RESUMEN
PURPOSE: Juvenile idiopathic arthritis-associated uveitis (JIAU) may run a chronic and treatment-resistant course, and occasionally, alterations of the iris vasculature may be observed clinically. METHODS: Iris tissue (IT), aqueous humor (AH) and serum samples from patients with clinically inactive JIAU (n = 30), acute anterior uveitis (AAU; n = 18), and primary open angle glaucoma (POAG; n = 20) were obtained during trabeculectomy or cataract surgery. Samples were analyzed by RNA-Seq, qRT-PCR, LC-IMS, Western-Blot, and LEGENDplex™ analysis. Pattern of iris vasculature in JIAU patients was assessed qualitatively via fluorescein and indocyanine green angiography (FLA/ICGA). RESULTS: RNA-Seq of IT showed significantly differential expression (DE) of 136 genes between JIAU and POAG, of which 15 were associated with angiogenesis. qRT-PCR, performed to validate RNA-Seq results, showed upregulation of the angiogenesis-related genes Kdr, Angpt-1, Tie-1, Tie-2 and Mmrn2 in IT (JIAU vs POAG, p > 0.05). LC-IMS of IT revealed a total number of 56 DE proteins (JIAU vs POAG), of which Angiopoetin, Lumican and Decorin were associated with angiogenesis and showed increased (p > 0.05) expression on Western-Blot analysis. LEGENDplex™ analysis showed upregulation of ANGPT-2 in AH from JIAU compared to AAU and POAG, whereas VEGF was upregulated in AAU. Iris vascular leakage, hypoperfusion and neovascularization were observed by FLA/ICGA in JIA patients with treatment-refractory complicated course of uveitis. CONCLUSION: Angiogenesis-related factors could play a role in long-standing complicated JIAU, leading to clinically visible alterations in selected cases.
Asunto(s)
Artritis Juvenil , Glaucoma de Ángulo Abierto , Trabeculectomía , Uveítis Anterior , Uveítis , Artritis Juvenil/complicaciones , Artritis Juvenil/genética , Humanos , Neovascularización Patológica/genética , Trabeculectomía/efectos adversos , Uveítis/complicaciones , Uveítis Anterior/complicacionesRESUMEN
Gene and protein expression profiles of iris biopsies, aqueous humor (AqH), and sera in patients with juvenile idiopathic arthritis-associated uveitis (JIAU) in comparison to control patients with primary open-angle glaucoma (POAG) and HLA-B27-positive acute anterior uveitis (AAU) were investigated. Via RNA Sequencing (RNA-Seq) and mass spectrometry-based protein expression analyses 136 genes and 56 proteins could be identified as being significantly differentially expressed (DE) between the JIAU and POAG group. Gene expression of different immunoglobulin (Ig) components as well as of the B cell-associated factors ID3, ID1, and EBF1 was significantly upregulated in the JIAU group as compared to POAG patients. qRT-PCR analysis showed a significantly higher gene expression of the B cell-related genes CD19, CD20, CD27, CD138, and MZB1 in the JIAU group. At the protein level, a significantly higher expression of Ig components in JIAU than in POAG was confirmed. The B cell-associated protein MZB1 showed a higher expression in JIAU patients than in POAG which was confirmed by western blot analysis. Using bead-based immunoassay analysis we were able to detect a significantly higher concentration of the B cell-activating and survival factors BAFF, APRIL, and IL-6 in the AqH of JIAU and AAU patients than in POAG patients. The intraocularly upregulated B cell-specific genes and proteins in iris tissue suggest that B cells participate in the immunopathology of JIAU. The intracameral environment in JIAU may facilitate local effector and survival functions of B cells, leading to disease course typical for anterior uveitis.
