HSV-1 persistently infects almost 90% of our population; however, only 30% of the infected subjects suffer from recurrent herpes lesions, most frequently herpes labialis (HL). Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Targeting this initial first step in HSV-1 pathogenesis, we generated different zinc oxide (ZnO) micro-nano structures (MNSs) that were capped with multiple nanoscopic spikes mimicking cell induced filopodia. Full text Full text is available as a scanned copy of the original print version. Short exposure of AzVDU-treated FM3A TK-/HSV-1 TK+ cells to irradiation at lambda = 254 nm enhanced the cytostatic activity of AzVDU by 5-fold. Full text Full text is available as a scanned copy of the original print version. The patients received nbUVB sessions, three times a week, for a total of 15 sessions or until the pain disappeared.
A 2008 study states that “non-thermal near infra-red…has been shown to have many beneficial photobiological effects on a range of cell types, including neurons. Herpes simplex virus-1 (HSV-1) is an alphaherpesvirus, which infects both dividing cells (epithelial cells) and non-dividing cells (neurons). In these studies, measurement of proinflammatory cytokines and type I IFN release from the infected DCs reflected the activation status of these transcription factors. Get a printable copy (PDF file) of the complete article (1.4M), or click on a page image below to browse page by page. Herpes simplex virus type 1 (HSV-1) is a highly successful human pathogen belonging to the Alphaherpesviridae subfamily of herpesviruses. Biological effects include: Increase in: arterial and venous oxygenation, blood pH, lymphocytes, electrical charge on the red blood cell, of bacteriocidal capacity of the blood, hemoglobin, white blood cell count, lymphocytes, phagocytosis ability; Reduction of : cholesterol, creatinine levels, blood lactate, clot formation tendencies, blood viscosity, uric acid levels, fibrin levels, plasma viscosity, surface tension of the blood; Normalization or modulation of: immune status, glucose tolerance , and fibrinolysis. Normal HSV infections are characterized by cycling between lytic infection at epithelial surfaces and stages of latency in neuronal cells (reviewed in reference 47).
The pathology of HSV infection is greatly influenced by the immune status of the host, which impacts both disease severity and the frequency of reactivation (21, 35, 42, 48, 69). Early during primary infection of the epithelium, HSV encounters a specialized type of immune cell, the dendritic cells (DCs). Occasionally, some people feel worse before they feel better. These cells survey peripheral tissues in an immature state and undergo a process referred to as maturation (or activation) upon encounter with virus-associated molecules (5, 32). DC maturation is initially characterized by the secretion of type I and III interferons (IFNs) and proinflammatory cytokines (e.g., interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α], and IL-12) and regulation of molecules necessary for migration to peripheral lymph nodes (32). It was suggested that type and extent of damage to DNA for virus in the presence of RB and light make it less likely to be repaired by normal repair pathways available in host cells 4. Numerous viral proteins are utilized by HSV to evade the host immune response at all stages of the virus life cycle (7, 9, 31, 33, 39, 63).
Immunomodulatory proteins are either produced during the virus replication cycle or prepackaged in viral particles in the tegument and deposited into the cell immediately following virus envelope-host cell membrane fusion. Some report that psychotropic medications (pain pills, anxiety and depression pills) should be used with caution as UVP may increase their effect. I. vhs has been reported to interfere with DC activation during both productive and nonproductive HSV infection (7, 49). The neurovirulent HSV-1 (McKrae) was grown in Vero (African green monkey kidney) cells in Eagle’s minimal essential medium 199 (EMEM:199) (Quality Biologicals, Inc., Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS) (Quality Biologicals, Inc.) and 1% glutamine-streptomycin-penicillin (Life Technologies Gibco BRL, Gaithersburg, Md.). The effect of ultraviolet radiation on the progression of skin disease has been the subject of investigation for almost a century. Moreover, vhs can block the activation of DCs triggered during coinfection of HSV-1 with RNA viruses.
