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Lethal Encephalitis in Myeloid Differentiation Factor 88-Deficient Mice Infected with Herpes Simplex Virus 1
2005; Elsevier BV; Volume: 166; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)62359-0
ISSN1525-2191
AutoresDaniel Santos Mansur, Erna Geessien Kroon, Maurício Lacerda Nogueira, Rosa Maria Esteves Arantes, Soraia C. O. Rodrigues, Shizuo Akira, Ricardo T. Gazzinelli, Marco Antônio Campos,
Tópico(s)NF-κB Signaling Pathways
ResumoHerpes simplex virus 1 (HSV-1), a large DNA virus from the Herpesviridae family, is the major cause of sporadic lethal encephalitis and blindness in humans. Recent studies have shown the importance of Toll-like receptors (TLRs) in the immune response to HSV-1 infection. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein that is downstream to mediated TLR activation and is essential for the production of inflammatory cytokines. Here, we studied the relationship between MyD88 and HSV-1 using a purified HSV-1 isolated from a natural oral recurrent human infection. We observed the activation of TLR-2 by HSV-1 in vitro using Chinese hamster ovary cells stably transfected with a reporter gene. Interestingly, we found that only peritoneal macrophages from MyD88−/− mice, but not macrophages from TRL2−/− or from wild-type mice, were unable to produce tumor necrosis factor-α in response to HSV-1 exposure. Additionally, although TLR2−/− mice showed no enhanced susceptibility to intranasal infection with HSV-1, MyD88−/− mice were highly susceptible to infection and displayed viral migration to the brain, severe neuropathological signs of encephalitis, and 100% mortality by day 10 after infection. Together, our results suggest that innate resistance to HSV-1 is mediated by MyD88 and may rely on activation of multiple TLRs. Herpes simplex virus 1 (HSV-1), a large DNA virus from the Herpesviridae family, is the major cause of sporadic lethal encephalitis and blindness in humans. Recent studies have shown the importance of Toll-like receptors (TLRs) in the immune response to HSV-1 infection. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein that is downstream to mediated TLR activation and is essential for the production of inflammatory cytokines. Here, we studied the relationship between MyD88 and HSV-1 using a purified HSV-1 isolated from a natural oral recurrent human infection. We observed the activation of TLR-2 by HSV-1 in vitro using Chinese hamster ovary cells stably transfected with a reporter gene. Interestingly, we found that only peritoneal macrophages from MyD88−/− mice, but not macrophages from TRL2−/− or from wild-type mice, were unable to produce tumor necrosis factor-α in response to HSV-1 exposure. Additionally, although TLR2−/− mice showed no enhanced susceptibility to intranasal infection with HSV-1, MyD88−/− mice were highly susceptible to infection and displayed viral migration to the brain, severe neuropathological signs of encephalitis, and 100% mortality by day 10 after infection. Together, our results suggest that innate resistance to HSV-1 is mediated by MyD88 and may rely on activation of multiple TLRs. Herpes simplex virus 1 (HSV-1), from the Herpesviridae family, is a complex virus containing a large 140-kb DNA, which encodes 84 proteins and is the ubiquitous neurotropic human pathogen most commonly associated with oro-labial and ocular infections.1Roizman B Whitley RJ The nine ages of herpes simplex virus.Herpes. 2001; 8: 23-27PubMed Google Scholar The most serious infection caused by HSV-1 is sporadic encephalitis,2Boivin G Coulombe Z Rivest S Intranasal herpes simplex virus type 2 inoculation causes a profound thymidine kinase dependent cerebral inflammatory response in the mouse hindbrain.Eur J Neurosci. 2002; 16: 29-43Crossref PubMed Scopus (46) Google Scholar which has a mortality rate of ∼70%, when not treated.3Hirsh HH Bossart W Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis.J Med Virol. 