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Humor Acuoso/inmunología , Artritis Juvenil/inmunología , Proteínas del Ojo/inmunología , Regulación de la Expresión Génica/inmunología , Iris/inmunología , Transcriptoma/inmunología , Uveítis/inmunología , Adolescente , Adulto , Anciano , Artritis Juvenil/complicaciones , Artritis Juvenil/patología , Niño , Preescolar , Femenino , Humanos , Iris/patología , Masculino , Persona de Mediana Edad , Proteómica , Uveítis/etiología , Uveítis/patologíaRESUMEN
In the present study, therapeutic effect of topically applied everolimus (EV)-loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly (lactic acid) (mPEGhexPLA) nanocarriers on experimental autoimmune uveoretinitis (EAU) were investigated. EAU was induced in B10.RIII mice via immunization with human interphotoreceptor retinoid-binding protein peptide 161-180 (hIRBPp161-180) in complete Freund's adjuvant. Everolimus-loaded mPEGhexPLA (EV/mPEGhexPLA) nanocarriers were prepared by using a solvent evaporation method. On days 12-21 postimmunization (p.i.), the right eyes were treated five times daily either with 10 µl of 0.5% everolimus formulation or PBS (control). The EAU score of the eyes was determined histologically. On day 21 p.i., the peripheral immune responses were measured in serum, cervical lymph nodes (LN), and spleens via hIRBPp161-180-specific serum antibodies, cytokine secretion (ELISA), lymphocyte proliferation, and FoxP3+ regulatory T cells (Treg; flow cytometry). Compared to the PBS-treated mice, unilateral topical everolimus treatment significantly reduced EAU severity in both eyes (p < .05). The treatment reduced the antigen (Ag)-specific hIRBPp161-180-induced proliferation (p < .05), IL-2, IL-17, and IFN-γ secretion from cells isolated from the left and right cervical LN (p < .05). Under everolimus treatment, IL-10 secretion and CD4+CD25+FoxP3+ Treg frequency from cervical LN were enhanced. The proliferative response and cytokine secretion as well as the frequency of splenic Treg were almost unchanged. Topical administration of an everolimus formulation improved EAU in both eyes. The effect might also be related to systemic immunosuppressive effects, as several systemic cellular immune responses were influenced.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Everolimus/administración & dosificación , Inmunosupresores/administración & dosificación , Nanocápsulas/uso terapéutico , Retinitis/tratamiento farmacológico , Animales , Autoanticuerpos/sangre , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interferón gamma/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Retinitis/inmunología , Bazo/metabolismoRESUMEN
In the present study, tissue distribution and the therapeutic effect of topically applied cyclosporine A (CsA)-loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly(lactic acid) (mPEGhexPLA) nanocarriers (ApidSOL) on experimental autoimmune uveitis (EAU) were investigated. The CsA-loaded mPEGhexPLA nanocarrier was tolerated well locally and showed no signs of immediate toxicity after repeated topical application in mice with EAU. Upon unilateral CsA treatment, CsA accumulated predominantly in the corneal and sclera-choroidal tissue of the treated eye and in lymph nodes (LN). This regimen reduced EAU severity in treated eyes compared to PBS-treated controls. This improvement was accompanied by reduced T-cell count, T-cell proliferation, and IL-2 secretion of cells from ipsilateral LN. In conclusion, topical treatment with CsA-loaded mPEGhexPLA nanocarriers significantly improves the outcome of EAU.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Ciclosporina/administración & dosificación , Portadores de Fármacos/química , Inmunosupresores/administración & dosificación , Uveítis/tratamiento farmacológico , Administración Oftálmica , Animales , Enfermedades Autoinmunes/inmunología , Modelos Animales de Enfermedad , Proteínas del Ojo/administración & dosificación , Proteínas del Ojo/inmunología , Femenino , Humanos , Ratones , Nanopartículas/química , Poliésteres/química , Polietilenglicoles/química , Proteínas de Unión al Retinol/administración & dosificación , Proteínas de Unión al Retinol/inmunología , Resultado del Tratamiento , Uveítis/inmunologíaRESUMEN
OBJECTIVE: We examined the role of behavioral conditioning of immune responses with cyclosporine A (CsA) on the development of Th1/Th17-driven experimental autoimmune uveoretinitis (EAU). METHODS: Mice received a 0.2% w/v saccharin solution as conditioned stimulus combined with CsA (20 mg/kg) in 6 association trials at 72-h intervals. For evocation periods, conditioned mice were reexposed to saccharin, whereas the conditioned but not reexposed group received water only. Animals were immunized with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBPp161-180) peptide in complete Freund adjuvant (CFA) and a concomitant injection of pertussis toxin. RESULTS: In naïve mice subjected to the behavioral conditioning regimen, mitogen-induced interleukin (IL)-2 production was decreased in conditioned mice compared to conditioned but not reexposed animals. Incidence and severity of EAU were not significantly lower in behaviorally conditioned and immunized mice. ELISA analysis of splenocytes revealed a reduced interferon (IFN)-γ/IL-17 ratio in CsA-treated, conditioned but not reexposed, and conditioned animals. The adoptive transfer of antigen-specific splenocytes from animals behaviorally conditioned with CsA to naïve mice decreased the severity of EAU in recipient mice compared to the control group. In vitro activation of splenocytes isolated from immunized mice with agonists targeting TLR2 and NOD2 together with ß2-adrenergic activation (induced by epinephrine, norepinephrine, or salbutamol) resulted in decreased IFN-γ but increased IL-17 immune responses. The ß2-adrenergic antagonist propranolol could restore IFN-γ production, whereas only the norepinephrine-induced increase in IL-17 production was abrogated. CONCLUSIONS: We conclude that CsA conditioning in the EAU model mitigates Th1 but enhances Th17 immune responses, and does not ameliorate disease. The results imply that in EAU the mechanism of immune conditioning interacts with CFA components during active immunization, most likely via the TLR2/NOD2 pathway, and induces differentiation of Th17 cells that drive autoimmune diseases.
