Insufficiently Defined Genetic Background Confounds Phenotypes in Transgenic Studies As Exemplified by Malaria Infection in Tlr9 Knockout Mice

The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO) and knock-in mice, has become an established tool to study gene function in many animal models for human diseases . However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS) malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP) analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT) control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential

The homeobox protein MSX2 interacts with tax oncoproteins and represses their transactivation activity.

Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to immortalize primary cells in vitro. To gain insight into the molecular pathways mediating the activities of this important gene, we identified cellular proteins interacting with Tax. By means of a two-hybrid approach, we show that Tax specifically interacts with MSX2, a general repressor of gene expression. GST pull-down experiments and co-immunoprecipitation assays further confirmed binding specificity. Furthermore, the N-terminal residues 1-79 of MSX2 are required for binding, whereas the C-terminal residues 201-267 of MSX2 do not play a critical role. Whereas the oncogenic potential of Tax in primary cells was only slightly affected by overexpression of MSX2, the other function of Tax, namely LTR-dependent transcriptional activation, was inhibited by MSX2 in human HeLa and bovine B-lymphoblastoid (BL3) cell lines. This MSX2 repression function can be counteracted by overexpression of transcription factors CREB2 and RAP74. The Tax/MSX2 interplay thus results in repression of viral transcriptional activation possibly acting as a regulatory feedback loop. Importantly, this viral gene silencing is not strictly associated with a concomitant loss of Tax oncogenicity as measured by its ability to immortalize primary cells. And interestingly, MSX2 also interacts with and inhibits the transactivation function of the related Tax1 protein encoded by the Human T-cell leukemia virus type 1 (HTLV-1)

Rat embryo fibroblasts immortalization by bovine leukemia virus Tax protein

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

IFN-gamma, IL-4, IL-10 and IL-12 gene expression in BCG-Leishmania vaccination of Trypanosoma cruzi-infected mice.

We have previously shown that vaccination of BALB/c mice with a combination of BCG plus killed Leishmania promastigotes, applied by the i.p. route 10 and 3 days before Trypanosoma cruzi inoculation, prolonged their survival and decreased their parasitaemia. In the present study we show that the BCG-Leishmania vaccine induced higher levels of circulating IFN-gamma in acute and chronic infection of mice [on day 25 and 40 post-infection (p.i.) respectively], in comparison to unvaccinated animals (PBS-treated). Though the IFN-gamma mRNA content of spleen cells of vaccinated and infected mice (on day 25 p.i.) was similar to that of unvaccinated animals, the BCG-Leishmania vaccine enhanced significantly the production of IFN-gamma by spleen cells stimulated with T. cruzi antigens. This effect was observed to a lower extent in BCG- and Leishmania-treated mice. The BCG-Leishmania vaccine reduced the expression of the IL-10 mRNA of splenocytes as soon as day 12 p.i. before the peak parasitaemia. Such this effect was not observed in BCG- or Leishmania-treated animals. On day 25 p.i. the BCG plus Leishmania- or BCG-treatment of mice abolished the capacity of spleen cells to produce IL-10 in response to T. cruzi antigens. The levels of mIL-4 RNA and protein production were not modified in any group of mice. T. cruzi infection in BCG-Leishmania-vaccined mice stimulated an early and high production of IL-12 transcripts in spleen cells during the acute phase of the infection, that was prolonged during the chronic phase of infection. This effect was weaker or absent in BCG- and Leishmania-treated animals, respectively. These results indicate that the BCG-Leishmania vaccine stimulates the production of IL-12 and IFN-gamma, but inhibits that of IL-10 and is without effect on IL-4 when mice are infected with T. cruzi. This highlights the key role of endogenously produced IFN-gamma, IL-10 and IL-12 in the control of T. cruzi acute and chronic infection in mice and the favorable modulation of their balance by a vaccination combining BCG and Leishmania.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Interferon-γ Orchestrates the Number and Function of Th17 Cells in Experimental Autoimmune Encephalomyelitis

Th17 cells are suggested to be pathogenic in mediating experimental autoimmune encephalomyelitis (EAE), but their relation to interferon (IFN)-γ-producing Th1 cells in vivo is not well understood. We studied the numbers and functions of Th17 cells in CREAE in Biozzi ABH mice, both in peripheral lymphoid organs and in the central nervous system. Th1 and Th17 cells alternated in secondary lymphoid organs and infiltrated into the central nervous system during chronic relapsing experimental autoimmune encephalomyelitis (CREAE). In the absence of IFN-γ the numbers and secretion of Th17 cells was enhanced, whereas exogenous administration of IFN-γ decreased the Th17 cells. In mice with intact IFN-γ genes, in vivo neutralization of interleukin (IL)-17 protected against EAE development by enhancing the number of IFN-γ-producing cells. IFN-γ knockout mice were partially protected by anti-IL-17 antibodies by decreasing cell numbers and production of IL-17. Our findings suggest that, whereas IFN-γ as such is not necessary for EAE development in the mouse, the lack of suppression of Th17 cells by IFN-γ enhances the susceptibility to develop EAE. IFN-γ thus orchestrates the number and function of Th17 cells.status: publishe

Protective effect of a single interleukin-12 (IL-12) predose against the toxicity of subsequent chronic IL-12 in mice: role of cytokines and glucocorticoids.

The mechanisms of interleukin-12 (IL-12) toxicity were studied in mice using a schedule (murine rIL-12, 400 ng/mouse, intraperitoneally [IP] once daily for 5 days) that markedly reduced body weight and food intake. On day 5, IL-12-treated mice had elevated serum and spleen IFN-gamma and tumor necrosis factor (TNF). Serum sTNFR-P75 and corticosterone (CS) were also elevated. IL-12 toxicity was partially prevented by anti-IFN-gamma antibodies or dexamethasone (DEX). A pre-dose of IL-12 (200 ng/mouse on day -14) completely prevented the toxicity of subsequent IL-12. The IL-12 predose also inhibited IL-12-induced IFN-gamma levels, but did not modify IL-12-induced CS, TNF or sTNFR-P75. A protective effect was observed with a predose of lipopolysaccharide (LPS) or murine recombinant (r)IL-10. The protective effect of the IL-12 predose was reduced by coadministration of anti-IFN-gamma, but a predose of murine rIFN-gamma was not protective, suggesting that IFN-gamma is necessary but not sufficient for the protective effect of IL-12. The IL-12 predose specifically protected against IL-12 toxicity and did not modify LPS toxicity. These data indicate that IL-12 can induce tolerance to its own toxicity, probably through a downregulation of IL-12-induced IFN-gamma but independently of endogenous glucocorticoids. IFN-gamma, and possibly IL-10, might be important in this tolerance.Journal Articleinfo:eu-repo/semantics/publishe