RESUMO
Existing flow cytometry approaches identify immune cells using the whole infected/inflamed cornea, which limits its ability to distinguish the immune cells infiltrating the corneal epithelium from the corneal stroma. Here, we present a protocol to analyze immune cells in the separated epithelium and stroma from naïve and herpes simplex virus-1 (HSV-1)-infected mouse corneas. We describe steps for viral infection, separation of corneal epithelium from stroma, preparation of a single-cell suspension of the individual epithelium and stroma, and flow cytometry assay.
Assuntos
Herpesvirus Humano 1 , Ceratite Herpética , Camundongos , Animais , Ceratite Herpética/diagnóstico , Citometria de Fluxo , Córnea , EpitélioRESUMO
PURPOSE: To characterize CXCR4-expressing cells in uninfected and herpes simplex virus-1 (HSV-1) infected corneas. METHODS: The corneas of C57BL/6J mice were infected with HSV-1 McKrae. The RT-qPCR assay detected CXCR4 and CXCL12 transcripts in uninfected and HSV-1-infected corneas. Immunofluorescence staining for CXCR4 and CXCL12 protein was performed in the frozen sections of herpes stromal keratitis (HSK) corneas. Flow cytometry assay characterized the CXCR4-expressing cells in uninfected and HSV-1-infected corneas. RESULTS: Flow cytometry data showed CXCR4 expressing cells in the separated epithelium and stroma of uninfected corneas. In the uninfected stroma, CD11b + F4/80+ macrophages are the predominant CXCR4-expressing cells. In contrast, most CXCR4 expressing cells in the uninfected epithelium were CD207 (langerin)+, CD11c+, and expressed MHC class II molecule, documenting the Langerhans cells (LCs) phenotype. After corneal HSV-1 infection, CXCR4 and CXCL12 mRNA levels increased significantly in HSK corneas than in uninfected corneas. Immunofluorescence staining showed CXCR4 and CXCL12 protein localization in the newly formed blood vessels in the HSK cornea. Furthermore, the infection resulted in LCs proliferation, causing an increase in their numbers in the epithelium at 4 days post-infection (p.i.). However, by 9-day p.i., the LCs numbers declined to the counts observed in naïve corneal epithelium. Our results also showed neutrophils and vascular endothelial cells as the prominent CXCR4-expressing cell types in the stroma of HSK corneas. CONCLUSIONS: Together, our data demonstrate the expression of CXCR4 on resident antigen presenting cells in the uninfected cornea and on infiltrating neutrophils and newly formed blood vessels in the HSK cornea.
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Herpesvirus Humano 1 , Ceratite Herpética , Animais , Camundongos , Córnea/metabolismo , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Reactive oxygen species (ROS) play an important role in tissue inflammation. In this study, we measured the intracellular level of ROS in herpes stromal keratitis (HSK) corneas and determined the outcome of manipulating ROS level on HSK severity. Our results showed the predominance of ROS generation in neutrophils but not CD4 T cells in HSK corneas. NADPH oxidase 2 (NOX2) enzyme is known to generate ROS in myeloid cells. Our results showed baseline expression of different NOX2 subunits in uninfected corneas. After corneal herpes simplex virus-1 (HSV-1) infection, an enhanced expression of NOX2 subunits was detected in infected corneas. Furthermore, flow cytometry results showed a higher level of gp91 (Nox2 subunit) protein in neutrophils from HSK corneas, suggesting the involvement of NOX2 in generating ROS. However, no significant decrease in ROS level was noticed in neutrophils from HSV-1-infected gp91-/- mice than in C57BL/6J (B6) mice, suggesting NOX2 is not the major contributor in generating ROS in neutrophils. Next, we used diphenyleneiodonium (DPI), a flavoenzyme inhibitor, to pharmacologically manipulate the ROS levels in HSV-1-infected mice. Surprisingly, the neutrophils from peripheral blood and corneas of the DPI-treated group exhibited an increased level of ROS than the vehicle-treated group of infected B6 mice. Excessive ROS is known to cause cell death. Accordingly, DPI treatment resulted in a significant decrease in neutrophil frequency in peripheral blood and corneas of infected mice and was associated with reduced corneal pathology. Together, our results suggest that regulating ROS levels in neutrophils can ameliorate HSK severity. IMPORTANCE Neutrophils are one of the primary immune cell types involved in causing tissue damage after corneal HSV-1 infection. This study demonstrates that intracellular ROS production in the neutrophils in HSK lesions is not NOX2 dependent. Furthermore, manipulating ROS levels in neutrophils ameliorates the severity of HSK lesions. Our findings suggest that excessive intracellular ROS in neutrophils disrupt redox homeostasis and affect their survival, resulting in a decrease in HSK lesion severity.
