BACKGROUND
gram-negative bacterium nontypeable haemophilus influenzae  and gram-positive bacterium streptococcus pneumoniae  are important bacterial pathogens causing lung infections  <cit> .
in children, they cause otitis media , the most common childhood infection and the leading cause of conductive hearing loss  <cit> , while in adults, they exacerbate chronic obstructive pulmonary diseases , the fourth leading cause of death in the united states  <cit> .
although a majority of om or copd is mainly associated with a single bacterial pathogen, there is a growing body of evidence that a portion of patients diagnosed with om or copd have mixed infections of nthi and s. pneumoniae  <cit> .
in the mixed infection of nthi and s. pneumoniae, two bacterial pathogens synergistically induce inflammation through activation of specific host signaling pathway  <cit> , which is initiated through the recognition of microbial pathogens by means of cell surface receptors.
toll-like receptors  are a class of pathogen recognition receptors that mediate recognition of pathogen-associated molecular pattern.
among  <dig> mammalian tlrs, tlr <dig> plays especially a critical role due to its capability of detecting the widest repertoire of pathogen-associated molecular patterns from a large variety of pathogens, including gram-positive or gram-negative bacteria, mycobacteria, fungi, viruses, and parasites  <cit> .
we previously reported that tlr <dig> is a key receptor recognizing nthi components  <cit>  and is highly inducible by multiple pro-inflammatory stimuli including nthi itself  <cit> .
given the fact that s. pneumoniae co-exists with nthi in lung and middle ear infections and synergistically enhances nthi-induced inflammation  <cit> , it is of particular interest to investigate the molecular mechanism by which tlr <dig> is likely induced by nthi and s. pneumoniae in a synergistic manner.
in the present study, we showed that s. pneumoniae induces tlr <dig> up-regulation via a cytoplasmic pneumolysin  through a tlr4-myd88-nf-κb-dependent signaling pathway.
moreover, s. pneumoniae induces expression of tumor suppressor cylindromoatosis , which in turn negatively regulates s. pneumoniae-induced nf-κb activation and tlr <dig> expression.
these studies thus bring new insights into synergistic regulation of host tlrs in mixed bacterial infections.
RESULTS
s. pneumoniae pneumolysin induces tlr <dig> expression in the epithelium of lung and middle ear in vitro and in vivo
we initially investigated the effect of s. pneumoniae on tlr <dig> expression in epithelial cells.
as shown in fig.
1a, tlr <dig> mrna expression was induced by s. pneumoniae treatment in human epithelial hela cells in a time-dependent manner.
tlr <dig> expression was markedly up-regulated at  <dig> h after treatment, peaked at  <dig> h and declined thereafter.
tlr <dig> up-regulation by s. pneumoniae was also confirmed in human respiratory epithelial a <dig> cells .
we next determined if tlr <dig> up-regulation by s. pneumoniae occurs at the transcriptional level.
as shown in fig.
1c, transcriptional activity of tlr <dig> promoter reporter was also up-regulated by s. pneumoniae in epithelial cells.
because pneumolysin  plays a key role in the pathogenesis of s. pneumoniae, we investigated whether ply is a crucial virulence factor for the tlr <dig> up-regulation by s. pneumoniae.
human respiratory epithelial cells were treated with either s. pneumoniae wt d <dig> or pneumolysin-deficient mutant pln, and mrna expression level of tlr <dig> was then measured by q-pcr analysis.
as shown in fig.
1d, inoculation of s. pneumoniae wt d <dig> induced tlr <dig> mrna expression, whereas s. pneumoniae pln did not induce it.
moreover, purified ply induced tlr <dig> expression to a similar extent to s. pneumoniae in middle ear hmeec- <dig> epithelial cells .
to further confirm if s. pneumoniae also induces tlr <dig> expression in vivo, we examined s. pneumoniae- and ply-induced tlr <dig> expression in vivo using s. pneumoniae-induced pneumonia and om model in mice.
as shown in fig.
