BACKGROUND
the gram-negative, anaerobic bacterium porphyromonas gingivalis is considered to be one of the key pathogens in periodontitis, an inflammatory polymicrobial condition leading to bone and tissue destruction and eventually tooth loss  <cit> .
increasing evidence associate periodontitis to systemic diseases  <cit>  and for instance, p. gingivalis has been found in atherosclerotic plaques  <cit>  as well as in non-healing ulcers .
p. gingivalis possess a number of pathogenic properties to enhance growth and survival such as fimbriae, lipopolysaccharides and gingipains.
the gingipains, which are grouped into lysine-specific  and arginine-specifik  gingipains due to their specificity for cleavage after lysyl and arginyl residues, respectively, are cysteine proteases that have been linked to the establishment and growth of p. gingivalis.
the gingipains are, like the fimbriae, important for the bacterial invasion and colonization.
they are reactive against an array of different proteins, e.g.
proteins of the complement and kallikrein system, coagulation factors and cytokines.
of particular interest, accumulating data shows that gingipains are involved in the regulation of host inflammatory responses.
p. gingivalis stimulates an innate immune response and induces expression of inflammatory mediators, but can at the same time downregulate the host response.
in other words, p. gingivalis has evolved several mechanisms to evade host immune system by invasion of host cells and disrupting signalling pathways by cytokine and receptor degradation  <cit> .
periodontitis is a chronic inflammation with associated bone-resorption and tissue destruction.
this degenerative process is mainly a consequence of the hosts attempt to eliminate the bacterial load rather than the bacteria themselves.
as a consequence to bacterial encounter, the host cells synthesize and release mediators attracting inflammatory cells to the site of infection, which in turn contribute to the characteristic tissue and bone destruction by release of proteolytic enzymes, induction of osteoclast formation and apoptosis of cells  <cit> .
one important chemokine that attracts neutrophils to the site of infection is cxcl <dig>  cxcl <dig> is expressed and produced by different cell types, including fibroblasts, neutrophils, endothelial cells, keratinocytes, epithelial cells and lymphocytes  <cit> .
innate immunity defence against invading pathogens involves their sensing through highly conserved pattern recognition receptors .
these receptors, including toll like receptors , are expressed by a variety of cells, both immune and none-immune cells.
for instance, human gingival fibroblasts  are likely to encounter microbial invasion at an early stage of periodontitis and interact with bacteria and bacterial products, and several studies report a role of hgfs in periodontal inflammation  <cit> .
hgfs have been shown to express tlrs and, although there are some inconsistencies in which tlrs that are expressed, the importance of fibroblasts in innate immunity has started to be revealed.
mahanonda and colleagues reported that hgfs express functional tlr  <dig>   <dig>   <dig> and  <dig>  and that ligand binding to these receptors lead to the secretion of cxcl <dig>  <cit> .
uehara et al.
demonstrated that hgfs express tlr 1– <dig>  and that stimulation of tlr 2/ <dig>   <dig>   <dig>  7/ <dig> and  <dig> caused production of several inflammatory mediators  <cit> .
however, increasing data suggest that fibroblasts are heterogeneous.
fibroblasts from different anatomic sites, and even subpopulations of fibroblasts from the same site, display distinct differences in morphology, extracellular matrix production, migratory phenotype and cell surface antigens  <cit> .
recently, our group showed that p. gingivalis target t cell derived interleukin   <dig> at the protein level and suppresses activator protein  <dig>  a mechanism by which p. gingivalis benefits its own establishment by altering adaptive immune responses  <cit> .
the aim of the present study is to characterize the effects of p. gingivalis on primary human fibroblasts and their derived inflammatory responses, with the hypothesis that initial establishment of p. gingivalis infection modulates immunoregulatory mechanisms of fibroblasts.
methods
isolation and culture of fibroblasts
primary human skin fibroblasts were isolated by explanting pieces of dermis obtained from elective abdominal or chest surgery from three young donors.
the tissue was removed using standard surgical procedures.
approval from the local ethical committee at Örebro county council, sweden, , and informed consent was obtained from each patient.
fibroblasts were propagated from dermal preparations pieces by the explant technique.
in brief, small pieces  of dermis were allowed to adhere to culture plastic for a few minutes followed by addition of culture medium  supplemented with 10% fetal bovine serum  and 1 mg/ml gentamicin .
gingival fibroblasts  were purchased from the american type collection .
the fibroblasts were cultured to confluence and removed from culture plastic surface by incubation in  <dig> % trypsin and 1 mm edta  at 37°c for 5 minutes.
the cells were plated in tissue culture flasks in dmem with 10% fbs.
fibroblasts were used at passages 3– <dig>
preparation of p. gingivalis
p. gingivalis atcc  <dig>  was cultured in fastidious anaerobe broth  under anaerobic conditions  at 37°c in an anaerobic chamber .
the bacteria were harvested by centrifugation, washed and resuspended in krebs-ringer glucose buffer  .
heat-killed p. gingivalis was prepared by incubation at 70°c for 1 h. to ensure that the bacteria were killed, 10 μl of the heat-killed suspension was spread on a fastidious anaerobe agar plate and incubated at 37°c for five days.
coculture of p. gingivalis and fibroblasts
in  <dig>  ml dmem supplemented with 10% fbs, primary dermal fibroblasts from each subject or gingival fibroblasts were seeded with a density of  <dig>  cells/well in a 24-wells plate .
after 24 hours, the fibroblasts were washed twice with phosphate buffered saline   and  <dig>  ml serumfree dmem was added.
after 24 hour of starvation, the medium was replaced with dmem supplemented with 1% fbs.
the cells were thereafter treated with viable p. gingivalis, at a multiplicity of infection  of 1: <dig>  1: <dig>  1: <dig> or 1: <dig>  or heat-killed p. gingivalis .
the cocultures were incubated for  <dig>   <dig>  or 24 hours in 37°c in 5% co <dig>
cxcl <dig> accumulation was induced by pre-stimulating fibroblasts with tumor necrosis factor-α   for 6 hours prior to infection with p. gingivalis.