Assuntos
Herpes Simples , Ceratite Herpética , Neutrófilos , Oniocompostos , Animais , Linfócitos T CD4-Positivos , Córnea , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1 , Ceratite Herpética/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/metabolismo , Neutrófilos/metabolismo , Oniocompostos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/metabolismoRESUMO
The involvement of leukocytes in the pathophysiology of DR has mostly examined the role of monocytes and neutrophils with little emphasis on other immune cell types. In this study, we determined the systemic alterations in T cell subsets, myeloid cell types, NK cells, and NKT cells in the streptozotocin (STZ) mouse model of diabetic retinopathy (DR), and the role of NKT cells on retinal leukostasis and permeability changes. C57BL/6 J mice were made diabetic with 60 mg/kg dose of STZ given for 5-days. Flow cytometry assay measured the frequency of leukocyte subsets in the peripheral blood, spleen, and bone marrow of STZ- and vehicle-treated C57BL/6 J mice. Our results showed an increased proportion of memory CD8 T cells and interferon-gamma (IFN-γ) secreting CD8 T cells in the bone marrow of STZ-treated compared to control mice. Subsequently, increased production of inflammatory monocytes in the bone marrow and an enhanced frequency of CD11b + cells in the diabetic retina were seen in STZ-treated compared to control mice. The diabetic mice also exhibited a decrease in total NKT and CD4+NKT cells. A monoclonal antibody-based approach depleted NKT cells from STZ-treated mice, followed by measurements of retinal vascular permeability and leukostasis. The depletion of NKT cells in STZ-treated mice resulted in a significant increase in vascular permeability in the retinal tissue. Together, our results strongly imply the involvement of NKT cells in regulating the pathophysiology of the diabetic retina.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética/patologia , Leucostasia/patologia , Células T Matadoras Naturais/patologia , Retina/patologia , Animais , Retinopatia Diabética/metabolismo , Leucostasia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , Retina/metabolismoRESUMO
Purpose: The goal of this study was to determine the role of insulin-like growth factor-binding protein-3 (IGFBP-3) in the pathogenesis of herpes stromal keratitis (HSK). Methods: In an unbiased approach, a membrane-based protein array was carried out to determine the level of expression of pro- and anti-angiogenic molecules in uninfected and HSV-1 infected corneas. Quantitative RT-PCR and ELISA assays were performed to measure the amounts of IGFBP-3 at mRNA and protein levels. Confocal microscopy documented the localization of IGFBP-3 in uninfected and infected corneal tissue. Flow cytometry assay showed the frequency of immune cell types in infected corneas from C57BL/6J (B6) and IGFBP-3 knockout (IGFBP-3-/-) mice. Slit-lamp microscopy was used to quantitate the development of opacity and neovascularization in infected corneas from both groups of mice. Results: Quantitation of protein array dot blot showed an increased level of IGFBP-3 protein in HSV-1 infected than uninfected corneas and was confirmed with ELISA and quantitative RT-PCR assays. Cytosolic and nuclear localization of IGFBP-3 were detected in the cells of corneal epithelium, whereas scattered IGFBP-3 staining was evident in the stroma of HSK developing corneas. Increased opacity and hemangiogenesis were noted in the corneas of IGFBP-3-/- than B6 mice during the clinical period of HSK. Furthermore, an increased number of leukocytes comprising of neutrophils and CD4 T cells were found in HSK developing corneas of IGFBP-3-/- than B6 mice. Conclusions: Our data showed that lack of IGFBP-3 exacerbates HSK, suggesting the protective effect of IGFBP-3 protein in regulating the severity of HSK.
