Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

Generation and Characterization of a SARS-CoV-2-Susceptible Mouse Model Using Adeno-Associated Virus (AAV6.2FF)-Mediated Respiratory Delivery of the Human ACE2 Gene.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic with millions of human infections. There continues to be a pressing need to develop potential therapies and vaccines to inhibit SARS-CoV-2 infection to mitigate the ongoing pandemic. Epidemiological data from the current pandemic indicates that there may be sex-dependent differences in disease outcomes. To investigate these differences, we proposed to use common small animal species that are frequently used to model disease with viruses. However, common laboratory strains of mice are not readily infected by SARS-CoV-2 because of differences in the angiotensin-converting enzyme 2 (ACE2), the cellular receptor for the virus. To overcome this limitation, we transduced common laboratory accessible strains of mice of different sexes and age groups with a novel a triple AAV6 mutant, termed AAV6.2FF, encoding either human ACE2 or luciferase via intranasal administration to promote expression in the lung and nasal turbinates. Infection of AAV-hACE2-transduced mice with SARS-CoV-2 resulted in high viral titers in the lungs and nasal turbinates, establishment of an IgM and IgG antibody response, and modulation of lung and nasal turbinate cytokine profiles. There were insignificant differences in infection characteristics between age groups and sex-related differences; however, there were significant strain-related differences between BALB/c vs. C57BL/6 mice. We show that AAV-hACE2-transduced mice are a useful for determining immune responses and for potential evaluation of SARS-CoV-2 vaccines and antiviral therapies, and this study serves as a model for the utility of this approach to rapidly develop small-animal models for emerging viruses.

Downregulation of Cell Surface Major Histocompatibility Complex Class I Expression Is Mediated by the Left-End Transcription Unit of Fowl Adenovirus 9.

Major histocompatibility complex class I (MHC-I) molecules play a critical role in the host's antiviral response by presenting virus-derived antigenic peptides to cytotoxic T lymphocytes (CTLs), enabling the clearance of virus-infected cells. Human adenoviruses evade CTL-mediated cell lysis, in part, by interfering directly with the MHC-I antigen presentation pathway through the expression of E3-19K, which binds both MHC-I and the transporter associated with antigen processing protein and sequestering MHC-I within the endoplasmic reticulum. Fowl adenoviruses have no homologues of E3-19K. Here, we show that representative virus isolates of the species Fowl aviadenovirus C, Fowl aviadenovirus D, and Fowl aviadenovirus E downregulate the cell surface expression of MHC-I in chicken hepatoma cells, resulting in 71%, 11%, and 14% of the baseline expression level, respectively, at 12 h post-infection. Furthermore, this work reports that FAdV-9 downregulates cell surface MHC-I through a minimum of two separate mechanisms-a lysosomal-independent mechanism that requires the presence of the fowl adenovirus early 1 (FE1) transcription unit located within the left terminal genomic region between nts 1 and 6131 and a lysosomal-dependent mechanism that does not require the presence of FE1. These results establish a new functional role for the FE1 transcription unit in immune evasion. These studies provide important new information about the immune evasion of FAdVs and will enhance our understanding of the pathogenesis of inclusion body hepatitis and advance the progress made in next-generation FAdV-based vectors.

A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions.

Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.

Generation and Characterization of Anti-Filovirus Nucleoprotein Monoclonal Antibodies.

Filoviruses cause lethal hemorrhagic fever in humans. The filovirus nucleoprotein (NP) is expressed in high abundance in infected cells and is essential for virus replication. To generate anti-filovirus monoclonal antibodies (mAbs) against the NP, mice were immunized with peptides known as B-cell epitopes corresponding to different filovirus NPs, and hybridomas were screened using FLAG-tagged filovirus NP constructs. Numerous mAbs were identified, isotyped, and characterized. The anti-NP mAbs demonstrated different ranges of binding affinities to various filovirus NPs. Most of the clones specifically detected both recombinant and wild-type NPs from different filoviruses, including Ebola (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Marburg (MARV), Tai Forest (TAFV), and Reston (RESTV) viruses in western blot analysis. The mAbs were also able to detect native NPs within the cytoplasm of infected cells by immunofluorescence confocal microscopy. Thus, this panel of mAbs represents an important set of tools that may be potentially useful for diagnosing filovirus infection, characterizing virus replication, and detecting NP⁻host protein interactions.