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Enfermedades Autoinmunes/inmunología , Ciclosporina/farmacología , Inmunosupresores/farmacología , Neuroinmunomodulación/fisiología , Uveítis/inmunología , Animales , Condicionamiento Clásico/fisiología , Modelos Animales de Enfermedad , Masculino , Ratones , Células Th17/inmunologíaRESUMEN
BACKGROUND: Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine known to participate in intraocular inflammatory disease. This study investigated whether treatment with intravitreal antisense-oligonucleotides (ASON) targeting TNF-α mRNA affects the progression of herpes simplex virus 1 (HSV-1) retinitis in mice. METHODS: The in vivo uptake of the oligonucleotid after intravitreal injection was determined with FITC-labeled TNF-α ASON. HSV-retinitis was induced on day 0 by the injection of HSV-1 (KOS strain) into the anterior chamber (AC) of the right eyes of BALB/c mice (von Szily model). The left contralateral eyes were injected intravitreally on day 7 with TNF-α ASON, sequence-unspecific control ASON (CON), or buffer. The clinical course of retinitis, ocular inflammatory cell-infiltration, TNF-α expression in the eye by ELISA, delayed-type hypersensitivity (DTH) reaction, virus-neutralizing antibody titers in the serum, uptake of [3H]thymidine from regional lymph node (rln) cells, and viral content in the eyes were determined. RESULTS: In vivo, strong fluorescence of FITC- TNF-α ASON was detected in the choroid and retina up to 3 days after intravitreal injection, but none in the rln. After treatment of eyes with ASON, decreased expression of TNF-α in the eye, and reduced incidence and severity of retinitis on day 10 after infection (P < 0.05) could be found. The other parameters were not significantly influenced after TNF-α ASON treatment. CONCLUSIONS: TNF-α participates in the pathology of HSV-1 retinitis. Local inhibition of TNF-α mRNA by intraocular TNF-α ASON injection did not influence the systemic HSV-specific immune response or the antiviral response in the eye, but reduced ocular inflammatory bystander damage.
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Infecciones Virales del Ojo/terapia , Herpes Simple/terapia , Herpesvirus Humano 1/fisiología , Oligonucleótidos Antisentido/uso terapéutico , Síndrome de Necrosis Retiniana Aguda/terapia , Factor de Necrosis Tumoral alfa/genética , Animales , Cámara Anterior/virología , Anticuerpos Neutralizantes , Ensayo de Inmunoadsorción Enzimática , Infecciones Virales del Ojo/patología , Infecciones Virales del Ojo/virología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Proteína Ácida Fibrilar de la Glía , Herpes Simple/patología , Herpes Simple/virología , Hipersensibilidad Tardía/inmunología , Inyecciones Intravítreas , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/genética , Síndrome de Necrosis Retiniana Aguda/patología , Síndrome de Necrosis Retiniana Aguda/virología , Resultado del Tratamiento , Ensayo de Placa ViralRESUMEN
The purpose of this study was to ascertain whether a correlation exists between glaucoma-associated alteration of ocular vascular haemodynamics and endothelin-1 (ET-1) levels exist. Eyes of patients with cataract (n = 30) or glaucoma (n = 68) were examined with optical coherence tomography (OCT) and OCT-angiography (OCT-A; AngioVue™-RTVue-XR; Optovue, Fremont, California, USA). The peripapillary and the macular vessel density (VD) values were measured. Inferior and superior retinal nerve fibre layer (RNFL) thickness loss was used for further OCT staging. Aqueous humour of the examined eye and plasma were sampled during cataract or glaucoma surgery and analysed by means of ELISA to determine their ET-1 level. Glaucoma eyes are characterised by reductions in RNFL thickness and VD that correlate significantly with the OCT GSS score. Peripheral and ocular ET-1 level were significantly elevated in patients with glaucoma and correlate positively with the OCT-GSS score of the entire study population. Peripapillary and macula VD of glaucoma patients correlates negatively with plasma ET-1 levels. Multivariable analysis showed a subordinate role of intraocular pressure predictive factor for impaired retinal blood flow compared with plasma ET-1 level in glaucoma. Peripheral ET-1 level serves as risk factor for detection of ocular blood flow changes in the optic nerve head region of glaucomatous eyes.