Assuntos
Córnea/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Ceratite Herpética/metabolismo , Animais , Neovascularização da Córnea/patologia , Opacidade da Córnea/patologia , Substância Própria/metabolismo , Herpesvirus Humano 1 , Ceratite Herpética/patologia , Ceratite Herpética/virologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Herpes stromal keratitis (HSK) is a chronic immunoinflammatory condition which develops in response to recurrent herpes simplex virus-1 (HSV-1) infection of the cornea. Patients with HSK often demonstrate the concurrence of corneal desiccation and the loss of blink reflex. However, the relationship between severity of HSK, level of basal tears and inflammation of the lacrimal gland is mostly unexplored. In this study, we compared these variables in extraorbital lacrimal gland (EoLG) after corneal HSV-1 infection in the C57BL/6J mouse model. Our results showed a significant reduction in the volume of tears in infected eyes during the development of HSK. Extensive architectural damage to EoLG, presumably caused by a massive influx of interferon-gamma secreting T cells, was observed during clinical disease period of HSK. A positive correlation between the decrease in tear volume, severity of HSK and the damage to EoLG were evident in infected mice. The presence of infectious virus measured in EoLG during pre-clinical, but not clinical disease period of HSK, suggested that viral cytopathic effects are not the major contributors of extensive damage seen in EoLG. Furthermore, topical administration of lacritin peptide delayed but did not prevent the decrease in tears in HSV-1 infected mice, and had no significant effect in either reducing the severity of HSK or T cell infiltration in EoLG of infected mice. Together, our results showed an interplay between the severity of HSK, inflammation of EoLG, and the reduced level of tears after corneal HSV-1 infection.
Assuntos
Substância Própria/patologia , Dacriocistite/fisiopatologia , Modelos Animais de Doenças , Ceratite Herpética/fisiopatologia , Animais , Linfócitos T CD4-Positivos/imunologia , Dacriocistite/tratamento farmacológico , Dacriocistite/imunologia , Dacriocistite/virologia , Feminino , Glicoproteínas/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/fisiopatologia , Inflamação/virologia , Ceratite Herpética/tratamento farmacológico , Ceratite Herpética/imunologia , Ceratite Herpética/virologia , Camundongos , Camundongos Endogâmicos C57BL , Lágrimas/metabolismoRESUMO
Chronic inflammation in tissues often causes the development of hypoxia. Herpes stromal keratitis (HSK) is a corneal chronic inflammatory condition that develops in response to recurrent HSV-1 infection. In this study, we investigated the development of hypoxia, the expression of hypoxia-associated glycolytic genes in HSV-1 infected corneas, and the outcome of blocking hypoxia-inducible factor (HIF) dimerization on the severity of HSK. Our results showed the development of hypoxia, an elevated expression of hypoxia-associated glycolytic genes, and an increased level of lactate in corneas with progressing HSK lesions. The magnitude of hypoxia correlated with the extent of neutrophils infiltrating the infected corneas, and the depletion of neutrophils reduced the development of hypoxia in infected corneas. Additionally, in progressing HSK lesions, nuclear localization of HIF-2α protein was detected in corneal epithelial cells, whereas HIF-1α protein stabilization was observed in infiltrating immune cells. Administration of acriflavine drug to HSV-1-infected mice inhibited nuclear accumulation of HIF-1α and HIF-2α protein in immune cell types and epithelial cells, respectively, in infected corneas. As a result, a decreased influx of CD4 T cells and nongranulocytic myeloid cells, but an increased influx of neutrophils, was noted in developing HSK lesions. Interestingly, acriflavine treatment given during the clinical disease period decreased neovascularization but increased the opacity in HSV-1-infected corneas. Taken together, the results of our study lay the foundation to dissect the role of inflammatory hypoxia and hypoxia-associated genes in the pathogenesis of HSK.