Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

VEGFR-3 Neutralization Inhibits Ovarian Lymphangiogenesis,Follicle Maturation,and Murine Pregnancy

Lymphatic vessels surround follicles within the ovary, but their roles in folliculogenesis and pregnancy, as well as the necessity of lymphangiogenesis in follicle maturation and health, are undefined. We used systemic delivery of mF4-31C1, a specific antagonist vascular endothelial growth factor receptor 3 (VEGFR-3) antibody to block lymphangiogenesis in mice. VEGFR-3 neutralization for 2 weeks before mating blocked ovarian lymphangiogenesis at all stages of follicle maturation, most notably around corpora lutea, without significantly affecting follicular blood angiogenesis. The numbers of oocytes ovulated, fertilized, and implanted in the uterus were normal in these mice; however, pregnancies were unsuccessful because of retarded fetal growth and miscarriage. Fewer patent secondary follicles were isolated from treated ovaries, and isolated blastocysts exhibited reduced cell densities. Embryos from VEGFR-3–neutralized dams developed normally when transferred to untreated surrogates. Conversely, normal embryos transferred into mF4-31C1–treated dams led to the same fetal deficiencies observed with in situ gestation. Although no significant changes were measured in uterine blood or lymphatic vascular densities, VEGFR-3 neutralization reduced serum and ovarian estradiol concentrations during gestation. VEGFR-3–mediated lymphangiogenesis thus appears to modulate the folliculogenic microenvironment and may be necessary for maintenance of hormone levels during pregnancy; both of these are novel roles for the lymphatic vasculature.Ovarian neovascularization provides a unique environment in which to study physiological adult vasculogenesis apart from the traditional settings of wound healing and cancer pathologies. Lymphatic circulation plays a central role in fluid, lipid, and cellular transport,¹ and lymphatic vessels are present within the ovary and surround follicles during development and maturation,^2–5 but the importance of the lymphatic vasculature and lymphangiogenesis in the ovary is unclear. Consequently, the potential roles of lymphatic vessels in follicle maturation and pregnancy, and the extent of involvement or even necessity of maternal lymphangiogenesis in reproduction, are undefined. This contrasts with ovarian blood angiogenesis, whose critical roles in follicular nourishment and maturation and in the formation and maintenance of the corpus luteum are well appreciated; indeed, oocyte fertilization, embryonic implantation, uterine expansion, and successful gestation all require blood angiogenesis.^6–8 Lymphangiogenesis, which is often concurrent with blood angiogenesis,⁹ may also play an important role in these processes.Adult blood angiogenesis requires signaling via vascular endothelial growth factor receptor 2 (VEGFR-2), most potently by VEGF ligation.^10,11 In murine ovaries, VEGF expression increases during angiogenic growth phases,¹² and blockade of VEGFR-2 signaling in mice effectively prevents angiogenesis, resulting in a marked decrease in ovarian weight, blood vessel density, and number of corpora lutea, and in infertility.^13–15 Because gonadotropin treatment apparently does not correct these deficiencies,¹⁶ it is likely that follicle maturation and successful pregnancy are highly dependent on VEGFR-2–mediated neovascularization in the ovary.^6,17 Vascularization also occurs in the uterine wall and decidua during pregnancy, and significant disruption of angiogenesis by VEGFR-2 blockade in these tissues after fertilization has been shown to greatly reduce pregnancy success.¹⁸VEGFR-3 is expressed primarily on lymphatic endothelial cells in adult tissue,^19,20 and its signaling, via ligation by VEGF-C or VEGF-D, is necessary for lymphangiogenesis by inducing lymphatic endothelial cell proliferation and migration.^19–23 Blockade of VEGFR-3 signaling using a function-blocking antibody such as mF4-31C1 (ImClone Systems; Eli Lilly, Indianapolis, IN) completely blocks the initiation of new lymphatic vessels in adult mice without affecting pre-existing lymphatic morphology or function and without apparently affecting blood angiogenesis.^18,21,22 The ovary contains a dense lymphatic network that has been morphologically assessed in large rodents.^24–26 Recent studies in which murine ovarian lymphatic vessel expansion was impaired during development found the dams to be infertile as adults.³We investigated VEGFR-3–mediated lymphangiogenesis and the roles of new lymphatic vessels and lymphangiogenesis in female reproduction and found that lymphangiogenesis occurs within the murine ovary during reproductive cycles and folliculogenesis and that VEGFR-3 neutralization prevents viable, full-term pregnancies. Using combined in vivo, ex vivo, and in vitro methods, we examined which aspects of female fertility are influenced by inhibited maternal lymphangiogenesis including oocyte and follicular development and maturation, uterine implantation, and embryonic development. After we had eliminated direct effects on fetal and uterine VEGFR-3–mediated neovascularization, our results suggested that the new ovarian lymphatic vessels specifically modulate follicle development and hormone production, demonstrating a critical and novel role for ovarian lymphangiogenesis in reproduction.     

ADAMTS9 Is a Cell-Autonomously Acting,Anti-Angiogenic Metalloprotease Expressed by Microvascular Endothelial Cells

The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9^+/− mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9^+/− mice developed spontaneous corneal neovascularization. β-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9^+/− mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor₁₆₅, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor₁₆₅. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1.Following de novo formation of the vasculature from undifferentiated mesodermal precursors during embryogenesis (vasculogenesis), endothelial cells (ECs) proliferate and migrate to generate new capillaries from pre-existing blood vessels, a process termed angiogenesis.^1,2 Angiogenesis is critical for embryonic development, subsequent organ growth, and physiological processes such as menstruation and wound healing.³ It is a prominent feature of age-related macular degeneration and diabetic retinopathy, and tumor growth is angiogenesis-dependent.^1,2 Owing to this considerable relevance for human pathology, the fundamental mechanisms that regulate angiogenesis have been the subject of intense investigation.Proteases are considered to be key participants in angiogenesis because of their traditional roles in remodeling of the vascular basement membrane during EC sprouting and migration.^4,5 However, proteases also influence EC behavior by shedding cell-surface receptors and growth factors, exposing cryptic adhesion sites, or releasing bioactive fragments.^3,6 Thus, a role for proteases as negative regulators of angiogenesis is also accepted.^3,7–9 The ADAMTS protease family contains 19 secreted mammalian metalloproteases that localize to the cell-surface and/or extracellular matrix.^10,11 Several ADAMTS metalloproteases have specialized physiological roles that were revealed by analysis of human genetic disorders, or naturally occurring and engineered animal mutations,^12–14 but the functions of other members of this family, such as ADAMTS9, are poorly understood. ADAMTS proteases share a complex modular structure, comprising a metalloprotease domain coupled to a large ancillary domain containing thrombospondin type-1 repeats (TSRs), which are the hallmark of this family.¹⁰ The TSRs of ADAMTS proteases are similar to those of the anti-angiogenic molecules thrombospondin-1 (TSP-1) and TSP-2.^15–17 Indeed, ADAMTS1 was previously shown to inhibit angiogenesis via both proteolytic and non-proteolytic mechanisms, ie, by cleavage of TSP-1 and TSP-2, releasing anti-angiogenic fragments, as well as by sequestration of the pro-angiogenic growth factor vascular endothelial growth factor (VEGF)₁₆₅ by the TSR-containing ancillary domain.^9,18 Adamts1 mRNA is widely expressed during embryogenesis and in adult tissues, but its expression in the vasculature appears to be restricted to smooth muscle cells, and is not reported in capillary endothelium.^19,20 Although Adamts1 null mice have significant perinatal lethality,^21,22 and Adamts1^−/− ovaries have fewer and larger ovarian blood vessels than normal, generalized defects in vascular development have not been reported.²³ However, ADAMTS1 produced by keratinocytes and skin fibroblasts regulates the migration of EC during wound healing.²⁴ADAMTS9 is the most highly conserved member of the ADAMTS family, being similar to Caenorhabditis elegans Gon-1, which is required for nematode morphogenesis.²⁵ ADAMTS9 not only has an identical active site sequence as ADAMTS1, but contains 15 potentially anti-angiogenic TSRs.²⁶ ADAMTS9 was previously identified as a tumor suppressor gene in esophageal and nasopharyngeal cancer.^27,28 Recent work demonstrated that ADAMTS9 worked cooperatively with another Gon-1-related protease, ADAMTS20, in the colonization of skin by neural crest-derived melanoblasts.²⁹ Using in situ hybridization during murine development, we previously found that ADAMTS9 was expressed in capillaries.³⁰ However, early embryonic lethality of ADAMTS9 null mice appeared to preclude analysis of its role in vascular development, as well as angiogenesis in the tumor context.²⁹ We demonstrate here that when congenic with the C57Bl/6 strain, ADAMTS9^+/− mice develop spontaneous corneal neovascularization within a few weeks of weaning and that heterotopic tumors in these mice attract more vasculature than wild-type mice. It is shown that ADAMTS9 acts via a cell-autonomous mechanism in microvascular endothelium and that it represents a nonredundant anti-angiogenic activity, since it does not share the mechanisms used by ADAMTS1, and differs from it in other fundamental respects.     

