RESUMO
Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.
Assuntos
Artefatos , Gânglios Sensitivos , Herpesvirus Humano 1 , Células Receptoras Sensoriais , Análise de Sequência de RNA , Análise da Expressão Gênica de Célula Única , Latência Viral , Animais , Camundongos , Morte Celular , Conjuntos de Dados como Assunto , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/patologia , Gânglios Sensitivos/virologia , Herpes Simples/imunologia , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , MicroRNAs/análise , MicroRNAs/genética , Reprodutibilidade dos Testes , RNA Viral/análise , RNA Viral/genética , Células Receptoras Sensoriais/patologia , Células Receptoras Sensoriais/virologiaRESUMO
BACKGROUND: Macrophages in the peripheral nervous system are key players in the repair of nerve tissue and the development of neuropathic pain due to peripheral nerve injury. However, there is a lack of information on the origin and morphological features of macrophages in sensory ganglia after peripheral nerve injury, unlike those in the brain and spinal cord. We analyzed the origin and morphological features of sensory ganglionic macrophages after nerve ligation or transection using wild-type mice and mice with bone-marrow cell transplants. METHODS: After protecting the head of C57BL/6J mice with lead caps, they were irradiated and transplanted with bone-marrow-derived cells from GFP transgenic mice. The infraorbital nerve of a branch of the trigeminal nerve of wild-type mice was ligated or the infraorbital nerve of GFP-positive bone-marrow-cell-transplanted mice was transected. After immunostaining the trigeminal ganglion, the structures of the ganglionic macrophages, neurons, and satellite glial cells were analyzed using two-dimensional or three-dimensional images. RESULTS: The number of damaged neurons in the trigeminal ganglion increased from day 1 after infraorbital nerve ligation. Ganglionic macrophages proliferated from days 3 to 5. Furthermore, the numbers of macrophages increased from days 3 to 15. Bone-marrow-derived macrophages increased on day 7 after the infraorbital nerve was transected in the trigeminal ganglion of GFP-positive bone-marrow-cell-transplanted mice but most of the ganglionic macrophages were composed of tissue-resident cells. On day 7 after infraorbital nerve ligation, ganglionic macrophages increased in volume, extended their processes between the neurons and satellite glial cells, and contacted these neurons. Most of the ganglionic macrophages showed an M2 phenotype when contact was observed, and little neuronal cell death occurred. CONCLUSION: Most of the macrophages that appear after a nerve injury are tissue-resident, and these make direct contact with damaged neurons that act in a tissue-protective manner in the M2 phenotype. These results imply that tissue-resident macrophages signal to neurons directly through physical contact.
Assuntos
Transplante de Medula Óssea/métodos , Crescimento Celular , Gânglios Sensitivos/patologia , Macrófagos/patologia , Traumatismos dos Nervos Periféricos/patologia , Células Receptoras Sensoriais/patologia , Animais , Gânglios Sensitivos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Traumatismos dos Nervos Periféricos/imunologia , Traumatismos dos Nervos Periféricos/terapia , Células Receptoras Sensoriais/imunologiaRESUMO
Reactivation of herpes simplex virus 1 (HSV-1) from neurons in sensory ganglia such as the trigeminal ganglia (TG) is influenced by virus-specific CD8+ T cells that infiltrate the ganglia at the onset of latency and contract to a stable activated tissue-resident memory population. In C57BL/6 mice, half of HSV-specific CD8+ T cells (gB-CD8s) recognize one dominant epitope (residues 498 to 505) on glycoprotein B (gB498-505), while the remainder (non-gB-CD8s) recognize 19 subdominant epitopes from 12 viral proteins. To address how expression by HSV-1 influences the formation and ganglionic retention of CD8+ T cell populations, we developed recombinant HSV-1 with the native immunodominant gB epitope disrupted but then expressed ectopically from different viral promoters. In mice, the epitope expressed from the gB promoter restored full gB-CD8 immunodominance to 50%. Intriguingly, earlier expression from constitutive, immediate-early, and early promoters did not significantly increase immunodominance, indicating that these promoters cannot elicit more than half of the CD8 compartment. Epitope expressed from candidate viral promoters of "true late" HSV-1 genes either delayed or reduced the priming efficiency of gB-CD8s and their levels in the TG at early times. HSV expressing the epitope from the full latency-associated transcript promoter did not efficiently prime gB-CD8s; however, gB-CD8s