Effect of Janus kinase inhibitors on T cell responses to herpes zoster in rheumatoid arthritis patients.

OBJECTIVES: The incidence of herpes zoster (HZ) in rheumatoid arthritis (RA) patients is greater than that in healthy controls (HC), particularly in RA patients treated with Janus kinase inhibitors (JAKi). Here, we examined the effect of JAKi on CD4+/CD8+ T cells, cytokine production, and regulation of transcriptional factors in RA patients and HC. METHODS: Peripheral blood mononuclear cells (PBMCs) obtained from RA patients (n=14) and HCs (n=7) were stimulated with varicella zoster virus lysates and exposed to three JAKi inhibitors (ruxolitinib [JAK1/2 inhibitor]; AG490 [JAK2 inhibitor]; and WHI-P154 [JAK3 inhibitor]) in the presence/absence of methotrexate. The CD4+ and CD8+ T cell populations were measured by flow cytometry. Cytokine levels in culture medium were measured by ELISA. Transcription factor expression was examined by RT-qPCR. RESULTS: There was a reduction in the CD4+IFN-γ+, CD4+CD69+IFN-γ+, CD8+IFN-γ+, and CD8+CD69+IFN-γ+ populations, and an increase in the CD4+CD25highFoxp3+ cell population, in PBMCs from RA patients and HCs after exposure to the three JAKi. ELISA revealed a reduction in IFN-γ and granzyme B levels in the presence of JAKi. JAKi reduced expression of mRNA encoding STAT1 and T-bet, but increased that of mRNA encoding STAT5 and Foxp3. Methotrexate plus the highest dose of each JAKi did not affect the Th1, cytotoxic T cell, or Treg populations, the levels of IFN-γ and granzyme B, or expression of transcription factors, significantly. CONCLUSIONS: JAKi reduce the Th1/cytotoxic T cell population and increase the Treg population in both RA patients and HC patients.

VZV Infection of Primary Human Adrenal Cortical Cells Produces a Proinflammatory Environment without Cell Death.

Virus infection of adrenal glands can disrupt secretion of mineralocorticoids, glucocorticoids, and sex hormones from the cortex and catecholamines from the medulla, leading to a constellation of symptoms such as fatigue, dizziness, weight loss, nausea, and muscle and joint pain. Specifically, varicella zoster virus (VZV) can produce bilateral adrenal hemorrhage and adrenal insufficiency during primary infection or following reactivation. However, the mechanisms by which VZV affects the adrenal glands are not well-characterized. Herein, we determined if primary human adrenal cortical cells (HAdCCs) infected with VZV support viral replication and produce a proinflammatory environment. Quantitative PCR showed VZV DNA increasing over time in HAdCCs, yet no cell death was seen at 3 days post-infection by TUNEL staining or Western Blot analysis with PARP and caspase 9 antibodies. Compared to conditioned supernatant from mock-infected cells, supernatant from VZV-infected cells contained significantly elevated IL-6, IL-8, IL-12p70, IL-13, IL-4, and TNF-α. Overall, VZV can productively infect adrenal cortical cells in the absence of cell death, suggesting that these cells may be a potential reservoir for ongoing viral replication and proinflammatory cytokine production, leading to chronic adrenalitis and dysfunction.

SIADH in the context of localised Herpes-Zoster infection.

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Exocytosis of Progeny Infectious Varicella-Zoster Virus Particles via a Mannose-6-Phosphate Receptor Pathway without Xenophagy following Secondary Envelopment.

The literature on the egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux is required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from that of either fibroblasts or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway (trans-Golgi network to late endosomes) was constrained in infected Pompe cells. When cells were analyzed with two different anti-M6PR antibodies, extensive colocalization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6P receptor (M6PR) was documented in the viral highways at the surfaces of non-Pompe cells after maximum-intensity projection of confocal z-stacks, but neither gE nor the M6PR was seen in abundance at the surfaces of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lack a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV particles in conventional cell substrates are transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane.IMPORTANCE The long-term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated and has a lower titer than other alphaherpesviruses, such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the trans-Golgi network within small vacuoles to the plasma membrane. In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRV's usage being closer to HSV1's usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included.

Kallikrein-Mediated Cytokeratin 10 Degradation Is Required for Varicella Zoster Virus Propagation in Skin.

Varicella zoster virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening, particularly in the immunocompromized. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and antiviral drugs. In VZV-infected skin, kallikrein 6 and the ubiquitin ligase MDM2 are upregulated concomitant with keratin 10 (KRT10) downregulation. MDM2 binds to KRT10, targeting it for degradation via the ubiquitin-proteasome pathway. Preventing KRT10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. KRT10 knockdown induced expression of NR4A1 and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels, and increased LAMP2 expression. We therefore describe a drug-able pathway whereby MDM2 ubiquitinates and degrades KRT10, increasing NR4A1 expression and allowing VZV replication and propagation.

