Unexpected role for the immunoproteasome subunit LMP2 in antiviral humoral and innate immune responses

Abstract

Proteasomes are multisubunit proteases that initiate degradation of many Ags presented by MHC class I molecules. Vertebrates express alternate forms of each of the three catalytic proteasome subunits: standard subunits, and immunosubunits, which are constitutively expressed by APCs and are induced in other cell types by exposure to cytokines. The assembly of mixed proteasomes containing standard subunits and immunosubunits is regulated in a tissue specific manner. In this study, we report that the presence of mixed proteasomes in immune cells in LMP2(-/-) mice compromises multiple components that contribute to the generation of antiviral Ab responses, including splenic B cell numbers, survival and function of adoptively transferred B cells, Th cell function, and dendritic cell secretion of IL-6, TNF-alpha, IL-1beta, and type I IFNs. These defects did not result from compromised overall protein degradation, rather they were associated with altered NF-kappaB activity. These findings demonstrate an important role for immunoproteasomes in immune cell function beyond their contribution to Ag processing.

FIGURE 1

B cells express immunoproteasomes. Immunoproteasome subunit expression was determined in C57BL/6 purified B cells. LMP2^−/−, LMP7^−/−MECL1^−/−, and MECL1^−/− purified B cells were used as negative controls.

FIGURE 2

LMP2^−/− mice have reduced amount of B cells. A, Flow cytometric analysis was completed on splenocytes isolated from C57BL/6 and immunoproteasome deficient mice. Cell numbers of CD4⁺ T cells (CD4+, TCRβ+), CD8⁺ T cells (CD8+, TCRβ+), and B cells (CD19+, B220+) were determined by multiplying the splenic frequencies by the total number of cells from each spleen. Cells isolated from bone marrow (B) or spleens (C) were phenotyped using markers of B cell development (B220, IgD, and CD43). For all experiments, n = 3 mice per group. Mean ± SEM is shown. Data are representative of two independent experiments. *p < 0.05.

FIGURE 3

LMP2^−/− B cells contain mixed proteasomes. Fractions of LMP2^−/− B cell lysate were isolated using a sucrose gradient and immunoblotting was performed. The first fraction contained the lightest material and the sixth fraction contained the heaviest.

FIGURE 4

Mixed proteasomes negatively affect antiviral Ab responses. Following vaccination with IAV, Ab responses were determined by HAI titers (A) and by ELISA (B, C; n = 10–12 mice per group). Data are representative of four independent experiments. Shown is mean ± SEM. *p < 0.05.

FIGURE 5

LMP2^−/− lymphocytes are defective. CD11c/CD11b depleted C57BL/6 or LMP2^−/− splenocytes were transferred into sublethally irradiated C57BL/6 or LMP2^−/− recipient mice. The mice were then vaccinated with IAV and Ab titers were determined by HAI (A, B) and ELISA (C–F; n = 3 mice per group). Data are mean ± SEM.

FIGURE 6

Immunoproteasome expression in B and CD4⁺ T cells is required for the generation of IAV Ab responses. Purified B and CD4⁺ T cells from C57BL/6 or LMP2^−/− mice were transferred into sublethally irradiated C57BL/6 or LMP2^−/− recipient mice. The mice were vaccinated with IAV and Ab titers were determined by HAI (A, B) and ELISA (C–F; n = 3 mice per group; n = 2 at some late time points). Data are mean ± SEM and are representative of two independent experiments.

FIGURE 7

LMP2^−/− lymphocytes proliferate normally but may have defects in survival. Purified B cells (A) and CD4⁺ T cells (B) were stimulated with LPS or Con A for a total of 3 d, and proliferation was determined by [³H] incorporation (n = 3 samples per group). Data are representative of two independent experiments. C, C57BL/6 or LMP2^−/− splenocytes were transferred into partially irradiated CD45.1⁺ mice; 7 d later, the number of surviving lymphocytes was determined by flow cytometry (n = 3 mice per group). Splenic frequencies were multiplied by total cell numbers to determine how many transferred cells were present in each spleen. Data are mean ± SEM and are representative of two independent experiments.

FIGURE 8

Mixed proteasomes negatively affect innate cytokine production. A, CD45.1⁺ splenocytes were transferred into partially irradiated C57BL/6 or LMP2^−/− mice; 7 d later, the number of CD45.1⁺ cells was determined by flow cytometry (n = 3 mice per group). Splenic frequencies were multiplied by total cell numbers to determine how many transferred cells were present in each spleen. Data are representative of two independent experiments. B, Numbers of cells per spleen were determined by flow cytometry as described in Fig. 2 (n = 3 mice per group). Data are representative of two independent experiments. Bone marrow-derived DCs were infected with IAV, and supernatant was tested for levels of cytokines by ELISA (C) or by Luminex technology (D–F; n = 3 samples per group). ELISA data are representative of three independent experiments, and Luminex data are representative of two independent experiments. All data are mean ± SEM.

FIGURE 9

Mixed proteasomes do not alter protein degradation. A, Purified B cells were stimulated with LPS, and levels of polyubiquitinated proteins were monitored by immunoblotting over time. Levels of polyubiquitinated proteins were normalized to actin. B, The experiment was repeated in triplicate, and levels of polyubiquitinated proteins were quantified using an Odyssey imaging system.

FIGURE 10

Mixed proteasomes dysregulate NF-κB signaling. Purified B cells were stimulated with LPS, and levels of IκB α were monitored by immunoblotting over time. Levels of IκB α were quantified and normalized to actin. Data are representative of three independent experiments.