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. 2010 Feb 5;397(1):130-8.
doi: 10.1016/j.virol.2009.10.036. Epub 2009 Nov 18.

Reversal of West Nile virus-induced blood-brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor

Saguna Verma  1 , Mukesh Kumar, Ulziijargal Gurjav, Stephanie Lum, Vivek R Nerurkar
Affiliations

Reversal of West Nile virus-induced blood-brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor

Saguna Verma et al. Virology. .

Abstract

Though compromised blood-brain barrier (BBB) is a pathological hallmark of WNV-associated neurological sequelae, underlying mechanisms are unclear. We characterized the expression of matrix metalloproteinases (MMP) in WNV-infected human brain microvascular endothelial cells (HBMVE) and human brain cortical astrocytes (HBCA), components of BBB and their role in BBB disruption. Expression of multiple MMPs was significantly induced in WNV-infected HBCA cells. Naïve HBMVE cells incubated with the supernatant from WNV-infected HBCA cells demonstrated loss of tight junction proteins, which were rescued in the presence of MMP inhibitor, GM6001. Further, supernatant from WNV-infected HBCA cells compromised the in vitro BBB model integrity. Our data suggest astrocytes as one of the sources of MMP in the brain, which mediates BBB disruption allowing unrestricted entry of immune cells into the brain, thereby contributing to WNV neuropathogenesis. Because of the unavailability of WNV antivirals and vaccines, use of MMP inhibitors as an adjunct therapy to ameliorate WNV disease progression is warranted.

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Figures

Fig. 1
Fig. 1
WNV replication kinetics in HBCA cells. (A) WNV titers in infected HBCA and HBMVE cells supernatants collected at various time points after infection were determined by plaque assay using Vero cells. Viral titers, expressed as plaque forming units (PFU)/mL of supernatant represent mean ± SD of data obtained from at least three independent experiments. (B) HBCA cells grown and fixed on coverslips at day 2 after infection were stained with anti-GFAP antibody or anti-WNV envelope antibody, and counterstained with DAPI (blue). (i) mock-infected HBCA cells demonstrate GFAP staining in the cytoplasm, (ii) WNV-infected HBCA cells demonstrate robust virus staining in the cytoplasm, and (iii) WNV-infected HBCA cells stained with secondary antibody, negative control, did not show any immunostaining.
Fig. 2
Fig. 2
WNV differentially modulates expression of MMP family genes in HBCA cells. qRT-PCR was conducted on cDNA templates from WNV- and mock-infected HBCA cells harvested from days 1 to 4 after infection to determine the fold-change of (A) MMP-1, -2, -3 and -9 and (B) TIMP-1, -2 and -3 genes expression. Change in the levels of each gene was first normalized to the GAPDH gene and then the fold-change in infected cells as compared to corresponding control cells was calculated. Data represents mean of at least four independent experiments conducted in duplicate.
Fig. 3
Fig. 3
WNV induces protein expression of MMPs in HBCA cells. (A) Confluent primary HBCA cells were infected with WNV at MOI-5 and MMP-9 expression was determined using immunofluorescence. (i) Mock-infected HBCA cells demonstrated very faint and diffused MMP-9 immunoreactivity (green), and (ii) Mock-infected cells incubated without MMP-9 antibody did not show any staining. (iii and iv) marked increase in MMP-9 staining (green) was observed at day 3 in WNV-infected HBCA cells. MMP-9 increase was evident in both, WNV-infected (red) as well as neighboring un-infected HBCA cells. The images shown are representative results of three independent experiments. Scale bar represents 10 μm at 20× magnification. (B) Western blot analysis of MMP-1, -3 and -9. Cellular proteins extracted from WNV- and mock-infected HBCA cells were separated on PAGE, transferred onto PVDF membranes and immunoblotted with antibodies specific to MMP-1, -3 and -9. The membranes were stripped and re-probed with anti-β-actin and the bands were detected by the chemiluminescence method. (C) Quantitative analysis of western blots represented as percentage increase in MMPs compared to respective control HBCA cells at the corresponding time point. Mean comparisons were based upon extrapolated CI for three data points. *p<0.05, compared to corresponding controls. C, mock- and I, WNV-infected HBCA cells.
Fig. 4
Fig. 4
WNV infection induces enzymatic activities of MMP-3 and -9 in HBCA cells. (A) The conditioned media from WNV- and mock-infected HBCA cells were analyzed for MMP -2/9 and -3 enzymes activities by gelatin and casein zymography, respectively. (B) The densitometric analysis demonstrates significant increase in MMP-3 and -9 activities in infected HBCA cells. (C) Total MMP-9 levels were measured by ELISA in the supernatant from mock- and WNV-infected HBCA cells at day 3 after infection. Mean comparisons were based upon extrapolated CI for three data points. Values represent mean ± S.D. *p<0.005, **p<0.05 compared to corresponding control cells.
Fig. 5
Fig. 5
WNV-induced MMPs lead to degradation of TJP of HBMVE cells. Confluent primary HBMVE cells were either infected with WNV or incubated with the supernatant from mock- or WNV-infected HBCA cells for six hours and TJPs expression was visualized using confocal immunofluorescence microscopy. (A) ZO-1 staining in (i and ii) naïve HBMVE cells characterized by staining at cell borders, did not change as a result of (iii and iv) WNV infection or (v and vi) after incubation of naïve HBMVE cells with supernatant media from mock-infected HBCA cells collected at days 3 and 4 after infection. Whereas there was a distinct loss of ZO-1 staining in naïve HBMVE cells incubated with supernatant media from WNV-infected HBCA cells at days 3 and 4 after infection (vii and viii). The treatment of supernatant media from mock-infected HBCA cells with GM6001 had no effect on ZO-1 staining (ix and x), however disrupted ZO-1 staining was markedly reversed in naïve HBMVE cells incubated with GM6001-treated supernatant from WNV-infected HBCA cells at days 3 and 4 after infection (xi and xii). The inset depicts, MMP-9 activity in (a) supernatant of infected HBCA cells and (b) in the presence of GM6001. (B) Immunostaining of claudin-1 in HBMVE cells by WNV-induced MMPs. Claudin-1 immunoreactivity which was characterized by intracellular and junctional pattern in (i and ii) naïve HBMVE cells, increased at days 3 and 4 after (iii and iv) WNV infection. (v and vi) incubation of naïve HBMVE cells with supernatant from mock-infected HBCA cells for six hours did not alter the claudin-1 staining pattern, however (vii and viii) incubation with supernatant from WNV-infected HBCA cells at days 3 and 4 after infection, disrupted the claudin-1 staining. Effect of GM6001 treatment on supernatant from (ix and x) mock-infected and (xi and xii) WNV-infected HBCA cells on the claudin-1 immunostaining. The images shown are representative results of at least three independent experiments in duplicate. Scale bar represents 20 μm at 63× magnification.
Fig. 6
Fig. 6
Supernatant from WNV-infected HBCA cells augment the paracellular permeability of the BBB model. At day 8 post seeding, the BBB models were incubated with supernatant from mock- and WNV-infected HBCA cells or synthetic MMP-9 enzyme as positive control for six hours and (A) The integrity of the BBB models was determined by measuring the TEER before and after the incubation. TEER values, presented as Ω/cm2, demonstrated a significant decrease only in the BBB model incubated with either supernatant from WNV-infected HBCA cells or synthetic MMP-9 enzyme. Presence of GM6001 rescued the BBB permeability. (B) FITC-dextran permeability assay of the BBB models. After six hours of incubation, the percentage of FITC-dextran that crossed the BBB models as compared to control BBB models did not vary significantly in BBB models incubated with supernatant from mock-infected HBCA cells. BBB models incubated with supernatant from WNV-infected HBCA cells demonstrated significant increase in the FITC-dextran transmigration and this increase was reversed in presence of GM6001. The data is representative of at least three independent experiments in duplicate. *p<0.005 percent change as compared to control inserts and **p<0.05 percent change of GM6001-treated supernatants as compared to corresponding BBB models without GM6001 treatment.
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References

