,3,4 In attempts to find putative antigens and mediators of disease, studies using gene array analyses revealed that osteopontin (OPN) was highly up-regulated in lesions from patients with MS compared with control subjects.⁵ This result has prompted the investigations of OPN expression and function in MS and experimental autoimmune encephalomyelitis (EAE).OPN, also known as early T cell activation gene 1 (Eta-1), is a calcium binding phosphorylated acidic glycoprotein.^6,7 OPN has pluripotent activity and is involved in various biological roles such as extracellular matrix remodeling, tumor invasion, angiogenesis, cell-mediated immunity, and the regulation of urokinase and matrix metalloproteinase (MMP) production.⁸ Chabas et al⁵ found that OPN was expressed during EAE in various cell types and that OPN^−/− mice had reduced EAE clinical disease, which was associated with a shift toward a Th2 cytokine profile. Jansson et al⁹ also found that OPN^−/− mice had reduced mean maximal score, fewer days of paralyzing disease, and no spontaneous relapses on proteolipid protein-induced EAE. In that study, OPN^−/− mice showed reduced production of pro-inflammatory cytokines, interferon-γ (IFN-γ), and tumor necrosis factor-α. In contrast to these studies, Blom et al¹⁰ found no difference between OPN^−/− and wild-type mice for EAE outcomes.The EAE results sparked an interest in the expression of OPN in MS. Increased levels of OPN protein is reported in the serum and plasma in patients with relapsing-remitting MS compared with controls,^11,12,13,14 particularly during relapses, and in their cerebrospinal fluid.^15,16 However, there is little evidence for a genetic link between OPN and MS disease susceptibility and disease course.^{11,13,17,18,19,20} Elevated OPN levels have also been documented by immunohistochemistry around MS lesions^21,22 although this was not observed by others.²³ The role that OPN plays in EAE and MS remains uncertain, but a prevailing view is that OPN plays a destructive role during EAE since it reduces apoptosis of effector T cells and because the exogenous administration of recombinant OPN exacerbates EAE clinical disease.²⁴The biological functions of OPN are heavily influenced by posttranslational modifications such as phosphorylation, glycosylation, sulfation, and proteolytic cleavage.^25,26 Currently, there is very little knowledge about the role that proteolytic cleavage of OPN has on MS and EAE. In other disease states, the cleavage of OPN by MMPs and thrombin alters the biological functions of OPN.^27,28 For example, OPN that is cleaved by MMP-3 and MMP-7 significantly increases adhesion of tumor cells compared with full length OPN.²⁹ Recombinant MMP-12 has been demonstrated to cleave OPN, a protein that strongly influences osteoclasts activities, including attachment, spreading, and resorption.³⁰The family of MMPs is implicated in MS and EAE as several MMP members are elevated in biological samples from these conditions. These include MMP-2, -3, -8, -9, -10, -11, -12, -13, -14, and -25.^{31,32,33,34,35,36,37,38} Although the majority of these MMP members appear to have detrimental roles in MS and EAE,^39,40,41 MMP-12 is unique since its absence causes an exacerbation in EAE disease scores that is associated with up-regulation of pro-inflammatory cytokines.^31,42 Since the MMP-12^−/− mice phenotype is opposite to the reduced levels of pro-inflammatory cytokines in OPN^−/− mice with EAE,⁵ we considered the possibility that MMP-12 and OPN are inversely associated. In this article, we have tested the hypothesis that MMP-12 processes OPN to reduce its pro-inflammatory potential in EAE. We describe OPN mRNA and protein expression during EAE and have used a combination of MMP-12 and OPN single and OPN/MMP-12 double null mice to profile their EAE phenotypes.     

Laminin α4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

Each extracellular matrix compartment in the kidney has a unique composition, with regional specificity in the expression of various laminin isoforms. Although null mutations in the majority of laminin chains lead to specific developmental abnormalities in the kidney, Lama4−/− mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor β in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to α4-containing laminins, but not other isoforms, results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. Given the known role of overexpression of PDGF-BB and PDGF-DD on glomerular and tubulointerstitial fibrosis, these data suggest that failure of laminin α4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin α4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans.Laminin (LN) is a large, heterotrimeric, cruciform molecule composed of α, β, and γ subunits.¹ Five distinct α (LAMA1-5), 3 β (LAMB1-3), and 3 γ (LAMC1-3) chains^1,2 variably assemble to create distinct isoforms³ that are temporally and spatially regulated, and each conveys a variety of biological functions.^{4,5,6,7,8,9,10,11} The LNα4-containing isoforms, LN411 (α4β1γ1) and LN421 (α4β2γ1), are abundant in microvessels. Studies of LNα4-deficient mutant mice (lama4−/−) reveal that although α4-LNs are not required for blood vessel formation, they play important roles in blood vessel maturation, and in stabilization of vessels that form with injury, inflammation and tumor growth.^7,12,13 In vitro studies indicate that α4LNs directly regulate endothelial cell proliferation and inhibit apoptosis.¹⁴ α4-LNs are produced by endothelial cells in most microvessels; however, endothelial cells in the renal glomerulus do not express LNα4-containing isoforms.¹⁵ Instead, LN411 and LN421 at the endothelial-mesangial interface are produced by the mesangial cells (MCs).¹⁵ Platelet-derived growth factor (PDGF) is the primary growth factor responsible for MC proliferation and migration during glomerulogenesis,¹⁶ and we have shown that PDGF-induced MC migration requires LNα4.¹⁵ This function could not be replaced by LN111 or LN511/521.¹⁵ Together these observations suggested the possibility that deficiency of LNα4 might impair the ability of the kidney microvasculature to mature or be repaired in lama4−/− adult mice, resulting in kidney disease despite normal initial development.Previous reports have documented a spectrum of developmental defects and tissue maintenance defects in lama4−/− mice. Early postal-natal hemorrhage from birth-related trauma to fragile blood vessels occurs in lama4−/− mice; yet, by three-weeks of age, accumulation of LNα5 stabilizes vessels, although they remain dilated.¹² Vessel fragility recurs when new vessels form in response to injury.¹² The heart forms normally, but lama4−/− mice develop cardiomyopathy with time.¹⁷ Neurological dysfunction occurs in lama4−/− mice, through independent defects in organizing presynaptic specializations at neuromuscular synapses,¹⁸ and in the ability of developing Schwann cells to properly sort and myelinate.^19,20 This report details the characteristics of kidney abnormalities, including the development of glomerulosclerosis and tubulointerstitial fibrosis over time in lama4−/− mice.     

Liver Xeno-Repopulation with Human Hepatocytes in Fah?/?Rag2?/? Mice after Pharmacological Immunosuppression

