Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)–bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I–expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.Notch signaling is known to play critical roles in development, tissue homeostasis, and disease.^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} Notch signaling is mediated via four known receptors, Notch 1, 2, 3, and 4, which serve as receptors for five membrane-bound ligands, Jagged 1 and 2 and Delta 1, 3, and 4.^{1, 11, 12, 13} The Notch receptors differ primarily in the number of epidermal growth factor-like repeats and C-terminal sequences.¹³ For instance, Notch 1 contains 36 of epidermal growth factor-like repeats, is composed of approximately 40 amino acids, and is defined largely by six conserved cysteine residues that form three conserved disulfide bonds.^{1, 13, 14, 15} These epidermal growth factor-like repeats can be modified by O-linked glycans at specific sites, which is important for their function.^{1, 14, 15} Modulation of Notch signaling by Fringe proteins,^{16, 17, 18} which are N-acetylglucosamine transferases, illustrates the importance of these carbohydrate residues.^{16, 18} Moreover, mutation of the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase causes defective fucosylation of Notch1, resulting in impairment of the Notch1 signaling pathway and myofibroblast differentiation.^{19, 20, 21} Because myofibroblasts are important in both lung development and fibrosis, elucidation of the role of Notch signaling in their genesis in vivo will provide insight into the significance of this signaling pathway in either context.The importance of Notch signaling in tissue fibrosis is suggested in multiple studies.^{10, 21, 22, 23, 24} As in other organs or tissues, pulmonary fibrosis is characterized by fibroblast proliferation and de novo emergence of myofibroblasts, which is predominantly responsible for the increased extracellular matrix production and deposition.^{25, 26, 27, 28, 29, 30, 31} Animal models, such as bleomycin-induced pulmonary fibrosis, are characterized by both acute and chronic inflammation with subsequent myofibroblast differentiation that mainly originated from the mesenchymal compartment.^{21, 25, 26, 27, 28} In vitro studies of cultured cells implicate Notch signaling in myofibroblast differentiation,²¹ which is mediated by induction of the Notch1 ligand Jagged1 when lung fibroblasts are treated with found in inflammatory zone 1.²¹ Moreover, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase knockout mice with defective fucosylation of Notch1 exhibit consequent impairment of Notch signaling and attenuated pulmonary fibrosis in studies using the bleomycin model.²¹ The in vivo importance of Notch signaling in myofibroblast differentiation during lung development has also been suggested by demonstration of impaired alveogenesis in mice deficient in lunatic fringe³² or Notch receptors.^{10, 33, 34, 35} These in vivo studies, however, do not pinpoint the cell type in which deficient Notch signaling is causing the observed impairment of myofibroblast differentiation. This is further complicated by the extensive evidence showing that, in addition to myofibroblast differentiation, Notch1 mediates multiple functional responses in diverse cell types, including inflammation and the immune system.^{21, 36, 37, 38} In the case of tissue injury and fibrosis, including the bleomycin model, the associated inflammation and immune response as well as parenchymal injury can affect myofibroblast differentiation via paracrine mechanisms.^{39, 40} Thus, although global impairment of Notch signaling can impair myofibroblast differentiation in vivo, it does not necessarily indicate a specific direct effect on the mesenchymal precursor cell. Furthermore, understanding the importance of Notch signaling in these different cell compartments is critical for future translational studies to develop effective drugs targeting this signaling pathway with minimal off-target or negative adverse effects.In this study, the effects of conditional selective Notch1 deficiency in the mesenchymal compartment on myofibroblast differentiation and bleomycin-induced pulmonary fibrosis were examined using a Cre-Lox strategy. The transgenic Cre mice bore the Cre-ER(T) gene composed of Cre recombinase and a ligand-binding domain of the estrogen receptor⁴¹ driven by a minimal promoter containing a far-upstream enhancer from the α2(I) collagen gene. When activated by tamoxifen, this enhancer enabled selective Cre expression only in type I collagen-expressing (mesenchymal) cells, such as fibroblasts and other mesenchymal cells,⁴² leading to excision of LoxP consensus sequence flanked target gene DNA fragment (floxed gene) of interest.^{41, 43, 44, 45, 46} To evaluate the importance of Notch1 in the mesenchymal compartment and discriminate its effects from those in the inflammatory and immune system and other compartments, the transgenic Cre-ER(T) mice [Col1α2-Cre-ER(T)^+/0] were crossed with mice harboring the floxed (containing loxP sites) Notch1 gene (Notch1^fl/fl). The resulting progeny mice [Notch1 conditional knockout (CKO)] that were homozygous for the floxed Notch1 allele and hemizygous for the Col1α2-Cre-ER(T) allele with genotype [Notch1^fl/fl,Col1α2-Cre-ER(T)^+/0] were Notch1 deficient in the mesenchymal compartment when injected with tamoxifen. Control Notch1 wild-type (WT) mice exhibited the expected pulmonary fibrosis along with induction of Jagged1 and Notch1 on treatment with bleomycin, consistent with previous observation of Notch signaling activation in this model.²¹ Isolated and cultured Notch1 CKO mouse lung fibroblasts were deficient in Notch1 and exhibited diminished myofibroblast differentiation compared with cells from the corresponding WT control mice. Most important, compared with WT control mice, the CKO mice exhibited diminished bleomycin-induced pulmonary fibrosis that was accompanied by significant reduction in α-smooth muscle actin (α-SMA) and type I collagen gene expression, consistent with defective myofibroblast differentiation. In contrast, enumeration of lung inflammatory and immune cells failed to show a significant difference in bleomycin-induced recruitment of these cells between control and CKO mice. Thus, selective Notch1 deficiency in mesenchymal cells caused impairment of fibrosis that is at least, in part, because of deficient myofibroblast differentiation, and without affecting the inflammatory and immune response in this animal model.     

Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.^{1, 2, 3} When inflammation is sustained, capillaries acquire venule-like properties that expand the sites of plasma leakage and leukocyte influx. Consistent with this transformation, the remodeled blood vessels express P-selectin, intercellular adhesion molecule 1 (ICAM-1), EphB4, and other venular markers.^{4, 5, 6} The changes are accompanied by remodeling of pericytes and disruption of pericyte-endothelial crosstalk involved in blood vessel quiescence.⁷ Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.^{6, 8, 9}Mycoplasma pulmonis infection causes sustained inflammation of the respiratory tract of rodents.¹⁰ This infection has proved useful for dissecting the features and mechanisms of vascular remodeling and lymphangiogenesis.^{6, 9, 10} At 7 days after infection, there is widespread conversion of capillaries into venules, pericyte remodeling, inflammatory cell influx, and lymphatic vessel sprouting in the airways and lung.^{4, 5, 6, 7, 8, 9} Many features of chronic M. pulmonis infection in mice are similar to Mycoplasma pneumoniae infection in humans.¹¹Angiopoietin-2 (Ang2) is a context-dependent antagonist of Tie2 receptors^{12, 13} that is important for prenatal and postnatal remodeling of blood vessels and lymphatic vessels.^{13, 14, 15} Ang2 promotes vascular remodeling,^{4, 5} lymphangiogenesis,^{15, 16, 17} and pericyte loss¹⁸ in disease models in mice. Mice genetically lacking Ang2 have less angiogenesis, lymphangiogenesis, and neutrophil recruitment in inflammatory bowel disease.³ Ang2 has proved useful as a plasma biomarker of endothelial cell activation in acute lung injury, sepsis, hypoxia, and cancer.¹⁹Like Ang2, tumor necrosis factor (TNF)-α is a mediator of remodeling of blood vessels and lymphatics.^{8, 9, 20, 21} TNF triggers many components of the inflammatory response, including up-regulation of expression of vascular cell adhesion molecule-1, ICAM-1, and other endothelial cell adhesion molecules.²² TNF inhibitors reduce inflammation in mouse models of inflammatory disease^{23, 24} and are used clinically in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn''s disease, psoriatic arthritis, and some other inflammatory conditions.^{24, 25} Indicative of the complex role of TNF in disease, inhibition or deletion of TNF can increase the risk of serious infection by bacterial, mycobacterial, fungal, viral, and other opportunistic pathogens.²⁶TNF and Ang2 interact in inflammatory responses. TNF increases Ang2 expression in endothelial cells in a time- and dose-dependent manner, both in blood vessels²⁷ and lymphatics.¹⁶ Administration of TNF with Ang2 increases cell adhesion molecule expression more than TNF alone.^{16, 28} Similarly, Ang2 can promote corneal angiogenesis in the presence of TNF, but not alone.²⁹ In mice that lack Ang2, TNF induces leukocyte rolling but not adherence to the endothelium.²⁸ Ang2 also augments TNF production by macrophages.^{30, 31} Inhibition of Ang2 and TNF together with a bispecific antibody can ameliorate rheumatoid arthritis in a mouse model.³²With this background, we sought to determine whether Ang2 and TNF act together to drive the remodeling of blood vessels and lymphatics in the initial inflammatory response to M. pulmonis infection. In particular, we asked whether Ang2 and TNF have synergistic actions in this setting. The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)^{33, 34} and the Database for Annotation, Visualization and Integrated Discovery (DAVID).³⁵ The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response.     

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.     