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Córnea/imunologia , Epitélio Corneano/metabolismo , Herpesvirus Humano 1/fisiologia , Hipóxia/imunologia , Inflamação/imunologia , Ceratite Herpética/imunologia , Neutrófilos/imunologia , Acriflavina/uso terapêutico , Transporte Ativo do Núcleo Celular , Animais , Anti-Infecciosos Locais/uso terapêutico , Movimento Celular , Células Cultivadas , Córnea/fisiologia , Córnea/virologia , Modelos Animais de Doenças , Progressão da Doença , Epitélio Corneano/patologia , Feminino , Glicólise/genética , Humanos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Although autacoids primarily derived from the cyclooxygenase-2 and 5-lipoxygenase (LOX) pathways are essential mediators of inflammation, endogenous specialized proresolving mediators (SPMs) act as robust agonists of resolution. SPM biosynthesis is initiated by the conversion of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid primarily via the 12/15-LOX pathway. Although 12/15-LOX activity is prominent in the cornea, the role of SPM pathway activation during infection remains largely unknown and is the focus of the current study. Pseudomonas keratitis was induced in resistant BALB/c and susceptible C57BL/6 (B6) mice. Biosynthetic pathways for proinflammatory autacoids and SPMs were assessed. Divergent lipid mediator profiles demonstrate the importance of 15-LOX pathways in the pathogenesis of ocular infectious disease. Results indicate that an imbalance of LOX enzymatic pathways contributes to susceptibility observed in B6 mice where deficient activation of SPM circuits, as indicated by reduced 15-hydroxy-eicosatetraenoic acid and 17-hydroxydocosahexaenoic acid levels, prevented transition toward resolution and led to chronic inflammation. In sharp contrast, BALB/c mice demonstrated a well-balanced axis of 5-LOX/12-LOX/15-LOX pathways, resulting in sufficient proresolving bioactive metabolite formation and immune homeostasis. Furthermore, a novel immunoregulatory role for 15-LOX was revealed in inflammatory cells (polymorphonuclear leukocytes and macrophages), which influenced phagocytic activity. These data provide evidence that SPM circuits are essential for host defense during bacterial keratitis.-Carion, T. W., Greenwood, M., Ebrahim, A. S., Jerome, A., Suvas, S., Gronert, K., Berger, E. A. Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Inflamação/tratamento farmacológico , Ceratite/tratamento farmacológico , Animais , Araquidonato 15-Lipoxigenase/efeitos dos fármacos , Araquidonato 15-Lipoxigenase/imunologia , Ácido Eicosapentaenoico/farmacologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismoRESUMO
Psbetaeudomonas (P.) aeruginosa infection of the cornea in BALB/c mice does not result in perforation and the mice have been classified as resistant. However, regulation of this response via inflammasome activation remained untested. Therefore, BALB/c mice were infected with P. aeruginosa ATCC strain 19660 and NLRP3 and NLRC4 protein tested by ELISA. Since NLRC4 vs NLRP3 protein levels were significantly higher in the corneas of BALB/c at 1 and 5 days postinfection we used silencing to knockdown NLRC4. Silencing NLRC4 vs scrambled siRNA treatment exacerbated disease in BALB/c mice, reduced myeloperoxidase levels and elevated bacterial plate counts at 5 days postinfection. It also increased pro IL-1beta, but reduced total protein for IL-1beta and IL-18 at 5 days postinfection. Flow cytometry to identify cells affected by silencing, showed reduced caspase-1 levels in a CD11blowLy6Glow population of cells, (but not PMN or macrophages) from the infected cornea of siNLRC4 treated mice that produced less mature IL-1beta. These data provide evidence that the NLRC4 inflammasome contributes to resistance through regulation of caspase-1, IL-1beta and IL-18 in a CD11blowLy6Glow population of cells.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Antígeno CD11b/imunologia , Proteínas de Ligação ao Cálcio/fisiologia , Caspase 1/biossíntese , Interleucina-1beta/biossíntese , Ceratite/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/isolamento & purificação , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Contagem de Colônia Microbiana , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/microbiologiaRESUMO
Substance P (SP) is an undecapeptide present in the CNS and the peripheral nervous system. SP released from the peripheral nerves exerts its biological and immunological activity via high-affinity neurokinin 1 receptor (NK1R). SP is also produced by immune cells and acts as an autocrine or paracrine fashion to regulate the function of immune cells. In addition to its proinflammatory role, SP and its metabolites in combination with insulin-like growth factor-1 are shown to promote the corneal epithelial wound healing. Recently, we showed an altered ocular surface homeostasis in unmanipulated NK1R-/- mice, suggesting the role of SP-NK1R signaling in ocular surface homeostasis under steady-state. This review summarizes the immunobiology of SP and its effect on immune cells and immunity to microbial infection. In addition, the effect of SP in inflammation, wound healing, and corneal epithelial homeostasis in the eye is discussed.