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury–induced donor hepatocyte selection: the uPA/SCID mice and Fah^−/− Rag2^−/−Il2rg^−/− mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah^−/−Rag2^−/− mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah^−/−Rag2^−/− mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.Human hepatocytes xeno-engrafted into the liver of immunodeficient mice could be used as a model to study human hepatitis virus infection in vivo as well as the efficacy of potential vaccines.^1,2,3,4,5,6 Engrafted human hepatocytes can be serially transplanted from primary mice into secondary mice without losing hepatic function.⁷ Mouse recipients of human liver cells must have two capabilities: robust liver repopulation and immune tolerance for human hepatocytes. Liver xeno-repopulation from human hepatocytes was first reported in uPA/Rag2^−/− mice¹ and uPA/SCID mice.^2,3,8 The levels of liver xeno-repopulation varies in several reports, ranging from 10% to as high as 90%.^1,8 Humanized livers in uPA/SCID mice are susceptible to hepatitis B virus (HBV)^1,2 and HCV^3,4 infection. However, uPA mice have several disadvantages: i) neonatal death during colony breeding; ii) transplantation of hepatocytes into newborn mice (within the second week of life) is technically difficult due to a bleeding disorder in the mice; iii) there is uncontrollable selection for donor cells; iv) there is autoreversion of endogenous hepatocytes; and v) kidney damage is induced by the human complement system.^1,2,8,9Recently, robust liver xeno-repopulation from human hepatocytes was found in Fah^−/−Rag2^−/−Il2rg^−/− mice, cross-bred from Fah^−/− mice and Rag2^−/−Il2rg^−/− mice.⁷ Fah^−/−Rag2^−/−Il2rg^−/− mice have advantages over previous immunodeficient uPA models.⁷ First, Rag2^−/−Il2rg^−/− mice lack B, T, and NK cells, rendering more complete immunodeficiency compared with either Rag2^−/− or SCID mice.¹⁰ Second, liver injury in Fah^−/− mice is controllable by switching on and off 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1, 3 cyclohexanedione (NTBC) administration.¹¹ NTBC inhibits accumulation of toxic metabolites in hepatocytes to maintain Fah^−/− mice in a healthy state. When the NTBC is removed, a powerful selection for fumaryl acetoacetate hydrolase (Fah) expressing cells is induced in the liver.¹² However, maintenance of Fah^−/−Rag2^−/−Il2rg^−/− mice during colony breeding, animal growing, and cell transplantation surgery are with high mortality in our experiments. The genotyping of animal offspring is overly elaborate. These concerns have not been discussed in previous publications^7,13 and may present a barrier to larger scale research projects.In comparison, Fah^−/−Rag2^−/− mice were much more tolerant of breeding and surgery procedures. However, Fah^−/−Rag2^−/− mice were thought to have no capacity for liver xeno-repopulation, because their NK cells are intact.⁷ We hypothesized that treatment of Fah^−/− Rag2^−/− mice with anti-asialo GM1 could result in complete depletion of NK cells as seen in Fah^−/−Rag2^−/− Il2rg^−/− mice.^14,15 We further tested the combined treatments of both anti-asialo GM1 and the immunosuppressor tacrolimus (FK506) to Fah^−/−Rag2^−/− mice.^16,17 The results indicated that the combined treatments enabled Fah^−/−Rag2^−/− recipients to have a high level of liver xeno-repopulation by human hepatocytes as seen in Fah^−/−Rag2^−/−Il2rg^−/− mice. Our results revealed a new and easily controlled mouse model with humanized liver. Using the same treatments, liver xeno-repopulation with human fetal liver progenitor cells was also achieved in Fah^−/−Rag2^−/− mice. Finally, for the first time, we were able to prove that human HBV actively replicated in the humanized Fah^−/−Rag2^−/− mice and that viral proteins were released in the serum of humanized Fah^−/−Rag2^−/− mice, which showed no significant difference with previous reports of human HBV infection in humanized uPA/SCID mice.^1,2,5     

Decay-Accelerating Factor Suppresses Complement C3 Activation and Retards Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

Decay-accelerating factor (DAF; CD55) is a membrane protein that regulates complement pathway activity at the level of C3. To test the hypothesis that DAF plays an essential role in limiting complement activation in the arterial wall and protecting from atherosclerosis, we crossed DAF gene targeted mice (daf-1^−/−) with low-density lipoprotein-receptor deficient mice (Ldlr^−/−). Daf-1^−/−Ldlr^−/− mice had more extensive en face Sudan IV staining of the thoracoabdominal aorta than Ldlr^−/− mice, both following a 12-week period of low-fat diet or a high-fat diet. Aortic root lesions in daf-1^−/−Ldlr^−/− mice on a low-fat diet showed increased size and complexity. DAF deficiency increased deposition of C3d and C5b-9, indicating the importance of DAF for downstream complement regulation in the arterial wall. The acceleration of lesion development in the absence of DAF provides confirmation of the proinflammatory and proatherosclerotic potential of complement activation in the Ldlr^−/− mouse model. Because upstream complement activation is potentially protective, this study underlines the importance of DAF in shielding the arterial wall from the atherogenic effects of complement.Complement, a complex cascade of serine proteases, is well characterized as playing a pivotal role in inflammation and in bridging innate and adaptive immunity.¹ Currently, complement is understood to be triggered by three proximal cascades, the classical, alternative, and mannose-binding lectin pathways, which converge on C3 at the central hub of the system. Cleavage of C3 leads to the generation of down-stream proinflammatory mediators, including the anaphylatoxins C3a and C5a and the membrane attack-complex C5b-9. Although the assembly and insertion of C5b-9 into cell membranes may lyse non-nucleated cells, sublytic levels can activate proliferation and/or proinflammatory gene expression.²There is increasing interest in dissecting the possible roles of complement in atherosclerosis in vivo.^3,4 Theoretically, many factors might activate complement in the arterial wall, including Igs, cholesterol crystals, enzymatically-modified low-density lipoprotein (LDL) and apoptotic cells.^{5,6,7,8,9,10,11,12} However, enzymatically-modified LDL is likely to be the most abundant stimulus for complement activation in atherosclerosis, and may act via the alternative pathway and also via direct binding of C1q and C-reactive protein.^8,9,13,14,15 In addition, the classical and alternative pathways are capable of low grade “tick-over” activity.^16,17Previous experimental work has focused on the effects of natural or experimental deficiency of individual complement pathway components. Relevant studies are as follows: (1) rabbits with natural deficiency of C6 have been shown to be protected from diet-induced atherosclerosis^18,19; (2) although C5 deficiency has been found to have no effect on lesion development in high fat diet-fed ApoE^−/− mice,²⁰ a recent study has shown protective effects of an anti-C5 antibody in ApoE^−/− mice deficient in both Cd59a and Cd59b genes²¹; (3) C3 deficient mice crossed with Ldlr^−/− single knock-out mice have been found to have increased aortic lipid deposition with impaired lesion development beyond the foam cell stage²²; (4) crossing Factor B deficient mice with ApoE^−/−Ldlr^−/− double-knock-outs had no effect, arguing against an important role for the alternative pathway in that model²³; and (5) more recently, we have reported that low fat diet-fed Ldlr^−/− mice deficient in classical pathway activity through gene-targeting of C1q (C1qa^−/−) show accelerated atherosclerosis with increased lesion complexity.²⁴ The increased lesion size and complexity in low fat diet-fed C1qa^−/−Ldlr^−/− mice was associated with an increase in lesional apoptotic cells, consistent with previous studies that have demonstrated a direct role for C1q in apoptotic cell clearance, independent of terminal pathway activation.^25,26 Recently, the role of the lectin pathway has also been shown to have atheroprotective functions in mice,²⁷ in line with the involvement of mannose-binding lectin in apoptotic cell clearance and also with the association of mannose-binding lectin deficiency with accelerated atherosclerosis in humans.^28,29Complement activity is tightly regulated by a number of fluid-phase and membrane-bound inhibitors, including the two glycosylphosphatidylinositol-anchored membrane proteins decay-accelerating factor (DAF, CD55) and protectin (CD59). Although CD59 inhibits insertion of C9 into cell membranes and thus the development of C5b-9 membrane attack complexes, DAF binds to C3, thereby accelerating the decay of the two C3 convertases, C3Bb (alternative pathway) and C4b2a (classical and mannose-binding lectin pathways).^30,31,32 Structurally, DAF is a multidomain protein comprising a proximal serine/threonine-rich region and four complement control protein (CCP) domains, of which CCP2 and CCP3 dissociate C3Bb and C4b2a oligomers into constituent proteins. The catalytic mechanism of DAF activity is not fully clear, but the crystal structure and substitution mutants identify Bb (Tyr338), DAF-CCP2 (Arg69, Arg96) and DAF-CCP3 (Phe148 Leu171) as key residues.^33,34The mouse has two DAF genes encoding glycosylphosphatidylinositol-anchored and trans-membrane forms, respectively, with the former being more representative of human DAF.³⁵ Gene-targeting of the glycosylphosphatidylinositol-anchored form has led to the generation of a knock-out strain that is healthy but shows exaggerated inflammation in models of renal, autoimmune, and nervous system diseases.^36,37,38,39 Recently, no protection or exacerbation of atherosclerosis was observed after crossing these mice with the ApoE^−/− strain.⁴⁰Observations that the classical pathway exerts atheroprotective effects without terminal pathway activation suggest the importance of a strong complement regulatory system in the arterial wall.^14,24 Consistent with this, we and others have recently published evidence that CD59 deficiency leads to an acceleration of atherosclerosis in Ldlr^−/− and ApoE^−/− mouse models, establishing the proatherogenic potential of the terminal complement pathway and highlighting the importance of CD59 in its regulation.^21,40,41 In this article, we show that DAF also plays a role in the regulation of atherosclerosis in the Ldlr^−/− model.     