    1. Afonso PV, Ozden S, Prevost MC, Schmitt C, Seilhean D, Weksler B, Couraud PO, Gessain A, Romero IA, Ceccaldi PE. Human blood-brain barrier disruption by retroviral-infected lymphocytes: role of myosin light chain kinase in endothelial tight-junction disorganization. J Immunol. 2007;179(4):2576–83. - PubMed
    1. Amantea D, Russo R, Gliozzi M, Fratto V, Berliocchi L, Bagetta G, Bernardi G, Corasaniti MT. Early upregulation of matrix metalloproteinases following reperfusion triggers neuroinflammatory mediators in brain ischemia in rat. Int Rev Neurobiol. 2007;82:149–69. - PubMed
    1. Anthony DC, Ferguson B, Matyzak MK, Miller KM, Esiri MM, Perry VH. Differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke. Neuropathol Appl Neurobiol. 1997;23(5):406–15. - PubMed
    1. Arjona A, Foellmer HG, Town T, Leng L, McDonald C, Wang T, Wong SJ, Montgomery RR, Fikrig E, Bucala R. Abrogation of macrophage migration inhibitory factor decreases West Nile virus lethality by limiting viral neuroinvasion. J Clin Invest. 2007a;117(10):3059–66. - PMC - PubMed
    1. Arjona A, Ledizet M, Anthony K, Bonafe N, Modis Y, Town T, Fikrig E. West Nile Virus Envelope Protein Inhibits dsRNA-Induced Innate Immune Responses. J Immunol. 2007b;179(12):8403–9. - PubMed

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