Distinct TLR- and NLR-Mediated Transcriptional Responses to an Intracellular Pathogen

How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNβ. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNβ was reduced in nod2−/− macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNβ induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens

<i>HAC1</i> Promoter Regulation Is Required to Survive Stress

<div><p>(A) Determination of Hac1p levels during either ER stress alone or during both ER stress and temperature shift. WT cells were either grown at 30 °C and treated with DTT (lanes 1–4) or grown at 23 °C and simultaneously shifted to 37 °C and treated with DTT (lanes 5–8). Protein lysates were prepared, and protein levels were analyzed by Western blot analysis. The relative Hac1p/Pgk1p ratio is normalized to the DTT-treated sample (lane 4).</p> <p>(B) Characterization of <i>HAC1</i> expression in strain used to approximate basal <i>HAC1</i> expression. Cells expressing <i>HAC1</i> from the endogenous promoter (lanes 1–4) or the <i>ADH1</i> promoter (lanes 5–8) were grown at 30 °C in synthetic medium supplemented with inositol and shifted to synthetic medium lacking inositol simultaneous with the addition of tunicamycin.</p> <p>(C) Reduced viability of strains unable to express <i>HAC1</i> at elevated levels. The strains described in (B) were plated in serial dilutions (left to right) on synthetic medium lacking inositol (“−ino”) and synthetic medium lacking inositol and containing tunicamycin (“−ino +TM”).</p></div

Activation of the <i>HAC1</i> Promoter Controls Increase in <i>HAC1</i> mRNA Abundance

<div><p>(A) Analysis of <i>HAC1</i> promoter activity during bipartite stress conditions. Δ<i>hac1</i> cells containing either a construct restoring <i>HAC1</i> expression (lanes 1–4) or a construct expressing <i>GFP</i> driven by the <i>HAC1</i> promoter (lanes 5–8) were grown at 23 °C and shifted to 37 °C concurrent with addition of DTT as indicated.</p> <p>(B) Determination of mRNA half-life during <i>HAC1</i>-mRNA-inducing conditions. <i>polII^ts</i> cells were grown at 23 °C and were shifted to 37 °C either in the absence (open symbols) or presence (filled symbols) of DTT. <i>HAC1</i> mRNA abundance (squares) and <i>ACT1</i> mRNA abundance (circles) are normalized to the abundance of the PolIII transcript <i>SCR1</i>.</p></div

ER-Distal Secretory Stress Boosts <i>HAC1</i> mRNA Abundance

<div><p>(A) Determination of <i>HAC1</i> mRNA abundance during the UPR. The UPR was induced in WT cells by addition of either 6 mM DTT (lanes 1–4) or 1 μg/ml tunicamycin (lanes 5–8) for the times indicated. Total RNA was harvested at the indicated intervals, and the relative abundance of <i>HAC1</i> and <i>ACT1</i> mRNAs was analyzed by Northern blot analysis (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020235#s4" target="_blank">Materials and Methods</a>). Splicing was calculated at the ratio of spliced <i>(HAC1ⁱ)</i> to total <i>(HAC1ⁱ + HAC1^u)</i> mRNA.</p> <p>(B) Determination of <i>HAC1</i> mRNA abundance during ER-distal secretory stress. WT, <i>sec12–1, sec14–3,</i> and <i>sec1–1</i> strains were grown at 23 °C and shifted to 37 °C.</p> <p>(C) Determination of <i>HAC1</i> mRNA abundance during ER-proximal secretory stress. WT, <i>sec14–3, sec61–101, sec62–101,</i> and <i>sec63–201</i> strains were grown at 23 °C and shifted to 37 °C.</p></div

<i>HAC1</i> mRNA Induction Requires a Bipartite Signal and Is <i>IRE1</i>-Independent

<div><p>(A) Determination of <i>HAC1</i> mRNA abundance during ER stress and temperature shift. WT cells were grown at 23 °C and shifted to 37 °C (lanes 1–4 and 9–12) or kept constant at 30 °C (lanes 5–8). DTT was added as indicated (lanes 5–8 and 9–12).</p> <p>(B) Determination of <i>HAC1</i> mRNA abundance during ER stress and inositol deprivation. WT cells were grown at 30 °C in synthetic medium supplemented with inositol and shifted to synthetic medium lacking inositol (lanes 1–4 and 9–12), or continuously grown in medium supplemented with inositol (lanes 5–8). Tunicamycin was added to a final concentration of 1 μg/ml as indicated (lanes 5–8 and 9–12).</p> <p>(C) Distinction between heat shock response and <i>HAC1-</i>mRNA-inducing conditions. WT (lanes 1–4 and 9–12) and <i>HSF1^c</i> (lanes 5–8) strains were grown at 23 °C and shifted to 37 °C (lanes 1–4 and 5–8) or continuously grown at 37 °C (lanes 9–12), and DTT added as indicated.</p> <p>(D) Analysis of <i>IRE1</i> pathway for a role in <i>HAC1</i> mRNA induction. Δ<i>ire1</i> cells were grown at 23 °C and shifted to 37 °C (lanes 1–4 and 9–12) or continuously grown at 30 °C (lanes 5–8), and DTT was added as indicated (lanes 5–8 and 9–12). Note that in Δ<i>ire1</i> cells, <i>HAC1</i> mRNA is modestly induced in response to DTT alone (lanes 5–8). This observation is indicative of feedback regulation, whereby a block in the UPR induces the I/T signal.</p></div

A Schematic of the Circuitry of the UPR

<p>The model depicts the circuitry of the UPR (red) and the S-UPR (blue). Transcriptional control of <i>HAC1</i> is indicated by an icon representing a rheostat affording gain control of the UPR; Ire1p-dependent <i>HAC1^u</i> mRNA splicing is indicated by an icon representing an on/off switch. The I/T and UP signals in the S-UPR are integrated by an AND gate (semicircle, top right), i.e., both conditions must be met to propagate the S-UPR signal. The putative UMF may collaborate with Hac1p to control transcription of UPR target genes (shown) and also be involved in regulating <i>HAC1</i> transcription (not shown); alternatively, different factors may be involved. The collaboration of Hac1p and UMF is indicated by the diamond-shaped icon, which integrates the information coming from both Hac1p and UMF concentration and activity.</p