Varicella zoster virus productively infects human peripheral blood mononuclear cells to modulate expression of immunoinhibitory proteins and blocking PD-L1 enhances virus-specific CD8+ T cell effector function.

Varicella zoster virus (VZV) is a lymphotropic alpha-herpesvirinae subfamily member that produces varicella on primary infection and causes zoster, vascular disease and vision loss upon reactivation from latency. VZV-infected peripheral blood mononuclear cells (PBMCs) disseminate virus to distal organs to produce clinical disease. To assess immune evasion strategies elicited by VZV that may contribute to dissemination of infection, human PBMCs and VZV-specific CD8+ T cells (V-CD8+) were mock- or VZV-infected and analyzed for immunoinhibitory protein PD-1, PD-L1, PD-L2, CTLA-4, LAG-3 and TIM-3 expression using flow cytometry. All VZV-infected PBMCs (monocytes, NK, NKT, B cells, CD4+ and CD8+ T cells) and V-CD8+ showed significant elevations in PD-L1 expression compared to uninfected cells. VZV induced PD-L2 expression in B cells and V-CD8+. Only VZV-infected CD8+ T cells, NKT cells and V-CD8+ upregulated PD-1 expression, the immunoinhibitory receptor for PD-L1/PD-L2. VZV induced CTLA-4 expression only in V-CD8+ and no significant changes in LAG-3 or TIM-3 expression were observed in V-CD8+ or PBMC T cells. To test whether PD-L1, PD-L2 or CTLA-4 regulates V-CD8+ effector function, autologous PBMCs were VZV-infected and co-cultured with V-CD8+ cells in the presence of blocking antibodies against PD-L1, PD-L2 or CTLA-4; ELISAs revealed significant elevations in IFNγ only upon blocking of PD-L1. Together, these results identified additional immune cells that are permissive to VZV infection (monocytes, B cells and NKT cells); along with a novel mechanism for inhibiting CD8+ T cell effector function through induction of PD-L1 expression.

Anti-varicella Zoster Virus IgG and hsCRP Levels Correlate with Progression of Coronary Artery Atherosclerosis.

The relationship between high levels of anti-Varicella Zoster Virus (VZV) IgG in cerebrospinal fluid (CSF) and cerebrovascular atherosclerosis commends a possible similar association in other vessels. We aimed to investigate the association of VZV-seropositivity with coronary artery atherosclerosis. We recruited 88 newly diagnosed patients with more than 50% stenosis in at least one of the main coronary arteries. As the control group, 99 age-matched individuals with normal/insignificant coronary artery findings were included. Clinical, paraclinical, and demographical data were gathered at the time of sampling. High-sensitivity C-reactive protein (hsCRP) levels were measured by nephelometry. VZV-seropositivity was determined by measuring of anti-VZV IgG level in plasma. Multivariable logistic regression was used to evaluate the correlation of data with coronary vascular atherosclerosis. The frequency of VZV-seropositivity was significantly higher in the atherosclerosis group compared to the controls (OR=1.88; 95%CI=1.03-3.44). The plasma levels of anti-VZV IgG were significantly higher in patients with atherosclerosis (Median=2.70, IQR=1.53-4.30 AU/mL) than in the controls (Median=2.10, IQR=1.70-3.10 AU/mL, p=0.034). The hsCRP levels in patients and controls were 5.19±2.00 and 1.51±1.07 mg/L, respectively. The correlation between hsCRP and anti-VZV IgG level in plasma was observed (r=0.40, p<0.001). The levels of hsCRP and anti-VZV IgG increased based on the number of diseased vessels but only the difference in hsCRP levels reached a significant level (p<0.001 and p=0.168, respectively). Our data suggest that VZV-seropositivity and hsCRP elevation jointly increase the risk of atherosclerosis. The multifactorial nature of atherosclerosis; however, leaves more options for the inflammatory milieu to be generated.

Valacyclovir-induced Neurotoxicity in a Patient with a Preserved Renal Function.

Valacyclovir, a prodrug of acyclovir, is the first-line treatment for herpes zoster, but the renal function must be monitored, because acyclovir is metabolized by the kidneys. We herein report a case of valacyclovir-induced neurotoxicity with no preceding renal impairment. An 88-year-old man was admitted because of an impaired consciousness after the administration of valacyclovir at 3,000 mg daily for herpes zoster on the chest. His consciousness level gradually improved with hydration and valacyclovir withdrawal. It was later confirmed that the level of acyclovir on admission had been 35.45 µg/mL in the blood and 36.45 µg/mL in the cerebrospinal fluid.

