ABSTRACT
Chikungunya virus (CHIKV) belongs to a group of mosquito-borne alphaviruses associated with acute and chronic arthropathy, with peripheral and limb joints most commonly affected. Using a mouse model of CHIKV infection and arthritic disease, we show that CHIKV replication and the ensuing foot arthropathy were dramatically reduced when mice were housed at 30°C, rather than the conventional 22°C. The effect was not associated with a detectable fever, but was dependent on type I interferon responses. Bioinformatics analyses of RNA-Seq data after injection of poly(I:C)/jetPEI suggested the unfolded protein response and certain type I interferon responses are promoted when feet are slightly warmer. The ambient temperature thus appears able profoundly to effect anti-viral activity in the periphery, with clear consequences for alphaviral replication and the ensuing arthropathy. These observations may provide an explanation for why alphaviral arthropathies are largely restricted to joints of the limbs and the extremities.
Subject(s)
Alphavirus Infections/immunology , Alphavirus Infections/virology , Arthritis, Experimental/immunology , Arthritis, Experimental/virology , Arthritis, Infectious/immunology , Arthritis, Infectious/virology , Interferon Type I/metabolism , Alphavirus Infections/pathology , Animals , Arthritis, Experimental/pathology , Arthritis, Infectious/pathology , Chikungunya Fever/immunology , Chikungunya Fever/pathology , Chikungunya Fever/virology , Chikungunya virus/immunology , Chikungunya virus/pathogenicity , Chikungunya virus/physiology , Female , Foot , Host-Pathogen Interactions/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Ross River virus/immunology , Ross River virus/pathogenicity , Ross River virus/physiology , Temperature , Viral Load , Virus Replication/immunology , Virus Replication/physiologyABSTRACT
Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/- and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/- mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281294.
Subject(s)
Arthritis/virology , Chikungunya Fever/genetics , Chikungunya Fever/immunology , Granzymes/immunology , Inflammation/virology , Animals , Chikungunya virus , Disease Models, Animal , Granzymes/analysis , Granzymes/biosynthesis , Humans , Immunohistochemistry , Macaca fascicularis , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/analysis , TranscriptomeABSTRACT
SerpinB2, also known as plasminogen activation inhibitor type 2 (PAI-2), is classically viewed as an inhibitor of fibrinolysis. However, we show herein a distinct, hitherto unrecognized role for SerpinB2 in hemostasis. Mice deficient in SerpinB2 expression and mice with an active site mutation in SerpinB2, both showed significant reductions in tail bleeding times. This hemostatic phenotype was associated with platelets, with SerpinB2 and SerpinB2-urokinase complexes clearly present in platelet fractions, and immunohistochemistry of blood clots suggesting SerpinB2 is associated with platelet aggregates. Thromboelastography illustrated faster onset of clot formation in blood from SerpinB2 deficient mice, whereas clotting of platelet-free plasma was unaffected. The results appear consistent with the low circulating SerpinB2 levels and hypercoagulation seen during pre-eclampsia; however, SerpinB2 was not detected in human platelets.
Subject(s)
Bleeding Time , Blood Coagulation/genetics , Blood Platelets/metabolism , Plasminogen Activator Inhibitor 2/deficiency , Platelet Activation , Animals , Biomarkers , Female , Immunohistochemistry , Mice , Mice, Knockout , Pregnancy , Thrombosis , Urokinase-Type Plasminogen ActivatorABSTRACT
Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for epidemics of debilitating arthritic disease. The recent outbreak (2004-2014) resulted in an estimated 1.4-6.5 million cases, with imported cases reported in nearly 40 countries. The development of CHIKV-specific diagnostics and research tools is thus highly desirable. Herein we describe the generation and characterization of the first mAbs specific for the capsid protein (CP) of CHIKV. The antibodies recognized isolates representing the major genotypes of CHIKV, as well as several other alphaviruses, and were reactive in a range of assays including ELISA, Western blot, immunofluorescence and immunohistochemistry (IHC). We have also used the anti-CP mAb 5.5G9 in IHC studies to show that capsid antigen is persistently expressed 30 days post-infection in cells with macrophage morphology in a mouse model of chronic CHIKV disease. These antibodies may thus represent useful tools for further research, including investigations into the structure and function of CHIKV CP, and as valuable reagents for CHIKV detection in a range of settings.
