BACKGROUND
streptococcus pneumoniae is an important cause of severe invasive infections, including pneumonia, bacteremia, and meningitis  <cit> .
nasopharyngeal carriage of s. pneumoniae is common among children, but in most cases bacterial invasion into the lungs or bloodstream is prevented by local host defenses in the upper airways  <cit> .
however, in a minority of individuals, pneumocooci gain access to deeper structures of the body and thereby cause invasive disease  <cit> , which are almost uniformly fatal if not treated with antibiotics.
even in the presence of appropriate treatment, invasive pneumococcal disease bears a relatively poor prognosis  <cit> .
pneumococci are known to elicit a very potent inflammatory response  <cit> , a key component of which is the production of cytokines and chemokines, which participate in the elimination of bacteria but may also result in undesirable excessive immunological responses, which may be harmful to the host  <cit> .
the cytokine tumor necrosis factor -α is produced by macrophages and dendritic cells as a primary response to infections and tissue damage, and is constitutively expressed in several autoimmune diseases  <cit> .
tnf-α plays an important role in activation and recruitment of leukocytes to inflamed tissue  <cit> , and has been demonstrated to be involved in the host-defense against a number of important human pathogens, including s. pneumoniae  <cit> .
however, tnf-α is also associated with excessive inflammation and immunopathology in infections and autoimmune diseases, and specifically, tnf-a has been suggested to be involved in breakage of the blood-brain barrier during development of hematogenous pneumococcal meningitis  <cit> .
production of tnf-α is regulated both at the level of transcription, mrna stability, and translation  <cit> .
the regulation of mrna stability and translation is mediated largely through adenylate uridylate -rich elements s present in the 3'-untranslated region  of mrna, which are targeted by are-binding proteins able to affect mrna stability and translation  <cit> .
the importance of ares in regulation of tnf-α production is evidenced by spontaneous development of chronic inflammatory arthritis and crohn's-like inflammatory bowel disease in transgenic mice devoid of ares in mrna encoding tnf-α  <cit> .
the inflammatory response to infection is triggered by pattern recognition receptors s, which recognize evolutionarily conserved pathogen-associated molecular patterns s and activate intracellular signaling, thereby up-regulating expression of genes with inflammatory activities, including tnf-α  <cit> .
the toll-like receptors s constitute an important class of prrs, and tlr <dig>   <dig>  and  <dig> have been shown to recognize pneumococcal pamps  <cit> .
the vast majority of tlr downstream signaling proceeds through the cytoplasmic adaptor protein myd <dig>  which is central for activation of the transcription factor nuclear factor  κb, and the mitogen-activated protein kinase  pathway, being involved in both transcriptional and post-transcriptional responses  <cit> .
however, several studies suggest that pneumococcal recognition and pathogenesis may be at least partly independent of tlr signaling  <cit> .
another class of prrs involved in recognition of s. pneumoniae is the nucleotide-binding oligomerization domain  proteins, which are intracellular prrs  <cit> .
the nod <dig> protein recognizing muramyl dipeptide has been reported to be important for activation of nf-κb during infection with s. pneumoniae  <cit> .
in this study, we have investigated the role of mrna stabilization in tnf-α production induced by s. pneunomiae, including the signaling mechanisms involved.
here we report that live s. pneunomiae strongly stabilizes tnf-α mrna by a mechanism dependent on the ares in the 3'-utr of the mrna.
importantly, increased tnf-α mrna stability induced by s. pneunomiae was not mediated by tlrs but was dependent on the p <dig> mapk, which was activated through a pathway involving transforming growth factor-activated kinase   <dig> and mapk kinase   <dig>
RESULTS
s. pneumoniae induces tnf-α production and stabilizes tnf-α mrna through ares in the 3'-utr
given the important role of the inflammatory response in the pathogenesis of streptococcal diseases  <cit> , we were interested in studying mechanisms involved in expression of inflammatory cytokines during infection with s. pneumoniae.
in a first set of experiments, we measured expression of tnf-α in murine macrophages after treatment with s. pneumoniae, n. meningitidis or pure ligands for tlr <dig> , tlr <dig> , and tlr <dig> .
as expected, cells responded to addition of bacteria or tlr ligands by secreting high amounts of tnf-α .
lps has been reported to stimulate tnf-α expression by both activating gene transcription, stabilizing the tnf-α mrna, and supporting translation  <cit> .
to examine whether s. pneumoniae stabilized tnf-α mrna, we treated cells with actinomycin d, which prevents de novo transcription of mrna, and stimulated the macrophages with s. pneumoniae, n. meningitidis or lps.
at the indicated time-points post-stimulation, rna was harvested and tnf-α mrna levels were determined by real-time pcr.
as shown in fig.