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5''-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Sialic acids (Sias) are negatively charged monosaccharides commonly found on the outer ends of glycan chains on glycoproteins and glycolipids in mammalian cells.¹ Although Sias are necessary for mammalian embryonic development,^1,2 they also have much structural diversity, with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) comprising the two most abundant Sia forms in most mammalian tissues. Neu5Gc differs from Neu5Ac by having an additional oxygen at the 5-N-acyl position.³ Neu5Gc synthesis requires the cytidine-5''-monophospho (CMP)-Neu5Ac hydroxylase gene, or CMAH, which encodes a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.^4,5 CMP-Neu5Ac and CMP-Neu5Gc can be utilized by the >20 sialyltransferases to attach Neu5Ac or Neu5Gc, respectively, onto glycoproteins and glycolipids.^1,3Humans cannot synthesize Neu5Gc, because of an inactivating deletion in the human CMAH gene that occurred approximately 2 to 3 million years ago.⁶ This event fundamentally changed the biochemical nature of all human cell membranes, eliminating millions of oxygen atoms on Sias on the glycocalyx of almost every cell type in the body, which instead present as an excess of Neu5Ac. Consistent with the proposed timing of this mutation at around the emergence of the Homo lineage, mice with a human-like inactivation of CMAH have an enhanced ability for sustained aerobic exercise,⁷ which may have provided an evolutionary advantage. In this regard, it is also interesting that the mild phenotype of X chromosome–linked muscular dystrophy (mdx) mice with a dystrophin mutation that causes Duchenne muscular dystrophy (DMD) in humans is exacerbated and becomes more human-like on mating into a human-like CMAH null state.⁸Inactivation of CMAH in humans also fundamentally changed the immunologic profile of humans. Almost all humans consume Neu5Gc from dietary sources (particularly the red meats beef, pork, and lamb), which can be taken up by cells through a salvage pathway, sometimes allowing for Neu5Gc expression on human cell surfaces.9, 10, 11, 12, 13 Meanwhile, most humans have some level of anti–Neu5Gc-glycan antibodies, defining Neu5Gc-bearing glycans as xeno-autoantigens recognized by the immune system.13, 14, 15, 16 Humans develop antibodies to Neu5Gc not long after weaning, likely triggered by Neu5Gc incorporation into lipo-oligosaccharides of commensal bacteria in the human upper airways.¹³ The combination of xeno-autoantigens and such xeno-autoantibodies generates xenosialitis, a process that has been shown to accelerate progression of cancer and atherosclerosis in mice with a human-like CMAH deletion in the mouse Cmah gene.^17,18 Inactivation of mouse Cmah also leads to priming of macrophages and monocytes¹⁹ and enhanced reactivity²⁰ that can hyperactivate immune responses. Cmah deletion in mice also causes hearing loss via increased oxidative stress,^21,22 diabetes in obese mice,²³ relative infertility,²⁴ delayed wound healing,²¹ mitochondrial dysfunction,²² changed metabolic state,²⁵ and decreased muscle fatigability.⁷Given that Cmah deletion can hyperactivate cellular immune responses, it is perhaps not surprising that the crossing of Cmah deletion in mouse models of various human diseases, to humanize their sialic acid repertoire, can alter pathogenic disease states and disease outcomes. This is true of cancer burden from transplantation of cancer cells into mice,¹⁷ infectious burden of induced bacterial infections in mice,^13,18,19 and muscle disease burden in response to Cmah deletion in the mdx model of Duchenne muscular dystrophy⁸ and the α sarcoglycan (Sgca) deletion model of limb girdle muscular dystrophy 2D.²⁶ The mdx mice possess a mutation in the dystrophin (Dmd) gene that prevents dystrophin protein expression in almost all muscle cells,²⁷ making it a good genetic model for DMD, which also arises from lack of dystrophin protein expression.^28,29 These mdx mice, however, do not display the severe onset of muscle weakness and overall disease severity found in children with DMD, suggesting that additional genetic modifiers are at play to lessen mouse disease severity, some of which have been described.30, 31, 32, 33, 34, 35, 36 Cmah deletion worsens muscle inflammation, in particular recruitment of macrophages to muscle with concomitant increases in cytokines known to recruit them, increases complement deposition, increases muscle wasting, and premature death in a fraction of affected mdx mice.⁸ Cmah-deficient mdx mice have changed cardiac function.³⁷ Prior studies⁸ show that about half of all mice display induced antibodies to Neu5Gc, which correlates well with the number of animals showing premature death in the 6- to 12-month period. Unpublished subsequent studies suggest that Cmah^−/−mdx mice that lack xeno-autoimmunity often have less severe disease, which likely causes selection for more efficient breeders lacking Neu5Gc immunity over time. Current studies were designed to re-introduce Neu5Gc xeno-autoimmunity into serum-naive Cmah^−/−mdx mice and describe the impact of xenosialitis on disease pathogenesis.     