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Catarata , Endotelina-1 , Ojo , Glaucoma , Catarata/metabolismo , Catarata/patología , Endotelina-1/metabolismo , Ojo/irrigación sanguínea , Glaucoma/metabolismo , Glaucoma/patología , Hemodinámica , Humanos , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Flujo Sanguíneo Regional , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Factores de Riesgo , Tomografía de Coherencia Óptica/métodosRESUMEN
Purpose: To analyze circulating immune cells in patients with anterior uveitis (AU) associated to axial spondyloarthritis (SpA), or juvenile idiopathic arthritis (JIA).Methods: Venous blood samples were collected from healthy controls (n = 16), and either SpA (n = 19) or JIA (n = 23) patients with associated anterior uveitis (AU) during active flare, or after ≥3 months of inactivity. Frequencies of CD56+, MHC-I+, and S100A9+ monocytes, CCR7+ dendritic cells, CD56+dim natural killer (NK) cells and CD3+CD56bright T-cells were analyzed via flow cytometry. Serum S100A8/A9 levels were determined via ELISA.Results: SpA patients showed a reduced frequency of CD56+dim NK cells during uveitis activity, a constitutively activated monocyte phenotype, and elevated S100A8/A9 serum levels. In contrast, JIAU patients showed elevated frequencies of CD56+ monocytes and CCR7+ DC.Conclusion: Phenotype of peripheral immune cells differ between patients, probably contributing to different courses of acute onset AU in SpA and insidious onset AU in JIAU patients.Abbreviations: AU: anterior uveitis, AR: arthritis, JIA: juvenile idiopathic arthritis, SpA: axial spondyloarthritis.
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Artritis Juvenil/inmunología , Espondiloartritis Axial/inmunología , Inmunidad Innata/fisiología , Uveítis Anterior/inmunología , Adolescente , Adulto , Calgranulina A/sangre , Calgranulina B/sangre , Niño , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Células Asesinas Naturales/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Fenotipo , Linfocitos T/inmunologíaRESUMEN
Uveitis describes a heterogeneous group of inflammatory eye diseases characterized by infiltration of leukocytes into the uveal tissues. Uveitis associated with the HLA haplotype B27 (HLA-B27) is a common subtype of uveitis and a prototypical ocular immune-mediated disease. Local immune mechanisms driving human uveitis are poorly characterized mainly due to the limited available biomaterial and subsequent technical limitations. Here, we provide the first high-resolution characterization of intraocular leukocytes in HLA-B27-positive (n = 4) and -negative (n = 2) anterior uveitis and an infectious endophthalmitis control (n = 1) by combining single-cell RNA-sequencing with flow cytometry and protein analysis. Ocular cell infiltrates consisted primarily of lymphocytes in both subtypes of uveitis and of myeloid cells in infectious endophthalmitis. HLA-B27-positive uveitis exclusively featured a plasmacytoid and classical dendritic cell (cDC) infiltrate. Moreover, cDCs were central in predicted local cell-cell communication. This suggests a unique pattern of ocular leukocyte infiltration in HLA-B27-positive uveitis with relevance to DCs.
Uveitis is a form of inflammation in the eye. It can occur in response to infection, or when the immune system mistakenly attacks the eye, in what is known as autoimmune uveitis. In approximately 80 percent of cases, the front part of the eye is affected. During an inflammatory episode, the liquid inside the front part of the eye fills with immune cells, but the nature of these cells remains unknown. This is because uveitis is rare, and doctors cannot routinely take samples from inside the eyes of affected individuals to diagnose the disease. This lack of samples makes research into this disease challenging. There are two main groups of immune cells that could be responsible for uveitis: myeloid cells and lymphoid cells. Myeloid cells form the first line of immune defense against infection by non-specifically attacking and removing pathogens . Lymphoid cells form the second line of immune defense, attacking specific pathogens. Lymphoid cells also have long-term memory, meaning they can 'remember' previous infections and fight them more effectively. Lymphoid cells receive instructions from a type of myeloid cell called a dendritic cell about what to attack. Dendritic cells relay their instructions to lymphoid cells using molecules called human leukocyte antigens (HLA). Autoimmune uveitis affecting the front part of the eye is common in individuals with an HLA type called HLA-B27, suggesting that communication between dendritic and lymphoid cells plays an important role in this type of inflammation. To make the most of limited patient samples, Kasper et al. used single cell techniques to examine the immune cells from the fluid inside the eye. Six samples came from people with autoimmune uveitis, and one from a person with an eye infection. The infection sample contained mainly myeloid cells that might attack bacteria responsible for the infection. In contrast, the autoimmune uveitis samples contained mainly lymphoid cells. Of these samples, four were from individuals with the gene that codes for the HLA-B27 molecule. These samples had a unique pattern of immune cells, with more dendritic cells than the samples from individuals that did not have this gene. This study included only a small number of individuals, but it shows that analysing single immune cells from the eye is possible in uveitis. This snapshot could help researchers understand the local immune response in the eye, and find an optimal treatment.