Conditional Knockout of Pik3c3 Causes a Murine Muscular Dystrophy

Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.Phosphatidylinositols (PIs) comprise a group of low-abundance lipids with hydroxylated inositol head groups that are capable of receiving phosphates at any of the three outer positions. Dynamic phosphorylation of the inositol ring influences many cellular and metabolic processes, including endocytosis, endosomal trafficking, and autophagy.^1–5 The quantity and localization of different PIs are regulated by a group of phosphoinositide kinases and phosphatases that function as key regulatory enzymes and that have been implicated in a number of human diseases, including especially oncologic and neurodegenerative diseases.^6–9Myotubular myopathy (MTM) is a severe childhood-onset disease of skeletal muscle caused by mutations in the phosphoinositide phosphatase myotubularin gene (MTM1).¹⁰ MTM is characterized by profound weakness and hypotonia at birth, persistent life-long disabilities including wheelchair and ventilator dependence, and early mortality.¹¹ As predicted by the fact that MTM1 has been shown in vitro to be responsible for dephosphorylation of PI(3)P, levels of PI(3)P are significantly elevated in animal models of MTM.^12–14 Despite growing knowledge of disease pathogenesis, there are currently no available treatments for MTM. One significant barrier toward therapy development for MTM is the fact that the normal function(s) of PI(3)P in skeletal muscle are unknown.PI(3)P is created through phosphorylation of PI at the D3 position by PI3 kinases,^15,16 or through dephosphorylation of PI(3,5)P₂ by the phosphatase FIG4.¹⁷ There are three classes of PI3 kinases that produce PI(3)P in mammals, with varying tissue expression and substrate specificity.^18,19 In skeletal muscle, the primary sources of PI(3)P are hypothesized (based on gene expression) to be the class III kinase, Pik3c3 (hVPS34), and the class II kinase Pik3c2β, with PIK3C3 considered the major enzymatic regulator of its production.^15,16,20 Previous studies of PIK3C3 have identified it as a regulator of several intracellular processes, including endosome-to-Golgi membrane traffic,⁷ endocytosis,^21,22 mTOR-S6K1 signaling,^23,24 and autophagy. Perhaps its best-studied function is in autophagy, where PIK3C3 and its regulatory subunit PIK3R4 (Vps15) form multiple complexes with other autophagy gene products to regulate several steps of autophagosome formation and maturation.^25–28The goal of the present study was to begin understanding the role of PI(3)P in skeletal muscle by evaluating the function of PIK3C3. As previously reported, whole-animal gene knockout of Pik3c3 in the mouse results in early embryonic lethality.^29,30 Therefore, to study PIK3C3 specifically in muscle, we have used the Cre-lox system. Cre-lox–mediated knockout of Pik3c3 has been performed in kidney,³¹ liver, and heart³²; sensory, cortical, and hippocampal neurons^30,33; and T cells,³⁴ but Pik3c3 has yet to be examined in skeletal muscle. We generated mice with conditional knockout of Pik3c3 in skeletal muscle by combining floxed Pik3c3³⁰ and Cre recombinase under the muscle creatine kinase promoter: Tg(Ckmm-Cre).³⁵ The resulting mice had normal embryonic and early postnatal development, but died by 2 months of age, presumably from severe cardiomyopathy.³² Somewhat surprisingly, examination of skeletal muscle in knockout animals revealed a murine muscular dystrophy. We present the comprehensive characterization of this dystrophic phenotype. In total, we report for the first time a requirement for Pik3c3 in skeletal muscle homeostasis, and further identify loss of Pik3c3 as a cause of muscular dystrophy in the mouse.     

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.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.     

A Murine Rp1 Missense Mutation Causes Protein Mislocalization and Slowly Progressive Photoreceptor Degeneration

Mutations in the RP1 gene can cause retinitis pigmentosa. We identified a spontaneous L66P mutation caused by two adjacent point mutations in the Rp1 gene in a colony of C57BL/6J mice. Mice homozygous for the L66P mutation exhibited slow, progressive photoreceptor degeneration throughout their lifespan. Optical coherence tomography imaging found abnormal photoreceptor reflectivity at 1 month of age. Histology found shortening and disorganization of the photoreceptor inner and outer segments and progressive thinning of the outer nuclear layer. Electroretinogram a- and b-wave amplitudes were decreased with age. Western blot analysis found that the quantity and size of the mutated retinitis pigmentosa 1 (RP1) protein were normal. However, immunohistochemistry found that the mutant Rp1 protein partially mislocalized to the transition zone of the shortened axonemes. This mutation disrupted colocalization with cytoplasmic microtubules in vitro. In conclusion, the L66P mutation in the first doublecortin domain of the Rp1 gene impairs Rp1 protein localization and function, leading to abnormalities in photoreceptor outer segment structure and progressive photoreceptor degeneration. This is the first missense mutation in Rp1 shown to cause retinal degeneration. It provides a unique, slowly progressive photoreceptor degeneration model that mirrors the slow degeneration kinetics in most patients with retinitis pigmentosa.As the most common inherited form of blindness, retinitis pigmentosa (RP) affects >100,000 persons in the United States and 1.5 million worldwide.¹ Clinically, it is characterized by night blindness, progressive loss of peripheral vision, and bone spicule-shaped pigmentary retinopathy. Mutations in the RP1 gene are a common cause of autosomal dominant RP and a less common cause of autosomal recessive RP.^2–5 Most pathological mutations in RP1 are either nonsense or frameshift mutations and are located at the beginning of exon 4 of the RP1 gene. A few missense variants in RP1, such as T373I,⁶ A669T,⁶ K663N,⁷ L1808P,⁷ D984G,⁸ R1652L,⁹ and K1370E,⁹ have been reported in patients with RP, but it is unclear whether they are disease-causing mutations.The RP1 gene located on chromosome 8q12 consists of four exons with an open reading frame of 6468 bp, which is primarily contained within exon 4 (788 to 6468 bp), and it encodes a predicted protein of 2156 amino acids. It has been determined by Northern blot analysis^3,10,11 and in situ hybridization³ that RP1 is expressed exclusively in rod and cone photoreceptor cells of the retina. In mice, retinitis pigmentosa 1 (Rp1) is localized to the axoneme of both rod and cone photoreceptors.¹² Targeted disruption of the Rp1 gene in mice results in disorganization of outer segments (OS) with progressive degeneration of photoreceptors.¹³ Further studies reported that Rp1 plays a role in controlling the orientation and organization of disks in the OS.¹⁴ The photoreceptor axoneme begins at the basal body in the inner segment (IS), passes through the transition zone between the IS and OS, and continues into the OS.¹⁵ It was suggested that the axoneme has a role in stabilizing the stack of disk membranes.¹⁵The N-terminus of RP1 shares significant homology with the protein doublecortin (DCX), whose mutation is associated with cerebral cortical abnormalities.^16,17 This domain binds microtubules and promotes their assembly.¹⁸ Our data show that the homozygous L66P substitution in the first DCX domain of the Rp1 gene alters axoneme binding and causes axoneme shortening, disorganization of OS, and slowly progressive photoreceptor death.     