1f &1g, s. pneumoniae induced tlr <dig> mrna expression in both lung and middle ear tissues.
similar results were also observed in ply-inoculated mice .
consistent with our in vitro findings, tlr <dig> expression was up-regulated by s. pneumoniae wt d <dig> and ply inoculation but not mutant pln .
furthermore, western blot analysis confirmed tlr <dig> up-regulation by s. pneumoniae at the protein level after intratracheal inoculation of s. pneumoniae in wt mice .
taken together, our data showed that s. pneumoniae ply induces tlr <dig> up-regulation in lung and middle ear in vitro and in vivo.
s. pneumoniae induces tlr <dig> expression through tlr4-myd88-nf-κb-dependent signaling pathway
tlrs play a critical role in recognizing extracellular pathogens and transducing the cell surface interaction between microbes and cells to intracellular components.
it was previously reported that cytolysins including bacillus anthracis anthrolysin o and s. pneumoniae ply act as tlr <dig> agonists  <cit> .
moreover, we reported that ply triggers tlr4-dependent intracellular signaling pathway in severe s. pneumoniae infection in lung  <cit> .
thus, we determined whether s. pneumoniae induced-tlr <dig> up-regulation is mediated by tlr <dig> in vitro and in vivo.
perturbing tlr <dig> signaling using tlr <dig> dn mutant plasmid inhibited both s. pneumoniae-induced tlr <dig> promoter activity  and tlr <dig> mrna expression  in vitro.
to further investigate whether tlr <dig> is also critical for s. pneumoniae-induced tlr <dig> up-regulation in vivo, wt and tlr <dig> ko mice were intratracheally inoculated with s. pneumoniae, and tlr <dig> mrna expression was measured in the lungs of inoculated mice.
as shown in fig.
2c, s. pneumoniae induced tlr <dig> expression in the lungs of wt mice but not tlr <dig> ko mice in vivo.
because myd <dig> is a well-known signaling adaptor for tlr <dig>  we determined whether myd <dig> is required for the s. pneumoniae-induced tlr <dig> expression in vivo.
as shown in fig.
2d, s, pneumoniae-induced tlr <dig> up-regulation was significantly reduced in myd <dig> ko mice compared to wt mice.
having demonstrated a marked decrease in tlr <dig> expression in tlr <dig> ko mice, we determined whether tlr4-deficiency caused this decrease via changing bacterial clearance in the lung tissues or inflammatory cell migration into airways.
we therefore measured bacteria number from the lungs of s. pneumoniae-inoculated wt and tlr <dig> ko mice  <dig> hrs after inoculation and also measured inflammatory cell migration into airway from the bal fluid of vehicle- or s. pneumoniae-inoculated wt and tlr <dig> ko mice  <dig> hrs after inoculation .
neither bacteria number of the lungs nor inflammatory cell migration into airways in tlr <dig> ko mice was significantly different from those in wt mice.
the results reported herein demonstrate that, under the conditions reported here, tlr4-deficiency does not affect the bacterial clearance of s. pneumoniae and inflammatory cell migration response to s. pneumoniae, and decrease in s. pneumoniae-induced tlr <dig> expression in tlr <dig> ko mice is independent of both bacteria number and inflammatory cells in the lung in vivo.
collectively, our data suggest that s. pneumoniae induces tlr <dig> up-regulation via tlr4-myd <dig> signaling pathway.
because nf-κb plays an important role in mediating ply-induced host responses, we next determined if nf-κb is also involved in mediating tlr <dig> up-regulation by ply  <cit> .
as shown in fig.
2g &2h, s. pneumoniae induced nf-κb activation in a dose-dependent manner.
similar result was also observed in ply-treated cells.
we further determined the involvement of ikkα-iκbα, the key signaling pathway upstream of nf-κb, in tlr <dig> induction  <cit> .
as shown in fig.
2i, perturbing ikkα and iκbα signaling by overexpressing dn mutant plasmid inhibited s. pneumoniae-induced tlr <dig> mrna up-regulation.
this finding was further confirmed by using ikkαsirna .
thus it is clear that s. pneumoniae-induced tlr <dig> up-regulation is mediated by ikkα and iκbα-dependent nf-κb activation.
s. pneumoniae induced-tlr <dig> up-regulation is negatively regulated by tumor suppressor cyld
bacteria modulates many innate immune responses via induction of negative regulatory molecules  <cit> .
we previously reported that s. pneumoniae induces tumor suppressor cyld, an important negative regulator of nf-κb signaling pathway, in severe s. pneumoniae infection  <cit> .
nthi induced cyld expression, which in turn leads to negative regulation of nthi-induced nf-κb-dependent inflammation  <cit> .
cyld expression is relatively low in lung but is highly induced under diseased condition  <cit> .
to determine the role of cyld in s. pneumoniae-induced tlr <dig> expression in the lung and middle ear, we first evaluated up-regulation of cyld in vitro.
as shown in fig.