Fowl Adenovirus 4 (FAdV-4)-Based Infectious Clone for Vaccine Vector Development and Viral Gene Function Studies.

Fowl adenovirus 4 (FAdV-4) is associated with economically important poultry diseases. Recent studies of fully sequenced genomes of FAdV-4 isolates suggest potential genomic regions associated with virulence and amenable for manipulation and vector development. Direct manipulation of viral genomes is cumbersome, as opposed to that of infectious clones-viral genomes cloned into plasmid or cosmid vectors. In this work, we generated an infectious clone, pFAdV-4 ON1, containing the entire viral genome of a nonpathogenic FAdV-4 (ON1 isolate). pFAdV-4 ON1 was used for targeted deletion of open reading frames (ORFs) 16 and 17 and replacement with the enhanced green fluorescence protein (EGFP) expression cassette to generate recombinant viruses. These viruses were viable, and EGFP was expressed in infected cells. Their replication, however, was significantly reduced with respect to that of the wild-type virus. These observations suggest the potential utility of FAdV-4 as a vaccine vector and the importance of ORFs 16 and 17 for virus replication at wild-type levels. To our knowledge, this is the first report of an infectious clone based on the FAdV-4 genome, and our results demonstrate its utility for studies of virulence determinants and as a platform for either vaccine or gene delivery vectors.

Fowl Aviadenovirus 9 dUTPase Plays a Role in Regulation of the Host Immune Response.

Fowl aviadenoviruses (FAdVs) are distributed worldwide in poultry farms. Some FAdVs are the causative agents of inclusion body hepatitis and hydropericardium syndrome that cause significant economic losses to the poultry industry. In contrast with human adenovirus, the study of the molecular biology of FAdV is still far behind. We previously showed that FAdV-9 open reading frame 1 (ORF1) is a dUTPase enzyme that contributes to the upregulation of type I interferons and is not required for virus replication in vitro. In the present study, we compared virus replication in vivo and the host immune response in chickens orally inoculated with a dUTPase knockout virus (ORF1stop), the rescued version of ORF1stop (resORF1), and wtFAdV-9. Our data showed that replication of ORF1stop was delayed on days 1 and 3 postinoculation compared with wtFAdV-9, as evidenced by significantly less virus shedding in feces and lower viral loads in tissues. Moreover, we found that there was a significant difference in the induction of cytokine gene mRNA expression in tissues and IgG antibody responses in ORF1stop versus wtFAdV-9-infected chickens, suggesting that ORF1 plays some roles in modulating the host immune response. Our study provides useful data on the mechanism of the host immune response against FAdV infection.

DNA vaccination protects mice against Zika virus-induced damage to the testes.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

EBV BILF1 evolved to downregulate cell surface display of a wide range of HLA class I molecules through their cytoplasmic tail.

Coevolution of herpesviruses and their hosts has driven the development of both host antiviral mechanisms to detect and eliminate infected cells and viral ploys to escape immune surveillance. Among the immune-evasion strategies used by the lymphocryptovirus (γ(1)-herpesvirus) EBV is the downregulation of surface HLA class I expression by the virally encoded G protein-coupled receptor BILF1, thereby impeding presentation of viral Ags and cytotoxic T cell recognition of the infected cell. In this study, we show EBV BILF1 to be expressed early in the viral lytic cycle. BILF1 targets a broad range of HLA class I molecules, including multiple HLA-A and -B types and HLA-E. In contrast, HLA-C was only marginally affected. We advance the mechanistic understanding of the process by showing that the cytoplasmic C-terminal tail of EBV BILF1 is required for reducing surface HLA class I expression. Susceptibility to BILF1-mediated downregulation, in turn, is conferred by specific residues in the intracellular tail of the HLA class I H chain. Finally, we explore the evolution of BILF1 within the lymphocryptovirus genus. Although the homolog of BILF1 encoded by the lymphocryptovirus infecting Old World rhesus primates shares the ability of EBV to downregulate cell surface HLA class I expression, this function is not possessed by New World marmoset lymphocryptovirus BILF1. Therefore, this study furthers our knowledge of the evolution of immunoevasive functions by the lymphocryptovirus genus of herpesviruses.

Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection.

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.

The Epstein-Barr virus G-protein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation.

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8(+) T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 gamma(1)-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV gamma(2)-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8(+) T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed.

Epstein-Barr virus evasion of CD8(+) and CD4(+) T cell immunity via concerted actions of multiple gene products.

Upon primary infection, EBV establishes a latent infection in B cells, characterized by maintenance of the viral genome in the absence of viral replication. The Epstein-Barr Nuclear Antigen 1 (EBNA1) plays a crucial role in maintenance of the viral DNA episome and is consistently expressed in all EBV-associated malignancies. Compared to other EBV latent gene products, EBNA1 is poorly recognized by CD8(+) T lymphocytes. Recent studies are discussed that shed new light on the mechanisms that underlie this unusual lack of CD8(+) T cell activation. Whereas the latent phase is characterized by the expression of a limited subset of viral gene products, the full repertoire of over 80 EBV lytic gene products is expressed during the replicative phase. Despite this abundance of potential T cell antigens, which indeed give rise to a strong response of CD4(+) and CD8(+) T lymphocytes, the virus can replicate successfully. Evidence is accumulating that this paradoxical situation is the result of actions of multiple viral gene products, inhibiting discrete stages of the MHC class I and class II antigen presentation pathways. Immediately after initiation of the lytic cycle, BNLF2a prevents peptide-loading of MHC class I molecules through inhibition of the Transporter associated with Antigen Processing, TAP. This will reduce presentation of viral antigens by the large ER-resident pool of MHC class I molecules. Synthesis of new MHC class I molecules is blocked by BGLF5. Viral-IL10 causes a reduction in mRNA levels of TAP1 and bli/LMP2, a subunit of the immunoproteasome. MHC class I molecules present at the cell surface are downregulated by BILF1. Also the antigen presenting capacity of MHC class II molecules is severely compromised by multiple EBV lytic gene products, including gp42/gH/gL, BGLF5, and vIL-10. In this review, we discuss how concerted actions of these EBV lytic proteins result in highly effective interference with CD8(+) and CD4(+) T cell surveillance, thereby providing the virus with a window for undisturbed generation of viral progeny.

In vivo binding of NF-kappaB to the IkappaBbeta promoter is insufficient for transcriptional activation.

Despite certain structural and biochemical similarities, differences exist in the function of the NF-kappaB (nuclear factor kappaB) inhibitory proteins IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. The functional disparity arises in part from variance at the level of gene regulation, and in particular from the substantial induction of IkappaBalpha, but not IkappaBbeta, gene expression post-NF-kappaB activation. In the present study, we probe the differential effects of IL (interleukin)-1beta on induction of IkappaBalpha and perform the first characterization of the human IkappaBbeta promoter. A consensus NF-kappaB-binding site, capable of binding NF-kappaB both in vitro and in vivo, is found in the IkappaBbeta gene 5' flanking region. However, the IkappaBbeta promoter was not substantially activated by pro-inflammatory cytokines, such as IL-1beta and tumour necrosis factor alpha, that are known to cause strong activation of NF-kappaB. Furthermore, in contrast with IkappaBalpha, NF-kappaB activation did not increase expression of endogenous IkappaBbeta as assessed by analysis of mRNA and protein levels. Unlike kappaB-responsive promoters, IkappaBbeta promoter-bound p65 inefficiently recruits RNA polymerase II, which stalls at the promoter. We present evidence that this stalling is likely due to the absence of transcription factor IIH engagement, a prerequisite for RNA polymerase II phosphorylation and transcriptional initiation. Differences in the conformation of promoter-bound NF-kappaB may underlie the variation in the ability to engage the basal transcriptional apparatus at the IkappaBbeta and kappaB-responsive promoters. This accounts for the differential expression of IkappaB family members in response to NF-kappaB activation and furthers our understanding of the mechanisms involved in transcription factor activity and IkappaBbeta gene regulation.

Persistent interleukin-1beta signaling causes long term activation of NFkappaB in a promoter-specific manner in human glial cells.