1999; 57: 31-35Crossref PubMed Scopus (14) Google Scholar HSV-1 is transmitted primarily by contact with oral secretions. On oral entry into skin and mucosal sites, HSV-1 replicates locally in epithelial cells, resulting in cell lysis and local inflammatory response. After primary infection, HSV-1 can travel along sensory nerve pathways and may become latent in the sensory ganglia, where it can eventually be reactivated.3Hirsh HH Bossart W Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis.J Med Virol. 1999; 57: 31-35Crossref PubMed Scopus (14) Google Scholar Animal models of human HSV encephalitis in mice using intranasal inoculation have been described.2Boivin G Coulombe Z Rivest S Intranasal herpes simplex virus type 2 inoculation causes a profound thymidine kinase dependent cerebral inflammatory response in the mouse hindbrain.Eur J Neurosci. 2002; 16: 29-43Crossref PubMed Scopus (46) Google Scholar This inoculation pathway leads to an inflammatory response that can be dangerous to the host. However, the precise mechanisms by which HSV-1 causes death are not clear. Toll-like receptors (TLRs) are innate immunity receptors linked with the response to pathogen-associated molecular patterns. Since the first description of TLRs in mammals, many TLR agonists have been described: peptidoglycans4Takeuchi O Hoshino K Kawai T Sanjo H Takada H Ogawa T Takeda K Akira S Differential roles of TLR2 and TLR4 in recognition of Gram negative and Gram positive bacterial cell wall components.Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2833) Google Scholar and Trypanasoma cruzi GPI anchor for TLR2,5Campos MA Almeida IC Takeuchi O Akira S Paganini E Procópio DO Travassos LR Smith JA Golenbock DT Gazzinelli RT Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.J Immunol. 2001; 167: 416-423PubMed Google Scholar lipopolysaccharide (LPS) for TLR4,6Poltorak A He X Smirnova I Liu MY Van Huffel C McNally O Birdwell D Alejos E Silva M Galanos C Freudenberg M Ricciardi-Castagnoli P Layton B Beutler B Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6583) Google Scholar, 7Lien E Means TK Heine H Yoshimura A Kusumoto S Fukase K Fenton MJ Oikawa M Qureshi N Monks B Finberg RW Ingalls RR Golenbock DT Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.J Clin Invest. 2000; 105: 497-504Crossref PubMed Scopus (696) Google Scholar, 8Campos MA Rosinha GMS Almeida IC Salgueiro XS Jarvis BW Splitter GA Bruna-Romero O Gazzinelli RT Oliveira SC The role of Toll-like receptor 4 in induction of cell-mediated immunity and resistance to Brucella abortus infection in mice.Infect Immun. 2004; 72: 176-186Crossref PubMed Scopus (100) Google Scholar dsRNA for TLR3,9Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NFKB by Toll-like receptor 3.Nature. 2001; 413: 432-438Crossref PubMed Scopus (5058) Google Scholar flagellin for TLR5,10Hayashi F Smith KD Ozinsky A Hawn TR Yi EC Goodlett DR Eng JK Akira S Underhill DM Aderem A The innate immune response to bacterial flagelin is mediated by Toll-like receptor 5.Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2886) Google Scholar and CpG DNA for TLR9.11Hemmi H Takeuchi O Kawai T Kaisho T Sato S Sanjo H Matsumoto M Hoshino K Wagner H Takeda K Akira S A Toll-like receptor recognizes bacterial DNA.Nature. 