This study provides novel insights into the macrophage response to GBS organisms. First, MyD88 is the key TLR adapter protein for the formation of inflammatory cytokines and chemokines by macrophages, whereas the TLR adapter proteins MAL/TIRAP, TRIF, and TRAM are dispensable for this response. Second, downstream of MyD88, MAPK p38 controls at least two modes of transcriptional activation, c-Fos/AP-1 and EGR-1, which essentially mediate cytokine formation. In contrast, Elk-1, which had previously been suggested as an essential signaling intermediate in TLR signaling, is dispensable for GBS-induced cytokine formation.
The discrete delineation of TLR adapter usage by whole streptococci seems important, since available studies of the TLR adapters (other than MyD88) are largely limited to purified bacterial toxins. The absolute dependence of GBS inflammatory signaling on MyD88 but not any other well-defined TLR adapter protein is consistent with the notion that GBS organisms activate TNF and IL-6 in macrophages via a pathway clearly distinct from that engaged by TLR2 and TLR4 (14
). With respect to the essential role of MyD88 alone, GBS mimics CpG DNA. Indeed, GBS has been reported to interact with the CpG receptor TLR9. However, the overall cytokine response to GBS is largely TLR9 independent, whereas it is entirely dependent on MyD88 (reference 14
and unpublished observations). It seems that this GBS-induced MyD88-dependent pathway, which does not involve any of the single TLRs commonly associated with gram-positive bacteria, is a common signaling route for many gram-positive bacteria, since recent data found similar modes of signal activation in response to Streptococcus pyogenes
, Streptococcus pneumoniae
, and Bacillus anthracis
Beyond the formation of inflammatory cytokines, MyD88 was essential for GBS-induced RANTES formation. This was intriguing, since, to the best of our knowledge, the role of MyD88 in the context of RANTES formation by gram-positive organisms had not been examined previously to this study. In contrast, RANTES formation in response to gram-negative bacteria occurs independently of MyD88 through engagement of TRIF and TRAM (7
Why MyD88 functionally interacts with MAL/TIRAP, TRIF, and TRAM for some ligands but not others is currently not understood. Docking studies suggest that TLRs, MyD88, and MAL/TIRAP form heterotetrameric complexes. Specifically, MAL and MyD88 bind to different regions in TLRs and interact at a third, nonoverlapping site (8
). It is unlikely that MyD88 can mediate TNF formation independently of other adapter proteins in the cases of GBS, flagellin, and CpG but requires interaction with one adapter protein (plus MAL/TIRAP) for the cytokine response to bacterial lipoproteins and four adapter proteins (plus MAL/TIRAP, TRIF, and TRAM) for the response to LPS. Thus, it is tempting to speculate on the existence of at least one further, currently unknown TLR adapter protein which serves as a signaling partner of MyD88 in the inflammatory response to GBS organisms. The current paradigm holds that, subsequently to TLR-MyD88 interaction, IL-1 receptor-associated kinase 1 (IRAK-1), IRAK-2, IRAK-4, and IRAK-M are recruited. Phosphorylation of IRAK-1 by IRAK-4 results in the dissociation of IRAK-1 from the receptor complex and interaction with TNF receptor-associated factor 6 (TRAF-6) through its COOH terminus. TRAF-6 activates the evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which in turn activates downstream MAPK cascades (20
). However, several technical issues, like the early lethality of the knockout mice, currently preclude absolute certainty as to the role of ECSIT between TRAF-6 and MAPK kinase kinases (33
). MAPKs are a family of serine/threonine protein kinases. Among these, p38 can be regarded as a signaling relay that funnels various upstream signals and allows downstream diversification depending on the immediate cellular environment. p38 itself is activated through phosphorylation on both threonine and tyrosine residues at a Thr-Gly-Tyr dual phosphorylation motif in the kinase catalytic domain. Of the four known p38 isoforms α, β, γ, and δ, only the first two are significantly inhibited by SB203580, a pyridinyl-imidazole that exerts its relatively high kinase specificity by binding to the ATP binding pocket of p38 (28
). Since SB203580 inhibits GBS-induced cytokine formation, p38 isoforms α and β are critical for GBS-induced inflammatory signaling, and the γ and δ isoforms cannot compensate for the inhibition of α and β.
