CD46-utilizing adenoviruses inhibit C/EBPbeta-dependent expression of proinflammatory cytokines

Abstract

The majority of adenovirus serotypes utilize the coxsackievirus-adenovirus receptor (CAR) for virus-host cell attachment, but subgroup B and subgroup D (adenovirus type 37 [Ad37]) viruses recognize CD46. CD46 is a ubiquitously expressed receptor that serves as a cofactor for the inactivation of the complement components C3b and C4b, and it also serves as a receptor for diverse microbial pathogens. A reported consequence of CD46 engagement is a reduced capability of human immune cells to express interleukin-12 (IL-12), a cytokine involved in both the innate and adaptive immune responses. Studies were thus undertaken to determine whether CD46-utilizing Ads alter the expression of proinflammatory cytokines. Subgroup B (Ad16 and -35) and Ad37, but not Ad2 or -5, significantly reduced IL-12 production by human peripheral blood mononuclear cells stimulated with gamma interferon (IFN-gamma) and lipopolysaccharide. IL-12 mRNA (p35 and p40 subunits) levels as well as other cytokine mRNA levels (IL-1alpha and -beta, IL-1Ra, and IL-6) were decreased upon interaction with CD46-utilizing Ads. Analysis of transcription factor activity required for cytokine expression indicated that CD46-utilizing Ads preferentially inhibited IFN-gamma-induced C/EBPbeta protein expression, consequently reducing its ability to form DNA complexes. Interference with IFN-gamma signaling events by CD46-utilizing Ads, but not CAR-utilizing Ads, reveals a potentially critical difference in the host immune response against distinct Ad vectors, a situation that has implications for gene delivery and vaccine development.

FIG. 1.

Inhibition of IL-12 expression by PBMCs upon infection with CD46-utilizing Ads. Fiber and penton base expression on the CAR-utilizing Ad5.F5 or CD46-utilizing Ad5-pseudotyped vectors Ad5.F37, Ad5.F16, and Ad5.F35 was analyzed by immunoblotting (A). An ELISA was used to measure IL-12 production in the culture supernates of PBMCs following stimulation with IFN-γ plus LPS in the absence or presence of 10⁴ Ad/cell (B). The ability of wild-type Ad37 or UV-inactivated Ad37 to inhibit IL-12 expression was also measured in an ELISA (C). The dose of Ad vector (multiplicity of infection) required to alter IL-12 production in PBMCs was assessed 3 days postinfection in an ELISA (D).

FIG. 2.

Relative efficiency of hematopoietic cell transduction by CAR- and CD46-utilizing Ad vectors. Transduction of unseparated PBMC cultures by different fiber-pseudotyped Ad vectors was determined 48 h postinfection by flow cytometry (green fluorescent protein [GFP]) (A). Transduction of the major hematopoietic cell populations in PBMCs was determined by FACS staining with antibodies specific for T (CD3), B (CD19), monocyte/macrophage (CD14), and NK (CD56) cell surface markers (B). IL-12 production was measured in an ELISA with PBMCs and monocyte-enriched cultures stimulated with IFN-γ plus LPS and infected with either Ad5.F5 or Ad5.F16 (C).

FIG. 3.

CD46-utilizing Ads suppress expression of proinflammatory cytokine mRNA. (A) Detection of cytokine mRNAs was performed by RPA using RNA harvested from cells stimulated with LPS for 3 h following an overnight incubation with IFN-γ in the presence of the indicated Ad vectors or wild-type virions. A multiprobe template set (P) was used to detect mRNAs for IL-12 p35, IL-12 p40, IL-10, IL-1α, IL-1β, IL-1Ra, IL-6, and IFN-γ as well as the control L32 and GAPDH mRNAs. *, mRNAs that were not detected. (B) IL-12 production in culture supernates from the same PBMC prep used for the RPA was measured on day 3 postinfection in an ELISA. The results are representative of two independent experiments from two separate donors.

FIG. 4.

Effect of Ads on NF-κB and AP-1 transcription factor activity. (A) Degradation of IκBα (inset) and the induction of TNF-α production in response to LPS stimulation of PBMCs in the presence of different Ad vectors were analyzed as described in Materials and Methods. (B and C) Cells were harvested 30 min after LPS addition for lκBα analysis by Western blotting, while replicate culture supernatants were collected and assayed for TNF-α. AP-1-driven luciferase reporter gene activity, expressed as relative light units (RLU), was measured in transfected HeLa (B) and THP-1 (C) cells following stimulation with PMA and ionomycin.

FIG. 5.

CD46-utilizing Ads suppress C/EBP DNA binding activity and transcription factor expression. Transcription factor binding to DNA probes following infection with different AD vectors was assessed by EMSA for C/EBPβ (A), NF-κB (B), and AP-1 (C). Nuclear extracts generated from IFN-γ/LPS-stimulated and Ad-infected PBMC cultures were used for these studies. To detect the presence of C/EBPβ in the DNA-protein complexes, samples in lanes 3 (αCβ) were incubated with antibody to C/EBPβ. SS, super shifted species; u, upper band; m, middle band; l, lower band. C/EBPβ protein levels were assessed by Western blot analysis from cells stimulated with IFN-γ/LPS (D) or IFN-γ only (E) in the presence of the various Ad vectors.

FIG. 6.

Model summarizing the events leading to inhibition of IFN-γ signaling and decreased C/EBPβ-mediated cytokine induction by adenoviruses utilizing CD46. Current analyses suggest that LPS signaling to NF-κB and AP-1 are not significantly impacted.