3a, s. pneumoniae induced cyld expression not only in lung epithelia cell a <dig>  but also in middle ear epithelial cell hmeec- <dig>  to further confirm whether s. pneumoniae induces cyld both in the lung and middle ear in vivo, wt mice were intratracheally or transtympanically inoculated with s. pneumoniae, and cyld mrna expression was then measured in the lung and middle ear tissues of inoculated mice, respectively.
as shown in fig.
3b, s. pneumoniae induced cyld expression in both lung and middle ear of the mice.
to further investigate the role of cyld in s. pneumoniae-induced nf-κb activation, the effect of cyld knockdown using cyld sirna was measured.
as shown in fig.
3c.
s. pneumoniae- and ply-induced nf-κb promoter activity was greatly enhanced by cyld sirna.
to investigate whether cyld acts as a negative regulator for s. pneumoniae-induced tlr <dig> expression, the effects of cyld sirna on s. pneumoniae-induced tlr <dig> mrna expression were measured.
as shown in fig.
3d, knockdown of cyld expression using cyld sirna greatly enhanced s. pneumoniae-induced tlr <dig> mrna expression.
to determine the in vivo role of cyld on s. pneumoniae-induced tlr <dig> expression, we measured tlr <dig> mrna expression in lung tissues of wt and cyld ko mice after s. pneumoniae inoculation.
consistent with our in vitro findings, s. pneumoniae-induced tlr <dig> mrna up-regulation was enhanced in cyld ko mice .
together, our data indicate that cyld acts as a negative regulator for s. pneumoniae-induced nf-κb activation and tlr <dig> up-regulation.
s. pneumoniae synergizes with nthi to induce inflammatory response via tlr2
to determine whether s. pneumoniae synergizes with nthi to induce inflammatory response, we investigated the effect of s. pneumoniae and nthi on the expression of il-1β, a key nf-κb-dependent pro-inflammatory cytokine in lung and middle ear.
as shown in fig.
4a, s. pneumoniae and nthi synergistically enhanced expression of il-1β ?
mrna in human lung epithelial cell.
moreover, s. pneumoniae and nthi also synergistically induced expression of il-1β mrna in the lungs of mice .
next we investigated the role of tlr <dig> in synergistic induction of nf-κb-dependent inflammatory response by s. pneumoniae and nthi.
as shown in fig.
4c, sirna mediated knockdown of tlr <dig> markedly inhibited synergistic induction of il-1β mrna expression by s. pneumoniae and nthi, thereby providing evidence for the involvement of tlr <dig> in this synergistic induction.
this finding was further confirmed in the cells from tlr <dig> ko mice in vitro .
having shown that s. pneumoniae-induced tlr <dig> up-regulation is inhibited by tlr4-deficiency, and tlr <dig> up-regulation is closely associated with synergistic response by s. pneumoniae with nthi, we determined if tlr4-deficiency affects synergistic activation of nf-κb and subsequent gene expression by s. pneumoniae with nthi.
we first measured nf-κb activation and mrna expressions of tlr <dig> and il-1β following co-treatment with s. pneumoniae and nthi in cells  and lung tissues  of wt and tlr <dig> ko mice.
as shown in fig.
4e, nf-κb activation by s. pneumoniae with or without nthi was significantly decreased in the cells from tlr <dig> ko mice compared with those in cells from wt mice .
in addition, mrna expressions of tlr <dig> and il-1β by s. pneumoniae with or without nthi in the lung tissues of tlr <dig> ko mice were also significantly decreased compared with those in wt mice .
taken together, these findings suggest that s. pneumoniae synergizes with nthi to induce nf-κb activation and subsequent gene expression via tlr <dig> up-regulation.
discussion
there is a growing body of evidence showing that a portion of patients diagnosed with om or copd have mixed infections of nthi and s. pneumoniae  <cit> .
under in vivo situations such as polymicrobial infections, mucosal epithelial surfaces are simultaneously exposed to a variety of bacteria and bacterial components, and one microbe synergizes with another one to induce the inflammatory response by activating intracellular signaling pathway  <cit> .