Matrilysin (Matrix Metalloproteinase-7) Regulates Anti-Inflammatory and Antifibrotic Pulmonary Dendritic Cells That Express CD103 (αEβ7-Integrin)

The E-cadherin receptor CD103 (α_Eβ₇-integrin) is expressed on specific populations of pulmonary dendritic cells (DC) and T cells. However, CD103 function in the lung is not well understood. Matrilysin (MMP-7) expression is increased in lung injury and cleaves E-cadherin from injured lung epithelium. Thus, to assess matrilysin effects on CD103-E-cadherin interactions in lung injury, wild-type, CD103^−/−, and Mmp7^−/− mice, in which E-cadherin isn’t cleaved in the lung, were treated with bleomycin or bleomycin with nFMLP to reverse the defect in acute neutrophil influx seen in Mmp7^−/− mice. Pulmonary CD103⁺ DC were significantly increased in injured wild-type compared with Mmp7^−/− mice, and CD103⁺ leukocytes showed significantly enhanced interaction with E-cadherin on injured wild-type epithelium than with Mmp7^−/− epithelium in vitro and in vivo. Bleomycin-treated CD103^−/− mice had persistent neutrophilic inflammation, increased fibrosis, and increased mortality compared with wild-type mice, a phenotype that was partially recapitulated in bleomycin/nFMLP-treated Mmp7^−/− mice. Soluble E-cadherin increased IL-12 and IL-10 and reduced IL-6 mRNA expression in wild-type bone marrow-derived DC but not in CD103^−/− bone marrow-derived DC. Similar mRNA patterns were seen in lungs of bleomycin-injured wild-type, but not CD103^−/− or Mmp7^−/−, mice. In conclusion, matrilysin regulates pulmonary localization of DC that express CD103, and E-cadherin cleavage may activate CD103⁺ DC to limit inflammation and inhibit fibrosis.Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute epithelial and endothelial damage, leakage of proteinaceous edema fluid into the alveolar space and interstitium, and a leukocytic cellular infiltrate, with polymorphonuclear neutrophils being the key inflammatory cell population in both humans and in experimental animals.¹ Unfavorable outcomes in patients with ALI/acute respiratory distress syndrome are associated with an exaggerated pulmonary inflammatory response that persists unabated over time.^2,3 Failure to resolve acute inflammation also contributes to chronic lung injury and pulmonary fibrosis, and the presence of extensive fibrosis may be an independent risk factor that correlates with poor outcome.⁴ Impaired epithelial repair contributes to fibrosis in the lung, liver, kidney, and other tissues,^5,6 and epithelial cell interactions with inflammatory and mesenchymal cells are central to both physiological lung repair and pathological lung remodeling.Important among the pulmonary responses to injury is the increased expression and activation of enzymes in the matrix metalloproteinase (MMP) family.⁷ MMPs are zinc-binding enzymes with activity against a wide range of extracellular proteins,⁸ and MMP expression is typically limited to tissue remodeling associated with development, involution, inflammation, tumor growth, and repair. Our laboratory found that matrilysin (MMP-7) is strongly induced in injured alveolar epithelium in emphysema, desquamative interstitial pneumonitis, cystic fibrosis, and acute respiratory distress syndrome.^9,10 In bleomycin-induced lung injury in mice, matrilysin expression is increased in alveolar epithelium early after injury and regulates acute neutrophil influx by controlling KC chemokine release into the alveolar compartment during the first 5 days following injury.¹¹ Beyond the acute phase of injury, matrilysin expression increases as neutrophilic inflammation subsides and fibrosis ensues,¹¹ and thus, matrilysin has been implicated in the progression of pulmonary fibrosis.¹² However, when acute neutrophil influx is restored in bleomycin-treated matrilysin-null (Mmp7^−/−) mice with the neutrophil chemotactic peptide nFMLP, mortality is higher in Mmp7^−/− mice than in wild-type mice.¹¹ Thus, observations of increased fibrosis in bleomycin-treated Mmp7^−/− mice likely reflect the early acute injury phenotype, and in chronic lung injury, matrilysin activity may regulate physiological functions that promote repair.E-cadherin regulates cell-cell adhesion in most epithelia and maintains epithelial integrity, restricts migration and proliferation, and promotes differentiation.¹³ The proteolytic cleavage of membrane proteins from the cell surface has been described as “ectodomain shedding,”^14,15,16,17 and we described a physiological role for matrilysin-dependent shedding of the E-cadherin ectodomain in airway mucosal repair.¹⁰ We also found that matrilysin cleaves E-cadherin from alveolar epithelium during the progression of bleomycin-induced pulmonary fibrosis, and Mmp7^−/− mice do not shed E-cadherin in the injured lung. A few in vitro studies have evaluated the function of E-cadherin shedding in cancer cells, suggesting potential roles in regulating cancer cell migration or gene expression.^18,19 However, to our knowledge, in vivo functions for E-cadherin shedding in chronic lung injury and fibrosis have not been previously assessed.The leukocyte-specific α_Eβ₇-integrin (CD103) is expressed on nearly all intraepithelial lymphocytes and on specific populations of dendritic cells (DC), and E-cadherin is the only known CD103 ligand.^20,21 Transforming growth factor-β1 (TGF-β1) induces CD103 expression, and increased TGF-β1 in injured tissues may up-regulate CD103 on infiltrating leukocytes.^22,23,24 Via interaction with E-cadherin, CD103 has been suggested to be an epithelial recognition molecule that retains CD103⁺ lymphocytes at epithelial surfaces, targets epithelial tumor cells for destruction by cytolytic T cells, or regulates kidney allograft rejection.^{23,25,26,27,28} CD103⁺ pulmonary DC arise from myeloid mononuclear precursors, do not express plasmacytoid DC markers,^29,30 and appear to have distinct cytokine and antigen presentation capabilities compared with CD103⁻ myeloid DC populations.^31,32 However, the function of CD103 in lung injury has not been defined. Therefore, we explored the possibility that E-cadherin shedding could be a mechanism controlling interactions between leukocytes that express CD103 and epithelial cells that express its ligand E-cadherin in bleomycin-induced lung injury in mice.     

IL-4/IL-13-Dependent Alternative Activation of Macrophages but Not Microglial Cells Is Associated with Uncontrolled Cerebral Cryptococcosis

Both interleukin (IL)-4- and IL-13-dependent Th2-mediated immune mechanisms exacerbate murine Cryptococcus neoformans-induced bronchopulmonary disease. To study the roles of IL-4 and IL-13 in cerebral cryptococcosis, IL-4 receptor α-deficient (IL-4Rα^−/−), IL-4-deficient (IL-4^−/−), IL-13-deficient (IL-13^−/−), IL-13 transgenic (IL-13^T/+), and wild-type mice were infected intranasally. IL-13^T/+ mice displayed a higher fungal brain burden than wild-type mice, whereas the brain burdens of IL-4Rα^−/−, IL-4^−/−, and IL-13^−/− mice were significantly lower as compared with wild-type mice. On infection, 68% of wild-type mice and 88% of IL-13-overexpressing IL-13^T/+ mice developed significant cerebral lesions. In contrast, only a few IL-4Rα^−/−, IL-4^−/−, and IL-13^−/− mice had small lesions in their brains. Furthermore, IL-13^T/+ mice harbored large pseudocystic lesions in the central nervous system parenchyma, bordered by voluminous foamy alternatively activated macrophages (aaMphs) that contained intracellular cryptococci, without significant microglial activation. In wild-type mice, aaMphs tightly bordered pseudocystic lesions as well, and these mice, in addition, showed microglial cell activation. Interestingly, in resistant IL-4^−/−, IL-13^−/−, and IL-4Rα^−/− mice, no aaMphs were discernible. Microglial cells of all mouse genotypes neither internalized cryptococci nor expressed markers of alternative activation, although they displayed similar IL-4Rα expression levels as macrophages. These data provide the first evidence of the development of aaMphs in a central nervous system infectious disease model, pointing to distinct roles of macrophages versus microglial cells in the central nervous system immune response against C. neoformans.The opportunistic pathogenic yeast Cryptococcus neoformans causes life-threatening fungal infections of most internal organs including the central nervous system (CNS), primarily in patients affected by immunodeficiency syndromes such as AIDS.¹ The pathogenesis of cryptococcosis is not fully understood, however, especially in cases of different levels of immunocompetence. It is generally accepted that the fungus first invades the respiratory system, where it leads to relatively mild or asymptomatic bronchopneumonia in the immunocompetent.^2,3,4,5 Fungemia with generalization of the infection may result from reduced immunological control mechanisms.^6,7,8,9 Invasion of the CNS with subsequent development of meningoencephalitis is the major cause of death during cryptococcosis.^10,11The precise reaction pattern of recruited inflammatory cells, especially monocytes/macrophages, due to fungal invasion of the CNS parenchyma has been addressed mainly via analysis of helper T cell (Th)1 responses.¹² In this context, in addition to protective Th1-driven immune responses, the role of Th2 cytokines has gained interest recently.¹³ The major Th2 cytokines interleukin (IL)-4 and IL-13 act via the IL-4Rα chain together with the γ_c chain or the IL-13Rα1/2 chains, and regulate macrophage functional status.¹⁴ IL-4 has been shown to be detrimental in murine models of systemic and pulmonary cryptococcosis,^{6,15,16,17,18} and we have recently illustrated the role of IL-13 in inducing the formation of alternatively activated macrophages (aaMphs) in murine pulmonary cryptococcosis.¹⁹The activation phenotype of macrophages may critically influence the regulatory mechanisms by which inflammation and infection in the CNS are controlled. According to the current paradigm, classically activated macrophages are primed by interferon-γ and produce tumor necrosis factor, IL-1, oxygen and nitrogen radicals,²⁰ thereby producing proinflammatory cytokines that regulate the Th1 immune response. In contrast, aaMph²¹ develop in response to Th2 cytokine stimulation such as IL-4 and IL-13 and are characterized by expression of genes associated with endocytosis and tissue repair such as arginase-1, mannose receptor (CD206), found-in-inflammatory-zone (FIZZ), and chitinase 3-like 3 (YM1) and largely fail to produce nitric oxide (NO) due to their induction of arginase.²² As such, they are thought to be involved in tissue repair and remodeling,^22,23 in protection against diet-induced obesity,^24,25 and schistosomiasis,²⁶ but they may also elicit adverse tissue processes such as pulmonary or liver fibrosis.^{27,28,29,30,31} In particular, their development renders the host vulnerable to infection with pathogens where macrophage activation and killing functions are required.³²In murine models of pulmonary C. neoformans infection, aaMph have been shown to be associated with uncontrolled lung infection.^18,19 The role of aaMph versus classically activated macrophage in the CNS due to pulmonary infection with the neurotropic pathogen C. neoformans has not been defined yet. In this study, we aimed to characterize the morphology and functional status of CNS macrophages in cerebral cryptococcosis following intranasal infection of susceptible wild-type and IL-13-transgenic BALB/c mice. Moreover, using mice unable to produce IL-4 or IL-13 or respond to both (IL-4Rα^−/− mice), we show that abrogation of CNS aaMph development is associated with controlled infection.     

