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1.
J Immunol ; 198(2): 681-690, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27986906

RESUMO

Th cells sensitized against autoantigens acquire pathogenicity following two sequential events, namely activation by their target Ag and a process named "licensing." In this study, we analyzed these processes in a transgenic mouse system in which TCR-transgenic Th cells specific to hen egg lysozyme (HEL) are adoptively transferred to recipients and induce inflammation in eyes expressing HEL. Our data show that the notion that the lung is the organ where "licensing" for pathogenicity takes place is based on biased data collected with cells injected i.v., a route in which most transferred cells enter via the lung. Thus, we found that when donor cells were activated in vitro and injected intraperitoneally, or were activated in vivo, they migrated simultaneously to the lung, spleen, and other tested organs. In all, tested organs donor cells undergo "licensing" for pathogenicity, consisting of vigorous increase in number and changes in expression levels of inflammation-related genes, monitored by both flow cytometry and microarray analysis. After reaching peak numbers, around day 3, the "licensed" donor cells migrate to the circulation and initiate inflammation in the HEL-expressing recipient eyes. Importantly, the kinetics of increase in number and of changes in gene expression by the donor cells were similar in lung, spleen, and other tested organs of the recipient mice. Furthermore, the total numbers of donor cells in the spleen at their peaks were 10- to 100-fold larger in the spleen than in the lung, contradicting the notion that the lung is the organ where "licensing" takes place.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva , Animais , Autoantígenos/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Pulmão/imunologia , Camundongos , Camundongos Transgênicos , Muramidase/imunologia , Baço/imunologia
2.
Nature ; 494(7435): 90-4, 2013 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-23242137

RESUMO

Chronic neuroinflammation is a common feature of the ageing brain and some neurodegenerative disorders. However, the molecular and cellular mechanisms underlying the regulation of innate immunity in the central nervous system remain elusive. Here we show that the astrocytic dopamine D2 receptor (DRD2) modulates innate immunity through αB-crystallin (CRYAB), which is known to suppress neuroinflammation. We demonstrate that knockout mice lacking Drd2 showed remarkable inflammatory response in multiple central nervous system regions and increased the vulnerability of nigral dopaminergic neurons to neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. Astrocytes null for Drd2 became hyper-responsive to immune stimuli with a marked reduction in the level of CRYAB. Preferential ablation of Drd2 in astrocytes robustly activated astrocytes in the substantia nigra. Gain- or loss-of-function studies showed that CRYAB is critical for DRD2-mediated modulation of innate immune response in astrocytes. Furthermore, treatment of wild-type mice with the selective DRD2 agonist quinpirole increased resistance of the nigral dopaminergic neurons to MPTP through partial suppression of inflammation. Our study indicates that astrocytic DRD2 activation normally suppresses neuroinflammation in the central nervous system through a CRYAB-dependent mechanism, and provides a new strategy for targeting the astrocyte-mediated innate immune response in the central nervous system during ageing and disease.


Assuntos
Astrócitos/imunologia , Astrócitos/metabolismo , Inflamação/imunologia , Receptores de Dopamina D2/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/imunologia , Fármacos Neuroprotetores/metabolismo , Quimpirol/farmacologia , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/deficiência , Receptores de Dopamina D2/genética , Substância Negra/citologia , Substância Negra/efeitos dos fármacos , Cadeia B de alfa-Cristalina/genética
3.
Exp Eye Res ; 166: 116-119, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29074386

RESUMO

In this study we compared polarized mouse T-helper (Th) lymphocytes of four populations, sensitized against an ocular antigen, for their patterns of migration and induction of inflammatory processes in recipient mouse eyes expressing the target antigen. Th1, Th2, Th9 and Th17 cells transgenically expressing T-cell receptor (TCR) specific against hen egg lysozyme (HEL) were adoptively transferred to recipient mice expressing HEL in their eyes. Recipient eyes collected 4 or 7 days post injection were analyzed for histopathological changes. Th1 and Th17 cells induced moderate to severe intraocular inflammation in the recipient mouse eyes, but essentially did not migrate into the conjunctiva. In contrast, Th2 and Th9 cells invaded minimally the intraocular space of recipient eyes, but accumulated in the limbus and migrated into the conjunctiva of the recipient mice and initiated allergy-like inflammatory responses, as indicated by remarkable eosinophil involvement. These data thus shed new light on the differences between the migration patterns and ocular pathogenic processes mediated by Th1/Th17 and by Th2/Th9 populations.