1b, addition of actinomycin d led to a rapid decline in the levels of remaining tnf-α mrna in cells receiving mock treatment.
however, treatment with s. pneumoniae profoundly augmented the half-life of tnf-α mrna .
lps and n. meningitidis also stabilized the tnf-α messenger but not to the same extent as s. pneumoniae.
to examine whether the ares of the tnf-α mrna were required for the observed effect, we turned to a cell system derived from raw <dig>  cells, consisting of two cell lines stably expressing a reporter gene , transcribed from a constitutive promoter.
in one of the cell lines  the 3'-utr of the mrna encoding the reporter gene is derived from the wild-type tnf-α mrna, and in the other cell line  the ares of the tnf-α 3'-utr are mutated.
when the raw tnf-α 3'-utr au+ cell line was incubated in the presence of s. pneumoniae, a very strong increase in cat levels was observed , whereas lps and also n. meningitidis affected accumulation of cat protein only to a more modest extent.
interestingly, the effect of s. pneumoniae on accumulation of cat was largely abrogated in the raw tnf-α 3'-utr au÷ cell line.
thus, s. pneumoniae stimulates production of tnf-α and stabilizes tnf-α mrna through a mechanism involving the ares in the 3'-utr of the mrna.
stabilization of tnf-α mrna by s. pneumoniae is dependent on the viability of the bacteria
we next examined the pneumococcal requirement for mediating tnf-α mrna stability.
to this end, we compared the ability of live versus killed bacteria to induce cat expression in the raw tnf-α 3'-utr au+/au- cell lines .
as also shown in fig.
1c, s. pneumoniae induced accumulation of cat, and this was dependent on the 3'-utr ares .
interestingly, s. pneumoniae killed either by heat-inactivation, uv light or penicillin displayed reduced ability to induce cat expression, with the reduction ranging from almost 100%  to about 65% .
thus, stabilization of tnf-α mrna by s. pneumoniae is dependent on the viability of the bacteria.
s. pneumoniae mediates tnf-α mrna stability independent of tlrs
the s. pneumoniae sk <dig> strain used in this study has previously been demonstrated to be recognized by tlr <dig> and  <dig>  <cit> , with the former recognizing bacterial peptidoglycan and lipoteichoic acid and the latter recognizing bacterial dna  <cit> .
we have previously demonstrated that tlr <dig> recognizes live but not heat-killed s. pneumoniae  <cit>  wherefore the data shown in fig.
<dig> prompted us to examine the role of tlr <dig> in stabilization of tnf-α mrna.
we first examined how pure tlr agonists affected cat expression in the raw-tnf-α 3'-utr au+ cell line and compared this with the response evoked by live s. pneumoniae.
the agonists for tlr <dig>   <dig>  and  <dig> all enhanced cat expression but to a rather moderate extent compared to what was observed in cells receiving live s. pneumoniae .
to directly assess the role of tlr <dig>  we treated cells with the tlr <dig> antagonist odn <dig> prior to addition of s. pneumoniae and measured cat expression in lysates at later time points.
although odn <dig> inhibited induction of tnf-α by the tlr <dig> agonist odn <dig> by more than 95% , the presence of the tlr <dig> antagonist had no effect on cat expression induced by s. pneumoniae , thus suggesting that mrna stabilization by s. pneumoniae is independent of tlr <dig>
to more broadly examine the role of tlrs in stabilization of tnf-α mrna during s. pneumoniae infection, we pretreated cells with a cell-permeable myd <dig> inhibitory peptide or an inactive control peptide prior to addition of bacteria.
however, this treatment did not affect s. pneumoniae-induced cat expression , although the inhibitory peptide did indeed inhibit tlr signaling both strongly and specifically .