primed by a concurrent wild-type flank infection infiltrated the TG and were retained long term, suggesting that latent epitope expression is sufficient to retain gB-CD8s. Taken together, the data indicate that viral promoters shape latent HSV-1-specific CD8+ T cell populations and should be an important consideration in future vaccine design.IMPORTANCE Latency of HSV-1 in host neurons enables long-term persistence from which reactivation may occur to cause recurrent diseases, such as blinding herpetic stromal keratitis. Latency is not antigenically silent, and viral proteins are sporadically expressed at low levels without full virion production. This protein expression is recognized by ganglion-resident HSV-1-specific CD8+ T cells that maintain a protective resident population. Since these T cells can influence lytic/latent decisions in reactivating neurons, we argue that improving their ganglionic retention and function may offer a strategy in vaccine design to reduce reactivation and recurrent disease. To understand factors driving the infiltration and retention of ganglionic CD8s, we examined several HSV recombinants that have different viral promoters driving expression of the immunodominant gB epitope. We show that the selection of epitope promoter influences CD8+ T cell population hierarchies and their function.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Gânglios/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Epitopos Imunodominantes/imunologia , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Gânglios Sensitivos/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Ceratite Herpética/imunologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Gânglio Trigeminal/virologia , Células Vero , Proteínas do Envelope Viral/genéticaRESUMO
Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.
Assuntos
Diabetes Mellitus Tipo 2/terapia , Retinopatia Diabética/prevenção & controle , Disbiose/terapia , Jejum , Microbioma Gastrointestinal , Retina/patologia , Vasos Retinianos/patologia , Animais , Bacteroidetes/crescimento & desenvolvimento , Bacteroidetes/imunologia , Bacteroidetes/isolamento & purificação , Ácidos e Sais Biliares/uso terapêutico , Colo/efeitos dos fármacos , Colo/imunologia , Colo/metabolismo , Colo/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/microbiologia , Diabetes Mellitus Tipo 2/patologia , Retinopatia Diabética/complicações , Retinopatia Diabética/imunologia , Retinopatia Diabética/patologia , Disbiose/complicações , Disbiose/microbiologia , Disbiose/patologia , Fezes/microbiologia , Firmicutes/crescimento & desenvolvimento , Firmicutes/imunologia , Firmicutes/isolamento & purificação , Gânglios Sensitivos/efeitos dos fármacos , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/metabolismo , Gânglios Sensitivos/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/patologia , Masculino , Camundongos Endogâmicos DBA , Camundongos Mutantes , Microvasos/efeitos dos fármacos , Microvasos/imunologia , Microvasos/metabolismo , Microvasos/patologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Retina/efeitos dos fármacos , Retina/imunologiaRESUMO
BNN27, a C17-spiroepoxy derivative of DHEA, was shown to have antiapoptotic properties via mechanisms involving the nerve growth factor receptors (tropomyosin-related kinase A [TrkA]/neurotrophin receptor p75 [p75NTR]). In this study, we examined the effects of BNN27 on neural/glial cell function, apoptosis, and inflammation in the experimental rat streptozotocin (STZ) model of diabetic retinopathy (DR). The ability of BNN27 to activate the TrkA receptor and regulate p75NTR expression was investigated. BNN27 (2,10, and 50 mg/kg i.p. for 7 days) administration 4 weeks post-STZ injection (paradigm A) reversed the diabetes-induced glial activation and loss of function of amacrine cells (brain nitric oxide synthetase/tyrosine hydroxylase expression) and ganglion cell axons via a TrkA receptor (TrkAR)-dependent mechanism. BNN27 activated/phosphorylated the TrkAY490 residue in the absence but not the presence of TrkAR inhibitor and abolished the diabetes-induced increase in p75NTR expression. However, it had no effect on retinal cell death (TUNEL+ cells). A similar result was observed when BNN27 (10 mg/kg i.p.) was administered at the onset of diabetes, every other day for 4 weeks (paradigm B). However, BNN27 decreased the activation of caspase-3 in both paradigms. Finally, BNN27 reduced the proinflammatory (TNFα and IL-1ß) and increased the anti-inflammatory (IL-10 and IL-4) cytokine levels. These findings suggest that BNN27 has the pharmacological profile of a therapeutic for DR, since it targets both the neurodegenerative and inflammatory components of the disease.