Assuntos
Movimento Celular , Túnica Conjuntiva/patologia , Eosinofilia/patologia , Limbo da Córnea/parasitologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Modelos Animais de Doenças , Cristalino/metabolismo , Camundongos , Muramidase , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia
4.
Biochim Biophys Acta ; 1862(6): 1214-27, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26851658

RESUMO

ßγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.215+1G>A splice mutation in the ßA3/A1-crystallin gene CRYBA1 was elucidated using three transgenic mice models. This mutation causes a splice defect in which the mutant mRNA escapes nonsense mediated decay by skipping both exons 3 and 4. Skipping these exons results in an in-frame deletion of the mRNA and synthesis of an unstable p.Ile33_Ala119del mutant ßA3/A1-crystallin protein. Transgenic expression of mutant ßA3/A1-crystallin but not the wild type protein results in toxicity and abnormalities in the maturation and orientation of differentiating lens fibers in c.97_357del CRYBA1 transgenic mice, leading to a small spherical lens, cataract, and often lens capsule rupture. On a cellular level, the lenses accumulated p.Ile33_Ala119del ßA3/A1-crystallin with resultant activation of the stress signaling pathway - unfolded protein response (UPR) and inhibition of normal protein synthesis, culminating in apoptosis. This highlights the mechanistic contrast between mild mutations that destabilize crystallins and other proteins, resulting in their being bound by the α-crystallins that buffer lens cells against damage by denatured proteins, and severely misfolded proteins that are not bound by α-crystallin but accumulate and have a direct toxic effect on lens cells, resulting in early onset cataracts.


Assuntos
Apoptose , Catarata/genética , Cristalino/patologia , Splicing de RNA , Resposta a Proteínas não Dobradas , Cadeia A de beta-Cristalina/genética , Animais , Sequência de Bases , Catarata/patologia , Linhagem Celular , Éxons , Humanos , Cristalino/citologia , Cristalino/metabolismo , Camundongos Transgênicos , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Deleção de Sequência
5.
Adv Exp Med Biol ; 854: 779-84, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26427489

RESUMO

We have previously demonstrated that ßA3/A1-crystallin, a member of the ß/γ-crystallin superfamily, is expressed in the astrocytes and retinal pigment epithelial (RPE) cells of the eye. In order to understand the physiological functions of ßA3/A1-crystallin in RPE cells, we generated conditional knockout (cKO) mice where Cryba1, the gene encoding ßA3/A1-crystallin, is deleted specifically from the RPE using the Cre-loxP system. By utilizing the cKO model, we have shown that this protein is required by RPE cells for proper lysosomal degradation of photoreceptor outer segments (OS) that have been internalized in phagosomes and also for the proper functioning of the autophagy process. We also reported that ßA3/A1-crystallin is trafficked to lysosomes, where it regulates endolysosomal acidification by modulating the activity of the lysosomal V-ATPase complex. Our results show that the V-ATPase activity in cKO RPE is significantly lower than WT RPE. Since, V-ATPase is important for regulating lysosomal pH, we noticed that endolysosomal pH was higher in the cKO cells compared to the WT cells. Increased lysosomal pH in cKO RPE is also associated with reduced Cathepsin D activity. Cathepsin D is a major lysosomal aspartic protease involved in the degradation of the OS and hence we believe that reduced proteolytic activity contributes to impaired degradation of OS in the cKO RPE. Reduced lysosomal activity in the cKO RPE also contributes to the incomplete degradation of the autophagosomes. Our results also suggest that ßA3/A1-crystallin regulates V-ATPase activity by binding to the V0 subunit of the V-ATPase complex. Taken together, these results suggest a novel mechanism by which ßA3/A1-crystallin regulates lysosomal function by modulating the activity of V-ATPase.