the fibroblasts were stimulated with the previously mentioned concentrations of viable or heat-killed bacteria, respectively, for 24 hours in 37°c in 5% co <dig>
to evaluate the role of gingipains, p. gingivalis was incubated with the arg-gingipain inhibitor leupeptin  or the lys-gingipain inhibitor cathepsin b inhibitor ii , for 1 hour prior to fibroblast stimulation.
after stimulation with viable and heat-killed p. gingivalis, and/or tnf-α, leupeptin as well as cathepsin b inhibitor ii, for  <dig>   <dig> or 24 hours, the supernatants were collected and stored in aliquots at −80°c prior to immunoassays.
fitc-labeling of p. gingivalis
p. gingivalis was washed three times with pbs by centrifugation at 12000 rpm for three minutes, whereby the bacteria were resuspended in buffered saline  containing  <dig>  mg/ml fluorescein isothiocyanate isomer  , and incubated in darkness at room-temperature for 45 minutes.
the bacteria were washed in pbs prior to fibroblast infection.
fluorescence microscopy
for fluorescence microscopy, fibroblasts were seeded on coverslips in multiwell plates and incubated for 24 hours.
the fibroblasts were stimulated with fitc-labeled p. gingivalis  for 6 hours.
the cells were washed twice with pbs, fixed with 4% paraformaldehyde  for 30 min at room temperature and washed with pbs.
f-actin was visualized by incubating the cells with  <dig> units alexa fluor®  <dig> phalloidin and 100 μg/ml lysophosphatidylcholine in darkness for 1 h at room temperature.
the nucleus was counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole, dihydrochloride  for 2 min .
determination of cytokine production
cxcl <dig> was measured by human il- <dig> elisa max deluxe set  according to the manufacturer’s instructions.
all samples were run in duplicates.
for the parallel determination of the relative levels of cytokines and chemokines, human cytokine array panel a  was performed according the manufacturer’s instructions.
briefly, cell culture supernatants from representative experiments were mixed with a cocktail of biotinylated detection antibodies and the sample/antibody mixture was incubated with the array where capture antibodies were spotted in duplicate on a nitrocellulose membrane.
any formed cytokine/detection antibody complex was then bound by its immobilized capture antibody on the membrane.
detection was performed by adding streptavidin-horseradish peroxidase and chemiluminescent detection reagents, and the signal produced was in proportion to the amount of cytokine bound.
chemiluminescence was detected in the same manner as a western blot .
the array determined the relative levels of  <dig> different cytokines, chemokines and acute phase proteins .
data analysis
cxcl <dig> experiments were performed in three independent experiments  in duplicates to confirm the reproducibility of the results.
experiments with human gingival fibroblasts were performed in three independent experiments.
statistical analysis with student’s t-test was performed using graphpad prism .
all data are presented as mean values with standard deviation.
a value of p <  <dig>  was considered statistically significant.
one experiment was performed for the cytokine array.
RESULTS
p. gingivalis invades fibroblasts
the morphology of fibroblasts following treatment with different concentrations of viable and heat-killed p. gingivalis was examined by light microscopy.
no obvious morphological changes induced by the bacteria were observed .
the interaction between p. gingivalis and fibroblasts was visualized by fluorescence microscopy.
we found that p. gingivalis after 6 h effectively adhered to and invaded the fibroblasts .
p. gingivalis affects the level of cxcl <dig> in a dose- and time-dependent manner
primary fibroblasts  were stimulated with different concentrations of viable p. gingivalis, as well as heat-killed p. gingivalis, for 1 h, 6 h or 24 h .
the highest concentration  of either viable or heat-killed p. gingivalis significantly increased cxcl <dig> expression after short-term exposure , whereas lower concentrations of viable p. gingivalis  did not change the cxcl <dig> level compared to the unstimulated control.
however, long-term treatment  with viable bacteria  resulted in a significant reduction in cxcl <dig> levels.
although not consistently statistically significant for all concentrations of viable bacteria tested, there is a tendency for decreasing cxcl <dig> levels with increasing moi.
heat-killed p. gingivalis  resulted in elevated cxcl <dig> production both after short- and long-term exposure of fibroblasts.
p. gingivalis is involved in the degradation of cxcl <dig> protein
we thereafter aimed to determine if the decreased levels of cxcl <dig>  in response to viable p. gingivalis, were due to protein degradation.
the fibroblasts were pre-treated with 50 ng/ml tnf-α for 6 hours to induce cxcl <dig> expression and accumulation.
thereafter, the fibroblasts were incubated with viable p. gingivalis  or heat-killed p. gingivalis  for 24 hours.
the fibroblasts synthesized high levels of cxcl <dig> in response to tnf-α, which was further enhanced in the presence of viable p. gingivalis at moi: <dig>  however, higher concentrations of viable p. gingivalis , completely abolished the tnf-α-induced accumulation of cxcl <dig> .
in contrast, however, heat-killed p. gingivalis did not suppress tnf-α triggered cxcl <dig> levels .
these results were further confirmed by using gingival fibroblasts stimulated with viable and heat-killed p. gingivalis, with and without tnf-α pre-stimulation.
cxcl <dig> basal levels were suppressed by viable p. gingivalis and by heat-killed p. gingivalis .
furthermore, tnf-α-induced cxcl <dig> expression was suppressed below basal levels by viable bacteria, while heat-killed bacteria showed no alteration in the pre-accumulated cxcl <dig> levels.
cxcl <dig> degradation is due to arginine-gingipains
to determine if p. gingivalis suppresses tnf-α induced cxcl <dig> release through kgp and rgp activities, viable p. gingivalis was incubated for 1 hour with increasing concentrations  of cathepsin b ii inhibitor or leupeptin, before fibroblast infection.
the fibroblasts were pre-stimulated with 50 ng/ml tnf-α for 6 hours and then incubated for 24 hours with treated or non-treated p.gingivalis.
