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Toll-Like Receptor (TLR) 2 and TLR9 Expressed in Trigeminal Ganglia are Critical to Viral Control During Herpes Simplex Virus 1 Infection
2010; Elsevier BV; Volume: 177; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2010.100121
ISSN1525-2191
AutoresGraciela Kunrath Lima, Guilherme Pimenta Zolini, Daniel Santos Mansur, Bráulio Henrique Freire Lima, Uschi Wischhoff, Ruiz G. Astigarraga, Marcela França Dias, Mariana das Graças Almeida Silva, Samantha Ribeiro Béla, Lis Ribeiro do Valle Antonelli, Rosa Maria Esteves Arantes, Ricardo T. Gazzinelli, André Báfica, Erna Geessien Kroon, Marco Antônio Campos,
Tópico(s)interferon and immune responses
ResumoHerpes simplex virus 1 (HSV-1) is a neurotropic DNA virus that is responsible for several clinical manifestations in humans, including encephalitis. HSV-1 triggers toll-like receptors (TLRs), which elicit cytokine production. Viral multiplication and cytokine expression in C57BL/6 wild-type (WT) mice infected with HSV-1 were evaluated. Virus was found in the trigeminal ganglia (TG), but not in the brains of animals without signs of encephalitis, between 2 and 6 days postinfection (d.p.i.). Cytokine expression in the TG peaked at 5 d.p.i. TLR9−/− and TLR2/9−/− mice were more susceptible to the virus, with 60% and 100% mortality, respectively, as opposed to 10% in the WT and TLR2−/− mice. Increased levels of both CXCL10/IP-10 and CCL2/MCP-1, as well as reduced levels of interferon-γ and interleukin 1-β transcripts, measured in both the TG and brains at 5 d.p.i., and the presence of virus in the brain were correlated with total mortality in TLR2/9−/− mice. Cytokine alterations in TLR2/9−/− mice coincided with histopathological changes in their brains, which did not occur in WT and TLR2−/− mice and occurred only slightly in TLR9−/− mouse brain. Increased cellularity, macrophages, CD8 T cells producing interferon-γ, and expression levels of TLR2 and TLR9 were detected in the TG of WT-infected mice. We hypothesize that HSV-1 infection is controlled by TLR-dependent immune responses in the TG, which prevent HSV-1 encephalitis. Herpes simplex virus 1 (HSV-1) is a neurotropic DNA virus that is responsible for several clinical manifestations in humans, including encephalitis. HSV-1 triggers toll-like receptors (TLRs), which elicit cytokine production. Viral multiplication and cytokine expression in C57BL/6 wild-type (WT) mice infected with HSV-1 were evaluated. Virus was found in the trigeminal ganglia (TG), but not in the brains of animals without signs of encephalitis, between 2 and 6 days postinfection (d.p.i.). Cytokine expression in the TG peaked at 5 d.p.i. TLR9−/− and TLR2/9−/− mice were more susceptible to the virus, with 60% and 100% mortality, respectively, as opposed to 10% in the WT and TLR2−/− mice. Increased levels of both CXCL10/IP-10 and CCL2/MCP-1, as well as reduced levels of interferon-γ and interleukin 1-β transcripts, measured in both the TG and brains at 5 d.p.i., and the presence of virus in the brain were correlated with total mortality in TLR2/9−/− mice. Cytokine alterations in TLR2/9−/− mice coincided with histopathological changes in their brains, which did not occur in WT and TLR2−/− mice and occurred only slightly in TLR9−/− mouse brain. Increased cellularity, macrophages, CD8 T cells producing interferon-γ, and expression levels of TLR2 and TLR9 were detected in the TG of WT-infected mice. We hypothesize that HSV-1 infection is controlled by TLR-dependent immune responses in the TG, which prevent HSV-1 encephalitis. 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The most well-characterized pathway involves myeloid differentiation factor 88 (MyD88), an adapter molecule composed of a Toll-interleukin-1 receptor domain and a death domain.18Takeda K Kaisho T Akira S Toll-like receptors.Annu Rev Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4731) Google Scholar MyD88 recruits the serine/threonine kinase interleukin (IL) receptor associated kinase-4, which activates tumor necrosis factor (TNF)-α receptor-associated factor-6 that, in turn, phosphorylates inhibitor NF kappa B kinase (IkB) and causes it to dissociate from and release nuclear factor κB in the cytoplasm. 