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Células Dendríticas/clasificación , Antígeno HLA-B27/inmunología , Uveítis Anterior/patología , Endoftalmitis/patología , Femenino , Humanos , Linfocitos , Masculino , Células Mieloides , Análisis de Secuencia de ARN , Uveítis Anterior/inmunologíaRESUMEN
The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.
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Acetilmuramil-Alanil-Isoglutamina/farmacología , Adyuvantes Inmunológicos/farmacología , Ácido Diaminopimélico/análogos & derivados , Microglía/efectos de los fármacos , Proteína Adaptadora de Señalización NOD1/agonistas , Proteína Adaptadora de Señalización NOD2/agonistas , Fenotipo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Forma de la Célula , Extensiones de la Superficie Celular/efectos de los fármacos , Células Cultivadas , Ácido Diaminopimélico/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microglía/citología , Microglía/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Patients with chronic anterior uveitis are at particularly high risk of developing secondary glaucoma when corticosteroids [e.g., dexamethasone (Dex)] are used or when inflammatory activity has regressed. Macrophage migration into the eye increases when secondary glaucoma develops and may play an important role in the development of secondary glaucoma. Our aim was to evaluate in vitro if increased hydrostatic pressure and corticosteroids could induce changes in macrophages phenotype. By using a pressure chamber cell culture system, we assessed the effect of increased hydrostatic pressure (HP), inflammation, and immunosuppression (Dex) on the M1/M2 phenotype of macrophages. Bone marrow-derived macrophages (BMDMs) were stimulated with medium, lipopolysaccharide (LPS, 100 ng/ml), Dex (200 ng/ml), or LPS + Dex and incubated with different HP (0, 20, or 60 mmHg) for 2 or 7 days. The numbers of CD86+/CD206- (M1 phenotype), CD86-/CD206+ (M2 phenotype), CD86+/CD206+ (intermediate phenotype), F4/80+/TNF-α+, and F4/80+/IL-10+ macrophages were determined by flow cytometry. TNF-α and IL-10 levels in cell culture supernatants were quantified by ELISA. TNF-α, IL-10, fibronectin, and collagen IV expression in BMDMs were detected by immunofluorescence microscopy. Higher HP polarizes macrophages primarily to an M1 phenotype (LPS, 60 vs. 0 mmHg, d2: p = 0.0034) with less extra cellular matrix (ECM) production and secondary to an M2 phenotype (medium, 60 vs. 0 mmHg, d7: p = 0.0089) (medium, 60 vs. 20 mmHg, d7: p = 0.0433) with enhanced ECM production. Dex induces an M2 phenotype (Dex, medium vs. Dex, d2: p < 0.0001; d7: p < 0.0001) with more ECM production. Higher HP further increased M2 polarization of Dex-treated macrophages (Dex, 60 vs. 0 mmHg, d2: p = 0.0417; d7: p = 0.0454). These changes in the M1/M2 phenotype by high HP or Dex treatment may play a role in the pathogenesis of secondary uveitic glaucoma- or glucocorticoid (GC)-induced glaucoma.