2000; 408: 740-745Crossref PubMed Scopus (5481) Google Scholar TLRs activate inflammatory responses and modulate immunity by several different signal transduction pathways. The most well known pathway involves myeloid differentiation factor 88 (MyD88), an adapter molecule composed of a Toll-interleukin-1 receptor domain and a death domain.12Takeda K Kaisho T Akira S Toll-like receptors.Annu Rev Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4829) Google Scholar MyD88 recruits the serine threonine kinase, interleukin receptor associated kinase-4, that activates tumor necrosis factor-α receptor-associated factor-6 (TRAF-6) which in turn phosphorylates IκB, causing it to dissociate from and leave nuclear factor (NF)-κB free in the cytoplasm. NF-κB then translocates to the nucleus and acts as a transcription factor of innate immunity-associated genes.12Takeda K Kaisho T Akira S Toll-like receptors.Annu Rev Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4829) Google Scholar, 13Yamamoto M Takeda K Akira S TIR domain-containing adaptors define the specificity of TLR signaling.Mol Immunol. 2004; 40: 861-868Crossref PubMed Scopus (308) Google Scholar In addition, TLR3 appears to activate the inflammatory response through another adapter molecule, named Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β.13Yamamoto M Takeda K Akira S TIR domain-containing adaptors define the specificity of TLR signaling.Mol Immunol. 2004; 40: 861-868Crossref PubMed Scopus (308) Google Scholar This pathway is MyD88-independent, and culminates with the translocation of interferon regulatory factor 3 (IRF-3) to the nucleus, leading to production of interferon (IFN)-β and IFN-inducible genes.13Yamamoto M Takeda K Akira S TIR domain-containing adaptors define the specificity of TLR signaling.Mol Immunol. 2004; 40: 861-868Crossref PubMed Scopus (308) Google Scholar A role for the TLR2, TLR3, TLR4, and TLR9 in the response to viruses has been previously established.9Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NFKB by Toll-like receptor 3.Nature. 2001; 413: 432-438Crossref PubMed Scopus (5058) Google Scholar, 12Takeda K Kaisho T Akira S Toll-like receptors.Annu Rev Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4829) Google Scholar, 14Krug A Luker GD Barchet W Leib DA Akira S Colonna M Herpes simplex virus type 1 (HSV-1) activates murine natural interferon-producing cells (IPC) through Toll-like receptor 9.Blood. 2004; 103: 1433-1437Crossref PubMed Scopus (581) Google Scholar, 15Kurt-Jones EA Popova L Kwinn L Haynes LM Jones LP Tripp RA Walsh EE Freeman MW Golenbock DT Anderson LJ Finberg RW Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.Nat Immunol. 2000; 1: 398-401Crossref PubMed Scopus (1368) Google Scholar, 16Kurt-Jones EA Chan M Zhou S Wang J Reed G Bronson R Arnold MM Knipe DM Finberg RW Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis.Proc Natl Acad Sci USA. 2004; 101: 1315-1320Crossref PubMed Scopus (532) Google Scholar, 17Lund J Sato A Akira S Medzhitov R Iwasaki A Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells.J Exp Med. 2003; 198: 513-520Crossref PubMed Scopus (1026) Google Scholar, 18Lundberg P Welander P Han X Cantin E Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo.J Virol. 2003; 77: 11158-11169Crossref PubMed Scopus (71) Google Scholar Lund and colleagues17Lund J Sato A Akira S Medzhitov R Iwasaki A Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells.J Exp Med. 2003; 198: 513-520Crossref PubMed Scopus (1026) Google Scholar showed that genomic HSV-2 DNA, which is closely related to HSV-1, was recognized by TRL9 and mediated activation through an MyD88-dependent endocytic pathway leading to type I IFN response. Using a recombinant HSV-1 KOS strain, Krug and colleagues14Krug A Luker GD Barchet W Leib DA Akira S Colonna M Herpes simplex virus type 1 (HSV-1) activates murine natural interferon-producing cells (IPC) through Toll-like receptor 9.Blood. 2004; 103: 1433-1437Crossref PubMed Scopus (581) Google Scholar confirmed the involvement of TLR9 in type I IFN response. Lundberg and colleagues18Lundberg P Welander P Han X Cantin E Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo.J Virol. 