Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use

Abstract

Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)-based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor-associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.

Fig. 1. A comprehensive siRNA screen of the TLR pathway in human and mouse macrophages

(A) Schematic of signal flow in the TLR pathway from initial signal encoding by TLR receptors, through complexes of TIR adaptor proteins, IRAKs, and TRAFs, to signal transmission through NF-κB, MAPK, and PI3K modules, and then signal decoding through gene transcription, cytokine secretion, and feedback regulation. Right: The 126 gene transcripts targeted in the screen by siRNAs. (B) Workflow for the RNAi screen using six siRNA sequences per gene distributed in separate regions of a 384-well plate. Red circles show examples of siRNA locations for a single gene. The blue region of the plate shows the locations of gene-specific siRNAs, whereas the orange region shows the positions of control siRNAs. Forty-eight to 72 hours after they were reverse-transfected with siRNAs, the mouse (RAW G9) and human (THP1 B5) reporter cell lines were assayed for their responses to simulation with different TLR ligands, and the effects of individual gene perturbations were measured (see fig. S1 and Materials and Methods for further details). (C and D) Calculation of false positive rates (C) and false negative rates (D) from negative and positive control siRNAs, respectively, in the mouse and human macrophage siRNA screens (see Materials and Methods). Data in (C) and (D) are presented as median z-scores from six siRNAs per gene. Individual siRNA scores were averaged from two (C) or three (D) independent experiments (see Materials and Methods for details).

Fig. 2. Effects of siRNA gene perturbations across the human and mouse TLR pathways

(A to G) Analysis of the effects of gene perturbations on TLR signaling. TLR pathway gene perturbation effects on (A) the TNF promoter–driven transcriptional response of the human THP1 cell line to LPS (10 ng/ml), 100 nM P3C, R848 (10 μg/ml), 1 nM P2C, 1 PGN (10 μg/ml), or FLG (10 ng/ml) and (D) on the NF-κB and Tnf promoter–dependent responses of the mouse RAW264.7 cell line to LPS (10 ng/ml), 100 nM P3C, or R848 (10 μg/ml). (B and E) Effects of the siRNA-mediated knockdown of the indicated TLRs on (B) the TNF promoter-dependent transcriptional response of the human THP1 cell line and (E) the NF-κB– and Tnf promoter–dependent responses of the mouse RAW264.7 cell line to the indicated TLR ligands. The top 10 genes with the most substantial effects on (C) the human TNF promoter–dependent response, (F) the mouse NF-κB response, and (G) the mouse Tnf promoter-driven response across all tested TLR ligands. Rankings for individual TLR ligands are shown, with overall gene order determined by the average rank calculated over all ligands (the full 126-gene ranking scores are shown in table S3). Data in (A), (B), (D), and (E) are presented as median z-scores from six siRNAs per gene. Individual siRNA scores were averaged from three (for A and B) or two (for D and E) independent experiments (see Materials and Methods).

Fig. 3. Human and mouse macrophages show both shared and distinct gene product dependencies in TLR signaling

(A) Hierarchical clustering analysis (Pearson uncentered, average linkage) of the TNF/Tnf promoter–driven responses to LPS, P3C, and R848 in human THP1 cells and mouse RAW cells. (B) Average z-scores calculated from the responses to the three TLR ligands shown in (A) were compared between the human and mouse reporter cell lines (see table S4). The z-score differences between the species were overlaid onto the canonical TLR pathway from KEGG. (C) Genes whose perturbation had the most substantial effects on the human and mouse TLR pathways. (D and E) The relative abundances of the mRNAs corresponding to the genes shown in (C) in human THP1 B5 cells (D) or mouse RAW G9 cells (E) after siRNA-mediated knockdown. Data in (D) and (E) are means ± SD from two independent experiments. Data in (A) and (C) are presented as median z-scores from six siRNAs for each gene. Individual siRNA z-scores were averaged from three (for the human TNF-α readout) or two (for the mouse NF-κB and TNF-α readouts) independent experiments (see Materials and Methods).