Assuntos
Células Amácrinas/efeitos dos fármacos , Anti-Inflamatórios/uso terapêutico , Desidroepiandrosterona/uso terapêutico , Retinopatia Diabética/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Receptor trkA/agonistas , Retina/efeitos dos fármacos , Células Amácrinas/imunologia , Células Amácrinas/metabolismo , Células Amácrinas/patologia , Animais , Anti-Inflamatórios/administração & dosagem , Axônios/efeitos dos fármacos , Axônios/imunologia , Axônios/metabolismo , Axônios/patologia , Desidroepiandrosterona/administração & dosagem , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/imunologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Relação Dose-Resposta a Droga , Proteínas do Olho/agonistas , Proteínas do Olho/metabolismo , Feminino , Gânglios Sensitivos/efeitos dos fármacos , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/metabolismo , Gânglios Sensitivos/patologia , Masculino , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/imunologia , Neuroglia/metabolismo , Neuroglia/patologia , Fármacos Neuroprotetores/administração & dosagem , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural/agonistas , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Retina/imunologia , Retina/patologia , Retina/fisiopatologia , EstreptozocinaRESUMO
Herpetic neuralgia is the most important symptom of herpes zoster disease, which is caused by Varicella zoster Nevertheless, the pathophysiological mechanisms involved in herpetic neuralgia are not totally elucidated. Here, we examined the neuroimmune interactions at the sensory ganglia that account for the genesis of herpetic neuralgia using a murine model of Herpes Simplex Virus Type-1 (HSV-1) infection. The cutaneous HSV-1 infection of mice results in the development of a zosteriform-like skin lesion followed by a time-dependent increase in pain-like responses (mechanical allodynia). Leukocytes composed mainly of macrophages and neutrophils infiltrate infected DRGs and account for the development of herpetic neuralgia. Infiltrating leukocytes are responsible for driving the production of TNF, which in turn mediates the development of herpetic neuralgia through downregulation of the inwardly rectifying K+ channel Kir4.1 in satellite glial cells. These results revealed that neuroimmune-glia interactions at the sensory ganglia play a critical role in the genesis of herpetic neuralgia. In conclusion, the present study elucidates novel mechanisms involved in the genesis of acute herpetic pain and open new avenues for its control.SIGNIFICANCE STATEMENT Acute herpetic neuralgia is the most important symptom of herpes zoster disease and it is very difficult to treat. Using a model of peripheral infection of mice with HSV-1, we have characterized for the first time the neuroimmune-glia interactions in the sensory ganglia that account for the development of acute herpetic neuralgia. Among these mechanisms, leukocytes composed mainly of macrophages and neutrophils infiltrate infected sensory ganglia and are responsible for driving the production of TNF. TNF, via TNFR1, mediates herpetic neuralgia development through downregulation of the inwardly rectifying K+ channel Kir4.1 in satellite glial cells. This study elucidates novel mechanisms involved in the genesis of acute herpetic neuralgia and open new avenues for its control.
