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. 2012 Dec 19:9:313.
doi: 10.1186/1743-422X-9-313.

Mouse macrophage innate immune response to Chikungunya virus infection

Shiril Kumar  1 , Marie-Christine Jaffar-Bandjee, Claude Giry, Léa Connen de Kerillis, Andres Merits, Philippe Gasque, Jean-Jacques Hoarau
Affiliations

Mouse macrophage innate immune response to Chikungunya virus infection

Shiril Kumar et al. Virol J. .

Abstract

Background: Infection with Chikungunya alphavirus (CHIKV) can cause severe arthralgia and chronic arthritis in humans with persistence of the virus in perivascular macrophages of the synovial membrane by mechanisms largely ill-characterized.

Findings: We herein analysed the innate immune response (cytokine and programmed cell death) of RAW264.7 mouse macrophages following CHIKV infection. We found that the infection was restrained to a small percentage of cells and was not associated with a robust type I IFN innate immune response (IFN-α4 and ISG56). TNF-α, IL-6 and GM-CSF expression were upregulated while IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-10 or IL-17 expression could not be evidenced prior to and after CHIKV exposure. Although CHIKV is known to drive apoptosis in many cell types, we found no canonical signs of programmed cell death (cleaved caspase-3, -9) in infected RAW264.7 cells.

Conclusion: These data argue for the capacity of CHIKV to infect and drive a specific innate immune response in RAW264.7 macrophage cell which seems to be polarized to assist viral persistence through the control of apoptosis and IFN signalling.

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Figures

Figure 1
Figure 1
CHIKV infects and replicates in RAW264.7 mouse macrophages. (A) Immunostainings using FDO (human anti-CHIKV polyclonal Ab, red fluorescence) and J2 (mouse anti-double stranded RNA, green) or ISG-15 or specific rabbit anti-phospho IRF3 at 24 hrs PI. Nuclei were counterstained with DAPI (blue). Only a limited number of total RAW264.7 cells were infected (< 5%) in form of clusters, as focused on here, compared to CLTT (> 95% of infected cells). (B) During the time course of infection, both structural and non-structural proteins of CHIKV were produced in infected cells as shown by double staining with FDO (green fluorescence) and rabbit anti cross-reacting SFV/CHIKV Nsp1 antibody (red fluorescence). Nuclei were stained with DAPI (blue fluorescence). Negative controls are shown as insets. * Cluster of infected cells.
Figure 2
Figure 2
RAW264.7 infected cells does not undergo apoptosis. (A& B) Immunofluorescence of RAW264.7 and CLTT at 48 hrs PI (mouse monoclonal anti-CHIKV E1, green fluorescence) and apoptosis markers (initiator cleaved caspase-9 or executor cleaved caspase-3, red fluorescence). Nuclei were stained with DAPI (blue fluorescence). (C) No DNA fragmentation (TUNEL assay) was observed in RAW264.7 cells (Inset, positive control of apoptosis) during the time course of infection.
Figure 3
Figure 3
CHIKV replication in RAW (macrophages) and CLTT (astrocyte control). (A) With time, CHIKV RNA raised in RAW264.7 cells and supernatants but remained low compared to CLTT as tested by RT-qPCR for E1 (n=3). The graph was plotted after adjusting the background level of the residual virus following the washing steps at T0 (2 hrs PI).
Figure 4
Figure 4
CHIKV positive and negative RNA strands expression. Positive (+) and negative (-) E2 RNAs were detected at very high levels in CLTT cells ( both in cells and supernatants) whereas RAW264.7 expressed the positive strand at level 1000 fold lower than CLTT (both in cells and supernatants) while the negative strand was detected at a low level and only in cell extract. The quantity of RNA strands (arbitrary unit) was estimated with the ΔCt method as 2-Δct with ΔCt = Ct (at 8 h or 48 h PI) – Ct (at T0). Ndt (Not detected).
Figure 5
Figure 5
Polarized innate immune response in CHIKV-infected RAW264.7. (A) During the time course of infection, RAW264.7 shows no significant change in IFNα4 mRNA expression by RT-qPCR compared to CLTT. However, a significant increase of ISG-56 at 24 hrs PI was measured for CLTT (fold increase = 121.5 ± 11.5) compare to RAW264.7 (0.7 ± 0.1). (B) A significant increase of TNFα for RAW264.7 at 24 hr PI (fold change = 17.9 ± 3.2) was observed by RT-qPCR and validated by ELISA (fold change = 3.3 ± 0.3). Fold change over non-infected control is expressed as mean ± SEM of three independent experiments.
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