- Conditional Deletion of Bmal1 Accentuates Microvascular and Macrovascular Injury. [Journal Article]
- AJAm J Pathol 2017 Apr 19
- The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditi...
The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1(fx/fx);Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, spinocerebellar ataxia type 1 protein homolog-positive, proto-oncogene c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1(fx/fx);Tek-Cre mice. The number of LSK cells from the Bmal1(fx/fx);Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1(fx/fx);Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.
- Human Effector Memory T Helper Cells Engage with Mouse Macrophages and Cause Graft-versus-Host-Like Pathology in Skin of Humanized Mice Used in a Nonclinical Immunization Study. [Journal Article]
- AJAm J Pathol 2017 Apr 19
- Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed...
Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced-dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4(+) T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4(+) cells near F4/80(+) mouse macrophages around hair follicles. In spleen, CD4(+) cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2-type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8(+) T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice.
- miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis. [Journal Article]
- AJAm J Pathol 2017 Apr 19
- miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We i...
miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We identified miRNAs with aberrant expression in Hodgkin lymphoma (HL) and investigated their role in pathogenesis. Small RNA sequencing revealed 84 significantly differentially expressed miRNAs in HL cell lines as compared to germinal center B cells. Three up-regulated miRNAs-miR-23a-3p, miR-24-3p, and miR-27a-3p-were derived from one primary miRNA transcript. Loss-of-function analyses for these miRNAs and their seed family members resulted in decreased growth on miR-24-3p inhibition in three and of miR-27a/b-3p inhibition in one HL cell line. Apoptosis analysis indicated that the effect of miR-24-3p on cell growth is at least in part caused by an increase of apoptotic cells. Argonaute 2 immunoprecipitation revealed 1142 genes consistently targeted by miRNAs in at least three of four HL cell lines. Furthermore, 52 of the 1142 genes were predicted targets of miR-24-3p. Functional annotation analysis revealed a function related to cell growth, cell death, and/or apoptosis for 15 of the 52 genes. Western blotting of the top five genes showed increased protein levels on miR-24-3p inhibition for CDKN1B/P27(kip1) and MYC. In summary, we showed that miR-24-3p is up-regulated in HL and its inhibition impairs cell growth possibly via targeting CDKN1B/P27(kip1) and MYC.
- This Month in AJP. [Journal Article]
- AJAm J Pathol 2017 Apr 19
- Interferon-γ Released by Activated CD8(+) T Lymphocytes Impairs the Calcium Resorption Potential of Osteoclasts in Calcified Human Aortic Valves. [Journal Article]
- AJAm J Pathol 2017 Apr 18
- Calcium content in patients with calcific aortic valve disease (CAVD) correlates with the severity of stenosis. In CAVD, activated T lymphocytes localize with osteoclast regions; however, the functio...
Calcium content in patients with calcific aortic valve disease (CAVD) correlates with the severity of stenosis. In CAVD, activated T lymphocytes localize with osteoclast regions; however, the functional consequences of this association remain unknown. We hypothesized that CD8(+) T cells modulate calcification in CAVD. Explanted CAVD valves (n = 52) dissected into noncalcified and calcified portions were subjected to mRNA extraction, real-time quantitative PCR, enzyme-linked immunosorbent assay, and immunohistochemical analyses. Compared with noncalcified portions, calcified regions exhibited significantly elevated transcripts for CD8, interferon (IFN)-γ, CXCL9, Perforin 1, Granzyme B, and heat shock protein 60. Osteoclast-associated receptor activator of NK-κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated receptor increased significantly, whereas Cathepsin K remained unchanged. The stimulation of tissue segments with phorbol-12-myristate-13-acetate and ionomycin resulted in IFN-γ release, recapitulating CAVD microenvironment. Real-time quantitative PCR detected signature mRNAs for CD8(+) T-cell activation (Perforin 1, Granzyme B). In stimulated versus unstimulated organoid cultures, elevated IFN-γ reduced the mRNAs encoding for RANKL, TRAP, and Cathepsin K. Molecular imaging showed increased calcium signal intensity in stimulated versus unstimulated parts. Human CD14(+) monocytes treated either with recombinant human IFN-γ or with conditioned media-derived IFN-γ exhibited low levels of Cathepsin K, TRAP, RANK, and tumor necrosis factor receptor-associated factor 6 mRNAs, whereas concentrations of the T-cell co-activators CD80 and CD86 increased in parallel with reduced osteoclast resorptive function, effects abrogated by neutralizing anti-IFN-γ antibodies. CD8(+) cell-derived IFN-γ suppresses osteoclast function and may thus favor calcification in CAVD.
- Divergent Function of Programmed Death-Ligand 1 in Donor Tissue versus Recipient Immune System in a Murine Model of Bronchiolitis Obliterans. [Journal Article]
- AJAm J Pathol 2017 Apr 17
- Costimulatory molecules, such as the programmed death ligand (PD-L1), might exert differential effects on T-cell function, depending on the clinical setting and/or immunological environment. Given th...