Subject(s)
Antibodies, Monoclonal/immunology , Capsid Proteins/immunology , Chikungunya virus/immunology , Animals , Antibody Specificity , Blotting, Western , COS Cells , Capsid Proteins/genetics , Capsid Proteins/metabolism , Chikungunya Fever/immunology , Chikungunya Fever/virology , Chikungunya virus/genetics , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Gene Expression Regulation, Viral , Humans , Immunohistochemistry , Macrophages/metabolism , Mice , Mice, Inbred BALB CABSTRACT
The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
Subject(s)
Chikungunya Fever/virology , Chikungunya virus/metabolism , Gene Expression Regulation/physiology , Unfolded Protein Response/physiology , Viral Nonstructural Proteins/metabolism , Animals , Arthritis, Infectious , Chikungunya Fever/pathology , Chikungunya virus/genetics , Chlorocebus aethiops , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum Chaperone BiP , Gene Expression Regulation/immunology , Mice , RNA Splicing , Regulatory Factor X Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Tunicamycin/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Virus Replication , X-Box Binding Protein 1ABSTRACT
UNLABELLED: Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1ß [IL-1ß], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides. IMPORTANCE: Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.
Subject(s)
Alphavirus Infections/immunology , Arthritis/immunology , Chikungunya virus/physiology , Neutrophils/immunology , Receptors, CCR2/deficiency , Alphavirus Infections/genetics , Alphavirus Infections/virology , Animals , Arthritis/genetics , Arthritis/virology , Chikungunya Fever , Chikungunya virus/genetics , Granulocyte Colony-Stimulating Factor/immunology , Humans , Interleukin-10/immunology , Interleukin-1beta/immunology , Mice , Mice, Knockout , Neutrophil Infiltration , Receptors, CCR2/genetics , Receptors, CCR2/immunologyABSTRACT
Chikungunya virus (CHIKV) recently caused the largest epidemic ever recorded for this virus involving an estimated 1.4-6.5million cases, with imported cased reported in over 40 countries. The number of monoclonal antibodies specific for this re-emerging alphavirus is currently limited. Herein we describe the generation and characterisation of five monoclonal antibodies specific for the E2 glycoprotein of CHIKV. The antibodies detected a range of CHIKV isolates in several assays including ELISA, Western blot, immunofluorescence assay (IFA) and immunohistochemistry (IHC) without evidence of cross-reactivity with other alphaviruses. Four antibodies also neutralised CHIKV in vitro, two of which provided complete protection against arthritis in a CHIKV mouse model when administered prior to infection. Given the current shortage of widely available reagents for CHIKV, these specific antibodies will be useful not only in research, but may also provide the basis for new diagnostics and treatments.
Subject(s)
Alphavirus Infections/prevention & control , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Arthritis, Experimental/prevention & control , Viral Envelope Proteins/immunology , Alphavirus Infections/immunology , Alphavirus Infections/virology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/biosynthesis , Antibodies, Viral/isolation & purification , Arthritis, Experimental/immunology , Arthritis, Experimental/virology , COS Cells , Chikungunya virus/drug effects , Chikungunya virus/immunology , Chlorocebus aethiops , Female , Humans , Hybridomas/immunology , Mice , Mice, Inbred BALB C , Vero Cells , Viral Envelope Proteins/geneticsABSTRACT
Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7(-/-)) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/ß) in serum, â¼50- and â¼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/ß receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/ß induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/ß responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.