the rgp inhibitor leupeptin significantly reversed the p. gingivalis-induced suppression of cxcl <dig> at all concentrations , whereas cathepsin b ii inhibitor at 1 mm only slightly changed the cxcl <dig> level .
p. gingivalis targets a wide range of fibroblast-derived inflammatory mediators
to examine if the immunomudulatory role of p. gingivalis accounts for inflammatory mediators other than cxcl <dig>  a parallel determination of cytokines and chemokines was performed with a cytokine array .
primary dermal fibroblasts  were stimulated with 50 ng/ml tnf-α for 6 h before the cells were incubated with viable or heat-killed p. gingivalis, , respectively .
non-stimulated fibroblasts were used as a control.
tnf-α alone, or in combination with heat-killed p. gingivalis, induced secretion of tnf-α itself, as well as serpin- <dig>  il- <dig>  ccl <dig>  ccl <dig>  cxcl <dig>  cxcl <dig> and cxcl <dig>  on the other hand, the levels of these inflammatory mediators, except tnf-α and serpine- <dig>  were markedly suppressed by viable p. gingivalis.
heat-killed p. gingivalis did not change the tnf-α induced expression of the different inflammatory mediators, except an inhibition of cxcl <dig> and an enhancement  of tnf-α.
the level of serpine- <dig> was consistently expressed at high levels independently of stimulation with tnf-α and/or bacteria.
discussion
the aim of the present study was to characterize the effects of p. gingivalis on human fibroblast inflammatory responses.
the connection between periodontitis and atherosclerosis, as well as other systemic diseases, has suggested a role for periodontitis-induced bacteremia, including p. gingivalis, in stimulating and maintaining a chronic state of inflammation  <cit> .
for instance, p. gingivalis dna has been detected in atherosclerotic plaques  <cit>  and in non-healing ulcers , however, to our knowledge, no previous studies on p. gingivalis infection of primary, human dermal fibroblasts have been performed.
the fibroblasts are a source of connective tissue that maintain tissue haemostasis and integrity, and play an important role in tissue generation after wounding as well as in the pathogenesis of fibrotic inflammatory diseases and excessive scarring involving extracellular matrix accumulation  <cit> .
likewise, these cells have an active role in the innate immunity, although the immunity properties of fibroblasts have just begun to be revealed and many characteristics remain to be established  <cit> .
in this study, we show that human skin fibroblasts, as well as human gingival fibroblasts, play an important part of the innate immune system by sensing microbial invasion and respond to it by producing and secreting inflammatory mediators, notably chemokines.
furthermore, we demonstrate that p. gingivalis has a direct modulatory function of the immune response of fibroblasts through the catalytic activities of gingipains targeting fibroblast-derived inflammatory mediators at the protein level.
fluorescent micrographs showed that viable p. gingivalis adhered to and invaded dermal fibroblasts, suggesting that p. gingivalis utilizes strategies to evade the host immune response.
this is in line with other studies that have shown p. gingivalis adhesion and invasion of oral epithelial cells, mainly mediated by gingipains and major fimbriae a. invasion of epithelial cells, as well as gingival fibroblasts, is probably a mechanism applied by the bacteria to evade the host immune system and cause tissue damage, an important part of the pathogenesis of periodontitis  <cit> .
for instance, this fimbriated strain of p. gingivalis has previously been shown to invade gingival epithelial cells after 90 minutes of incubation  <cit> .
in this study we observed that p. gingivalis invaded dermal fibroblasts and had established an infection after six hours of incubation.
in addition, after six hours of incubation was the cxcl <dig> level significantly reduced by p. gingivalis.
consistent with previous observations  <cit> , we show that short-term exposure of viable or heat-killed p. gingivalis  induces cxcl <dig> production in fibroblasts.
however, after  <dig> and 24 hours of incubation, viable p. gingivalis suppressed basal cxcl <dig> accumulation.
on the contrary, heat-killed p. gingivalis increased cxcl <dig> levels, indicating that p. gingivalis possess heat-instable structures that are responsible for the degradation of cxcl <dig>  in correlation, previous studies have shown that heat-killed p. gingivalis induces higher levels of inflammatory mediators, in particular il- <dig> and cxcl <dig>  than viable bacteria, suggesting degradation by the heat-instable gingipains  <cit> .
to further investigate the effect of p. gingivalis on cxcl <dig>  the fibroblasts were pre-stimulated with tnf-α, a well known inducer of inflammatory mediators.
lower doses of viable p. gingivalis  in combination with tnf-α did not alter cxcl <dig> levels when compared to the positive tnf-α-stimulated control.
however, higher concentrations  completely abolished the tnf-α-induced cxcl <dig> accumulation, while corresponding concentration of heat-killed p. gingivalis  did not cause the same effects.
this further implies that the suppression of cxcl <dig> is due to the proteolytic capacities of the gingipains.
to test this theory and evaluate the importance of gingipains, we used cathepsin b inhibitor ii and leupeptin, inhibitors of kgp and rgp, respectively.
we found that p. gingivalis-mediated degradation is mainly dependent on rgp.
these findings are consistent with our previous findings, as well as results from others, showing that the gingipains from p. gingivalis degrades il- <dig> and cxcl <dig>  respectively  <cit> .
however, inhibition of rgp could only partially restore the cxcl <dig> levels, suggesting involvement of other proteolytic enzymes.
it is also possible that a combination of rgp and kgp has a synergistic degradative effect, mediated by their specificity for cleavage after arginyl and lysyl residues, respectively.
furthermore, dias and colleagues showed that there are two main types of cxcl <dig>  a  <dig> amino acid variant, secreted by immune cells, and a  <dig> amino acid variant, secreted by non-immune cells.
the latter was shown to have a lower chemotactic activity than the immune cell derived variant.
however, upon cleavage by gingipains this shifted, and the  <dig> amino acid variant increased the chemotactic activity of neutrophils compared to the  <dig> amino acid variant  <cit> .
although gingipains degrade cxcl <dig>  this consequently suggests that the chemotactic activity of cxcl <dig> increases upon stimulation of fibroblasts with p. gingivalis.
