Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production
March 2nd, 2014
We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (γHV68) that is closely-related to human Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo
The overall goal of the following experiment is to dissect key host innate immune molecules regulating antiviral cytokine production upon gamma herpes virus 68 or gamma HV 68 injection. This is achieved by comparing antiviral cytokines, secreted by gamma HV 68 injected Mavs wild type and knockout mice. The second step is to quantify cytokines produced by Mavs Wild type and Mavs knockout MEF cells after gamma HV 68 infection, which confirms Mavs is utilized to suppress antiviral cytokine secretion.
The next step is to restore Mavs and Mavs knockout MEF cells in order to assess whether cytokine expression levels are rescued by exogenous Mavs. Results are obtained that show Gamma HV 68 hijacks Mavs, a key innate immune signaling component to abrogate antiviral cytokine production in vivo and ex vivo based on western blotting analysis. Eliza and Q-R-T-P-C-R in Mavs Wild type and knockout mice and corresponding MEF cells.
This measure can help to answer the key questions in the innate field, such as host innate immune response upon gum hepworth infection demonstrating the procedure will be leaning to a postdoc in Dr.P'S lab. Intranasally inoculate the anesthetized C 57 black six mice with 40 microliters of 40 plaque forming units of the natural mirroring pathogen gamma herpes virus 68. After sacrificing the mice at experimental time points post-infection, open the chest with a left lateral cut along the sternum, cut through the ribs with sharp scissors and collect the lung tissues.
Place the left lobe into a sterile 1.5 milliliter screw caps tube containing 500 microliters of 1.0 millimeters. Zirconia silica beads. Add one milliliter of cold serum free DMEM and homogenize the lung tissue by bead beading for 30 seconds.
Chill the tubes on ice for two minutes and repeat the bead beading process. Once swans, then centrifuge the tubes at four degrees Celsius and collect the supernatant. Now measure the desired cytokines using commercially available cytokine ELIZA kits according to the manufacturer's instructions.
Next, place the right lobe of the lung tissue into another bead containing tube and add one milliliter of triol homogenize and collect the supernat as described earlier. Then extract the RNA according to the manufacturer's instructions. To determine the RNA concentration measure absorbance readings at 260 and 280 nanometers.
Next, perform a reverse transcription reaction with one microgram of total RNA templates in a total final volume of 20 microliters. Now for the quantitative real-time PCR dilute the resulting CD NA 50 times with DNAs and RNAs free water for each reaction at 0.5 microliters of a pair of gene specific or control primers. Five microliters of the cyber master mix and four microliters of the diluted CD NA.Calculate the threshold cycles to determine the relative quantity of mRNA.
Transcripts of select antiviral cytokines grow the wild type and knockout mouse embryonic fibroblasts to 80%co fluency. Split the MEF cells into 12 well plates at 100, 000 cells per well to synchronize and maximize cytokine induction. Prepare a working stock of gamma herpes virus for infecting the cells in triplicate with a multiplicity of infection of five to 10.
Now remove the medium and add gamma HV 68 containing suspension to the MEF cells. Place the plates in a tissue culture incubator rocking every 30 minutes over the course of a two hour incubation. Next, remove the gamma HV 68 containing medium.
Wash the cells once with PBS and then add fresh complete DMEM at various time points. Post-infection, harvest a medium into 1.5 milliliter einor tubes. Wash the cells with cold PBS and harvest the infected cells by trypsin digestion.
Measure the antiviral cytokines in medium by Eliza as shown earlier. Also extract RNA, prepare CDNA and perform Q-R-T-P-C-R to determine the antiviral cytokine gene expression profile as shown earlier. Split confluent 2 9 3 T cells one day before transfection and allow the cells to reach 40%co fluency at the time of transfection.
Transfect the 2 9 3 T cells with the plasmids using the calcium phosphate precipitation method. After six to eight hours post transfection, replace the media with fresh complete DMEM at 72 hours post transfection. Collect the medium containing the lentivirus centrifuge at 4, 000 GS for 15 minutes and filter the super names through a 0.22 micrometer membrane.