Subject(s)
Alphavirus Infections/immunology , Alphavirus Infections/prevention & control , Chikungunya virus/pathogenicity , Interferon Regulatory Factor-3/physiology , Interferon Regulatory Factor-7/physiology , Adaptor Proteins, Vesicular Transport/deficiency , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/physiology , Alphavirus Infections/pathology , Animals , Chikungunya Fever , Chikungunya virus/immunology , Chikungunya virus/physiology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interferon Regulatory Factor-3/deficiency , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-7/deficiency , Interferon Regulatory Factor-7/genetics , Interferon-alpha/biosynthesis , Interferon-alpha/pharmacology , Interferon-beta/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/physiology , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/physiology , Shock, Hemorrhagic/immunology , Shock, Hemorrhagic/prevention & control , Virus Replication/drug effectsABSTRACT
OBJECTIVE: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a chronic debilitating polyarthralgia/polyarthritis, for which current treatments are often inadequate. To assess whether new drugs being developed for rheumatoid arthritis (RA) might find utility in the treatment of alphaviral arthritides, we sought to determine whether the inflammatory gene expression signature of CHIKV arthritis shows any similarities with RA or collagen-induced arthritis (CIA), a mouse model of RA. METHODS: Using a recently developed animal model of CHIKV arthritis in adult wild-type mice, we generated a consensus CHIKV arthritis gene expression signature, which was used to interrogate publicly available microarray studies of RA and CIA. Pathway analyses were then performed using the overlapping gene signatures. RESULTS: Gene set enrichment analysis showed that there was a highly significant overlap in the differentially expressed genes in the CHIKV arthritis model and in RA. This concordance also increased with the severity of RA, as measured by the inflammation score. A highly significant overlap was also seen between CHIKV arthritis and CIA. Pathway analysis revealed that the overlap between these arthritides was spread over a range of different inflammatory processes. Involvement of T cells and interferon-γ (IFNγ) in CHIKV arthritis was confirmed in studies of MHCII-deficient mice and IFNγ-deficient mice, respectively. CONCLUSION: These results suggest that RA, a chronic autoimmune arthritis, and CHIKV disease, usually a self-limiting viral arthropathy, share multiple inflammatory processes. New drugs and biologic therapies being developed for RA may thus find application in the treatment of alphaviral arthritides.
Subject(s)
Alphavirus Infections , Arthritis, Infectious , Arthritis, Rheumatoid , Chikungunya virus/immunology , Transcriptome/immunology , Alphavirus Infections/complications , Alphavirus Infections/genetics , Alphavirus Infections/immunology , Animals , Arthritis, Infectious/genetics , Arthritis, Infectious/immunology , Arthritis, Infectious/virology , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/virology , Asia , Chikungunya Fever , Disease Models, Animal , Gene Regulatory Networks/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Reunion , Synovial Membrane/immunologyABSTRACT
SerpinB2 (plasminogen activator inhibitor-2) is widely described as an inhibitor of urokinase plasminogen activator; however, SerpinB2(-/-) mice show no detectable increase in urokinase plasminogen activator activity. In this study, we describe an unexpected immune phenotype in SerpinB2(-/-) mice. After immunization with OVA in CFA, SerpinB2(-/-) mice made approximately 6-fold more IgG2c and generated approximately 2.5-fold more OVA-specific IFN-gamma-secreting T cells than SerpinB2(+/+) littermate controls. In SerpinB2(+/+) mice, high inducible SerpinB2 expression was seen at the injection site and in macrophages low levels in draining lymph nodes and conventional dendritic cells, and no expression was seen in plasmacytoid dendritic, B, T, or NK cells. SerpinB2(-/-) macrophages promoted greater IFN-gamma secretion from wild-type T cells in vivo and in vitro and, when stimulated with anti-CD40/IFN-gamma or cultured with wild-type T cells in vitro, secreted more Th1-promoting cytokines than macrophages from littermate controls. Draining lymph node SerpinB2(-/-) myeloid APCs similarly secreted more Th1-promoting cytokines when cocultured with wild-type T cells. Regulation of Th1 responses thus appears to be a physiological function of inflammation-associated SerpinB2; an observation that may shed light on human inflammatory diseases like pre-eclampsia, lupus, asthma, scleroderma, and periodontitis, which are associated with SerpinB2 polymorphisms or dysregulated SerpinB2 expression.