thus, the ability of s. pneumoniae to stabilize tnf-α mrna seems to be independent of bacterial signaling through tlrs.
p <dig> mapk is important for stabilization of tnf-α mrna by s. pneumoniae
the signaling pathways responsible for mediating mrna stability have recently been studied extensively, and it appears that p <dig> mapk plays a central role in this process  <cit> .
to examine the role of p <dig> in stabilization of tnf-α mrna in response to s. pneumoniae infection, we treated the raw tnf-α 3'-utr au+ cell line with the p <dig> inhibitor sb <dig>  <dig> min prior to addition of s. pneumoniae.
cat was subsequently measured in cell lysates.
as shown in fig.
4a, the strong elevation of cat protein levels observed after infection with live s. pneumoniae was significantly inhibited by the presence of sb <dig>  it has been reported that the p <dig> mapk inhibitor sb <dig>  which is structurally very similar to sb <dig>  not only inhibits p <dig> but also the kinase receptor-interacting protein   <dig>  <cit> , which plays an important role in signaling downstream of the s. pneumoniae-activated prr nod <dig>  <cit> .
therefore, we also examined the effect of another rip <dig> inhibitor, pp <dig>  <cit> , on induction of cat protein in our reporter system however, as illustrated in fig.
4a, no effect of pp <dig> treatment towards bacteria-induced cat production was observed.
in fig.
<dig> we demonstrated that live but not heat-killed s. pneumoniae mediated stabilization of tnf-α mrna.
therefore, we hypothesized that live versus heat-killed s. pneumoniae may differentially activate p <dig>  raw <dig>  cells were stimulated with live or heat-killed s. pneumoniae for the indicated time intervals, whole-cell lysates were harvested and the levels of phosphorylated p <dig> were measured.
indeed, the results showed that live bacteria potently stimulated phosphorylation of p <dig>  heat-killed bacteria were not able to elicit such a response .
to more thoroughly study the role of p <dig> in s. pneumoniae-mediated mrna stabilization, we wanted to examine how inactivation of tlr signaling, which did not affect tnf-α mrna stabilization , might influence p <dig> activation.
therefore, we pretreated raw <dig>  cells and primary macrophages with the myd <dig> inhibitory peptide or an inactive control peptide prior to addition of s. pneumoniae.
the cells were lysed  <dig> min post-infection and levels of phospho-p <dig> were measured.
in both cell types we observed strong activation of p <dig> in response to s. pneumoniae treatment and this was independent of the presence of the myd <dig> inhibitory peptide .
thus, stabilization of tnf-α mrna by s. pneumoniae is dependent on p <dig>  and this kinase is activated by live bacteria through a mechanism independent of tlrs.
p <dig> mediates mrna stabilization in response to s. pneumoniae independently of the mapk-activated protein kinases mk <dig>  msk1/ <dig>  and mnk
p38-mediated mrna stabilization can occur through several mechanisms, including involvement of members of the mapk-activated protein kinase family, in particular mk <dig>  <cit> .
to examine the role of this family of kinases in stabilization of tnf-α mrna, we compared macrophages from c57bl/ <dig> versus mk2-/- or msk1/2-/- mice with respect to stabilization of endogenous tnf-α mrna after challenge with s. pneumoniae.
furthermore, we also examined the effect of the mnk inhibitor cgp <dig> on production of cat protein in the reporter cell line system.
in all cases, no role for the mapk-activated protein kinases was found .
activation of p <dig> mapk in response to s. pneumoniae is dependent on tak <dig> and involves mkk3
in order to gain more insight into the mechanism of p <dig> activation during infection with s. pneumoniae, we first examined the role of tak <dig>  which has been ascribed an important role in this process by activating a number of different pathways  <cit> .
raw <dig>  cells were seeded and treated with the tak <dig> inhibitor -7-oxozeaenol  <dig> min prior to stimulation with s. pneumoniae or anisomycin.
lysates were prepared  <dig> min post-stimulation, and levels of phospho-p <dig> were measured.
as shown in fig.