2003; 77: 11158-11169Crossref PubMed Scopus (71) Google Scholar also showed that HSV-1 DNA is stimulatory both in vitro and in vivo. Recently, Kurt-Jones and colleagues16Kurt-Jones EA Chan M Zhou S Wang J Reed G Bronson R Arnold MM Knipe DM Finberg RW Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis.Proc Natl Acad Sci USA. 2004; 101: 1315-1320Crossref PubMed Scopus (532) Google Scholar demonstrated that TLR2 mediates the induction of inflammatory cytokines in response to intravenous inoculation with the HSV-1 KOS strain, whereas in mice lacking functional TLR2, they detected a reduction in encephalitis symptoms. Here we used a HSV-1 isolated from a natural oral recurrent human infection, expanded in Vero cells, and purified in sucrose gradient.19Joklik WK The purification of four strains of poxvirus.Virology. 1962; 18: 9-18Crossref PubMed Scopus (395) Google Scholar We demonstrate the activation of TLR2 by HSV-1 in vitro using Chinese hamster ovary (CHO) cells stably transfected with human TLR2 and a reporter gene. We also show for the first time, using an in vivo mouse model of intranasal inoculation,3Hirsh HH Bossart W Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis.J Med Virol. 1999; 57: 31-35Crossref PubMed Scopus (14) Google Scholar which is a natural route of infection, that HSV-1 leads to lethal encephalitis in 100% of the mice lacking the functional MyD88 protein. These results further suggest the importance of TLRs and innate immunity in host resistance to HSV-1. HSV-1 strain EK,20Nogueira ML Siqueira RC Freitas N Amorim JB Bonjardim CA Ferreira PC Orefice F Kroon EG Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.J Clin Pathol. 2001; 54: 103-106Crossref PubMed Scopus (32) Google Scholar isolated from a human case of recurrent oral herpes with blisters and Vaccinia virus Western Reserve (VV) were allowed to multiply in Vero cells, maintained with minimal essential medium (GIBCO, Grand Island, NY) containing 5% fetal bovine serum (FBS) (GIBCO) and 25 μg/μl of ciprofloxacin (Fesenius, Pune, India) at 37°C in a 5% CO2 atmosphere. HSV-1 and VV were purified in sucrose gradients,19Joklik WK The purification of four strains of poxvirus.Virology. 1962; 18: 9-18Crossref PubMed Scopus (395) Google Scholar and the titers determined in Vero cells as previously described.21Campos MA Kroon EG Critical period of irreversible block of vaccinia virus replication.Rev Bras Microbiol. 1993; 24: 104-110Google Scholar The virus titers obtained were: 1.1 × 108 PFU/ml for HSV-1 and 2 × 1010 PFU/ml for VV. LPS from Escherichia coli O55:B5 was obtained from Sigma (St. Louis, MO) and UV-inactivated S. aureus was described before.5Campos MA Almeida IC Takeuchi O Akira S Paganini E Procópio DO Travassos LR Smith JA Golenbock DT Gazzinelli RT Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.J Immunol. 2001; 167: 416-423PubMed Google Scholar Vero cells were maintained in minimal essential medium supplemented with 5% heat-inactivated FBS and antibiotics in 5% CO2 at 37°C. These cells were used for multiplication and titration of virus and in neutralization tests. The CHO reporter cell lines,22Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (792) Google Scholar, 23Delude RL Yoshimura A Ingalls RR Golenbock DT Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction.J Immunol. 1998; 161: 3001-3009PubMed Google Scholar a kind gift from Douglas T. Golenbock (University of Massachusetts Medical School, Worcester, MA), were maintained as adherent monolayers in Ham's F-12/Dulbecco's modified Eagle's medium supplemented with 5% FBS and antibiotics at 37°C, 5% CO2. All of the cell lines were derived from clone 3E10, a CHO/CD14 cell line that has been stably transfected with a reporter construct containing the structural gene for CD25 under the control of the human E-selectin promoter. This promoter contains a NF-κB binding site; CD25 expression is completely dependent on NF-κB translocation to the cell nucleus.23Delude RL Yoshimura A Ingalls RR Golenbock DT Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction.J Immunol. 