Subject(s)
Adaptive Immunity/immunology , Inflammation/immunology , Plasminogen Activator Inhibitor 2/physiology , Th1 Cells/immunology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Blotting, Western , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Flow Cytometry , Immunization , Immunoglobulin G/immunology , Inflammation/physiopathology , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-6/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Plasminogen Activator Inhibitor 2/genetics , Plasminogen Activator Inhibitor 2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Th1 Cells/metabolism , Tumor Necrosis Factor-alpha/metabolism , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
Many malignant tissues, including human papilloma virus (HPV)-associated cancers, express SerpinB2, also known as plasminogen activator inhibitor type-2 (PAI-2). Whether SerpinB2 is expressed by the HPV-transformed cancer cells, and if so, whether SerpinB2 is mutated or behaves aberrantly remains unclear. Here we show that HPV-transformed CaSki cells express high levels of constitutive wild-type SerpinB2, with cellular distribution, glycosylation, secretion, cleavage, induction and urokinase binding similar to that reported for primary cells. Neutralization of secreted SerpinB2 failed to affect CaSki cell migration or growth. Lentivirus-based over-expression of SerpinB2 also had no effect on growth, and we were unable to confirm a role for SerpinB2 in binding or regulating expression of the retinoblastoma protein. CaSki cells thus emerge as a useful tool for studying SerpinB2, with the physiological function of SerpinB2 expression by tumor cells remaining controversial. Using CaSki cells as a source of endogenous SerpinB2, we confirmed that SerpinB2 efficiently binds the proteasomal subunit member ß1.
Subject(s)
Neoplasms/metabolism , Papillomaviridae , Plasminogen Activator Inhibitor 2/analysis , Cell Line, Transformed , Cell Movement , Cell Proliferation , Cell Transformation, Viral , Humans , Neoplasm Proteins , Neoplasms/pathology , Neoplasms/virology , Plasminogen Activator Inhibitor 2/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Retinoblastoma Protein/metabolismABSTRACT
Recombinant Kunjin replicon virus-like particle (VLP), vaccinia virus (rVV) and DNA vaccines were tested in a large series of prime-boost vaccinations using interferon (IFN)γ ELISPOT assays that reflected effector (E), effector memory (EM) and central memory (CM) responses. All vaccine constructs encoded the murine polytope immunogen and responses to four CD8 T-cell epitopes (TYQRTRALV, SYIPSAEKI, YPHFMPTNL and RPQASGVYM) were measured. VLP/rVV out performed (by 14- to 20-fold) DNA/rVV for induction of CM responses, whereas EM responses were only marginally increased. DNA/VLP induced more EM, but not CM responses, than VLP alone, illustrating that DNA priming is not universally beneficial. rVV/VLP gave comparable results to VLP/rVV combinations, although the former induced approximately threefold more E responses, illustrating the utility of poxvirus priming in this setting. Although higher doses of VLP and rVV increased responses after single immunizations, such dose increases provided only marginal benefit in heterologous prime-boost settings. Triple combinations also provided no benefit over two vaccinations. DNA vaccination was associated with broad CM, but not EM responses, and the breadth of EM and E responses was significantly improved by increasing viral vector dose. VLP/rVV, rather than DNA priming, induced T cells with consistently high IFNγ secretion profiles across all ELISPOT measures. Vector-specific CD8 T-cell responses generally correlated well with immunogen-specific responses, although, as expected, single use of each vector reduced the relative levels of vector-specific responses. These experiments illustrate the utility of replicons in heterologous prime-boost vaccinations, and illustrate the diversity of data that can be obtained from ELISPOT analyses.