Extracellular Generation of Adenosine by the Ectonucleotidases CD39 and CD73 Promotes Dermal Fibrosis

Adenosine has an important role in inflammation and tissue remodeling and promotes dermal fibrosis by adenosine receptor (A_2AR) activation. Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential phosphohydrolysis of released ATP by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73). Because the role of these ecto-enzymes in fibrosis appears to be tissue specific, we determined whether these ectonucleotidases were directly involved in diffuse dermal fibrosis. Wild-type and mice globally deficient in CD39 knockout (CD39KO), CD73 (CD73KO), or both (CD39/CD73DKO) were challenged with bleomycin. Extracellular adenosine levels and dermal fibrosis were quantitated. Adenosine release from skin cultured ex vivo was increased in wild-type mice after bleomycin treatment but remained low in skin from CD39KO, CD73KO, or CD39/CD73DKO bleomycin-treated mice. Deletion of CD39 and/or CD73 decreased the collagen content, and prevented skin thickening and tensile strength increase after bleomycin challenge. Decreased dermal fibrotic features were associated with reduced expression of the profibrotic mediators, transforming growth factor-β1 and connective tissue growth factor, and diminished myofibroblast population in CD39- and/or CD73-deficient mice. Our work supports the hypothesis that extracellular adenosine, generated in tandem by ecto-enzymes CD39 and CD73, promotes dermal fibrogenesis. We suggest that biochemical or biological inhibitors of CD39 and/or CD73 may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.Tissue damage leads to the release of the signaling nucleoside adenosine, which, by engaging specific adenosine receptors (A₁R, A_2AR, A_2BR, and A₃R), exhibits both tissue-protective and tissue-destructive effects.^{1, 2, 3, 4} In particular, adenosine is a potent regulator of tissue repair, and we have previously reported that adenosine promotes dermal fibrosis via the A_2AR receptor, as shown in vitro,⁵ in a bleomycin-induced dermal injury model of scleroderma,⁶ and in a model of elevated tissue adenosine.⁷ Similarly, we found that pharmacological blockade of A_2AR diminishes skin scarring.⁸Elevations in extracellular adenosine can result from either an increase in intracellular adenosine, followed by release into the extracellular space, or the release of adenine nucleotides, followed by their extracellular catabolism into adenosine.⁹ The main source of extracellular adenosine stems from the enzymatic phosphohydrolysis of precursor nucleotides to adenosine.^{10, 11, 12, 13} This is achieved by a two-step enzymatic process involving the ecto-apyrase, CD39 (conversion of ATP/ADP to AMP) and the ecto-5′-nucleotidase, CD73 (conversion of AMP to adenosine).¹⁴ It is widely accepted that CD39 and CD73 promote anti-inflammatory effects of adenosine in the immune system,^{15, 16, 17} and both enzymes have been previously shown to attenuate acute injury and inflammation in models of ambient hypoxia,^{18, 19} cyclic mechanical stretch,²⁰ and bleomycin-induced lung injury.² However, CD39 and CD73 promote fibrosis in murine models of pancreatitis²¹ and hepatic fibrosis,²² respectively, suggesting an important role for CD39 and CD73 in the regulation of fibrogenesis in vivo.We hypothesized that limiting extracellular adenosine levels by CD39 and/or CD73 gene deletion may protect against bleomycin-induced dermal fibrosis, a model of scleroderma. CD39-deficient, CD73-deficient, and CD39/73 double-deficient mice were subjected to bleomycin-induced skin injury, and the extent of skin fibrosis was compared with the wild-type (WT) mice. Our results show that, after bleomycin injection, mice globally null for CD39 and/or CD79 released lower levels of adenosine and concurrently developed less dermal fibrosis, indicating that adenosine generation by CD39 and CD73 is highly likely to be a critical regulator of fibrogenesis in skin.     

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.     

