Human macrophage activation programs induced by bacterial pathogens

Abstract

Understanding the response of innate immune cells to pathogens may provide insights to host defenses and the tactics used by pathogens to circumvent these defenses. We used DNA microarrays to explore the responses of human macrophages to a variety of bacteria. Macrophages responded to a broad range of bacteria with a robust, shared pattern of gene expression. The shared response includes genes encoding receptors, signal transduction molecules, and transcription factors. This shared activation program transforms the macrophage into a cell primed to interact with its environment and to mount an immune response. Further study revealed that the activation program is induced by bacterial components that are Toll-like receptor agonists, including lipopolysaccharide, lipoteichoic acid, muramyl dipeptide, and heat shock proteins. Pathogen-specific responses were also apparent in the macrophage expression profiles. Analysis of Mycobacterium tuberculosis-specific responses revealed inhibition of interleukin-12 production, suggesting one means by which this organism survives host defenses. These results improve our understanding of macrophage defenses, provide insights into mechanisms of pathogenesis, and suggest targets for therapeutic intervention.

Figures

Figure 1

Macrophage activation program elicited in response to a variety of bacteria and bacterial components. (A) The macrophage activation program. Macrophage gene expression was measured at 1, 2, 6, 12, and 24 h after the introduction of bacteria or latex beads. One hundred ninety-one genes met criteria for significant changes (see Materials and Methods) on exposure to six of eight bacteria studied. The genes are ordered along the y axis by category, with the degree of change indicated by color intensity in the color bar. The display has been described in detail elsewhere (8). Minimal gene expression changes occurred in macrophages cultured in media over 24 h (Fig. 6, which is published as supporting information on the PNAS web site). Gene expression changes attributable to individual donors were excluded from this display (Fig. 7, which is published as supporting information on the PNAS web site). (B) A subset of bacterial components elicits much of the activation program (induced genes shown only). Control experiments demonstrated that residual endotoxin contamination of the recombinant hsps was not responsible for the extent of cytokine expression induced by hsps (Fig. 8, which is published as supporting information on the PNAS web site). The idea that the hsps were acting via TLR was supported by the observation that a specific TLR-4 antagonist, Rhodobacter sphaeroides lipid A (32), blocked cytokine production by macrophages exposed to E. coli, LPS, and hsp70 (Fig. 9, which is published as supporting information on the PNAS web site).

Figure 2

Differences in expression profiles distinguish between bacteria. (A) Differential gene expression in macrophages exposed to M. tuberculosis, E. coli, or S. aureus. The difference index represents how expression levels induced by the three bacteria differ from the average expression level induced by all bacteria studied: large values are assigned to genes whose expression levels exhibit the greatest differences in specific bacterial infections. Multiple time course experiments were used (M. tuberculosis, two repeats; E. coli, three repeats; and S. aureus, two repeats). For every gene within each profile, the responses of macrophages to each of the bacterial species in question were compared with the average response to all bacterial species studied. Statistically significant differences were identified by using Student's t test (P < 0.005). The difference index was calculated as follows: D.I. =  log2 (fold-change of gene X in infection A) − log2 (avg fold-change of gene X in all infections) . (B) M. tuberculosis induced lower levels of macrophage IL-12 and IL-15 gene expression than the average expression measured across all data sets. The average fold-change values observed after exposure to M. tuberculosis (▴) are displayed for the time course. In comparison, the average fold-change values across all data sets was significantly greater (■).

Figure 3

M. tuberculosis induced repression of IL-12 protein production. (A) M. tuberculosis induced less IL-12 protein than E. coli and suppressed IL-12 production induced by E. coli. IL-12 p40 accumulation in supernatants of macrophages 12 h after exposure to either M. tuberculosis, E. coli, or both bacteria was measured by ELISA (R & D Systems). Additional experiments demonstrated that this response was reproducible and not donor dependent. TNF-α ELISA was performed on supernatants from the same cultures after 4 h (peak expression of this cytokine preceding the wash step). Values are means ± SD of replicate ELISA measurements. Neither IL-12 nor TNF-α was detected in macrophages treated with media alone.