Retinal Dystrophy and Optic Nerve Pathology in?the Mouse Model of Mucolipidosis IV

Mucolipidosis IV is a debilitating developmental lysosomal storage disorder characterized by severe neuromotor retardation and progressive loss of vision, leading to blindness by the second decade of life. Mucolipidosis IV is caused by loss-of-function mutations in the MCOLN1 gene, which encodes the transient receptor potential channel protein mucolipin-1. Ophthalmic pathology in patients includes corneal haze and progressive retinal and optic nerve atrophy. Herein, we report ocular pathology in Mcoln1^−/− mouse, a good phenotypic model of the disease. Early, but non-progressive, thinning of the photoreceptor layer, reduced levels of rhodopsin, disrupted rod outer segments, and widespread accumulation of the typical storage inclusion bodies were the major histological findings in the Mcoln1^−/− retina. Electroretinograms showed significantly decreased functional response (scotopic a- and b-wave amplitudes) in the Mcoln1^−/− mice. At the ultrastructural level, we observed formation of axonal spheroids and decreased density of axons in the optic nerve of the aged (6-month-old) Mcoln1^−/− mice, which indicates progressive axonal degeneration. Our data suggest that mucolipin-1 plays a role in postnatal development of photoreceptors and provides a set of outcome measures that can be used for ocular therapy development for mucolipidosis IV.Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by severe psychomotor retardation and visual loss. MLIV is classified as a lysosomal storage disease because of abnormal accumulation of storage material in lysosomes of all cells and tissues of the body.¹ Corneal clouding because of accumulation of lysosomal storage is an early pathological hallmark of the disease that, when present with developmental delay in infanthood, is highly suggestive of MLIV.Ophthalmic manifestations in patients generally have a progressive course and, in addition to corneal clouding, include optic nerve atrophy and outer retinal degeneration.^{2, 3, 4} In most of the patients, MLIV leads to blindness in the second decade of life.⁵Mutations in MCOLN1, which encodes the transient receptor potential cation channel TRPML1 (alias mucolipin-1) cause the disease.^{6, 7, 8, 9} More than 20 mutations in MCOLN1 have been identified to date.⁵ More than 75% of known MLIV patients are Ashkenazi Jewish, and the two founder mutations, present in 95% of Ashkenazi Jewish patients, result in complete loss of mRNA and protein.¹⁰ MLIV is a rare disease with carrier frequency of 1:100 in Ashkenazi Jewish and 1:10,000 in the general population. Many patients with MLIV remain undiagnosed or are misdiagnosed with cerebral palsy. Thus, bringing awareness of this disease to pediatric ophthalmologists and neurologists is important to improve diagnosis as new therapies are developed.Mucolipin-1 has six transmembrane domains and is permeable to Ca²⁺, Na⁺, K⁺, Fe²⁺, Mn²⁺, and Zn²⁺.^{11, 12, 13} Its channel activity is regulated by both calcium concentration and pH, and mucolipin-1 has been shown to have lipase activity.¹⁴ Previous studies by our group and others showed mucolipin-1 localization to the late endosomes and lysosomes.^{15, 16, 17, 18} The transient receptor potential channel protein mucolipin-1 is required for transport of lipids from the late endosomes-lysosomes to the trans-Golgi compartment,^{19, 20} Ca²⁺-dependent late endosome-lysosome fission-fusion events,²⁰ reformation of lysosomes from endosome-lysosome hybrids^{18, 21} and autolysosomes,^{22, 23} and lysosomal exocytosis.^{24, 25} Mucolipin-1 is strongly expressed in the mouse retina, with the highest mRNA levels in the outer plexiform layer and outer nuclear layer.²⁶The Mcoln1 knockout (KO) mouse model recapitulates the main features of the human disease, and is a good phenotypic platform for investigating MLIV disease mechanisms.^{27, 28, 29, 30} At the ultrastructural level, typical MLIV storage inclusions have been found in the brain during embryonic development.³¹ Histochemical analysis in the young adult (2 months of age) Mcoln1^−/− mice has shown pronounced glial activation, reduced myelination, and no neuronal loss in the cerebrum in the regions most affected by gliosis.²⁸In this study, we used Mcoln1^−/− mice to characterize the consequences of mucolipin-1 loss on retinal morphology, optic nerve myelination, and visual function in the course of the disease. Major manifestations of ophthalmic pathology in Mcoln1^−/− mice were as follows: non-progressive thinning of the photoreceptor layer; profound accumulation of storage inclusions throughout the retina and in the optic nerve, including formation of large axonal spheroids; axonal degeneration in the optic nerve in older mice; hypertrophied lysosomes in photoreceptors and other retinal cells; and reduced visual function.     

Complement Regulatory Protein CD46 Protects against Choroidal Neovascularization in Mice

Dysregulation of the complement system is increasingly recognized as a contributing factor in age-related macular degeneration. Although the complement regulator CD46 is expressed ubiquitously in humans, in mouse it was previously thought to be expressed only on spermatozoa. We detected CD46 mRNA and protein in the posterior ocular segment (neuronal retina, retinal pigment epithelium, and choroid) of wild-type (WT) C57BL/6J mice. Cd46^−/− knockout mice exhibited increased levels of the membrane attack complex and of vascular endothelial growth factor (VEGF) in the retina and choroid. The Cd46^−/− mice were also more susceptible to laser-induced choroidal neovascularization (CNV). In Cd46^−/− mice, 19% of laser spots were positive for CNV at day 2 after treatment, but no positive spots were detected in WT mice. At day 3, 42% of laser spots were positive in Cd46^−/− mice, but only 11% in WT mice. A fully developed CNV complex was noted in both Cd46^−/− and WT mice at day 7; however, lesion size was significantly (P < 0.05) increased in Cd46^−/− mice. Our findings provide evidence for expression of CD46 in the mouse eye and a role for CD46 in protection against laser-induced CNV. We propose that the Cd46^−/− mouse has a greater susceptibility to experimental CNV because of insufficient complement inhibition, which leads to increased membrane attack complex deposition and VEGF expression.Age-related macular degeneration (AMD) is a leading worldwide cause of central vision loss in individuals over the age of 50.^1–5 The prevalence of AMD is growing because of increased longevity. The disease brings negative changes in life style. AMD patients often cannot perform daily tasks of living, such as reading or driving. Current estimates are that it requires 575 to 733 million dollars annually to treat AMD in the United States.⁵ Thus, AMD profoundly affects the quality of life, creating a serious social and public health problem.^3,4Two major clinical phenotypes of AMD are recognized: nonexudative (dry type) and exudative (wet type). Wet AMD is frequently associated with central blindness, and choroidal neovascularization (CNV) is the hallmark of this type in humans. Agents available for treating exudative AMD reduce the rate of vision loss, but they do not reverse damage; furthermore, they are associated with a variety of ocular complications, require repetitive administration, and are expensive.^1–4AMD is a disease with numerous risk factors, and multiple pathological mechanisms have been reported.^6–10 Evidence accumulated during the past decade, primarily through genetic studies, strongly indicates that the alternative pathway of the complement system plays an important role in AMD pathogenesis in humans.^3,11–15 Reports from multiple investigators have established that the membrane attack complex (MAC) C5b-9, formed as a result of alternative pathway complement engagement, participates in mediating animal models of CNV.^{11,12,16–23} Complement regulatory proteins control the complement cascades,^24,25 and their deficiency has been reported to predispose to the development of experimental CNV.^18,20CD46 [alias membrane cofactor protein (MCP)] is a widely expressed transmembrane glycoprotein in primates that serves as a complement regulatory protein^26,27 and is present in the normal human eye.^28–31 It binds C3b and C4b and then serves as a cofactor protein for the cleavage of these two substrates by serine protease factor I. In rodents, however, CD46 expression has been firmly established only for the inner acrosomal membrane of spermatozoa.^32–34 Our goal in the present study was to investigate whether CD46 is expressed in the mouse eye. After this was unequivocally shown, we explored its role in a murine model of laser-induced CNV.^11,12,35,36     

Platelets Control Leukocyte Recruitment in a Murine Model of Cutaneous Arthus Reaction