among many other innate immune components, tlrs are critical for initiating and mediating immune response.
thus, regulation of tlrs expression plays an important role in modulating host response to microbes.
however, little is known about the molecular mechanisms underlying regulation of tlrs expression in mixed infection.
as we previously reported, tlr <dig> expression is relatively low in unstimulated epithelial cells but is markedly up-regulated in response to invading microbes  <cit> .
however, the link between tlr <dig> up-regulation and mixed infection in otitis media and pneumonia still remains unknown.
from what we have shown in the present data, it is evident that tlr <dig> expression is up-regulated by s. pneumoniae via a tlr4-myd88-nf-κb-dependent signaling pathway through its well-known virulence factor ply.
we further show that s. pneumoniae and nthi synergize with each to induce nf-κb activation and il-1β expression dependently of tlr <dig> up-regulation.
in the present study, we provided evidence for the first time that s. pneumoniae synergizes with nthi to induce nf-κb-dependent inflammatory response likely via tlr <dig> up-regulation in lung and middle ear epithelia cells, thereby bringing new insight into our understanding of the complex regulatory mechanisms underlying inflammation in polymicrobial infections.
in addition to tlr <dig> and tlr <dig>  tlr <dig>   <dig>  and  <dig> have also been shown to be involved in s. pneumoniae infection  <cit> .
therefore we evaluated lung epithelial cell expression of  <dig> tlrs  and cd <dig>  myd <dig>  md <dig> and  <dig> following s. pneumoniae treatment in a <dig> cells.
in untreated cells, the expression level of tlr <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> was relatively high compared to those of tlr <dig>   <dig>  and  <dig>  interestingly, the expression of tlr <dig>   <dig> and  <dig> at the mrna level was highly up-regulated following s. pneumoniae treatment , implying a possible important role for tlr <dig> up-regulation in bacterial infections.
indeed, the data we presented in this study demonstrated that the synergistic activation of nf-κb and inflammation by s. pneumoniae and nthi is dependent on tlr <dig> up-regulation.
although there is strong evidence to support that tlr <dig> is a critical receptor for recognizing pneumolysin and pneumolysin is a major virulence factor of pneumococcal infection, the role of tlr <dig> in pneumococcal infection is not fully understood  <cit> .
it remains somewhat controversial whether tlr4-deficiency confers susceptibility or tolerance to pneumococcal infection in vivo  <cit> .
controversial phenotype of tlr4-deficiency mice in s. pneumoniae infection may be partly due to the strain differences of the mice used in each experiment, e.g.
using c3h/hej, balb/c and c57bl/ <dig> as background strains.
it is well known that different mouse strains exhibit a very different susceptibility to pneumococcal infection  <cit> .
balb/c mice, which were used as a background strain of tlr <dig> ko mice in our current experiment, were known as resistant strain in pneumococcal infection compared to other strains  <cit> .
tlr4-deficiency in this strain thus may have no significant effect on pneumococcal clearance.
another interesting finding in this study is the experimental evidence for the negative regulation of s. pneumoniae-induced tlr <dig> up-regulation by tumor suppressor cyld.
cyld was previously identified as an important regulator not only in tumor development  <cit>  but also in adaptive immune response  <cit> .
recent evidence demonstrated that cyld also serves as a key regulator for innate immune response via negative-crosstalk with mapks and nf-κb  <cit> .
moreover we recently identified an important role for cyld in modulating host antiviral response by regulating tlr <dig> expression in mixed infection of bacteria and virus  <cit> .
however, still unknown is how cyld regulates inflammatory response in mixed bacterial infection.
CONCLUSIONS
in the present study we showed for the first time that s. pneumoniae synergizes with nthi to induce nf-κb activation and the subsequent inflammatory response via up-regulation of tlr <dig>  moreover, we showed that s. pneumoniae-induced tlr <dig> up-regulation is mediated by tlr4-myd88-ikkα-iκbα signaling pathway.
interestingly, tumor suppressor cyld acts as a negative regulator for the synergistic induction of inflammation by s. pneumoniae and nthi, thereby implying a critical role for cyld in preventing overactive inflammatory response in mixed infections.
this study may bring new insights into the molecular mechanisms underlying polymicrobial infections, especially mixed infection of gram-positive and negative bacteria.

