JEV infection induced the translocation and secretion of HMGB1 in HBMEC
The HBMECs were infected with JEV at 1 MOI, and the expression of JEV-E protein was measured by Western blotting. JEV replicates in HBMEC in a time-dependent manner (Fig. 1A), abundant JEV-E protein presented intracellularly at 24 h and 48 h (Fig. 1B). JEV-infection induced a dramatical increase of HMGB1 both in mRNA (Fig. 1C), and protein level (Fig. 1D). Meanwhile, HMGB1 was also highly expressed in mouse brain microvascular endothelial cell line bEnd.3 during JEV infection (S.1). These results demonstrate that brain microvascular endothelial cells are susceptible to JEV that particularly induces HMGB1 production in brain microvascular endothelial cells.
The biological functions of HMGB1 are dominated by its expression and subcellular location [51]. Thus, the HMGB1 intracellular distribution and release after JEV infection on HBMEC was determined. HMGB1 was predominantly located in the nucleus of uninfected cells at a low level detected by confocal immunofluorescence microscopy (Fig. 1E). At 6 h, 12 h and 24 h, there was a significant increase of HMGB1 at the cytoplasm in HBMEC, which suggested the translocation of HMGB1 from the nucleus to the cytoplasm (Fig. 1E). To further confirm this phenomenon, the protein was extracted from the different cell compartment, the nuclear and the cytoplasmic. And then, the expression of HMGB1 was measured with JEV infection at 1 MOI. The results showed a significant increase of HMGB1 in the cytoplasm after JEV infection, and reached a peak at 12 h, and then gradually slowed-down from 24 h to 48 h. The expression of HMGB1 in the nucleus concomitantly increased at 6 h and slipped to the lower level from 12 h to 48 h after JEV infection (Fig. 1F). It further confirmed that JEV-infection stimulated the cellular expression of HMGB1, and HMGB1 translocated from the nucleus to the cytoplasm. The accumulation of HMGB1 in the cytoplasm could actively trigger the autocrine, which is governed by post-translational modifications. Brefeldin A, an inhibitor of intracellular protein transport, was applied to HBMEC after 12 h postinfection. The intracellular distribution of HMGB1 was observed by the confocal laser scanning microscope. Inhibition of vesicles by Brefeldin A dramatically suppressed HMGB1 release accompanied with an increase of the cytoplasmic HMGB1 at 24 h (Fig. 1G), which suggested that the increased intracellular expression of HMGB1 could be released to the extracellular space.
Taken together, these data suggested that JEV induced HMGB1 upregulation and translocation from the nucleus to the cytoplasm in brain microvascular endothelial cells, and then subsequently released from the cells.
JEV infection induced activation of BMECs and increased adhesion molecules
Brain microvascular endothelial cells are critical in forming BBB and maintaining the barrier function. High expression of adhesion molecules and integrin ligands is necessary for immune cells adhesion to BBB endothelium, which facilitates the immune cell infiltration into the CNS. To identify the underlying mechanism of leukocytes crossing BBB, yeast cells which highly express GFP-LFA-1 (ICAM-1 ligand) were used to detect the interaction with JEV-activated endothelial cells [52]. It was found that more GFP-LFA-1 yeast cells were attached to JEV-infected bEnd.3 monolayer than control cells (Fig. 2A, B), which suggested that JEV caused an upregulation of ICAM-1 on bEnd.3 monolayer. Western blotting also confirmed the upregulation of adhesion molecules on endothelial cells after JEV infection, including VCAM-1, ICAM-2, E-Selectin, VE-Cadherin and beta-catenin (Fig. 2C, D). As expected, recombinant HMGB1(100 ng/ml) induced upregulation of LFA-1 and VLA-4 on mice splenocytes, which act as the receptor of ICAM-1 and VCAM-1 (Fig. 2E, F). Furthermore, we found an upregulation of ICAM-1 and VCAM-1 in the brain, and a corresponding increase of LFA-1 and VLA-4 in the spleen of the JEV-infected mouse model (S. 3A, B, C, D).
Meanwhile, the expression of adhesion molecules on HBMEC was determined with real-time PCR, which showed an increase of ICAM-1, VCAM-1 after JEV infection (S. 2A, B). With the treatment of recombinant HMGB1, an upregulation was also observed in the expression of LFA-1 (CD11a and CD18) and VLA-4 (CD49d and CD29) on human THP-1 cells (S. 2C, D).
