Adenovirus expression of IL-1 and NF-kappaB inhibitors does not inhibit acute adenoviral-induced brain inflammation, but delays immune system-mediated elimination of transgene expression

Abstract

Despite their ability to provide long-term transgene expression in the central nervous system of naïve hosts, the use of first-generation adenovirus (Ad) vectors for the treatment of chronic neurological disorders is limited by peripheral immunization, which stimulates anti-adenovirus immune responses and causes severe inflammation in the central nervous system (CNS) and elimination of transgene expression. The purpose of this study was to investigate the roles of NF-kappaB and interleukin-1 (IL-1) during inflammatory responses to Ads in the CNS of naïve and preimmunized rats. We assessed activation of macrophages/microglia, up-regulation of MHC I expression, infiltration of leukocytes, and transgene expression following delivery of Ads to the rat striatum. After delivery of increasing doses of adenoviral vectors expressing various anti-inflammatory agents (e.g., NF-kappaB or IL-1 inhibitors) to naïve rats, no reduction in Ad-mediated CNS inflammation was seen 1 week after delivery of Ads, compared to a control Ad.hCMV.beta-galactosidase (RAd.35) virus. We then assessed CNS inflammation and transgene expression at a time when control transgene expression would be completely eliminated, i.e., 1 month post-vector injection into the brain. This would optimize the assessment of an anti-inflammatory agent expressed by an adenoviral vector that could either delay or diminish immune system-mediated elimination of transgene expression. As expected, at 1 month postinfection, control preimmunized rats receiving Ad.mCMV.beta-galactosidase (RAd.36)/saline or RAd.36/Ad.null (RAd.0) showed complete elimination of beta-galactosidase expression in the brain and levels of inflammation comparable to those of naïve animals. However, animals injected with RAd.36 in combination with Ads expressing NF-kappaB or IL-1 inhibitors showed a delayed elimination of beta-galactosidase compared to controls. As predicted, the extended presence of transgene expression was accompanied by increased levels of CNS inflammation. This suggests that blocking NF-kappaB or IL-1 delays, albeit partially, transgene elimination in the presence of a preexisting systemic immune response. Prolonged transgene expression is predicted to extend concurrent brain inflammation, as noted earlier. Taken together these data demonstrate a role for NF-kappaB and IL-1 in immune system-mediated elimination of Ad-mediated CNS transgene expression.

FIG. 1

In vitro inhibition of NF-κB activation or IL-1 signaling. (A) Luciferase activity in pNRE-Luc-transfected HeLa cells following activation of NF-κB by administration of rhIL-1β. (B) Luciferase activity in pNRE-Luc-transfected HeLa cells following infection with RAd.GFP. (C) Inhibition of NF-κB activation in pNRE-Luc-transfected HeLa cells (±rhIL-1β 30 pg/ml) following infection with RAd.IL-1ra, RAd.IL-1RII, RAd.p65RHD, and RAd.IκBα (m.o.i. 100). (D) Inhibition of NF-κB activation in pNRE-Luc-transfected HeLa cells (±rhIL-1β 30 pg/ml) following incubation with conditioned medium from RAd.IL-1ra- and RAd.IL-1RII-infected Cos-7 cells. ns, not significant; **, P < 0.0001 (n = 3).

FIG. 2

ED1 immunoreactivity following delivery of 10⁷, 10⁸, and 10⁹ iu of RAd.35, RAd.IκBα, RAd.p65RHD, RAd.IL-1ra, and RAd.IL-1RII to the rat striatum. Areas of activated macrophages/microglia in the ipsilateral hemisphere are indicated by yellow arrows. Scale bar (bottom left), 1 mm.

FIG. 3

MHC I immunoreactivity following delivery of 10⁷, 10⁸, and 10⁹ iu of RAd.35, RAd.IκBα, RAd.p65RHD, RAd.IL-1ra, and RAd.IL-1RII to the rat striatum. Areas of MHC I-positive cells in the ipsilateral hemisphere are indicated by yellow arrows. Scale bar (bottom left), 1 mm.

FIG. 4

CD43 immunoreactivity following delivery of 10⁷, 10⁸, and 10⁹ iu of RAd.35, RAd.IκBα, RAd.p65RHD, RAd.IL-1ra, and RAd.IL-1RII to the rat striatum. Areas of CD43-positive cells are indicated by yellow arrows. Red arrows indicate perivascular cuffing of leukosialin-positive cells. Scale bar (bottom left), 1 mm.

FIG. 5

Quantification of ED1, MHC I, or CD43 immunoreactivity following delivery of 10⁷, 10⁸, and 10⁹ iu of RAd.35, RAd.IκBα, RAd.p65RHD, RAd.IL-1ra, and RAd.IL-1RII to the rat striatum. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3).

FIG. 6

Transgene expression and inflammation following delivery of RAds to the striatum of adenovirus-sensitized rats. RAds were delivered to the striatum 14 days after intradermal priming and rats were sacrificed after a further 30 days. RAds were delivered at a dose of 1 × 10⁷ iu per virus. (A) Low-power β-galactosidase expression in the ipsilateral hemisphere is shown on the left. High-power composite images show β-galactosidase-positive cells plus ED1-, MHC I-, and CD8-positive cells in serial sections. Scale bars (bottom left two images), both 1 mm. (B) Quantification of ED1, MHC I, and CD8 immunoreactivity 30 days after delivery of saline/RAd.36, RAd.0/RAd.36, RAd.IκBα/RAd.36, and RAd.IL-1ra/RAd.36 to the striatum of immunized or nonimmunized rats. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5).