Progressive Secondary Neurodegeneration and Microcalcification Co-Occur in Osteopontin-Deficient Mice

In the brain, osteopontin (OPN) may function in a variety of pathological conditions, including neurodegeneration, microcalcification, and inflammation. In this study, we addressed the role of OPN in primary and secondary neurodegeneration, microcalcification, and inflammation after an excitotoxic lesion by examining OPN knock-out (KO) mice. Two, four, and ten weeks after injection of the glutamate analogue ibotenate into the corticostriatal boundary, the brains of 12 mice per survival time and strain were evaluated. OPN was detectable in neuron-shaped cells, in microglia, and at the surface of dense calcium deposits. At this primary lesion site, although the glial reaction was attenuated in OPN-KO mice, lesion size and presence of microcalcification were comparable between OPN-KO and wild-type mice. In contrast, secondary neurodegeneration at the thalamus was more prominent in OPN-KO mice, and this difference increased over time. This was paralleled by a dramatic rise in the regional extent of dense microcalcification. Despite these differences, the numbers of glial cells did not significantly differ between the two strains. This study demonstrates for the first time a genetic model with co-occurrence of neurodegeneration and microcalcification, mediated by the lack of OPN, and suggests a basic involvement of OPN action in these conditions. In the case of secondary retrograde or transneuronal degeneration, OPN may have a protective role as intracellular actor.Co-occurrence of neurodegeneration, parenchymal (micro-) calcification, and inflammation can be observed in a number of brain diseases, including Fahr’s, Alzheimer’s, diffuse Lewy body and Parkinson’s disease, Down’s syndrome, and hypoxia.^1,2,3,4,5,6Osteopontin (OPN) is a glycophosphoprotein with intra- and extracellular functions influencing cell survival, inflammation, microcalcification and the maintenance of tissue integrity after an injury.⁷ The manifold higher abundance of OPN in cerebrospinal fluid than in blood^8,9 argues for a crucial role of this protein in central nervous system (CNS) physiology and pathology. In the developing and adult (rodent) brain, neurons of the olfactory bulb, retina, striatum, and brainstem are OPN-positive.^10,11,12,13 In the aging human brain, OPN is found in pyramidal neurons—more pronounced in Alzheimer’s disease¹⁴—and in dopaminergic neurons of Parkinson’s disease patients.⁸ Transient expression of neuronal OPN has been observed under experimental conditions like cryolesioning¹⁵ and status epilepticus.¹⁶ In addition, OPN is detectable in microglial cells of lesioned CNS tissue after ischemia,¹⁷ excitotoxicity,¹² spinal cord contusion,¹⁸ as well as in multiple sclerosis plaques¹⁹ and in microglial cells of the substantia nigra of Parkinson’s disease patients.⁸ OPN may also be located extracellularly.^8,17 The role of OPN in CNS diseases remains controversial. OPN has been shown to be protective in models of stroke^17,20,21 and spinal cord contusion.¹⁸ However, OPN inhibited axonal regeneration after injury in the optic nerve,²² and the absence of the protein led to a better outcome in models of multiple sclerosis¹⁹ and Parkinson’s disease.⁸OPN inhibits calcification in bone and at ectopic sites.^{23,24,25,26,27} To our knowledge, the role of OPN in brain microcalcification is unknown. In addition, the co-occurrence of neurodegeneration and microcalcification has not yet been investigated with a genetic model. In our present study, we were interested in whether OPN deficiency–induced neurodegeneration is paralleled by microcalcification, and which functions of the protein may be primarily involved.     

Carbon Monoxide Rescues Heme Oxygenase-1-Deficient Mice from Arterial Thrombosis in Allogeneic Aortic Transplantation

Heme oxygenase-1 (HO-1) catalyzes the conversion of heme into carbon monoxide (CO), iron, and biliverdin. In preliminary studies, we observed that the absence of HO-1 in aortic allograft recipients resulted in 100% mortality within 4 days due to arterial thrombosis. In contrast, recipients normally expressing HO-1 showed 100% graft patency and survival for more than 56 days. Abdominal aortic transplants were performed using Balb/cJ mice as donors and either HO-1^+/+ or HO-1^−/− (C57BL/6×FVB) mice as recipients. Light and electron microscopy revealed extensive platelet-rich thrombi along the entire length of the graft in HO-1^−/− recipients at 24 hours. Treatment of recipients with CORM-2, a CO-releasing molecule (10 mg/kg of body weight intravenously), 1 hour prior and 1, 3, and 6 days after transplantation, significantly improved survival (62% at >56 days, P < 0.001) compared with HO-1^−/− recipients treated with inactive CORM-2 (median survival 1 day). Histological analyses revealed that CO treatment markedly reduced platelet aggregation within the graft. Adoptive transfer of wild-type platelets to HO-1^−/− recipients also conferred protection and increased survival. Aortic transplants from either HO-1^−/− or HO-1^+/+ C57BL/6 donors into HO-1^+/+ (Balb/cJ) mice did not develop arterial thrombosis, surviving more than 56 days. These studies demonstrate an important role for systemic HO-1/CO for protection against vascular arterial thrombosis in murine aortic allotransplantation.Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in heme degradation, leading to the generation of equimolar amounts of iron, biliverdin, and carbon monoxide (CO). Biliverdin is then converted to bilirubin by biliverdin reductase.^1,2 HO-1 is highly up-regulated in mammalian tissues in response to a wide variety of conditions including vascular injury, ischemia, inflammation, immune injury, oxidative stress, cell cycle dysregulation, and sublethal and lethal cell damage.^3,4,5 The wide range of inducers of HO-1 provides support for a vital role in maintenance of cellular homeostasis under different pathophysiological conditions including inflammatory diseases such as septic shock and asthma,^6,7 cardiovascular diseases such as myocardial infarction and atherosclerosis,^8,9 ischemia-reperfusion injury in multiple organ systems,^8,10 and transplant rejection.^11,12One of the products of HO-1-mediated heme degradation, CO, is known to be toxic at high concentrations due to its high affinity for hemoglobin. However, there is substantial evidence that lower concentrations of CO endogenously generated from the breakdown of heme by HO serves essential regulatory roles in a variety of physiological and pathophysiological processes.¹³ Exogenous or endogenous CO can confer some of the cytoprotective effects attributed to HO-1.^14,15Transitional metal carbonyls, CO-releasing molecules (CORMs), have been used to deliver CO in a controlled manner without altering carboxyhemoglobin levels.^16,17,18 A wide range of CORMs containing manganese (CORM-1), ruthenium (CORM-2 and −3), boron (CORM-A1), and iron (CORM-F3) are currently being investigated to facilitate the pharmaceutical use of CO for the prevention of vascular dysfunction, inflammation, ischemia-reperfusion injury, and transplant rejection.^{19,20,21,22,23}Thrombosis is a major complication during multiple vascular pathological conditions during which HO-1 and its byproduct CO could provide significant protection through attenuation of inflammation, endothelial cell damage, and apoptosis, as well as modulation of vascular tone.^6,8,9 However, very little is known regarding the potential roles of HO-1 and CO in modulating platelet-dependent effects after vascular injury in the setting of transplantation. In these studies, we show that expression of HO-1 plays a critical role in the development of post-transplant arterial thrombosis immediately following abdominal aortic transplantation. We tested the hypothesis that CO, a product of the HO-1 reaction, mediates anti-thrombotic effects in vivo by inhibition of platelet mediated thrombus formation within the graft. We found that HO-1-deficient mice develop vascular thrombosis following aortic transplantation and that the development of thrombosis can be prevented by systemic administration of CORM-2.     