5a, both s. pneumoniae and anisomycin potently induced phosphorylation of p <dig>  whereas the tak <dig> inhibitor did not affect activation of p <dig> in response to anisomycin, the ability of s. pneumoniae to induce p <dig> phosphorylation was abolished in the presence of the tak <dig> inhibitor.
mkk <dig>  as well as mkk <dig>  and  <dig> are p <dig> upstream kinases and can be activated through tak <dig>  <cit> .
to evaluate the role of mkk <dig> in the s. pneumoniae-induced p <dig> activation pathway, we compared the ability of spleen cells from c57bl/ <dig> and mkk3-/- mice to activate p <dig> in response to s. pneumoniae treatment.
anisomycin was included as a control, and was found to trigger p <dig> activation independently of mkk <dig> .
by contrast, mkk3-/- cells responded to s. pneumoniae with significantly reduced, although not abolished, activation of p <dig>  taken together, these data demonstrate that s. pneumoniae activates p <dig> through a tlr-independent pathway involving the upstream kinases tak <dig> and mkk <dig>
discussion
in this work we demonstrate that s. pneumoniae mediates stabilization of tnf-α mrna in macrophages by a mechanism dependent on the ares in the 3'-utr of tnf-α mrna.
the pathway stimulating this mechanism is independent of tlr signaling and involves tak <dig>  mkk <dig>  and p <dig> mapk.
an important finding of this work is that recognition systems independent of tlrs are responsible for triggering s. pneumoniae-induced signal transduction leading to stabilization of tnf-α mrna.
here we did not identify an alternative prr responsible for activating the p38-dependent signal.
it has been reported that s. pneumoniae is recognized by nod <dig>  which does indeed signal to p <dig>  <cit> .
however, since inactivated s. pneumoniae should retain the capacity to activate nod <dig>  and we found that the rip <dig> inhibitor pp <dig> did not affect expression of the cat reporter, our data suggest that the observed stabilization of tnf-α mrna by live s. pneumoniae does not depend on nod <dig>  in addition, there is evidence that other receptor systems may be involved in shaping the inflammatory cytokine response during bacterial infections.
for instance, it has been reported that borrelia burgdorferi can induce intracellular signaling and inflammatory gene expression independent of tlrs through direct binding to integrin α <dig> and β <dig>  <cit> .
the present finding of only live s. pneumoniae being able to induce tnf-α mrna stability suggests that a product or activity of the bacterial life cycle is required to trigger the p38-dependent signal mediating mrna stability.
similar findings have been previously reported for other bacteria, since kumar et al.
demonstrated that live as opposed to heat-killed staphylococcus aureus were able to stimulate phosphorylation of the mapks p <dig> and jnk, as well as the nf-κb inhibitory protein iκb  <cit> .
in that study, however, the effects of live bacteria may have been attributable to bacterial exoproducts, since the conditioned medium of s. aureus also stimulated the signalling pathways  <cit> .
the ability of s. pneumoniae to induce mrna stabilization was abrogated in the presence of the p <dig> mapk inhibitor sb <dig>  suggesting a role for this kinase.
in addition, the signaling pathway to p <dig> was entirely dependent on tak <dig> and partially dependent on mkk <dig>  p <dig> mapk is a well-described mediator of mrna stabilization affecting the activity of a number of are-binding proteins  <cit> .
for instance, p <dig> activates the kinase mk <dig>  which phosphorylates tristetraprolin , an are-binding protein with mrna-destabilizing activity  <cit> .
this phosphorylation event decreases the are affinity of ttp, hence leading to increased mrna stability.
another mechanism involves hur, which is induced by p <dig> mapk and binds to the ares, thus stabilizing tnf-α mrna  <cit> .
in this work we did not systematically investigate the molecular mechanism of mrna stabilization in response to s. pneumoniae infection, although we did find that it was independent of the mapk-activated protein kinases mk <dig>  msk1/ <dig>  and mnk.
CONCLUSIONS
here we report that live s. pneumoniae stabilizes tnf-α mrna and that this is dependent on the ares in the 3'-utr of the mrna.
the bacteria-induced signals mediating this function were not transduced from tlrs but were dependent on the p <dig> mapk, the upstream activation of which was dependent on tak <dig> and mkk <dig>  given the important role of tnf-α in the inflammatory response, the present study provides new insight into the mechanisms that govern production of inflammatory mediators in macrophages during pneumococcal infection.