however, more research is needed to determine the effects of p. gingivalis-derived proteolytic enzymes on the activity of these cxcl <dig> variants.
to investigate whether the gingipain-mediated effects of p. gingivalis also include other fibroblast-derived inflammatory mediators, we performed a relative cytokine assay which measured various cytokines and chemokines.
this assay revealed that tnf-α stimulated primary, human skin fibroblasts produce cxcl <dig>  tnf-α, il- <dig>  ccl <dig>  ccl <dig>  cxcl <dig> and cxcl <dig>  remarkably, the fibroblasts produced mostly chemokines, indicating that fibroblasts might play an important role as a link between the innate and the acquired immunity.
all tnf-α induced inflammatory mediators, except tnf-α, were suppressed by viable p. gingivalis, strongly suggesting an effect of the gingipains per se.
this shows that gingipains have a broad proteolytic capacity and targets a wide array of cytokines and chemokines, thereby interrupting several signaling pathways.
the chemokines ccl <dig>  ccl <dig>  cxcl <dig> as well as cxcl <dig> are all important for recruiting immune cells to the site of infection, and by inhibiting their biological activity, p. gingivalis is able to modulate and diminish the level of infiltrating immune cells.
in contrast, viable p. gingivalis was not able to suppress tnf-α which is one of the most important inflammatory mediators.
in fact, the level of tnf-α increased nearly two-fold by heat-killed bacteria, showing that p. gingivalis induce tnf-α expression in fibroblasts and, at the same time, degrade the tnf-α protein, although not extensively.
periodontitis is associated with a decreased abundance of fibroblasts  <cit>  and tnf-α has been shown to be an important mediator of p. gingivalis-induced apoptosis.
graves et al.
demonstrated that the numbers of apoptotic fibroblasts were significantly reduced in the absence of the tnf-receptor, suggesting that tnf-α-signalling is an important part in apoptosis of fibroblasts  <cit> .
thus, our results may indicate that p. gingivalis stimulates apoptosis of fibroblasts through a less extensive degradation of tnf-α and this could account for the fibroblast apoptosis that is a distinctive feature of periodontitis.
nevertheless, the degree of apoptotic fibroblasts after p. gingivalis infection need to be further investigated.
in addition, it has been shown that the first nine residues of tnf-α n terminus are not needed for tnf-α protein to exhibit its biological activity  <cit> .
calkins and colleagues demonstrated that the two types of gingipains are able to individually degrade tnf-α, and also eliminate the biological activity  <cit> .
cxcl <dig> is a chemokine with pleiotropic functions.
it works as a chemoattractant for its cxcr <dig>  positive cells such as t cells, eosinophils, monocytes and nk cells, and it has also the capacity to induce apoptosis and regulate cell growth and proliferation, as well as angiogenesis  <cit> .
of interest, cxcl <dig> was the only chemokine that was suppressed by heat-killed as well as viable p. gingivalis, indicating that this chemokine is regulated by some additional mechanism beside that of heat-instable gingipains.
for instance, a study by ohno et al.
showed that cxcl <dig> and ccl <dig> gene is induced by p. gingivalis in osteoblasts and st <dig> mouse stromal cells and that nfκb inhibitor suppressed ccl <dig> expression but not cxcl <dig>  <cit> .
this suggest that p. gingivalis modulates cxcl <dig> gene expression through an nfκb-independent pathway.
of further interest, the expression of cxcl <dig> is increased in autoimmunity diseases like rheumatoid arthritis and multiple sclerosis.
for instance, lee et al.
reported that cxcl <dig> contributes to bone-resorption by inducing osteoclast formation in rheumatoid arthritis via induction of receptor activator of nfκb ligand   <cit> .
however, since bone-resorption is a hallmark of progressive periodontitis, our results may indicate that cxcl <dig> plays a minor role when it comes to bone-resorption since even heat-killed p. gingivalis totally suppressed cxcl <dig>  besides, high levels of cxcl <dig> have receptor-independent anti-microbial properties.
even though it is questionable if such high levels , i.e.
concentrations 100-fold higher than needed for chemotactic function, are realistic in vivo, prost and colleagues showed that this antimicrobial activity is achievable in vitro and may be an important response against bacterial infection  <cit> .
thus, the strong suppressive effect of cxcl <dig> by both viable and heat-killed p. gingivalis may in this case be beneficial for a sustained p. gingivalis infection.
anyway, further research is needed about the regulation of cxcl <dig> and its signaling pathways as well as its role in bacterial infection.
serpin- <dig> , was continuously expressed regardless of stimulation with tnf-α and/or bacteria.
serpin- <dig> plays an integrated part of the plasmin system, working as an inhibitor of tissue plasminogen activator as well as urokinase plasminogen activator, both of which converts plasminogen to plasmin.
interestingly, serpin- <dig> has been implicated in fibroblast senescent.
serpin- <dig> is induced by various growth factors and has been suggested to be a down-stream target of p <dig>  where p <dig> controls growth factor-dependent proliferation by upregulating serpin- <dig>  <cit> .
however, the fibroblast strains in our experiments were used at low passages.
CONCLUSIONS
in conclusion, our results show that a broad range of fibroblast-derived inflammatory mediators are inactivated by p. gingivalis due to proteolytic activities of gingipains, whereby the bacteria can create a more favourable milieu where it can evade the host immune system and promote its own growth and establishment.
also, by differentially regulate the inflammatory mediators, such as cxcl <dig> and tnf-α, p. gingivalis may have an impact on the composition of inflammatory cells infiltrate and the inflammatory process itself.
increased understanding of the role of fibroblasts in innate and acquired immunity and their interaction with periodontal bacteria is crucial for developing new strategies for preventing and treating periodontitis and related chronic inflammatory diseases.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
hk, ep and tb designed the study.
ep wrote the manuscript with hk and tb.
ep and hk performed the experiments.
all authors read and approved the final manuscript.