 gram-negative bacterium nontypeable haemophilus influenzae and gram-positive bacterium streptococcus pneumoniae are important bacterial pathogens causing lung infections. in children, they cause otitis media, the most common childhood infection and the leading cause of conductive hearing loss, while in adults, they exacerbate chronic obstructive pulmonary diseases, the fourth leading cause of death in the united states. although a majority of om or copd is mainly associated with a single bacterial pathogen, there is a growing body of evidence that a portion of patients diagnosed with om or copd have mixed infections of nthi and s. pneumoniae. in the mixed infection of nthi and s. pneumoniae, two bacterial pathogens synergistically induce inflammation through activation of specific host signaling pathway, which is initiated through the recognition of microbial pathogens by means of cell surface receptors. toll-like receptors are a class of pathogen recognition receptors that mediate recognition of pathogen-associated molecular pattern. among mammalian tlrs, tlr plays especially a critical role due to its capability of detecting the widest repertoire of pathogen-associated molecular patterns from a large variety of pathogens, including gram-positive or gram-negative bacteria, mycobacteria, fungi, viruses, and parasites. we previously reported that tlr is a key receptor recognizing nthi components and is highly inducible by multiple pro-inflammatory stimuli including nthi itself. given the fact that s. pneumoniae co-exists with nthi in lung and middle ear infections and synergistically enhances nthi-induced inflammation, it is of particular interest to investigate the molecular mechanism by which tlr is likely induced by nthi and s. pneumoniae in a synergistic manner. in the present study, we showed that s. pneumoniae induces tlr up-regulation via a cytoplasmic pneumolysin through a tlr4-myd88-nf-κb-dependent signaling pathway. moreover, s. pneumoniae induces expression of tumor suppressor cylindromoatosis, which in turn negatively regulates s. pneumoniae-induced nf-κb activation and tlr expression. these studies thus bring new insights into synergistic regulation of host tlrs in mixed bacterial infections.  s. pneumoniae pneumolysin induces tlr expression in the epithelium of lung and middle ear in vitro and in vivo we initially investigated the effect of s. pneumoniae on tlr expression in epithelial cells. as shown in fig. 1a, tlr mrna expression was induced by s. pneumoniae treatment in human epithelial hela cells in a time-dependent manner. tlr expression was markedly up-regulated at h after treatment, peaked at h and declined thereafter. tlr up-regulation by s. pneumoniae was also confirmed in human respiratory epithelial a cells. we next determined if tlr up-regulation by s. pneumoniae occurs at the transcriptional level. as shown in fig. 1c, transcriptional activity of tlr promoter reporter was also up-regulated by s. pneumoniae in epithelial cells. because pneumolysin plays a key role in the pathogenesis of s. pneumoniae, we investigated whether ply is a crucial virulence factor for the tlr up-regulation by s. pneumoniae. human respiratory epithelial cells were treated with either s. pneumoniae wt d or pneumolysin-deficient mutant pln, and mrna expression level of tlr was then measured by q-pcr analysis. as shown in fig. 1d, inoculation of s. pneumoniae wt d induced tlr mrna expression, whereas s. pneumoniae pln did not induce it. moreover, purified ply induced tlr expression to a similar extent to s. pneumoniae in middle ear hmeec- epithelial cells. to further confirm if s. pneumoniae also induces tlr expression in vivo, we examined s. pneumoniae- and ply-induced tlr expression in vivo using s. pneumoniae-induced pneumonia and om model in mice. as shown in fig. 1f &1g, s. pneumoniae induced tlr mrna expression in both lung and middle ear tissues. similar results were also observed in ply-inoculated mice. consistent with our in vitro findings, tlr expression was up-regulated by s. pneumoniae wt d and ply inoculation but not mutant pln. furthermore, western blot analysis confirmed tlr up-regulation by s. pneumoniae at the protein level after intratracheal inoculation of s. pneumoniae in wt mice. taken together, our data showed that s. pneumoniae ply induces tlr up-regulation in lung and middle ear in vitro and in vivo. s. pneumoniae induces tlr expression through tlr4-myd88-nf-κb-dependent signaling pathway tlrs play a critical role in recognizing extracellular pathogens and transducing the cell surface interaction between microbes and cells to intracellular components. it was previously reported that cytolysins including bacillus anthracis