All these results suggested that JEV infection induced the upregulation of adhesion molecules on BMEC, and HMGB1 also increased the integrin ligands of immune cells during early JEV infection, which could facilitate the interaction between immune cells and the BBB endothelium.
Extracellular HMGB1 promoted immune cells adhesion to endothelium
Leukocyte-endothelium adhesion is indispensable for immune cell CNS infiltration. 293 T cells were used to overexpress HMGB1, and the supernatant was collected to treat the THP-1 cells. The results showed an increase of THP-1 cell adhesion to JEV-infected HBMEC monolayer with the treatment of supernatant containing HMGB1 (Fig. 3A). Trichostatin A (TSA), a deacetylase inhibitor, have been shown to stimulate the hyperacetylation of HMGB1 as well as histones which facilitates the release of HMGB1 from chromatin. Besides, the secreted HMGB1 (sHMGB1) dramatically increased THP-1 cells adhering to the JEV-activated HBMEC monolayer than that of the uninfected group in vitro (Fig. 3A). Moreover, the adhesion was measured between GFP+-leukocytes from JEV-infected mice and bEnd.3 cell preincubated with JEV-P3 virus. More GFP+ cells were found adhering on bEnd.3 cells primed with alive JEV-P3 virus, but not found on the cells treated with UV-deactivated virus (Fig. 3B). To further study the function of HMGB1, mouse splenocytes were treated with recombinant HMGB1 (rHMGB1), and co-cultured with bEnd.3 monolayers. It was found that rHMGB1 increased the adhesion of Ly6C+CD11b+ monocytes to the JEV-primed bEnd.3 monolayer (Fig. 3C, D). However, there was no significant change of the HMGB1-treated adhering CD3+ T cells and CD19+ B cells to the JEV-primed endothelial monolayer (Fig. 3C, D). Furthermore, JEV infection enhanced the purified Ly6C+ monocyte adhesion to the BMEC monolayer in vitro (Fig. 3E). These results indicated that JEV-activated endothelium promoted the adhesion of immune cells and the presentation of extracellular HMGB1 enhanced monocytes adhering to BMEC monolayer, which may potentiate immune cells crossing BBB entry into the CNS.
Furthermore, a rearrangement in F-actin in BMECs was noticed F-actin in BMEC in the early stage of JEV-infection, while no significant change was found with the treatment of UV-inactivated JEV-P3 (UV-P3) (S. 4A). Notably, there was no significant effect on the endothelium integrity with JEV-treatment in vitro; with no difference between the live virus or UV-inactivated virus (S. 4B). However, JEV-infected mouse brain supernatant (10%) caused fluctuation to endothelial cells integrity in vitro with the dramatic decrease of RB value (from 15 to 50 h) of BMEC monolayer (S. 4B), which suggested that JEV per se does not disrupt the BBB. However, JEV infection induced the inflammatory responses to break down the BBB.
Extracellular HMGB1 facilitated transendothelial migration of JEV-infected monocytes
Data above suggested that the presence of HMGB1 increased the transmigration of THP-1 cells in the JEV-infected monolayer model. To emphasize, the BMEC monolayer was employed to elucidate the role of extracellular HMGB1 in leucocytes migration during JEV infection. We used 10 kD and 70 kD dextrans to visualize the spatially variable, size-dependent permeability of the monolayer (Fig. 4A). Without JEV stimulation, the TEER value, which reflects the integrity of the BBB, were kept stable over 24 hours (>200 Ω×cm2), which implied the integrity of the BBB monolayer in vitro (Fig. 4B). Accompanied with the decrease of TEERs, more Ly6C+CD11b+ monocytes, CD3+ T cells, and CD19+ B cells transmigrated from the upper chamber to the lower chamber in the JEV-infected monolayer models (Fig. 4B, C, D). A high concentration of rHMGB1 exacerbated the destruction of the monolayer models during the early infection (Fig. 4B). Moreover, rHMGB1 increased mice Ly6C+CD11b+ monocytes transmigrating from the upper chamber to the lower chamber, while there was no significant change of CD3+ T cells, and CD19+ B cells compared to JEV-infected group (Fig. 4B, C, D). These results indicated that JEV infection caused BBB fluctuation and increased immune cell CNS infiltration during early infection, which was aggravated by HMGB1. Furthermore, infection-induced HMGB1 enhanced monocyte transendothelial migration, which was based on leukocyte-endothelium adhesion, resulting in BBB breakdown during early infection.
