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Japanese Encephalitis Virus-Infected Macrophages Induce Neuronal Death

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Abstract

Inflammation in the central nervous system (CNS) in Japanese encephalitis (JE) is shown to be the result of microglial activation that leads to the release of various proinflammatory mediators. Peripheral macrophages have been reported to infiltrate into the CNS in JE, though their contribution to the inflammatory process is yet to be elucidated. In this study, using an in vitro macrophage model, we have shown that upon JE virus infection, these cells secrete various soluble factors which may significantly add to the existing inflammatory milieu and lead to apoptotic or necrotic death of neurons. However, it is difficult to quantify the extent of involvement of either the microglia or infiltrating macrophages in the inflammatory processes.

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Acknowledgements

The authors would like to thank Vinod Babu for his help and Manish Kumar Dogra and Kanhaiya Lal Kumawat for their technical assistance.

Financial disclosure

This work is funded by a grant from the Council of Scientific and Industrial Research (CSIR), Govt. of India [27(1638)/10] to A.B. A.N. is a recipient of the Junior Research Fellowship from the Council of Scientific and Industrial Research, Government of India; K.D. is a recipient of a Research Associateship in Biotechnology and Life Sciences from the Department of Biotechnology, Government of India; S.D. is a recipient of a Senior Research Fellowship from University Grants Commission, Government of India.

Conflict of interest

The authors declare that there are no conflicts of interests with this manuscript.

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Authors and Affiliations

  1. National Brain Research Centre, Manesar, Haryana, 122050, India

    Arshed Nazmi, Kallol Dutta, Sulagna Das & Anirban Basu

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  1. Arshed Nazmi
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  2. Kallol Dutta
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Correspondence to Anirban Basu.

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Fig. S1

Internalization and persistence of JEV within macrophages. Immunocytochemical staining of mock-infected and JEV-infected primary macrophages with CD11b and JEV antigen showed that the virus was present within the macrophages even after 48 h post infection. Magnification ×40; scale bar corresponds to 50 μm (GIF 132 kb)

High resolution image file (TIFF 1030 kb)

Fig. S2

Plaque assay performed with conditioned media obtained from JEV-infected macrophages showed no visible cytopathic effect on a monolayer of Vero cells at various dilutions. Following incubation with the conditioned media, Vero cell monolayers were stained with crystal violet solution. However, no plaque formation could be visualized (GIF 8 kb)

High resolution image file (TIFF 127 kb)

Fig. S3

After 12 h of infection with JEV (J), the release of IL-12p70, MCP-1, TNF-α, IL-6 and IFN-γ (a–e) were significantly increased from macrophages. Application of NFκB and p38MAPK inhibitors MG-132 and SKF-86002 onto JEV-infected macrophages (J+MG and J+S, respectively) resulted in marked reduction in their levels (*p < 0.01 for J compared to mock-infected; # p < 0.01 for J+MG and J+S compared to J only) (GIF 39 kb)

High resolution image file (TIFF 248 kb)

Fig. S4

Immunoblot showing Bcl-2 and Bax levels in N2a that were treated with 12-h post-incubation conditioned media from mock-infected (M), JEV-infected (J), JEV-infected and MG-132 treated (J+MG) and JEV-infected and SKF-86002-treated (J+S) macrophages (a). Bcl-2 levels were significantly increased and Bax levels were decreased, following the addition of media from J+MG and J+S macrophages, in comparison to those treated with conditioned media from macrophages that were infected with JEV only (*p < 0.01 for J compared to mock-infected; # p < 0.01 for J+MG and J+S compared to J only) (GIF 35 kb)

High resolution image file (TIFF 290 kb)

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Nazmi, A., Dutta, K., Das, S. et al. Japanese Encephalitis Virus-Infected Macrophages Induce Neuronal Death. J Neuroimmune Pharmacol 6, 420–433 (2011). https://doi.org/10.1007/s11481-011-9271-x

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