Assuntos
Inflamação/imunologia , Sistema Nervoso , Neuroimunomodulação , Neurotransmissores/imunologia , Substância P/imunologia , Animais , Córnea/metabolismo , Córnea/patologia , Homeostase , Humanos , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais , CicatrizaçãoRESUMO
BACKGROUND: Hyperglycemia is a significant risk factor for diabetic retinopathy and induces increased inflammatory responses and retinal leukostasis, as well as vascular damage. Although there is an increasing amount of evidence that miRNA may be involved in the regulation in the pathology of diabetic retinopathy, the mechanisms by which miRNA mediate cellular responses to control onset and progression of diabetic retinopathy are still unclear. The purpose of our study was to investigate the hypothesis that miR-15a/16 inhibit pro-inflammatory signaling to reduce retinal leukostasis. METHODS: We generated conditional knockout mice in which miR-15a/16 are eliminated in vascular endothelial cells. For the in vitro work, human retinal endothelial cells (REC) were cultured in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC in high glucose with miRNA mimic (hsa-miR-15a-5p, hsa-miR-16-5p). Statistical analyses were done using unpaired Student t test with two-tailed p value. p < 0.05 was considered significant. Data are presented as mean ± SEM. RESULTS: We demonstrated that high glucose conditions decreased expression of miR-15a/16 in cultured REC. Overexpression of miR-15a/16 with the mimic significantly decreased pro-inflammatory signaling of IL-1ß, TNFα, and NF-κB in REC. In vivo data demonstrated that the loss of miR-15a/16 in vascular cells led to increased retinal leukostasis and CD45 levels, together with upregulated levels of IL-1ß, TNFα, and NF-κB. CONCLUSIONS: The data indicate that miR-15a/16 play significant roles in reducing retinal leukostasis, potentially through inhibition of inflammatory cellular signaling. Therefore, we suggest that miR-15a/16 offer a novel potential target for the inhibition of inflammatory mediators in diabetic retinopathy.
Assuntos
Citocinas/metabolismo , Células Endoteliais/metabolismo , Leucostasia/terapia , MicroRNAs/metabolismo , Transdução de Sinais/fisiologia , Animais , Citocinas/genética , Células Endoteliais/efeitos dos fármacos , Citometria de Fluxo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Leucostasia/metabolismo , Leucostasia/patologia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , RNA Mensageiro/metabolismo , Retina/citologia , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Substance P neuropeptide and its receptor, neurokinin-1 receptor (NK1R), are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation and increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R-/- mice. After ocular HSV-1 infection, the number of conventional DCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R-/- than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R-/- mice. As a result, the number of IFN-γ-secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R-/- mice was much higher than in infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R-/- mice was associated with an early development of severe herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R-/- mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R-/- mice.
Assuntos
Córnea/imunologia , Córnea/patologia , Herpesvirus Humano 1/imunologia , Ceratite Herpética/virologia , Receptores da Neurocinina-1/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/patologia , Túnica Conjuntiva/virologia , Córnea/virologia , Células Dendríticas/imunologia , Herpesvirus Humano 1/fisiologia , Homeostase , Interferon gama/imunologia , Ceratite Herpética/imunologia , Ceratite Herpética/fisiopatologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Receptores da Neurocinina-1/deficiência , Receptores da Neurocinina-1/imunologia , Carga ViralRESUMO
INTRODUCTION: Ocular infections remain an important cause of blindness worldwide and represent a challenging public health concern. In this regard, microbial keratitis due to fungal, bacterial, or viral infection can result in significant vision loss secondary to corneal scarring or surface irregularity. Left untreated corneal perforation and endophthalmitis can result, leading to loss of the eye. Rigorously studied animal models of disease pathogenesis have provided novel information that suggests new modes of treatment that may be efficacious clinically and emerging clinical data is supportive of some of these discoveries. AREAS COVERED: This review focuses on advances in our understanding of disease pathogenesis in animal models and clinical studies and how these relate to improved clinical treatment. We also discuss a novel approach to treatment of microbial keratitis due to infection with these bacterial pathogens using PACK-CXL and recommend increased basic and clinical studies to address and refine the efficacy of this procedure. EXPERT COMMENTARY: Because resistance to antibiotics has developed over time to these bacterial pathogens, caution must be exercised in treatment. Attractive novel modes of treatment that hold new promise for further investigation include lipid based therapy, as well as use of small molecules that bind deleterious specific host responsive molecules and use of microRNA based therapies.