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In the same year, Hochrein et al21Hochrein H Schlatter B O'Keeffe M Wagner C Schmitz F Schiemann M Bauer S Suter M Wagner H Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways.Proc Natl Acad Sci USA. 2004; 101: 11416-11421Crossref PubMed Scopus (378) Google Scholar and Krug et al22Krug 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 (569) Google Scholar showed that TLR9 was important for the dendritic cell response to HSV-1. However, Krug et al22Krug 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 (569) Google Scholar could not find differences in viral replication or in susceptibility in TLR9−/− and MyD88−/− mice infected in the footpad or in the corneas with HSV-1. Nevertheless, our group23Mansur DS Kroon EG Nogueira ML Arantes RME Rodrigues SCO Akira S Gazzinelli RT Campos MA Lethal encephalitis in myeloid differentiation factor 88: deficient mice infected with herpes simplex virus 1.Am J Pathol. 2005; 166: 1419-1426Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar demonstrated that TLRs (and/or IL-1β) are essential to control the virus in an intranasal model of HSV-1 infection because 100% of MyD88−/− mice developed lethal encephalitis after viral inoculation. We also showed that 50% of the inoculated IFN-γ knockout (KO) mice died from encephalitis.23Mansur DS Kroon EG Nogueira ML Arantes RME Rodrigues SCO Akira S Gazzinelli RT Campos MA Lethal encephalitis in myeloid differentiation factor 88: deficient mice infected with herpes simplex virus 1.Am J Pathol. 2005; 166: 1419-1426Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Additionally, cooperation between TLR2 and TLR9 in HSV control has been demonstrated in HSV-1 infected dendritic cells24Sato A Linehan MM Iwasaki A Dual recognition of herpes simplex viruses by TLR2 and TLR9 in dendritic cells.Proc Natl Acad Sci USA. 2006; 103: 17343-17348Crossref PubMed Scopus (225) Google Scholar and, more recently, in an HSV-2 mouse model of vaginal and intraperitoneal infection.25Sorensen LN Reinert LS Malmgaard L Bartholdy C Thomsen AR Paludan SR TLR2 and TLR9 synergistically control herpes simplex virus infection in the brain.J Immunol. 2008; 181: 8604-8612PubMed Google Scholar TLRs have also been proposed to be important in Herpes simplex encephalitis in humans.26Casrouge A Zhang S Eidenschenk C Jouanguy E Puel A Yang K Alcais A Picard C Mahfoufi N Nicolas N Lorenzo L Plancoulaine S Sénéchal B Geissmann F Tabeta K Hoebe K Du X Miller RL Héron B Mignot C de Villemeur TB Lebon P Dulac O Rozenberg F Beutler B Tardieu M Abel L Casanova JL Herpes simplex virus encephalitis in human UNC-93B deficiency.Science. 2006; 314: 308-312Crossref PubMed Scopus (615) Google Scholar, 27Zhang S Jouanguy E Ugolini S Smahi A Elain G Romero P Segal D Sancho-Shimizu V Lorenzo L Puel A Picard C Chapgier A Plancoulaine S Titeux M Cognet C von Bernuth H Ku CL Casrouge A Zhang XX Barreiro L Leonard J Hamilton C Lebon P Héron B Vallée L Quintana-Murci L Hovnanian A Rozenberg F Vivier E Geissmann F Tardieu M Abel L Casanova JL TLR3 deficiency in patients with herpes simplex encephalitis.Science. 2007; 317: 1522-1527Crossref PubMed Scopus (875) Google Scholar How innate immunity and which TLRs contribute to the control of HSV-1 and related diseases are still unknown. In this study, we investigated how HSV-1 infection is controlled in a murine model of intranasal infection by using the HSV-1 EK strain, which was isolated from a human case of recurrent oral herpes with blisters. Our experiments indicated that in C57BL/6 wild-type (WT) mice, control of virus infection seemed to be highly regulated at the level of the TG. The levels of cytokine transcripts were directly related to the viral load in TG, and once the virus was controlled, the cytokine levels were reduced. Additionally, we found