Assuntos
Gânglios Sensitivos/imunologia , Leucócitos/imunologia , Neuralgia Pós-Herpética/imunologia , Neuroglia/imunologia , Neuroimunomodulação/imunologia , Células Receptoras Sensoriais/imunologia , Animais , Células Cultivadas , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Varicella zoster virus (VZV) causes varicella during acute infection and establishes latency in the sensory ganglia. Reactivation of VZV results in herpes zoster, a debilitating and painful disease. It is believed that VZV reactivates due to a decline in cell-mediated immunity; however, the roles that CD4 versus CD8 T cells play in the prevention of herpes zoster remain poorly understood. To address this question, we used a well-characterized model of VZV infection where rhesus macaques are intrabronchially infected with the homologous simian varicella virus (SVV). Latently infected rhesus macaques were thymectomized and depleted of either CD4 or CD8 T cells to induce selective senescence of each T cell subset. After T cell depletion, the animals were transferred to a new housing room to induce stress. SVV reactivation (viremia in the absence of rash) was detected in three out of six CD8-depleted and two out of six CD4-depleted animals suggesting that both CD4 and CD8 T cells play a critical role in preventing SVV reactivation. Viral loads in multiple ganglia were higher in reactivated animals compared to non-reactivated animals. In addition, reactivation results in sustained transcriptional changes in the ganglia that enriched to gene ontology and diseases terms associated with neuronal function and inflammation indicative of potential damage as a result of viral reactivation. These studies support the critical role of cellular immunity in preventing varicella virus reactivation and indicate that reactivation results in long-lasting remodeling of the ganglia transcriptome.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Gânglios Sensitivos/imunologia , Herpes Zoster/veterinária , Herpesvirus Humano 3/imunologia , Proteínas do Tecido Nervoso/genética , Ativação Viral/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Feminino , Gânglios Sensitivos/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Herpes Zoster/genética , Herpes Zoster/imunologia , Depleção Linfocítica/métodos , Macaca mulatta , Masculino , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/imunologia , Estresse Psicológico , Timectomia , Timo/imunologia , Timo/cirurgia , Timo/virologiaRESUMO
Primary infection with varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, results in varicella. VZV establishes latency in the sensory ganglia and can reactivate later in life to cause herpes zoster. The relationship between VZV and its host during acute infection in the sensory ganglia is not well understood due to limited access to clinical specimens. Intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV) recapitulates the hallmarks of VZV infection in humans. We leveraged this animal model to characterize the host-pathogen interactions in the ganglia during both acute and latent infection by measuring both viral and host transcriptomes on days postinfection (dpi) 3, 7, 10, 14, and 100. SVV DNA and transcripts were detected in sensory ganglia 3 dpi, before the appearance of rash. CD4 and CD8 T cells were also detected in the sensory ganglia 3 dpi. Moreover, lung-resident T cells isolated from the same animals 3 dpi also harbored SVV DNA and transcripts, suggesting that T cells may be responsible for trafficking SVV to the ganglia. Transcriptome sequencing (RNA-Seq) analysis showed that cessation of viral transcription 7 dpi coincides with a robust antiviral innate immune response in the ganglia. Interestingly, a significant number of genes that play a critical role in nervous system development and function remained downregulated into latency. These studies provide novel insights into host-pathogen interactions in the sensory ganglia during acute varicella and demonstrate that SVV infection results in profound and sustained changes in neuronal gene expression. IMPORTANCE: Many aspects of VZV infection of sensory ganglia remain poorly understood, due to limited access to human specimens and the fact that VZV is strictly a human virus. Infection of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, provides a robust model of the human disease. Using this model, we show that SVV reaches the ganglia early after infection, most likely by T cells, and that the induction of a robust innate immune response correlates with cessation of virus transcription. We also report significant changes in the expression of genes that play an important role in neuronal function. Importantly, these changes persist long after viral replication ceases. Given the homology between SVV and VZV, and the genetic and physiological similarities between rhesus macaques and humans, our results provide novel insight into the interactions between VZV and its human host and explain some of the neurological consequences of VZV infection.
Assuntos
Gânglios Sensitivos/metabolismo , Gânglios Sensitivos/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Varicellovirus/patogenicidade , Doença Aguda , Animais , Transporte Axonal , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Varicela/virologia , DNA Viral/genética , DNA Viral/metabolismo , Modelos Animais de Doenças , Gânglios Sensitivos/imunologia , Expressão Gênica , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 3/patogenicidade , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macaca mulatta , Neurogênese , Varicellovirus/genética , Varicellovirus/fisiologia , Replicação ViralRESUMO
Primary simian varicella virus (SVV) infection in non-human primates causes varicella, after which the virus becomes latent in ganglionic neurons and reactivates to cause zoster. The host response in ganglia during establishment of latency is ill-defined. Ganglia from five African green monkeys (AGMs) obtained at 9, 13, and 20 days post-intratracheal SVV inoculation (dpi) were analyzed by ex vivo flow cytometry, immunohistochemistry, and in situ hybridization. Ganglia at 13 and 20 dpi exhibited mild inflammation. Immune infiltrates consisted mostly of CD8(dim) and CD8(bright) memory T cells, some of which expressed granzyme B, and fewer CD11c(+) and CD68(+) cells. Chemoattractant CXCL10 transcripts were expressed in neurons and infiltrating inflammatory cells but did not co-localize with SVV open reading frame 63 (ORF63) RNA expression. Satellite glial cells expressed increased levels of activation markers CD68 and MHC class II at 13 and 20 dpi compared to those at 9 dpi. Overall, local immune responses emerged as viral DNA load in ganglia declined, suggesting that intra-ganglionic immunity contributes to restricting SVV replication.