Fig. 4. IRAK4 is required for human TLR responses, but the human and mouse IRAK4 proteins are not functionally interchangeable

(A and B) Analysis of the relative abundances of IRAK4 mRNA (A) and protein (B) in RAW G9 cells, THP1 B5 cells, and hMDMs transfected with control or IRAK4-specific siRNAs. The relative fractions of IRAK4 protein abundance in cells treated with IRAK4-specific siRNA compared to that in control cells were as follows: 0.30 ± 0.07 for RAW G9 cells (P < 0.001); 0.28 ± 0.10 for THP1 B5 cells (P < 0.01); and 0.49 ± 0.06 for hMDMs (P < 0.01). Statistical analysis was by two-tailed t test. Western blots are representative of three independent experiments. (C) hMDMs transfected with control siRNA or IRAK4-specific siRNA were stimulated with LPS (10 ng/ml), 100 nM P3C, PGN (10 μg/ml), R848 (10 μg/ml), or FLG (10 ng/ml) for 24 hours and the amounts of TNF-α secreted by the cells were measured by ELISA. (D to F) hMDMs isolated from control and IRAK4-deficient patients were left untreated (Unt) or were stimulated with LPS (10 ng/ml), 100 nM P3C, PGN (10 μg/ml), R848 (10 μg/ml), or FLG (10 ng/ml) for 24 hours. The amounts of TNF-α (D), IL-6 (E), and IL-8 (F) secreted by the cells were measured by Bioplex assay. (G) Immortalized bone marrow–derived mouse macrophages (IMM) from IRAK4 KO mice were stably transduced with retrovirus expressing either mouse IRAK4-mCitrine or human IRAK4-mCherry. The cells were then stimulated for 24 hours with LPS (10 ng/ml), 100 nM P3C, PGN (30 μg/ml), or R848 (10 μg/ml) before the amount of TNF-α secreted by the cells was measured by ELISA. (H) IRAK4 KO THP1 cells were stably transduced with retrovirus expressing either human IRAK4-mCherry or mouse IRAK4-mCitrine. The cells were then stimulated for 24 hours with LPS (10 ng/ml) before the amount of TNF-α that they secreted was measured by ELISA. Data are means ± SD of two representative experiments for (A), (C), (G), and (H) or are means ± SD of two independent biological replicates from one set of patient blood samples for (D) to (F). **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed t test.

Fig. 5. Differential characteristics of human and mouse IRAK4 signaling properties

(A and B) Mouse IRAK4 KO IMMs were stably transduced with retrovirus expressing either (A) mouse IRAK4-mCitrine or (B) human IRAK4-mCherry before being treated with LPS (10 ng/ml) for the indicated times. Cell lysates were then subjected to immunoprecipitation (IP) with an antibody against Myd88 and samples were analyzed by Western blotting (IB) with antibody specific for IRAK4. RhoGDI was used as an input control for the Myd88 immunoprecipitation. (C and D) Human IRAK4 KO THP1 cells were stably transduced with retrovirus expressing either (C) mouse IRAK4-mCitrine or (D) human IRAK4-mCherry before being treated with LPS (10 ng/ml) for the indicated times. Cell lysates were then subjected to immunoprecipitation with an antibody specific for Myd88 and analyzed by Western blotting with antibody specific for IRAK4. Myd88 was used as an input control for the immunoprecipitation. (E and F) The nuclear intensity of phosphorylated ATF2 (pATF2) was measured by high-content imaging in either (E) mouse IRAK4 KO IMMs or (F) human IRAK4 KO THP1 cells stably transduced with retrovirus expressing either mouse IRAK4-mCitrine or human IRAK4-mCherry. Cells were left untreated or were stimulated for 20 (IMM) or 60 min (THP1) with 500 nM P3C. Data are shown for the central 98^th percentile of cells. (G) IRAK4 KO IMMs were stably transduced with retrovirus expressing either wild-type (WT) or kinase-deficient (Kin⁻) mouse IRAK4. Cells were stimulated with LPS (10 ng/ml) for 24 hours and the amount of TNF-α secreted was measured by ELISA. (H) IRAK4 KO THP1 cells were stably transduced with retrovirus expressing either WT or kinase-deficient (Kin⁻) human IRAK4. Cells were stimulated with LPS (10 ng/ml) for 24 hours and the amount of TNF-α secreted by the cells was measured by ELISA. Western blots in (A) to (D) are representative of at least two independent experiments. Single-cell data in (E) and (F) are representative of two independent experiments (100 to 500 cells imaged per condition, error bars indicate mean ± 95% CI). Data in (G) and (H) are means ± SD of two independent experiments. **P < 0.01, ***P < 0.001 by Kolmogorov-Smirnov test (for E and F) or by two-tailed t test (for G and H).