Platelets have been shown to be important in inflammation, but their role in the cutaneous Arthus reaction remains unclear. To assess the role of platelets in this pathogenetic process, the cutaneous Arthus reaction was examined in wild-type mice and mice lacking E-selectin, P-selectin, or P-selectin glycoprotein ligand-1 (PSGL-1) with or without platelet depletion by busulfan, a bone marrow precursor cell-specific toxin. Edema and hemorrhage induced by immune complex challenge significantly decreased in busulfan-treated wild-type mice compared with untreated mice. Busulfan treatment did not affect edema and hemorrhage in P-selectin- or PSGL-1-deficient mice, suggesting that the effect by busulfan is dependent on P-selectin and PSGL-1 expression. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells and reduced levels of circulating platelets. Increased cutaneous production of interleukin-6, tumor necrosis factor-α, and platelet-derived chemokines during Arthus reaction was inhibited in busulfan-treated wild-type mice relative to untreated mice, which paralleled the reduction in cutaneous inflammation. Flow cytometric analysis showed that immune complex challenge generated blood platelet-leukocyte aggregates that decreased by busulfan treatment. In thrombocytopenic mice, the cutaneous inflammation after immune complex challenge was restored by platelet infusion. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates and secreting chemokines at inflamed sites, mainly through the interaction of P-selectin on platelets with PSGL-1 on leukocytes.The pathogenesis of autoimmune diseases frequently involves the formation of IgG-containing immune complexes (ICs) inducing inflammatory responses with significant tissue injury, commonly referred to as type III hypersensitivity reaction. This IC injury has been implicated in the pathogenesis of vasculitis syndrome, systemic lupus erythematosus, rheumatoid arthritis, and cryoglobulinemia.¹ The mechanisms by which the immune system controls effector responses to ICs are of central importance for developing therapeutic strategies. The standard animal model for the inflammatory response in these IC-mediated diseases is the Arthus reaction.² Analyses using gene knockout mice have revealed that activation of the complement system, especially C5a and its interaction with C5a receptor, and of Fc receptors for IgG on inflammatory cells, particularly mast cells, are both required to initiate the Arthus reaction.^3–8 In addition, accumulation of neutrophils and mast cells is necessary for the progression of the IC-mediated vascular tissue damage, which results in edema and hemorrhage.^3–8Leukocyte recruitment from the circulation to a site of inflammation is an essential process in the inflammatory response. Leukocytes first tether and roll on vascular endothelial cells, before they are activated to adhere firmly and subsequently immigrate into the extravascular space. This multistep process is highly regulated by multiple cell-surface adhesion molecules.^9,10 The selectins cooperate to support leukocyte tethering and rolling along inflamed vascular walls by mediating leukocyte interactions with glycoconjugated counter-receptors expressed by endothelium, adherent platelets, or leukocytes. The selectin family consists of three cell-surface molecules expressed by leukocytes (L-selectin), vascular endothelium (E- and P-selectins), and platelets (P-selectin).¹¹ Although the adhesive mechanisms underlying the capture and immobilization of circulating leukocytes in inflamed blood vessels have been well described, factors triggering and controlling the leukocyte recruitment into inflamed sites are poorly understood.The multistep process of leukocyte tethering and rolling, followed by leukocyte activation and firm adhesion, also occurs on activated platelets.¹² Platelets are essential for primary hemostasis, but they also play an important pro-inflammatory role.^13,14 Platelets normally circulate in a quiescent state, protected from untimely activation by inhibitory mediators released from intact endothelial cells. Endothelial dysfunction and changes in release of antiplatelet factors lead to increased platelet activation followed by their interaction with leukocytes, and increased platelet adhesion and aggregation.^15,16 On activation, platelets can change their shapes as well as the expression pattern of adhesion molecules, and secrete neutrophil and endothelial activators inducing production of pro-inflammatory cytokines.¹⁷ These changes are associated with the adhesion of platelets to leukocytes and endothelium.¹⁴ Thus, platelets are important amplifiers of acute inflammation.Platelets accumulate in inflammatory lesions concomitantly with leukocytes and regulate a variety of inflammatory responses by secreting or activating adhesion proteins, growth factors, and coagulation factors.^18,19 These proteins induce widely differing biological activities, including cell adhesion, chemotaxis, cell survival, and proliferation, all of which accelerate the inflammatory process.²⁰ In vitro and in vivo studies have shown that platelets bind to leukocytes through their surface protein.^12,14,20,21 Indeed, previous studies have reported that platelet-leukocyte aggregates are formed in circulating blood of asthmatic patients.²² Platelets express much amounts of P-selectin than endothelium and also bind endothelium via selectin dependent and independent mechanisms.^23–25 In addition to classical leukocyte recruitment process, platelets bound to activated endothelial cells can interact with leukocytes, which results in secondary capture that induces interactions of leukocytes with platelets first, followed by leukocyte-endothelial cell interaction.²⁶ Leukocytes within platelet-leukocyte complexes have increased adhesive capacity to the activated endothelium.²⁷ Therefore, platelet can function as a bridge between the circulating leukocyte and endothelium.We previously showed that mice lacking P-selectin (P-selectin^−/−) or mice treated with anti- P-selectin glycoprotein ligand-1 (PSGL-1) antibody (Ab) exhibited reduced Arthus reaction that is associated with decreased infiltration of neutrophils and mast cells.^28,29 In addition to interacting with selectins and selectin ligands on endothelial cells, leukocytes can also interact with selectins and selectin ligands presented by platelets or their microparticle fragments, which are all found at sites of inflammation.³⁰ This indicates that observations of altered leukocyte recruitment in selectin- and selectin ligand-deficient mice must be discussed in light of altered selectin and selectin-ligand expression not only by endothelial cells, but also by platelets. Recently, involvement of platelets has been demonstrated in the pathogenesis of inflammatory disorders, including asthma,^22,31 arthritis,¹⁸ inflammatory bowel disease,³² and chronic allergic dermatitis.³³ Although the role of platelets in inflammatory process is being increasingly recognized, it remains unknown how platelets induce leukocyte recruitment in the cutaneous Arthus reaction. A recent report has identified a role of platelets in promoting IC-induced leukocyte recruitment to the cremaster muscle in a murine model of reverse passive Arthus reaction.³⁴ However, the relative role of each leukocyte and adhesion molecule in the inflammation varies according to the tissue site and the nature of inflammatory stimuli.²⁹ Therefore, to clarify the importance of platelets, their surface adhesion molecule expression, and platelet-derived chemokines on leukocyte recruitment, we examined the cutaneous Arthus reaction in wild-type, P-selectin^−/−, E-selectin^−/−, and PSGL-1^−/− mice, with or without treatment with busulfan, a bone marrow precursor cell-specific toxin.     

Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model

We investigated human cytomegalovirus pathogenesis by comparing infection with the low-passage, endotheliotropic strain VR1814 and the attenuated laboratory strain AD169 in human placental villi as explants in vitro and xenografts transplanted into kidney capsules of SCID mice (ie, mice with severe combined immunodeficiency). In this in vivo human placentation model, human cytotrophoblasts invade the renal parenchyma, remodel resident arteries, and induce a robust lymphangiogenic response. VR1814 replicated in villous and cell column cytotrophoblasts and reduced formation of anchoring villi in vitro. In xenografts, infected cytotrophoblasts had a severely diminished capacity to invade and remodel resident arteries. Infiltrating lymphatic endothelial cells proliferated, aggregated, and failed to form lymphatic vessels. In contrast, AD169 grew poorly in cytotrophoblasts in explants, and anchoring villi formed normally in vitro. Likewise, viral replication was impaired in xenografts, and cytotrophoblasts retained invasive capacity, but some partially remodeled blood vessels incorporated lymphatic endothelial cells and were permeable to blood. The expression of both vascular endothelial growth factor (VEGF)-C and basic fibroblast growth factor increased in VR1814-infected explants, whereas VEGF-A and soluble VEGF receptor-3 increased in those infected with AD169. Our results suggest that viral replication and paracrine factors could undermine vascular remodeling and cytotrophoblast-induced lymphangiogenesis, contributing to bleeding, hypoxia, and edema in pregnancies complicated by congenital human cytomegalovirus infection.Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, with an incidence in the United States of approximately 1% to 3% of live births.¹ Primary maternal HCMV infection during gestation poses a 40% to 50% risk of intrauterine transmission, whereas recurrent infection in seropositive mothers rarely causes disease.^2,3 Symptomatic infants (25%) have intrauterine growth restriction (IUGR) and permanent birth defects, including neurological deficiencies, retinopathy, and sensorineuronal deafness.^4–6 Congenital disease is more severe when primary maternal infection occurs in the first trimester.⁷ IUGR and spontaneous abortion in the absence of fetal HCMV infection can result from placental pathology.^8–10 Placentas infected in early gestation show long-standing damage and fibrosis at the uterine-placental interface, which impairs critical functions and results in a hypoxic intrauterine environment.^10–15 Despite the prevalence and the medical and societal impact of congenital HCMV infection, the mechanisms of virus replication, pathogenesis, and transplacental transmission are still unresolved because of the complex nature of placental development and extreme species specificity of HCMV, which replicates only in human tissues.Differentiating/invading cytotrophoblasts switch to an endothelial phenotype in a process that is similar to vasculogenesis.¹⁶ The cells up-regulate novel adhesion molecules and proteinases that enable their attachment to and invasion of the uterus. Interstitial invasion requires down-regulation of integrins characteristic of epithelial cells and novel expression of the integrins α1β1, α5β1, and αvβ3.¹⁷ Endovascular cytotrophoblasts that remodel uterine blood vessels transform their adhesion receptor phenotype to resemble that of endothelial cells, expressing vascular-endothelial cadherin, platelet-endothelial adhesion molecule-1, and vascular endothelial adhesion molecule-1.^16,18 Like endothelial cells, cytotrophoblasts express substances that influence vasculogenesis and angiogenesis, including the vascular endothelial growth factor (VEGF) family ligands VEGF-A and VEGF-C and receptors VEGFR-1 [fms-like tyrosine kinase 1 (Flt-1)] and VEGFR-3.^19–21 Expression of these molecules changes as the cells differentiate/invade, and they regulate cytotrophoblast survival in the remodeled uterine vasculature. Finally, as hemiallogeneic embryonic/fetal cells, invasive cytotrophoblasts must avoid maternal immune responses. Their expression of the nonclassical major histocompatibility complex (MHC) class I molecules HLA-G^22,23 and HLA-C, which have limited polymorphisms,^24,25 contributes to their lack of immunogenicity.Previous studies led to a rudimentary understanding of HCMV infection of the human placenta and identified several molecular mechanisms that impair functions of differentiating/invading cytotrophoblasts. HCMV infection dysregulates the expression of key integrins required for cell invasiveness,^26,27 reduces the expression of matrix metalloproteinase-9,²⁷ and down-regulates cell-cell and cell-matrix adhesion molecules,²⁸ including those required for pseudovasculogenesis¹⁶ and vascular remodeling.¹⁸ The immunosuppressive viral cytokine cmv IL-10 further reduces cytotrophoblast invasion through paracrine effects that increase IL-10 expression.^27,28 Peroxisome proliferator-activated receptor γ activation by infection also compromises cytotrophoblast functions.^29,30 In chorionic villi, the neonatal Fc receptor for IgG, expressed in syncytiotrophoblasts that contact maternal blood, transcytoses circulating maternal antibodies.^31–33 In conjunction with neutralizing titers, developmental expression of HCMV receptors, EGFR, and integrins^34–37 determines susceptibility to infection.^33,38–40 HCMV infects spatially distinct populations of cytotrophoblasts that express α1β1 and αvβ3 integrins used as surface receptors.⁴¹How HCMV disseminates to the placenta and the early stages of pathogenesis in pregnancy are still unresolved because of the virus'' extreme host range restriction. A successful approach to overcome the obstacle to studies of HCMV in vivo has been to infect SCID mice (ie, mice with severe combined immunodeficiency) that have received xenografts of human tissues. Infection of human fetal thymus/liver under the mouse kidney capsule showed that medullary epithelial cells are prominent targets of HCMV replication.⁴² Thus, dramatic interstrain differences were evident in replication of low-passage clinical isolates and laboratory strains in thymus/liver xenografts in vivo.^42,43 The strain Toledo replicates to high titers in implants, whereas the laboratory strains AD169 and Towne, serially passaged in fibroblasts, are attenuated and fail to propagate in tissues in vivo.⁴³ AD169 lacks a 15-kb segment of viral genome that encodes at least 19 open reading frames present in the genomes of all pathogenic clinical strains.⁴⁴ A deletion mutant of Toledo lacking these sequences, although exhibiting only a minor growth defect in fibroblasts, fails to replicate in thymus/liver implants in SCID mice, evidence that genes in this region are central to infection in vivo.⁴⁵ HCMV replication in endothelial and epithelial cells correlates with determinants specified by ORFs UL128-131A,^46,47 which are highly conserved in clinical isolates⁴⁸ and which elicit neutralizing antibodies in humans.^49,50Herein, we investigated HCMV pathogenesis in infected human placental villous explants and in xenografts maintained in vivo. We used a model of human placentation to investigate the vascular effects of fetal cytotrophoblasts in human placental villi transplanted beneath the kidney capsules of SCID mice.²¹ VR1814, a clinical isolate, infected cell column cytotrophoblasts in placental explants and impaired the formation of anchoring villi in vitro. In xenografts, VR1814-infected placental cells had a severely diminished capacity to invade and form lymphatic vessels. In striking contrast, AD169 replicated poorly in villous explants. In SCID mice, AD169-infected cytotrophoblasts remodeled the resident arteries, but these were faulty. Our results show, for the first time to our knowledge, that HCMV genes dispensable for growth in culture function as determinants of pathogenesis that could contribute to vascular anomalies that originate in early placentation.     

Junctional Adhesion Molecule A Promotes Epithelial Tight Junction Assembly to Augment Lung Barrier Function

Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A^−/− mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A^−/− mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton.To support efficient gas exchange, the lung must maintain a barrier between the atmosphere and fluid-filled tissues. Without this crucial barrier, the air spaces would flood, and gas exchange would be severely limited.^{1, 2} In acute lung injury and acute respiratory distress syndrome, fluid leakage into the lung air space is associated with increased patient mortality and morbidity.^{3, 4} Lung fluid clearance is maintained, in part, by tight junctions that regulate paracellular flux between cells.^{5, 6, 7}Tight junctions are multiprotein complexes located at sites of cell-cell contact and are composed of transmembrane, cytosolic, and cytoskeletal proteins that together produce a selective barrier to water, ions, and soluble molecules. Among the transmembrane proteins required for epithelial barrier function is the Ig superfamily protein junctional adhesion molecule A (JAM-A).^{8, 9, 10, 11} JAM-A is ubiquitously expressed and regulates several processes related to cell-cell and cell-matrix interactions, including cell migration and proliferation in addition to barrier function regulation. Specific mechanistic roles for JAM-A in regulating tight junctions continue to be elucidated.JAM-A signaling is stimulated by cis-dimerization, which provides a platform for multiple proteins to cluster in close apposition.¹² In particular, JAM-A has been shown to recruit scaffold proteins, such as zonula occludens 1 (ZO-1), ZO-2, and Par3, to tight junctions, where these proteins enhance the assembly of multiprotein junctional complexes.^{13, 14} More recently, it was demonstrated that JAM-A directly interacts with ZO-2, which then recruits other scaffold proteins, including ZO-1.¹⁵ This nucleates a core complex that includes afadin, PDZ-GEF1, and Rap2c and that stabilizes filamentous actin by repressing rhoA.¹⁵ Together, all of these activities of JAM-A promote tight junction formation and barrier function.Although JAM-A is part of the tight junction complex, the main structural determinants of the paracellular barrier are proteins known as claudins. Claudins are a family of transmembrane proteins that interact to form paracellular channels that either promote or limit paracellular ion and water flux.^{16, 17, 18} Claudins that promote flux are known collectively as pore-forming claudins, whereas claudins that limit flux are known as sealing claudins.¹⁹ In fact, there is a link between JAM-A and claudin expression because it was demonstrated that JAM-A–deficient intestinal epithelium has increased expression of two pore-forming claudins, claudin-10 and claudin-15.²⁰ Critically, increased claudin-10 and claudin-15 leads to a compromised intestinal barrier, as demonstrated by an enhanced susceptibility of JAM-A^−/− mice to dextran sulfate sodium–induced colitis.²⁰ However, it is not known whether this relationship between JAM-A and claudin expression occurs in other classes of epithelia.Several claudins are expressed by the alveolar epithelium. The most prominent alveolar claudins are claudin-3, claudin-4, and claudin-18; several additional claudins are expressed by alveolar epithelium and throughout the lung as well.^{21, 22} A central role for claudin-18 in regulating lung barrier function was demonstrated in two independently derived strains of claudin-18–deficient mice that showed altered alveolar tight junction morphologic features and increased paracellular permeability.^{23, 24} Claudin-4 also is an important part of the lung response to acute lung injury because it improves barrier function by limiting alveolar epithelial permeability and promoting lung fluid clearance.^{25, 26} Although claudin-4–deficient mice show a relatively mild baseline phenotype, these mice have impaired fluid clearance in response to ventilator-induced lung injury.²⁷ An analysis of ex vivo perfused human donor lungs revealed that increased claudin-4 was linked to increased rates of alveolar fluid clearance and decreased physiologic respiratory impairment,²⁸ further underscoring the importance of claudin regulation in promoting efficient barrier function in response to injury.Although JAM-A has a clear role in regulating gut permeability,²⁰ a recent report that wild-type and JAM-A^−/− mice show comparable levels of pulmonary edema in response to intratracheal endotoxin challenge²⁹ raises questions about potential roles for JAM-A in lung barrier function. Herein we used a combination of in vivo and in vitro approaches to assess the contributions of JAM-A to alveolar barrier function. Using a model of mild systemic endotoxemia induced by i.p. injection of Escherichia coli–derived lipopolysaccharide (LPS), we found that JAM-A^−/− mice showed greater lung edema than comparably treated wild-type mice. Greater sensitivity to injury was due to aberrant regulation of tight junction protein expression, which was recapitulated by JAM-A–depleted alveolar epithelial cells. JAM-A depletion also resulted in decreased β1 integrin protein levels and disrupted cytoskeletal assembly. Together, these effects indicated that the loss of JAM-A impaired tight junction formation, thus rendering the lung more susceptible to edema and injury.     

Alterations of Retinal Vasculature in Cystathionine–β-Synthase Heterozygous Mice: A Model of Mild to Moderate Hyperhomocysteinemia

Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine–β-synthase (Cbs^+/−) to evaluate retinal vascular integrity. The Cbs^+/+ (wild type) and Cbs^+/− (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti–zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti–pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs^+/− mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs^+/− retinas. Retinal vein occlusion was observed in some Cbs^+/− mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs^+/− mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.Homocysteine (Hcy), a sulfur-containing amino acid, is an intermediate in methionine metabolism. It is converted to either methionine, via the remethylation pathway, or cysteine for permanent disposal via the trans-sulfuration pathway. Hyperhomocysteinemia (HHcy) results from increased Hcy dietary loading (eg, diet rich in methionine), decreased rates of Hcy metabolism due to deficiency of vitamin cofactors (folic acid and vitamins B₆ and B₁₂), or genetic mutations of enzymes involved in remethylation or trans-sulfuration pathways. Mild to moderate HHcy is prevalent in 5% to 10% of the general population, up to 90% of patients undergoing hemodialysis, and approximately 40% of patients with peripheral vascular disease.^1,2 HHcy is a risk factor for human cardiovascular diseases (eg, stroke and venous thrombosis) and neurodegenerative diseases.³ It has been implicated in eye-related diseases, including ectopia lentis,⁴ glaucoma (primary and secondary open-angle glaucoma, exfoliation glaucoma, and pigmentary glaucoma),^5–7 macular degeneration, maculopathy, retinal degeneration,^8–10 diabetic retinopathy,^11,12 and retinal vascular diseases (ie, central retinal vein occlusion, branch retinal vein occlusion, and central retinal artery occlusion^13–16), although its role in these diseases is inconsistent.Our laboratory and others have used in vitro and in vivo experimental models to understand the mechanisms by which HHcy affects retina. Apoptotic neuronal cell death was induced in primary mouse retinal ganglion cells incubated with elevated, but physiologically relevant, levels of Hcy (50 μmol/L).¹⁷ In vivo studies showed that intravitreal injection of high dosages of d,l-Hcy-thiolactone led to marked ganglion cell loss and disruption of the inner retina in mice within 5 days of injection.¹⁸ Lower-dosage d,l-Hcy-thiolactone intravitreal injections led to marked loss of photoreceptor cells within 15 days and ablation of the outer retinal nuclear layer within 90 days.¹⁹ Mice deficient or lacking the gene encoding cystathionine–β-synthase (CBS)²⁰ have proved useful for analysis of the effects of mild to severe endogenous elevation of Hcy on several tissues, including retina.^21–26 CBS is a key enzyme in the trans-sulfuration pathway, and its deficiency is the most common cause of inherited homocystinuria.⁴The Cbs^−/− mice have a 30- to 40-fold increase in plasma Hcy²⁰ and a shortened life span of 3 to 5 weeks. Retinal Hcy levels are increased by approximately sevenfold in Cbs^−/− mice compared with age-matched wild-type littermates.²² Functional studies of the visual system in these mice reveal significantly reduced amplitudes of the dark- and light-adapted electroretinogram (ERG) and a marked delay in the N1 implicit time of the visual-evoked potential, which reflects signal transmission to the visual cortex.²⁵ Comprehensive histological assessment of Cbs^−/− retinas demonstrates profound loss of cells in the retinal ganglion cell layer, marked disruption of the inner/outer nuclear retinal layers, and hypertrophy of the retinal pigment epithelial cell layer.²²Functional studies of retinas of Cbs^+/− mice, which have a much milder HHcy with an approximately fourfold to sevenfold increase in plasma Hcy (and a twofold increase in retinal Hcy), reveal a gradual reduction of the light and dark amplitudes of the ERG, reduced direct-coupled ERG light peak, and a slight increase in the implicit times of the visual-evoked potential.²⁵ Histological analyses of Cbs^+/− mouse retinas show a much milder retinal phenotype than Cbs^−/− mice, characterized by moderate cell loss in the ganglion cell layer and decreased thickness of the inner plexiform and inner and outer nuclear layers.²² Altered mitochondria of the nerve fiber layer and increased expression of the mitochondrial proteins, Opa1 and Fis1, accompanied the retinal ganglion cell loss.²⁴ The functional deficits and histological alterations in the Cbs^+/− mouse become evident by approximately 30 weeks of age.^22,25Recently, we analyzed the retinal vasculature of Cbs^−/− mice to determine whether the profound neuronal degeneration of the retina was accompanied by a vascular phenotype.²⁶ The study was restricted to mice aged 3 weeks owing to the short life span of the homozygous (Cbs^−/−) mice. Angiography revealed considerable vascular leakage in Cbs^−/− retinas, and the immunohistochemical analysis revealed vascular patterns consistent with ischemia. Vascular endothelial growth factor (VEGF), a marker of new blood vessels, was increased in Cbs^−/− retinas, and the blood-retinal barrier was compromised. In this same study, the vasculature of the heterozygous (Cbs^+/−) mice at 3 weeks showed minimal evidence of vasculopathy, but raised the question as to whether retinal vascular alterations would be detectable with advancing age. Because mild HHcy is common in the human population, the present study investigated the retinal vasculature of the Cbs^+/− mice as a function of age. Our data demonstrate subtle vascular changes early that become more prominent by 4 months of age and progress to ischemia and neovascularization by 6 to 12 months of age.     