 streptococcus pneumoniae is an important cause of severe invasive infections, including pneumonia, bacteremia, and meningitis. nasopharyngeal carriage of s. pneumoniae is common among children, but in most cases bacterial invasion into the lungs or bloodstream is prevented by local host defenses in the upper airways. however, in a minority of individuals, pneumocooci gain access to deeper structures of the body and thereby cause invasive disease, which are almost uniformly fatal if not treated with antibiotics. even in the presence of appropriate treatment, invasive pneumococcal disease bears a relatively poor prognosis. pneumococci are known to elicit a very potent inflammatory response, a key component of which is the production of cytokines and chemokines, which participate in the elimination of bacteria but may also result in undesirable excessive immunological responses, which may be harmful to the host. the cytokine tumor necrosis factor -α is produced by macrophages and dendritic cells as a primary response to infections and tissue damage, and is constitutively expressed in several autoimmune diseases. tnf-α plays an important role in activation and recruitment of leukocytes to inflamed tissue, and has been demonstrated to be involved in the host-defense against a number of important human pathogens, including s. pneumoniae. however, tnf-α is also associated with excessive inflammation and immunopathology in infections and autoimmune diseases, and specifically, tnf-a has been suggested to be involved in breakage of the blood-brain barrier during development of hematogenous pneumococcal meningitis. production of tnf-α is regulated both at the level of transcription, mrna stability, and translation. the regulation of mrna stability and translation is mediated largely through adenylate uridylate -rich elements s present in the 3'-untranslated region of mrna, which are targeted by are-binding proteins able to affect mrna stability and translation. the importance of ares in regulation of tnf-α production is evidenced by spontaneous development of chronic inflammatory arthritis and crohn's-like inflammatory bowel disease in transgenic mice devoid of ares in mrna encoding tnf-α. the inflammatory response to infection is triggered by pattern recognition receptors s, which recognize evolutionarily conserved pathogen-associated molecular patterns s and activate intracellular signaling, thereby up-regulating expression of genes with inflammatory activities, including tnf-α. the toll-like receptors s constitute an important class of prrs, and tlr and have been shown to recognize pneumococcal pamps. the vast majority of tlr downstream signaling proceeds through the cytoplasmic adaptor protein myd which is central for activation of the transcription factor nuclear factor κb, and the mitogen-activated protein kinase pathway, being involved in both transcriptional and post-transcriptional responses. however, several studies suggest that pneumococcal recognition and pathogenesis may be at least partly independent of tlr signaling. another class of prrs involved in recognition of s. pneumoniae is the nucleotide-binding oligomerization domain proteins, which are intracellular prrs. the nod protein recognizing muramyl dipeptide has been reported to be important for activation of nf-κb during infection with s. pneumoniae. in this study, we have investigated the role of mrna stabilization in tnf-α production induced by s. pneunomiae, including the signaling mechanisms involved. here we report that live s. pneunomiae strongly stabilizes tnf-α mrna by a mechanism dependent on the ares in the 3'-utr of the mrna. importantly, increased tnf-α mrna stability induced by s. pneunomiae was not mediated by tlrs but was dependent on the p mapk, which was activated through a pathway involving transforming growth factor-activated kinase and mapk kinase  s. pneumoniae induces tnf-α production and stabilizes tnf-α mrna through ares in the 3'-utr given the important role of the inflammatory response in the pathogenesis of streptococcal diseases, we were interested in studying mechanisms involved in expression of inflammatory cytokines during infection with s. pneumoniae. in a first set of experiments, we measured expression of tnf-α in murine macrophages after treatment with s. pneumoniae, n. meningitidis or pure ligands for tlr, tlr, and tlr. as expected, cells responded to addition of bacteria or tlr ligands by secreting high amounts of tnf-α. lps has been reported to stimulate tnf-α expression by both activating gene transcription, stabilizing the tnf-α mrna, and supporting translation. to examine whether s. pneumoniae stabilized tnf-α mrna, we treated cells with actinomycin d, which prevents de novo transcription of mrna, and stimulated the macrophages with