that TLR2 and, more importantly, TLR9 play a role in immune responses and immune control in the TG and mouse brain. Thus, it seems that HSV-1 infection control in the intranasal murine model occurs in the TG and brain, and TLR deficiencies may cause deregulated inflammation in these organs, which consequently allows virus entry into the brain and raises the susceptibility of mice to infection. HSV-1 strain EK,28Nogueira 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, was multiplied in Vero cells as previously described23Mansur DS Kroon EG Nogueira ML Arantes RME Rodrigues SCO Akira S Gazzinelli RT Campos MA Lethal encephalitis in myeloid differentiation factor 88: deficient mice infected with herpes simplex virus 1.Am J Pathol. 2005; 166: 1419-1426Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar and purified as previously described.29Campos 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 3.0 × 109 plaque forming units (PFU)/ml. Vero cells (American Type Culture Collection, Manassas, VA) were maintained in minimal essential medium supplemented with 5% heat-inactivated fetal bovine serum and antibiotics in 5% CO2 at 37°C. These cells were used for multiplication and titration of the virus. Human embryo kidney (HEK) 293 cells stably transfected with the pcDNA3 plasmid (Invitrogen, Carlsbad, CA) containing the human TLR2, TLR4MD.2, or TLR9 sequences or the empty vector, each fused with yellow fluorescent protein, were a kind gift from Dr. Douglas T. Golenbock (Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA). Cells were stimulated with HSV-1 (105 PFU/ml or at multiplicities of infection [MOIs] of 2 or 10), LPS (100 ng/ml; from Escherichia coli, 055:B5; Sigma, St. Louis, MO), Malp-2 (10 ng/ml; Alexis Biochemicals, San Diego, CA), CpG 2006 and 1826 (5 μmol/L; Alexis Biochemicals), or E. coli (100 units/cell) for 6 or 24 hours. Human IL-8 was measured by enzyme-linked immunosorbent assay (ELISA; BD, Franklin Lakes, NJ) in the supernatants of HEK293, HEK TLR4, and HEK TLR2 cells 24 hours after stimulation, and the relative increase in luciferase activity was measured in HEK TLR9 cells 6 hours after stimulation. Thioglycollate-elicited peritoneal macrophages were obtained from either C57BL/6, TLR2−/−, TLR9−/−, or TLR2/9−/− mice by peritoneal washing, activated with murine IFN-γ as previously described,23Mansur DS Kroon EG Nogueira ML Arantes RME Rodrigues SCO Akira S Gazzinelli RT Campos MA Lethal encephalitis in myeloid differentiation factor 88: deficient mice infected with herpes simplex virus 1.Am J Pathol. 2005; 166: 1419-1426Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar and then stimulated with HSV-1 (MOI of 10) for 24 hours. Murine TNF α and IL-12 p40 were measured in the supernatants by ELISA (BD). TLR2−/− and TLR9−/− mice were generated at Osaka University (Osaka, Japan) and were kind gifts from Shizuo Akira, and the TLR2/9−/− mice were obtained by crossing TLR2−/− and TLR9−/− mice at the National Institutes of Health (Bethesda, MD) and were kind gifts from Alan Sher. The mice were backcrossing to the C57BL/6 background for eight generations. The C57BL/6 (wild-type, control) and the knockout mice were maintained in a pathogen-free, barrier environment in the Centro de Pesquisas René Rachou, Oswaldo Cruz Foundation (CPqRR/FIOCRUZ; Belo Horizonte, Minas Gerais, Brazil). Six- to ten-week-old male mice were anesthetized with ketamine (Agribrands do Brasil Ltda, Brazil), and 106 PFU of purified HSV-1 in 10 μl was inhaled by the mice as described previously.30Boivin 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 (44) Google Scholar The control mice inhaled PBS. The mouse colonies and all experimental procedures were performed according to the institutional animal care and use guidelines from the CPqRR/FIOCRUZ. The project was previously approved by the Ethics Committee on Animal Experimentation (Comitê de Ética em Experimentação Animal (CETEA) from Universidade Federal de Minas Gerais (UFMG) and Comitê de Ética em Utilizaçào de Animais (CEUA) from CPqRR/FIOCRUZ). Frozen mouse tissues were ground with sterile sand and 300 μl (trigeminal ganglia) or 500 μl (brains) of Dulbecco's modified Eagle's medium containing 1% fetal bovine serum and antibiotics. Then the samples were centrifuged at 6700 g for 10 minutes at 4°C, and the supernatants were used for titration in a standard tissue culture infectious dose (TCID50) assay.31Schmidt NJ Lennette EH Schmidt NJ Cell culture techniques for diagnostic virology. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. American Public Health Association Inc, Washington DC1979: 100Google Scholar Trigeminal ganglia and brains were aseptically removed and stored at −70°C until processing. RNA extraction was performed by using the TRIzol reagent (Invitrogen) according to the manufacturer's procedures. One microliter of the extracted RNA was quantified with a Nanodrop ND-1000 spectrophotometer at wavelengths of 260 and 280 nm. Reverse transcription was performed according to the procedures provided by the manufacturer of the M-MLV RT enzyme (Promega, Madison, WI). Real-time quantitative PCR (Applied Biosystems, Carlsbad, CA) was performed to measure mRNA expression in the trigeminal ganglia and brains of mice infected with HSV-1. The reactions were performed by using the SYBR Green PCR Master Mix (Applied Biosystems) in an Applied Biosystems 7000 Sequence Detection System and at 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 minutes and 60°C for 1 minute, followed by a final dissociation stage. The following oligonucleotides were used in the reactions: Hypoxanthine-guanine phosphoribosyltransferase (forward: 5′-GTTGGATACAGGCCAGACTTTGTTG-3′; reverse: 5′-GATTCAACTTGCGCTCATCTTAGGC-3′); IFN β (forward: 5′-CTGGAGCAGCTGAATGGAAA-3′; reverse: 5′-TGTCTGCTGGTGGAGTTCAT-3′); IP-10 (CXCL10; forward: 5′-GCCGTCATTTTCTGCCTCAT-3′; reverse: 5′-GCTTCCCTATGGCCCTCATT-3′); MCP-1 (CCL2; forward: 5′-CTTCTGGGCCTGCTGTTCA-3′; reverse: 5′-CCAGCCTACTCATTGGGATCA-3′); MIP-1α (CCL3; forward: 5′-ACTGCCTGCTGCTTCTCCTA-3′; reverse: 5′-TTGGAGTCAGCGCAGATCTG-3′32Giulietti A Overbergh L Valckx D Decallonne B Bouillon R Mathieu C An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.Methods. 2001; 25: 386-401Crossref PubMed Scopus (1098) Google Scholar); IL-1β (forward: 5′-CGCAGCAGCACATCAACAAGAGC-3′; reverse: 5′-TGTCCTCATCCTGGAAGGTCCACG-3′); α trans-inducing factor (forward: 5′-TTTGACCCGCGAGATCCTAT-3′; reverse: 5′-GCTCCGTTGACGAACATGAA-3′33Broberg EK Nygardas M Salmi AA Hukkanen V Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by light cycler quantitative real time PCR.J Virol Methods. 2003; 112: 53-65Crossref PubMed Scopus (29) Google Scholar); TLR2 (forward: 5′-TTGCTCCTGCGAACTCCTAT-3′; reverse: 5′-AGCCTGGTGACATTCCAAGA-3′); and TLR9 (forward: 5′-ACCTCAGCCACAACATTCTC-3′; reverse: 5′-TGCACCTCCAACAGTAAGTC-3′.34Kuhlicke J Frick JS Morote-Garcia JC Rosenberger P Eltzschig HK Hypoxia inducible factor (HIF)-1 coordinates induction of toll-like receptors TLR2 and TLR6 during hypoxia.PLoS One. 2007; 2: e1364Crossref PubMed Scopus (144) Google Scholar The comparative Ct method with the formula 2−ΔΔCt was used to analyze the data. Gene expression was normalized to the expression of the constitutively expressed gene Hypoxanthine-guanine phosphoribosyltransferase. All reactions were replicated. Supernatants from HEK293 cells (empty vector and TLR2 and TLR4MD.2 transfected cells) were tested for the presence of human IL-8 (BD) according to the manufacturer's protocols. Supernatants from macrophages stimulated with HSV-1 were tested for the presence of murine TNF α and murine IL-12 p40 (BD) according to the manufacturer's protocols. Mice sera were tested for the presence of murine IL-1β and CXCL10 (IP10) by using ELISA kits (R&D Systems, Minneapolis, MN) and for murine IFN-γ and CCL2 (MCP1) by using the cytometric bead array (CBA) mouse inflammation kit (BD) according to the manufacturer's protocols. HEK TLR9 cells were cultured in 96-well plates (2 × 104 cells/well). After incubation for 1 day, cells were transiently transfected (using Genejuice [Novagen, Darmstadt, Germany] according to manufacturer's instructions) with a plasmid containing an artificial promoter preceding the firefly luciferase gene with five binding sites for nuclear factor κB. Cells were also co-transfected with a plasmid containing a constitutively expressed Renilla-luciferase reporter gene (Promega). After 24 hours, the cells were stimulated as described in Human Embryo Kidney Cells. After 6 hours, the cells were lysed, and 20 μl of each protein extract was used for the activity measurement. Extracts were distributed in 96-well plates, and 100 μl of luciferin was added to each well at room temperature immediately before the reading. Luciferase activity levels were detected in a Lumat LB 9501 over 10 seconds of luminosity. Brain samples were fixed with 10% formaldehyde in phosphate buffer, routinely processed, and embedded in paraffin as previously described.23Mansur DS Kroon EG Nogueira ML Arantes RME Rodrigues SCO Akira S Gazzinelli RT Campos MA Lethal encephalitis in myeloid differentiation factor 88: deficient mice infected with herpes simplex virus 1.Am J Pathol. 2005; 166: 1419-1426Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar For each group, topographically matched temporal lobe and periventricular white matter consecutive sections were scored in 14 microscopic fields (20× objective; n = 3 per group). The histopathological aspects of parenchyma and meninges were evaluated by vascular reactivity (endothelial reactivity and proliferation and level of perivascular cell infiltration) and scored as mild (+), moderate (++), or intense (+++). Vascular changes, edema, and cell infiltration of leptomeninges were scored as mild (+), moderate (++), or intense (+++). For trigeminal ganglia immunostaining, samples were frozen in Tissue-Tek O.C.T. compound (Sakura, Finetek, Torrance, CA), and 5-μm slices were cut with a HM505N microtome cryostat (Mikron, Vista, CA). Tissues were stained as previously described35Préhaud C Mégret F Lafage M Lafon M Virus infection switches TLR3 positive human neurons to become strong producers of beta interferon.J Virol. 2005; 79: 12893-12904Crossref PubMed Scopus (291) Google Scholar with modifications (see Supplemental Figure 1, A–E, at http://ajp.amjpathol.org). Briefly, the tissue sections were incubated with primary antibodies for 2 hours, washed, and incubated with labeled secondary antibody. The sections were counterstained with Hoechst and mounted in Hydromount aqueous medium (National Diagnostics, Atlanta, GA). The primary antibodies used were CD3 (1:100) and CD8 (1:100; Serotec, Raleigh, NC). The secondary antibody was Alexa Fluor 488 goat anti-rat IgG (1:500; Molecular Probes, Carlsbad, CA). Nuclei counterstaining was performed by using Hoechst (0.2 μg/ml; Molecular Probes). The stained sections were observed and photographed on an Olympus BX51 microscope (Olympus, Tokyo, Japan) by using a Megacybernetics color digital camera and the Image Pro-Express software. Single-cell suspensions were prepared from the TG of WT mice that were divided into control and infected groups. Tissues were digested with collagenase for 30 minutes at 37°C. After incubation, the TGs were disrupted by grinding with a syringe plunger. The cells were washed, suspended in RPMI with 10% fetal bovine serum, and counted by trypan blue exclusion with high viability. A total of 106 cells were plated for macrophage and dendritic cell measurements, and 5 × 105 for CD4, CD8, and natural killer (NK) cell measurements. Cells were incubated for 1 hour in 5% CO2 at 37°C. Then the BD GolgiPlug Protein Transport Inhibitor (BD Biosciences, San Jose, CA) was added to the wells where IFN-γ production was evaluated, and the plates were incubated for 8 hours. Plates were centrifuged at 532 × g for 5 minutes at 4°C and suspended in Fluorescence activated cell sorting (FACS) buffer solution with anti-mouse CD11c (FITC; BD Pharmingen, San Diego, CA), anti-mouse CD11b (PECy7; eBioscience, San Diego, CA), and an
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