Assuntos
Cristalinas/metabolismo , Células Epiteliais/metabolismo , Epitélio Pigmentado da Retina/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Catepsina D/metabolismo , Cristalinas/genética , Concentração de Íons de Hidrogênio , Immunoblotting , Lisossomos/metabolismo , Camundongos Knockout , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/citologia , Cadeia A de beta-Cristalina
6.
J Immunol ; 191(1): 415-23, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23720812

RESUMO

The interaction between TLRs and their cognate ligands triggers both the innate and adaptive immune systems, and thus can play a pivotal role in the defense against pathogen invasion. This work investigates the differentiation of naive CD4 cells into Th1 or Th17 phenotypes in mice treated with different TLR ligands. We use a model system in which naive transgenic cells specific to hen egg lysozyme are adoptively transferred into recipients that express hen egg lysozyme in the lens of the eye. The transferred naive T cells induce ocular inflammation only in recipients treated with TLR ligands. Treatment with LPS preferentially stimulated IL-17 production, whereas CpG oligodeoxynucleotide and polyinosinic:polycytidylic acid primarily stimulated Th1 cells. Peptidoglycan stimulated the two Th subpopulations equally. The preferential induction of Th1 or Th17 by the four ligands was detected in the spleen (where a major portion of the adoptively transferred cells homed) and in the eyes, where activated Th cells initiate inflammation. Analysis of the cytokines present in recipient mice suggests that Th1 induction is elicited by IL-12 and/or IFN-α, whereas Th17 generation is preferentially mediated by IL-6. Importantly, we show in this article that treatment with LPS selectively promoted in the recipient mice the generation of IL-6-producing activated B cells. An inverse correlation was found between the level of regulatory T cells and severity of inflammation induced by the donor cells. Taken together, our data show that specific TLR ligands differentially activate the immune system as evidenced by the generation of distinct Th phenotypes from naive CD4 cells.


Assuntos
Doenças Autoimunes/imunologia , Células Th1/imunologia , Células Th17/imunologia , Receptores Toll-Like/metabolismo , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Galinhas , Ligantes , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muramidase/genética , Peptidoglicano/metabolismo , Poli I-C/metabolismo , Células Th1/metabolismo , Células Th1/patologia , Células Th17/metabolismo , Células Th17/patologia , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/fisiologia
7.
J Proteome Res ; 13(3): 1177-89, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24450463

RESUMO

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. To study the developmental processes that require intact ubiquitin, we executed the most extensive characterization of the lens proteome to date. We quantified lens protein expression changes in multiple replicate pools of P1 wild-type and K6W-Ub-expressing mouse lenses. Lens proteins were digested with trypsin, peptides were separated using strong cation exchange and reversed-phase liquid chromatography, and tandem mass (MS/MS) spectra were collected with a linear ion trap. Transgenic mice that expressed low levels of K6W-Ub (low expressers) had normal, clear lenses at birth, whereas the lenses that expressed high levels of K6W-Ub (higher expressers) had abnormal lenses and cataracts at birth. A total of 2052 proteins were identified, of which 996 were reliably quantified and compared between wild-type and K6W-Ub transgenic mice. Consistent with a delayed developmental program, fiber-cell-specific proteins, such as γ-crystallins (γA, γB, γC, and γE), were down-regulated in K6W-Ub higher expressers. Up-regulated proteins were involved in energy metabolism, signal transduction, and proteolysis. The K6W-Ub low expressers exhibited delayed onset and milder cataract consistent with smaller changes in protein expression. Because lens protein expression changes occurred prior to lens morphological abnormalities and cataract formation in K6W-Ub low expressers, it appears that expression of K6W-Ub sets in motion a process of altered protein expression that results in developmental defects and cataract.


