Persistent expression of the full genome of hepatitis C virus in B cells induces spontaneous development of B-cell lymphomas in vivo

Abstract

Extrahepatic manifestations of hepatitis C virus (HCV) infection occur in 40%-70% of HCV-infected patients. B-cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection. The mechanism by which HCV infection of B cells leads to lymphoma remains unclear. Here we established HCV transgenic mice that express the full HCV genome in B cells (RzCD19Cre mice) and observed a 25.0% incidence of diffuse large B-cell non-Hodgkin lymphomas (22.2% in males and 29.6% in females) within 600 days after birth. Expression levels of aspartate aminotransferase and alanine aminotransferase, as well as 32 different cytokines, chemokines and growth factors, were examined. The incidence of B-cell lymphoma was significantly correlated with only the level of soluble interleukin-2 receptor α subunit (sIL-2Rα) in RzCD19Cre mouse serum. All RzCD19Cre mice with substantially elevated serum sIL-2Rα levels (> 1000 pg/mL) developed B-cell lymphomas. Moreover, compared with tissues from control animals, the B-cell lymphoma tissues of RzCD19Cre mice expressed significantly higher levels of IL-2Rα. We show that the expression of HCV in B cells promotes non-Hodgkin-type diffuse B-cell lymphoma, and therefore, the RzCD19Cre mouse is a powerful model to study the mechanisms related to the development of HCV-associated B-cell lymphoma.

Figure 1

Establishment of RzCD19Cre mice. (A) Schematic diagram of the transgene structure comprising the complete HCV genome (HCR6-Rz). HCV genome expression was regulated by the Cre/loxP expression cassette (top diagram). The Cre transgene was located in the CD19 locus (bottom diagram). (B) Expression of HCV core protein in the liver, spleen, and plasma of RzCD19Cre mice was quantified by core ELISA. Data represent the mean ± SD (n = 3). (C) Detection of HCV RNA in PBLs by RT-PCR. Samples that included the RT reaction are indicated by +, and those that did not include the RT reaction are indicated by –. (D) Survival rates of male and female RzCD19Cre mice (males, n = 45; females, n = 40), Rz mice (males, n = 20; females, n = 19), CD19Cre mice (males, n = 16; females, n = 22), and WT mice (males, n = 5; females, n = 10).

Figure 2

Histopathologic analysis of B-cell lymphomas in RzCD19Cre mouse tissues. (A) Histologic analysis of tissues from a normal mouse (i, iii, v; CD19Cre mouse, ID No. 47-4, male) and a B-cell lymphoma from a RzCD19Cre mouse (ii, iv, vi; ID No. 69-5, male). Paraformaldehyde-fixed and paraffin-embedded tumor tissues were stained with hematoxylin and eosin (iii-vi) or immunostained using anti-CD45R (vii; bottom right, inset) and anti-CD3 (viii; bottom right, inset). Also shown is a macroscopic view of the lymphoma from a mesenchymal lymph node (ii, indicated by forceps), which is not visible in the normal mouse (i). Mitotic cells are indicated with arrowheads (vi). Scale bars: 100 μm (iii-iv, vii-viii) and 20 μm (v-vi, insets in vii-viii). (B) Expression of HCV RNA in B-cell lymphomas from RzCD19Cre mice was examined by RT-PCR. The first round of PCR amplification yielded a 123-base pair fragment of HCV cDNA (upper panel), and a second round of PCR amplification yielded a 65-base pair fragment (lower panel). The β-actin mRNA was a control. As an additional control, the first and second rounds of amplification were performed using samples that had not been subjected to reverse transcription. NTC, no-template control. (C) Ig gene rearrangements in the tumors of RzCD19Cre mice. Genomic DNA isolated from B-cell lymphoma tissues of RzCD19Cre mice (ID Nos. 24-1, 24-4, 54-1, 56-5, 69-5, 31-4, 42-4, 43-4) and spleen tissues of a WT mouse (ID No. 21-2) was PCR amplified using primers specific for Vκ-Jκ genes. Amplification of controls was performed using genomic DNA isolated from a GANP transgenic mouse (GANP Tg#3) and in the absence of template DNA (no-template control, NTC). M, DNA ladder marker.

Figure 3

Analysis of serum cytokine levels using a multisuspension array system. The serum concentration levels of IL-2, IL-4, IL-6, IL-10, IL-12(p70), and IFN-γ were measured in RzCD19Cre mice with B-cell lymphomas (B), T-cell lymphomas (T), and other tumors (mammary tumor, sarcoma, and hepatocellular carcinoma) and in tumor-free RzCD19Cre, Rz, CD19Cr,e and WT mice.

Figure 4

Serum titers of AST, ALT and soluble IL-2Rα in transgenic and control mice lacking or harboring B-cell lymphomas. (A-B) The AST (A) and ALT (B) assays were performed on serum samples from tumor-free control mice and the RzCD19Cre, Rz, CD19Cre and WT mice with or without B-cell lymphomas or other tumors. (C) ELISA analysis was performed to determine the sIL-2Rα concentration in serum samples from tumor-free control mice and the RzCD19Cre, Rz, CD19Cre, and WT mice with or without B-cell lymphomas or other tumors. (D) Concentration of soluble IL-2Rα in sera from transgenic (MxCre/CN2-29 or CN2-29) mice with or without B-cell lymphomas (*P < .05).

Figure 5

Levels of IL-2Rα in transgenic and control mice lacking or harboring B-cell lymphomas. The expression level of IL-2Rα in splenocytes and PBLs from CD19Cre and RzCD19Cre mice and in B-cell lymphomas from RzCD19Cre mice was measured by ELISA. IL-2Rα levels per total protein are indicated (picograms per milligram). Data from quadruplicate samples are shown as the mean ± SD (*P < .05).