1998; 161: 3001-3009PubMed Google Scholar Cells expressing TLRs were constructed by stable transfection of the CHO/CD14 reporter cell line with the cDNA for human TLR2 or expressing endogenous TLR4 as described.22Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (792) Google Scholar In addition to the LPS-responsive cell lines described above, we also tested a LPS nonrespondent cell line derived from 3E1022Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (792) Google Scholar designated clone 7.19, as well as a clonal line derived from this mutant that was transfected with CD14 and TLR2 (7.19/CD14/TLR2). The LPS nonresponsive phenotype of the 7.19 cell lines is due to a mutation in the MD-2 gene, and thus is defective in signaling via TLR4.22Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (792) Google Scholar These cell lines report NF-κB activation via surface expression of CD25, similarly to the other CHO cell lines described. CHO reporter cells were plated at a density of 1 × 105 cells/well in a 24-well tissue culture dish. After 20 hours, UV-inactivated bacteria, HSV-1 or VV were added in a total volume of 250 μl of medium/well for 18 hours. The cells were then harvested with trypsin-ethylenediamine-tetraacetic acid (Sigma, St. Louis, MO) and washed once with medium containing 5% FBS and then with phosphate-buffered saline (PBS). Cells harvested physically without trypsin displayed similar results. Subsequently, the cells were counted and 1 × 105 cells stained with phycoerythrin-labeled anti-CD25 (mouse monoclonal antibody to human CD25, R-PE conjugate; Caltag Laboratories, Burlingame, CA) 1:200 in PBS, on ice in the dark, for 30 minutes. After labeling, the cells were washed twice with PBS containing 1 mmol/L sodium azide (Sigma), and 10,000 cells were examined by flow cytometry (BD Biosciences, San Jose, CA) for the expression of surface CD25 as described.5Campos MA Almeida IC Takeuchi O Akira S Paganini E Procópio DO Travassos LR Smith JA Golenbock DT Gazzinelli RT Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.J Immunol. 2001; 167: 416-423PubMed Google Scholar, 8Campos MA Rosinha GMS Almeida IC Salgueiro XS Jarvis BW Splitter GA Bruna-Romero O Gazzinelli RT Oliveira SC The role of Toll-like receptor 4 in induction of cell-mediated immunity and resistance to Brucella abortus infection in mice.Infect Immun. 2004; 72: 176-186Crossref PubMed Scopus (100) Google Scholar, 22Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (792) Google Scholar After excluding dead cells by gating with forward and side scatter parameters, an average of 8750 ± 312 live cells, were analyzed for the expression of CD25. Analysis was performed using CellQuest software (BD Biosciences). TLR2−/− and MyD88−/− mice were generated at Osaka University (Osaka, Japan) and backcrossed in the C57BL/6 background for eight generations. IFNγ−/− mice in the C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). The knockout mice were transferred to the Federal University of Minas Gerais, Institute of Biological Sciences (Belo Horizonte, Minas Gerais, Brazil) and maintained in a pathogenic-free, barrier environment. C57BL/6 mice, used as wild-type (WT) control, were obtained from the Centro de Pesquisas René Rachou, Oswaldo Cruz Foundation (Belo Horizonte, Minas Gerais, Brazil). Four-week-old male mice were anesthetized with ketamine (Agribrands do Brasil Ltda, Paulinia, Brazil), and 104 PFU of the purified HSV-1 contained in 10 μl were inhaled by mice as described previously.5Campos MA Almeida IC Takeuchi O Akira S Paganini E Procópio DO Travassos LR Smith JA Golenbock DT Gazzinelli RT Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.J Immunol. 