Assuntos
Gânglios Sensitivos/imunologia , Herpesvirus Humano 3/imunologia , Doenças dos Primatas/imunologia , Células Receptoras Sensoriais/imunologia , Infecção pelo Vírus da Varicela-Zoster/veterinária , Ativação Viral , Latência Viral , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Chlorocebus aethiops , DNA Viral/genética , DNA Viral/imunologia , Gânglios Sensitivos/virologia , Regulação da Expressão Gênica/imunologia , Granzimas/genética , Granzimas/imunologia , Herpesvirus Humano 3/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/imunologia , Memória Imunológica , Doenças dos Primatas/genética , Doenças dos Primatas/patologia , Células Receptoras Sensoriais/virologia , Infecção pelo Vírus da Varicela-Zoster/genética , Infecção pelo Vírus da Varicela-Zoster/imunologia , Infecção pelo Vírus da Varicela-Zoster/patologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Carga Viral/genética , Carga Viral/imunologiaRESUMO
BACKGROUND: A neuroimmune crosstalk between dendritic cells (DCs) and airway nerves in the lung has recently been reported. However, the presence of DCs in airway sensory ganglia under normal and allergic conditions has not been explored so far. Therefore, this study aims to investigate the localisation, distribution and proliferation of DCs in airway sensory ganglia under allergic airway inflammation. METHODS: Using the house dust mite (HDM) model for allergic airway inflammation BALB/c mice were exposed to HDM extract intranasally (25 µg/50 µl) for 5 consecutive days a week over 7 weeks. With the help of the immunohistochemistry, vagal jugular-nodose ganglia complex (JNC) sections were analysed regarding their expression of DC-markers (MHC II, CD11c, CD103), the neuronal marker PGP 9.5 and the neuropeptide calcitonin gene-related peptide (CGRP) and glutamine synthetase (GS) as a marker for satellite glia cells (SGCs). To address the original source of DCs in sensory ganglia, a proliferation experiment was also carried in this study. RESULTS: Immune cells with characteristic DC-phenotype were found to be closely located to SGCs and vagal sensory neurons under physiological conditions. The percentage of DCs in relation to neurons was significantly increased by allergic airway inflammation in comparison to the controls (HDM 51.38 ± 2.38% vs. control 28.16 ± 2.86%, p < 0.001). The present study also demonstrated that DCs were shown to proliferate in jugular-nodose ganglia, however, the proliferation rate of DCs is not significantly changed in the two treated animal groups (proliferating DCs/ total DCs: HDM 0.89 ± 0.38%, vs. control 1.19 ± 0.54%, p = 0.68). Also, increased number of CGRP-positive neurons was found in JNC after allergic sensitisation and challenge (HDM 31.16 ± 5.41% vs. control 7.16 ± 1.53%, p < 0.001). CONCLUSION: The present findings suggest that DCs may migrate from outside into the ganglia to interact with sensory neurons enhancing or protecting the allergic airway inflammation. The increase of DCs as well as CGRP-positive neurons in airway ganglia by allergic airway inflammation indicate that intraganglionic DCs and neurons expressing CGRP may contribute to the pathogenesis of bronchial asthma. To understand this neuroimmune interaction in allergic airway inflammation further functional experiments should be carried out in future studies.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/fisiologia , Gânglios Sensitivos/imunologia , Pneumonia/imunologia , Pyroglyphidae/imunologia , Hipersensibilidade Respiratória/imunologia , Animais , Feminino , Gânglios Sensitivos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/patologia , Hipersensibilidade Respiratória/patologiaRESUMO
Infection induces the expression of inflammatory chemokines that recruit immune cells to the site of inflammation. Whereas tissues such as the intestine and skin express unique chemokines during homeostasis, whether different tissues express distinct chemokine profiles during inflammation remains unclear. With this in mind, we performed a comprehensive screen of the chemokines expressed by two tissues (skin and sensory ganglia) infected with a common viral pathogen (herpes simplex virus type 1). After infection, the skin and ganglia showed marked differences in their expression of the family of Cxcr2 chemokine ligands. Specifically, Cxcl1/2/3, which in turn controlled neutrophil recruitment, was up-regulated in the skin but absent from the ganglia. Within the ganglia, Cxcl2 expression and subsequent neutrophil recruitment was inhibited by type I interferon (IFN). Using a combination of bone marrow chimeras and intracellular chemokine staining, we show that type I IFN acted by directly suppressing Cxcl2 expression by monocytes, abrogating their ability to recruit neutrophils to the ganglia. Overall, our findings describe a novel role for IFN in the direct, and selective, inhibition of Cxcr2 chemokine ligands, which results in the inhibition of neutrophil recruitment to neuronal tissue.