anthrolysin o and s. pneumoniae ply act as tlr agonists. moreover, we reported that ply triggers tlr4-dependent intracellular signaling pathway in severe s. pneumoniae infection in lung. thus, we determined whether s. pneumoniae induced-tlr up-regulation is mediated by tlr in vitro and in vivo. perturbing tlr signaling using tlr dn mutant plasmid inhibited both s. pneumoniae-induced tlr promoter activity and tlr mrna expression in vitro. to further investigate whether tlr is also critical for s. pneumoniae-induced tlr up-regulation in vivo, wt and tlr ko mice were intratracheally inoculated with s. pneumoniae, and tlr mrna expression was measured in the lungs of inoculated mice. as shown in fig. 2c, s. pneumoniae induced tlr expression in the lungs of wt mice but not tlr ko mice in vivo. because myd is a well-known signaling adaptor for tlr we determined whether myd is required for the s. pneumoniae-induced tlr expression in vivo. as shown in fig. 2d, s, pneumoniae-induced tlr up-regulation was significantly reduced in myd ko mice compared to wt mice. having demonstrated a marked decrease in tlr expression in tlr ko mice, we determined whether tlr4-deficiency caused this decrease via changing bacterial clearance in the lung tissues or inflammatory cell migration into airways. we therefore measured bacteria number from the lungs of s. pneumoniae-inoculated wt and tlr ko mice hrs after inoculation and also measured inflammatory cell migration into airway from the bal fluid of vehicle- or s. pneumoniae-inoculated wt and tlr ko mice hrs after inoculation. neither bacteria number of the lungs nor inflammatory cell migration into airways in tlr ko mice was significantly different from those in wt mice. the results reported herein demonstrate that, under the conditions reported here, tlr4-deficiency does not affect the bacterial clearance of s. pneumoniae and inflammatory cell migration response to s. pneumoniae, and decrease in s. pneumoniae-induced tlr expression in tlr ko mice is independent of both bacteria number and inflammatory cells in the lung in vivo. collectively, our data suggest that s. pneumoniae induces tlr up-regulation via tlr4-myd signaling pathway. because nf-κb plays an important role in mediating ply-induced host responses, we next determined if nf-κb is also involved in mediating tlr up-regulation by ply. as shown in fig. 2g &2h, s. pneumoniae induced nf-κb activation in a dose-dependent manner. similar result was also observed in ply-treated cells. we further determined the involvement of ikkα-iκbα, the key signaling pathway upstream of nf-κb, in tlr induction. as shown in fig. 2i, perturbing ikkα and iκbα signaling by overexpressing dn mutant plasmid inhibited s. pneumoniae-induced tlr mrna up-regulation. this finding was further confirmed by using ikkαsirna. thus it is clear that s. pneumoniae-induced tlr up-regulation is mediated by ikkα and iκbα-dependent nf-κb activation. s. pneumoniae induced-tlr up-regulation is negatively regulated by tumor suppressor cyld bacteria modulates many innate immune responses via induction of negative regulatory molecules. we previously reported that s. pneumoniae induces tumor suppressor cyld, an important negative regulator of nf-κb signaling pathway, in severe s. pneumoniae infection. nthi induced cyld expression, which in turn leads to negative regulation of nthi-induced nf-κb-dependent inflammation. cyld expression is relatively low in lung but is highly induced under diseased condition. to determine the role of cyld in s. pneumoniae-induced tlr expression in the lung and middle ear, we first evaluated up-regulation of cyld in vitro. as shown in fig. 3a, s. pneumoniae induced cyld expression not only in lung epithelia cell a but also in middle ear epithelial cell hmeec- to further confirm whether s. pneumoniae induces cyld both in the lung and middle ear in vivo, wt mice were intratracheally or transtympanically inoculated with s. pneumoniae, and cyld mrna expression was then measured in the lung and middle ear tissues of inoculated mice, respectively. as shown in fig. 3b, s. pneumoniae induced cyld expression in both lung and middle ear of the mice. to further investigate the role of cyld in s. pneumoniae-induced nf-κb activation, the effect of cyld knockdown using cyld sirna was measured. as shown in fig. 3c. s. pneumoniae- and ply-induced nf-κb promoter activity was greatly enhanced by cyld sirna. to investigate whether cyld acts as a negative regulator for s. pneumoniae-induced tlr expression, the effects of cyld sirna on s. pneumoniae-induced tlr mrna expression were measured. as shown in fig. 3d, knockdown of cyld expression using cyld sirna greatly enhanced s. pneumoniae-induced tlr