Then centrifuge the containing medium at 110, 000 Gs for two and a half hours at four degrees Celsius. Carefully resuspend the viral pellets in a small volume of the medium of interest. Next, infect knockout MEF cells with one milliliter of lentivirus and two milliliters of fresh complete DMEM supplemented with poly brain centrifuge.
The plates at 500 GS at 30 degrees Celsius for 30 minutes. Then transfer the cells to a tissue culture incubator at six hours post infection. Replace the medium with fresh complete DMEM, split the infected MEF cells at 24 hours post infection and at 48 hours post infection, add pur mycin to a final concentration of one microgram per milliliter to select for cells that stably express the viral construct.
Maintain the MEF cells in pur mycin containing media and verify the protein expression by immuno blotting with corresponding antibodies. These cells can now be used for viral infection, cytokine gene expression, and secretion experiments. As shown earlier, the MAVS gene codes for a mitochondrial antiviral signaling protein that plays a key role in viral immunity.
These results show that gamma HV 68 infection of Mavs knockout mice leads to significantly higher production of CCL five in vivo. MA embryonic fibroblasts were then used to investigate the mechanism of regulated cytokine production. Ex vivo.
Here the Mavs knockout cells produced more antiviral cytokine than wild type during gamma HV 68 infection, which recapitulates the en vivo phenotype, reconstituted expression of Mavs and Mavs knockout MEFs decreased CCL five production. Collectively, these EISA and quantitative PCR results demonstrate that Mavs is necessary for gamma HV 68 to suppress antiviral cytokine production, both in vivo and ex vivo After its development. This technique paved the way for researchers in the field of innate immunity to explore host antiviral response in transgenic mice infected by herpes virus.
View the full transcript and gain access to thousands of scientific videos
Overview
This study investigates the role of innate immune molecules in regulating antiviral cytokine production in response to gamma herpesvirus 68 (纬HV68) infection. By utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), the research aims to elucidate the mechanisms by which 纬HV68 affects cytokine secretion.
Key Study Components
Area of Science
- Immunology
- Virology
- Infectious Diseases
Background
- 纬HV68 is a model for studying herpesvirus infections.
- Understanding cytokine production is crucial for developing antiviral strategies.
- Innate immune responses play a key role in controlling viral infections.
- Previous studies have shown that 纬HV68 can manipulate host immune responses.
Purpose of Study
- To identify key innate immune molecules involved in antiviral cytokine production.
- To compare cytokine responses in wild type and knockout mice.
- To assess the impact of 纬HV68 on cytokine secretion mechanisms.
Methods Used
- In vivo and ex vivo analysis of cytokine production.
- Use of genetically modified Mavs wild type and knockout mice.
- Quantification of cytokines using ELISA and Q-R-T-P-C-R.
- Western blotting to analyze signaling pathways affected by 纬HV68.
Main Results
- 纬HV68 infection alters cytokine production in Mavs knockout mice.
- Restoration of Mavs in MEF cells influences cytokine expression levels.
- Results indicate that 纬HV68 hijacks Mavs to suppress antiviral responses.
- Significant differences in cytokine levels were observed between wild type and knockout models.
Conclusions
- The study highlights the importance of Mavs in regulating antiviral cytokine production.
- Understanding these mechanisms could inform therapeutic strategies against herpesvirus infections.
- Further research is needed to explore the implications of these findings in human herpesvirus infections.
Frequently Asked Questions
Executive Industry Relevance
Understanding how viruses hijack innate immune signaling to suppress antiviral cytokine production is critical for de-risking antiviral target validation. This study reveals a mechanism by which gamma herpesvirus 68 subverts MAVS-dependent NF-κB activation, providing a disease-relevant system to evaluate host-pathogen interface disruptions. Such insights support mechanistic de-risking in early discovery by clarifying how viral evasion strategies can be intercepted to restore immune signaling and inform therapeutic design.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypothesis by demonstrating how viral proteins target MAVS and IKKε to abrogate NF-κB signaling and cytokine production.
