Identification of Host Cytosolic Sensors and Bacterial Factors Regulating the Type I Interferon Response to Legionella pneumophila (original) (raw)
Figure 5
L. pneumophila DNA and RNA stimulate type I IFN production in macrophages.
(A) Purified genomic DNA and RNA from L. pneumophila induces type I interferon independently of MyD88 and Trif. Bone marrow derived _Myd88_−/−_Trif_−/− macrophages were stimulated by transfection of 3.3 µg/ml purified L. pneumophila DNA, L. pneumophila RNA, pA:T (DNA), and pI:C (RNA). Nucleic acids were treated with DNase and/or RNase A before transfection. Macrophage supernatants were harvested 8 hours post stimulation and analyzed for IFNβ levels by L929-ISRE luciferase bioassay. IFNβ production by DNase-treated L. pneumophila RNA was statistically significantly higher compared to DNase-treated L. pneumophila DNA (**, p<0.005). In addition, RNase A-treated _L. pneumophila_ RNA produced statistically significant lower levels of IFNβ (***, p<0.0001, Student's t-test) than _L. pneumophila_ RNA and RNase-treated _L. pneumophila_ DNA. (B) Genomic _L. pneumophila_ DNA does not induce type I interferon in a _Ips-1_-dependent manner. _Ips-1_−/− and heterozygous littermate bone marrow derived macrophages were stimulated by transfection of 1.0 µg/ml pA:T and purified genomic _L. pneumophila_ DNA. Macrophages were infected with Δ_flaA L. pneumophila_ at an MOI of 1. Sendai virus (SeV) was overlaid onto _Ips-1_−/− and heterozygous littermate macrophages. Transcriptional activation of _Ifnb_ was determined by quantitative RT-PCR as described in Figure 1. (C) The viral RNA sensor _Mda5_ is not required for induction of type I interferon by _L. pneumophila_ DNA. WT (C57BL/6) and _Mda5_−/− bone marrow derived macrophages were stimulated by transfection of 1.0 µg/ml pA:T and purified genomic _L. pneumophila_ DNA. Sendai virus (SeV) and Theiler's virus (TMEV) were overlaid onto WT and _Mda5_−/− macrophages. Quantitative RT-PCR was used to determine _Ifnb_ gene expression. (D) Non-CpG containing DNA (pA:T) does not induce _Ips-1-_dependent _Ifnb_ at all concentrations tested. _Ips-1_−/− and heterozygous littermate bone marrow derived macrophages were stimulated with a titration of pA:T by transfection of 10, 1.0, 0.1, 0.01 µg/ml pA:T. The difference between _Ips-1_+/− and _Ips-1_−/− macrophages transfected with pA:T was not statistically significant (ns, p>0.1, Student's t-test). Sendai virus (SeV) was overlaid onto _Ips-1_−/− and heterozygous littermate macrophages (**, p<0.005). Cell supernatants were collected 8 hours post stimulation/infection. Induction of type I interferon was determined by L929-ISRE luc bioassay. Units are relative light units (RLU). (E) Genomic _L. pneumophila_ DNA induces type I interferon independently of _Ips-1_ at all concentrations tested. _Ips-1_−/− and heterozygous littermate bone marrow derived macrophages were stimulated with a titration of purified genomic _L. pneumophila_ DNA by transfection of 10, 1.0, 0.1, 0.01 µg/ml _L. pneumophila_ DNA. No statistically significant difference was found between _Ips-1_+/− and _Ips-1_−/− macrophages transfected with genomic _L. pneumophila_ DNA (ns, p>0.1, Student's t-test). Sendai virus (SeV) was overlaid onto _Ips-1_−/− and heterozygous littermate controls (**, p<0.005). Macrophage supernatants were collected 8 hours post stimulation/infection. Type I interferon levels were determined by L929-ISRE luc bioassay, units are relative light units (RLU).