Assuntos
Quimiocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Interferon Tipo I/imunologia , Neutrófilos/imunologia , Receptores de Interleucina-8B/metabolismo , Animais , Quimiocinas/genética , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/virologia , Indóis , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Pele/imunologia , Pele/virologiaRESUMO
UNLABELLED: Varicella-zoster virus (VZV) is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes latency within the sensory ganglia and can reactivate to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had active herpes zoster. Ganglia innervating the site of the cutaneous herpes zoster rash showed evidence of necrosis, secondary to vasculitis, or localized hemorrhage. Despite this, there was limited evidence of VZV antigen expression, although a large inflammatory infiltrate was observed. Characterization of the infiltrating T cells showed a large number of infiltrating CD4(+) T cells and cytolytic CD8(+) T cells. Many of the infiltrating T cells were closely associated with neurons within the reactivated ganglia, yet there was little evidence of T cell-induced neuronal apoptosis. Notably, an upregulation in the expression of major histocompatibility complex class I (MHC-I) and MHC-II molecules was observed on satellite glial cells, implying these cells play an active role in directing the immune response during herpes zoster. This is the first detailed characterization of the interaction between T cells and neuronal cells within ganglia obtained from patients suffering herpes zoster at the time of death and provides evidence that CD4(+) and cytolytic CD8(+) T cell responses play an important role in controlling VZV replication in ganglia during active herpes zoster. IMPORTANCE: VZV is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes a life-long dormant infection within the sensory ganglia and can reawaken to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had active herpes zoster. We found that specific T cell subsets are likely to play an important role in controlling VZV replication in ganglia during active herpes zoster.
Assuntos
Gânglios Sensitivos/imunologia , Gânglios Sensitivos/virologia , Herpes Zoster/imunologia , Herpesvirus Humano 3/fisiologia , Subpopulações de Linfócitos T/imunologia , Ativação Viral/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Caspase 3/metabolismo , Criança , Feminino , Gânglios Sensitivos/metabolismo , Gânglios Sensitivos/patologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/imunologia , Neurônios/patologia , Neurônios/virologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Adulto JovemRESUMO
A large proportion of the world population harbors HSV type 1 (HSV-1) in a latent state in their trigeminal ganglia (TG). TG-resident CD8(+) T cells appear important for preventing HSV-1 reactivation from latency and recurrent herpetic disease. In C57BL/6J mice, half of these cells are specific for an immunodominant epitope on HSV-1 glycoprotein B, whereas the other half are specific for 18 subdominant epitopes. In this study, we show that the CD8(+) T cell dominance hierarchy in the TG established during acute infection is maintained during latency. However, CD8(+) T cells specific for subdominant epitopes lose functionality, whereas those specific for the immunodominant epitope exhibit increased functionality in latently infected TG. Furthermore, we show that IL-10 produced by 16.4 ± 2.8% of TG-resident CD4(+) T cells maintains the immunodominance hierarchy in part through selective inhibition of subdominant CD8(+) T cell proliferation. Upon systemic anti-IL-10R Ab treatment, we observed a significant expansion of functional subdominant CD8(+) T cells, resulting in significantly improved protection from viral reactivation. In fact, systemic anti-IL-10R Ab treatment prevented viral reactivation in up to 50% of treated mice. Our results not only demonstrate that HSV-1 reactivation from latency can be prevented by expanding the repertoire of functional TG-resident CD8(+) T cells, but also that IL-10R blockade might have therapeutic potential to reduce or eliminate recurrent herpetic disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Gânglios Sensitivos/virologia , Herpes Simples/imunologia , Ativação Viral/imunologia , Latência Viral/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Citometria de Fluxo , Gânglios Sensitivos/imunologia , Herpesvirus Humano 1/fisiologia , Epitopos Imunodominantes/imunologia , Interleucina-10/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Varicella-zoster virus (VZV) causes chickenpox, establishes latency in trigeminal (TG) and dorsal root ganglia (DRG), and can lead to herpes zoster upon reactivation. The VZV proteome expressed during latency remains ill-defined, and previous studies have shown discordant data on the spectrum and expression pattern of VZV proteins and transcripts in latently infected human ganglia. Recently, Zerboni and colleagues have provided new insight into this discrepancy (Zerboni et al. in J Virol 86:578-583, 2012). They showed that VZV-specific ascites-derived monoclonal antibody (mAb) preparations contain endogenous antibodies directed against blood group A1 proteins, resulting in false-positive intra-neuronal VZV staining in formalin-fixed human DRG. The aim of the present study was to confirm and extend this phenomenon to snap-frozen TG (n=30) and DRG (n=9) specimens of blood group genotyped donors (n=30). The number of immunohistochemically stained neurons was higher with mAb directed to immediate early protein 62 (IE62) compared with IE63. The IE63 mAb-positive neurons always co-stained for IE62 but not vice versa. The mAb staining was confined to distinct large intra-neuronal vacuoles and restricted to A1(POS) donors. Anti-VZV mAb staining in neurons, but not in VZV-infected cell monolayers, was obliterated after mAb adsorption against blood group A1 erythrocytes. The data presented demonstrate that neuronal VZV protein expression detected by ascites-derived mAb in snap-frozen TG and DRG of blood group A1(POS) donors can be misinterpreted due to the presence of endogenous antibodies directed against blood group A1-associated antigens present in ascites-derived VZV-specific mAb preparations.
Assuntos
Gânglios Sensitivos/química , Herpes Zoster/diagnóstico , Herpesvirus Humano 3/metabolismo , Proteínas Imediatamente Precoces/análise , Neurônios/química , Transativadores/análise , Proteínas do Envelope Viral/análise , Sistema ABO de Grupos Sanguíneos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Eritrócitos/imunologia , Reações Falso-Positivas , Feminino , Congelamento , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/virologia , Herpes Zoster/imunologia , Herpes Zoster/virologia , Herpesvirus Humano 3/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/imunologia , Imuno-Histoquímica , Masculino , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Neurônios/imunologia , Neurônios/virologia , Transativadores/genética , Transativadores/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Latência ViralRESUMO
Persisting infections are often associated with chronic T cell activation. For certain pathogens, this can lead to T cell exhaustion and survival of what is otherwise a cleared infection. In contrast, for herpesviruses, T cells never eliminate infection once it is established. Instead, effective immunity appears to maintain these pathogens in a state of latency. We used infection with HSV to examine whether effector-type T cells undergoing chronic stimulation retained functional and proliferative capacity during latency and subsequent reactivation. We found that latency-associated T cells exhibited a polyfunctional phenotype and could secrete a range of effector cytokines. These T cells were also capable of mounting a recall proliferative response on HSV reactivation and could do so repeatedly. Thus, for this latent infection, T cells subjected to chronic Ag stimulation and periodic reactivation retain the ability to respond to local virus challenge.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/toxicidade , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/imunologia , Proteínas do Envelope Viral/toxicidade , Ativação Viral/imunologia , Latência Viral/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/transplante , Linfócitos T CD8-Positivos/virologia , Doença Crônica , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/imunologia , Gânglios Sensitivos/enzimologia , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/patologia , Granzimas/biossíntese , Herpes Simples/patologia , Herpesvirus Humano 1/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Envelope Viral/administração & dosagemRESUMO
There is debate over whether effective T-cell mediated protection against a second infection, or post-vaccination, is better done by central memory cells or effector memory cells. The former may have greater powers of expansion, whereas the latter may be closer to the site of pathogen entry and faster to respond. This review focuses on memory T cells which are not recirculating but which remain at the peripheral site of initial pathogen or vaccine encounter, so-called tissue-resident memory cells. They may play key roles in protection against re-eruption of latent viral infections and at mucosal surfaces.