Koebner's sheep in Wolf's clothing: does the isotopic response exist as a distinct phenomenon?

Until 1995, a case of psoriasis developing within the dermatome of a healed herpes zoster was taken as a Koebner phenomenon. In this year, however, the term 'isotopic response' was introduced by Wolf et al. to describe 'the occurrence of a new skin disorder at the site of another, unrelated and already healed skin disease', thus appearing 'on apparently unaffected and healthy skin'. Initially, the term was mainly related to herpes zoster, but today the name 'Wolf's isotopic response' is used to include a plethora of other triggering factors such as healed cutaneous leishmaniasis, tinea or varicella. For obvious reasons, such triggering factors cannot be taken as examples of 'unaffected and healthy skin'. Notably, the authors themselves have categorized the dermatome of a healed herpes zoster as a 'vulnerable area'. In a recent commentary, Wolf et al. have expanded the definition of healed skin diseases triggering an 'isotopic response'. They now included 'scars, pigment changes, color changes or various other minimal changes by the first disease'. Hence, there is no clear-cut criterion to distinguish the isotopic response from a Koebner reaction. Wolf et al. even argue that, if the triggered disorder precedes the appearance of generalized skin lesions, then it is not a Koebner reaction but 'Wolf's isotopic response'. In our view, such definition is unacceptable. All reactions of this kind represent examples of a Koebner phenomenon. Accordingly, the 'isotopic response' should today be taken as a historical error.

Induction of varicella zoster virus DNA replication in dissociated human trigeminal ganglia.

Varicella zoster virus (VZV), a human neurotropic alphaherpesvirus, becomes latent after primary infection and reactivates to produce zoster. To study VZV latency and reactivation, human trigeminal ganglia removed within 24 h after death were mechanically dissociated, randomly distributed into six-well tissue culture plates and incubated with reagents to inactivate nerve growth factor (NGF) or phosphoinositide 3-kinase (PI3-kinase) pathways. At 5 days, VZV DNA increased in control and PI3-kinase inhibitor-treated cultures to the same extent, but was significantly more abundant in anti-NGF-treated cultures (p = 0.001). Overall, VZV DNA replication is regulated in part by an NGF pathway that is PI3-kinase-independent.

Transient Receptor Potential Vanilloid-1 in Epidermal Keratinocytes May Contribute to Acute Pain in Herpes Zoster.

The role of transient receptor potential vanilloid-1 (TRPV1) in the initiation of neurogenic inflammation and transduction of pain is well established. In this study 33 patients with herpes zoster (HZ) were recruited from a single centre and underwent a questionnaire interview at their first visit. Punch biopsies from the HZ lesions and the contralateral unaffected skin were performed to localize and quantify the expression of TRPV1. Immunofluorescent staining for TRPV1 was most prominent in the epidermal keratinocytes. Both TRPV1 mRNA and protein levels were significantly higher in the HZ epidermis than in control epidermis (relative ratio 1.62 ± 0.27, p = 0.033 and 2.55 ± 0.51, p = 0.005, respectively). Protein TRPV1 ratio (HZ lesion/control) correlated with the degree of pain (measured on a visual analogue scale; VAS) (p = 0.017) and was significantly lower in patients who had taken either HZ medication or painkillers prior to their visit. These results suggest that non-neuronal TRPV1 may contribute to acute pain in herpes zoster.

Differential regulation of matrix metalloproteinases in varicella zoster virus-infected human brain vascular adventitial fibroblasts.

Upon reactivation, varicella zoster virus (VZV) spreads transaxonally, infects cerebral arteries and causes ischemic or hemorrhagic stroke, as well as aneurysms. The mechanism(s) of VZV-induced aneurysm formation is unknown. However, matrix metalloproteinases (MMPs), which digest extracellular structural proteins in the artery wall, play a role in cerebral and aortic artery aneurysm formation and rupture. Here, we examined the effect of VZV infection on expression of MMP-1, -2, -3, and -9 in primary human brain vascular adventitial fibroblasts (BRAFS). At 6 days post-infection, VZV- and mock-infected BRAFs were analyzed for mRNA levels of MMP-1, -2, -3 and -9 by RT-PCR and for corresponding total intra- and extracellular protein levels by multiplex ELISA. The activity of MMP-1 was also measured in a substrate cleavage assay. Compared to mock-infected BRAFs, MMP-1, MMP-3 and MMP-9 transcripts, cell lysate protein and conditioned supernatant protein were all increased in VZV-infected BRAFs, whereas MMP-2 transcripts, cell lysate protein and conditioned supernatant protein were decreased. MMP-1 from the conditioned supernatant of VZV-infected BRAFs showed increased cleavage activity on an MMP-1-specific substrate compared to mock-infected BRAFs. Differential regulation of MMPs in VZV-infected BRAFs may contribute to aneurysm formation in VZV vasculopathy.