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Presión Hidrostática/efectos adversos , Macrófagos/inmunología , Animales , Supervivencia Celular , Células Cultivadas , Colágeno Tipo IV/metabolismo , Dexametasona/farmacología , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Glaucoma/etiología , Glaucoma/inmunología , Inflamación , Interleucina-10/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Fenotipo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Microglia represent the primary resident immune cells of the central nervous system (CNS) and modulate local immune responses. Depending on their physiological functions, microglia can be classified into pro- (M1) and anti-inflammatory (M2) phenotype. Interleukin (IL)-10 is an important modulator of neuronal homeostasis, with anti-inflammatory and neuroprotective functions, and can be released by microglia. Here, we investigated how IL-10 deficiency affected the M1/2 polarization of primary microglia upon lipopolysaccharide (LPS) stimulation in vitro. Microglia phenotypes were analyzed via flow cytometry. Cytokine and chemokine secretion were examined by ELISA and bead-based multiplex LEGENDplexTM. Our results showed that genetic depletion of IL-10 led to elevated M1 like phenotype (CD86+ CD206-) under pro-inflammatory conditions associated with increased frequency of IL-6+, TNF-α+ cells and enhanced release of several pro-inflammatory chemokines. Absence of IL-10 led to an attenuated M2 like phenotype (CD86- CD206+) and a reduced secretion of TGF-ß1 upon LPS stimulation. In conclusion, IL-10 deficiency may promote the polarization of microglia into M1-prone phenotype under pro-inflammatory conditions.
RESUMEN
The purpose of the current study was to analyze the binding patterns of serum autoantibodies from juvenile idiopathic arthritis (JIA) and JIA-associated uveitis (JIAU) patients to proteomes from different ocular tissues and to identify potential ocular autoantigens in JIAU. Proteomes from porcine iris, ciliary body, or retina tissue were isolated, separated using 2D-gel electrophoresis, and transferred to a blotting membrane. The binding pattern of serum antibodies from JIA or JIAU patients or healthy controls to ocular proteins was visualized by using anti-human IgG secondary antibodies and chemiluminescence reaction. Selected protein spots were excised from silver-stained 2D gels and subjected to mass spectrometry. Serum antibodies binding to ocular proteins were detected in all patient groups and healthy controls. Irrespective of the patient groups, serum antibodies bound to 49 different protein spots of the retina proteome, to 53 of the ciliary body proteome, and to 44 of the iris proteome. The relative binding frequency of sera to these iris protein spots was significantly higher in JIAU than in JIA patients or healthy controls. Particularly in JIAU patients, cluster analyses indicated a broad range of serum antibodies directed against ocular antigens, mostly in the iris proteome. Iris proteins frequently bound by serum antibodies in all groups were identified as tubulin beta chain, vimentin, ATP synthase subunit beta, actin, and L-lactate dehydrogenase B chain. Iris proteins exclusively bound by JIAU serum antibodies were heat shock cognate 71 kDa protein and keratin. Although serum autoantibody binding to ocular antigens was not disease-specific, a significant diversity of autoantibodies against a broad range of antigens, particularly from the iris tissue, was detected in JIAU patients. As the iris is a major site of inflammation in JIAU, the present data give further evidence that autoantibodies may be involved in JIAU immunopathology.
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Artritis Juvenil/complicaciones , Autoantígenos/análisis , Ojo/inmunología , Uveítis/inmunología , Adolescente , Animales , Autoanticuerpos/inmunología , Niño , Preescolar , Análisis por Conglomerados , Femenino , Humanos , Masculino , Proteoma , Porcinos , Uveítis/etiologíaRESUMEN
Patients with juvenile idiopathic arthritis often develop chronic anterior uveitis (JIAU). JIAU patients possess a particularly high risk for developing secondary glaucoma when inflammatory inactivity has been achieved. By using multiplex bead assay analysis, we assessed levels of pro- and anti-inflammatory cytokines, chemokines, or metalloproteinases in the aqueous humor (AH) of patients with clinically inactive JIAU with (JIAUwG) or without secondary glaucoma (JIAUwoG), or from patients with senile cataract as controls. Laser-flare photometry analysis prior to surgery showed no significant differences between JIAUwG or JIAUwoG. Compared with the control group, levels of interleukin-8, matrix metalloproteinase-2, -3, -9, serum amyloid A (SAA), transforming growth factor beta-1, -2, -3 (TGFß-1, -2, -3), and tumor necrosis factor-alpha in the AH were significantly higher in patients with clinically inactive JIAUwG or JIAUwoG. Samples from JIAwoG patients displayed significantly higher levels of SAA (P < 0.0116) than JIAUwG patients. JIAUwG patients showed an increased level of TGFß-2 in AH samples compared with JIAUwoG (P < 0.0009). These molecules may contribute to the clinical development of glaucoma in patients with JIAU.