Subject(s)
Enzyme-Linked Immunospot Assay , Vaccination , Vaccinia virus/immunology , Viral Vaccines/immunology , West Nile virus/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Dose-Response Relationship, Immunologic , Female , Genetic Vectors/genetics , Genetic Vectors/immunology , Immunologic Memory/immunology , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Vaccines, Virus-Like Particle/immunology , Vaccinia virus/genetics , West Nile virus/geneticsABSTRACT
Chikungunya virus is a mosquito-borne arthrogenic alphavirus that has recently reemerged to produce the largest epidemic ever documented for this virus. Here we describe a new adult wild-type mouse model of chikungunya virus arthritis, which recapitulates the self-limiting arthritis, tenosynovitis, and myositis seen in humans. Rheumatic disease was associated with a prolific infiltrate of monocytes, macrophages, and NK cells and the production of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma). Infection with a virus isolate from the recent Reunion Island epidemic induced significantly more mononuclear infiltrates, proinflammatory mediators, and foot swelling than did an Asian isolate from the 1960s. Primary mouse macrophages were shown to be productively infected with chikungunya virus; however, the depletion of macrophages ameliorated rheumatic disease and prolonged the viremia. Only 1 microg of an unadjuvanted, inactivated, whole-virus vaccine derived from the Asian isolate completely protected against viremia and arthritis induced by the Reunion Island isolate, illustrating that protection is not strain specific and that low levels of immunity are sufficient to mediate protection. IFN-alpha treatment was able to prevent arthritis only if given before infection, suggesting that IFN-alpha is not a viable therapy. Prior infection with Ross River virus, a related arthrogenic alphavirus, and anti-Ross River virus antibodies protected mice against chikungunya virus disease, suggesting that individuals previously exposed to Ross River virus should be protected from chikungunya virus disease. This new mouse model of chikungunya virus disease thus provides insights into pathogenesis and a simple and convenient system to test potential new interventions.
Subject(s)
Alphavirus Infections/pathology , Alphavirus Infections/virology , Arthritis/pathology , Arthritis/virology , Chikungunya virus/isolation & purification , Chikungunya virus/pathogenicity , Disease Models, Animal , Alphavirus Infections/immunology , Alphavirus Infections/prevention & control , Animals , Arthritis/immunology , Arthritis/prevention & control , Chikungunya virus/immunology , Cytokines/metabolism , Female , Foot/pathology , Foot/virology , Histocytochemistry , Humans , Immunohistochemistry , Killer Cells, Natural/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Microscopy , Monocytes/immunology , Ross River virus/immunology , Vaccines, Inactivated/immunology , Viral Load , Viral Vaccines/immunology , ViremiaABSTRACT
The 'Nanopatch' (NP) comprises arrays of densely packed projections with a defined geometry and distribution designed to physically target vaccines directly to thousands of epidermal and dermal antigen presenting cells (APCs). These miniaturized arrays are two orders of magnitude smaller than standard needles-which deliver most vaccines-and are also much smaller than current microneedle arrays. The NP is dry-coated with antigen, adjuvant, and/or DNA payloads. After the NP was pressed onto mouse skin, a protein payload co-localized with 91.4 + or - 4.1 APC mm(-2) (or 2925 in total) representing 52% of the delivery sites within the NP contact area, agreeing well with a probability-based model used to guide the device design; it then substantially increases as the antigen diffuses in the skin to many more cells. APC co-localizing with protein payloads rapidly disappears from the application area, suggesting APC migration. The NP also delivers DNA payloads leading to cutaneous expression of encoded proteins within 24 h. The efficiency of NP immunization is demonstrated using an inactivated whole chikungunya virus vaccine and a DNA-delivered attenuated West Nile virus vaccine. The NP thus offers a needle-free, versatile, highly effective vaccine delivery system that is potentially inexpensive and simple to use.