One implication of this earlier study is that vhs may target the RIG-I-like receptor (RLR) family of cytosolic sensors, the pattern recognition receptors that detect these RNA viruses. Type I IFNs (IFN-α/β) are critical antiviral factors produced during virus infection (reviewed in reference 60). An initial induction phase results in modest levels of IFN-β expression driven by activation of the transcription factors NF-κB, IRF3, and AP-1. We suggest that the balance between activation via these two pathways and inhibition by viral products is a key factor in determining the immunological outcomes of HSV-1 infection. Adelung, C. In addition to limiting virus infection, activation of the type I IFN pathway is important for DCs to undergo the maturation process (55, 60). Infection by certain viruses, such as Newcastle disease virus (NDV) and murine cytomegalovirus (MCMV), fail to mature DCs when these cells are generated from IFN receptor (IFNR) knockout (KO) mice, highlighting a crucial role for type I IFN signaling in this process (8, 18).
The replication of HSV-1 viruses in which the gene for vhs (UL41) is either mutated or absent is only moderately affected in both transformed and primary cell lines (7, 41). The patients included nine patients with acute herpetic neuralgia, six patients with subacute herpetic neuralgia, and two patients with established PHN. There is much interest in employing vhs-null viruses as live, attenuated vaccines for HSV due in large part to this replication defect (15, 22, 23, 64). The VP16 promoter construct was constructed same as RR using the forward primer: 5`tgggtggggtt3` and the reverse primer: 5`ccaaggaagagcgtccg3`. Consistent with this idea are the in vitro findings that vhs mutant infections of DCs results in increased STAT1 phosphorylation and more ISG transcripts than wild-type (WT) virus infections (10, 27, 41). Here, we sought to better understand the early events governing the interaction between HSV-1 and DCs. To achieve this, we utilized a recombinant HSV deficient in host shutoff activity (which strongly activates DCs) as a tool to aid in understanding which molecular pathways are involved in the early activation of DCs following HSV-1 infection.
In addition, we utilized this vhs-null virus to understand mechanistically how the vhs-mediated repression of DC function occurs. Our data show that the vhs protein carried in the virion and delivered into DCs blocks the early replication-independent activation of NF-κB during HSV-1 infection. KOS 1.1, referred to in this study as KOS, was the wild-type strain of HSV-1 used in all experiments. The vhs Δsma mutant, referred to in this study as vhs−, was generously provided by G. The biological cycle that P.O.T. It contains a deletion of the 588-bp region between SmaI restriction enzyme sites in the UL41 gene. Importantly, this vhs− deletion virus does not incorporate the mutant vhs polypeptide into virions (45).
The solution was overwrapped in an opaque foil pouch to protect it from the light. For experiments requiring UV-inactivated viruses, viral stocks were exposed to UV irradiation at 10 cm from a germicidal lamp (UVP Multiple-Ray 8-WUV lamp [60 Hz]; Fisher) for 4 min. This amount of UV irradiation has previously been shown to prevent viral transcription and replication without inactivating the function of a representative tegument protein, VP16 (50). Naswitis irradiated the blood directly through a shunt. Further, no virus growth was observed during Vero cell plaque assay. Recombinant NDV B1 was generated from the B1 Hitchner avian vaccine strain as previously described (40). On the indicated days, both eyes were swabbed with a moistened Dacron polyester-tipped applicator (Baxter Healthcare Corp., Deerfield, Ill.).
Leishmanial disease occurs on every continent except Australia and Antarctica. Lipopolysaccharide (LPS) was obtained from Alexis Biochemicals and utilized at a concentration of 300 ng/ml. For all experiments, HSV-1 (both wild-type and vhs− mutant viruses) was added to DCs at a multiplicity of infection (MOI) of 5 (PFU/cell). RNA viruses were added at an MOI of 1.5. For cotreatment of HSV-1-infected cells with TLR agonists and coinfections with RNA viruses, following HSV-1 treatment, the TLR agonist (or RNA virus) was added to the cells immediately. The virus stock used in this entire study had a titer of 1 × 109 PFU/ml as determined by plaque assay. The enzymatic activity was monitored at 410 nm by spectrophotometry (BioTek Instruments Inc.