Regulator of calcineurin 1 (Rcan1) is required for the development of pulmonary eosinophilia in allergic inflammation in mice

The presence of eosinophils in the lung is often regarded as a defining feature of asthma. On allergen stimulation, numbers of eosinophils and their progenitors are increased in both the bone marrow and lungs. Eosinophil progenitors provide an ongoing supply of mature eosinophils. Here, we report that deficiency in the regulator of calcineurin 1 gene (Rcan1) leads to a near-complete absence of eosinophilia in ovalbumin-induced allergic asthma in mice. In the absence of Rcan1, bone marrow cells produce significantly fewer eosinophils in vivo and in vitro on interleukin-5 stimulation. Importantly, eosinophil progenitor populations are significantly reduced in both naïve and ovalbumin-challenged Rcan1^−/− mice. Bone marrow cells from Rcan1^−/− mice are capable of developing into fully mature eosinophils, suggesting that Rcan1 is required for eosinophil progenitor production but may not be necessary for eosinophil maturation. Thus, Rcan1 represents a novel contributor in the development of eosinophilia in allergic asthma through regulation of eosinophil progenitor production.A nationwide survey found that more than half (54.6%) of the U.S. population test positive to one or more allergens.¹ Allergic asthma is a chronic inflammatory disease that is characterized by eosinophil infiltration. Eosinophils are prominent effector cells in allergic asthma.^2–4 Several studies have established a causative link between eosinophils and allergic lung diseases.^5–8 Targeting eosinophils using anti-IL-5 antibodies has been considered as a therapeutic approach for the treatment of asthma. In steady state, eosinophil progenitors constantly egress from the bone marrow into the blood and circulate to peripheral tissues. In allergic diseases, the bone marrow releases increased numbers of eosinophil progenitor cells that migrate to the site of allergic inflammation, where they provide a constant supply of mature eosinophils.^9–13 Molecular mechanisms governing eosinophil progenitor production remain incompletely defined.Down syndrome patients who overexpress regulator of calcineurin 1 (Rcan1) have an association with immune disorders including allergy and asthma.^14–16 Hypereosinophilic syndrome was reported in a fetus harboring trisomy 21.¹⁷ Eosinophilic pericardial effusion was also documented in a neonate with Down syndrome.¹⁸ The proform of eosinophil major basic protein has been identified as a maternal serum marker for Down syndrome.¹⁹ Increased incidence of allergic asthma in patients with Down syndrome has been reported in a recent large survey study.¹⁵ These findings suggest an association of Rcan1 with eosinophils and asthma.The human RCAN1 gene was previously known as DSCR1 (Down syndrome critical region 1).²⁰ Additional names for Rcan1 include Adapt78 (the gene is transiently induced during cell adaptation),²¹ myocyte-enriched calcineurin interacting protein 1 (MCIP1),²² calcipressin 1,²³ and calcineurin binding protein 1 (CBP1).²⁴ The human RCAN1 gene is located on chromosome 21. Rcan1 is widely expressed in various tissues, including heart, lung, kidney, brain, muscle, liver, and testis.^22,25,26 The RCAN1 gene consists of seven exons, of which exons 1 to 4 can be alternatively transcribed.²⁷ Deletion of exons 5 and 6 from the mouse Rcan1 gene leads to deficiency of the Rcan1 protein.²⁸ These Rcan1^−/− mice are viable and fertile and provide a useful tool for the study of Rcan1 function.²⁸Published reports on Rcan1 function are concerned largely with calcineurin activity. Experiments in different organisms and cell types have showed a dual function for Rcan1, which can act as either an inhibitor^29,30 or a facilitator^{24,28,31–33} of calcineurin activity, depending on the cellular context. In yeast and in Cryptococcus neoformans, Rcan1 has the characteristics of a calcineurin activator,^24,32,33 and in a cardiac hypertrophic mouse model Rcan1 functions as a calcineurin facilitator.^28,31 In contrast, in other experimental models, Rcan1 inhibits calcineurin activity in vitro and in vivo.^23,29,30 Recently, we found that Rcan1 is an inhibitor of calcineurin in mast cells.³⁴Our finding that Rcan1 is a negative regulator of IgE-mediated mast cell activation³⁴ prompted us to determine what role Rcan1 has in allergic asthma. Surprisingly, we found that Rcan1 deficiency leads to near-complete absence of eosinophilia in ovalbumin-induced asthma in mice. The number of eosinophil progenitors was significantly reduced in Rcan1^−/− mice, and calcineurin activity was reduced in Rcan1^−/− eosinophil progenitors. Thus, Rcan1 represents a novel mechanism in the development of eosinophilia in allergic asthma, likely by regulating eosinophil progenitor cell numbers.     

CUL4high Lung Adenocarcinomas Are Dependent on the CUL4-p21 Ubiquitin Signaling for Proliferation and Survival

Cullin (CUL) 4A and 4B ubiquitin ligases are often highly accumulated in human malignant neoplasms and are believed to possess oncogenic properties. However, the underlying mechanisms by which CUL4A and CUL4B promote pulmonary tumorigenesis remain largely elusive. This study reports that CUL4A and CUL4B are highly expressed in patients with non–small cell lung cancer (NSCLC), and their high expression is associated with disease progression, chemotherapy resistance, and poor survival in adenocarcinomas. Depletion of CUL4A (CUL4A^k/d) or CUL4B (CUL4B^k/d) leads to cell cycle arrest at G₁ and loss of proliferation and viability of NSCLC cells in culture and in a lung cancer xenograft model, suggesting that CUL4A and 4B are oncoproteins required for tumor maintenance of certain NSCLCs. Mechanistically, increased accumulation of the cell cycle–dependent kinase inhibitor p21/Cip1/WAF1 was observed in lung cancer cells on CUL4 silencing. Knockdown of p21 rescued the G₁ arrest of CUL4A^k/d or CUL4B^k/d NSCLC cells, and allowed proliferation to resume. These findings reveal that p21 is the primary downstream effector of lung adenocarcinoma dependence on CUL4, highlight the notion that not all substrates respond equally to abrogation of the CUL4 ubiquitin ligase in NSCLCs, and imply that CUL4A^high/CUL4B^high may serve as a prognostic marker and therapeutic target for patients with NSCLC.