Nuclear factor-kappaB (NFkappaB) is an inducible transcription factor that plays a key role in regulating the expression of a wide range of immune and inflammatory response genes. The activity of NFkappaB is controlled at multiple levels, including cytoplasmic retention with inhibitor of kappaB (IkappaB) proteins in the basal state. Persistent activation of the transcription factor is seen in numerous chronic inflammatory disease states, and we have previously demonstrated sustained activation of NFkappaB in human glial cells upon stimulation with interleukin (IL)-1beta. In these cells, NFkappaB retains DNA binding activity for up to 72 h despite the presence of resynthesized IkappaBalpha and in the absence of IkappaBbeta. Here we characterized the apparent inability of newly synthesized IkappaBalpha to terminate activation of NFkappaB in glial cells. We showed unexpectedly that newly synthesized IkappaBalpha can enter the nucleus, interact with the NFkappaB subunit p65, and export it to the cytoplasm. However, in vitro analysis of enzyme activity demonstrates that IL-1beta causes the long term activation of the IkappaB kinase complex leading to chronic phosphorylation of the newly synthesized IkappaBalpha signal response domain and persistent activation of NFkappaB. Such sustained activation of NFkappaB is dependent on the continuous presence and activity of IL-1beta. Interestingly, the sustained nature of NFkappaB activity is promoter type-specific. Chromatin immunoprecipitation studies revealed that p65 is detected at the promoters of both intercellular adhesion molecule-1 and IL-8 1 h following IL-1beta stimulation but is only found at the latter at 24 h. The functional significance of this finding is indicated by the transient induction of intercellular adhesion molecule-1 mRNA, but more sustained induction of IL-8 expression, by IL-1beta. These studies thus demonstrated that persistent IL-1 signaling causes sustained activation of NFkappaB in a promoter-specific manner in human glial cells, leading to prolonged induction of selective pro-inflammatory genes. This is likely to make a key contribution to chronic inflammatory conditions of the brain.

The synthetic cannabinoid R(+)WIN 55,212-2 inhibits the interleukin-1 signaling pathway in human astrocytes in a cannabinoid receptor-independent manner.

R(+)WIN 55,212-2 is a synthetic cannabinoid that controls disease progression in models of multiple sclerosis. This is associated with its ability to reduce migration of leukocytes into the central nervous system. Because leukocyte migration is dependent on induction of adhesion molecules and chemokines by pro-inflammatory cytokines, we examined the effects of R(+)WIN 55,212-2 on their expression. Using 1321N1 astrocytoma and A-172 glioblastoma as cell models we show that R(+)WIN 55,212-2, but not its inactive chiral form S(-)WIN 55,212-2, strongly inhibits the interleukin-1 (IL-1) induction of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the chemokine IL-8. This inhibition is not mediated via the CB1 or CB2 cannabinoid receptors, because their selective antagonists and pertussis toxin failed to affect the inhibitory effects of R(+)WIN 55,212-2. Furthermore reverse transcription-PCR analysis did not detect the expression of either receptor in 1321N1 cells. R(+)WIN 55,212-2 was shown to inhibit adhesion molecule and chemokine expression at the level of transcription, because it strongly inhibited the IL-1 induction of ICAM-1, VCAM-1, and IL-8 mRNAs and blocked the IL-1 activation of their promoters. The NFkappaB pathway was then assessed as a lead target for R(+)WIN 55,212-2. NFkappaB was measured by expression of a transfected NFkappaB-regulated reporter gene. Using this assay, R(+)WIN 55,212-2 strongly inhibited IL-1 activation of NFkappaB. Furthermore R(+)WIN 55,212-2 inhibited the ability of overexpressed Myd88, Tak-1, and IKK-2 to induce the reporter gene suggesting that R(+)WIN 55,212-2 acts at or downstream of IKK-2 in the IL-1 pathway. However R(+)WIN 55,212-2 failed to inhibit IL-1-induced degradation of IkappaBalpha, excluding IKK-2 as a direct target. In addition electrophoretic mobility shift and chromatin immunoprecipitation assays showed that R(+)WIN 55,212-2 does not regulate the IL-1-induced nuclear translocation of NFkappaB or the ability of the latter to bind to promoters regulating expression of ICAM-1 and IL-8. These data suggest that R(+)WIN 55,212-2 blocks IL-1 signaling by inhibiting the transactivation potential of NFkappaB.