Assuntos
Catarata/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/metabolismo , Proteoma/genética , Ubiquitina/genética , Substituição de Aminoácidos , Animais , Animais Recém-Nascidos , Catarata/metabolismo , Catarata/patologia , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica , Cristalino/patologia , Lisina/metabolismo , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma/metabolismo , Triptofano/metabolismo , Ubiquitina/metabolismo
8.
FASEB J ; 27(1): 151-62, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23033322

RESUMO

The molecular chaperone αB-crystallin has emerged as a target for cancer therapy due to its expression in human tumors and its role in regulating tumor angiogenesis. αB-crystallin also reduces neuroinflammation, but its role in other inflammatory conditions has not been investigated. Here, we examined whether αB-crystallin regulates inflammation associated with tumors and ischemia. We found that CD45(+) leukocyte infiltration is 3-fold increased in tumors and ischemic myocardium in αB-crystallin-deficient mice. Notably, αB-crystallin is prominently expressed in CD11b(+) Gr-1(+) immature myeloid cells (IMCs), known as regulators of angiogenesis and immune responses, while lymphocytes and mature granulocytes show low αB-crystallin expression. αB-Crystallin deficiency results in a 3-fold higher accumulation of CD11b(+) Gr-1(+) IMCs in tumors and a significant rise in CD11b(+) Gr-1(+) IMCs in spleen and bone marrow. Similarly, we noted a 2-fold increase in CD11b(+) Gr-1(+) IMCs in chronically inflamed livers in αB-crystallin-deficient mice. The effect of αB-crystallin on IMC accumulation is limited to pathological conditions, as CD11b(+) Gr-1(+) IMCs are not elevated in naive mice. Through ex vivo differentiation of CD11b(+) Gr-1(+) cells, we provide evidence that αB-crystallin regulates systemic expansion of IMCs through a cell-intrinsic mechanism. Our study suggests a key role of αB-crystallin in limiting expansion of CD11b(+) Gr-1(+) IMCs in diverse pathological conditions.


Assuntos
Células da Medula Óssea/imunologia , Antígeno CD11b/imunologia , Cristalinas/fisiologia , Teratocarcinoma/patologia , Animais , Sequência de Bases , Diferenciação Celular , Primers do DNA , Progressão da Doença , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Teratocarcinoma/imunologia
9.
J Immunol ; 189(3): 1220-7, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22745380

RESUMO

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-ß, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-ß and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23-dependent Th17 cells.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular/imunologia , Linhagem da Célula/imunologia , Muramidase/metabolismo , Células Th17/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Oftalmopatias/enzimologia , Oftalmopatias/imunologia , Oftalmopatias/patologia , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-23/metabolismo , Camundongos , Camundongos Transgênicos , Muramidase/efeitos adversos , Muramidase/imunologia , Células Th17/enzimologia , Células Th17/patologia
10.
Nature ; 448(7152): 474-9, 2007 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-17568699

RESUMO

alphaB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/prevenção & controle , Esclerose Múltipla/metabolismo , Esclerose Múltipla/prevenção & controle , Fármacos Neuroprotetores/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Animais , Apoptose , Astrócitos/metabolismo , Caspase 3/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/prevenção & controle , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/imunologia , Bainha de Mielina/imunologia , Bainha de Mielina/patologia , NF-kappa B/metabolismo , Neuroglia/patologia , Fármacos Neuroprotetores/uso terapêutico , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/uso terapêutico
11.
Blood ; 115(16): 3398-406, 2010 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-20023214

RESUMO

alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF-A but not transforming growth factor-beta in cultured retinal pigment epithelial (RPE) cells. alphaB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin(-/-) RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin(-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin(-/-) mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.


Assuntos
Neovascularização de Coroide/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Neovascularização de Coroide/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Cadeia B de alfa-Cristalina/genética
12.
Transgenic Res ; 21(5): 1033-42, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22427112

RESUMO

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for ßA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express ßA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant ßA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that ßA3/A1-crystallin is essential for normal astrocyte function in the retina.