Costimulatory molecules, such as the programmed death ligand (PD-L1), might exert differential effects on T-cell function, depending on the clinical setting and/or immunological environment. Given the impact of T cells on bronchiolitis obliterans (BO) in lung transplantation, we used an established tracheal transplant model inducing BO-like lesions to investigate the impact of PD-L1 on alloimmune responses and histopathological outcome in BO. In contrast to other transplant models in which PD-L1 generally shows protective functions, we demonstrated that PD-L1 has divergent effects depending on its location in donor versus recipient tissue. Although PD-L1 deficiency in donor tissue worsened histopathological outcome, and increased systemic inflammatory response, recipient PD-L1 deficiency induced opposite effects. Mechanistic studies revealed PD-L1-deficient recipients were hyporesponsive toward alloantigen, despite increased numbers of CD8(+) effector T cells. The function of PD-L1 on T cells after unspecific stimulation was dependent on both cell type and strength of stimulation. This novel function of recipient PD-L1 may result from the high degree of T-cell activation within the highly immunogenic milieu of the transplanted tissue. In this model, both decreased T-cell alloimmune responses and the reduction of BO in PD-L1-deficient recipients suggest a potential therapeutic role of selectively blocking PD-L1 in the recipient. Further investigation is warranted to determine the impact of this finding embedded in the complex pathophysiological context of BO.
- Pathologic Active mTOR Mutation in Brain Malformation with Intractable Epilepsy Leads to Cell-Autonomous Migration Delay. [Journal Article]
- AJAm J Pathol 2017; 187(5):1177-1185
- The activation of phosphatidylinositol 3-kinase-AKTs-mammalian target of rapamycin cell signaling pathway leads to cell overgrowth and abnormal migration and results in various types of cortical malf...
The activation of phosphatidylinositol 3-kinase-AKTs-mammalian target of rapamycin cell signaling pathway leads to cell overgrowth and abnormal migration and results in various types of cortical malformations, such as hemimegalencephaly (HME), focal cortical dysplasia, and tuberous sclerosis complex. However, the pathomechanism underlying abnormal cell migration remains unknown. With the use of fetal mouse brain, we performed causative gene analysis of the resected brain tissues from a patient with HME and investigated the pathogenesis. We obtained a novel somatic mutation of the MTOR gene, having approximately 11% and 7% mutation frequency in the resected brain tissues. Moreover, we revealed that the MTOR mutation resulted in hyperphosphorylation of its downstream molecules, S6 and 4E-binding protein 1, and delayed cell migration on the radial glial fiber and did not affect other cells. We suspect cell-autonomous migration arrest on the radial glial foot by the active MTOR mutation and offer potential explanations for why this may lead to cortical malformations such as HME.
- Local Group 2 Innate Lymphoid Cells Promote Corneal Regeneration after Epithelial Abrasion. [Journal Article]
- AJAm J Pathol 2017 Apr 15
- Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new ther...
Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2(-) corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin.
- Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice. [Journal Article]
- AJAm J Pathol 2017 Apr 14
- Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily ...
Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6(-/-) mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6(-/-) mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6(-/-) mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.
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- Exposure of the Amino Terminus of Tau Is a Pathological Event in Multiple Tauopathies. [Journal Article]
- AJAm J Pathol 2017 Apr 13
- Pathological changes to the tau protein, including conformational changes and aggregation, are major hallmarks of a group of neurodegenerative disorders known as tauopathies. Among the conformational...
Pathological changes to the tau protein, including conformational changes and aggregation, are major hallmarks of a group of neurodegenerative disorders known as tauopathies. Among the conformational changes are alterations involving the extreme amino terminus of the protein, known as the phosphatase-activating domain (PAD). Aberrant PAD exposure induces a signaling cascade that leads to disruption of axonal transport, a critical function for neuronal survival. Conformational display of PAD is an early marker of pathological tau in Alzheimer disease (AD), but its role in other tauopathies has yet to be firmly established. We used a relatively novel N-terminal, conformation-sensitive antibody, TNT2, to determine whether misfolding in the amino terminus (ie, PAD exposure) occurs in non-AD tauopathies. We found that TNT2 specifically labeled pathological tau in post-mortem human brain tissue from Pick disease, progressive supranuclear palsy, corticobasal degeneration, and chronic traumatic encephalopathy, but did not label nonpathological, parenchymal tau. Tau13, another N-terminal antibody, was not sensitive to pathological N-terminal conformations. Tau13 did not readily distinguish between normal (ie, parenchymal tau) and pathological tau species and showed a range of effectiveness at identifying tau pathologies in the non-AD tauopathies. These findings demonstrate that the conformational display of the PAD in tau represents a common pathological event in many tauopathies.