RESUMO
The IL-2/anti-IL-2 Ab immunocomplex has recently been shown to expand the naturally occurring pool of CD4(+)Foxp3(+) regulatory T cells (Tregs). In this study, we show that administration of the IL-2/anti-IL-2 Ab immunocomplex to C57BL/6 mice, prior to corneal HSV-1 infection, significantly increased the pool of Foxp3(+) Tregs when measured at early time points postinfection. Increased numbers of Foxp3(+) Tregs on days 2 and 4 postinfection resulted in a marked reduction in the development of severe herpetic stromal keratitis (HSK). When compared with corneas from the control group, corneas from the immunocomplex-treated group showed a significant reduction in the amount of infectious virus on day 2 but not on day 4 postinfection. Reduced viral load was associated with a 2-fold increase in NK cell numbers in corneas from the immunocomplex-treated group of mice. Moreover, a dramatic reduction in the influx of CD4 T cells in inflamed corneas was determined on days 7 and 16 postinfection in the immunocomplex-treated group of infected mice. Immunocomplex treatment given on days 5, 6, and 7 postinfection significantly increased Foxp3(+) Tregs in draining lymph nodes and in the spleen but failed to reduce the severity of HSK. In terms of the influx of CD4 T cells and granulocytes into inflamed corneas, no significant differences were noted between both groups of mice on day 16 postinfection. Our findings demonstrate that increasing Foxp3(+) Tregs early but not late postinfection in secondary lymphoid tissues is more efficacious in controlling the severity of HSK.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Córnea/imunologia , Herpesvirus Humano 1/imunologia , Interleucina-2/uso terapêutico , Ceratite Herpética/terapia , Animais , Anticorpos Monoclonais/imunologia , Movimento Celular/imunologia , Córnea/patologia , Córnea/virologia , Progressão da Doença , Feminino , Fatores de Transcrição Forkhead/biossíntese , Granulócitos/imunologia , Imunoterapia , Interferon gama/metabolismo , Interleucina-2/imunologia , Ceratite Herpética/patologia , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Carga Viral/imunologiaRESUMO
In this paper, an estimation of model parameters is performed by using the Alternative Regression (AR) approach on an experimental data set of Herpes Simplex Virus type-1 (HSV-1) infection with innate immune response. Throughout the specified course of time, the measurements of monocytes, neutrophils, and viral load were obtained from the corneas of infected mice. C57BL/6 (B6) mice were used at Oakland University, Department of Biological Sciences, and the outcome measurements were divided into training and testing data sets. The HSV-1 nonlinear dynamic model is proposed based on the observed data patterns and biological system information. The simulation results of the proposed model showed that they consistently fit the experimental data set. In addition, the sensitivity test and model validation diagnostics are considered to determine the most significant key parameters that affect the dynamics of the HSV-1 system.
RESUMO
The blocking of programmed death ligand-1 (PDL-1) has been shown to enhance virus-specific CD8 T cell function during chronic viral infections. Though, how PDL-1 blocking at the time of priming affects the quality of CD8 T cell response to acute infections is not well understood and remains controversial. This report demonstrates that the magnitude of the primary and secondary CD8 T cell responses to herpes simplex virus-1 (HSV-1) infection is subject to control by PDL-1. Our results showed that after footpad HSV-1 infection, PD-1 expression increases on immunodominant SSIEFARL peptide specific CD8 T cells. Additionally, post-infection, the level of PDL-1 expression also increases on CD11c+ dendritic cells. Intraperitoneal administration of anti-PDL-1 monoclonal antibody given one day prior to and three days after cutaneous HSV-1 infection, resulted in a marked increase in effector and memory CD8 T cell response to SSIEFARL peptide. This was shown by measuring the quantity and quality of SSIEFARL-specific CD8 T cells by making use of ex-vivo assays that determine antigen specific CD8 T cell function, such as intracellular cytokine assay, degranulation assay to measure cytotoxicity and viral clearance. Our results are discussed in terms of the beneficial effects of blocking PDL-1 interactions, while giving prophylactic vaccines, to generate a more effective CD8 T cell response to viral infection.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Herpes Simples/tratamento farmacológico , Doença Aguda , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Expressão Gênica/imunologia , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Humanos , Imunoensaio , Epitopos Imunodominantes/administração & dosagem , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Memória Imunológica/efeitos dos fármacos , Injeções Intraperitoneais , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/administração & dosagem , Oligopeptídeos/genética , Oligopeptídeos/imunologiaRESUMO
PURPOSE: To determine whether substance P (SP) in herpes simplex virus-1 (HSV-1) infected cornea regulates the severity of herpetic stromal keratitis (HSK) lesions in a mouse model. METHODS: C57BL/6 mice were infected ocularly with HSV-1 (RE). The corneas with HSK lesions, on Day 15 postinfection, were grouped on the basis of the corneal opacity as mild (≤2) or severe (>2). The amount of SP was determined in the corneas with mild or severe HSK lesions by enzyme immunosorbent assay (EIA) and confocal microscopy. Subconjunctival inoculation of spantide I, SP receptor antagonist, was carried out during the clinical phase of HSK. ELISA and flow cytometry were used to determine the level of cytokines, chemokines, and influx of immune cell types in the corneal lesions. RESULTS: The authors determined a significantly higher level of SP in the corneas with severe HSK lesions in comparison with mild lesions. The corneas with a higher level of SP also exhibited higher amounts of proinflammatory cytokines (IL-6, IFN-γ) and chemokines (CCL3, CXCL2) when compared with the corneas with a lower level of SP. SP receptor NK1R expression was determined in CD45- and CD45+ cells in infected cornea. SP present in the corneal stroma of the eyes with severe HSK lesions colocalized with ß-III tubulin(+) and IA(b+) cell types. Subconjunctival inoculation of spantide I during the clinical phase of HSK resulted in significant reduction in the corneal opacity and angiogenesis. CONCLUSIONS: Collectively, these results demonstrate the relative contribution of substance P in regulating the clinical severity of HSK lesions in a mouse model.