Assuntos
Astrócitos/fisiologia , Cristalinas/metabolismo , Retina/crescimento & desenvolvimento , Vasos Retinianos/crescimento & desenvolvimento , Animais , Astrócitos/patologia , Western Blotting , Movimento Celular , Forma Celular , Cristalinas/genética , Angiofluoresceinografia , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Regiões Promotoras Genéticas , Ratos , Retina/patologia , Vasos Retinianos/patologia , Transgenes
13.
J Immunol ; 185(11): 6795-801, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20971929

RESUMO

Recently reported lines of Th9 cells, producing IL-9 and IL-10, were generated by polarization with IL-4 and TGF-ß and activation with Abs against CD3 and CD28. In this paper, we analyzed features of Th9 lines similarly polarized but activated by the "natural mode" (i.e., exposure of CD4 cells to their target Ag, hen egg lysozyme [HEL] and APCs). Main observations are the following: 1) both IL-9 and IL-10 were expressed by the line cells, but with strikingly different kinetics, with IL-9 being produced rapidly, reaching a peak on day 3 in culture and declining sharply thereafter, whereas IL-10 production increased gradually, resembling IL-4 and IL-17 production by their corresponding lineage cells; 2) reactivation of Th9, following expansion, triggered faster and higher production of both IL-9 and IL-10; 3) incubating Th9 cells in polarizing media specific for other phenotypes stimulated moderate levels of phenotype switching to Th1 or Th17 but a massive switching to Th2; 4) Th9 cells induced moderate inflammation in HEL-expressing recipient eyes but only when producing high levels of IL-9; and 5) IL-9-producing donor cells were detected in the blood of Th9 recipients but not in their inflamed eyes, suggesting that similar to findings in culture, exposure to HEL in these eyes arrested the IL-9 production in Th9 cells. Collectively, these data provide new information concerning Th9 cells and reveal their uniqueness, in particular with regard to the unusual production kinetics of IL-9 and the short retention of these cells in affected target tissues.


Assuntos
Proteínas do Olho/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-9/biossíntese , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Linhagem Celular , Galinhas , Relação Dose-Resposta Imunológica , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/imunologia , Proteínas do Olho/fisiologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/fisiologia , Interleucina-10/biossíntese , Interleucina-10/metabolismo , Interleucina-9/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muramidase/biossíntese , Muramidase/genética , Muramidase/imunologia , Especificidade de Órgãos/imunologia , Receptores de Antígenos de Linfócitos T/biossíntese , Linfócitos T Auxiliares-Indutores/patologia , Fatores de Tempo
14.
Proc Natl Acad Sci U S A ; 106(15): 6152-7, 2009 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-19369214

RESUMO

VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a "survival," rather than an "angiogenic" factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.


Assuntos
Neovascularização Patológica , Fator B de Crescimento do Endotélio Vascular/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genoma , Membro Posterior/irrigação sanguínea , Isquemia/genética , Isquemia/metabolismo , Camundongos , Camundongos Knockout , Ratos , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Regulação para Cima , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genética
15.
Hum Mol Genet ; 18(7): 1190-9, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19129171

RESUMO

Alexander disease (AxD) is a primary disorder of astrocytes caused by dominant mutations in the gene for glial fibrillary acidic protein (GFAP). These mutations lead to protein aggregation and formation of Rosenthal fibers, complex astrocytic inclusions that contain GFAP, vimentin, plectin, ubiquitin, Hsp27 and alphaB-crystallin. The small heat shock protein alphaB-crystallin (Cryab) regulates GFAP assembly, and elevation of Cryab is a consistent feature of AxD; however, its role in Rosenthal fibers and AxD pathology is not known. Here, we show in AxD mouse models that loss of Cryab results in increased mortality, whereas elevation of Cryab rescues animals from terminal seizures. When mice with Rosenthal fibers induced by over-expression of GFAP are crossed into a Cryab-null background, over half die at 1 month of age. Restoration of Cryab expression through the GFAP promoter reverses this outcome, showing the effect is astrocyte-specific. Conversely, in mice engineered to express both AxD-associated mutations and elevated GFAP, which despite natural induction of Cryab also die at 1 month, transgenic over-expression of Cryab results in a markedly reduced CNS stress response, restores expression of the glutamate transporter Glt1 (EAAT2) and protects these animals from death. In its most common form, AxD is a devastating neurodegenerative disease, with early onset, characterized by seizures, spasticity and developmental delays, ultimately leading to death. Cryab plays a critical role in tempering AxD pathology and should be investigated as a therapeutic target for this and other diseases with astropathology.