Wild-type C57BL/6 and Sv129 mice were obtained from Taconic Farms (Germantown, NY). Type I IFNR KO mice were obtained from B&K Universal on the Sv129 genetic background, STAT1 KO mice were obtained from Taconic Farms on the Sv129 background, and MAVS (IPS-1) KO mice were generously provided by Kate Fitzgerald (University of Massachusetts) on the C57BL/6 background with the permission of Zhijian Chen (University of Texas Southwestern). All mice were bred and maintained in our facility consistent with regulations for animal care standard protocols as described by the Mount Sinai School of Medicine IACUC. In addition to suppression of LCs, UVB modifies the T-cell response to persistent VZV particles in nerve fibers, which might be involved in the pathogenesis of PHN. The inflammatory response of the body to VZV is predominantly of Th-1 type with the release of IFN-γ and IL-2, whereas UVB induces a shift from a Th-1 immune response to a Th-2 response in different ways. All cells were grown at 37°C at 5 to 7% CO2. The cellular DNA damage pathway induction by dosage treatment with camptothecin () and etoposide () were measured through determination of the phosphorylation of histone H2Ax (γ-H2Ax). Briefly, bone marrow was extracted from mouse femurs and tibias.
Red blood cells were lysed with ammonium chloride buffer. MHC class II-expressing cells and lymphocytes were removed using a cocktail of antibodies and magnetic-bead separation. The purified precursor cells were cultured in 24-well dishes in RPMI medium containing 10% fetal bovine serum (FBS), 50 μg/ml gentamicin, 2 mM glutamine, 1 mM sodium pyruvate (NaPy), and 50 U/ml recombinant granulocyte macrophage colony-stimulating factor (PreproTech EC) at 1 × 106 cells/ml. Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-6 (DuoSet ELISA development systems) were obtained from R&D Systems. ELISAs for mouse IL-12p40 were conducted using capture and secondary antibodies from BD Pharmingen. ELISA kits for IFN-β were obtained from PBL. Manufacturer instructions were followed for all ELISA methods used.
Plates were read on an ELISA reader from Bioteck Instruments. Protein extracts were generated from infected cell cultures by lysing cells in an NP-40-based lysis buffer containing protease and phosphatase inhibitors and 0.5 M EDTA (Thermo Scientific). For this purpose, PC was diluted in PBS to a concentration of 3×105platelets/µl. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody was obtained from Sigma. Mouse monoclonal and rabbit polyclonal secondary antibodies (horseradish peroxidase [HRP] linked) were obtained from Jackson Immunologicals. Lumi-light Western blotting substrate was used for HRP detection (Roche). Phycoerythrin (PE)-CD86, fluorescein isothiocyanate (FITC)-CD80, and PE-MHC II antibodies were obtained from BD Pharmingen.
Cells were stained with antibodies and subjected to flow cytometry using the FC500 flow cytometer from Beckman Coulter. Means, medians, and distributions were compared by the Wilcoxon two-sample test. Susie Mathews for expert technical assistance. RNA was concentrated via precipitation with ammonium acetate. An equal amount of RNA was reverse transcribed to generate the cDNA template to be used in qRT-PCRs using oligo(dT) (Roche) and Affinity Script Reverse Transcriptase (Stratagene). cDNA was diluted 50-fold in water. PCRs were conducted in triplicate using specific primers, Platinum Taq Polymerase (Roche), and SYBR green (Roche).
Results are expressed as median fluorescence intensity (MFI). The concentration of ZnO MNSs was kept below the toxic levels throughout the course of this study. We have previously reported that coinfection of HSV-infected cells with RNA viruses (SeV and NDV) resulted in a significant vhs-dependent block to cytokine secretion (7). In contrast, when HSV-1-infected cells were treated with TLR agonists, the presence or absence of the vhs protein had no effect on the accumulation of proinflammatory cytokines in the supernatants (7), suggesting that mRNA for these cytokines is not a target for the nuclease activity of vhs. We began this study by utilizing this coinfection model of HSV-1 infection and directly measured the mRNA levels for proinflammatory cytokines and type I IFN. We infected BM-DCs with wild-type HSV-1 and then immediately added LPS, CpG, or NDV to the cells. The transcription of cytokines and type I IFN was measured by qRT-PCR ().
In contrast, infection alone could not induce a DNA damage response in the C1300 cells. We compared this level of mRNA expression to the level induced when the DCs were solely treated with either the TLR agonists or NDV (gray bars). We did, however, observe a significant decrease in IFN-β and cytokine mRNA expression during coinfection of HSV with NDV. These results highlight the sensitivity of the qRT-PCR assay to detect changes in cytokine and IFN-β mRNA expression. Moreover, these findings demonstrate that these select mRNA transcripts are not universally targeted for degradation by vhs and suggest that vhs may act by an as yet undescribed mechanism to inhibit DC activation. Based on the ability of wild-type HSV-1 to block NDV-induced proinflammatory cytokine mRNA expression, we next systematically evaluated what role known cellular signaling pathways critical for DC activation in response to NDV play during HSV-1 infection. Coinfection of DCs with HSV-1 and an RNA virus, but not cotreatment with TLR agonists, significantly impairs proinflammatory gene mRNA expression.