Subject(s)
Chikungunya virus/immunology , Nanostructures/chemistry , Vaccination/methods , Viral Vaccines/administration & dosage , West Nile Virus Vaccines/administration & dosage , Administration, Cutaneous , Alphavirus Infections/prevention & control , Animals , Antigen-Presenting Cells/immunology , Chikungunya Fever , Mice , Mice, Inbred BALB C , Vaccines, DNA/administration & dosage , West Nile Fever/prevention & control , West Nile Virus Vaccines/genetics , West Nile Virus Vaccines/immunology , West Nile virus/immunologyABSTRACT
SerpinB2 (plasminogen activator inhibitor type 2) has been called the "undecided serpin" with no clear consensus on its physiological role, although it is well described as an inhibitor of urokinase plasminogen activator (uPA). In macrophages, pro-inflammatory stimuli usually induce SerpinB2; however, expression is constitutive in Gata6+ large peritoneal macrophages (LPM). Interrogation of expression data from human macrophages treated with a range of stimuli using a new bioinformatics tool, CEMiTool, suggested that SerpinB2 is most tightly co- and counter-regulated with genes associated with cell movement. Using LPM from SerpinB2-/- and SerpinB2R380A (active site mutant) mice, we show that migration on Matrigel was faster than for their wild-type controls. Confocal microscopy illustrated that SerpinB2 and F-actin staining overlapped in focal adhesions and lamellipodia. Genes associated with migration and extracellular matrix interactions were also identified by RNA-Seq analysis of migrating RPM from wild-type and SerpinB2R380A mice. Subsequent gene set enrichment analyses (GSEA) suggested SerpinB2 counter-regulates many Gata6-regulated genes associated with migration. These data argue that the role of SerpinB2 in macrophages is inhibition of uPA-mediated plasmin generation during cell migration. GSEA also suggested that SerpinB2 expression (likely via ensuing modulation of uPA-receptor/integrin signaling) promotes the adoption of a resolution phase signature.
Subject(s)
Cell Movement , Macrophages, Peritoneal/metabolism , Plasminogen Activator Inhibitor 2/metabolism , Animals , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Macrophages, Peritoneal/cytology , Mice , Mice, Knockout , Plasminogen Activator Inhibitor 2/genetics , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus associated with a robust systemic infection and an acute inflammatory rheumatic disease. A high fiber diet has been widely promoted for its ability to ameliorate inflammatory diseases. Fiber is fermented in the gut into short chain fatty acids such as acetate, propionate, and butyrate, which enter the circulation providing systemic anti-inflammatory activities. Herein we show that mice fed a high fiber diet show a clear exacerbation of CHIKV arthropathy, with increased edema and neutrophil infiltrates. RNA-Seq analyses illustrated that a high fiber diet, in this setting, promoted a range of pro-neutrophil responses including Th17/IL-17. Gene Set Enrichment Analyses demonstrated significant similarities with mouse models of inflammatory psoriasis and significant depression of macrophage resolution phase signatures in the CHIKV arthritic lesions from mice fed a high fiber diet. Supplementation of the drinking water with butyrate also increased edema after CHIKV infection. However, the mechanisms involved were different, with modulation of AP-1 and NF-κB responses identified, potentially implicating deoptimization of endothelial barrier repair. Thus, neither fiber nor short chain fatty acids provided benefits in this acute infectious disease setting, which is characterized by widespread viral cytopathic effects and a need for tissue repair.
