Therefore, early T-cell apoptosis observed during L. monocytogenes infection appears to hamper bacterial clearance and strategies

to prevent lymphocyte apoptosis may be beneficial for therapy. In support of this notion, SCID and nude mice, which lack T cells, exhibit a severe increase in L. monocytogenes accumulating in the liver [35]. c-FLIP is known to inhibit death receptor-mediated apoptosis but not other cell death pathways [36]. Of note, lymphocytes were depleted upon L. monocytogenes infection in TNF-R1-deficient mice and lpr mice [37], suggesting that these two death receptors are not involved in L. monocytogenes-induced T-cell apoptosis. In contrast, TRAIL-deficient www.selleckchem.com/products/Metformin-hydrochloride(Glucophage).html mice show a similar phenotype as vavFLIPR mice with reduced splenocyte apoptosis and reduced bacterial load [38]. Therefore, c-FLIPR might be important for inhibiting TRAIL-mediated apoptosis during L. monocytogenes infection. Alternatively, TNF-R1 and CD95 but not TRAIL may exert redundant functions during pathogen-induced cell death. Regardless of which death receptor system is crucial during L. monocytogenes CH5424802 infection, approaches, which aim at increasing c-FLIP expression in T cells, will target all death receptor pathways and, thus, might be a promising

therapeutic option. Taken together, endogenous murine c-FLIPR is induced on the protein level upon lymphocyte activation. Strikingly, vavFLIPR mice show better control of L. monocytogenes infection than WT littermates. These data strongly suggest that c-FLIPR is the murine functional counterpart of human c-FLIPS, but differs from viral FLIP. c-FLIPR cDNA was cloned into the vector HS21/45 vav-hCD4 [18] using the SfiI and NotI (New England Biolabs, Ipswich, MA, USA) restriction sites. The transgenic construct was injected into B6C3F1 zygotes and positive mice were identified via PCR on tail biopsies with following primers specific for the SV40 poly A sequence: c-FLIP forward 5′-GCCTGAAGAACATCCACAGAATAG-3′, Poly A reverse 5′-CTCATCAATGTATCTTATCATGTC-3′.

Positive lines were identified PLEKHM2 and backcrossed for more than ten generations to C57BL/6 mice. Expression of the transgene was analyzed via RT PCR and immunoblot (see later). WT littermates were used as control animals. All of the animals were kept under pathogen-free conditions in the animal facilities of the Heinrich-Heine University, Duesseldorf, and the Helmholtz Centre for Infection Research, Braunschweig. C57BL/6 mice were purchased from Harlan Laboratories (Indianapolis, IN, USA) and Charles River (Wilmington, MA, USA). All breeding and experiments were performed in accordance with the guidelines of national and local authorities. Lymphoid organs (thymus, spleen, lymph nodes) were taken from sex-matched vavFLIPR mice and nontransgenic littermate controls. Single cell suspensions were prepared by filtering organ suspensions through a nylon mesh.