Síndrome de Horner: una rara complicación del herpes zóster cérvico-torácico / Horner Syndrome: A Rare Complication of Cervical and Thoracic Herpes Zoster Infection

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Direct transfer of viral and cellular proteins from varicella-zoster virus-infected non-neuronal cells to human axons.

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.

Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells.

Cellular transcription factor YY1 mediates the varicella-zoster virus (VZV) IE62 transcriptional activation.

Several cellular transcription factors have been shown to be involved in IE62-mediated activation. The YY1 cellular transcription factor has activating and repressive effects on gene transcription. Analysis of the VZV genome revealed 19 postulated YY1 binding sites located within putative promoters of 16 VZV genes. Electrophoretic mobility shift assays (EMSA) confirmed the binding of YY1 to ORF10, ORF28/29 and gI promoters and the mutation of these binding sites inhibited YY1 binding and the promoter activation by IE62 alone or following VZV infection. Mutation of the ORF28/29 YY1 site in the VZV genome displayed insignificant influence on virus growth in melanoma cells; but it inhibited the virus replication significantly at day 5 and 6 post infection in HELF cells. This work suggests a novel role for the cellular factor YY1 in VZV replication through the mediation of IE62 activation of viral gene expression.

Human antimicrobial peptides LL-37 and human ß-defensin-2 reduce viral replication in keratinocytes infected with varicella zoster virus.

BACKGROUND: There is mounting evidence that antimicrobial peptides have an important role in cutaneous defence, but the expression of these antimicrobial peptides in atopic eczema (AE) is still unclear. There are several families of antimicrobial peptides, including cathelicidins and human ß-defensins. Patients with AE are more susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV). AIM: To characterize the functional activity of the antimicrobial peptides LL-37 (human cathelicidin) and human ß-defensin (hBD)-2 keratinocytes were infected with VZV, in a skin-infection model. METHODS: Flow-cytometry analysis was used to investigate LL-37 expression in normal human keratinocytes, and quantitative PCR was used to determine viral loads in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and hBD-2. RESULTS: LL-37 expression was present in keratinocytes, and both exogenous LL-37 and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Pre-incubation of VZV with LL-37, but not hBD-2, further reduced VZV load. CONCLUSIONS: Both LL-37 and hBD-2 have an antiviral effect on VZV replication in the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is different. Evidence of the relationship between antimicrobial peptide expression and higher susceptibility to infections in AE skin is still emerging. Developing novel antiviral therapies based on antimicrobial peptides may provide improved treatment options for patients with AE.

Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host.

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek's disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well.

Varicella-zoster virus inhibition of the NF-κB pathway during infection of human dendritic cells: role for open reading frame 61 as a modulator of NF-κB activity.

Dendritic cells (DC) are antigen-presenting cells essential for initiating primary immune responses and therefore an ideal target for viral immune evasion. Varicella-zoster virus (VZV) can productively infect immature human DCs and impair their function as immune effectors by inhibiting their maturation, as evidenced by the expression modulation of functionally important cell surface immune molecules CD80, CD86, CD83, and major histocompatibility complex I. The NF-κB pathway largely regulates the expression of these immune molecules, and therefore we sought to determine whether VZV infection of DCs modulates the NF-κB pathway. Nuclear localization of NF-κB p50 and p65 indicates pathway activation; however, immunofluorescence studies revealed cytoplasmic retention of these NF-κB subunits in VZV-infected DCs. Western blotting revealed phosphorylation of the inhibitor of κBα (IκBα) in VZV-infected DCs, indicating that the pathway is active at this point. We conclude that VZV infection of DC inhibits the NF-κB pathway following protein phosphorylation but before the translocation of NF-κB subunits into the nucleus. An NF-κB reporter assay identified VZV open reading frame 61 (ORF61) as an inhibitor of tumor necrosis factor alpha-induced NF-κB reporter activity. Mutational analysis of ORF61 identified the E3 ubiquitin ligase domain as a region required for NF-κB pathway inhibition. In summary, we provide evidence that VZV inhibits the NF-κB signaling pathway in human DCs and that the E3 ubiquitin ligase domain of ORF61 is required to modulate this pathway. Thus, this work identifies a mechanism by which VZV modulates host immune function.