s. pneumoniae, n. meningitidis or lps. at the indicated time-points post-stimulation, rna was harvested and tnf-α mrna levels were determined by real-time pcr. as shown in fig. 1b, addition of actinomycin d led to a rapid decline in the levels of remaining tnf-α mrna in cells receiving mock treatment. however, treatment with s. pneumoniae profoundly augmented the half-life of tnf-α mrna. lps and n. meningitidis also stabilized the tnf-α messenger but not to the same extent as s. pneumoniae. to examine whether the ares of the tnf-α mrna were required for the observed effect, we turned to a cell system derived from raw cells, consisting of two cell lines stably expressing a reporter gene, transcribed from a constitutive promoter. in one of the cell lines the 3'-utr of the mrna encoding the reporter gene is derived from the wild-type tnf-α mrna, and in the other cell line the ares of the tnf-α 3'-utr are mutated. when the raw tnf-α 3'-utr au+ cell line was incubated in the presence of s. pneumoniae, a very strong increase in cat levels was observed, whereas lps and also n. meningitidis affected accumulation of cat protein only to a more modest extent. interestingly, the effect of s. pneumoniae on accumulation of cat was largely abrogated in the raw tnf-α 3'-utr au÷ cell line. thus, s. pneumoniae stimulates production of tnf-α and stabilizes tnf-α mrna through a mechanism involving the ares in the 3'-utr of the mrna. stabilization of tnf-α mrna by s. pneumoniae is dependent on the viability of the bacteria we next examined the pneumococcal requirement for mediating tnf-α mrna stability. to this end, we compared the ability of live versus killed bacteria to induce cat expression in the raw tnf-α 3'-utr au+/au- cell lines. as also shown in fig. 1c, s. pneumoniae induced accumulation of cat, and this was dependent on the 3'-utr ares. interestingly, s. pneumoniae killed either by heat-inactivation, uv light or penicillin displayed reduced ability to induce cat expression, with the reduction ranging from almost 100% to about 65%. thus, stabilization of tnf-α mrna by s. pneumoniae is dependent on the viability of the bacteria. s. pneumoniae mediates tnf-α mrna stability independent of tlrs the s. pneumoniae sk strain used in this study has previously been demonstrated to be recognized by tlr and, with the former recognizing bacterial peptidoglycan and lipoteichoic acid and the latter recognizing bacterial dna. we have previously demonstrated that tlr recognizes live but not heat-killed s. pneumoniae wherefore the data shown in fig. prompted us to examine the role of tlr in stabilization of tnf-α mrna. we first examined how pure tlr agonists affected cat expression in the raw-tnf-α 3'-utr au+ cell line and compared this with the response evoked by live s. pneumoniae. the agonists for tlr and all enhanced cat expression but to a rather moderate extent compared to what was observed in cells receiving live s. pneumoniae. to directly assess the role of tlr we treated cells with the tlr antagonist odn prior to addition of s. pneumoniae and measured cat expression in lysates at later time points. although odn inhibited induction of tnf-α by the tlr agonist odn by more than 95%, the presence of the tlr antagonist had no effect on cat expression induced by s. pneumoniae, thus suggesting that mrna stabilization by s. pneumoniae is independent of tlr to more broadly examine the role of tlrs in stabilization of tnf-α mrna during s. pneumoniae infection, we pretreated cells with a cell-permeable myd inhibitory peptide or an inactive control peptide prior to addition of bacteria. however, this treatment did not affect s. pneumoniae-induced cat expression, although the inhibitory peptide did indeed inhibit tlr signaling both strongly and specifically. thus, the ability of s. pneumoniae to stabilize tnf-α mrna seems to be independent of bacterial signaling through tlrs. p mapk is important for stabilization of tnf-α mrna by s. pneumoniae the signaling pathways responsible for mediating mrna stability have recently been studied extensively, and it appears that p mapk plays a central role in this process. to examine the role of p in stabilization of tnf-α mrna in response to s. pneumoniae infection, we treated the raw tnf-α 3'-utr au+ cell line with the p inhibitor sb min prior to addition of s. pneumoniae. cat was subsequently measured in cell lysates. as shown in fig. 4a, the strong elevation of cat protein levels observed after infection with live s. pneumoniae was significantly inhibited by the presence of sb it has been reported that the p mapk inhibitor sb which is structurally very similar to sb not only inhibits p but also the kinase receptor-interacting protein, which plays an important role in signaling downstream of the s. pneumoniae-activated prr