 the gram-negative, anaerobic bacterium porphyromonas gingivalis is considered to be one of the key pathogens in periodontitis, an inflammatory polymicrobial condition leading to bone and tissue destruction and eventually tooth loss. increasing evidence associate periodontitis to systemic diseases and for instance, p. gingivalis has been found in atherosclerotic plaques as well as in non-healing ulcers. p. gingivalis possess a number of pathogenic properties to enhance growth and survival such as fimbriae, lipopolysaccharides and gingipains. the gingipains, which are grouped into lysine-specific and arginine-specifik gingipains due to their specificity for cleavage after lysyl and arginyl residues, respectively, are cysteine proteases that have been linked to the establishment and growth of p. gingivalis. the gingipains are, like the fimbriae, important for the bacterial invasion and colonization. they are reactive against an array of different proteins, e.g. proteins of the complement and kallikrein system, coagulation factors and cytokines. of particular interest, accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. p. gingivalis stimulates an innate immune response and induces expression of inflammatory mediators, but can at the same time downregulate the host response. in other words, p. gingivalis has evolved several mechanisms to evade host immune system by invasion of host cells and disrupting signalling pathways by cytokine and receptor degradation. periodontitis is a chronic inflammation with associated bone-resorption and tissue destruction. this degenerative process is mainly a consequence of the hosts attempt to eliminate the bacterial load rather than the bacteria themselves. as a consequence to bacterial encounter, the host cells synthesize and release mediators attracting inflammatory cells to the site of infection, which in turn contribute to the characteristic tissue and bone destruction by release of proteolytic enzymes, induction of osteoclast formation and apoptosis of cells. one important chemokine that attracts neutrophils to the site of infection is cxcl cxcl is expressed and produced by different cell types, including fibroblasts, neutrophils, endothelial cells, keratinocytes, epithelial cells and lymphocytes. innate immunity defence against invading pathogens involves their sensing through highly conserved pattern recognition receptors. these receptors, including toll like receptors, are expressed by a variety of cells, both immune and none-immune cells. for instance, human gingival fibroblasts are likely to encounter microbial invasion at an early stage of periodontitis and interact with bacteria and bacterial products, and several studies report a role of hgfs in periodontal inflammation. hgfs have been shown to express tlrs and, although there are some inconsistencies in which tlrs that are expressed, the importance of fibroblasts in innate immunity has started to be revealed. mahanonda and colleagues reported that hgfs express functional tlr and and that ligand binding to these receptors lead to the secretion of cxcl. uehara et al. demonstrated that hgfs express tlr 1– and that stimulation of tlr 2/ 7/ and caused production of several inflammatory mediators. however, increasing data suggest that fibroblasts are heterogeneous. fibroblasts from different anatomic sites, and even subpopulations of fibroblasts from the same site, display distinct differences in morphology, extracellular matrix production, migratory phenotype and cell surface antigens. recently, our group showed that p. gingivalis target t cell derived interleukin at the protein level and suppresses activator protein a mechanism by which p. gingivalis benefits its own establishment by altering adaptive immune responses. the aim of the present study is to characterize the effects of p. gingivalis on primary human fibroblasts and their derived inflammatory responses, with the hypothesis that initial establishment of p. gingivalis infection modulates immunoregulatory mechanisms of fibroblasts. methods isolation and culture of fibroblasts primary human skin fibroblasts were isolated by explanting pieces of dermis obtained from elective abdominal or chest surgery from three young donors. the tissue was removed using standard surgical procedures. approval from the local ethical committee at Örebro county council, sweden,, and informed consent was obtained from each patient. fibroblasts were propagated from dermal preparations pieces by the explant technique. in brief, small pieces of dermis were allowed to adhere to culture plastic for a few minutes followed by addition of culture medium supplemented with 10% fetal bovine serum and 1 mg/ml gentamicin. gingival fibroblasts were purchased from the american type collection. the fibroblasts were cultured to confluence and removed from culture plastic surface by incubation in % trypsin and 1 mm edta at 37°c for 5 minutes. the cells were plated in tissue culture flasks in dmem with 10% fbs. fibroblasts were used at passages 3– preparation of p. gingivalis p. gingivalis atcc was cultured in fastidious anaerobe broth under anaerobic conditions at 37°c in an anaerobic chamber. the bacteria were harvested by centrifugation, washed and resuspended in krebs-ringer glucose buffer. heat-killed p. gingivalis was prepared by incubation at 70°c for 1 h. to ensure that the bacteria were killed, 10 μl of the heat-killed suspension was spread on a fastidious anaerobe agar plate and incubated at 37°c for five days. coculture of p. gingivalis and fibroblasts in  ml dmem supplemented with 10% fbs, primary dermal fibroblasts from each subject or gingival fibroblasts were seeded with a density of cells/well in a 24-wells plate. after 24 hours, the fibroblasts were washed twice with phosphate buffered saline and  ml serumfree dmem was added. after 24 hour of starvation, the medium was replaced with dmem supplemented with 1% fbs. the cells were thereafter treated with viable p. gingivalis, at a multiplicity of infection of 1: 1: 1: or 1: or heat-killed p. gingivalis. the cocultures were incubated for or 24 hours in 37°c in 5% co cxcl accumulation was induced by pre-stimulating fibroblasts with tumor necrosis factor-α for 6 hours prior to infection with p. gingivalis. the fibroblasts were stimulated with the previously mentioned concentrations of viable or heat-killed bacteria, respectively, for 24 hours in 37°c in 5% co to evaluate the role of gingipains, p. gingivalis was incubated with the arg-gingipain inhibitor leupeptin or the lys-gingipain inhibitor cathepsin b inhibitor ii, for 1 hour prior to fibroblast stimulation. after stimulation with viable and heat-killed p. gingivalis, and/or tnf-α, leupeptin as well as cathepsin b inhibitor ii, for or 24 hours, the supernatants were collected and stored in aliquots at −80°c prior to immunoassays. fitc-labeling of p. gingivalis p. gingivalis was washed three times with pbs by centrifugation at 12000 rpm for three minutes, whereby the bacteria were