Assuntos
Doença de Alexander/metabolismo , Doença de Alexander/patologia , Proteína Glial Fibrilar Ácida/toxicidade , Supressão Genética , Cadeia B de alfa-Cristalina/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutação/genética , Fenótipo , Convulsões/metabolismo , Convulsões/patologia , Estresse Fisiológico , Análise de Sobrevida
16.
J Immunol ; 183(11): 7547-56, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19890052

RESUMO

Both Th1 and Th17 T cell subsets can mediate inflammation, but the kinetics of the pathogenic processes mediated by these two subsets have not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme induce ocular inflammation in recipient mice expressing eye-restricted hen egg lysozyme, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly. By contrast, Th17 cells accumulated and remained the majority of the infiltrating CD4(+) cells in the eye for as long as 25 days after transfer, mediating more long-lasting pathological changes. Unlike Th1, Th17 cells were highly resistant to restimulation-induced apoptosis, a major pathway by which autoimmune and chronically restimulated Th1 cells are eliminated. Th17 cells had reduced Fas ligand production and resistance to Fas-induced apoptosis, relative to Th1 cells, despite similar surface expression of Fas. Th17-induced ocular inflammation also differed from Th1-induced inflammation by consisting of more neutrophils, whereas Th1-induced disease had higher proportions of CD8 cells. Taken together, our data show that pathogenic processes triggered by Th17 lag behind those induced by Th1, but then persist remarkably longer, apparently due to the relative resistance of Th17 cells to restimulation-induced cell death. The long-lasting inflammation induced by Th17 cells is in accord with these cells being involved in chronic conditions in humans.


Assuntos
Apoptose/imunologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Inflamação/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos , Divisão Celular , Olho/imunologia , Proteína Ligante Fas/imunologia , Proteína Ligante Fas/metabolismo , Citometria de Fluxo , Interleucina-17/biossíntese , Interleucina-17/imunologia , Camundongos , Camundongos Transgênicos , Muramidase/imunologia , Células Th1/imunologia , Receptor fas/imunologia , Receptor fas/metabolismo
17.
J Immunol ; 181(10): 7205-13, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18981142

RESUMO

Th1 and Th17 cells are characterized by their expression of IFN-gamma or IL-17, respectively. The finding of Th cells producing both IL-17 and IFN-gamma suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-gamma was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-gamma or both IFN-gamma and IL-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-gamma or IFN-gamma/IL-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with 20-25% nonpolarized cells than in recipients of 35-40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-gamma or IFN-gamma/IL-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-gamma t was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-gamma or IFN-gamma/IL-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.


Assuntos
Inflamação/imunologia , Interferon gama/biossíntese , Interleucina-17/biossíntese , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Animais , Oftalmopatias/imunologia , Oftalmopatias/metabolismo , Citometria de Fluxo , Imunofenotipagem , Inflamação/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Muramidase/imunologia , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/metabolismo , Células Th1/metabolismo
18.
Infect Immun ; 76(4): 1781-90, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18227158

RESUMO

Bacterial infections of the eye highlight a dilemma that is central to all immune-privileged sites. On the one hand, immune privilege limits inflammation to prevent bystander destruction of normal tissue and loss of vision. On the other hand, bacterial infections require a robust inflammatory response for rapid clearance of the pathogen. We demonstrate that the retina handles this dilemma, in part, by activation of a protective heat shock protein. During Staphylococcus aureus-induced endophthalmitis, the small heat shock protein alphaB-crystallin is upregulated in the retina and prevents apoptosis during immune clearance of the bacteria. In the absence of alphaB-crystallin, mice display increased retinal apoptosis and retinal damage. We found that S. aureus produces a protease capable of cleaving alphaB-crystallin to a form that coincides with increased retinal apoptosis and tissue destruction. We conclude that alphaB-crystallin is important in protecting sensitive retinal tissue during destructive inflammation that occurs during bacterial endophthalmitis.