Lung cancer is the most common cause of cancer mortality worldwide,¹ accounting for 19.4% of all cancer-related deaths and representing a significant clinical burden.² Among the subtypes of lung cancer, non–small cell lung cancer (NSCLC) accounts for 80% to 85% of cases.3, 4, 5 Although multimodality treatments, including targeted therapies and immunotherapies, have been applied to NSCLCs, with high rates of local and distant failure, the overall cure and survival rates for NSCLC remain low.^6,7 Thus, understanding the molecular mechanisms underlying NSCLC development and progression is of fundamental importance for the development of new therapeutic strategies for patients with NSCLC.Cullin (CUL) 4, a molecular scaffold of the CUL4-RING ubiquitin ligase (CRL4), plays an important role in regulating key cellular processes through modulating the ubiquitylation and degradation of various protein substrates.⁸ Two CUL4 proteins, CUL4A and CUL4B, share an 82% sequence homology, with similar but distinct functions.⁹ CUL4 has been extensively studied in the process of nucleotide excision repair (NER) after UV irradiation.10, 11, 12, 13 Loss of CUL4A, but not CUL4B, elevates global genomic NER activity and confers increased protection against UV-induced skin carcinogenesis.¹¹ In addition to DNA repair, CUL4 also plays a significant role in a wide spectrum of physiologic processes, such as the cell cycle, cell signaling, and histone methylation, which have direct relevance to the development of human cancers.14, 15, 16 Accumulating studies have found that CUL4A is amplified or expressed at abnormally high levels in multiple cancers, including breast cancer, squamous cell carcinoma, hepatocellular carcinomas, and lung cancer.^9,17, 18, 19 More importantly, CUL4A and 4B overexpression is implicated in tumor progression, metastasis, and a poorer survival rate for patients with cancer.^9,20,21 CUL4A, but not CUL4B, is inversely correlated with the NER protein xeroderma pigmentosum, complementation group C and the G₁/S DNA damage checkpoint protein p21 in patients with lung squamous cell carcinoma, highlighting a reduced DNA damage response⁹ as well as promoting cell growth and tumorigenesis.^22,23 Increased expression of CUL4A caused hyperplasia as well as lung adenocarcinomas in mice.²⁴ However, the mechanistic basis and clinical significance of CUL4A dysregulation in NSCLC remain unclear.The CUL4A paralog CUL4B shares extensive sequence homology and redundant functions with CUL4A.⁹ To date, research on CUL4B has been focused mainly on its genetic association with human X-linked mental retardation.25, 26, 27, 28 Recently, CUL4B was found to be overexpressed in colon cancer and correlated with tumor stage, histologic differentiation, vascular invasion, and distant metastasis.²⁹ Patients with lung and colon cancer with high levels of CUL4B had lower overall survival (OS) and disease-free survival (DFS) rates than those with low CUL4B expression.^9,29 CUL4B is also overexpressed in cervical, esophageal, and breast cancers and associated with tumor invasion and lymph node metastasis.^16,30,31 Furthermore, CUL4B overexpression promotes the development of spontaneous liver tumors at a high rate and enhances diethylnitrosamine-induced hepatocarcinogenesis in transgenic mice.³²The molecular mechanisms underlying the capacity of CUL4 to promote pulmonary tumorigenesis remain largely elusive. CUL4A promotes NSCLC cell growth.²² CUL4 targets a panel of cell cycle regulators for ubiquitination and degradation, including Cdc6, Cdt1, p21, cyclin E, minichromosome maintenance 10 replication initiation factor, and forkhead box M1.³³ However, which of the cell cycle substrates of CUL4 play a key role in tumor dependence on dysregulated CUL4A or CUL4B remains to be defined. This study found that attenuation of CUL4, especially CUL4B, inhibited NSCLC cell proliferation and tumorigenesis through increased accumulation of p21 and cell cycle arrest in G₁.     