resuspended in buffered saline containing  mg/ml fluorescein isothiocyanate isomer, and incubated in darkness at room-temperature for 45 minutes. the bacteria were washed in pbs prior to fibroblast infection. fluorescence microscopy for fluorescence microscopy, fibroblasts were seeded on coverslips in multiwell plates and incubated for 24 hours. the fibroblasts were stimulated with fitc-labeled p. gingivalis for 6 hours. the cells were washed twice with pbs, fixed with 4% paraformaldehyde for 30 min at room temperature and washed with pbs. f-actin was visualized by incubating the cells with units alexa fluor® phalloidin and 100 μg/ml lysophosphatidylcholine in darkness for 1 h at room temperature. the nucleus was counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole, dihydrochloride for 2 min. determination of cytokine production cxcl was measured by human il- elisa max deluxe set according to the manufacturer’s instructions. all samples were run in duplicates. for the parallel determination of the relative levels of cytokines and chemokines, human cytokine array panel a was performed according the manufacturer’s instructions. briefly, cell culture supernatants from representative experiments were mixed with a cocktail of biotinylated detection antibodies and the sample/antibody mixture was incubated with the array where capture antibodies were spotted in duplicate on a nitrocellulose membrane. any formed cytokine/detection antibody complex was then bound by its immobilized capture antibody on the membrane. detection was performed by adding streptavidin-horseradish peroxidase and chemiluminescent detection reagents, and the signal produced was in proportion to the amount of cytokine bound. chemiluminescence was detected in the same manner as a western blot. the array determined the relative levels of different cytokines, chemokines and acute phase proteins. data analysis cxcl experiments were performed in three independent experiments in duplicates to confirm the reproducibility of the results. experiments with human gingival fibroblasts were performed in three independent experiments. statistical analysis with student’s t-test was performed using graphpad prism. all data are presented as mean values with standard deviation. a value of p <  was considered statistically significant. one experiment was performed for the cytokine array.  p. gingivalis invades fibroblasts the morphology of fibroblasts following treatment with different concentrations of viable and heat-killed p. gingivalis was examined by light microscopy. no obvious morphological changes induced by the bacteria were observed. the interaction between p. gingivalis and fibroblasts was visualized by fluorescence microscopy. we found that p. gingivalis after 6 h effectively adhered to and invaded the fibroblasts. p. gingivalis affects the level of cxcl in a dose- and time-dependent manner primary fibroblasts were stimulated with different concentrations of viable p. gingivalis, as well as heat-killed p. gingivalis, for 1 h, 6 h or 24 h. the highest concentration of either viable or heat-killed p. gingivalis significantly increased cxcl expression after short-term exposure, whereas lower concentrations of viable p. gingivalis did not change the cxcl level compared to the unstimulated control. however, long-term treatment with viable bacteria resulted in a significant reduction in cxcl levels. although not consistently statistically significant for all concentrations of viable bacteria tested, there is a tendency for decreasing cxcl levels with increasing moi. heat-killed p. gingivalis resulted in elevated cxcl production both after short- and long-term exposure of fibroblasts. p. gingivalis is involved in the degradation of cxcl protein we thereafter aimed to determine if the decreased levels of cxcl in response to viable p. gingivalis, were due to protein degradation. the fibroblasts were pre-treated with 50 ng/ml tnf-α for 6 hours to induce cxcl expression and accumulation. thereafter, the fibroblasts were incubated with viable p. gingivalis or heat-killed p. gingivalis for 24 hours. the fibroblasts synthesized high levels of cxcl in response to tnf-α, which was further enhanced in the presence of viable p. gingivalis at moi: however, higher concentrations of viable p. gingivalis, completely abolished the tnf-α-induced accumulation of cxcl. in contrast, however, heat-killed p. gingivalis did not suppress tnf-α triggered cxcl levels. these results were further confirmed by using gingival fibroblasts stimulated with viable and heat-killed p. gingivalis, with and without tnf-α pre-stimulation. cxcl basal levels were suppressed by viable p. gingivalis and by heat-killed p. gingivalis. furthermore, tnf-α-induced cxcl expression was suppressed below basal levels by viable bacteria, while heat-killed bacteria showed no alteration in the pre-accumulated cxcl levels. cxcl degradation is due to arginine-gingipains to determine if p. gingivalis suppresses tnf-α induced cxcl release through kgp and rgp activities, viable p. gingivalis was incubated for 1 hour with increasing concentrations of cathepsin b ii inhibitor or leupeptin, before fibroblast infection. the fibroblasts were pre-stimulated with 50 ng/ml tnf-α for 6 hours and then incubated for 24 hours with treated or non-treated p.gingivalis. the rgp inhibitor leupeptin significantly reversed the p. gingivalis-induced suppression of cxcl at all concentrations, whereas cathepsin b ii inhibitor at 1 mm only slightly changed the cxcl level. p. gingivalis targets a wide range of fibroblast-derived inflammatory mediators to examine if the immunomudulatory role of p. gingivalis accounts for inflammatory mediators other than cxcl a parallel determination of cytokines and chemokines was performed with a cytokine array. primary dermal fibroblasts were stimulated with 50 ng/ml tnf-α for 6 h before the cells were incubated with viable or heat-killed p. gingivalis,, respectively. non-stimulated fibroblasts were used as a control. tnf-α alone, or in combination with heat-killed p. gingivalis, induced secretion of tnf-α itself, as well as serpin- il- ccl ccl cxcl cxcl and cxcl on the other hand, the levels of these inflammatory mediators, except tnf-α and serpine- were markedly suppressed by viable p. gingivalis. heat-killed p. gingivalis did not change the tnf-α induced expression of the different inflammatory mediators, except an inhibition of cxcl and an enhancement of tnf-α. the level of serpine- was consistently expressed at high levels independently of stimulation with tnf-α and/or bacteria. discussion the aim of the present study was to characterize the effects of p. gingivalis on human fibroblast inflammatory responses. the connection between periodontitis and atherosclerosis, as well as other systemic diseases, has suggested a role for periodontitis-induced bacteremia, including p. gingivalis, in stimulating and maintaining a chronic state of inflammation. for instance, p. gingivalis dna has been