Notably, the signaling pathways of p38 and other MAPKs are interrelated on several levels. As an example, Elk-1 can be phosphorylated by both p38 and JNK. However, our data indicate that, in the absence of p38 activity, JNK is not sufficient for GBS-induced Elk-1 phosphorylation. JNK and p38 signaling events furthermore converge on the level of AP-1 transcription factors, which are homo- or heterodimers of the Jun family, Fos proteins (c-Fos, FosB, Fra-1, and Fra-2), and members of subfamilies of the activating transcription factor family (ATF2, LRF1/ATF3, B-ATF, JDP1, and JDP2). While c-Jun proteins are mainly activated by the JNK, the Fos and ATF proteins are phosphorylated downstream of p38 and other MAPKs. In our hands, AP-1 activation is similarly reduced by the p38 inhibitor SB203580 and the JNK inhibitor SP600125 (Fig. and unpublished observations).
Two transcription factor heteromers (c-Jun/c-Fos and c-Jun/ATF2) have previously been shown in more detail to have particularly high affinities for preferentially binding to the AP-1 binding site within the TNF promoter. Hence, c-Jun and therefore JNK activity is essential for the activation of AP-1 as a transcription factor for tnf
. Cross-inhibition of JNK substrates by p38 inhibition is unlikely to be relevant in this context, since JNK (19
) but not p38 (current study) mediates transactivation of NF-κB in response to GBS. It seems notable that GBS differs from IL-1β in this context, since the latter engages p38 for the transactivation of the NF-κB subunit p65 (through amino-terminal phosphorylation) (16
). Accordingly, GBS engages a specific MyD88-dependent pathway that comprises the MAPKs p38 and JNK as essential inflammatory intermediates with overlapping but clearly distinct regulatory functions.
Several publications suggest that the microbial engagement of TLRs leads to the activation of Elk-1 (5
) and that this event in turn mediates transcriptional activation of TNF (36
). However, conclusive data on the cytokine response to TLR agonists, such as data generated for Elk−/−
mice, were not available. We report here that GBS and LPS induced normal amounts of TNF in Elk−/−
macrophages. Accordingly, Elk-1 is dispensable for the responses to both stimuli. Furthermore, and in contrast to the upstream kinase p38, Elk-1 was not essential for GBS phagocytosis (19
). We conclude that signaling events upstream of or parallel to Elk-1 mediate the p38 effect on transcriptional activation of tnf
Several lines of evidence suggest that, next to Elk-1, neither AP-1 nor NF-κB accounts for the antimicrobial effects of p38 activation. First, JNK mediates AP-1 activation, but its inhibition does not interfere with phagocytosis or bacterial killing. Furthermore, GBS-induced NF-κB activation is not dependent on p38 activation. Therefore, antimicrobial activity and TNF formation are independently regulated events downstream of p38. The Rab proteins are attractive intermediates in the p38-dependent antimicrobial pathway, since they modulate the endocytic traffic downstream of p38 (2
In summary, MyD88 and p38 are parts of a distinct and functionally pivotal pathway activated by streptococci. AP-1 and Egr-1 inhabit key downstream positions in this highly inflammatory process (Fig. ). Since p38 exerts desirable antimicrobial functions, interventional strategies that aim to target cytokine formation need to modulate molecules further downstream in the signaling cascade.
FIG. 7. The signaling pathway MyD88 p38 EGR-1/AP-1 is essential for GBS-induced transcriptional activation of TNF. In response to GBS, MyD88 serves as a master signaling molecule, which essentially mediates p38 phosphorylation. In turn, p38 (more ...)