Table 1.
Fig 1.
Nitrosporeusine treatment reduced the severity of infection and increases survivability in BALB/c mouse.
A) Kaplan-Meier plot showed an increase in survivability of animals after (-)-25b treatment by 48–72 hours (2–3 days). B) Growth plot showed a comparatively delayed decrease in weight of (-)-25b treated group against only CHPV infected group. C) Plaque assay was performed on Vero cells inoculated with brain homogenate obtained from CHPV infected and CHPV infected-(-)-25b treated groups. Significant low number of plaques was observed in Vero cell culture infected with (-)-25b treated group when compared to the untreated group. D) qPCR analysis showed a significant reduction in CHPV mRNA level after (-)-25b treatment when compared to only CHPV infected group. E) TUNEL assay showed a decrease in TUNEL positive cells after minocycline treatment in CHPV infected, and (-)-25b treated brain sections. Bar graph was plotted denoting number of TUNEL positive cells in different samples. F) Western Blot for Viral M-protein and cleaved Caspase 3 showed significant decrease in expression after minocycline treatment in CHPV infected brain samples, which was quantified by densitometry graph. Validation of results were done by 3 independent experiments with at least 4 animals in each group.*p < 0.05, **p < 0.01.
Fig 2.
Nitrosporeusine inhibits CHPV induced microglia activation and reduces inflammatory molecules.
A). CBA analysis was done from N9 supernatant and mouse brain protein to analyse expression level of cytokines. TNF-a, CCL2 and IL-6 shows enhanced expression in supernatant (6hpi) of N9. Our data suggest treatment of cells with (-)-25b compound is effective in inhibiting microglia mediated inflammation. Similarly, in vivo validation was performed in mouse brain (2dpi) and data was concomitant with in vitro result. B). Immunofluorescence staining for Iba-1 shows activated morphology of microglia. The star shaped morphology was more prominent in CHPV infected samples. The (-)-25b treated group shows decrease in activated morphology of microglia. C). ROS level using DCFDA shows decrease level in (-)-25b treated samples as compared to CHPV treated samples. NO generation was measured from N9 cells using Griess reagent. NO was measured from N9 supernatant shows increase level of NO in CHPV infected samples as compared to (-)-25b treated group at 6 hpi. D). Western blot analysis of Cox-2 and iNOS shows decrease expression level in (-)-25b treated group in in vitro as well as in vivo samples which was found to be upregulated in CHPV infected group.
Fig 3.
Chemokine expression increases post CHPV infection.
A). mRNA expression level was checked for CXCL10 and CCL5 from N9 cell sample. The data shows significant increase in expression level of these chemokines in CHPV infected microglia which was found to be similar with control in (-)-25b treated group. B). Further to confirm the protein expression level, ELISA was performed. ELISA data shows upregulation in expression level in CHPV infected microglia. C &D). Chemokine expression was further validated in brain sample and was found to be consistent with N9 data showing increase in CHPV infected sample and decrease in (-)-25b treated sample. Experiments were performed at thrice before reaching to conclusion. In vivo experiment were performed with at least four animal in each group. *p < 0.05, **p < 0.01.
Fig 4.
Leukocyte infiltration and BBB breaching during CHPV infection.
A). Peripheral immune cells were stained to check their presence in brain post infection. CD3 positive cell were more prominent in CHPV infected samples. Similarly CD68 and CD11b positive cells were present in CHPV infected sample suggesting infiltration of peripheral cells in brain. B). To examine BBB breaching, we checked expression level of tight junction protein. Claudin-1, occludin and β-catenine was checked for western blot. Data suggest increase in expression of these proteins in CHPV infected samples. (-)-25b treated brain shows significant decrease in expression level of the proteins. C). mRNA expression was checked to analyse the status of ICAM, VCAM and MMP-9. Validation of results were done by 3 independent experiments with at least 4 animals in each group.*p < 0.05, **p < 0.01.
Fig 5.
CHPV infection activates MAPK signaling pathways in microglia.
(A) MAPK activation in microglia following CHPV infection. Cells were mock infected or infected with CHPV or (-)-25b treated followed by CHPV infection were processed for western blot. The protein levels of phosphorylated MAPKs and NF-kappaB were analyzed by Western blotting as described in the text. The amounts of β-actin were also assessed to monitor the equal loadings of protein extracts. Data shows upregulation in expression of p-p38, p-JNK, p-Iκκb, p-NF-kappaB proteins. (B) Further brain lysate were processed to check expression of these proteins. Our data shows increase in p-p38, p-JNK, p-Iκκa/b, p-NF-kappaB in CHPV infected group which decreases in (-)-25b treated samples. Experiments were performed at thrice before reaching to conclusion. In vivo experiment were performed with atleast four animal in each group. *p < 0.05, **p < 0.01.
Fig 6.
CHPV induces microglial activation through NF-kappaB pathway.
A). p38 (SB239063), JNK (SP600125), Dexamethasone (Dexa) an inhibitor of NF-kappaB was used at concentration of 10μm to check their effect on CHPV induced cytokines and chemokines production. CBA analysis of cytokines expression shows decrease in expression level in Dexa treated inhibitors whereas other inhibitors were not very effective. Though SB239063 showed significance decrease in CCL2, but was not effective in downregulating TNF-α and IL-6. B). Chemokine expression level was checked in presence of inhibitors. Dexa was found to be most effective in inhibiting chemokine expression. C) CBA was performed in presence of inactivated virus to asses if live virus is required for virus dependent inflammation. CBA data shows incapability of inactivated virus in inducing cytokine expression. D). Western blot experiment shows role of Akt phosphorylation in activation of NF-kappaB. The data shows treatment of PI3K inhibitor (LY294002) at 10μM inhibited p-Akt level and thus NF-kappaB was inhibited.
Fig 7.
Bystander killing of neurons using supernatants from activated microglial cells.
(A) Immunoblot image shows expression of caspase 3 in supernatant treated HT-22 cells. CHPV protein was checked to confirm complete absence of live virus particle in cells β -actin was used as loading control. (B) In vitro culture of HT-22 neuron showed TUNEL positive cells in supernatant treated panel confirming bystander killing of neurons. Bar graph shows number of positive cells in each sample. (C) CBA data revels presence of cytokines after UV-inactivation of supernatant suggesting role of cytokines in neuronal death.