mrna expression. to determine the in vivo role of cyld on s. pneumoniae-induced tlr expression, we measured tlr mrna expression in lung tissues of wt and cyld ko mice after s. pneumoniae inoculation. consistent with our in vitro findings, s. pneumoniae-induced tlr mrna up-regulation was enhanced in cyld ko mice. together, our data indicate that cyld acts as a negative regulator for s. pneumoniae-induced nf-κb activation and tlr up-regulation. s. pneumoniae synergizes with nthi to induce inflammatory response via tlr2 to determine whether s. pneumoniae synergizes with nthi to induce inflammatory response, we investigated the effect of s. pneumoniae and nthi on the expression of il-1β, a key nf-κb-dependent pro-inflammatory cytokine in lung and middle ear. as shown in fig. 4a, s. pneumoniae and nthi synergistically enhanced expression of il-1β? mrna in human lung epithelial cell. moreover, s. pneumoniae and nthi also synergistically induced expression of il-1β mrna in the lungs of mice. next we investigated the role of tlr in synergistic induction of nf-κb-dependent inflammatory response by s. pneumoniae and nthi. as shown in fig. 4c, sirna mediated knockdown of tlr markedly inhibited synergistic induction of il-1β mrna expression by s. pneumoniae and nthi, thereby providing evidence for the involvement of tlr in this synergistic induction. this finding was further confirmed in the cells from tlr ko mice in vitro. having shown that s. pneumoniae-induced tlr up-regulation is inhibited by tlr4-deficiency, and tlr up-regulation is closely associated with synergistic response by s. pneumoniae with nthi, we determined if tlr4-deficiency affects synergistic activation of nf-κb and subsequent gene expression by s. pneumoniae with nthi. we first measured nf-κb activation and mrna expressions of tlr and il-1β following co-treatment with s. pneumoniae and nthi in cells and lung tissues of wt and tlr ko mice. as shown in fig. 4e, nf-κb activation by s. pneumoniae with or without nthi was significantly decreased in the cells from tlr ko mice compared with those in cells from wt mice. in addition, mrna expressions of tlr and il-1β by s. pneumoniae with or without nthi in the lung tissues of tlr ko mice were also significantly decreased compared with those in wt mice. taken together, these findings suggest that s. pneumoniae synergizes with nthi to induce nf-κb activation and subsequent gene expression via tlr up-regulation. discussion there is a growing body of evidence showing that a portion of patients diagnosed with om or copd have mixed infections of nthi and s. pneumoniae. under in vivo situations such as polymicrobial infections, mucosal epithelial surfaces are simultaneously exposed to a variety of bacteria and bacterial components, and one microbe synergizes with another one to induce the inflammatory response by activating intracellular signaling pathway. among many other innate immune components, tlrs are critical for initiating and mediating immune response. thus, regulation of tlrs expression plays an important role in modulating host response to microbes. however, little is known about the molecular mechanisms underlying regulation of tlrs expression in mixed infection. as we previously reported, tlr expression is relatively low in unstimulated epithelial cells but is markedly up-regulated in response to invading microbes. however, the link between tlr up-regulation and mixed infection in otitis media and pneumonia still remains unknown. from what we have shown in the present data, it is evident that tlr expression is up-regulated by s. pneumoniae via a tlr4-myd88-nf-κb-dependent signaling pathway through its well-known virulence factor ply. we further show that s. pneumoniae and nthi synergize with each to induce nf-κb activation and il-1β expression dependently of tlr up-regulation. in the present study, we provided evidence for the first time that s. pneumoniae synergizes with nthi to induce nf-κb-dependent inflammatory response likely via tlr up-regulation in lung and middle ear epithelia cells, thereby bringing new insight into our understanding of the complex regulatory mechanisms underlying inflammation in polymicrobial infections. in addition to tlr and tlr tlr and have also been shown to be involved in s. pneumoniae infection. therefore we evaluated lung epithelial cell expression of tlrs and cd myd md and following s. pneumoniae treatment in a cells. in untreated cells, the expression level of tlr and was relatively high compared to those of tlr and interestingly, the expression of tlr and at the mrna level was highly up-regulated following s. pneumoniae treatment, implying a possible important role for tlr up-regulation in bacterial