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7^−/−), or dystrophin and utrophin (mdx/utr^−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr^−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr^−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr^−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease that affects 1 in every 3500 live male births. Patients with DMD have impaired mobility, are restricted to a wheelchair by their teens, and die from cardiopulmonary failure in their early twenties.^1,2 Currently, there is no cure or effective treatment for this devastating disease. Mutations in the dystrophin gene resulting in loss of the dystrophin protein are the cause of disease in DMD patients and the mdx mouse model.^3,4,5,6,7The dystrophin glycoprotein complex links laminin in the extracellular matrix to the actin cytoskeleton. The N-terminal region of dystrophin interacts with cytoskeletal F-actin⁸ and the C-terminal region associates with the dystrophin-associated protein complex, which include α- and β-dystroglycan, α- and β-syntrophin, the sarcoglycans, and sarcospan.⁹ In DMD, the absence of dystrophin leads to disruption of the dystrophin glycoprotein complex, resulting in increased muscle fragility and altered cell signaling.⁹ Loss of this critical transmembrane linkage complex in DMD patients and mdx mice results in progressive muscle damage and weakness, inflammation, necrosis, and fibrosis. Lack of dystrophin also leads to abnormalities at myotendinous and neuromuscular junctions (MTJ and NMJ), which further contribute to skeletal muscle damage.^{10,11,12,13,14,15,16,17} In addition, defective muscle repair in DMD patients eventually results in muscle degeneration exceeding the rate of regeneration.¹⁸ Overall, dystrophin is critical for muscle function, structure, and stability, and its absence results in progressive muscle wasting and severe muscular dystrophy. In the absence of dystrophin two additional laminin-binding receptors, the α7β1 integrin and utrophin, are up-regulated in the skeletal muscle of DMD patients and mdx mice, which may compensate for the loss of the dystrophin glycoprotein complex.^19,20,21The α7β1 integrin is a heterodimeric laminin receptor involved in bidirectional cell signaling and is localized at junctional and extrajunctional sites in skeletal muscle.^22,23 At least six α7 integrin isoforms produced by developmentally regulated RNA splicing are expressed in skeletal muscle.²⁴ Mutations in the α7 integrin gene (ITGA7) cause myopathy in humans.²⁵ Mice lacking the α7 integrin develop myopathy, exhibit vascular smooth muscle defects and have altered extracellular matrix deposition.^{26,27,28,29,30} The observation that the α7β1 integrin is elevated in the muscle of DMD patients and mdx mice led to the hypothesis that the α7β1 integrin may compensate for the loss of dystrophin.¹⁹ Enhanced expression of the α7 integrin in the skeletal muscle of severely dystrophic mice reduced muscle pathology and increased lifespan by threefold.^10,11 In contrast, loss of both dystrophin and α7 integrin in mice results in severe muscular dystrophy and premature death by 4 weeks of age.^28,31 The α7β1 integrin is therefore a major modifier of disease progression in DMD.The utrophin glycoprotein complex is a third major laminin receptor in skeletal muscle. Utrophin has significant sequence homology to dystrophin.^32,33 In normal adult muscle utrophin is restricted to neuromuscular and myotendinous junctions.³⁴ During development or in damaged or diseased muscle, utrophin expression is increased and becomes localized at extrajunctional sites.^35,36 Utrophin interacts with the same proteins as dystrophin, but binds to actin filaments at different sites.³⁷ In mice, loss of utrophin results in a mild form of myasthenia with reduced sarcolemmal folding at the postsynaptic membrane of the neuromuscular junction.^12,15 Transgenic overexpression of utrophin has been shown to rescue mdx mice.³⁸ Mice that lack both dystrophin and utrophin exhibit severe muscular dystrophy and die by 14 weeks of age.^13,14 Thus, utrophin is also a major laminin receptor that modifies disease progression in DMD.To understand the functional overlap between the α7β1 integrin and utrophin in skeletal muscle, we produced mice that lack both α7 integrin and utrophin (α7/utr^−/−). Since both complexes are highly enriched at the MTJ and NMJ, we hypothesized that α7/utr^−/− mice may have severe abnormalities at these critical junctional sites. Our study demonstrates α7/utr^−/− mice exhibit partial embryonic lethality comparable with that observed in α7^−/− mice. Dystrophin is increased in these animals and enriched at the NMJ but not the MTJ. α7/utr^−/− mice display ultrastructural defects in their MTJ and compromised force transmission. Together, these results indicate that the α7β1 integrin, dystrophin and utrophin laminin binding complexes provide continuity between laminin in the extracellular matrix and the cell cytoskeleton, which are necessary for the normal structural and functional properties of skeletal muscle.     

Increased Activity and Altered Subcellular Distribution of Lysosomal Enzymes Determine Neuronal Vulnerability in Niemann-Pick Type C1-Deficient Mice