Sphingosine Kinase 1 Deficiency Confers Protection against Hyperoxia-Induced Bronchopulmonary Dysplasia in a Murine Model: Role of S1P Signaling and Nox Proteins

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1^−/− (Sphk1^−/−), sphingosine kinase 2^−/− (Sphk2^−/−), and S1P lyase^+/− (Sgpl1^+/−) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1^−/−, but not Sphk2^−/− or Sgpl1^+/−, mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling–regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.Bronchopulmonary dysplasia (BPD) is a chronic lung disease occurring as a consequence of injury to the rapidly developing premature lungs of a preterm newborn infant.¹ Preterm neonates receive ventilator care and inhaled oxygen supplementation for variable periods after delivery; prolonged exposure of preterm lungs to hyperoxia results in inflammation, pulmonary edema, lung injury, and, ultimately, death.^2,3 BPD is characterized by decreased secondary septation of alveoli, resulting in the formation of enlarged simplified alveoli and reduced area for gas exchange.^4,5 More than 25% of premature infants with birth weights <1500 g develop BPD.^5,6 Infants with BPD have higher rehospitalization rates because of asthma, infection, pulmonary hypertension, and other respiratory tract ailments.^7,8 Many surviving neonatal BPD patients reaching adulthood show a sharp decline in lung capacity, indicating that the adverse effects of insult in the neonatal stage can be long lasting.^9,10 There is no effective treatment for BPD, and strategies to prevent BPD by administering gentler ventilation and other therapeutic approaches have not been effective.¹¹ The identification of novel signaling pathways linking hyperoxia-induced lung injury in neonatal BPD is necessary for new therapeutic approaches.Sphingolipids and their metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate (S1P), are important bioregulators, capable of modulating acute lung injury in a variety of lung disorders.^12–14 S1P plays an important role in vascular development and endothelial barrier function.^14,15 It is generated by the phosphorylation of sphingosine catalyzed by sphingosine kinases (SphKs) 1 and 2 and metabolized by S1P phosphatases and lipid phosphatases to yield sphingosine or by S1P lyase (S1PL; Sgpl1) that generates Δ2-hexadecenal and ethanolamine phosphate in mammalian cells.¹⁶ In addition to the previously mentioned enzymes, serine palmitoyltransferase (SPT) initiates the biosynthesis of sphingolipids by catalyzing condensation of serine and palmitoyl-CoA to form 3-ketosphinganine.¹⁷ S1P acts extracellularly and intracellularly, and most effects of extracellular S1P are mediated via a family of five highly specific G-protein–coupled S1P_1-5 receptors.^18,19 Significantly lower levels of S1P in plasma and lung tissues were reported in a murine model of lipopolysaccharide (LPS)–induced lung injury, most likely because of elevated expression of S1PL,²⁰ and infusion of S1P ameliorated LPS-induced acute lung injury in murine and canine models.^21,22 Taken together, these results suggest a protective role for S1P in LPS-mediated lung injury. Hyperoxia is also known to cause lung injury; however, the underlying pathological characteristics are not similar to those observed in the LPS-treated mouse model.^20,23The goal of the present study was, therefore, to elucidate the role of S1P in the development of lung injury and BPD in the murine neonatal model. Our results showed that hyperoxia-induced accumulation of S1P is detrimental and linked to BPD because SphK1-, but not SphK2-, deficient mice exhibited significantly less hyperoxia-induced reactive oxygen species (ROS) formation, lung injury, and BPD, such as morphological characteristics, whereas S1P lyase–deficient heterozygous mice showed the opposite. Furthermore, by using human lung microvascular endothelial cells (HLMVECs), we observed that exogenous S1P stimulated ROS production, and down-regulation of SphK1 with siRNA blocked hyperoxia-induced ROS generation. We also present herein evidence in support of an inflammatory role for S1P in BPD as it relates to increased expression of NADPH oxidase (NOX) proteins, such as NOX2 and NOX4, and the proinflammatory cytokine, IL-6.     

Homozygosity and Heterozygosity for Null Col5a2 Alleles Produce Embryonic Lethality and a Novel Classic Ehlers-Danlos Syndrome–Related Phenotype

Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2^−/− homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2^−/− embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2^−/− and Col5a1^−/− fibril formation and embryonic survival suggest that α1(V)₃ homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of α2(V) chains, serve functional roles that partially compensate for loss of the most common collagen V isoform. Col5a2^+/− adults have skin with marked hyperextensibility and reduced tensile strength at high strain but not at low strain. Col5a2^+/− adults also have aortas with increased compliance and reduced tensile strength. Results thus suggest that COL5A2^+/− humans, although unlikely to present with frank classic Ehlers-Danlos syndrome, are likely to have fragile connective tissues with increased susceptibility to trauma and certain chronic pathologic conditions.Collagen V is a low-abundance fibrillar collagen widely distributed in vertebrate tissues as α1(V)₂α2(V) heterotrimers,¹ which are incorporated into growing fibrils with the more abundant collagen I and involved in regulating the geometry and tensile strength of the resulting collagen I/V heterotypic fibrils.^2,3 Mutations in the genes encoding either the α1(V)⁴ or α2(V)⁵ chain can result in the human heritable connective tissue disorder classic Ehlers-Danlos syndrome (cEDS), clinical hallmarks of which include skin hyperextensibility, atrophic scarring, and joint hypermobility, with patients also often presenting with easy bruising and bleeding.⁶At the molecular level, the collagen fibrils of cEDS skin have variability in diameter not seen in normal skin and include large diameter collagen fibril aggregates with abnormal cauliflower-like shapes when viewed in cross section.⁶ Deficits in the tensile strength of cEDS collagen fibrils are inferred from the hyperextensibility and fragility of cEDS skin and the hypermobility of cEDS joints.Most cEDS cases that have been characterized at the molecular level are heterozygous for null alleles of the α1(V) chain gene COL5A1,⁷ resulting in the deposition of haploinsufficient levels of normal collagen V in tissues, with excess α2(V) chains unable to form stable triple helical molecules or be incorporated into the extracellular matrix (ECM).⁸A lesser number of cEDS cases are associated with COL5A1 missense mutations, and a number of these [eg, signal peptide and C-propeptide mutations that reduce secretion or incorporation of α1(V) chains into heterotrimers, respectively] may result in de facto functional haploinsufficiency rather than structurally abnormal collagen V in the ECM.⁷ An even smaller number of cEDS cases have been associated with missense mutations in the α2(V) chain gene COL5A2 and probably involve incorporation of aberrant collagen 1/V heterotypic fibrils, containing abnormal α2(V) chains, into the ECM.⁷ Interestingly, COL5A2 null alleles have yet to be detected in cEDS patients, leading to the suggestion that haploinsufficiency for the α2(V) chain may not lead to cEDS or, perhaps, to any clinically abnormal phenotype.⁷Previously, knockout of the α1(V) (Col5a1) gene produced a mouse model in which Col5a1^+/− adults exhibit a skin phenotype similar to that of cEDS.^9,10 Col5a1^+/− mice also have decreased aortic stiffness and tensile strength,¹⁰ presumably corresponding to the easy bleeding and somewhat increased prevalence of aortic root dilation, thought to result from increased aortic compliance, in cEDS patients.^11–13 The homozygous null Col5a1^−/− phenotype is embryonic lethal at approximately embryonic day 10, with a seeming absence of collagen fibril formation suggesting an early role for collagen V in a nucleation event necessary to collagen fibril formation.⁹ In another study, mice heterozygous for a small in-frame deletion in the N-telopeptide domain of the α2(V) chain were phenotypically normal, but homozygotes, which developed spinal abnormalities not characteristic of cEDS and most of which died before weaning, had skin with some features reminiscent of cEDS.¹⁴ A subsequent study on the same mice claimed the mutated allele to be functionally null.¹⁵We report the creation and characterization of mice with the first true null Col5a2 allele. Contrary to mice homozygous for the previously described Col5a2 mutant allele,¹⁴ Col5a2^−/− homozygous null mice are early embryonic lethal, consistent with the early embryonic lethality of the previously described Col5a1^−/− mice.⁹ Differences in length of embryonic survival and in collagen fibril density and morphology between the Col5a2^−/− embryos described here and the previously described Col5a1^−/− mice⁹ provide insights into the roles of different forms of collagen V in fibrillogenesis. Col5a2^+/− adults have changes to the extensibility and tensile strength of skin and aortae. Implications of the data for α2(V) and collagen V function and for the possible phenotype of humans heterozygous null for COL5A2 are discussed.     

The Microbiota Protects against Ischemia/Reperfusion-Induced Intestinal Injury through Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Signaling

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular pattern recognition receptor, induces autophagy on detection of muramyl dipeptide (MDP), a component of microbial cell walls. The role of bacteria and NOD2 signaling toward ischemia/reperfusion (I/R)–induced intestinal injury response is unknown. Herein, we report that I/R-induced intestinal injury in germ-free (GF) C57BL/6 wild-type (WT) mice is worse than in conventionally derived mice. More important, microbiota-mediated protection against I/R-induced intestinal injury is abrogated in conventionally derived Nod2^−/− mice and GF Nod2^−/− mice. Also, WT mice raised in specific pathogen-free (SPF) conditions fared better against I/R-induced injury than SPF Nod2^−/− mice. Moreover, SPF WT mice i.p. administered 10 mg/kg MDP were protected against injury compared with mice administered the inactive enantiomer, l-MDP, an effect lost in Nod2^−/− mice. However, MDP administration failed to protect GF mice from I/R-induced intestinal injury compared with control, a phenomenon correlating with undetectable Nod2 mRNA level in the epithelium of GF mice. More important, the autophagy-inducer rapamycin protected Nod2^−/− mice against I/R-induced injury and increased the levels of LC3⁺ puncta in injured tissue of Nod2^−/− mice. These findings demonstrate that NOD2 protects against I/R and promotes wound healing, likely through the induction of the autophagy response.The epithelium lining the intestinal track is composed of a single layer sheet of epithelial cells that provides nutrient absorption, hormone secretion, and innate immune sampling of luminal contents.^1,2 In addition, the epithelium provides a physical barrier between the host and gut microbes, where intestinal epithelial cells (IECs) are stitched together by tight junctions that maintain the architecture of the epithelial sheet and prevent uncontrolled access of luminal content (eg, microbes and dietary toxins) to subepithelial tissues.³ The epithelium is preserved by the homeostatic migration and proliferation of IECs from the base of the intestinal crypts to tips of the villi. Events that disrupt this equilibrium could have deleterious consequences for the host, as seen in patients experiencing intestinal ischemia.⁴Ischemia occurs when blood supply to the small bowel is occluded, which is followed by reperfusion, the return of blood flood flow, and simultaneous re-oxygenation of the tissue. During ischemia, an imbalance of metabolic demand and supply results in hypoxic response with activation of hypoxia-inducible factor-1 as well as cell death programs,⁵ autophagy,^6–8 and necrosis (organelle swelling and plasma membrane rupturing).^4,9 Paradoxically, the restoration of blood flow causes the release of inflammatory mediators, such as IL-6, tumor necrosis factor-α, and IL-1β, which exacerbate the injury.⁴ As a result, extra-intestinal organs, such as liver and the lung, may experience inflammatory activation and fatal multiorgan dysfunction syndrome. In the clinic, causes of intestinal ischemia/reperfusion (I/R)–induced injury include atherosclerosis, hypotension, blood clots, hernias, cardiac and mesenteric surgery, venous thrombosis, and necrotizing enterocolitis.Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a pattern-recognition receptor whose function is the intracellular reconnaissance of pathogen-associated molecular patterns. NOD2 is important for the recognition of muramyl dipeptide, MDP, a component of peptidoglycan that is present in the cell walls of Gram-positive and Gram-negative bacteria. Loss-of-function mutations of NOD2 have been associated with Crohn''s disease and, recently, NOD2-associated autoinflammatory disease.¹⁰ The mechanism by which NOD2 maintains intestinal homeostasis has yet to be clearly defined, although a current paradigm suggests an involvement of this innate sensor in controlling microbial composition,^11,12 likely through expression of antimicrobial peptides from Paneth cells.¹³ In addition, NOD2 is implicated in other important biological responses, such as inflammasome activation¹⁴ and autophagy.⁶ More important, in a preclinical model of necrotizing enterocolitis, NOD2 signaling was shown to protect against hypoxic stress through down-regulation of the Toll-like receptor (TLR) 4 pathway.¹⁵ However, the role of commensal bacteria and NOD2 signaling in intestinal I/R injury response has not been elucidated.A balance between innate inflammatory responses and cytoprotective mechanisms dictates the extent of end-organ damage during I/R injury. During injury-induced hypoxic stress, cells undergo a prosurvival process called autophagy.¹⁶ This autophagic response occurs on inhibition of mammalian target of rapamycin, thereby inducing the encapsulation of cytoplasmic components in a double membrane (autophagosome), which is delivered to the lysosome for degradation.¹⁶ In hepatic ischemia, autophagy has been shown to be a protective mechanism that favors cell survival and proliferation,⁸ two key processes in epithelial injury response. Interestingly, NOD2 recruits ATG16L1 to the plasma membrane to initiate autophagosome formation in response to MDP and at the site of Shigella flexneri entry.¹⁷ However, the role of commensal bacteria–induced autophagy in the context of hypoxic stress and intestinal damage is currently unknown.Herein, we investigated the role of microbes and NOD2 signaling in I/R-induced intestinal injury using germ-free (GF) and conventionally derived (CONV-D) Nod2^−/− mice. We demonstrate that microbes are important for optimal intestinal response to injury, an effect mediated by NOD2 signaling.