Parasite-mediated nuclear factor kappaB regulation in lymphoproliferation caused by Theileria parva infection

Abstract

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor kappaB (NFkappaB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFkappaB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IkappaB molecules which normally sequester NFkappaB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IkappaBalpha. However, IkappaBalpha reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFkappaB-mediated positive feedback loop which restores cytoplasmic IkappaBalpha. In contrast, T. parva mediated continuous degradation of IkappaBbeta resulting in persistently low cytoplasmic IkappaBbeta levels. Normal IkappaBbeta levels were only restored following T. parva killing, indicating that viable parasites are required for IkappaBbeta degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IkappaB degradation and consequent enhanced expression of NFkappaB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IkappaB levels or NFkappaB activation, indicating that the parasite subverts the normal IkappaB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.

Figure 1

T. parva infection induces increased levels of IκBα and phosphorylated IκBα and decreased total IκBβ levels. T. parva-infected T cells were left untreated (−) or treated daily with BW720C to clear the parasite (+). Lysates were prepared from 10⁷ cells collected following 1, 2, or 3 days in culture and IκBα and IκBβ levels determined by immunoblot analysis. *P-IκBα designates phosphorylated IκBα.

Figure 2

T. parva infection induces nuclear expression of IκBβ normally restricted to the cytoplasm. Nuclear and cytoplasmic extracts were prepared from T. parva infected cells (TpM) or the same cloned cell line cleared of the parasite (BW720C), either unstimulated (−) or stimulated with Con A (+). Nuclear and cytoplasmic distribution of the IκBβ molecular size forms was determined by immunoblot analysis using an anti-IκBβ specific antibody. The position of the constitutive nuclear form of IκBβ (∗) and the constitutive cytoplasmic phosphorylated (labeled 1), and unphosphorylated (labeled 2) forms are designated in the left margin.

Figure 3

T. parva infection does not abrogate IκBα and IκBβ binding to p65 RelA and p50/p65 RelA heterodimers. The specific antibodies used in immunoprecipitation are designated above each lane in the figures. The immunoprecipitated complexes were then analyzed using immunoblots to identify coprecipitated components. (a) IκBα and IκBβ bind p65 RelA. Immunoblot analysis of IκBα and IκBβ immunoprecipitates with anti-p65 RelA antibodies detected bound p65 RelA. The immunoblot with antibody against Grb2 was used as a negative control for nonspecific complex formation. (b) p50 is bound to both IκBα and p65 RelA. Immunoblot analysis of p65 and IκBα immunoprecipitates with anti-p50 antibodies detected bound p50. The p50 and Grb2 immunoprecipitates were included as positive and negative controls, respectively. (c) p65 RelA is bound to both IκBβ and p50. Immunoblot analysis of p50 and IκBβ immunoprecipitates with anti-p65 antibodies detected bound p65. The p65 immunoprecipitate was included as a positive control. (d) IκBβ is bound to both p50 and p65 RelA. Immunoblot analysis of p50 and p65 RelA immunoprecipitates with anti-IκBβ antibodies detected bound IκBβ. The IκBβ immunoprecipitate was included as a positive control.

Figure 4

T. parva infection mediates continuous degradation of IκBα and IκBβ. T. parva infected T cells (−) or the same cloned cell line cleared of the parasite using BW720C (+) were treated with cycloheximide (CHX) to inhibit protein synthesis. The duration of CHX treatment in hr (h) prior to cell harvest is designated at the top of each figure. Okadaic acid (OA), which blocks the serine/threonine phosphatase inhibitors types PP1 and PP2A, was included as a positive control for IκBα degradation. IκBα (a) and IκBβ levels (b) were determined by immunoblot analysis.

Figure 5

T. parva-mediated continuous degradation of IκBα and IκBβ is inhibitable by PDTC but not by the antioxidant NAc. T. parva-infected cells were left untreated or treated with PDTC or NAc for 5 hr and then incubated with cycloheximide (CHX). IκB levels were determined by immunoblot analysis.

Figure 6

T. parva-mediated NFκB dependent gene expression is inhibitable by PDTC but not NAc. Results are expressed as the percentage of CAT activity in untreated HIV-CAT transfected cells. Results include SDs.