Subject(s)
Butyrates/adverse effects , Chikungunya Fever/immunology , Chikungunya virus/physiology , Dietary Fiber/adverse effects , Inflammation/etiology , Neutrophils/immunology , Rheumatic Diseases/etiology , Animals , Butyrates/administration & dosage , Chikungunya Fever/complications , Diet , Dietary Fiber/administration & dosage , Disease Models, Animal , Disease Progression , Edema , Humans , Joint Diseases , Mice , Mice, Inbred C57BL , Neutrophil InfiltrationABSTRACT
The dichotomous effects of the protein kinase C (PKC) modulatory compounds 12-myristate 13-acetate (PMA), prostratin, and ingenol 3-angelate (I3A) on HIV-1 infection were investigated. PKC modulatory compounds were shown to be potent activators of cells latently infected with HIV-1 (I3A > prostratin). Conversely, PKC modulatory compounds inhibited infection of indicator cells (MAGI) with CXCR4-tropic HIV-1 (PMA > I3A > prostratin), and I3A also inhibited infection with CCR5-tropic virus (AD8-1). Pretreatment with the PKC inhibitors prior to treatment with either I3A or PMA resulted in increased infection, indicating inhibition is PKC mediated. Cell infections suggested that I3A rapidly inhibited the virus from infecting cells at an early point in infection. This observation was supported by the demonstration of inhibition at or before the synthesis of early reverse transcription products, and the inability of these compounds to block vesicular stomatitis virus (VSV) pseudotyped HIV-1 particles. As has already been shown with prostratin, treatment with I3A resulted in down-regulation of the CD4 receptor and CXCR4 coreceptor suggesting that this was a contributor to the infection inhibition. Intriguingly, 48 h pretreatment of unstimulated peripheral blood mononuclear cells (PBMC) prior to infection resulted in abrogation of virus production at concentrations where receptor/ coreceptor levels were not significantly reduced. This result hints at the possibility of inhibition by a PKC modulatory compound of an early pathway of viral entry in PBMC.
Subject(s)
HIV Infections/prevention & control , HIV-1/drug effects , Leukocytes, Mononuclear/virology , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Virus Activation/drug effects , CD4 Antigens/metabolism , Cells, Cultured/cytology , Cells, Cultured/drug effects , Diterpenes/pharmacology , Down-Regulation/drug effects , Gene Expression Regulation/drug effects , HIV Infections/drug therapy , HeLa Cells , Humans , In Vitro Techniques , Leukocytes, Mononuclear/drug effects , Phorbol Esters/pharmacology , Receptors, Chemokine/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Virus LatencyABSTRACT
Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodessication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 micro g) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; n = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; n = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Foxn1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD(90) for PEP005 for a panel of tumor cell lines was 180-220 micro M. Electron microscopy showed that treatment with PEP005 both in vitro (230 micro M) and in vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. (51)Cr release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 micro M) treatment of tumor cells in vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.
Subject(s)
Antineoplastic Agents/pharmacology , Diterpenes/pharmacology , Esters/pharmacology , Mitochondria/drug effects , Administration, Topical , Animals , Cell Death/drug effects , Cell Line, Tumor , Cell Membrane/drug effects , Female , Humans , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Membrane Potentials/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mitochondria/physiology , Neoplasms, Experimental/drug therapyABSTRACT
Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7-/-) mice, which are deficient for interferon α/ß responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities.
Subject(s)
Chikungunya Fever/pathology , Chikungunya virus/genetics , Saliva/virology , Adolescent , Animals , Chikungunya Fever/transmission , Chikungunya Fever/virology , Chikungunya virus/isolation & purification , Child , Disease Models, Animal , Female , Haplorhini , Humans , Interferon Regulatory Factor-3/deficiency , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-7/deficiency , Interferon Regulatory Factor-7/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Olfactory Mucosa/pathology , Olfactory Mucosa/virology , RNA, Viral/metabolism , Viral LoadABSTRACT
Foroderm is a new cutaneous delivery technology that uses high-aspect ratio, cylindrical silica microparticles, that are massaged into the skin using a 3D-printed microtextured applicator, in order to deliver payloads across the epidermis. Herein we show that this technology is effective for delivery of a non-adjuvanted, inactivated, whole-virus chikungunya virus vaccine in mice, with minimal post-vaccination skin reactions. A single topical Foroderm-based vaccination induced T cell, Th1 cytokine and antibody responses, which provided complete protection against viraemia and disease after challenge with chikungunya virus. Foroderm vaccination was shown to deliver fluorescent, virus-sized beads across the epidermis, with beads subsequently detected in draining lymph nodes. Foroderm vaccination also stimulated the egress of MHC II(+) antigen presenting cells from the skin. Foroderm thus has potential as a simple, cheap, effective, generic, needle-free technology for topical delivery of vaccines.