NOD2, RIP2 and IRF5 Play a Critical Role in the Type I Interferon Response to Mycobacterium tuberculosis (original) (raw)
Figure 7
Mtb-induced type I IFN response is Tbk1-dependent and mediated through both Irf3 and Irf5.
A. _M. tuberculosis_-induced type I IFN response is Tbk1-dependent and only partially mediated through Irf3. BMDM derived from wt, irf3−/− and tbk1−/−tnfr1−/− mice and littermate controls were infected with virulent Mtb H37Rv (Rv) at an MOI of 10 for 4 h. RNA was harvested and IFNβ mRNA level was quantified using real time PCR. Gene expression is reported as copy number per 1,000 copies of β-actin. Samples were assayed in triplicate; error bars represent the standard deviation. The experiment shown is representative of at least three. B. Co-expression of RIP2 and IRF5 stimulate IFNβ luciferase reporter activity. HEK293T cells were co-transfected with IFNβ-luciferase reporter plasmid (40 ng) together with the indicated concentrations of MyD88, IRF5 and RIP2 expression plasmids. Luciferase activity was measured 24 h later using Dual Luciferase reporter assay system (Promega). Renilla luciferase gene (40 ng) was co-transfected and used as an internal control. Each experiment was repeated three times. Data are expressed as mean±s.d. of three replicates. C. Co-expression of RIP2 and IRF3 does not stimulate IFNβ luciferase reporter activity. HEK293T cells were co-transfected with IFNβ-luciferase reporter plasmid (40 ng) together with the indicated concentrations of IRF5, IRF3 and RIP2 expression plasmids. Luciferase activity was measured 24 h later using Dual Luciferase reporter assay system (Promega). The Renilla luciferase gene (40 ng) was co-transfected and used as an internal control. Each experiment was repeated three times. Data are expressed as mean±s.d. of three replicates. D. Irf5 is required for an optimal type I IFN response upon Mtb infection. BMDM from irf5−/− or control littermates were infected with virulent Mtb H37Rv (Rv) at an MOI of 10, or with Listeria monocytogenes (Lm) strain 10403S (MOI 10) for 4 hours. RNA was harvested and IFNβ mRNA level was quantified by real time-PCR. IFNβ mRNA levels are reported as copy number per 1,000 copies of β-actin. Samples were assayed in triplicate; error bars represent standard deviation. Data shown is representative of at least three independent experiments.