detected in atherosclerotic plaques and in non-healing ulcers, however, to our knowledge, no previous studies on p. gingivalis infection of primary, human dermal fibroblasts have been performed. the fibroblasts are a source of connective tissue that maintain tissue haemostasis and integrity, and play an important role in tissue generation after wounding as well as in the pathogenesis of fibrotic inflammatory diseases and excessive scarring involving extracellular matrix accumulation. likewise, these cells have an active role in the innate immunity, although the immunity properties of fibroblasts have just begun to be revealed and many characteristics remain to be established. in this study, we show that human skin fibroblasts, as well as human gingival fibroblasts, play an important part of the innate immune system by sensing microbial invasion and respond to it by producing and secreting inflammatory mediators, notably chemokines. furthermore, we demonstrate that p. gingivalis has a direct modulatory function of the immune response of fibroblasts through the catalytic activities of gingipains targeting fibroblast-derived inflammatory mediators at the protein level. fluorescent micrographs showed that viable p. gingivalis adhered to and invaded dermal fibroblasts, suggesting that p. gingivalis utilizes strategies to evade the host immune response. this is in line with other studies that have shown p. gingivalis adhesion and invasion of oral epithelial cells, mainly mediated by gingipains and major fimbriae a. invasion of epithelial cells, as well as gingival fibroblasts, is probably a mechanism applied by the bacteria to evade the host immune system and cause tissue damage, an important part of the pathogenesis of periodontitis. for instance, this fimbriated strain of p. gingivalis has previously been shown to invade gingival epithelial cells after 90 minutes of incubation. in this study we observed that p. gingivalis invaded dermal fibroblasts and had established an infection after six hours of incubation. in addition, after six hours of incubation was the cxcl level significantly reduced by p. gingivalis. consistent with previous observations, we show that short-term exposure of viable or heat-killed p. gingivalis induces cxcl production in fibroblasts. however, after and 24 hours of incubation, viable p. gingivalis suppressed basal cxcl accumulation. on the contrary, heat-killed p. gingivalis increased cxcl levels, indicating that p. gingivalis possess heat-instable structures that are responsible for the degradation of cxcl in correlation, previous studies have shown that heat-killed p. gingivalis induces higher levels of inflammatory mediators, in particular il- and cxcl than viable bacteria, suggesting degradation by the heat-instable gingipains. to further investigate the effect of p. gingivalis on cxcl the fibroblasts were pre-stimulated with tnf-α, a well known inducer of inflammatory mediators. lower doses of viable p. gingivalis in combination with tnf-α did not alter cxcl levels when compared to the positive tnf-α-stimulated control. however, higher concentrations completely abolished the tnf-α-induced cxcl accumulation, while corresponding concentration of heat-killed p. gingivalis did not cause the same effects. this further implies that the suppression of cxcl is due to the proteolytic capacities of the gingipains. to test this theory and evaluate the importance of gingipains, we used cathepsin b inhibitor ii and leupeptin, inhibitors of kgp and rgp, respectively. we found that p. gingivalis-mediated degradation is mainly dependent on rgp. these findings are consistent with our previous findings, as well as results from others, showing that the gingipains from p. gingivalis degrades il- and cxcl respectively. however, inhibition of rgp could only partially restore the cxcl levels, suggesting involvement of other proteolytic enzymes. it is also possible that a combination of rgp and kgp has a synergistic degradative effect, mediated by their specificity for cleavage after arginyl and lysyl residues, respectively. furthermore, dias and colleagues showed that there are two main types of cxcl a amino acid variant, secreted by immune cells, and a amino acid variant, secreted by non-immune cells. the latter was shown to have a lower chemotactic activity than the immune cell derived variant. however, upon cleavage by gingipains this shifted, and the amino acid variant increased the chemotactic activity of neutrophils compared to the amino acid variant. although gingipains degrade cxcl this consequently suggests that the chemotactic activity of cxcl increases upon stimulation of fibroblasts with p. gingivalis. however, more research is needed to determine the effects of p. gingivalis-derived proteolytic enzymes on the activity of these cxcl variants. to investigate whether the gingipain-mediated effects of p. gingivalis also include other fibroblast-derived inflammatory mediators, we performed a relative cytokine assay which measured various cytokines and chemokines. this assay revealed that tnf-α stimulated primary, human skin fibroblasts produce cxcl tnf-α, il- ccl ccl cxcl and cxcl remarkably, the fibroblasts produced mostly chemokines, indicating that fibroblasts might play an important role as a link between the innate and the acquired immunity. all tnf-α induced inflammatory mediators, except tnf-α, were suppressed by viable p. gingivalis, strongly suggesting an effect of the gingipains per se. this shows that gingipains have a broad proteolytic capacity and targets a wide array of cytokines and chemokines, thereby interrupting several signaling pathways. the chemokines ccl ccl cxcl as well as cxcl are all important for recruiting immune cells to the site of infection, and by inhibiting their biological activity, p. gingivalis is able to modulate and diminish the level of infiltrating immune cells. in contrast, viable p. gingivalis was not able to suppress tnf-α which is one of the most important inflammatory mediators. in fact, the level of tnf-α increased nearly two-fold by heat-killed bacteria, showing that p. gingivalis induce tnf-α expression in fibroblasts and, at the same time, degrade the tnf-α protein, although not extensively. periodontitis is associated with a decreased abundance of fibroblasts and tnf-α has been shown to be an important mediator of p. gingivalis-induced apoptosis. graves et al. demonstrated that the numbers of apoptotic fibroblasts were significantly reduced in the absence of the tnf-receptor, suggesting that tnf-α-signalling is an important part in apoptosis of fibroblasts. thus, our results may indicate that p. gingivalis stimulates apoptosis of fibroblasts through a less extensive degradation of tnf-α and this could account for the fibroblast apoptosis that is a distinctive feature of periodontitis. nevertheless, the degree of apoptotic fibroblasts after p. gingivalis infection need to be further investigated. in addition, it has been shown that the first nine residues