infections. indeed, the data we presented in this study demonstrated that the synergistic activation of nf-κb and inflammation by s. pneumoniae and nthi is dependent on tlr up-regulation. although there is strong evidence to support that tlr is a critical receptor for recognizing pneumolysin and pneumolysin is a major virulence factor of pneumococcal infection, the role of tlr in pneumococcal infection is not fully understood. it remains somewhat controversial whether tlr4-deficiency confers susceptibility or tolerance to pneumococcal infection in vivo. controversial phenotype of tlr4-deficiency mice in s. pneumoniae infection may be partly due to the strain differences of the mice used in each experiment, e.g. using c3h/hej, balb/c and c57bl/ as background strains. it is well known that different mouse strains exhibit a very different susceptibility to pneumococcal infection. balb/c mice, which were used as a background strain of tlr ko mice in our current experiment, were known as resistant strain in pneumococcal infection compared to other strains. tlr4-deficiency in this strain thus may have no significant effect on pneumococcal clearance. another interesting finding in this study is the experimental evidence for the negative regulation of s. pneumoniae-induced tlr up-regulation by tumor suppressor cyld. cyld was previously identified as an important regulator not only in tumor development but also in adaptive immune response. recent evidence demonstrated that cyld also serves as a key regulator for innate immune response via negative-crosstalk with mapks and nf-κb. moreover we recently identified an important role for cyld in modulating host antiviral response by regulating tlr expression in mixed infection of bacteria and virus. however, still unknown is how cyld regulates inflammatory response in mixed bacterial infection.  in the present study we showed for the first time that s. pneumoniae synergizes with nthi to induce nf-κb activation and the subsequent inflammatory response via up-regulation of tlr moreover, we showed that s. pneumoniae-induced tlr up-regulation is mediated by tlr4-myd88-ikkα-iκbα signaling pathway. interestingly, tumor suppressor cyld acts as a negative regulator for the synergistic induction of inflammation by s. pneumoniae and nthi, thereby implying a critical role for cyld in preventing overactive inflammatory response in mixed infections. this study may bring new insights into the molecular mechanisms underlying polymicrobial infections, especially mixed infection of gram-positive and negative bacteria. 
interestingly, tumor suppressor cyld acts as a negative regulator of s. pneumoniae-induced tlr up-regulation via negative-crosstalk with nf-κb signaling. here we provide direct evidence that s. pneumoniae synergizes with nthi to upregulate tlr expression in lung and middle ear of the mice. toll-like receptor plays a critical role in mediating inflammatory/immune responses against bacterial pathogens in lung. despite the relatively known intracellular signaling pathways involved in the synergistic induction of inflammation, it is still unclear if both bacterial pathogens also synergistically induce expression of surface tlr our study thus provides novel insights into the regulation of tlr expression in mixed bacterial infections. streptococcus pneumoniae and nontypeable haemophilus influenzae were previously reported to synergize with each other to induce inflammatory responses. moreover, s. pneumoniae ply induces tlr expression via a tlr4-myd88-nf-κb-dependent signaling pathway. pneumolysin appears to be the major virulence factor involved in this synergism.
 toll-like receptor plays a critical role in mediating inflammatory/immune responses against bacterial pathogens in lung. streptococcus pneumoniae and nontypeable haemophilus influenzae were previously reported to synergize with each other to induce inflammatory responses. despite the relatively known intracellular signaling pathways involved in the synergistic induction of inflammation, it is still unclear if both bacterial pathogens also synergistically induce expression of surface tlr  here we provide direct evidence that s. pneumoniae synergizes with nthi to upregulate tlr expression in lung and middle ear of the mice. pneumolysin appears to be the major virulence factor involved in this synergism. moreover, s. pneumoniae ply induces tlr expression via a tlr4-myd88-nf-κb-dependent signaling pathway. interestingly, tumor suppressor cyld acts as a negative regulator of s. pneumoniae-induced tlr up-regulation via negative-crosstalk with nf-κb signaling.  our study thus provides novel insights into the regulation of tlr expression in mixed bacterial infections. 