Fig. 6. Differences in IRAK1 and IRAK2 protein use in mouse and human TLR responses in primary macrophages

(A and B) Bone marrow-derived macrophages (BMDMs) from WT, IRAK1 KO, and IRAK2 KO mice were stimulated with LPS (10 ng/ml), 100 nM P3C, PGN (30 μg/ml), or R848 (10 μg/ml) for 24 hours before the amounts of TNF-α (A) and IL-6 (B) that were secreted by the cells were measured by ELISA. (C) THP1 cells transfected with control, IRAK1-specific, or IRAK2-specific siRNAs were stimulated with LPS (10 ng/ml) for 24 hours before the amounts of TNF-α that they secreted were measured by ELISA. (D) hMDMs transfected with control, IRAK1-specific, or IRAK2-specific siRNAs were stimulated with LPS (10 ng/ml), 100 nM P3C, PGN (10 μg/ml), R848 (10 μg/ml), or FLG (10 ng/ml) for 24 hours before the amounts of TNF-α that they secreted were measured by ELISA. Data are means ± SD of four (for A and B) or three experiments (for C and D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed t test.

Fig. 7. IRAK1 and IRAK2 have different functionalities in the human and mouse macrophage TLR signaling pathways

(A) The WT and indicated IRAK1 KO and IRAK2 KO THP1 cell lines were analyzed by Western blotting with antibody against IRAK proteins. RhoGDI was used as a loading control. Western blots are representative of two independent experiments. (B) The indicated control and IRAK KO THP1 cell lines were stimulated for 4 hours with LPS (10 ng/ml) before the amounts of TNF-α that they secreted were measured by ELISA. (C and D) The WT and indicated IRAK KO THP1 cell lines (C) and mouse IMMs (D) were stimulated with 500 nM P3C for the indicated times before the relative abundances of phosphorylated p38α MAPK (left) and p65 (right) were measured by phosphoflow cytometry. (E and F) The WT and indicated IRAK KO THP1 cell lines (E) and mouse IMMs (F) were stimulated with 500 nM P3C for the indicated times before the relative abundances of Nfkbiz (left), Tnf (middle), and Il6 (right) mRNAs were measured by qRT-PCR analysis. Data in (B) to (F) are means ± SD of two independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed t test.

Fig. 8. Differential human and mouse IRAK dependencies are reflected in responses to gram-negative bacterial infection and in ligand-induced signaling complexes

(A and B) IMMs from WT mice and the indicated IRAK KO mice (A) and the WT and indicated IRAK KO THP1 cell lines (B) were infected with B. cenocepacia at an MOI of 1. Twenty hours later, the amounts of IL-6 secreted by the cells were measured by ELISA. (C and D) IMMs from IRAK4 KO mice (C) and IRAK4 KO THP1 cells (D) were stably transduced with retrovirus expressing either mouse IRAK4-mCitrine or human IRAK4-mCherry, respectively. The cells were then treated with LPS (10 ng/ml) for the indicated times before cell lysates were subjected to immunoprecipitation (IP) with antibodies against Myd88 and analyzed by Western blotting (IB) with antibodies against either IRAK1 or IRAK2. RhoGDI is shown as an input control for the Myd88 immunoprecipitation. Western blots are representative of three independent experiments. Data in (A) and (B) are means ± SD of two independent experiments. ***P < 0.001 by two-tailed t test.