Niemann-Pick disease type C (NPC), caused by mutations in the Npc1 or Npc2 genes, is a progressive neurodegenerative disorder characterized by intracellular accumulation/redistribution of cholesterol in a number of tissues including the brain. This is accompanied by a severe loss of neurons in selected brain regions. In this study, we evaluated the role of lysosomal enzymes, cathepsins B and D, in determining neuronal vulnerability in NPC1-deficient (Npc1^−/−) mouse brains. Our results showed that Npc1^−/− mice exhibit an age-dependent degeneration of neurons in the cerebellum but not in the hippocampus. The cellular level/expression and activity of cathepsins B and D are increased more predominantly in the cerebellum than in the hippocampus of Npc1^−/− mice. In addition, the cytosolic levels of cathepsins, cytochrome c, and Bax2 are higher in the cerebellum than in the hippocampus of Npc1^−/− mice, suggesting a role for these enzymes in the degeneration of neurons. This suggestion is supported by our observation that degeneration of cultured cortical neurons treated with U18666A, which induces an NPC1-like phenotype at the cellular level, can be attenuated by inhibition of cathepsin B or D enzyme activity. These results suggest that the increased level/activity and altered subcellular distribution of cathepsins may be associated with the underlying cause of neuronal vulnerability in Npc1^−/− brains. Therefore, their inhibitors may have therapeutic potential in attenuating NPC pathology.Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral disorder caused by mutations in the Npc1 or Npc2 gene. NPC1 is a membrane protein that contains a sterol-sensing domain and resides primarily in late endosomes/lysosomes, whereas NPC2 is a soluble protein that resides primarily in lysosomes.^1,2,3,4 The loss of function of either protein results in intracellular accumulation of unesterified cholesterol and glycosphingolipids within the endosomal-lysosomal (EL) system in a number of tissues including the brain. In addition, there is evidence that homeostatic responses to exogenously supplied cholesterol and activation of cholesterol esterification are severely impaired in cells lacking functional NPC1. These defects in cholesterol accumulation/homeostasis trigger abnormal liver and spleen function as well as widespread neurological deficits including ataxia, dystonia, seizures, and dementia that eventually lead to premature death.^5,6,7,8,9 Interestingly, BALB/cNctr-Npc^N/N mice, which do not express NPC1 protein because of a spontaneous deletion/insertion mutation in the Npc1 gene, have been shown to recapitulate pathological features associated with NPC disease. These Npc1^−/− mice are asymptomatic at birth but gradually develop tremor and ataxia, dying prematurely at ∼3 months.^10,11,12,13 As in the human disease, Npc1^−/− mice show accumulation of unesterified cholesterol in the EL system and exhibit activation of microglia and astrocytes as well as degradation of the myelin sheath throughout the central nervous system. Progressive loss of neurons is particularly evident in the prefrontal cortex, thalamus, brainstem, and cerebellum but not in the hippocampal formation.^{13,14,15,16,17,18} However, at present, very little is known about the underlying mechanisms associated with the vulnerability of select populations of neurons in Npc1^−/− mice.A number of earlier studies have shown that the EL system, the major site of cholesterol accumulation in NPC pathology, consists of two dynamic interrelated cellular pathways: the endocytic pathway and the lysosomal system. Under normal conditions, the EL system serves as an important site for intracellular protein turnover and proteolytic processing of certain proteins mediated by lysosomal hydrolases termed cathepsins.^19,20,21 After their synthesis in the endoplasmic reticulum, cathepsins bind to the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (M6P) receptor on the trans face of the Golgi complex and are transported in vesicles to the EL system.^22,23,24 The importance of lysosomal enzymes in the proper functioning of the EL system is underscored by the fact that altered synthesis, sorting, or targeting of lysosomal enzymes is the molecular basis of more than 40 inherited disorders associated with extensive neurodegeneration, mental retardation and often progressive cognitive decline.^19,25,26,27There is evidence that increased endosome volumes and/or levels of cathepsins, such as cathepsins B and D, can mediate cell death by inducing lysosomal destabilization and enzyme leakage into cell cytosol, as is observed during oxidative stress²⁸ and experimental brain ischemia in primates.²⁹ Conversely, a number of recent studies have shown that lysosomal enzyme expression/levels can be up-regulated in the absence of cell death as a compensatory mechanism to repair damage/injury.^30,31,32,33 Thus, it seems that lysosomal enzymes are not only involved in the degeneration of neurons but also in the protection of neurons against toxicity in a variety of experimental as well as pathological paradigms. Although the EL system, the major site of cholesterol accumulation in NPC1-deficient cells, has been suggested to play a critical role in the development of NPC pathology,^6,7,8 very little is known about the significance of lysosomal cathepsins in determining neuronal vulnerability associated with the disease. To address this issue, we measured age-related changes in the levels, distribution, and activity of cathepsins B and D in the hippocampus and cerebellum of Npc1^−/− and age-matched control mice. In parallel, we evaluated the levels and distribution of the IGF-II/M6P receptor in Npc1^−/− and control mice to establish whether factors regulating cathepsin bioavailability can also influence the development of pathological changes. In addition, using cultured mouse cortical neurons we determined the significance of cathepsins B and D in the degeneration of neurons after accumulation of cholesterol. Our results reveal that alterations in the levels/activity as well as subcellular distribution of the lysosomal enzymes may be one of the underlying mechanisms associated with the selective neuronal vulnerability observed in NPC pathology.     

Aggregation and Binding Substances Enhance Pathogenicity in Rabbit Models of Enterococcus faecalis Endocarditis

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models of Enterococcus faecalis cardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 10⁸ CFU and observed for 2 days. No clinical signs of illness developed in animals given AS⁻ EBS⁻ organisms, and all survived. All rabbits given AS⁻ EBS⁺ organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS⁺ EBS⁻ organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS⁺ EBS⁺ organisms developed signs of illness and died. None of the rabbits receiving AS⁺ EBS⁺ organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS⁺ EBS⁺ organisms. In contrast, cell extracts from AS⁻ EBS⁻ organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS⁺/EBS⁺ enterococci contained high levels of gamma interferon and tumor necrosis factor alpha (TNF-α) and TNF-β, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 × 10⁹ CFU of test organisms. None of the animals given AS⁻ EBS⁻ organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS⁻ EBS⁺ or AS⁺ EBS⁻ organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS⁺ EBS⁺ organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for 3 days revealed that all rabbits given AS⁻ EBS⁻ or AS⁺ EBS⁺ organisms developed vegetations, but animals given AS⁺ EBS⁺ organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.Recently, Enterococcus faecalis and other enterococci have become increasingly recognized as significant causes of nosocomial infections (23, 26, 27, 30). They are important causes of bacteremia, endocarditis, and urinary tract infections. These organisms are also important because of their increasing incidence of resistance to vancomycin and other antibiotics and because of the potential of transferring antibiotic resistance to other bacteria.An important mechanism for horizontal transfer of antibiotic resistance in enterococci is pheromone-inducible conjugation (10, 12, 34). The expression of conjugative transfer functions of plasmids such as pCF10 (58 kb; encodes tetracycline resistance [12, 14]) and pAD1 (60 kb; encodes hemolysin and bacteriocin production [10, 31]) is induced by peptide pheromones produced by recipient cells (13, 31, 34). The conjugation gene products induced by pheromones include a cell surface adhesin, aggregation substance (AS). This protein mediates the formation of mating aggregates between donor and recipient cells by binding to a cognate ligand on the recipient cell, enterococcal binding substance (EBS) (13, 14). The prgB gene of pCF10 encodes the AS protein, Asc10 (25), whose nucleotide and amino acid sequences are highly similar to those of AS proteins encoded by other pheromone plasmids (19, 20). The genetics of EBS are complex, with multiple, unlinked insertion mutations required to generate an EBS-negative phenotype (5, 12, 32). Lipoteichoic acid (LTA) appears to be an important component of EBS (7, 15, 32).Previous studies of the pathogenicity of E. faecalis have shown that hemolysin contributes to the virulence of the organism in animal models, including murine peritonitis, rabbit endophthalmitis, and rabbit endocarditis (9, 11, 21, 25, 26). In their study, Chow et al. (9) also showed that AS contributed significantly to the production of experimental endocarditis. Hemolysin and AS were associated with increased mortality, and AS was associated with increased vegetation weight.AS proteins of E. faecalis are thought to be virulence factors in enterococcal infections by promoting binding to a variety of eukaryotic cell surfaces (21, 25, 26). AS expression may be induced in vivo by eukaryotic factors in serum (6). AS contains amino acid motifs, Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val, which are found in fibronectin and other proteins and which mediate binding to eukaryotic cell adhesion molecules of the integrin superfamily (18, 21, 25). Soluble LTA inhibits aggregate formation and may function as EBS (26). LTA from E. faecalis has previously been shown to induce both interleukin 1β and tumor necrosis factor alpha (TNF-α) production from macrophages (7).This study was undertaken to evaluate the role of both AS and EBS in two rabbit models of E. faecalis cardiac infections. Greater insight into the role of these two factors in virulence may lead to alternative methods of prophylaxis and treatment of resistant enterococcal infections. Our studies indicate that the presence of both cell surface components is associated with both increased mortality and formation of vegetations.(This work was presented in part at the 13th Lancefield International Symposium on Streptococci and Streptococcal Diseases, Paris, France, 16 to 20 September 1996.)     