BM-DCs were generated from C57BL/6 mice and infected with wild-type HSV (KOS) at an MOI of 5 and then immediately … DC activation by RNA viruses is largely governed by members of the RLR family of cytosolic proteins (reviewed in reference 61). These receptors recognize the RNA of these viruses and signal through the mitochondrial adaptor protein MAVS to activate downstream transcription factors necessary for the expression of proinflammatory cytokines and IFNs. Based on the capacity for HSV to interfere with the activation of DCs in response to RNA virus infection (NDV and SeV [ and reference 7]), we investigated the role of MAVS in the DC response to HSV. We generated BM-derived DCs from MAVS KO mice and infected them with SeV, NDV, and wild-type HSV-1. In contrast to SeV and NDV, HSV-1 could induce the expression of cytokines and type I IFN independently of MAVS (A). Further, when we compared wild-type HSV-1 to the vhs− mutant virus, which fails to incorporate the mutated vhs polypeptide into virion particles (45), we observed enhanced cytokine production for the vhs− mutant in both wild-type DCs and MAVS KO DCs (B).
The data shown in eliminate MAVS and, by extension, the RLR signaling pathway as critical for the activated DC phenotype observed during in vitro wild-type HSV or vhs− virus infection. The HSV-1 vhs protein modulates an RLR-independent pathway to proinflammatory release during HSV-1 infection. (A) BM-DCs were generated from C57BL/6 control and MAVS KO mice and infected with wild-type HSV (KOS) (MOI = 5), SeV (MOI = 1.5), and NDV (MOI … Consistent with prior reports (3), the presence of HMBA increased the rate of virus reactivation (Table ). These prior observations led us to hypothesize that, through its potential inhibition of type I IFN signaling, vhs might exert its block to the activation of DCs observed during HSV infection. This would also explain the phenotype observed during HSV-1 coinfection with NDV (), as type I IFN signaling is required for this RNA virus to trigger DC activation (18, 67, 68). Moreover, the presence of elevated secretion of IFN-α/β in the vhs− mutant-infected DC cultures compared to wild-type-infected DC cultures (A) would fit with a model where inhibition of type I IFN signaling by vhs impairs the amplification phase of type I IFNs.
HSV-induced DC maturation has type I IFN signaling-dependent and -independent requirements. (A) BM-DCs were generated from C57BL/6 mice and infected with the following: wild-type HSV-1 (strain KOS) (MOI = 5), vhs− mutant virus (MOI = 5), and SeV-HD … SupHSV-DC did not contain significant levels of the proinflammatory cytokines IL-1, IL-6, TNF-α, or IFN-γ (Y. In parallel, the control untreated effector cells co-cultured with target cells showed expected fusion (; black bars in panel A). As shown in C, the production of cytokines associated with DC activation was robustly induced in the vhs−-infected IFNR KO DC cultures to levels similar to that observed in control B6 BM-DCs. In contrast to the vhs− virus-infected cells, wild-type HSV infection required type I IFN signaling for the production of the same cytokines. Importantly, vhs exerted its inhibitory function in the absence of exogenous IFN signaling.
We also measured the cell surface expression of costimulatory molecules (CD80 and CD86) and MHC II. In contrast to cytokines, all surface markers tested required type I IFN signaling during HSV infection (both wild-type and vhs− mutant viruses) for optimal expression (E). was supported by NIH training grant T32 DK-007748. vhs blocks a pathway to proinflammatory cytokine release independently of type I IFN signaling.vhs has been reported to negatively regulate the type I IFN signaling pathway by blocking the phosphorylation of STAT1, as well as interfering with the expression of ISGs (10, 27, 41). These results demonstrate that the vhs protein packaged within virion particles is responsible for inhibition of an IFN signaling-independent pathway to cytokine release during HSV infection. Moreover, activation of this initial DC response is also independent of virus replication.