Assuntos
Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Viroses/imunologia , Animais , Sistema Nervoso Central/imunologia , Gânglios Sensitivos/imunologia , Mucosa Intestinal/imunologia , Camundongos , Pele/imunologia , VacinaçãoRESUMO
Immune cells and glia interact with neurons to alter pain sensitivity and to mediate the transition from acute to chronic pain. In response to injury, resident immune cells are activated and blood-borne immune cells are recruited to the site of injury. Immune cells not only contribute to immune protection but also initiate the sensitization of peripheral nociceptors. Through the synthesis and release of inflammatory mediators and interactions with neurotransmitters and their receptors, the immune cells, glia and neurons form an integrated network that coordinates immune responses and modulates the excitability of pain pathways. The immune system also reduces sensitization by producing immune-derived analgesic and anti-inflammatory or proresolution agents. A greater understanding of the role of the immune system in pain processing and modulation reveals potential targets for analgesic drug development and new therapeutic opportunities for managing chronic pain.
Assuntos
Sistema Imunitário/imunologia , Sistema Nervoso/imunologia , Sistema Nervoso/patologia , Dor/imunologia , Animais , Citocinas/metabolismo , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Neuroglia/metabolismo , Nociceptores/imunologia , Nociceptores/patologia , Manejo da DorRESUMO
Herpes simplex virus type 1 (HSV-1) latent persistence in human trigeminal ganglia (TG) is accompanied by a chronic CD8 T-cell infiltration. Thus far, during HSV-1 latency only a single transcript, namely the latency-associated transcript (LAT), has been identified to be synthesized but not translated into a protein. In contrast, the chronic CD8 T-cell infiltration suggests that an antigen trigger must be present. The focus of the current work was to look for HSV-1 transcription activity as a potential trigger of the immune response and to demonstrate whether the immune cells are clonally expanded and have a phenotype that suggests that they have been triggered by viral antigen. By combining in situ hybridization, laser cutting microscopy, and single-cell real time RT-PCR, we demonstrated expression of the HSV-1 immediate early (IE) genes ICP0 and ICP4 in human trigeminal neurons. Using CDR3 spectratyping, we showed that the infiltrating T cells are clonally expanded, indicating an antigen-driven immune response. Moreover, the persisting CD8(+) T cells had prominent features of the memory effector phenotype. Chemokines CCL5 and CXCL10 were expressed by a subpopulation of infiltrating cells and the corresponding chemokine receptors CCR5 and CXCR3 were co-expressed on virtually all T cells bearing the CD8 phenotype. Thus, HSV-1 IE genes are expressed in human TG, and the infiltrating T cells bear several characteristics that suggest viral antigenic stimulation.
Assuntos
Gânglios Sensitivos/imunologia , Gânglios Sensitivos/virologia , Herpesvirus Humano 1/genética , Memória Imunológica , RNA Mensageiro/genética , Linfócitos T/imunologia , Genes Precoces , Genes Virais , Humanos , Hibridização In Situ , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Effective immunity is dependent on long-surviving memory T cells. Various memory subsets make distinct contributions to immune protection, especially in peripheral infection. It has been suggested that T cells in nonlymphoid tissues are important during local infection, although their relationship with populations in the circulation remains poorly defined. Here we describe a unique memory T cell subset present after acute infection with herpes simplex virus that remained resident in the skin and in latently infected sensory ganglia. These T cells were in disequilibrium with the circulating lymphocyte pool and controlled new infection with this virus. Thus, these cells represent an example of tissue-resident memory T cells that can provide protective immunity at points of pathogen entry.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Gânglios Sensitivos/imunologia , Herpes Simples/imunologia , Memória Imunológica , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Quimiotaxia de Leucócito/imunologia , Citometria de Fluxo , Gânglios Sensitivos/citologia , Gânglios Sensitivos/virologia , Imuno-Histoquímica , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Simplexvirus/imunologia , Pele/citologia , Pele/virologiaRESUMO
The tissue sites of long-term herpes simplex virus type 2 (HSV-2)-specific antibody production in mice and guinea pigs were identified. In addition to secondary lymphoid tissue and bone marrow, HSV-specific plasma cells were detected in spinal cords of mice up to 10 months after intravaginal inoculation with a thymidine kinase-deficient HSV-2 strain and in lumbosacral ganglia and spinal cords of guinea pigs inoculated with HSV-2 strain MS. The long-term retention of virus-specific plasma cells in the peripheral and central nervous systems following HSV infection may be important for resistance to reinfection of neuronal tissues or may play a role in modulation of reactivation from latency.