We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

ABSTRACT

CD40͞CD40L interaction is essential for multiple biological events in T dependent humoral immune responses, including B cell survival and proliferation, germinal center and memory B cell formation, and antibody isotype switching and affinity maturation. By using high-density microarrays, we examined gene expression in primary mouse B lymphocytes after multiple time points of CD40L stimulation. In addition to genes involved in cell survival and growth, which are also induced by other mitogens such as lipopolysaccharide, CD40L specifically activated genes involved in germinal center formation and T cell costimulatory molecules that facilitate T dependent humoral immunity. Next, by examining the roles of individual CD40-activated signal transduction pathways, we dissected the overall CD40-mediated response into genes independently regulated by the individual pathways or collectively by all pathways. We also found that gene down-regulation is a significant part of the overall response and that the p38 pathway plays an important role in this process, whereas the NF-B pathway is important for the up-regulation of primary response genes. Our finding of overlapping independent control of gene expression modules by different pathways suggests, in principle, that distinct biological behaviors that depend on distinct gene expression subsets can be manipulated by targeting specific signaling pathways. C D40 is a member of the pleiotropic tumor necrosis factor receptor (TNFR) superfamily, which also includes such functionally diverse molecules as CD27, CD30, OX40, RANK, LT-␤R, the p75 low-affinity nerve growth factor receptor, Fas, and members of the death receptor family as well as TNFR1 and TNFR2 (1). CD40 stimulation activates B cells and promotes various aspects of a functional humoral immune response, including enhancement of survival and proliferation (2). Like many TNFR family members, CD40 activates the JNK͞SAPK and NF-B pathways (3, 4). Both of these pathways involve serine͞threonine kinases that regulate gene expression through activation of AP1 and Rel transcription factors, respectively. Another stress-responsive pathway that has also been reported to be activated by CD40 (5) is the p38 kinase pathway, which leads to the phosphorylation and activation of transcription factors such as ATF2 (6). Previous work has also linked CD40 to the activation of the extracellular signal-regulated kinase (ERK)͞mitogen-activated protein kinase (MAPK) pathway (7).In addition to serine͞threonine kinases, a link between CD40 signaling and tyrosine kinase activation has been suggested (8).One pathway shown to be activated by CD40 and to play an important role in CD40 signaling is that initiated by PI-3 kinase (PI-3K) (8-10). PI-3K phosphorylates membrane phospholipids (11), which can recruit and activate pleckstrin homology domaincontaining molecules such as the kinase protein kinase B. This kinase may then regulate the activity of forkhead-related transcription factors (12, 13).In each of the above pathways, one of the major final ...

No abstract