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Humor Acuoso/metabolismo , Artritis Juvenil/complicaciones , Citocinas/metabolismo , Glaucoma/etiología , Glaucoma/metabolismo , Uveítis Anterior/etiología , Uveítis Anterior/metabolismo , Adolescente , Factores de Edad , Anciano , Anciano de 80 o más Años , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Artritis Juvenil/tratamiento farmacológico , Artritis Juvenil/metabolismo , Biomarcadores , Niño , Preescolar , Humanos , Mediadores de Inflamación , Factores de Riesgo , Uveítis Anterior/complicaciones , Uveítis Anterior/tratamiento farmacológicoRESUMEN
HLA-B27 is the allele most frequently associated with human anterior uveitis. The majority of HLA-B27-positive [acute anterior uveitis (AAU)] patients develop clinically distinct symptoms with acute symptomatic onset of flare and a recurrent disease course characterized by a massive cellular ocular infiltrate during uveitis relapse. By contrast, uveitis in HLA-B27-negative [idiopathic anterior uveitis (IAU)] patients tends to develop a clinically less fulminant, more chronic, and typically asymptomatic disease course. To analyze systemic immune responses in the different uveitis entities, we analyzed peripheral blood cells by flow cytometry. In addition, as a pro-inflammatory biomarker serum, S100A8/A9 levels were quantified by ELISA from patients with AAU (n = 27) and IAU (n = 21), and in healthy controls (n = 30). Data were obtained either during active uveitis flare or after 3 months of inactivity. IAU patients showed a transiently increased frequency of CD56- and CD163-positive monocytes and of both granulocytic myeloid-derived suppressor cells and Th17 cells during active uveitis. By contrast, AAU patients showed an elevated frequency of monocytes, activated T cells, and elevated S100A8/A9 serum levels during clinically quiescent disease. The differentially regulated response of both innate and adaptive immune cells in the blood may be related to the clinically distinct characteristics of the two different uveitis entities.
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Antígeno HLA-B27/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Uveítis Anterior/etiología , Uveítis Anterior/metabolismo , Adulto , Autoinmunidad , Biomarcadores , Calgranulina A/sangre , Calgranulina B/sangre , Estudios de Casos y Controles , Citocinas/metabolismo , Femenino , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Fenotipo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Uveítis Anterior/diagnóstico , Adulto JovenRESUMEN
The increasing incidence of aciclovir- (ACV) resistant strains in patients with ocular herpes simplex virus (HSV) infections is a major health problem in industrialized countries. In the present study, the humanized monoclonal antibody (mAb) hu2c targeting the HSV-1/2 glycoprotein B was examined for its efficacy towards ACV-resistant infections of the eye in the mouse model of acute retinal necrosis (ARN). BALB/c mice were infected by microinjection of an ACV-resistant clinical isolate into the anterior eye chamber to induce ARN and systemically treated with mAb hu2c at 24h prior (pre-exposure prophylaxis) or at 24, 40, and 56h after infection (post-exposure immunotherapy). Mock treated controls and ACV-treated mice showed pronounced retinal damage. Mice treated with mAb hu2c were almost completely protected from developing ARN. In conclusion, mAb hu2c may become a reliable therapeutic option for drug/ACV-resistant ocular HSV infections in humans in order to prevent blindness.
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Aciclovir/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/inmunología , Herpesviridae/efectos de los fármacos , Inmunoterapia , Retinitis/virología , Animales , Antivirales/farmacología , Farmacorresistencia Viral , Femenino , Herpesviridae/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Retinitis/inmunologíaRESUMEN
Herpes simplex virus (HSV) is a leading cause of blindness and viral encephalitis in the developed world. Upon reactivation from sensory neurons, HSV returns via axonal transport to peripheral tissues where it causes, e.g., severe, potentially blinding ocular diseases. In the present study we investigated whether the HSV-1/2 glycoprotein B-specific antibody mAb 2c or its humanized counterpart mAb hu2c can protect from ocular disease in a mouse model of HSV-1-induced acute retinal necrosis (ARN). In this model the viral spread from the initially infected to the contralateral eye resembles the routes taken in humans upon HSV reactivation. Systemic antibody treatment prior or early after infection effectively protected the mice from the development of ARN. These observations suggest that the antibody potently neutralized the infection and inhibited the viral transmission, since there was almost no virus detectable in the contralateral eyes and trigeminal ganglia of antibody treated mice. Besides of neutralizing free virus or limiting the infection via activating the complement or cellular effector functions, blocking of the anterograde directed neuron-to-cell spread of HSV represents a viable mode of action how mAb 2c protected the mice from ARN. We proved this hypothesis using a microfluidic chamber system. Neurons and epithelial cells were cultured in two separate compartments where the neurons sent axons via connecting microgrooves to the epithelial cells. Neurons were infected with a reporter HSV-1 strain expressing mCherry, and the co-culture was treated with neutralizing antibodies. In contrast to commercial polyclonal human HSV-neutralizing immunoglobulins, mAb 2c effectively blocked the anterograde directed neuron-to-cell transmission of the virus. Our data suggest that the humanized HSV-1/2-gB antibody protects mice from ocular disease by blocking the neuronal spread of HSV. Therefore, mAb hu2c may become a potent novel therapeutic option for severe ocular HSV infections.