Assuntos
Endoftalmite/microbiologia , Retina/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus , Cadeia B de alfa-Cristalina/metabolismo , Animais , Apoptose/fisiologia , Proteínas de Bactérias/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Peptídeo Hidrolases/metabolismo , Retina/citologia , Retina/microbiologia , Infecções Estafilocócicas/microbiologia , Regulação para Cima , Cadeia B de alfa-Cristalina/genética
19.
BMC Dev Biol ; 8: 88, 2008 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-18803847

RESUMO

BACKGROUND: alphaA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12-24 hours, compared to the expression of the endogenous Cryaa gene. RESULTS: Here, we used bacterial artificial chromosome (BAC) and standard transgenic approaches to examine temporal and spatial regulation of the mouse Cryaa gene. Two BAC transgenes, with EGFP insertions into the third coding exon of Cryaa gene, were created: the intact alphaA-crystallin 148 kb BAC (alphaA-BAC) and alphaA-BAC(DeltaDCR3), which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the Cryaa gene in lens, but not outside of the lens. The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of alphaA-crystallin locus derived from alphaA-BAC(DeltaDCR3), 15 kb Cryaa/EGFP. A 15 kb region of Cryaa/EGFP supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of alphaA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing alphaA-crystallin in the lens pit. CONCLUSION: We conclude that a 148 kb alphaA-BAC likely contains all of the regulatory regions required for alphaA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb Cryaa/EGFP region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic regions may play modulatory functions in regulating extralenticular alphaA-crystallin expression. Finally, deletion of DCR3 in either alphaA-BAC(DeltaDCR3) or Cryaa (15 kb) transgenic mice result in EGFP expression patterns that are consistent with DCR's previously established role as a distal enhancer active in "late" primary lens fiber cells.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transcrição Gênica/fisiologia , Cadeia A de alfa-Cristalina/genética , Animais , Genes Reporter , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cristalino/embriologia , Cristalino/metabolismo , Camundongos , Transgenes , Cadeia A de alfa-Cristalina/biossíntese , Cadeia A de alfa-Cristalina/fisiologia
20.
Invest Ophthalmol Vis Sci ; 49(3): 1161-71, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18326745

RESUMO

PURPOSE: During the early phase of experimental autoimmune uveitis (EAU), before macrophages infiltrate the retina and uvea, photoreceptor mitochondrial oxidative stress, nitration of photoreceptor mitochondrial proteins, and release of cytochrome c have been observed. However, no apoptosis has been detected during this phase. In this study, alphaA-crystallin upregulation in the retina and its antiapoptotic protective role were evaluated in early EAU. METHODS: Gene microarrays were first used to identify upregulated genes in retinas with early EAU. Among highly upregulated crystallins, alphaA was confirmed by real-time polymerase chain reaction and Western blot, and the site of upregulation was localized by immunohistochemistry. The association of alphaA-crystallin to nitrated cytochrome c and interaction with a procaspase-3 subunit was assayed. Photoreceptor apoptosis in alphaA knockout mice was compared with that in wild-type animals with EAU, by using the terminal transferase dUTP nick-end labeling assay and polymerase chain reaction. RESULTS: In early EAU, alphaA-crystallin was increased 33-fold, and the site of increase was localized to the photoreceptor inner segments. This crystallin suppressed apoptosis by associating with the nitrated cytochrome c and p24. The association with nitrated cytochrome c, in particular, appeared to be restricted to nitrated cytochrome c, and thus, no association of non-nitrated cytochrome c was detected. The knockout mice showed signs of EAU development early and showed apoptosis in the retina; no such changes were seen in the wild-type control animals. CONCLUSIONS: alphaA-Crystallin is highly upregulated in the retina during early EAU. This upregulation is localized primarily in the photoreceptor inner segments, the site of mitochondrial oxidative stress. Further, in early EAU, the photoreceptors preferentially use alphaA-crystallin to suppress mitochondrial oxidative stress-mediated apoptosis.


Assuntos
Doenças Autoimunes/prevenção & controle , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico/genética , Retina/metabolismo , Uveíte/prevenção & controle , Cadeia A de alfa-Cristalina/genética , Animais , Apoptose , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Western Blotting , Caspase 3/metabolismo , Citocromos c/metabolismo , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Choque Térmico/metabolismo , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Células Fotorreceptoras de Vertebrados/fisiologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Uveíte/metabolismo , Uveíte/patologia , Cadeia A de alfa-Cristalina/metabolismo
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