Vascular Permeability and Pathological Angiogenesis in Caveolin-1-Null Mice

Caveolin-1, the signature protein of endothelial cell caveolae, has many important functions in vascular cells. Caveolae are thought to be the transcellular pathway by which plasma proteins cross normal capillary endothelium, but, unexpectedly, cav-1^−/− mice, which lack caveolae, have increased permeability to plasma albumin. The acute increase in vascular permeability induced by agents such as vascular endothelial growth factor (VEGF)-A occurs through venules, not capillaries, and particularly through the vesiculo-vacuolar organelle (VVO), a unique structure composed of numerous interconnecting vesicles and vacuoles that together span the venular endothelium from lumen to ablumen. Furthermore, the hyperpermeable blood vessels found in pathological angiogenesis, mother vessels, are derived from venules. The present experiments made use of cav-1^−/− mice to investigate the relationship between caveolae and VVOs and the roles of caveolin-1 in VVO structure in the acute vascular hyperpermeability induced by VEGF-A and in pathological angiogenesis and associated chronic vascular hyperpermeability. We found that VVOs expressed caveolin-1 variably but, in contrast to caveolae, were present in normal numbers and with apparently unaltered structure in cav-1^−/− mice. Nonetheless, VEGF-A-induced hyperpermeability was strikingly reduced in cav-1^−/− mice, as was pathological angiogenesis and associated chronic vascular hyperpermeability, whether induced by VEGF-A¹⁶⁴ or by a tumor. Thus, caveolin-1 is not necessary for VVO structure but may have important roles in regulating VVO function in acute vascular hyperpermeability and angiogenesis.Caveolae (also referred to as plasmalemmal vesicles) were described by Palade and Bruns in capillary endothelial cells as 50- to 100-nm diameter smooth membrane-bound vesicles.^1,2 Palade and Bruns proposed that caveolae shuttled across capillary endothelium from lumen to ablumen, carrying with them “cargoes” of plasma and in this manner provided the small amounts of plasma proteins that are required for maintaining tissue health. Later work demonstrated that caveolae could also form short chains of two to three linked vesicles that spanned the short distance across the capillary endothelium.³ Together these studies implied that, whether shuttling or interconnected into short chains, capillary caveolae were de facto the elusive “large pores” that physiologists had postulated to account for plasma protein extravasation.^4,5Since their initial discovery, much has been learned about caveolae and their signature protein, caveolin.^6,7,8,9,10 Caveolin is thought to be necessary for caveolae formation and overexpression of caveolin can induce caveolae in cells that normally lack them.¹¹ Caveolin exists in three isoforms.^12,13,14 The first two isoforms, cav-1 and cav-2, are highly expressed in vascular endothelium, pericytes and smooth muscle, among other cell types, whereas cav-3 is confined to muscle.¹⁵ Caveolae and caveolin have many functions besides plasma protein transport, including regulation of cholesterol homeostasis and sorting of signaling molecules such as endothelial nitric oxide synthase, heterotrimeric G proteins, and nonreceptor tyrosine kinases.^7,9,14,16,17Cav-1^−/− mice have contributed much to our understanding of caveolin and caveolae. Cav-1^−/− mice are viable and fertile but lack caveolae and exhibit various types of vascular dysfunction, including impaired nitric oxide and Ca²⁺ signaling.^{12,18,19,20,21,22} However, there is controversy on some other points, such as whether tumor growth and angiogenesis are altered in cav-1^−/− mice, and, if so, in what direction and by what mechanism.^{22,23,24,25,26,27,28,29} Recently, cav-1^−/− mice have been found to be systemically hyperpermeable to plasma albumin^25,30,31; this finding was unexpected in that caveolae have been thought to be necessary for transporting plasma proteins across capillary endothelium under basal conditions.^1,2,3However, vascular permeability is not of a single type.³² In contrast to the normal, low level basal vascular permeability (BVP) of normal tissues, two distinctly different types of increased vascular permeability are found in pathological conditions.³² Vascular permeabilizing factors, such as vascular endothelial growth factor (VEGF)-A, histamine, and others, induce acute vascular hyperpermeability (AVH), a characteristic feature of acute inflammation. Chronic vascular hyperpermeability (CVH), on the other hand, is found in the pathological angiogenesis induced by tumors, healing wounds, and chronic inflammatory diseases; as its name implies, CVH persists for long periods of time—days to weeks and sometimes indefinitely. AVH and CVH differ from BVP not only in terms of the much greater amounts of plasma that extravasate but also with respect to the microvessels that leak. BVP takes place in capillaries.^2,3,4 In contrast, AVH takes place primarily in postcapillary venules^{33,34,35,36,37} and is thought to involve an organelle, the vesiculo-vacuolar organelle (VVO), that is uniquely present in venular endothelial cells. VVOs are grapelike clusters of hundreds of uncoated, trilaminar unit membrane-bound, interconnecting vesicles and vacuoles that extend across the relatively tall cytoplasm of venular endothelium from lumen to ablumen. The relationship of VVOs to caveolae is uncertain.^{36,37,38,39,40} Unlike caveolae, which are of relatively uniform size, the vesicles and vacuoles that comprise VVOs vary widely in size from caveolae-sized vesicles to those with a cross-sectional areas more than 10-fold greater.³⁷ They attach to each other and to the endothelial plasma membrane by stomata that are normally closed by thin diaphragms. In this respect, VVO stomata and diaphragms closely resemble the analogous structures by which caveolae attach to each other and to the luminal and abluminal plasma membranes of capillary endothelium.^36,37,38,41 On exposure to acute permeabilizing agents such as VEGF or histamine, the diaphragms interconnecting VVO vesicles and vacuoles open to provide a trans-endothelial cell pathway for plasma extravasation.^36,37,42 Others have reported leakage through a paracellular route, independent of VVOs.^43,44 In CVH, yet another type of blood vessel, the “mother” vessel, accounts for the bulk of vascular hyperpermeability.⁴⁵ Mother vessels are greatly enlarged, thin-walled, pericyte-poor sinusoids that derive from pre-existing normal venules after longer exposures to VEGF and other angiogenic stimuli.⁴⁶ VVOs participate in mother vessel formation and associated CVH.^45,47The experiments reported here made use of cav-1^−/− mice to investigate the relationship between caveolae and VVOs and the role of caveolin-1 in VVO structure, in AVH and CVH, and in pathological angiogenesis. We report here that some, but not all, VVO vesicles and vacuoles express caveolin-1. Nonetheless, VVOs are present in normal numbers and with unaltered structure in cav-1^−/− mice. Further, we find that AVH and CVH are strikingly reduced in cav-1^−/− mice. Angiogenesis is also reduced in cav-1^−/− mice, whether induced by an adenoviral vector expressing VEGF-A¹⁶⁴ (Ad-VEGF-A¹⁶⁴) or by a tumor, the B16 melanoma.     

T-Cell Activation Leads to Reduced Collagen Maturation in Atherosclerotic Plaques of Apoe−/− Mice

Rupture of the collagenous, fibrous cap of an atherosclerotic plaque commonly causes thrombosis. Activated immune cells can secrete mediators that jeopardize the integrity of the fibrous cap. This study aimed to determine the relationship between T-cell-mediated inflammation and collagen turnover in a mouse model of experimental atherosclerosis. Both Apoe^−/− × CD4dnTβRII mice with defective transforming growth factor-β receptors in T cells (and hence released from tonic suppression of T-cell activation) and lesion size-matched Apoe^−/− mice were used. Picrosirius red staining showed a lower content of thick mature collagen fibers in lesions of Apoe^−/− × CD4dnTβRII mice, although both groups had similar levels of procollagen type I or III mRNA and total collagen content in lesions. Analysis of both gene expression and protein content showed a significant decrease of lysyl oxidase, the extracellular enzyme needed for collagen cross-linking, in aortas of Apoe^−/− − CD4dnTβRII mice. T-cell-driven inflammation provoked a selective and limited increase in the expression of proteinases that catabolize the extracellular matrix. Atheromata of Apoe^−/− − CD4dnTβRII mice had increased levels of matrix metalloproteinase-13 and cathepsin S mRNAs and of the active form of cathepsin S protein but no increase was detected in collagen fragmentation. Our results suggest that exaggerated T-cell-driven inflammation limits collagen maturation in the atherosclerotic plaque while having little effect on collagen degradation.A physical disruption of atherosclerotic plaques causes many acute thrombotic complications such as myocardial infarction and stroke.^1,2 Pathologists have identified a number of characteristics of vulnerable atherosclerotic plaques that have ruptured³ including large lipid cores and thin fibrous caps harboring activated macrophages and T cells.^1,4 The resistance of the atherosclerotic plaque to disruption depends in part on the integrity of its fibrous cap, which prevents contact between the highly thrombogenic lipid core and the circulating blood.^1,2 The fibrous cap is composed of smooth muscle cells (SMCs) and a collagen-rich extracellular matrix. The fibrillar collagens types I and III synthesized by SMCs primarily determine the tensile strength of the cap. Sites of plaque rupture characteristically display signs of inflammatory cell activation accompanied by dissolution of matrix.Inflammation may impair plaque stability because macrophages and mast cells release a set of collagen-degrading matrix metalloproteinases (MMPs) and cysteine proteases.^5,6,7 Additional possible mechanisms include inhibited expression of procollagen genes and death or reduced renewal of the collagen-producing SMC population, both phenomena promoted by T-cell-derived interferon (IFN)-γ.⁸ Several studies suggest that T cells may modulate plaque stability. Activated T cells accumulate in vulnerable plaques.⁴ Th1 cells, which dominate in plaques, secrete IFN-γ, a powerful inhibitor of endothelial and SMC proliferation.⁹ IFN-γ also inhibits differentiation and collagen gene expression in SMCs^10,11 and modulates expression of several MMPs and cathepsins.^12,13 In vivo treatment with IFN-γ increases atherosclerosis and transplant arteriosclerosis,^14,15 whereas targeted gene deletion in the IFN-γ receptor leads to reduced atherosclerosis in hypercholesterolemic mice.¹⁶ Mice with defective control of T-cell activity show modulation of atherogenesis: interleukin-10 targeted mice display increased plaque formation with reduced collagen accumulation,¹⁷ and Apoe^−/− mice lacking transforming growth factor (TGF)-β inhibition of T cells rapidly develop large, hyperinflammatory lesions.¹⁸ These data point to a proatherosclerotic and possibly destabilizing role for activated T cells.The present study aimed to assess the effect of inflammation on the fibrous component of the atherosclerotic plaque. We used Apoe^−/− × CD4dnTβRII mice, which lack functional TGF-β receptors on T cells. Therefore, uncontrolled T-cell activation leads to rampant inflammation and accelerated atherosclerosis in hypercholesterolemic animals.¹⁸ Therefore, Apoe^−/− × CD4dnTβRII mice provide an opportunity to study the effect of inflammation on atherosclerotic lesions. Our data show reduced enzyme-dependent collagen maturation in hyperinflamed lesions, whereas effects on procollagen expression and collagenolytic enzymes were modest. These results suggest a novel mechanism by which adaptive immunity can modulate plaque stability—impairment of collagen maturation by T-cell-dependent inflammation.     

ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing

Wound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1^−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1^−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1^−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium.Intercellular adhesion molecule-1 (ICAM-1, CD54)¹ is a conserved member of the immunoglobulin supergene family² and is expressed by many cell types in response to stimuli such as cytokines,^3,4 and oxidative and physical stress.^5,6 It has been extensively studied in the context of adhesion and transmigration of leukocytes through endothelium⁷ and epithelium,^8,9 and it also serves as an adhesive ligand for leukocyte-mediated cytotoxic activity.^9,10,11 ICAM-1 is recognized by members of the β2 (CD18) integrin family, especially lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18),¹² and this adhesion is critical to many of the migratory and cytotoxic events in which ICAM-1 participates.^7,10,11 ICAM-1 also functions as a signaling molecule, dependent on its cytoplasmic tail interacting with cytoskeletal elements.⁷ This capability influences functions such as leukocyte transendothelial migration⁷ and vascular permeability.¹³Of importance to the current study is the fact that ICAM-1 can be expressed by corneal epithelial cells and limbal vessel endothelial cells.^{14,15,16,17,18,19,20,21} It appears to be expressed in conditions associated with inflammation, but its role in this context is poorly understood, especially its expression by the epithelial cells. Using a murine model of central corneal epithelial abrasion, we observed ICAM-1 on corneal epithelial cells in the periphery of the cornea, a region not directly injured by the abrasion.¹⁴ Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,^22,23 it was of interest to determine whether ICAM-1 is necessary for these processes. To this end we studied wound healing in mice that do not express ICAM-1.^24,25As a part of this evaluation, we focused attention on γδ T cells. We observed in earlier studies that epithelial expression of ICAM-1 occurred at a time when γδ T cells increased within the corneal epithelium,^14,26 and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,²⁷ and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,^20,27 it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1.     

Accelerated Lipofuscinosis and Ubiquitination in Granulin Knockout Mice Suggest a Role for Progranulin in Successful Aging

Progranulin (PGRN) is involved in wound repair, inflammation, and tumor formation, but its function in the central nervous system is unknown. Roles in development, sexual differentiation, and long-term neuronal survival have been suggested. Mutations in the GRN gene resulting in partial loss of the encoded PGRN protein cause frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions. We sought to understand the neuropathological consequences of loss of PGRN function throughout the lifespan of GRN-deficient (^−/+ and ^−/−) mice. An aged series of GRN-deficient and wild-type mice were compared by histology, immunohistochemistry, and electron microscopy. Although GRN-deficient mice were viable, GRN^−/− mice were produced at lower than predicted frequency. Neuropathologically, GRN^−/+ were indistinguishable from controls; however, GRN^−/− mice developed age-associated, abnormal intraneuronal ubiquitin-positive autofluorescent lipofuscin. Lipofuscin was noted in aged GRN^+/+ mice at levels comparable with those of young GRN^−/− mice. GRN^−/− mice developed microgliosis, astrogliosis, and tissue vacuolation, with focal neuronal loss and severe gliosis apparent in the oldest GRN^−/− mice. Although no overt frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions type- or TAR DNA binding protein-43-positive lesions were observed, robust lipofuscinosis and ubiquitination in GRN^−/− mice is strikingly similar to changes associated with aging and cellular decline in humans and animal models. Our data suggests that PGRN plays a key role in maintaining neuronal function during aging and supports the notion that PGRN is a trophic factor essential for long-term neuronal survival.Progranulin (PGRN) is a 593-amino acid cysteine-rich protein that is heavily glycosylated.¹ Encoded by a single gene (GRN) on human chromosome 17q21, PGRN contains seven granulin-like domains, which consist of highly conserved tandem repeats of a rare 12-cysteinyl motif.^2,3 PGRN is secreted and proteolytically cleaved by extracellular proteases giving rise to smaller peptide fragments termed granulins (GRNs).¹ Full-length PGRN and its proteolytically derived GRNs are biologically active with distinct properties. In the periphery PGRN and GRNs have been implicated in wound repair, inflammation, tumor formation, and trophic support.^4,5PGRN has gained the attention of the neuroscience community with the recent discovery that mutations in GRN cause autosomal-dominant forms of frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U).^6,7 Pathogenic mutations in GRN mostly create null alleles, with premature termination of the coding sequence and nonsense-mediated decay of the mutant mRNA, resulting in haploinsufficiency of the protein.⁶ Recent studies have confirmed that patients with GRN mutations have significantly reduced levels of PGRN, at least in their plasma and in lymphoblastoid cells derived from mutation carriers.^8,9,10Neuropathologically, FTLD-U (both with and without GRN mutations) is characterized by ubiquitin-positive intracytoplasmic and sometimes intranuclear neuronal inclusions.¹¹ The major constituent of these inclusions in the majority of cases is a seemingly unrelated protein, TAR DNA binding protein-43 (TDP-43).¹² In affected neurons, TDP-43 is absent from its normal nuclear location and forms ubiquitinated and hyperphosphorylated aggregates in the cytoplasm. The neocortex, hippocampus, amygdala, and basal ganglia seem to be most vulnerable to ubiquitin/TDP-43-positive inclusions and are often associated with atrophy, severe neuronal loss, gliosis, and tissue vacuolation.^11,13 Despite these recent discoveries, how PGRN haploinsufficiency in FTLD-U with GRN mutations results in redistribution and aggregation of TDP-43 and neurodegeneration is currently unknown.Little is known about the role of PGRN in the central nervous system (CNS). The expression of PGRN is regulated during CNS development,¹ and in the adult CNS, PGRN is expressed in neurons and microglia^6,14,15,16 with low or no expression in other glial cells. Consistent with its known functions in the periphery and cell type expression in the CNS, PGRN has also been implicated as a neurotrophic factor for long-term neuronal survival^4,16,17 and is increased in the inflammatory response to CNS injury, infection, and neurodegeneration.⁴Kayasuga and colleagues^18,19 used gene targeting to knock out the murine GRN gene and demonstrated that PGRN was important for establishing some sexual dimorphic behaviors. Surprisingly, these mice were viable without any obvious disorders in reproductive function, suggesting that PGRN was not a critical factor for fertilization and development. Subsequently, it was suggested that the volumetric difference in the locus ceruleus of progranulin-deficient mice compared with controls was a possible cause of sexually dimorphic anxiety.²⁰ Given that progranulin deficiency in humans leads to FTLD-U, it was surprising that C-terminal cleavage products of the TDP-43 protein that are characteristic for human FTLD-U were not observed in these progranulin-deficient mice.²¹The objective of the current study was to characterize an aged series (1, 7, 12, and 23 months) of GRN-deficient (^−/+ and ^−/−) mice to determine the neuropathological consequences of having partial or complete loss of PGRN function throughout life as a means to understand the biological role of PGRN in the CNS. Despite a partial loss of PGRN protein expression reminiscent of that seen in FTLD-U, GRN^−/+ mice were neuropathologically identical to wild-type controls. In contrast, GRN^−/− mice, which completely lacked PGRN protein, exhibited a progressive neuropathological phenotype consistent with accelerated aging, suggesting a novel role for PGRN in successful aging.