Genome-Wide RNAi Screen in IFN-γ-Treated Human Macrophages Identifies Genes Mediating Resistance to the Intracellular Pathogen Francisella tularensis

Interferon-gamma (IFN-γ) inhibits intracellular replication of Francisella tularensis in human monocyte-derived macrophages (HMDM) and in mice, but the mechanisms of this protective effect are poorly characterized. We used genome-wide RNA interference (RNAi) screening in the human macrophage cell line THP-1 to identify genes that mediate the beneficial effects of IFN-γ on F. tularensis infection. A primary screen identified ∼200 replicated candidate genes. These were prioritized according to mRNA expression in IFN-γ-primed and F. tularensis-challenged macrophages. A panel of 20 top hits was further assessed by re-testing using individual shRNAs or siRNAs in THP-1 cells, HMDMs and primary human lung macrophages. Six of eight validated genes tested were also found to confer resistance to Listeria monocytogenes infection, suggesting a broadly shared host gene program for intracellular pathogens. The F. tularensis-validated hits included ‘druggable’ targets such as TNFRSF9, which encodes CD137. Treating HMDM with a blocking antibody to CD137 confirmed a beneficial role of CD137 in macrophage clearance of F. tularensis. These studies reveal a number of important mediators of IFN-γ activated host defense against intracellular pathogens, and implicate CD137 as a potential therapeutic target and regulator of macrophage interactions with Francisella tularensis

CD137 modulates clearance of <i>F. tularensis</i>.

<p>Adherent HMDMs were pre-treated with anti-CD137 antibody or isotype control prior to infection with <i>F. tularensis</i> GFP-LVS, and analyzed for bacterial load at 2 h and 24 h by flow cytometry, scanning cytometry and CFU assay. (A) Flow cytometry fluorescence histograms for a single experiment at 2 and 24 h. Gray histograms represent uninfected cells. Green histograms represent cells infected with GFP-LVS. (B) Representative scanning cytometry images of GFP-LVS-containing HMDMs at 24 h post infection. (C–E) Quantitative results at 2 h and 24 h for (C) flow cytometry (N = 4), (D) scanning cytometry (N = 5), and (E) CFU assay (N = 3). *<i>P</i><0.05, **<i>P</i><0.01, ** <i>P</i><0.001 (symbols directly above bars indicate significance relative to untreated control; symbols between bars indicate significance between the two bars indicated by horizontal lines).</p

Network Analysis.

<p>(A) Top hits involved in ten significant networks identified by Ingenuity pathway analysis. The numbers surrounding the pie charts represent the number of the top hits involved in each network. (B) 16 hits (highlighted in green) are in a network linked to a category designated as ‘hematological disease, immunological disease, cell death’, which includes TNFRSF9/CD137. Connection lines ⁃⁃, represent direct interactions; ----, represent indirect interactions.</p

Top 10 genes with increased expression in all 3 human macrophage types.

<p>Top 10 genes with increased expression in all 3 human macrophage types.</p

Effect of TNFRSF9 and SERPINI1 knockdown on Listeria growth in THP-1 macrophages.

<p>Flow cytometry analysis of THP-1 macrophages (transduced with TNFRSF9 and SERPINI1 shRNA or non-target control shRNA) 20 hours following infection with GFP-<i>Listeria</i> (MOI = 150∶1). Y-axis indicates mean green fluorescence index (GFL). *<i>P</i><0.05 compared to Non-target shRNA+IFN-γ group.</p

Validation of TNFRSF9 and SERPINI1 genes using virulent <i>F. tularensis</i>.

<p>(A) IFN-γ-activated THP-1 cells inhibited SchuS4 growth at 4 h and 24 h infection. IFN-γ, 40 U/ml (SchuS4, MOI = 30∶1). (B) CFU assay of TNFRSF9 and SERPINI1 shRNA knockdown THP-1 macrophages infected with SchuS4 strain. Control (THP-1 cells transduced with negative control lentiviral shRNA), TNFRSF9 knockdown cells (clone D) and SERPINI1 knockdown cells (clone1) were infected with virulent SchuS4 strain for 24 h (MOI = 30∶1). *<i>P</i><0.05 compared to +IFN-γ group.</p

Summary of functional validation results for top candidates.

<p><b>Note</b>: Following shRNA or siRNA knockdown of the target genes, IFN-γ pretreated THP-1 and HMDM cells were infected with <i>F. tularensis</i> GFP-LVS. After 24 hours, the intracellular GFP-LVS was measured by flow cytometry and compared to control. Results are summarized as ++, GFP greater than control by ≥40%; +, GFP greater than control by ≥20%; −, GFP greater than control by <20%. NT, not tested.</p

Protocol used for genome-wide RNAi screen: sorting for high GFP-positives.

<p>Step 1: construct THP-1 library cells by transduction of human THP-1 macrophage cell line with GeneNet™ Human 50 k shRNA library. Step 2: differentiate THP-1 library cells with PMA, and activate the cells with IFN-γ. Step 3: infect the library cells with GFP-labeled <i>F. tularensis</i> live vaccine strain (GFP-LVS). Step 4: harvest cells at indicated time points, and fix the cells for biosafety prior to flow cytometry. Step 5: FACS sorting to isolate small population of cells with the highest green fluorescence (containing the most GFP-LVS). Step 6: identify siRNA targets and genes by PCR, cloning and DNA sequencing.</p

Validation of TNFRSF9 and SERPINI1 gene expression and function with gene knockdown by siRNA.

<p>(A) Comparison of TNFRSF9 gene expression using qPCR assay after treatment of THP-1 cells with 100 nM specific or control siRNA for 48 hours and 72 hours. (B) Flow cytometry analysis of siRNA TNFRSF9 knockdown in THP-1 cell with <i>F. tularensis</i> GFP-LVS infection for 24 h. THP-1 macrophages were treated with 50 nM or100 nM of siRNA for 72 h, and pre-activated with IFN-γ (40 U/ml) overnight prior to GFP-LVS infection (MOI = 35∶1). Y-axis represents mean green fluorescence level (GFL). *<i>P</i><0.05 compared to control Ctr_siRNA+IFN-γ group. (C) Flow cytometry analysis of GFP-LVS infection for 24 h in HMDMs following 100 nM siRNA knockdown of TNFRSF9 and SERPINI1, respectively. *<i>P</i><0.05 compared to control Ctr_siRNA+IFN-γ group.</p