CXCL9 Is Important for Recruiting Immune T?Cells into the Brain and Inducing an Accumulation of the T Cells to the Areas of Tachyzoite Proliferation to Prevent Reactivation of Chronic Cerebral Infection with Toxoplasma gondii

T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non–glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4⁺ and CD8⁺ T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3⁺ T cells into the areas of tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.Toxoplasma gondii is an obligate intracellular parasite in humans and animals. Interferon (IFN)-γ–mediated immune responses^{1, 2} and, to a lesser degree, humoral immunity^{3, 4, 5} control the tachyzoite growth during the acute stage of infection, but the parasite establishes a chronic infection by forming cysts preferentially in the brain. Chronic infection with T. gondii is ubiquitous in humans, and 500 million to 2 billion people worldwide are estimated to be chronically infected with this parasite.^{6, 7} This chronic infection can be reactivated and develop life-threatening toxoplasmic encephalitis (TE) in immunocompromised persons such as those with AIDS, neoplastic diseases, and organ transplants.^{8, 9} This fact clearly indicates an importance of the protective immunity to maintain the latency of chronic infection with T. gondii in the brain. However, the mechanisms by which the immune responses prevent reactivation of the chronic infection are not well understood.Although T. gondii has three major genotypes (types I, II, and III), type II is predominant in the strains isolated from patients with TE in North America and Europe.^{10, 11, 12} Therefore, for investigating the mechanisms by which the immune system maintains the latency of chronic T. gondii infection and prevents TE, animals that establish a latent, chronic infection with type II parasite in their brains provide an excellent model. BALB/c mice are one of those animals.^{13, 14} IFN-γ is essential to maintain the latency of chronic cerebral infection with T. gondii.^{15, 16} This cytokine can activate microglia^{17, 18} and astrocytes^{19, 20, 21} to prevent tachyzoite proliferation. In addition, IFN-γ plays an important role in regulating recruitment of immune T cells into the brain of BALB/c mice during both the acute and chronic stages of infection.^{22, 23} Induction of vascular cell adhesion molecule 1 (VCAM-1) expression on the cerebral vessels during the chronic infection is largely mediated by IFN-γ,²² and the binding of α4β1 integrin expressed on the surface of T cells to VCAM-1 expressed on the cerebrovascular endothelial cells is important for inducing prompt recruitment of immune T cells into their brains during the early stage of reactivation of chronic T. gondii infection to prevent TE.²⁴Chemokines, in addition to adhesion molecules, are crucial for T-cell entry into various organs.^{25, 26} In BALB/c mice chronically infected with T. gondii, CXCL9, CXCL10, and CCL5 are the chemokines predominantly expressed in their brains.^{27, 28} In the present study, we examined the role of IFN-γ in cerebral expression of these three chemokines during reactivation of the chronic infection in BALB/c-background immunodeficient mice, and found that the CXCL9 expression requires IFN-γ. On the basis of this observation, we examined the role of CXCL9 in recruiting immune T cells into the brain for maintaining the latency of chronic infection with T. gondii with the use of a model of reactivation of the infection in severe combined immunodeficient (SCID) mice with adoptive transfer of immune T cells from infected wild-type animals. By applying anti-CXCL9 antiserum in this animal model, the present study revealed that CXCL9 is crucial for recruiting immune T cells into the brain and for inducing an accumulation of the T cells around the areas associated with tachyzoites to prevent reactivation of cerebral infection with T. gondii.     

Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.Macrophages are versatile cells that have been long recognized as immune effectors where their recruitment to sites of injury is a fundamental feature of inflammation. Although their role in host defense has been well documented, macrophages and their precursors are also important during embryogenesis, normal tissue maintenance, and postnatal organ repair.^1,2 Almost all developing organs contain a population of resident monocytes that infiltrate very early during organogenesis and persist throughout adult life.^3–6 In addition to their phagocytic capabilities during tissue remodeling-associated apoptosis,^5,7 fetal macrophages have many trophic effects that promote tissue and organ growth.^6,8,9Colony-stimulating factor (CSF)-1 controls the differentiation, proliferation, and survival of macrophages by binding to a high-affinity cell-surface tyrosine kinase receptor (CSF-1R), encoded by the c-fms proto-oncogene that is expressed on macrophages and their progenitors.⁶ CSF-1 is critical for both adult and embryonic macrophage development. This is manifested by multiple organ growth deficiencies observed in osteopetrotic (Csf1^op/Csf1^op) mice that have a spontaneous mutation in the csf-1 gene. These mice show growth restriction and developmental abnormalities of the bones, brain, and reproductive and endocrine organs,^10–13 a phenotype that can be rescued by injection of exogenous CSF-1 or insertion of a csf-1 transgene.^14–16In adult organs, there is considerable heterogeneity of monocytes and macrophages with distinct subsets defined by phenotype, function, and the differential expression of cell surface markers.^17–19 Subpopulations of macrophages directly contribute to wound healing and tissue repair, supporting the concept that some macrophage phenotypes can promote organ regeneration after a pro-inflammatory state of injury.²⁰ The concept of macrophage polarization states has emerged; the M1 “classically activated” pro-inflammatory cell type apparently opposed by an M2 “alternatively activated” immune regulatory macrophage.¹⁸ In general, these two states are thought to be analogous to the opposing T helper 1 and T helper 2 immune responses, although in both cases this model is probably too simplistic. Functionally, it is more likely that distinct subpopulations of macrophages may exist in the same tissue and play critical roles in both the injury and recovery phases of inflammatory scarring.²⁰Our previous study provided evidence that the addition of CSF-1 to a developing murine kidney promotes a growth and differentiation response that is accompanied by increased numbers of macrophages.³ Furthermore, with the use of expression profiling we demonstrated that fetal kidney, lung, and brain macrophages share a characteristic gene expression profile that includes the production of factors important in the suppression of inflammation and the promotion of proliferation.³ Embryonic macrophages appear to play a positive trophic role that may have parallel reparative functions in many adult tissues undergoing repair and cellular replacement.^1,20 A number of studies have suggested that infiltrating macrophages along with the trophic factors they release participate in tissue repair of the kidney,^20–22 brain,²³ skin,^24,25 lung,²⁶ liver,²⁷ heart,²⁸ gastrointestinal tract,^29,30 and skeletal muscle.^31,32 Indeed, the pleiotrophic roles for CSF-1 in reproduction, development of multiple organ systems, and maternal-fetal interactions during pregnancy by macrophage-mediated processes have also been well defined.^2,33,34To determine the physiological relevance of CSF-1 as a component of the mammalian growth regulatory axis, CSF-1 was administered to neonatal mice. We report that CSF-1 administration to newborn mice increased body weight and kidney weight and volume and was associated with increased numbers of macrophages. Our results also establish that CSF-1 injection into mice after ischemia-reperfusion (IR) injury promoted endogenous repair with characteristic rapid re-epithelialization of the damaged tubular epithelium, leading to functional recovery. Flow cytometric and gene expression analyses were used to delineate the macrophage profile present in the kidneys during the early and resolution phase of IR injury with and without CSF-1 therapy. We thus provide evidence that CSF-1 recruits macrophages to the reparative site and influences their phenotype, partly through an insulin-like growth factor (IGF)-1 signaling response. Therefore, macrophages under the stimulus of CSF-1 in an acute setting of renal disease markedly accelerate renal cell replacement and tissue remodeling while attenuating downstream interstitial extracellular matrix accumulation.     