2001; 167: 416-423PubMed Google Scholar Control mice inhaled PBS. Nine mice from each knockout or WT group were used in the survival experiments shown in Figure 2. Eight days after infection, brain, lung, liver, and spleen were removed from three animals per group and either frozen or fixed in formalin (Sciavicco Comercio e Industria Ltda, Belo Horizonte, Brazil). Each experiment was repeated three times. Mice presenting symptoms such as total paralysis and/or seizures were sacrificed. The mouse colonies and all experimental procedures were performed according to the institutional animal care and use guidelines from the Centro de Pesquisas René Rachou, Oswaldo Cruz Foundation. Thioglycollate-elicited peritoneal macrophages were obtained from either C57BL/6, TLR2−/−, or MyD88−/− mice by peritoneal washing. Adherent peritoneal macrophages were cultured in 96-well plates (2 × 105 cells/well) at 37°C/5% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Paisley, UK) supplemented with 5% heat-inactivated FBS (Life Technologies), 2 mmol/L l-glutamine (Sigma) and 40 μg/ml of gentamicin (Schering do Brasil, Rio de Janeiro, Brazil). Cells were then stimulated with HSV-1 (multiplicity of infection, 40), LPS, or S. aureus for 24 hours to evaluate TNF-α production. TNF-α was quantified using a DuoSet ELISA kit from R&D Systems (Minneapolis, MN). Frozen mice tissues were ground with sterile sand and 200 μl of minimal essential medium, centrifuged, and the supernatant was used for titration in a standard tissue culture infectious dose (TCID50) assay24Schmidt NJ Lennette EH Schmidt NJ Cell culture techniques for diagnostic virology. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. American Public Health Association, Inc., Washington1979: 100Google Scholar and for nested PCR. The primers and conditions used for the first reaction of PCR were described previously by Nogueira and colleagues.20Nogueira ML Siqueira RC Freitas N Amorim JB Bonjardim CA Ferreira PC Orefice F Kroon EG Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.J Clin Pathol. 2001; 54: 103-106Crossref PubMed Scopus (32) Google Scholar The nested PCR was developed using the primers specific for HSV-1 thymidine kinase: TKI3 CCA GCA TAG CCA GGT CAA GC and TKI5 GCG AAC ATC TAC ACC ACA CAA CA. The reaction was performed at 95°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute for 40 cycles. Brain samples were fixed with 10% formaldehyde in phosphate buffer and then embedded in paraffin. Sections were mounted on glass slides, deparaffinized, and then treated with 3.5% H2O2 in PBS. Tissue sections were blocked with 7% normal goat serum in PBS for 30 minutes at room temperature and incubated overnight with polyclonal rabbit anti-herpes simplex I antibody p0175 (DAKO, NH) diluted 1:50 or monoclonal anti-gC HSV-1 diluted 1:500 in PBS containing 0.4% Triton X-100. After incubation with primary antibody, tissue sections were washed three times with PBS and incubated for 90 minutes at room temperature with the secondary biotinylated antibody solution (DAKO, NH), washed with PBS, and treated with the tertiary solution, containing peroxidase-conjugated streptavidin (DAKO, NH), for 60 minutes at room temperature. Sections were then rinsed in PBS and with 3,3′-diaminobenzidine tetrahydrochloride (Sigma) (0.05%) and hydrogen peroxide (0.03%). The sections were then rinsed in PBS and stained with hematoxylin and eosin (H&E) (Reagen, Rio de Janeiro, Brazil). Sera of mice were serially diluted from 1:10 to 1:1280 in minimal essential medium in a total volume of 100 μl. One hundred TCID50 of HSV-1 was added to each dilution and incubated for 1 hour at 37°C in a 5% CO2 atmosphere. The mixture was added to a 96-well plate containing Vero cells. The plates were incubated and observed during 5 days. All of the samples were titrated in duplicate. The titer was determined as the inverse of the highest dilution of serum that protected Vero cells from cytopathic effect of HSV-1. Statistical analyses were performed with Student's t-test using the software program Minitab (Minitab Inc., State College, PA). We investigated if a recently isolated strain of HSV-1, purified by sucrose gradient, induces activation of TLR2 in vitro. Flow cytometry analysis of the expression of a CD25 reporter gene in CHO cells, stably transfected with the TLR constructions described previously22Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (792) Google Scholar, 23Delude RL Yoshimura A Ingalls RR Golenbock DT Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction.J Immunol. 