nod. therefore, we also examined the effect of another rip inhibitor, pp, on induction of cat protein in our reporter system however, as illustrated in fig. 4a, no effect of pp treatment towards bacteria-induced cat production was observed. in fig. we demonstrated that live but not heat-killed s. pneumoniae mediated stabilization of tnf-α mrna. therefore, we hypothesized that live versus heat-killed s. pneumoniae may differentially activate p raw cells were stimulated with live or heat-killed s. pneumoniae for the indicated time intervals, whole-cell lysates were harvested and the levels of phosphorylated p were measured. indeed, the results showed that live bacteria potently stimulated phosphorylation of p heat-killed bacteria were not able to elicit such a response. to more thoroughly study the role of p in s. pneumoniae-mediated mrna stabilization, we wanted to examine how inactivation of tlr signaling, which did not affect tnf-α mrna stabilization, might influence p activation. therefore, we pretreated raw cells and primary macrophages with the myd inhibitory peptide or an inactive control peptide prior to addition of s. pneumoniae. the cells were lysed min post-infection and levels of phospho-p were measured. in both cell types we observed strong activation of p in response to s. pneumoniae treatment and this was independent of the presence of the myd inhibitory peptide. thus, stabilization of tnf-α mrna by s. pneumoniae is dependent on p and this kinase is activated by live bacteria through a mechanism independent of tlrs. p mediates mrna stabilization in response to s. pneumoniae independently of the mapk-activated protein kinases mk msk1/ and mnk p38-mediated mrna stabilization can occur through several mechanisms, including involvement of members of the mapk-activated protein kinase family, in particular mk. to examine the role of this family of kinases in stabilization of tnf-α mrna, we compared macrophages from c57bl/ versus mk2-/- or msk1/2-/- mice with respect to stabilization of endogenous tnf-α mrna after challenge with s. pneumoniae. furthermore, we also examined the effect of the mnk inhibitor cgp on production of cat protein in the reporter cell line system. in all cases, no role for the mapk-activated protein kinases was found. activation of p mapk in response to s. pneumoniae is dependent on tak and involves mkk3 in order to gain more insight into the mechanism of p activation during infection with s. pneumoniae, we first examined the role of tak which has been ascribed an important role in this process by activating a number of different pathways. raw cells were seeded and treated with the tak inhibitor -7-oxozeaenol min prior to stimulation with s. pneumoniae or anisomycin. lysates were prepared min post-stimulation, and levels of phospho-p were measured. as shown in fig. 5a, both s. pneumoniae and anisomycin potently induced phosphorylation of p whereas the tak inhibitor did not affect activation of p in response to anisomycin, the ability of s. pneumoniae to induce p phosphorylation was abolished in the presence of the tak inhibitor. mkk as well as mkk and are p upstream kinases and can be activated through tak. to evaluate the role of mkk in the s. pneumoniae-induced p activation pathway, we compared the ability of spleen cells from c57bl/ and mkk3-/- mice to activate p in response to s. pneumoniae treatment. anisomycin was included as a control, and was found to trigger p activation independently of mkk. by contrast, mkk3-/- cells responded to s. pneumoniae with significantly reduced, although not abolished, activation of p taken together, these data demonstrate that s. pneumoniae activates p through a tlr-independent pathway involving the upstream kinases tak and mkk discussion in this work we demonstrate that s. pneumoniae mediates stabilization of tnf-α mrna in macrophages by a mechanism dependent on the ares in the 3'-utr of tnf-α mrna. the pathway stimulating this mechanism is independent of tlr signaling and involves tak mkk and p mapk. an important finding of this work is that recognition systems independent of tlrs are responsible for triggering s. pneumoniae-induced signal transduction leading to stabilization of tnf-α mrna. here we did not identify an alternative prr responsible for activating the p38-dependent signal. it has been reported that s. pneumoniae is recognized by nod which does indeed signal to p. however, since inactivated s. pneumoniae should retain the capacity to activate nod and we found that the rip inhibitor pp did not affect expression of the cat reporter, our data suggest that the observed stabilization of tnf-α mrna by live s. pneumoniae does not depend on nod in addition, there is evidence that other receptor systems may be involved in shaping the inflammatory cytokine response during