Role of IL-4 in an experimental model of encephalitis induced by intracranial inoculation of herpes simplex virus-1 (HSV-1)

Article Arq Neuropsiquiatr 2011;69(2-A):237-241 Role of IL-4 in an experimental model of encephalitis induced by intracranial inoculation of herpes simplex virus-1 (HSV-1) Márcia Carvalho Vilela1, Roberta Dayrell de Lima Campos1, Daniel Santos Mansur1, David Henrique Rodrigues1, Norinne Lacerda-Queiroz1, Graciela Kunrath Lima2, Milene Alvarenga Rachid1, Erna Geessien Kroon2, Marco Antônio Campos3, Antônio Lúcio Teixeira1 ABSTRACT Herpes simplex virus-1 (HSV-1) is a pathogen that may cause severe encephalitis in humans. In this study, we aimed to investigate the role of interleukin-4 (IL-4) in a model of HSV-1 brain infection. IL-4 knockout (IL-4 –/–) and wild type (WT) C57BL/6 mice were inoculated with 104 plaque-forming units of HSV-1 by the intracranial route. Histopathologic analysis revealed a distinct profile of infiltrating cells at 3 days postinfection (dpi). Infected WT mice presented mononuclear inflammatory cells while IL4–/– mice developed meningoencephalitis with predominance of neutrophils. IL-4–/– mice had diminished leukocyte adhesion at 3 dpi when compared to infected WT animals in intravital microscopy study. Conversely no differences were found in cerebral levels of CXCL1, CXCL9, CCL3, CCL5 and TNF-α between WT and IL-4–/– infected mice. IL-4 may play a role in the recruitment of cells into central nervous system in this acute model of severe encephalitis caused by HSV-1. Key words: herpes simplex virus type 1, IL-4, neuroinflammation. Papel da IL-4 em modelo experimental de encefalite induzida pela inoculação intracraniana do herpes simplex vírus-1 (HSV-1) Correspondence Antônio Lúcio Teixeira Laboratório de Imunofarmacologia Depto. de Bioquímica e Imunologia Instituto de Ciências Biológicas (UFMG) Av. Antônio Carlos, 6627 31270-901 Belo Horizonte MG - Brasil E-mail: Support This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig), Brazil Received 22 June 2010 Received in final form 13 October 2010 Accepted 20 October 2010 RESUMO O vírus herpes simplex-1 (HSV-1) é um patógeno que pode causar encefalite grave em humanos. Neste estudo, buscamos investigar o papel da interleucina-4 (IL-4) no modelo de infecção intracerebral por HSV-1. Camundongos C57BL/6 selvagens (WT) e deficientes no gene IL-4 (IL-4–/–) foram inoculados com 104 unidades formadoras de placas de HSV-1 por via intracraniana. A análise histopatológica revelou um padrão distinto de infiltrado leucocitário. Camundongos WT infectados apresentaram infiltrado de células mononucleares, enquanto camundongos IL-4 –/– desenvolveram meningoencefalite com predomínio de neutrófilos 3 dias pós-infecção (dpi). Animais IL-4–/– tiveram menor adesão de leucócitos 3 dpi quando comparados aos animais WT infectados à microscopia intravital. Em contrapartida, não foram encontradas diferenças nos níveis cerebrais de CXCL1, CXCL9, CCL3, CCL5 e TNF-α entre camundongos WT e IL-4–/– infectados. Esses resultados sugerem que IL-4 pode desempenhar um papel no recrutamento de células no sistema nervoso central neste modelo agudo de encefalite grave causada pelo HSV-1. Palavras-chave: vírus herpes simplex tipo 1, IL-4, neuroinflamação. 1 Laboratório de Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas (ICB), UFMG, Belo Horizonte MG, Brazil; 2Departamento de Microbiologia, ICB, UFMG; 3Centro de Pesquisas René Rachou, Belo Horizonte MG, Brazil. 237 IL-4: experimental encephalitis Vilela et al. Interleukin-4 (IL-4) is a pleiotropic cytokine synthesized primarily by CD4+ T lymphocytes in response to their activation1,2. Studies have reported that IL-4 may have either detrimental or protective effects during viral infection3-6. For instance, the expression of IL-4 by mousepox virus, due to the insertion of a copy of mouse IL-4 cDNA in viral genome, turns this virus lethal to mice that are usually resistant to the infection3. Similarly, the expression of IL-4 by myxoma virus enhances virulence and overcomes genetic resistance of rabbits to viral infection4. IL-4 knockout (IL-4–/–) mice challenged with herpes simplex virus type 1 (HSV-1) by ocular route had reduced virus load in their eyes when compared with wild type (WT) mice5. Conversely one study demonstrated that a recombinant HSV-1 expressing IL-4 had a great decrease in its pathogenic potential6. HSV-1 is a neurotropic virus known to cause infection in the central nervous system (CNS). Herpes simplex encephalitis is a common sporadic viral disease of the brain7,8. Although antiviral treatment has greatly reduced mortality due to herpetic encephalitis, the majority of survivors presents residual neuropsychological deficits and/or neuropsychiatric symptoms9. Our group has developed an experimental model of severe HSV-1 encephalitis10-12. We observed an increase in the levels of rolling and adhered leukocytes in meningeal vessels of infected mice in parallel with the increase of the expression of several cytokines in the CNS12. Nonetheless, the role of IL-4 on the early inflammatory response to HSV-1 brain infection has not been investigated yet. In the present study we aimed to investigate the possible involvement of IL-4 in the inflammatory response to HSV-1, assessing the recruitment of leukocytes by intravital microscopy, the chemokine and cytokine profile and the histopathological changes in IL-4–/– and WT mice infected with an intracerebral inoculum of HSV-1. METHOD Mouse strains Male C57BL/6 mice and IL-4–/– mice on a C57BL/6 background, aged 6-9 weeks, were obtained from Animal Care Facilities of the Institute of Biological Sciences (ICB), Federal University of Minas Gerais (UFMG). All experiments were approved by the Animal Ethics Committee of UFMG. Virus HSV-1 strain EK13 was allowed to multiply in Vero cells and was maintained with minimal essential medium (GIBCO, Grand Island, NY) containing 5% fetal bovine serum (FBS) (GIBCO) and 25 μg/μL of ciprofloxacin (Cellofarm, Carapina, ES, Brazil) at 37ºC in 5% CO2. Virus was purified in sucrose gradient and the titers de238 Arq Neuropsiquiatr 2011;69(2-A) termined in Vero cells as previously described14,15. The virus titers obtained were 1.1×108 plaque-forming cells (PFU)/mL for HSV-1. Vero cells Vero cells were maintained in minimal essential medium (GIBCO) supplemented with 5% heat-inactived FBS and antibiotics in 5% CO2 at 37ºC. These cells were used for virus multiplication. Infection with HSV-1 Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (150 mg/kg) and xylazine (10 mg/kg). A 104 plaque-forming units (PFU) inoculum of HSV-1 resuspended in 10 μL of phosphate-buffered saline (PBS) was injected intracranially in the right side of sagittal suture at the level of the eye10-12. Control mice received PBS. Intravital microscopy At 1 and 3 days post-infection (dpi) intravital microscopy of the mouse brain microvasculature was pe (...truncated)