1998; 161: 3001-3009PubMed Google Scholar is shown in Figure 1A. We used cells stably transfected with CD14 alone (CHO/CD14) or with CD14 and TLR2 (CHO/CD14/TLR2), both expressing endogenous TLR4, as well as the clone 7.19, which does not have a functional TLR4 signaling pathway, but are stably transfected with CD14 (7.19) or CD14 and TLR2 (7.19/CD14/TLR2). These cells were exposed for 18 hours to 104 PFU of HSV-1 or Vaccinia virus (VV). VV served as a negative control,25Bowie A Kiss-Toth E Symons JA Smith GL Dower SK O'Neil LAJ A46R and A52R from vaccinia virus are antagonists of host IL-1 and Toll-like receptor signaling.Proc Natl Acad Sci USA. 2000; 97: 10162-10175Crossref PubMed Scopus (398) Google Scholar because it was produced in cell cultures and purified by the same process19Joklik WK The purification of four strains of poxvirus.Virology. 1962; 18: 9-18Crossref PubMed Scopus (395) Google Scholar used to purify HSV-1. We observed that the cells stimulated with HSV-1 were activated through TLR2, but not through TLR4. Figure 1B shows an increased percentage of CD25-positive cells in TLR2/CHO or TLR2/TLR4/CHO cells stimulated with HSV-1. These data indicate that purified HSV-1 triggers NF-κB through TLR2/CD14, but not through TLR4/CD14. To compare the host innate immune response after HSV-1 challenge ex vivo, we measured the levels of TNF-α in culture supernatants of macrophages from WT, TLR2−/−, and MyD88−/− mice (Figure 1C). Both WT and TLR2−/− produced significant amounts of this cytokine when challenged by HSV-1, whereas the production in MyD88−/− was totally abrogated. LPS and S. aureus were used as controls. LPS,6Poltorak A He X Smirnova I Liu MY Van Huffel C McNally O Birdwell D Alejos E Silva M Galanos C Freudenberg M Ricciardi-Castagnoli P Layton B Beutler B Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6583) Google Scholar, 7Lien E Means TK Heine H Yoshimura A Kusumoto S Fukase K Fenton MJ Oikawa M Qureshi N Monks B Finberg RW Ingalls RR Golenbock DT Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.J Clin Invest. 2000; 105: 497-504Crossref PubMed Scopus (696) Google Scholar differently from S. aureus,22Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (792) Google Scholar still activated macrophages from TLR2−/− mice, whereas none of the microbial stimuli were effective on macrophages from MyD88−/− mice.13Yamamoto M Takeda K Akira S TIR domain-containing adaptors define the specificity of TLR signaling.Mol Immunol. 2004; 40: 861-868Crossref PubMed Scopus (308) Google Scholar Our next step was to evaluate the importance of TLR2 and MyD88 during infection with HSV-1 in an in vivo model. We used the intranasal model2Boivin G Coulombe Z Rivest S Intranasal herpes simplex virus type 2 inoculation causes a profound thymidine kinase dependent cerebral inflammatory response in the mouse hindbrain.Eur J Neurosci. 2002; 16: 29-43Crossref PubMed Scopus (46) Google Scholar because it is a natural route of infection with HSV-1. Four-week-old C57BL/6, TLR2−/−, and MyD88−/− mice were inoculated with 104 PFU of HSV-1 intranasally. Because it has been previously demonstrated that IFN-γ receptor-deficient mice are more susceptible to infection with HSV-126Smith PS Wolcott RM Chervenak R Jennings SR Control of acute Herpes simples virus infection: T-cell-mediated viral clearance is dependent upon interferon-γ (IFN-γ).Virology. 1
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