- Operational Value: Uses MAVS wild-type and knockout models to validate target necessity in viral immune evasion, enabling causal inference in pathway interrogation.
- Predictive Value: Establishes a disease-relevant system to assess whether restoring MAVS function rescues antiviral cytokine secretion, supporting target confidence in immunomodulatory approaches.
Screening & Assay Development
- Scientific Value: Quantifies antiviral cytokines (e.g., CCL5) via ELISA and qRT-PCR in lung tissue and MEFs, delivering reproducible, quantitative readouts for pathway modulation.
- Operational Value: Standardizes ex vivo infection assays using primary MEFs and lentiviral reconstitution to enable scalable screening of compounds or genetic interventions that restore cytokine production.
- Assay Readiness: Provides a validated biological system (MAVS-deficient MEFs) that recapitulates in vivo phenotypes, supporting platform reuse for antiviral compound evaluation.
Translational & Preclinical Research
- Translational Continuity: Uses murine γHV68 as a tractable model to study mechanisms conserved in human KSHV and EBV, enabling preclinical extrapolation of innate immune evasion strategies.
- Mechanistic De-risking: Defines a clear molecular intervention point—MAVS restoration—to test whether blocking viral hijacking rescues immune function, informing lead identification.
- Predictive Confidence: Links upstream IKKε activation to downstream RelA degradation and NF-κB suppression, offering a biomarker-aligned readout for pathway activity in therapeutic screening.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation to lead identification, where mechanistic insights into viral immune evasion inform assay development and compound screening efforts focused on restoring innate signaling.
- Discovery Biology: Supports hypothesis testing by isolating MAVS as a necessary factor for γHV68-mediated suppression of antiviral cytokines, clarifying causal relationships in immune signaling.
- Screening: Delivers standardized, quantitative cytokine measurements (ELISA, qRT-PCR) from infected WT and KO MEFs, enabling reproducible evaluation of modulators that restore signaling.
- Analytics: Generates correlative data from western blotting (IKKε phosphorylation, RelA degradation) and functional cytokine outputs, allowing multi-parametric assessment of pathway modulation.
- Translational Research: Connects murine γHV68 findings to human herpesvirus pathogenesis through conserved MAVS-IKKε-NF-κB axis, supporting preclinical validity.
- Enterprise Reuse: Establishes a modular platform—MAVS KO MEFs + lentiviral rescue—that can be adapted to other viruses or pathways, reducing redundant model development.
Operational & Enterprise Impact
- Scientific Value: Provides mechanistic clarity on how viral pathogens uncouple upstream kinase activation from downstream transcriptional responses, reducing ambiguity in target validation.
- Operational Value: Employs standardized, quantifiable assays (ELISA, qRT-PCR, western blot) across in vivo and ex vivo systems, enhancing reproducibility and cross-team consistency.
- Strategic Value: Enables better go/no-go decisions by distinguishing whether compounds act upstream (e.g., IKKε inhibition) or downstream (e.g., MAVS stabilization) of viral evasion points.
- Portfolio Impact: Supports risk-adjusted prioritization of immunomodulatory candidates that demonstrate rescue of cytokine production in validated evasion models.
Implementation Considerations
- Requires expertise in virology, immunology, and molecular biology to handle γHV68 infection, MEF culture, and lentiviral transduction.
- Dependent on access to biosafety level 2 facilities, ELISA/qRT-PCR platforms, and western blotting infrastructure for cytokine and signaling analysis.
- Necessitates standardization across teams for mouse infection timing, tissue harvest, and MEF transfection protocols to ensure data comparability.
- Adaptation to other model systems (e.g., human cells) may require validation of MAVS-IKKε-NF-κB axis conservation and viral tropism.
- Practical limitations include variability in primary MEF responses and the need for clonal validation of lentiviral reconstitution efficiency.