RESUMEN
PURPOSE: To study matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the corneas from mice with ulcerative herpes stromal keratitis (HSK) treated with amniotic membrane transplantation (AMT). METHODS: The corneas from BALB/c mice were infected with HSV-1. Mice with ulcerative HSK on postinfection (PI) day 14 were used for the experiments. In one group of mice, the corneas were treated with transplantation of amniotic membrane (AMT) that was secured with a tarsorrhaphy, and a control group underwent tarsorrhaphy alone. After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically. Corneal sections were studied by immunohistochemistry for the expression of MMP-2, -8, and -9 and TIMP-1 and -2. MMP activity in the corneas was investigated by zymography, and the expression of the enzymes was measured by the Western blot technique. RESULTS: At day 14 PI, the ulcers stained intensely positive for MMP-2, -8, and -9 and TIMP-1 and -2. Ulceration (P < 0.001), stromal inflammation (P < 0.01) and inflammatory cell infiltration (P < 0.001) markedly improved by day 2 after AMT. This was associated with reduced expression (P < 0.01) and activity of MMP-8, and -9 and increased localization of TIMP-1 (P < 0.01), whereas TIMP-2 was not affected. In contrast, high levels of expression of MMP-8 and -9 remained in the cornea after tarsorrhaphy, and the TIMP-1 expression was only slightly upregulated. CONCLUSIONS: Rapid improvement of HSV-1-induced ulcerative keratitis is noted after amniotic membrane transplantation. This may be caused by reduced expression and activity of MMP-8 and -9, increased expression of TIMP-1, and sustained expression of TIMP-2.
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Amnios/trasplante , Sustancia Propia/enzimología , Queratitis Herpética/cirugía , Metaloproteinasas de la Matriz/metabolismo , Animales , Western Blotting , Sustancia Propia/virología , Párpados/cirugía , Femenino , Herpesvirus Humano 1/patogenicidad , Técnicas para Inmunoenzimas , Interleucina-1/metabolismo , Queratitis Herpética/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.
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Anticuerpos Monoclonales/inmunología , Glicoproteínas/inmunología , Queratitis Herpética/inmunología , Queratitis Herpética/prevención & control , Simplexvirus/inmunología , Aciclovir/inmunología , Animales , Antivirales/inmunología , Chlorocebus aethiops , Sustancia Propia/inmunología , Sustancia Propia/virología , Femenino , Herpes Simple/complicaciones , Herpes Simple/virología , Inmunoglobulinas/inmunología , Queratitis Herpética/etiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células VeroRESUMEN
PURPOSE: Tumor necrosis factor (TNF)-alpha is a pleiotropic factor that is critical for the development of inflammation. The authors investigated whether topical application of TNF-alpha-antagonizing molecules, antisense oligonucleotides (ASON), might be an effective way of modifying the course of immune-mediated herpetic stromal keratitis (HSK). METHODS: ASON targeting TNF-alpha mRNA were examined for their efficiency in interfering with the production of this cytokine in vitro. In vivo uptake was determined by FITC-labeled ASON. HSV-1 corneally infected mice were injected three times with ASON. Mice from the control groups received unrelated control oligonucleotides (CON) or buffer. The clinical course of HSK, the delayed-type hypersensitivity (DTH) reaction, the uptake of [(3)H]thymidine from cells derived from the spleen, virus-neutralizing antibody titers in the serum, and viral replication in the infected eyes were determined. The eyes were examined histologically. The corneal TNF-alpha content was measured by ELISA. RESULTS: The TNF-alpha ASON reduced the lymphocytic cytokine expression in vitro. In vivo, the FITC-labeled molecules were detected in the cornea even after 10 days. In the TNF-alpha ASON mice the incidence of HSK decreased, and the severity of the disease was diminished. The corneal content of TNF-alpha was reduced, and the number of inflammatory cells was decreased. The other investigated parameters were not significantly altered by TNF-alpha ASON treatment. CONCLUSIONS: The data suggest that TNF-alpha ASON diminishes the release of TNF-alpha from cultured lymphocytes and from lymphocytes in the HSV-1-infected cornea. This topical treatment mitigates the course of HSK, whereas the systemic antiviral effector functions were not impaired.