bacterial infections. for instance, it has been reported that borrelia burgdorferi can induce intracellular signaling and inflammatory gene expression independent of tlrs through direct binding to integrin α and β. the present finding of only live s. pneumoniae being able to induce tnf-α mrna stability suggests that a product or activity of the bacterial life cycle is required to trigger the p38-dependent signal mediating mrna stability. similar findings have been previously reported for other bacteria, since kumar et al. demonstrated that live as opposed to heat-killed staphylococcus aureus were able to stimulate phosphorylation of the mapks p and jnk, as well as the nf-κb inhibitory protein iκb. in that study, however, the effects of live bacteria may have been attributable to bacterial exoproducts, since the conditioned medium of s. aureus also stimulated the signalling pathways. the ability of s. pneumoniae to induce mrna stabilization was abrogated in the presence of the p mapk inhibitor sb suggesting a role for this kinase. in addition, the signaling pathway to p was entirely dependent on tak and partially dependent on mkk p mapk is a well-described mediator of mrna stabilization affecting the activity of a number of are-binding proteins. for instance, p activates the kinase mk which phosphorylates tristetraprolin, an are-binding protein with mrna-destabilizing activity. this phosphorylation event decreases the are affinity of ttp, hence leading to increased mrna stability. another mechanism involves hur, which is induced by p mapk and binds to the ares, thus stabilizing tnf-α mrna. in this work we did not systematically investigate the molecular mechanism of mrna stabilization in response to s. pneumoniae infection, although we did find that it was independent of the mapk-activated protein kinases mk msk1/ and mnk.  here we report that live s. pneumoniae stabilizes tnf-α mrna and that this is dependent on the ares in the 3'-utr of the mrna. the bacteria-induced signals mediating this function were not transduced from tlrs but were dependent on the p mapk, the upstream activation of which was dependent on tak and mkk given the important role of tnf-α in the inflammatory response, the present study provides new insight into the mechanisms that govern production of inflammatory mediators in macrophages during pneumococcal infection. 
streptococcus pneumoniae is a human pathogenic bacteria and a major cause of severe invasive diseases, including pneumonia, bacteremia, and meningitis. secretion of tnf-α was strongly induced by s. pneumoniae, which was able to stabilize tnf-α mrna through a mechanism dependent on the viability of the bacteria as well as the adenylate uridylate-rich elements in the 3'untranslated region of tnf-α mrna. the ability of s. pneumoniae to stabilize tnf-α mrna was dependent on the mitogen-activated protein kinase p whereas inhibition of toll-like receptor signaling via myd did not affect s. pneumoniae-induced mrna stabilization. infections with s. pneumoniae evoke a strong inflammatory response, which plays a major role in the pathogenesis of pneumococcal disease. production of tnf-α may contribute significantly to the inflammatory response raised during pneumococcal infection. thus, s. pneumoniae stabilizes tnf-α mrna through a pathway dependent on p but independent of toll-like receptors. in this study, we have examined how s. pneumoniae affects expression of the inflammatory cytokine tumor necrosis factor α, and the molecular mechanisms involved.
 streptococcus pneumoniae is a human pathogenic bacteria and a major cause of severe invasive diseases, including pneumonia, bacteremia, and meningitis. infections with s. pneumoniae evoke a strong inflammatory response, which plays a major role in the pathogenesis of pneumococcal disease.  in this study, we have examined how s. pneumoniae affects expression of the inflammatory cytokine tumor necrosis factor α, and the molecular mechanisms involved. secretion of tnf-α was strongly induced by s. pneumoniae, which was able to stabilize tnf-α mrna through a mechanism dependent on the viability of the bacteria as well as the adenylate uridylate-rich elements in the 3'untranslated region of tnf-α mrna. the ability of s. pneumoniae to stabilize tnf-α mrna was dependent on the mitogen-activated protein kinase p whereas inhibition of toll-like receptor signaling via myd did not affect s. pneumoniae-induced mrna stabilization. p was activated through a pathway involving the upstream kinase transforming growth factor-activated kinase and mapk kinase  thus, s. pneumoniae stabilizes tnf-α mrna through a pathway dependent on p but independent of toll-like receptors. production of tnf-α may contribute significantly to the inflammatory response raised during pneumococcal infection. 