Assuntos
Substância Própria/metabolismo , Herpesvirus Humano 1 , Ceratite Herpética/metabolismo , Índice de Gravidade de Doença , Substância P/metabolismo , Analgésicos/farmacologia , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Quimiocina CCL3/metabolismo , Quimiocina CXCL2/metabolismo , Substância Própria/imunologia , Substância Própria/patologia , Modelos Animais de Doenças , Feminino , Interleucina-6/metabolismo , Ceratite Herpética/tratamento farmacológico , Ceratite Herpética/imunologia , Ceratite Herpética/patologia , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Neutrófilos/patologia , Neutrófilos/virologia , Receptores da Neurocinina-1/metabolismo , Substância P/análogos & derivados , Substância P/farmacologia , Tubulina (Proteína)/metabolismo , Carga Viral/fisiologiaRESUMO
Decline in CD4 T cell function is the hallmark of aging. In this study, we compared the proportion and absolute number of inhibitory receptor expressing splenic CD4 T cells in unprimed young (2 months) and aged (20 months) C57BL/6 mice. Our results showed a predominance of PD-1, ICOS and CTLA-4 expressing conventional and regulatory CD4 T cells in aged mice. Furthermore, the expression of these molecules was localized to memory but not naïve CD4 T cell subset. Since aging is associated with decline in naïve but accumulation of hyporesponsive memory phenotype (MP) CD4 T cell, we hypothesize that inhibitory receptors can account for the senescence noted in MP subset of CD4 T cell.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD4-Positivos/imunologia , Baço/imunologia , Fatores Etários , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígeno CTLA-4 , Tolerância Imunológica , Memória Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1 , Baço/citologiaRESUMO
Using a viral-induced immunopathology model, we showed that when CD4(+) T cells were allowed to undergo homeostatic expansion prior to ocular herpes simplex virus infection, mice developed more severe inflammatory lesions with the increased severity associated with enhanced effector function of ocular CD4(+) T cells, and blocking their functional activity reduced the lesion severity. Additionally, homeostatically expanded CD4(+) T cells upregulated VLA-4, and in vivo administration of anti-VLA-4 mAb significantly decreased the homeostatic proliferation. Furthermore, blocking of VLA-4 interaction also diminished the infiltration of CD4(+) T cells into the cornea and decreased lesion severity. Our results imply that homeostatic expansion of T cells, as could occur in a virus-induced lymphopenia, may generate cells with enhanced effector function that can contribute to tissue damage.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Herpesvirus Humano 1/patogenicidade , Integrina alfa4beta1/metabolismo , Ceratite Herpética/imunologia , Ceratite Herpética/patologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Córnea/imunologia , Córnea/patologia , Feminino , Homeostase , Integrina alfa4beta1/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Ceratite Herpética/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Regulação para CimaRESUMO
Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor beta (TGF-beta) and interleukin-2 could induce up to 90% of CD4(+) T cells to become Foxp3(+) and able to mediate suppression in vitro. CD11c(+) dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-beta, could convert Foxp3(-) CD4(+) cells into Foxp3(+) CD4(+)cells by producing TGF-beta. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.