Stereotyped and specific gene expression programs in human innate immune responses to bacteria

Abstract

The innate immune response is crucial for defense against microbial pathogens. To investigate the molecular choreography of this response, we carried out a systematic examination of the gene expression program in human peripheral blood mononuclear cells responding to bacteria and bacterial products. We found a remarkably stereotyped program of gene expression induced by bacterial lipopolysaccharide and diverse killed bacteria. An intricately choreographed expression program devoted to communication between cells was a prominent feature of the response. Other features suggested a molecular program for commitment of antigen-presenting cells to antigens captured in the context of bacterial infection. Despite the striking similarities, there were qualitative and quantitative differences in the responses to different bacteria. Modulation of this host-response program by bacterial virulence mechanisms was an important source of variation in the response to different bacteria.

Figure 1

Stereotyped host responses to diverse heat-killed bacteria. PBMCs obtained by apheresis from a single healthy donor were treated with B. pertussis LPS (BpLPS; 1 μg/ml), and heat-killed B. pertussis virulent laboratory strain 338 (Bp338), B. pertussis Minnesota 1 clinical isolate (BpMinn1), E. coli clinical isolate, and S. aureus clinical isolates 1 and 2, each at a ratio of ≈1 microbial cell to 1 human cell, and ionomycin (1 μM) plus PMA (25 ng/ml). For the mock time course, PBMCs were treated with sterile liquid media. (a) Genes displayed are those exhibiting at least 2.5-fold change in level of expression from baseline (t = 0) in at least 4 (of 64) experiments. The expression pattern of the corresponding 920 cDNAs is displayed in hierarchical cluster format. Each row represents a single array element and each column, a separate experimental mRNA sample. Experiments are organized by increasing time within each time course as indicated by the key at the bottom. Each expression measurement represents the ratio of fluorescence from the hybridized experimental sample to the reference sample, and is displayed as relative to the averaged zero time point. Missing or excluded data are represented by gray squares. The gray vertical bar identifies a cluster of genes whose expression pattern varies between individuals, as discussed in the text. (b and c) The common induction (b) and common repression (c) clusters, which are expanded portions of the larger cluster (a). A blue/yellow version of this figure is published as Fig. 10 in the supporting information on the PNAS web site.

Figure 2

Host responses to dose escalation. PBMCs isolated from an apheresis sample from a second healthy donor were treated with a dose titration of B. pertussis LPS at 0.01 μg/ml, 0.1 μg/ml, and 1 μg/ml, a dose titration of heat-killed Bp338 [0.004 bacteria per human cell (1×) to 4 bacteria per human cell (1000×)], and a dose titration of heat-killed S. aureus isolate 1 [0.002 bacteria per human cell (1×) to 2 bacteria per human cell (1000×)]. Cells were harvested at 0, 0.5, 2, 4, 6, and 12 hr (100× time courses also have 1-hr and 24-hr time points). Data were filtered as in Fig. 1, and the resulting 463 genes are displayed as a six-node self-organizing map (38, 39). The common induction, common repression, and donor variant clusters are again marked. A blue/yellow version of this figure is published as Fig. 11 in the supporting information on the PNAS web site.

Figure 3

Dose–response patterns reveal discrimination. The modeled expression profile of CD64 is shown in response to B. pertussis (a) and S. aureus (b) treatments as an example (surfaces fitted by using four independent measurements). The x axis represents time; the y axis, exposure dose; and the z axis, the expression level (log₂) of the zero-transformed data. The surface represents the least-squares fit of the expression data to the model. The correlation of fitted to observed responses was 0.83. (c) The subtraction of the two expression surfaces, Δ(d, t). (d) The same fitted response difference as in c, but in the form of a two-dimensional color image and contour plot, in which a red to blue color scale represents high to low absolute difference, respectively. Further examples are shown in Fig. 8 in the supporting information.

Figure 4

B. pertussis toxins modify the host response. (a) PBMCs isolated from whole blood from a third healthy donor were treated with live (≈20 bacteria per human cell) and heat-killed (≈20 bacteria per human cell) Bp338 from the same bacterial culture. Data were collected at 0, 0.5, 2, 4, 6, and 12 hr. Data were selected as in previous figures and for each gene, and the Euclidean distance between responses to the two treatment conditions was calculated. Genes with Euclidean distances greater than 2 (≈400 cDNAs), are displayed in hierarchical cluster format. The 0.5-hr time point is not displayed in the cluster because of lower data quality. The names of selected genes are displayed next to the cluster. (b) The average of eight independent measurements of TNFα expression during treatment with both live and killed Bp338 are displayed with error bars representing the SEM. (c) The average induction ratio of the TNFα expression measurements are displayed for PBMCs treated with live and killed B. pertussis (corresponding to b), and for U937 cells, treated with isogenic mutants of B. pertussis that lack toxins that influence levels of intracellular cAMP. Error bars represent the SEM of measurements made by independent TNFα cDNAs (n = 8 for PBMC, n = 3 for U937). A blue/yellow version of this figure is published as Fig. 12 in the supporting information on the PNAS web site.