of tnf-α n terminus are not needed for tnf-α protein to exhibit its biological activity. calkins and colleagues demonstrated that the two types of gingipains are able to individually degrade tnf-α, and also eliminate the biological activity. cxcl is a chemokine with pleiotropic functions. it works as a chemoattractant for its cxcr positive cells such as t cells, eosinophils, monocytes and nk cells, and it has also the capacity to induce apoptosis and regulate cell growth and proliferation, as well as angiogenesis. of interest, cxcl was the only chemokine that was suppressed by heat-killed as well as viable p. gingivalis, indicating that this chemokine is regulated by some additional mechanism beside that of heat-instable gingipains. for instance, a study by ohno et al. showed that cxcl and ccl gene is induced by p. gingivalis in osteoblasts and st mouse stromal cells and that nfκb inhibitor suppressed ccl expression but not cxcl. this suggest that p. gingivalis modulates cxcl gene expression through an nfκb-independent pathway. of further interest, the expression of cxcl is increased in autoimmunity diseases like rheumatoid arthritis and multiple sclerosis. for instance, lee et al. reported that cxcl contributes to bone-resorption by inducing osteoclast formation in rheumatoid arthritis via induction of receptor activator of nfκb ligand. however, since bone-resorption is a hallmark of progressive periodontitis, our results may indicate that cxcl plays a minor role when it comes to bone-resorption since even heat-killed p. gingivalis totally suppressed cxcl besides, high levels of cxcl have receptor-independent anti-microbial properties. even though it is questionable if such high levels, i.e. concentrations 100-fold higher than needed for chemotactic function, are realistic in vivo, prost and colleagues showed that this antimicrobial activity is achievable in vitro and may be an important response against bacterial infection. thus, the strong suppressive effect of cxcl by both viable and heat-killed p. gingivalis may in this case be beneficial for a sustained p. gingivalis infection. anyway, further research is needed about the regulation of cxcl and its signaling pathways as well as its role in bacterial infection. serpin-, was continuously expressed regardless of stimulation with tnf-α and/or bacteria. serpin- plays an integrated part of the plasmin system, working as an inhibitor of tissue plasminogen activator as well as urokinase plasminogen activator, both of which converts plasminogen to plasmin. interestingly, serpin- has been implicated in fibroblast senescent. serpin- is induced by various growth factors and has been suggested to be a down-stream target of p where p controls growth factor-dependent proliferation by upregulating serpin-. however, the fibroblast strains in our experiments were used at low passages.  in conclusion, our results show that a broad range of fibroblast-derived inflammatory mediators are inactivated by p. gingivalis due to proteolytic activities of gingipains, whereby the bacteria can create a more favourable milieu where it can evade the host immune system and promote its own growth and establishment. also, by differentially regulate the inflammatory mediators, such as cxcl and tnf-α, p. gingivalis may have an impact on the composition of inflammatory cells infiltrate and the inflammatory process itself. increased understanding of the role of fibroblasts in innate and acquired immunity and their interaction with periodontal bacteria is crucial for developing new strategies for preventing and treating periodontitis and related chronic inflammatory diseases. competing interests the authors declare that they have no competing interests. authors’ contributions hk, ep and tb designed the study. ep wrote the manuscript with hk and tb. ep and hk performed the experiments. all authors read and approved the final manuscript. 
p. gingivalis possesses a number of pathogenic properties to enhance growth and survival, including proteolytic gingipains. the aim of this study was to determine if p. gingivalis infection modulates the inflammatory response of fibroblasts, including the release of chemokines and cytokines. human gingival fibroblasts or primary dermal fibroblasts were pre-stimulated with tumor-necrosis factor-α and cocultured with p. gingivalis. tnf-α-triggered cxcl levels were completely abolished by viable p. gingivalis, whereas heat-killed p. gingivalis did not suppress cxcl accumulation of cxcl was partially restored by an arginine-gingipain inhibitor. furthermore, fibroblasts produced several inflammatory mediators, notably chemokines, all of which were suppressed by viable p. gingivalis. cxcl levels were determined with elisa and the relative levels of various inflammatory mediators were determined by a cytokine assay. these findings provide evidence that fibroblast-derived inflammatory signals are modulated by heat-instable gingipains, whereby the bacteria can escape killing by the host immune system and promote its own growth and establishment. accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. in addition, we show that fibroblasts are important mediators of inflammation in response to infection and thereby play a crucial role in determining the nature and magnitude of the invasion of immune cells.
 porphyromonas gingivalis is a key pathogen in periodontitis, an inflammatory disease leading to destruction of bone and tooth-supporting tissue. p. gingivalis possesses a number of pathogenic properties to enhance growth and survival, including proteolytic gingipains. accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. the aim of this study was to determine if p. gingivalis infection modulates the inflammatory response of fibroblasts, including the release of chemokines and cytokines. human gingival fibroblasts or primary dermal fibroblasts were pre-stimulated with tumor-necrosis factor-α and cocultured with p. gingivalis. gingipain inhibitors were used to explore the effect of gingipains. cxcl levels were determined with elisa and the relative levels of various inflammatory mediators were determined by a cytokine assay.  tnf-α-triggered cxcl levels were completely abolished by viable p. gingivalis, whereas heat-killed p. gingivalis did not suppress cxcl accumulation of cxcl was partially restored by an arginine-gingipain inhibitor. furthermore, fibroblasts produced several inflammatory mediators, notably chemokines, all of which were suppressed by viable p. gingivalis.  these findings provide evidence that fibroblast-derived inflammatory signals are modulated by heat-instable gingipains, whereby the bacteria can escape killing by the host immune system and promote its own growth and establishment. in addition, we show that fibroblasts are important mediators of inflammation in response to infection and thereby play a crucial role in determining the nature and magnitude of the invasion of immune cells. porphyromonas gingivalisfibroblastschemokinescytokines 