Differential Effects of Myocilin and Optineurin,Two Glaucoma Genes,on Neurite Outgrowth

Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC_WT-EGFP, pMYOC_P370L-EGFP, pMYOC_1-367-EGFP, pOPTN_WT-EGFP, and pOPTN_E50K-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTN_WT-EGFP and pOPTN_E50K-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.Glaucoma, one of the leading causes of irreversible blindness worldwide, is characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. Primary open-angle glaucoma (POAG), the most common form of glaucoma, is frequently associated with increased intraocular pressure (IOP). The IOP is controlled by a balance between the production and outflow of the aqueous humor contained in the anterior chamber. The trabecular meshwork (TM), a specialized eye tissue neighboring the cornea, is the major site for regulation of the aqueous humor outflow.¹Recent studies have revealed that POAG is genetically heterogeneous, caused by several susceptibility genes and environmental factors.² To date, a total of 14 chromosomal loci have been mapped and designated as GLC1A to GLC1N.^3,4 Three candidate genes have been identified that include myocilin as the GLC1A,^5,6 optineurin as the GLC1E,^7,8 and WDR36 as the GLC1G⁹ gene.Myocilin is the first identified gene for both juvenile- and adult-onset POAG.⁶ More than 70 myocilin mutations have been found in a number of families.¹⁰ Patients with glaucoma with myocilin mutation tend to have high IOP.^11,12 Among the myocilin mutations, the Gln368Stop (Q368X) mutation is the most frequent¹⁰ and the Pro370Leu (P370L) mutation is responsible for one of the most severe glaucoma phenotypes.^13–15The human myocilin gene encodes a protein of 504 amino acids, containing a nonmuscle myosin-like domain near the amino (N)-terminus and an olfactomedin-like domain at the carboxyl (C)-terminus.¹⁶ Myocilin interacts with itself and a number of other proteins, mainly through the leucine zipper motif and a coiled-coil region in the myosin-like domain.^17–19 The wild-type myocilin is a secreted protein. Mutants with mutations in the olfatomedin-like domain, however, are not secreted. They are retained in the cells, aggregating to cause endoplasmic reticulum stress.^20–22Optineurin, identified in 2002,⁸ is a gene that links principally to normal tension glaucoma, a subtype of POAG.²³ Optineurin mutations were noted to vary with ethnic background.^24,25 The Glu50Lys (E50K) mutation, found in Caucasian and Hispanic populations,²⁵ seems to be associated with a more progressive and severe disease in patients with normal tension glaucoma.²⁶The human optineurin gene codes for a 577-amino acid protein that contains multiple coiled-coil domains and a C-terminal zinc finger.^27,28 The optineurin protein from different species has high amino acid homology.²⁸ The amino acid 50 glutamic acid residue is conserved in mice, rats, chickens, and cows.²³ Optineurin is expressed in many tissues including the brain and the retina.^27,28Despite intense interest, the roles of myocilin and optineurin in cellular functions still remain largely undefined. Because glaucoma is a group of diseases known as progressive optic neuropathies, we undertook the current study to investigate the effects of myocilin and optineurin on neurite outgrowth. We demonstrated that overexpression of wild-type myocilin or P370L and Q368X mutants caused an inhibition of neurite outgrowth, whereas forced expressed wild-type or E50K optineurin did not result in any changes in neuronal rat pheochromocytoma PC12²⁹ and RGC5³⁰ cells.