To discover whether transmigrated immune cells act as virus carriers, JEV with EGFP tag (EGFP-JEV) was applied to the bEnd.3 monolayer to visualize the transmigration of immune cells (Fig. 5A). On the contrary, during JEV infection, the TEER value decreased with time, which means that the integrity of the monolayer was compromised, and the permeability accordingly increased (Fig. 5B). More importantly, the decrease of TEER coincided with an increased number of immune cells transmigrating into the lower chamber. The migrated cells, including Ly6C+CD11b+ monocytes, CD3+ T cells and CD19+ B cells, were measured in the absence or presence of the virus. The migratory capability was enhanced in the presence of EGFP-JEV (Fig. 5E). In addition, more EGFP+Ly6C+CD11b+ monocytes infected transmigrated from the upper chamber to the lower chamber than CD3+ T cells with EGFP-JEV infection, and only a few transmigrated CD19+ B cells were detected in the lower chamber (Fig. 5F). These results showed that the disruption of the BBB monolayer model increases transmigration of immune cells, especially Ly6C+CD11b+ monocytes after JEV infection.
These data suggested that extracellular HMGB1 promotes the leukocytes transendothelial migration, especially for monocyte during early infection. Meanwhile, the infected monocytes, T cells and B cells carried JEV participating in transendothelial migration, which acts as “Trojan horse”, may enhance JEV neuroinvasion and aggravate JE.
Transmigration of JEV-infected immune cells correlated with the onset of JE in mice
The correlation between JEV-infected transmigrated splenocytes and the JE incidence in vivo needs further confirmation. Hence, Ly6C+ monocytes, CD3+ T cells and CD19+ B cells were purified by flow cytometry, which were exposed with JEV at MOI of 1. There was a high replication of JEV in splenocytes at 24 h (Fig. 6A). Western blot demonstrated that there was inclusive JEV inside the isolated splenocytes (Fig. 6B). Then, these cells were incubated with the anti-JEV serum to neutralize the non-specific adhesive viruses and injected into the brain of healthy adult mice at 1×105 cells respectively. Furthermore, the initial onset of JE was much earlier in the intracranial injection of JEV-infected Ly6C+ monocyte and CD3+ T cells than the injection of JEV-infected CD19+ B cells (Fig. 6C). No doubt, all the mice delivered JEV-infected immune cells resulted in JE ultimately, which were further supported by the detection of JEV in the brain by immunofluorescence (Fig. 6D, E, F). Moreover, there was a positive association between the onset time of JE and the quantity of transformed JEV-infected immune cells. These data indicated that the transmigration of JEV-infected immune cells from the circulation to the brain were precisely associated with JE onset in the early stage.
The natural infection route was also mimicked by JEV footpad injection in C57BL/6 mice. Tissue samples were collected from the cerebrum, olfactory bulb and spinal cord after infection, and the viral loads were determined by real-time PCR. The results showed that JEV tends to accumulate early in the cerebrum than that of olfactory bulbs and spinal cords (S. 6A, B), suggesting that the hematogenous route, rather than the olfactory nerve or long-range retrograde axonal transport, is more feasible in the JEV neuroinvasion. To further confirm that the hematogenous route is one of those pathways for JEV spreading into the CNS; Immunofluorescence was conducted to determine the JEV distribution in the different neuronal tissues, including the cerebrum, olfactory bulb, and spinal cord. A high count of the JEV virus existed in cerebrum in comparison, to the fewer count entered the CNS via the olfactory bulb or retrograded axonal transportation (S. 6D). These results demonstrated that cerebrum was the primary target of the JEV and the hematogenous route was one of the major pathways in the JEV neuroinvasion.
Together, these data indicated that the hematogenous pathway could be utilized by JEV, while the transmigration of JEV-infected monocytes, T cells and B cells were relevant to the onset of JE in mice.